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Special Edition n Cleaning Validation Editor and Publisher
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4 Institute of Validation Technology
CONTENTS
TABLE OF

Special Edition n Cleaning Validation III

Equipment Cleaning Validation: Microbial Control Issues . . . . . . . . . . . . . . . . . . . . . . . 6
by Destin A. LeBlanc, M.A.

Cleaning Validation: Maximum Allowable Residue: Question and Answer . . . . . . . 13
by William E. Hall, Ph.D.

Development of Total Organic Carbon (TOC) Analysis
for Detergent Residue Verification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
by James G. Jin and Cheryl Woodward

Total Organic Carbon Analysis for Cleaning Validation
in Pharmaceutical Manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
by Karen A. Clark

Detergent Selection – A First Critical Step in Developing
a Validated Cleaning Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
by Mark Altier

Analysis Cleaning Validation Samples: What Method? . . . . . . . . . . . . . . . . . . . . . . . . . . 35
by Herbert J. Kaiser, Ph.D., Maria Minowitz, M.L.S.

Control and Monitoring of Bioburden in
Biotech/Pharmaceutical Cleanrooms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
by Raj Jaisinghani, Greg Smith and Gerald Macedo

A Cleaning Validation Program for the ELIFA System . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
by LeeAnne Macaulay, Jeff Morier, Patti Hosler and Danuta Kierek-Jaszczuk, Ph.D.

A Cleaning Validation Master Plan for Oral Solid Dose
Pharmaceutical Manufacturing Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
by Julie A. Thomas
BONUS

Proposed Validation standard — VS-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Special Edition: Cleaning Validation III 5
Equipment Cleaning Validation:
Microbial Control Issues
By Destin A. LeBlanc, M.A.
Cleaning Validation Technologies

v

T
he PDA spring conference taminants.” How­­ever, Section 6.7 of
was held in Las Vegas, }…it is this document that covers “Micro­­bio­
Nevada in March 20, 2001.
The conference showcased clean­
becoming more logical As­pects” focuses exclusively
on the same issue discussed in the
ing validation, residue limits, bio­ common for FDA guidance document, namely
burden, micro­bial limits, and sani­ the issue of preventing microbial pro­
tization. This paper is based on a
regulatory liferation during storage.
pre­sentation at that conference. authorities As a practical matter, microbial
The initial focus of regulatory to cite residues on equipment surfaces are
documents relating to cleaning part of the contaminants that should
validation for process equipment manufacturers be reduced to an acceptable level;
in pharmaceutical manufacturing for deficiencies that acceptable level being what is
in­volved measuring residues of the safe for the manufacture of the sub­
drug active and the cleaning agent. related to sequently manufactured pro­duct.
For example, the introduction to microbial Unfortunately, very little has been
the Food and Drug Ad­mini­stra­tion written on what is a safe level for
(FDA) guidance document on clean­­ control in microorganisms following cleaning
ing validation1 states: “This guide is cleaning and/or sanitation.3,4 Part of the reason
intended to cover equipment clean­ for this is that microbial resi­dues are
ing for chemical residues only.” validation significantly different from chemi­
While admitting that microbial re­s­ programs.~ cal re­sidues. Chemical resi­dues are
i­dues are beyond the scope of the “in­ert” in the sense that it is easy to
guideline, that guidance document cal­culate (especially using scenarios
further states, “microbiological aspects of equip­ of uniform contamination in the subsequently manu­
ment cleaning should be considered,” particularly factured product) the potential levels and effects of
with reference to preventive measures so that micro­ those chemical residues in the subsequently manu­
bial proliferation does not occur during storage. The factured pro­duct should they be transferred to that
European PIC/S document,2 that was issued several subsequently manufactured pro­duct. With microbial
years later, does explicitly mention microbial re­si­ residues left after the cleaning process, the situation
dues. In Section 6.2.1, contaminants to be re­moved is somewhat different. Because microorganisms are
in­clude “the previous products, residues of cleaning living organisms, those left as residues on equipment
agents as well as the control of potential microbial con­ may change in number after the cleaning process, but

6 Institute of Validation Technology
Destin A. LeBlanc, M.A.

before the manufacture of the subsequently manu­ the cleaned equipment. However, many times this
factured pro­duct. Those microbes transferred to the does not include any assessment as to the effect of
subsequently manufactured product may also change that unchanged bioburden level on the subsequently
in number after they are incorporated into the subse­ manufactured product.
quently manufactured product in the manufacturing This paper will address issues covering ap­proaches
step. This change may be a significant reduction in to control of microorganisms in process equipment,
bioburden, either due to drying of the equipment or setting of acceptance limits, sampling techniques, and
due to a preservative in the finished drug product, approaches to providing acceptable documentation.
for example. This change may also involve rapid
proliferation, either due to suitable growth conditions Microbial Control Measures
in wet equipment during storage, or due to suitable
growth conditions in the finished drug product. Or, Control measures to reduce the bioburden on
they may result in no significant change in microbial cleaned process equipment include control of bio­
level, because the bioburden was due to bacterial burden of raw materials, the cleaning process itself,
spores (that will survive readily in
dried equipment), or because the
subsequently manufactured product }Some companies will measure the
was a dry product (with low water change in microbial levels on
activity). There­fore, knowing the
levels of microorganisms left on the equipment surfaces during storage
equipment following cleaning does of the cleaned equipment. However,
not necessarily give one the full
story of the po­ten­tial hazards of those many times this does not include any
microbial residues. Addi­tional in­for­ assessment as to the effect
mation is required to assess those
potential hazards. of that unchanged bioburden
Why has microbial evaluation level on the subsequently
during cleaning of process equip­
ment been a little discussed topic? manufactured product.~
Part of the reason is that it is not a
significant problem in process man­
ufacturing. Yes, it could conceivably be a problem if a separate sanitizing step, and drying of the equip­
cleaning and storage were inadequate. How­ever, for ment following cleaning. Bioburden of raw materials
the most part, cleaning and storage of pro­cess equip­ in­cludes the active, excipients, water, and any process­
ment, in so far as it applies to microbial residues, ing aids. In many cases, the manufacturer may have
probably is done relatively well in most pharmaceu­ little control over the bioburden of raw materials other
tical manufacturing facilities. On the other hand, it is than to accept a specification by the raw material sup­
becoming more common for regulatory authorities to plier. The most critical raw materials probably will be
cite manufacturers for deficiencies related to micro­ natural products, in which there may be considerable
bial control in cleaning validation programs. One variation in the levels and types of microorganisms.
reason for this seeming anom­aly is that while firms A solid monitoring program to control in­coming bio­
are adequately controlling microbial contamination of burden of raw material is necessary. If there could be
process equipment, there may be little documentation significant variation in bioburden, then that should
to support this. This lack of documentation includes be addressed in the cleaning validation Performance
any measurement of microbial residues during the Qualification (PQ) trials. At least one PQ trial should
cleaning validation and/or during routine monitoring. utilize the worst-case incoming bioburden of raw
Some companies will measure the change in micro­ materials to demonstrate adequate cleaning and micro­
bial levels on equipment surfaces during storage of bial control under those conditions.

Special Edition: Cleaning Validation III 7
Destin A. LeBlanc, M.A.

A second means of microbial control is the cleaning conditions (“initial” bioburden, time, temperature, and
process itself. The conditions of aqueous cleaning humidity). Needless to say, if the chemical sanitizing
are often hostile to microbial survival. These con­ditions step is performed im­mediately prior to manufacture of
include high temperature (commonly 60-80ºC), pH the subsequently manufactured product, then removal
extremes (>11 and <4), and the presence of oxidizers of the sanitizer chemical residues to an acceptable level
(such as sodium hypochlorite in biotechnology manu­ should also be demonstrated.
facture). In addition, the presence of surfactants in the A fourth consideration for control of microor­
cleaning solution can assist in providing good physical ganisms is drying the process equipment surfaces
removal of microbes (without necessarily killing them). following the final rinse. Drying the surfaces will
Good cleaning is also beneficial to microbial control in further reduce the levels of vegetative organisms on
that chemical residues left behind can provide a physi­ the surface. In addition, drying will assist in prevent­
cal “microbial trap” to allow microorganisms to survive ing microbial proliferation during storage. Drying
even in the presence of chemical sanitizers. Those can be achieved by heated air, heated nitrogen, or
chemical residues left behind might also serve as a by rinsing with alcohol. In all cases, the process can
nutrient source that allows microbes to proliferate dur­ be assisted by application of a vacuum (to speed the
ing improper storage. Based on the author’s experience, evaporation of the water or, in the case of an alcohol
in most cases, effective control of microorganisms in rinse, of the alcohol itself).
pharmaceutical process equipment can be achieved
with the use of an effective cleaning process, without Limits for Microbes
the need for a separate chemical sanitizing step.
In some cases, a separate sanitizing step may be As mentioned earlier, it is possible to reasonably
necessary. This may include sanitation by steam or by predict levels of chemical residues in subsequently
chemical sanitizers. Suitable chemical sanitizers for manufactured products based on the levels present on
process equipment include sodium hypochlorite (chlo­ equipment surfaces.5,6 With microorganisms, it is pos­
rine bleach), quaternary ammonium compounds, alco­ sible to measure levels on equipment surfaces; how­
hol (ethyl or isopropyl), hydrogen peroxide, and per­ ever, the effect of those residues will depend on what
acetic acid. It should be noted that, with the exception happens to those microorganisms once they come in
of alcohol and hydrogen peroxide, additional rinses contact with the subsequently manufactured product.
would be necessary to remove any chemical residues Areas that may have to be evaluated include the species
of the sanitizer from the equipment. Those chemical (including the so-called “objectionable” organisms),
residues may also have to be evaluated as residues to type of organism (vegetative bacteria versus bacterial
be measured in the cleaning validation protocol. For spore, for ex­ample), the presence of preservatives in that
such chemical treatments, it is not an expectation that subsequently manufactured product, the water activity
the equipment be sterile. Unless the final rinse is with of the subsequently manufactured product, as well as
sterile water, microorganisms will be reintroduced any subsequent sterilization process performed on that
into the equipment from the use of Water-for-Injection product. As a general rule, if the water activity is less
(WFI) or purified water as the final rinse. than 0.6, then it can be expected that microorganisms
Some companies will use an alternative to sanitizing will not proliferate (although they may continue to sur­
immediately after cleaning. This usually involves sani­ vive without reproducing).7 Water activity is a physical-
tizing after storage and immediately before use. This chemical measurement that ex­presses the water vapor
may be used in situations where it is difficult to control pressure above the test sample as a fraction of the water
microbial recontamination or proliferation during stor­ vapor pressure of pure water at the same temperature
age. It should be noted that control of storage condi­ as the test sample. For aqueous products with a neutral
tions, if possible, is preferable. The practice of relying pH, microbial proliferation can generally be expected
solely on a separate sanitizing step immediately before unless there is a preservative in the product. If there
manufacture should be discouraged. If this is practiced, is a possibility of microbial proliferation because the
then the sanitization step should be shown to be effec­ product is unpreserved and neutral, then that should be
tive in reducing bioburden under the worst-case storage addressed in setting limits.

8 Institute of Validation Technology
Destin A. LeBlanc, M.A.

Three methods to set microbial limits will be ment surfaces are not the only source of bioburden.
ad­dressed. The first (Case I) involve limits where the One must also consider the raw materials themselves,
sub­sequent product does not allow microbial prolif­ as well as the primary packaging, as potential sources
eration and is not subject to any further sterilization of microorganisms. The best way to deal with this
process. The second (Case II) involves subsequently issue is to develop information on the bio­burden of the
manufactured products that are terminally sterilized. raw materials and the primary packaging, and factor
The third (Case III) involves subsequently manufac­ these into the limits calculation. For example, if one
tured products that are processed aseptically. were dealing with an oral liquid, one might calculate
the contribution from the raw materials (assuming
Case I Limits the upper limit bioburden for each raw material) as a
If the subsequently manufactured product does not maximum of 27 CFU/g. At the same time the contribu­
allow microbial proliferation, then the determination tion from the primary packaging is determined to be 3
of acceptable microbial limits in the cleaned equip­ CFU/g. Therefore, the amount allowed from equipment
ment can be calculated using the same principles used surfaces would be 70 CFU/g (100 minus 27 minus 3).
for chemical residues with one important exception. An additional safety factor should be used to account
This process involves first determining the accep­ for the significant variability in microbiological enu­
tance limit in the subsequently manufactured product. meration. An appropriate factor may be on the order
This limit is typically given in Colony Forming Units of 5. There­fore, in this case, the limit (in CFU/g) that
(CFU) per gram of product. Once this is determined, would be allowed solely due to the cleaned equipment
then the limit per surface area of equipment (assum­ surfaces would be 14 CFU/g (obtained by dividing 70
ing uniform contamination) can be calculated based by 5). Higher safety factors also could be considered.
on the batch size of the subsequently manufactured These numbers are given for illustration purposes only.
product and the equipment surface area. It should be realized that the contribution percentage
How is the limit in the subsequently manufactured allowed from cleaned equipment would vary depend­
product determined? For chemical residues, it is based ing on the contributions from the raw materials and the
on dosing information for actives or toxicity in­for­mation primary packaging.
for cleaning agents. Such concepts cannot be directly Once the limit in the subsequently manufactured
applied to microbes. Fortunately, there are two good product allowed from the cleaned equipment sur­
sources of information relating to levels of microorgan­ faces is determined, the next step is to determine the
isms in products. One is the manufacturer’s own Quality limit per surface area (CFU/cm2). This is calculated
Control (QC) specifications for the product, that may exactly as it would be for chemical residues:
include a limit for bioburden in the product. A second
source is information given in the proposed United Limit per surface area =  LSP x MBS
States Pharmacopeia (USP) <1111> relating to SA
“Microbial Attributes of Non­sterile Pharma­copeial
Articles.”8 Examples of those limits are given below: where
LSP = Limit in the subsequent product
Solid oral: ≤1000 CFU/g MBS = Minimum batch size
Liquid oral; ≤100 CFU/g SA = Product contact surface area
Topicals: ≤100 CFU/g
In the example above, if the batch size is 200 kg
Note: Although these limits were discussed and and the product contact surface area is 260,000 cm2,
proposed in the Pharmacopeial Forum, these spe­ then the microbial surface limit of the cleaned equip­
cific recommendations were not adopted officially ment is:
as part of the 24th edition of the USP.
Unfortunately, this is where the one exception to Limit per surface area = (70 CFU/g)(200,000g) = 54 CFU/ cm2
the conventional treatment arises. When one looks at (260,000 cm2)
the bioburden in a finished drug product, the equip­

Special Edition: Cleaning Validation III 9
Destin A. LeBlanc, M.A.

If sampling were done with a typical contact plate ment surfaces where the subsequently manufactured
of 25 cm2, this would correspond to a limit of over product is aseptically produced. This case is slightly
1300 CFU per contact plate. Since it is reasonable different from Case II in that it is the equipment itself,
to count a maximum of only 250 CFU on a typical and not the product, which is subsequently sterilized.
contact plate, this would clearly be in the TNTC (too This case is relatively straightforward, because the
numerous to count) category. Needless to say, this will microbial limits on the surfaces of cleaned equipment
vary with the limit in the subsequently manufactured are established based on the assumed bioburden of the
product, the portion allowed from cleaned surfaces, the equipment surfaces for sterilization validation of that
safety factor used, batch size, and the shared surface equipment. No information on batch sizes or surface
area. However, under most reasonable scenarios, the areas is necessary. The assumed bioburden for the
calculated limit due to microorganisms on the cleaned sterilization validation can be used directly for limit
equipment surfaces will be significantly above what purposes. The only adjustment may be the incorpora­
should be (and can be) achieved by proper cleaning. tion of a safety factor (to accommodate normal varia­
As a general rule, a good cleaning process should tion in microbiological enumeration).
produce surfaces that contain no more than 25 CFU
per contact plate (<1 CFU/cm2). When failures occur, Measurement Techniques
generally they will be gross failures, with counts gen­
erally above 100 CFU per-plate. Conventional tools used for microbial enumeration
from surfaces can be used. These include rinse water
Case II Limits sampling (usually with membrane filtration), swab­
This involves setting limits for cleaned equipment bing (with desorption of the swab into a sterile solu­
when the product subsequently manufactured in that tion and then a pour plate count), and use of a con­tact
equipment is to be sterilized. In this case, the microbial plate. The choice of recovery medium and incubation
limit in the subsequently manufactured product can be conditions is usually dictated by the expected organ­
established based on the assumed bioburden of that isms. As a general rule, the initial focus is on aerobic
product at the time of sterilization. In other words, any bacteria. However, if anaerobic bac­teria or molds/
validated sterilization process depends on an assumed yeasts are suspected problems, these should be also
bioburden of the item being sterilized. That assumed evaluated.
bioburden then becomes the limit in the subsequently One issue that does not translate directly from
manufactured product. Once that limit in the subse­ chemical residue measurements is the idea of deter­
quently manufactured product is established, then the mining percent recovery using the sampling method.
calculations are the same as for Case I – a certain por­ In the measurement of chemical residues, the target
tion of that total limit is allowed from cleaned equip­ residue is spiked onto a model surface and the quan­
ment surfaces, a safety factor is applied, and then the titative percent recovery is determined. The amount
limit per surface area is calculated using the minimum re­covered as a percent of the amount spiked is consid­
subsequent product batch size and the product contact ered the sampling method percent recovery. Per­cent
surface area. It is significant that this issue is actually recoveries in chemical sampling measurement are
addressed in the FDA’s cleaning validation guidance generally above 50 percent. This percent recovery is
document; that states: then used to convert an analyzed sample value; for
example, if a chemical residue measured by a swab­
“…it is important to note that control of bio­ bing technique gives 0.6 µg of residue, then with a 50
burden through adequate cleaning and storage of percent recovery, this actually represents the possibil­
equipment is important to ensure that subsequent ity of 1.2 µg being on that surface. This concept can­
sterilization or sanitization procedures achieve not be applied directly to microbiological sampling.
the necessary assurance of sterility.” 9 The reason for this is partly the inherent variability in
microbiological testing. If one measured 10 CFU in
Case III Limits one test and 5 CFU in a duplicate test (a 50 percent
This third case involves setting limits on equip­ difference), one would be hard pressed to say that

10 Institute of Validation Technology
Destin A. LeBlanc, M.A.

those numbers are significantly different. In addition, limits should be included in the validation protocol,
how would one actually measure the percent recovery and measured as part of the three PQ trials. One
in a microbiological test? If a model surface is spiked should also include the absence of “ob­jectionable”
with a specific number of a certain bacterium, and organisms as part of the acceptance criteria.
then that surface is allowed to dry and is sampled, To deal with processes for which cleaning valida­
just the process of drying might cause a low recovery tion has already been completed, but for which no
of bacteria (due to the dying of vegetative bacteria by microbial evaluation has been done, there are two
drying). In addition, what species of bacteria would strategies available. The objective of each is to devel­
be used for the recovery study? op documentation that the cleaning process consis­
It is recognized that microbiological sampling tently provides equipment surfaces with acceptable
methods may understate the number of microbes on bioburden. One option is to perform a cleaning
a surface (indeed the concept of a CFU, that may validation PQ, measuring only bioburden on sur­
faces for comparison to calculated
acceptance limits. The other option
}One issue that does not translate is to initiate a routine microbiologi­
directly from chemical residue cal mon­itoring program as part of
the monitoring of cleaning. This
measurements is the idea of may involve something as simple
determining percent recovery as monitoring the bioburden in the
final rinse water to demonstrate con­
using the sampling method.~ sistency. This data, combined with
product QC data on bioburden, may
satisfy the need for adequate docu­
contain any number of bacteria, also clouds the issue). mentation.
There are two ways to view such an issue. One is to One should also consider one’s motivation for
make it clear that whatever variation exists in measur­ wanting to obtain assur­ance that the bioburden is
ing micro­organisms on surfaces is probably equally an ac­ceptably low after cleaning. If the im­petus for action
issue when one sets limits based on product limits or is due to lack of data, one should resist the impulse to
sterilization bioburden limits. Therefore, the variabili­ immediately add a sanitizer into the cleaning program.
ty issue becomes a “wash.” The other perspective is to The focus should be on developing data to demonstrate
ac­count for such variation by choosing extremely high the sufficiency of the current cleaning process. Adding
safety factors. In the calculation example for Case I, a separate sanitizing step only complicates matters by
a factor of 5 was used as a safety factor. Even if that adding additional residue concerns. If the impetus for
safety factor were increased to 10 or 20, the calculated action is due to observed high microbial counts on
acceptance limits would have still been ex­tremely equipment surfaces or (more likely) in manufactured
high, and still beyond what one should achieve with a product, then it is important to determine by careful
well-designed cleaning program. investigation whether that unacceptable contamination
is due to issues with the cleaning process, with stor­
Documentation Strategies age, or to both. In such a case, a separate sanitizing
step should only be added if the data fully support it.
How these issues will be addressed will depend on
the stage of the cleaning process development. For a Conclusion
new process being designed, the best strategy is to pre­
pare a calculation of microbial limits, and then design Bioburden on cleaned equipment is an impor­
the cleaning process to meet those acceptance criteria. tant concern in the cleaning process. Fortunately,
Included in that evaluation should be any change in most aqueous cleaning processes, properly designed,
bioburden (in particular, any increase or proliferation) should provide low and acceptable bioburden levels
on storage of the equipment. The micro­bial acceptance on equipment surfaces following the cleaning pro­cess.

Special Edition: Cleaning Validation III 11
Destin A. LeBlanc, M.A.

Proper drying and storage should provide assurance
Article Acronym Listing
that microbial proliferation does not occur be­fore
the manufacture of the subsequently manufactured CFU: Colony Forming Units
product in that equipment. Any scientifically justi­ FDA: Food and Drug Ad­mini­stra­tion
fied determination of acceptable bioburden levels, PQ: Performance Qualification
particularly for non-sterile products, is generally far QC: Quality Control
higher than what should be achieved in conventional USP: United States Pharmacopeia
practice. This is becoming more of a regulatory and WFI: Water-For-Injection
compliance issue, not because microbial contami­
nation is a widespread pro­blem, but rather because
pharmaceutical manufacturers may lack appropriate
documentation to support their practices. This can
easily be remedied by a separate validation protocol
to address microbial issues, or by routine monitoring
to demonstrate consistency. o

About the Author
Destin A. LeBlanc, M.A., is with Cleaning Validation
Technologies, providing consulting in the area of
pharmaceutical cleaning validation. He has 25
years experience with cleaning and microbial con-
trol technologies. He is a graduate of the University
of Michigan and the University of Iowa. He can be
reached by phone at 210-481-7865, and by e-mail
at destin@cleaningvalidation.com.

References
1. FDA. “Guide to Inspections of Validation of Cleaning Pro­
cesses.” 1993.
2. Pharmaceutical Inspection Cooperation Scheme. Recom­men­
da­tions on Cleaning Validation. Document PR 1/99-2. Geneva,
Switzerland. April 1, 2000.
3. A.M. Cundell. Microbial Monitoring. Presented at the 4th IIR
Cleaning Validation Conference, October 20-22, 1997. (http://
microbiol.org/files/PMFList/clean.ppt, accessed May 29, 2001).
4. S.E. Docherty. “Establishing Microbial Cleaning Limits for Non-
sterile Manufacturing Equipment.” Pharmaceutical En­gineering.
Vol. 19 No. 3. May/June 1999. Pp. 36-40.
5. G.L. Fourmen and M.V. Mullen. “Determining Cleaning
Validation Acceptance Limits for Pharmaceutical Manufact­uring
Operations.” Pharmaceutical Technology. Vol. 17 No. 4. 1993.
Pp. 54-60.
6. D.A. LeBlanc. “Establishing Scientifically Justified Ac­ceptance
Criteria of Finished Drug Products.” Pharma­ceutical Technology.
Vol. 19 No. 5. October 1998. Pp. 136-148.
7. R.R. Friedel. “The Application of Water Activity Measurements
to Microbiological Attributes Testing of Raw Materials Used
in the Manufacture of Nonsterile Pharma­ceutical Products.”
Pharmacopoeial Forum. Vol. 25 No. 5. September-October
1999. pp. 8974-8981.
8. <1111> Microbial Attributes of Nonsterile Pharmacopoeial
Articles (proposed). Pharmacopoeial Forum. Vol. 25 No. 2.
March-April 1999. Pp. 77857791.
9. FDA. “Guide to Inspections of Validation of Cleaning Pro­
cesses.” 1993.

12 Institute of Validation Technology
Cleaning Validation:
Maximum Allowable Residue
Question and Answer
W
e are involved in the pro­ The choice of which ingredient in
duction of soft gel­atin }…sometimes a multi-ingredient product should
capsules and tablets in serve as the focus of the cleaning
our newly built facility. Our prod­ the many validation is often a difficult one for
ucts consist of at least 17 minerals possible vitamin and mineral products. For
and multivitamins in a single pro­ classical pharmaceutical products,
duct, while other products consist of combinations the choice is usually based on choos­
the same ingredients having some of products and ing the most potent ingredient, or the
quantity (in MG) varying with the least water soluble ingredient, or a
previous one. In some products, equipment would combination of these two factors.
some vitamins are not present. I want result in so many For vitamins and minerals the choice
to know how to conduct a cleaning may be more difficult because of
validation study of each product. studies that the the many ingredients present in the
Again, I want to know which ingre­ company would formulation and the relatively small
dients I have to check after cleaning amounts present. Coup­led with these
of the equipment to determine the never be able to difficulties is often the difficulty in
residues? complete them assaying the very small amounts of
active re­sidues that might be pres­
• What will the limit be for the micro­­ during a ent after cleaning. My suggestion
bial contamination for the cleaning reasonable would be to identify an ingredient for
validation studies, and what will be which there is a good sensitive assay
the rationale for the same? period of time.~ available. For example, if one of the
• If I’m using some cleaning agent, in­gredients hap­pens to show good
then what rationale is used for de­tectable levels of fluorescence
keeping the limit the same? (e.g., riboflavin, folic acid, and certain B vitamins
show good fluorescence) in water, then this material

A: Thank you for your question. It is a very good could be selected as the “marker” material, and could
one because it represents cleaning from the serve as the ingredient to focus on during the analysis
point of view of a manufacturer of vitamins and min­ of the rinse samples. In the case of vitamins and min­
erals, which in some countries, are considered drugs, erals, it may be necessary, and even highly desirable,
and in other countries, are considered as “nutraceuti­ to take this ap­proach because of the extremely low
cals,” an important and emerging part of our business. levels of residues present after cleaning. It may also
The first specific question you asked related to be possible to examine equipment in a dark room with
how to conduct a cleaning validation for each prod­ the use of an ultraviolet light to identify areas of equip­
uct, and how to select which ingredient to check ment that are not cleaned sufficiently (an enhanced
after cleaning to verify that the cleaning is adequate. visual examination), again utilizing the known fluo­

Special Edition: Cleaning Validation III 13
William E. Hall, Ph.D.

rescent behavior of certain vitamins. A brief study will cases, I would sug­gest that you refer to the Internet,
need to be carried out to determine if this approach is and conduct a search on the toxicity or potency of these
appropriate and adequate for your particular situation. materials. You may be surprised to find that a vita­
I would suggest that you not try to con­duct cleaning min, such as folic acid, is quite potent in terms of its
validation for every product. The reason I say that medical effect and dosage.
is be­cause sometimes the many possible combina­ The limits for these products can be calculated
tions of products and equipment would result in so by allowing a certain small fraction of vitamins or
many studies that the company would never be able minerals to carry over to each dose of the following
to complete them during a reasonable period of time. product. Again, you will need basic information, such
If, for example, you have 50 products, and each could as the medical dosage of the initial product, the batch
be run on ten (10) different pieces of equipment, then size and dosage of the next or subsequently manufac­
you would need 500 studies to cover all the possible tured product. In terms of the safety factor, i.e., the
combinations and permutations. That is simply too factor that is used to reduce the allowable dosage, I
much of a re­source and cost issue for the average suggest that you use a factor of 1/100th for vitamin
company to face. It would be much better to divide and mineral products. A factor of 1/1000th is often
your products into groups or families, and choose one used for pharmaceuticals, but I feel a more generous
or two representatives from each group to conduct full factor of 1/100th is appropriate for vitamin and min­
cleaning validation. The assumption is that you can eral products. You could refer to some of the articles
pick some “worst-case,” most difficult to clean, potent published in the Journal of Validation Technology for
products from each group. The first step is to divide the details of how to calculate specific limits.
the products into groups. I don’t know the names and Your last question related to what rationale should
ingredients of the products your company manufactur­ be used for the cleaning agent itself. The basic
ers; however, you did mention that some products are re­quirement is that you be able to provide data that
vitamin products and others are mineral products. So I de­monstrates that the cleaning agent itself is re­moved
think there would be two major groups – vitamins and during the cleaning process, usually by the final rinse.
minerals. Then each of these groups might be further You will need to go through the same rationale for
divided, if necessary. For example, in the vitamin cat­ the product residue limits, i.e., establish a scientific
egory you may have some products that contain water basis or justification that shows that the most potent
sol­uble vitamins, and some that contain fat soluble ingredient in the cleaning agent is reduced to a medi­
vitamins. So now we have three (3) major groups cally insignificant level. It is beyond the scope of this
(water soluble vitamins, fat soluble vitamins, and answer to go into the mathematical details of how to
mineral pro­­ducts). So you begin to see our approach. calculate this data, but again the details can be found
It might be that if you have vastly different types of in the various articles published in the Journal of
mineral products you might want to also further divide Validation Technology. You will need to know about
that group into smaller groups. In any event, you want the ingredients in your cleaning agent, as they are
to have pro­bably four (4) to ten (10) products in each typically multi-ingredient formulations, just like our
group, and then pick a worst-case representative from pharmaceutical products, and you will need to get that
each group. So by choosing this “grouping approach,” information from your supplier of cleaning agents.
you have re­duced the work from a very large resource The good news is that if you use the same cleaning
requirement to a doable or achievable project. agent and cleaning procedure for many products, then
The choice of the worst-case representative should you only have to do a single cleaning validation study
be based on a combination of aqueous solubility and (three runs) for the cleaning agent. o
po­tency. The potency can be determined for some
pro­ducts by determining the amount present in the
product from the label or package insert. Sometimes This answer was provided by an Editorial Advisory
this may be a little confusing for vitamin products Board Member, William E. Hall, Ph.D. Dr. Hall be
because the amounts are listed in units instead of reached by phone at 910-458-5068, or by fax at 910-
quantitative amounts, such as milligrams. In these 458-1087, and by e-mail at cleandoct@aol.com.

14 Institute of Validation Technology
Development of Total Organic Carbon
(TOC) Analysis for Detergent
Residue Verification
By James G. Jin
and Cheryl Woodward
Boehringer Ingelheim Pharmaceuticals, Inc.

T
v
he 1993 FDA Guideline for concluded that the visual detection
cleaning validation states }…the of foam was the best method for the
that the removal of deter­ detergents they tested.4 The method
gent residues should be evaluated biotechnology and of visual detection of foam is only
and there should be no or very low pharmaceuti- effective for foaming detergents,
detergent levels left after cleaning. 1
but is invalid for low foaming deter­
Currently, the pharmaceutical in­dus­ cal industry has gents. From a user’s point of view,
try employs varieties of detergents become this paper documents that TOC is an
for cleaning and different clean­­ing effective and quantitative method
validation programs. Many com­ increasingly for detergent residue verification.
panies have not included detergent interested in
residue evaluation as part of their Total Organic Carbon
cleaning validation programs main­ the use of Methodology
ly due to unavailability of ef­fective TOC [Total
methodologies or lack of aware­ness TOC is a non-specific method for
of the requirement by man­agement. Organic Carbon] the compound analyzed. How­ever,
In the late 1970s, To­tal Organic as an analytical TOC analysis is sensitive to very
Carbon (TOC) analysis had been low levels of 0.002-0.8 ppm carbon,
used for monitoring water quality in tool in cleaning depending on whether the sample is
pharmaceuticals and en­viron­mental validation a water sample or a swab sample.
controls. More re­cent­ly, the biotech­ Cur­rent­ly, two major oxidation tech­
nology and pharmaceutical industry programs.~ nologies dominate the TOC market:
has be­come in­creasingly interested combustion and Ultra Violet (UV)/
in the use of TOC as an analytical persulfate. There has been debate
tool in cleaning validation programs. TOC analy­ about which technique is better suited for TOC testing
sis has been used as an analytical tool for cleaning since the late 1980s. The major differences for each
validation in the biotechnology industry for years.2,3 technique5 are described in Figure 1, and give the user
Westman and Karlson recently conducted a compari­ appropriate information to make an informed deci­
son study for different analytical methods – visual sion as to which technique better serves their needs.
detection of foam, pH, conductivity measurements, The best TOC oxidation technology is the one
and TOC for detergent residue evaluation. They that meets the application and analytical needs of the

Special Edition: Cleaning Validation III 15
James G. Jin

Figure 1
Types of Total Organic Carbon Techniques
Oxidation Detection Technique Analytical Range (TOC) Official Methods
Combustion Thermal Conductivity Detector (TCD) 0.5 – 100% AOAC 955.07
Combustion Coulometric 1 – 100% ASTM D4129
UV/Persulfate Non-Dispersive Infrared Detector (NDIR) 0.002 – 10,000 mg/L USP 643
Heated Persulfate NDIR 0.002 to 1,000 mg/L USP 643
Combustion NDIR 0.004 – 25,000 mg/L USP 643
UV/Persulfate Membrane/Conductivity 0.0005 – 50 mg/L USP 643
UV Conductivity or NDIR 0.0005 – 0.5 mg/L USP 643

user’s situation. The UV/Persulfate method meets degradation of all carbon species to carbon dioxide,
precision and accuracy requirements for low-level water, and other oxides of heteroelements. The UV
cal­ibration check standards such as 0.5 ppm carbon light alone induces breakdown of many carbon spe­
in detergent residue evaluation. However, if captur­ cies with the persulfate providing additional help to
ing the particulate organic matter in the TOC value attack compounds difficult to oxidize. The radical
is important, then combustion would be the better reactions are aggressive and indiscriminate in their
oxidation technology. The instrument we chose is a attack.
Tekmar-Dohrmann Phoenix 8000 with the UV/Per­
sul­fate oxidation technique. S O -2 → SO -1 + R → H O + CO
2 8 4 2 2

Chemistry of Oxidation and Total Organic The NDIR is constructed in such a way as to be
Carbon Analysis of UV/Persulfate sensitive and selective for carbon dioxide present
in the gas flow. An infrared beam from the source
Wet chemistry oxidation of carbon compounds is passed through a chopper and down the sample
utilizes two chemical reactions to complete the chamber to a dual chamber detector. Each chamber is
analysis. A 21 percent solution of phosphoric acid filled with carbon dioxide and is separated by a thin
is utilized in converting inorganic carbon species. membrane. Varying intensity of the light hitting the
Acid­ification of the sample allows for attack on inor­ cell causes fluctuation in temperature and thus the
ganic species such as carbonates and bicarbonates pressure of the gas inside the detector. This causes
to convert them to carbon dioxide. This, along with the membrane to deflect, which is ultimately read as
any dissolved carbon dioxide in the sample is then a millivolt output signal from the detector.
sparged out, and either exhausted to vent or routed
to the Non-­Dispersive Infrared detection (NDIR) for Detergent Evaluation
quantification when analyzing for Inorganic Carbon
(IC) or TOC by difference (TC-IC). Three detergents (CIP-100, CIP-200, and Sparquat
256) were tested both in-house using the Tekmar
H+ + CO -2 → H O + CO
3 2 2
Dohrmann Phoenix 8000 TOC Analyzer and at a
contract lab, Quantitative Technologies Inc. (QTI),
Persulfate is used to do the rest of the oxidation to ver­ify the total amount of organic carbon in each
chemistry that is required for analysis. Sodium persul­ de­tergent at its original concentration. The method
fate, at a concentration of 10 percent, and phosphoric and instrument used at QTI was a Perkin-Elmer CHN
acid, five percent are added to the UV chamber for Analyzer 2400. This experiment was performed to
analysis. The persulfate species in the presence of make a comparison between our instrument and the
UV light breaks down at a weak oxygen-oxygen instrument in a qualified contract laboratory for infor­
bond yielding two radicals per molecule. These radi­ mation purposes only. One detergent (Chlor-Mate)
cals start chain reactions that ultimately lead to the was tested in-house and compared with the available

16 Institute of Validation Technology
James G. Jin

vendor’s specification. The TOC results for all the and TX2418 show acceptable results with respect to
detergents are shown in Figure 2. result consistency. The average of the seven TOC
The differences between the in-house and QTI results from TX2412 and TX2418 found in Figure
results with respect to the TOC assay for CIP-100 and 3 is 0.8327 ± 0.1860 ppm carbon. The variation is
CIP-200 are 5.0 percent and 9.6 percent, respectively. acceptable compared to the acceptance criterion of
These differences are relatively low compared to the three ppm carbon. These two swabs with the same
20 percent recovery criteria during recovery studies. material were selected to be our TOC swabs (cut to
The difference between the in-house and QTI results 5x5 cm2) for detergent residue verification.
with respect to the TOC assay for Sparquat 256 is The TX3340 TOC cleaning validation kit including
28.4 percent. The in-house result was reviewed and Eagle EP Picher 03464-40mL clear vials, Tex­wipe®
no error was noted in the performance of the test­ TX714L-large SnapSwabsTM, and blank vial labels
ing procedure. The major differences may be due to may be chosen since it is specially de­signed for TOC
in­strument and testing method variations. The result swabbing purposes.
for Chlor-Mate is within the vendor’s specification.
Detergent Recovery Evaluation from Stainless
Swab Selection Steel Surface
Ten stainless steel templates were spiked with
It has been known for years that polyester is a detergent solution and swabbed using the polyester
suitable material for TOC swabbing analysis. Over wipers AlphaSorb® HC TX2418 (5x5 cm2) for the
20 different kinds of polyester swab samples were detergent recovery study. The spiking and swabbing
received from The Texwipe Company LLC. Five procedures were the same as those used for drug
of them were chosen for TOC evaluation based on substance recovery studies. Forty mL of ultra puri­
sample design and the convenience for use. The fied water was added to each test tube as the extrac­
purpose of this experiment was to select a type of tion solution, vortexed about one minute, and then
swab that has little TOC background interference and sonicated for five minutes for testing. The results are
with consistent TOC results over time. Ultra purified shown in Figure 4.
water with 0.05 to 0.08 ppm carbon was used for The recoveries for CIP-100, CIP-200, and Chlor-
swab analysis. The TOC results obtained from our Mate are over 80 percent and no correction factor is
TOC analyzer are shown in Figure 3. necessary.
Swabs TX761 and TX741A showed increasing For Sparquat 256, a correction factor of 0.61 will
TOC results from 0.0813 to 0.9692 ppm carbon and be used. For example, if a result of 0.5 ppm carbon
from 0.1724 to 1.1246 ppm carbon over five days, is obtained from the TOC analyzer, the final reported
re­spectively. Swab TX700 showed an unacceptably result would be 0.82 (0.5 ÷ 0.61) ppm carbon.
high TOC result of 46.1991 ppm carbon at the begin­
ning of the experiment, and was therefore not tested Detergent Recovery Evaluation from Non-Stain­
further. None of these swabs are suitable for our less Steel Surfaces
TOC analysis. The aforementioned study was repeated using
Both polyester wipers AlphaSorb® HC TX2412 non-stainless steel templates. Two or three non-stain­

Figure 2
Total Organic Carbon Results for Detergent Evaluation
Detergent Manufacturer/Lot Total Organic Carbon Result TOC Results
Identification From BIPI* From QTI/Vendor
CIP-100 Vestal Convac lot 211097 4.0208 ± 0.0139% 4.22%
CIP-200 Convac lot 213915 2.4986 ± 0.0114% 2.26%
Sparquat 256 ISSA (lot: n/a) 14.0232 ± 0.9336% 18.0%
Chlor-Mate WestAgro® lot J8G0489AR 1.29% ± 0.0086% 1 – 1.5%
*Boehringer Ingelheim Pharmaceuticals, Inc.

Special Edition: Cleaning Validation III 17
James G. Jin

Figure 3
Total Organic Carbon Results (ppm C) for Swab Selection
Swab TOC/Two Hours TOC/Four Hours TOC/One Day TOC/Two Days TOC/Five Days
Description in H O 2
in H O in H O in H O
2
in H O 2 2 2

Polyester Alpha 0.0813 0.3221 0.3926 0.9410 0.9692
swab TX761 ± 0.0041 ± 0.0853 ± 0.0166 ± 0.0288 ± 0.0299
Polyester Alpha 0.1724 0.2509 0.5330 0.8091 1.1246
swab TX741 A ± 0.0144 ± 0.0068 ± 0.0250 ± 0.0200 ± 0.0394
Polyester wipers 1.1665 0.6091 0.8602 0.7535 0.9723
AlphaSorb® ± 0.0406 ± 0.0490 ± 0.0264 ± 0.0328 ± 0.0668
HC TX2412
Polyester wipers 0.7406 0.7269 N/A(1) N/A(1) N/A(1)
AlphaSorb
®
± 0.0056 ± 0.0297
HC TX2418
Polyester Alpha 46.1991 N/A N/A N/A N/A
swab TX700 ± 8.0761
1. Polyester wipers AlphaSorb ® HC TX2412 and polyester wipers AlphaSorb ® HC. TX2418 is same material cut to different sizes.

less steel templates were spiked with each detergent Figure 4
solution and swabbed using the polyester wipers
AlphaSorb® HC TX2418 (5x5 cm2). The results are Total Organic Carbon Recovery
shown in Figure 5. Results from a Stainless
For CIP-100 and CIP-200, the recoveries from Steel Surface
each non-metal surface are over 80 percent. There­ Detergent Percent Number Percent
fore, no correction factor is needed with respect to Recovery of Relative
the TOC recovery. For Sparquat 256, the recoveries Samples Standard
vary with different surfaces. The correction factors Deviation
are as follows: CIP-100 111.7 30 5.92
CIP-200 92.4 10 4.10
For Delrin surface: correction factor = 0.74 Sparquat 256 61.0 20 8.47
For Glass surface: correction factor = 0.75 Chlor-Mate 99.1 10 2.76
For Nylon surface: correction factor = 0.43 Note: R
 esults were automatically corrected for the
For Lexan surface: correction factor = 1.0 instrument blank effect.

Evaluation of Detergent Residue After Rinsing Figure 5
The purpose of this experiment was to evaluate: Total Organic Carbon Recovery
Results from a Non-Stainless
∂ The suitability of the Acceptance Criterion
(AC) of three ppm carbon
Steel Surface
∑ The effect of detergent concentration on deter­ Detergent Lexan Delrin Glass Nylon
Surface Percent Percent Percent Percent
gent residue after rinsing Recovery Recovery Recovery Recovery
∏ Recovery of detergent from different surfaces
with and without rinsing CIP-100 106.9 113.8 107.6 127.0
π Rinsing efficiency and rinse time CIP-200 90.3 92.3 97.4 93.2
Sparquat 83.3 74.0 75.1 42.5
256
Four detergents (CIP-100, CIP-200, Sparquat
256, and Chlor-Mate) were used in both a concen­ was pipetted and spiked onto the templates with
trated form and at a working concentration of 0.5 different materials of construction and dried with
oz/gal. Approximately one mL of detergent solution ventilation under a hood in the research and devel­

18 Institute of Validation Technology
James G. Jin

opment manufacturing area for a minimum of four The Tekmar Dohrmann Phoenix 8000 TOC ana­
hours. The templates were swabbed per standard lyzer was easily able to detect the non-rinse samples
swabbing procedure either before or after rinsing, with the results of 3.911 ppm carbon, 2.0928 ppm
using the polyester wipers AlphaSorb® HC TX2412 carbon, and 10.0868 ppm carbon for CIP-100, CIP-
cut to 5x5 cm2. The rinse was first conducted using 200, and Sparquat 256, respectively. The results
tap water and then purified water United States indicate that the AC of three ppm carbon is still high
Pharmacopoeia (USP), both at room temperature for detergents CIP-100, CIP-200, and Sparquat 256.
and with a slow flow rate of approximately 2.7 L/ The AC of one ppm carbon is acceptable. There
min. Two different rinse times (30 seconds and 60 were no differences in detectable residue for all four
seconds) were evaluated for different detergents on detergents (both concentrated and at 0.5 oz/gal) on
different templates to simulate the final rinse step in stainless steel after a 30-second tap water rinse fol­
our manual cleaning process. The recovery results lowed by a 30-second purified water, USP rinse.
are reported in Figure 6. Delrin was chosen for a typical material of construc­
Figure 6
Total Organic Carbon Results on Detergent Residue by Rinsing
Sample Concentration Templates Rinse Time Area TOC Results
Identification Swabbed (ppm C)d
CIP-100 0.5 oz/gal SS a No rinse 100 cm2 3.9111
CIP-100 0.5 oz/gal SS
a
30”/30”
b
100 cm2 Less than blank
CIP-100 Concentrated SS a 30”/30” b 100 cm2 Less than blank
CIP-100 0.5 oz/gal Delrin 30”/30”
b
100 cm
2
Less than blank
CIP-100 0.5 oz/gal Delrin 60”/60” b 100 cm2 Less than blank
CIP-100 0.5 oz/gal Nylon 30”/30” b 100 cm2 0.6682
CIP-100 0.5 oz/gal Glass 30”/30” b 100 cm2 0.0001
CIP-100 0.5 oz/gal Lexan 30”/30”
b
100 cm
2
Less than blank

CIP-200 0.5 oz/gal SS a No rinse 100 cm2 2.0928
CIP-200 0.5 oz/gal SS a 30”/30” b 100 cm2 Less than blank
CIP-200 Concentrated SS a 30”/30” b 100 cm2 Less than blank
CIP-200 0.5 oz/gal Delrin 30”/30” b 100 cm2 Less than blank
CIP-200 0.5 oz/gal Delrin 60”/60” b 100 cm2 Less than blank
CIP-200 0.5 oz/gal Nylon 30”/30” b 100 cm2 0.7720
CIP-200 0.5 oz/gal Glass 30”/30” b 100 cm2 0.0133
CIP-200 0.5 oz/gal Lexan 30”/30” b 100 cm2 Less than blank

Sparquat 256 0.5 oz/gal SS a No rinse 100 cm2 10.0868 c
Sparquat 256 0.5 oz/gal SS a 30”/30” b 100 cm2 0.2693 c
Sparquat 256 Concentrated SS a 30”/30” b 100 cm2 Less than blank
Sparquat 256 0.5 oz/gal Delrin 30”/30” b 100 cm2 Less than blank
Sparquat 256 0.5 oz/gal Delrin 60”/60” b 100 cm2 Less than blank
Sparquat 256 0.5 oz/gal Nylon 30”/30” b 100 cm2 0.3866 c
Sparquat 256 0.5 oz/gal Glass 30”/30” b 100 cm2 Less than blank
Sparquat 256 0.5 oz/gal Lexan 30”/30” b 100 cm2 Less than blank

Chlor-Mate 0.5 oz/gal SS a 30”/30” b 100 cm2 Less than blank
Chlor-Mate Concentrated SS a 30”/30” b 100 cm2 Less than blank
Notes: a. Stainless steel.
b. 30”/30” or 60”/60” – rinse time in seconds, tap water/purified water United States Pharmacopoeia (USP).
c. Result without correction factor.

Special Edition: Cleaning Validation III 19
James G. Jin

tion and 30/60 seconds were chosen for evaluation of = 0.79 mL of Chlor-Mate to be left on 100 cm2 of
the rinse time. There was no difference in detectable equipment surface after cleaning, respectively.
residue for CIP-100, CIP-200, and Sparquat 256 on
the Delrin surface after 30-second and 60-second Detergent Residue Verification Program
rinse times. The results also show that it is more dif­ Our detergent verification program is designed
ficult to remove residues of CIP-100, CIP-200, and to be a one-time verification for each detergent
Sparquat 256 from a Nylon surface than from other used. This was based on the rinse experiment and
materials. the assumption that our routine rinsing procedures
performed by well trained operators are sufficient to
Acceptance Criterion for Detergent Residue remove detergent residues to the level of less than
There is no universal AC for detergent residue the AC. This assumption has been verified from the
allowed to be left on GMP equipment surfaces. In results shown in Figure 6 that all the residues are eas­
our detergent residue verification program, the AC ily removed by a 30-second tap water rinse followed
for each detergent residue left on equipment surfaces by a 30-second purified water, USP rinse with very
depends on the sensitivity of the instrument used for low spray rate. Verification rather than validation is
analysis. This means we must set a low AC that is still currently required by the 1993 FDA, Guide to In­spec­­
quantifiable and applicable. Toxicity of the detergent tions of Validation of Cleaning Procedures due to
is not a concern at these trace amounts de­tergent the fact that detergent residue is less significant than
level. Effects on human health from re­sidue left on drug substance residue left after cleaning.
equipment surfaces should be insignificant at a low
concentration such as 0.5 oz/gal and with a routine Summary
rinse procedure. Our objective in this program is to
demonstrate that we are able to verify whether or not The detergent residue verification program has
the detergent residues are removed to an acceptable been successfully established using the Tekmar
low-level we can achieve. Dohrmann Phoenix 8000 TOC analyzer. This paper
Therefore, the AC should be established as close has shown the program development, and presents
to the instrument’s level of detection as possible. We critical data to support the detergent verification
tighten the initial limit of three ppm carbon to AC = reports for each detergent used.
1.0 ppm carbon (net reading automatically corrected The instrument Installation Qualification (IQ),
with blank by the instrument in a 40 mL solution), Operational Qualification (OQ), system calibration,
which is less than two times the blank baseline. The and the TOC analysis method development were
AC can also be expressed as AC ≤ 10 ppb carbon/ performed but not discussed in this paper. The poly­
cm2. This AC is practical and verifiable. ester wipers AlphaSorb® HC TX2412 and TX2418
The significance of the 1.0 ppm carbon AC for cut to 5x5 cm2 have been selected as the swabs for
each detergent can be explained in Figure 7. sampling detergent residue from equipment surface
We can see from the above calculations that AC for TOC analysis. The AC for the detergents CIP-
= 1.0 ppm carbon means, for all detergents at 0.5 100, CIP-200, Sparquat 256, and Chlor-Mate with
oz/gal, that we allow the maximum of 1 ÷ 3.92 = respect to TOC has been established as AC ≤ 10 ppb
0.26 mL of CIP-100, 1 ÷ 2.44 = 0.41 mL of CIP-200, car­bon/cm2. Two different rinse times, 30 seconds
1 ÷ 13.68 = 0.07 mL of Sparquat 256, and 1 ÷ 1.26 and 60 seconds, were evaluated. The results show
Figure 7
Significance of Total Organic Carbon Results for Detergent at 0.5 oz/gal
CIP-100 CIP-200 Sparquat 256 Chlor-mate
1 mL at 0.5 oz/gal 3.92 ppm 2.44 ppm 13.68 ppm 1.26 ppm
diluted to 40 mL
1.0 ppm C per 100 cm2 0.26 mL 0.41 mL 0.07 mL 0.79 mL
corresponding to

20 Institute of Validation Technology
James G. Jin

that 30-second/30-second rinse time (30-second rinse
with tap water and then 30-second rinse with puri­
fied water, USP) is sufficient to remove the detergent
re­sidues from different material templates including
stainless steel, Delrin, Glass, Nylon, and Lexan to a
level below the AC. The correction factors were de­ter­
mined based on the results of the recovery studies and
will be used by analytical sciences to report the final
TOC results for the detergent residue verification. o

About the Authors
James G. Jin is Chairman of the Cleaning Validation
Committee for Boehringer Ingelheim Pharma­ceuti­
cals, Inc., which is responsible for clean­ing valida-
tion program development and implementation. He
has more than ten years experience in pharmaceuti-
cal science and business arenas. He can be reach­
ed by phone at 203-798-5309.
Cheryl Woodward is Associate Director of Research
and Development (R&D) Manufacturing, for Boeh­
ringer Ingelheim Pharmaceuticals, Inc. She is
re­sponsible for all aspects of GMP manufacturing
for clinical supplies and has over 18 years experi-
ence in the pharmaceutical and related industries.
She can be reached by phone at 203-798-5367.

References
1. FDA. Guide to Inspections of Validation of Cleaning Pro­ce­
dures. July, 1993.
2. Jenkins K.M., Vanderwielen A.J, Armstrong J.A, Leonard L.M,
Murphy G.P, Piros N.A. 1996. “Application of Total Organic
Carbon Analysis to Cleaning Validation.” PDA. Journal of
Pharma­ceutical Science and Technology. 50. Pp 6-15.
3. Guazzaroni M., Yiin B., Yu J., 1998. “Application of Total
Or­ganic Carbon Analysis for Cleaning Validation in Pharma­ceuti­
cal Manufacturing.” American Biotechnology Laboratory. Septem­
ber. Pp. 66-67.
4. Westman L., Karlsson G., 2000. “Methods for Detecting Re­si­
dues of Cleaning Agents During Cleaning Validation.” Re­search
Article, Vol. 54, No. 5. September/October.
5. Furlong J., Booth B., Wallace B. 1999. “Selection of a TOC
Analyzer: Analytical Considerations.” Tekmar-Dohrmann
Ap­pli­cation Note. Vol. 9.20.

Special Edition: Cleaning Validation III 21
Total Organic Carbon Analysis
for Cleaning Validation in
Pharmaceutical Manufacturing
By Karen A. Clark
Anatel Corporation

v
I
n the pharmaceutical industry, specific methods like TOC is that
Good Manufacturing Practice }TOC analysis they cannot identify exactly what
(GMP) requires that the clean­ the residue material is. Depending
ing of drug manufacturing equip­ can be adapted on the chosen cleaning process and
ment be validated.1 Many different established acceptance limits, a
validation techniques can demon­
to any drug non-­specific method may be all that
strate that the manufacturing equip­ compound or is needed to validate the process.
ment is cleaned and essentially free TOC analysis can be adapted
from residual active drug substanc­ cleaning agent to any drug compound or clean­
es and all cleaning agents. that contains ing agent that contains carbon and
Common analytical techniques is “adequately” soluble in water.
in the validation process include carbon and is Studies have been conducted to
High Performance Liquid Chrom­ ‘adequately’ demonstrate that TOC methods can
atography (HPLC), spectrophotom­ also be applied to carbon containing
etry Ultraviolet/Visible (UV/Vis) soluble in water.~ compounds that have limited water
and Total Organic Carbon (TOC). solubility, and recovery results are
HPLC and UV/Vis are classified as equal to those achieved by HPLC.6
specific methods that identify and measure appropri­ TOC methods are sensitive to the parts per billion
ate active substances. TOC is classified as a non- (ppb) range and are less time consuming than HPLC
specific method and is ideal for detecting all carbon- or UV/Vis. United States Pharmacopoeia (USP)
containing compounds, including active species, TOC methods are standard for Water-for-Injection
excipients, and cleaning agent(s).2,3,4,5 and Purified Water,7 and simple modifications of
The disadvantage of specific methods, particular­ these methods can be used for cleaning validation.
ly HPLC, is that a new procedure must be developed
for every manufactured active drug substance. This Methodology
development process can be very time consuming
and tedious, plus important sampling issues must TOC analysis involves the oxidation of carbon and
also be considered. In addition, HPLC analyses must the detection of the resulting carbon dioxide. A num­
be performed in a relatively short time period after ber of different oxidation techniques exist, including
sampling to avoid any chemical deterioration of the photocatalytic oxidation, chemical oxidation, and
active substance. Finally, the sensitivity of HPLC high-temperature combustion. In this study, an Anatel
methods can be limited by the presence of degrada­ A-2000 Wide-Range TOC Analyzer, equipped with
tion products. Of course the disadvantage to non- an autosampler, was used. The Anatel A-2000 Wide-

22 Institute of Validation Technology
Karen A. Clark

Range Analyzer measures TOC in accordance with Figure 1
American Society for Testing and Materials (ASTM)
methods D 4779-88 and D 4839-88. It measures Linearity of CIP-100
TOC directly by adding phosphoric acid to the water 9000
sample to reduce the pH from approximately two to y=39.254x + 1.462
8000 R2=0.9997
three. At this low pH any inorganic carbon that is

Measured TOC (ppb)
7000
present is liberated as CO into a nitrogen carrier gas
2
6000
and is directly measured by a non-dispersive infrared 5000
(NDIR) detector. Any remaining carbon in the sample 4000
is assumed to be TOC. A sodium persulfate oxidant is 3000
then added to the sample, and in the presence of UV 2000
radiation, the remaining carbon is oxidized to CO . 2
1000
The amount of CO generated is then measured by
2
0
the NDIR to determine the amount of TOC originally 0 50 100 150 200 250
present in the water. CIP 100 Concentration (ppm)
For equipment cleaning validation there are two
types of TOC sampling techniques. One is the direct
surface sampling of the equipment using a swab. Figure 2
The second consists of a final rinse of the equipment Linearity of CIP- 200
with high-purity water (typically <500 ppb TOC)
and collecting a sample of the rinse for analysis. In 9000
y=19.132x + 51.042
general, direct surface sampling indicates how clean 8000 R2=0.9998
Measured TOC (ppb)

the actual surface is. This study demonstrates how 7000
to develop and validate a TOC method to measure 6000
a variety of different organic residues on stain­ 5000
less steel surfaces. Performance parameters tested 4000
include linearity, method detection limit (MDL), 3000
limit of quantitation (LOQ), accuracy, precision, and 2000
swab recovery. 1000
0
0 100 200 300 400 500
Linearity CIP 200 Concentration (ppm)

TOC analysis should provide a linear relationship
between the measured compound concentration and Figure 3
the TOC response of the analyzer. We evaluated
Linearity of Alconox
four different types of cleaning agents for linearity:
45
y=0.0355x + 1.1983
∂ CIP-100 (alkaline)
®
40 R2=0.9787
Measured TOC (ppm)

∑ CIP-200 ® (acidic) 35
∏ Alconox ® (emulsifier) 30
π Triton-X 100 (wetting agent) 25
20
Results are shown in Figures 1-4. Correlation 15
coefficients ranged from 0.9787 to 0.9998. Alconox 10
and Triton-X 100 have a tendency to foam, depend­ 5
ing on the concentrations that are analyzed and this 0
foaming phenomena can have a negative effect on 0 200 400 600 800 1000
the accuracy of the TOC result (reduced R2). Three Alconox Concentration (ppm)

Special Edition: Cleaning Validation III 23
Karen A. Clark

Figure 4 Figure 7
Linearity of Triton-X 100 Linearity of Endotoxin
12500 8000
y=415.76x + 16.997 y=0.9287x + 30.8
R2=0.9982 7000 R2=0.9998
Measured TOC (ppb)

Measured TOC (ppb)
10000
6000
7500 5000
4000
5000
3000
2500 2000
1000
0
0 5 10 15 20 25 0
Triton-X 100 Concentration (ppm) 0 2000 4000 6000 8000
Endotoxin Concentration (ppb)

Figure 5 representative examples of active substances were
also tested for linearity: an excipient (sucrose), an
Linearity of Sucrose antibiotic (vancomycin), and endotoxin. Results
12000
are shown in Figures 5-7. All three compounds
y=1.003x + 45.185 demonstrated excellent linearity with correlation
R2=0.9996
10000 coefficients (R2) ranging from 0.9996 to 0.9998.
Measured TOC (ppb)

8000
Method Detection Limit and
6000 Limit of Quantitation
4000
We determined the Method Detection Limit
2000 (MDL) by measuring the TOC response of the meth­
od blank.
0
0 2000 4000 6000 8000 10000 12000 A method blank consists of the sampling vial,
Sucrose Concentration (ppb) swab, and recovery solution. In this study, the
recovery solution was low TOC (< 25 ppb) water.
Ten pre-cleaned vials were filled with the low TOC
Figure 6 water. One swab was placed in each vial (Texwipe
Linearity of Vancomycin Alpha Swab TX761; tips cut off). Solutions were
vortexed and allowed to stand for one hour prior to
8000
y=0.8758x + 62.133 analysis. Four replicates from each vial were ana­
R2=0.9998 lyzed. The four replicates from each of the ten blank
Measured TOC (ppb)

6000 vials were averaged. These ten values were averaged
again and a standard deviation was calculated. The
4000
standard deviation was multiplied by the Student t
number for n-1 degrees of freedom (3.25 for n=10),
at 99% confidence levels to determine the method
2000
detection limit. The MDL was calculated to be 50
ppb. The Limit of Quantitation (LOQ) was calcu­
0 lated by multiplying the MDL by three. A value of
0 2000 4000 6000 8000 150 ppb was obtained (see Figure 8).
Vancomycin Concentration (ppb)
Precision and Accuracy

24 Institute of Validation Technology
Karen A. Clark

Figure 8
To demonstrate the precision and accuracy for this
Calculated TOC Averages TOC method, a representative solution of CIP-100 as
from 10 Blank Vials 1000 ppb, or one ppm as carbon, was analyzed sequen­
Vial Number Average TOC (ppb) tially ten times. This carbon concentration was chosen
1 58 to evaluate these method parameters because, in gen­
2 72 eral, TOC residual limits are typically around one ppm.
3 75
Results are listed in Figure 9. At this TOC level, the
4 93
5 79
precision was ± 1% and the accuracy was ± 5%.
6 102
7 60 Swab Recovery
8 83
9 67 Stainless steel plates were used in the swab recov­
10 54 ery test to simulate manufacturing equipment. One
Average 74.3 side of each plate was spiked with a solution of active
15.5
Standard Deviation
substance or cleaning agent. The plates were allowed
MDL (Student t, n=10) 50 ppb
LOQ 151 ppb
to completely dry overnight at room temperature. A
Texwipe alpha swab TX761 was moistened with low
Figure 9 TOC (< 25 ppb) water and the spiked plate surface was
swabbed both vertically and horizontally. The swab
Calculated Accuracy and Precision end was cut off, placed into a vial to which we added
from 10 Replicates of a 1ppm CIP- 40-mL of low TOC water. The vial was capped tight,
100 Solution as Carbon vortexed, and allowed to stand for one hour prior to
Vial Number Measured TOC (ppb) analysis. The same volume of each solution that was
1 1041 spiked onto the plates was separately spiked directly
1 1025 into 40-mL of low TOC water and analyzed. The per­
1 1039 cent recoveries of the different substances are listed in
1 1057
Figure 10. Reported values are the average of three
1 1054
2 1034
individual swab samples for each substance. The swab
2 1042 recoveries varied between 79.3% to 95.9%
2 1048
2 1054 Conclusion
2 1055
Average 1045 This study demonstrates that TOC analysis is
Standard Deviation 10.5 suitable for measuring organic residues on stain­
1.0%
% CV (precision)
less steel surfaces, and that it is a reliable method
% Recovery based on 105%
1 ppm C (accuracy) for cleaning validation as demonstrated by surface
residue recoveries of 79%-96%. This methodology
Figure 10
Representative Examples of Swab Recoveries from Cleaning Agents
and Active Substances
Substance ppm C of Spike ppm C of Spiked % Recovery % RSD
Standard Solution Plate
CIP-100 1810 1710 94.5 1.8
Sucrose 2663 2112 79.3 4.9
Vancomycin 661 634 95.9 3.0
Endotoxin 902 736 80.0 2.8

Special Edition: Cleaning Validation III 25
Karen A. Clark

shows that low limits of detection, excellent linear­
ity, precision, and accuracy can be obtained. All of
these TOC results, with the exception of Alconox
and Triton-X 100, were generated using the same
TOC method, making TOC analysis a low cost and
less time consuming alternative for cleaning valida­
tion. o

About the Author
Karen A. Clark is a Product Manager at Anatel
Corporation. She has over 15 years experience in
the pharmaceutical/biotechnology industry focus-
ing on drug formulations, analytical methods devel-
opment and validation, and GLP/GMP laboratory
management. Clark holds a B.S. in Biochemistry
from Millersville University and an M.S. in Chemical
Engineering from the University of Colorado. She
can be reached by e-mail at kclark@anatel.com or at
Anatel Corporation, 2200 Central Avenue, Boulder,
CO 80301.

References
1. FDA. Current Good Manufacturing Practice Regulations, 21
CFR 211.220.
2. Baffi, R. et al. 1991. “A Total Organic Carbon Analysis Method
for Validating Cleaning Between Products in Bio­pharmaceutical
Manufacturing.” Journal of Parenteral Science and Technology
45, no. 1: 13-9.
3. Jenkins, K. M. et al. 1996. “Application of Total Organic Carbon
Analysis to Cleaning Validation.” PDA Journal of Pharm­
aceutical Science and Technology 50, no. 1: 6-15.
4. Strege, M. A. et al. 1996. “Total Organic Carbon Analysis of Swab
Samples for the Cleaning Validation of Bioprocess Fer­men­tation
Equipment.” BioPharm (April).
5. Guazzaroni, M. et al. 1998. “Application of Total Organic Car­
bon Analysis for Cleaning Validation in Pharmaceutical Man­
ufacturing.” American Biotechnology Laboratory 16, no. 10
(September).
6. Walsh, A. 1999. “Using TOC Analysis for Cleaning Val­idation.”
Presented at The Validation Council’s Conference on Cleaning
Validation, 27 October, Princeton, New Jersey.
7. USP 23, Fifth Supplement, 15 November 1996.

26 Institute of Validation Technology
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Special Edition: Cleaning Validation III 27
Detergent Selection – A First
Critical Step in Developing a
Validated Cleaning Program
By Mark Altier
Ecolab, Inc.

v

T
he FDA recognizes the detergent for a given ap­plication
im­portance of effective }This article will should be based on sound, scientific
cleaning and sanitizing pro­ discuss the key reasoning.
tocols as a proactive measure in A sound rationale for detergent
preventing cross-contamination in factors that selection begins at the man­­ufactur­
the pharma­ceutical and cosmetic must be ing site, where the process and
in­dus­tries: clean­­ing program will take place. A
ad­dres­sed when full evaluation of the pro­cess, clean­
21CFR 211.67: “Equip­ment selecting a ing strategies, potential contaminant
and utensils shall be cleaned, main­
tained, and sanitized at appropriate
detergent. Each levels, and available utilities is a
good first step. Follow­ing this step,
intervals to prevent malfunctions factor will be laboratory testing is re­quired to
or contamination that would alter discussed in de­termine the exact nature of the
the safety, identity, strength, qual­ po­tential contaminant. Next, ident­
ity, or purity of the drug product detail and ifi­ca­tion and testing of various clean­
beyond the official or other estab­ examples are ing chem­istries against the potential
lished requirements.” contaminant is performed to deter­
given when mine which de­tergent type is best
In order to comply with this reg­ appropriate. suit­ed for con­taminant re­moval. The
ulatory requirement, sound clean­ next step is to return to the manufac­
ing and sanitizing protocols must
The roles of turing site, test the cleaning chem­
be developed and followed. One laboratory testing istry, and optimize the program.
of the most critical components of and plant This ap­proach provides a sound,
any cleaning program is detergent scientific rationale for the detergent
se­lection. Different pro­­cesses and optimization are selection and lays a firm foundation
po­tential contaminants may require also addressed.~ to the formal cleaning protocol, once
different de­tergents that are appro­ de­veloped.
priate for the application. In certain This article will discuss the key
cleaning ap­plications, a neutral foaming de­tergent factors that must be ad­dres­sed when selecting a
might be appropriate, where­as in others, a non-foam­­ detergent. Each factor will be discussed in detail
ing alkaline detergent is de­sirable. The choice of and examples are given when appropriate. The roles

28 Institute of Validation Technology
Mark Altier

of laboratory testing and plant optimization are also residue types will fall into one of the following three
addressed. categories: organic, inorganic, or a combination of
The Five Factors for Determining these. Most potential contaminants are a combina­
Detergent Suitability
Figure 1
There are five key factors that must be ad­dres­sed Common Residue Types in the
when determining which detergent is most suitable
Pharmaceutical Industry
for a cleaning application. These are:
Organic Residues Inorganic Residues
∂ Nature of the residue (or potential contami­ Eudragit Titanium Dioxide
nant) Acetaminophen Zinc Oxide
∑ Surface to be cleaned
Carbopols Iron Oxide
∏ Method of application
π Role of water Albuterol Sulfate Calcium Carbonate
∫ Environmental factors Neomycin Sulfate Inorganic Salts
Water/Oil – Oil/Water Silicon Dioxide
All five of these factors must be addressed when Emulsions
developing a cleaning program. Failure to address
Glyburide
any of these issues in sufficient detail can result in a
less than desirable cleaning program and could place
the successful completion of the cleaning validation tion of organic and inorganic components. Com­mon
at serious risk. residue types in the pharmaceutical industry are
given in Figure 1.
The Nature of the Residue A number of powerful analytical instruments are
A residue can be defined as any unwanted matter available that can provide tremendous insight into
or potential contaminant on the surface of the ob­ject the nature and composition of almost any unknown
or equipment being cleaned. Oftentimes, what is potential contaminant type. Some of the more useful
re­ferred to as a “residue,” is in fact a finished prod­ tools include:
uct, drug active, or other component that is produced
us­ing the process equipment that is being cleaned. • Fourier Transform Infrared Spectroscopy
The terms “residue,” “contaminant,” and “potential (FTIR)
con­taminant” will be used interchangeably through­ • Energy Dispersive X-Ray Spectroscopy (EDS)
out this article. • Scanning Electron Microscopy (SEM)
Determination of the nature of a residue is a funda­ • Compound microscopic imaging
mental component in the development of any clean­­ing • Nuclear Magnetic Resonance imaging (NMR)
program. In some cases, the exact nature and com­ • Inductively Coupled Plasma detector (ICP)
position of a residue is known. For example, if the • Atomic Absorption Analyzer (AA)
residue is a finished product, the exact composition
and physical properties are almost always known. Often, a combination of two or more of these tools
However, the identity and nature of the re­sidue may is required to provide a full picture of a potential
be completely unknown if the re­sidue is composed contaminant in question. For example, Fig­ure 2 and
of an intermediate, byproduct, or result of thermal, Fig­ure 3 are typical images generated to help char­
chemical, or other degradation of a previously known acterize unknown potential contaminant samples.
substance. This type of analysis is invaluable in determining the
The nature of the potential contaminant plays a ex­act residue type and breakdown of the organic and
central role in determining what type of detergent inorganic portions of a residue.
is most appropriate for the application. Individual Figure 2 is an FTIR image of an unknown re­sidue.
re­si­dues require different detergent chemistries. All This characterizes and gives a general breakdown of

Special Edition: Cleaning Validation III 29
Mark Altier

Figure 2
FTIR Scan of Unknown Sample. This Analysis Indicates the Presence of
Alkyl and Amide Protein Components and Possible Inorganic Content

.35

694.924
1631.22
1545.75
.30

1454.66
1396.07
1311.04
1251.26

1044.64
980.456
.25

1077.51

870.204
Absorbance

.20

2926.38
.15 2958.86

2854.86
.10

2155.88
2033.97
2013.07
2165.99
2287.55
2257.66
2211.86
3933.71

.05

0

3500 3000 2500 2000 1500 1000
Wave Number (cm-1)

Figure 3
EDS Scan of Unknown Sample
This Analysis Confirms the Presence of Inorganic Components Such as Silicon,
Aluminum, and Iron, in Addition to Organic Compounds
600
580 C
560
540
520
500
480
460
440
420
400
380
360
340
320 Fe
300
Counts

280
260
Si
240
220
200
180
160 Al
140
120
O
100
80 Mg Fe
60 Fe
40
20
0

0.000 1.000 2.000 3.000 4.000 5.000 6.000 7.000 8.000
Key

30 Institute of Validation Technology
Mark Altier

the organic portion of the residue. FTIR imaging may tolerate high pH, but may not tolerate chlorine or
gives valuable insight into the functional groups that chlorides. It is important to have a clear understand­
may be present in the organic component of a re­si­due. ing of how the substrate being cleaned will interact
Figure 3 confirms the presence of inorganic ma­terial with the detergent system, otherwise serious dam­
and identifies the specific inorganic components pres­ age to equipment surfaces can result. A SEM image,
ent in an unknown sample. This information is useful shown in Figure 4, is a stainless steel surface that has
when determining which chelant or surfactant family been pitted by using an in­compatible detergent. The
is most suitable for re­moving or tying up the free prospective customer in this case felt that the residue
metal ions and other inorganic material. was becoming more tenacious with time and was
Combined, FTIR and EDS imaging can give a using higher detergent concentrations to remove the
com­plete picture of most unknown residues. These residue.
analyses provide the information needed to select a A close look at the surface revealed that the surface
group of detergent chemistries that are formulated was actually being pitted by the detergent, providing
and known to be effective against the residue type. microscopic crevices where the residue was able to
harbor during the cleaning cycle. This problem was
Surfaces To Be Cleaned aggravated by the fact that the customer continued to
Different substrates (i.e., product contact surfaces, increase the detergent concentration, which acceler­
such as stainless steel, glass, or plastic) will interact ated the rate and degree of corrosion, and provided
differently with the contaminant and the de­tergent the residue with even more locations to harbor during
system. Some materials, such as glass, and alumi­num, the cleaning cycle.
are not tolerant to high pH systems. Other substrates These images clearly demonstrate the problems

Figure 4
Microscopic Corrosion of a Stainless Steel Surface Caused by Improper
Detergent Selection. Inset Shows Boxed Region at 1000 Times Magnification

Special Edition: Cleaning Validation III 31
Mark Altier

that can be caused by improper detergent selection. CIP application, as both create high-shear and thus
In this case, the customer was advised to discontinue are prone to foam formation. The result of this is a
the use of the incompatible detergent, and a compat­ detergent solution that foams out-of-process or CIP
ible detergent chemistry was identified and tested. vessels, cavitates pumps, and pro­vides inefficient
The customer was also required to re­place or re­pair surface coverage when sprayed on the inside of a ves­
damaged equipment. sel through a spray ball. Con­versely, a high foaming
When developing a cleaning protocol, it is neces­ detergent is desirable in a manual application, as this
sary to identify all components of the process that will gives the operator a visual indication of where the
be exposed to the cleaning chemical(s). This includes detergent solution has been applied to the surface.
equipment surfaces, gasket materials, nozzles, piping, Some cleaning application technologies exist
pumps, etc. It is also important to consider surfaces that are widely used in other industries, but have
that will be exposed to the vapor phase of the cleaning not taken hold in the pharmaceutical industry. These
solution, such as overhead spaces in enclosed vessels application methods include:
and pipes. A common mistake is to concentrate only
on items that will have direct contact with the liquid • Thin film cleaning
solution, neglecting the vapor phase. • Stabilized foam generators
• Built, solvated detergents (Generally Recog­
Method of Application nized As Safe [GRAS])
There are several common methods of applying
a detergent to equipment surfaces. Some are more Discussion regarding these application methods
common than others in the pharmaceutical industry. are outside of this article and will not be addressed.
Some of the more common methods of application
in the pharmaceutical industry include: The Role of Water
In general, 95-99% of a cleaning solution is
• Clean-in-Place (CIP) com­­posed of water. It is important to know the
• Clean Out-of-Place (COP) purity level of the water being used for cleaning and
• Manual scrubbing/wiping sanitizing. In many pharmaceutical applications, the
• High and low pressure spray water being used for cleaning and sanitizing is high
• Soaking/immersion purity water. However, this is not the case in every
application and in these cases, know­ing and under­
Each of these application methods dictate cer­ standing how the purity level of the process water
tain desirable or undesirable detergent properties. affects the cleaning process is critical. Some of the
For example, a high pH detergent is ideal in a CIP factors that can affect the cleaning process include
application where little, or no direct contact is made water hardness, pH, metals, salts, and microbial con­
be­tween the detergent and the operator. In a manual tamination. Refer to Figure 5.
application, however, a high pH detergent creates Of the factors listed above, water hardness has the
a significant safety risk to an operator handling the most significant impact on cleaning and sanitizing
de­tergent concentrate and use solutions. In a manual solutions. Water hardness can be classified as tem­
application, a neutral or mildly alkaline de­tergent porary or permanent hardness. Temporary hard­ness
(pH 7.0 – 10.0) is much more desirable as it sig­ indicates the presence of bicarbonates of mag­nesium
nificantly reduces the risk for accidental chemical or calcium. Both of these compounds are readily
burn to the operator’s eyes, skin, and mucous mem­ water soluble and can be present at high levels. When
branes. heated, these compounds react to form the carbonate
Other detergent characteristics, such as foam salts, which are water insoluble. Permanent hardness
properties, are important considerations in light of the refers to a condition where the chloride or sulfate
method by which the cleaning solution will be applied salts of magnesium and/or calcium are present in the
to a surface. A moderate-to-high foaming detergent is water. These compounds are also very water soluble,
not desirable when used in an agitated immersion or but are unaffected by temperature.

32 Institute of Validation Technology
Mark Altier

Figure 5 must not fall below four. If a strong acid or alkaline
detergent is used, the pH restrictions could be violated.
Typical Water Impurities That Can In this case, choosing a neutral, mildly alkaline or
Impact a Cleaning Process mildly acidic detergent may be the solution. However,
Component Chemical Problem in some cases, a strongly acidic or alkaline detergent
Formula Caused might be re­quired to effectively re­move the potential
Barium Sulfate BaSO 4
Scale contaminant from equipment surfaces. If a strong
Carbon Dioxide CO 2
Corrosion alkaline detergent is re­quired, the cleaning cycle could
Calcium Bicarbonate Ca(HCO ) Scale and be designed to include an acid rinse. The acid rinse
will help reduce the amount of rinse water re­quired to
3 2

Corrosion
Calcium Sulfate CaSO 4
Scale and neutralize residual alkalinity in the system, will help
Corrosion remove any inorganic residues, and can be captured
Iron Fe Scale and mixed with the alkaline wash water to neutralize.
Manganese Mn Scale In general, detergents will have the greatest
Magnesium Bicarbonate Mg(HCO ) 3 2
Scale im­pact on pH and phosphate levels. Relative to the
Magnesium Chloride MgCl 2
Scale and residue load, detergents generally have little im­pact
Corrosion on BOD, COD, TOC, or solids levels.
Magnesium Sulfate MgSO 4
Scale and If effluent restrictions exist, these should be
Corrosion ad­dressed in the early stages of the development of
Oxygen O 2
Corrosion a cleaning program to avoid compounded problems
Sodium Chloride NaCl Corrosion later on when the cleaning protocol is implemented.
Silica Si Scale At this point, five key factors that should be
Suspended Solids r Deposit and considered when selecting a detergent to be used
Corrosion as a part of a validated cleaning program have been
Both temporary and permanent hardness cause discussed. Once these factors are addressed and an
problems in alkaline solutions, as they both pre­ appropriate detergent chemistry is identified, labora­
cipitate in high pH solutions and cause scaling on tory testing should be done to verify that the chem­
equipment surfaces. Water hardness is responsible istry is effective against the potential contaminant.
for scaling, film formation, excessive detergent con­ Other cleaning parameters such as cleaning time,
sumption, and formation of precipitate. Water hard­ temperature, and concentration can be evaluated in
ness can be addressed by installing a water softening the laboratory as well.
system, or by using a detergent that is formulated to
handle hard water. Laboratory Testing

Environmental Factors Cleaning studies conducted in the laboratory can
Many pharmaceutical plants have some type of be designed to closely mimic the actual applica­
effluent restrictions mandated by local municipalities, tion method, such as a CIP system, or they can be
or by the plant’s internal effluent treatment facility.
Common factors that must be considered are pH, Figure 6
phosphate levels, Biological Oxygen De­mand (BOD) Water Hardness
or Chemical Oxygen Demand (COD) loading, Total
(Reported as CaCO ) Rating
Organic Carbon (TOC) levels, and solids levels. In
3

many cases, the correct choice of detergent can help Hardness Grains Parts Per
Per Gallon Million (PPM)
reduce the impact on components of the effluent
stream that are a concern. For example, if phos­phates Soft 0 – 3.5 0 – 60
are a concern, a detergent that contains low levels of Moderately Hard 3.5 – 7.0 60 – 120
phosphate can be used. Another example is a situation Hard 7.0 – 10.5 120 – 180
where the pH of the effluent must not exceed 10 and Very Hard >10.5 >180

Special Edition: Cleaning Validation III 33
Mark Altier

de­signed to stress the system to differentiate between
similar cleaning chemistries. An example of the lat­ Once a cleaning chemistry has been identified
ter is a designed study that removes all mechanical and verified in the laboratory and other cleaning
action from the system, forcing the chemistry type parameters such as cleaning time, temperature and
and concentration, thermal energy, and contact concentration have been established, testing and
time to act on the residue. This ap­proach is espe­ optimization must be carried out at the production
cially effective in differentiating between similar plant. Initial optimization and testing is usually done
chemi­stries that appear to be equally effective when on a pilot scale, prior to scaling up. The re­sults of the
ap­plied using some type of mechanical action. laboratory cleaning studies should be used as a guide
An important component of designed clean­ or a starting point for the optimization process at the
ing studies is the preparation of the residue being plant site.
tested.
Typically, the residue is applied to a 304 or 316 Conclusion
stainless steel coupon and the treated coupon is then
subjected to the cleaning solution. The application of Process cleaning is an integral component of any
the residue to the coupon is critical to obtain results pharmaceutical process. The five key factors that
that can be directly applied to the actual system in must be addressed to help identify a detergent when
the plant. For example, a manufacturing process developing a cleaning program have been defined
may involve a heating step that causes some of the and discussed. The interaction of these factors with
finished product to “bake” onto a vessel side wall. each other and with the development of a cleaning
To obtain re­sults that are ap­plicable to this situation, program must be understood. Lab­oratory testing
the residue should be applied to the coupon surface, is critical for documenting the ap­propriateness of
heated, and then allowed to bake for an equivalent the detergent selection for the cleaning applica­
amount of time as is experienced in the actual pro­ tion. Plant optimization is a final critical step prior
cess. If this is not done, the results of the study will to starting the validation process at the production
have little relevance to the development of a cleaning facility. When these steps are taken, a complete,
program aimed at removing a baked on residue from scientifically sound approach to the development of
equipment surfaces. a cleaning program can be documented. o
Prior to implementing any cleaning studies, a
set of success criteria must be established. Once the
cleaning studies have been completed, quantitative About the Author
measurements against the success criteria should be Mark Altier is a Principal Chemical Engineer for
made. Based on the results of this work, a final deter­ Ecolab Inc., where he manages their pharmaceu-
gent chemistry recommendation is derived. Ideally, tical and cosmetic programs. Mark has worked
the detergent chemistry should meet or exceed all for Ecolab for seven years and has held positions
established success criteria. in quality assurance, process engineering, and
The end result of the laboratory work will be a re­search and development. He can be reached at
scientifically sound recommendation of detergent 651-306-5876, by fax at 651-552-4899, or by e-mail
chemistry and other important cleaning parameters at mark.altier@ecolab.com.
such as cleaning time, temperature, and concentra­
tion. This is the basis for the overall cleaning pro­
gram that will be tested at the production facility.
The importance of performing preliminary labora­
tory testing is that it provides a sound, scientific
rationale of why the selected chemistry is appropri­
ate for the cleaning application.

Plant Optimization

34 Institute of Validation Technology
Analyzing Cleaning
Validation Samples:
What Method?
By Herbert J. Kaiser, Ph.D.
and
Maria Minowitz, M.L.S.
Steris Corporation

C
v
leaning validations are very and cons of each technique will be
difficult to perform. They ex­am­ined. Validating the methods
can be made easier if an }This article will will be discussed, as well. The ref­
ap­propriate method for analyzing erences included with this paper can
the samples is used. The method describe various be used to provide more in-depth
used should be based on the previ­ information to the reader and act as
ously established residue limits of
analytical guides to the available literature.
the active and cleaning agents. There technologies Choosing the appropriate ana­
are many choices of an­alyt­i­cal tech­ lytical tool depends on a variety of
niques that can potentially be used. available for use, factors.5,6 The most important fac­
This article will de­scribe various
analytical technologies avail­able for
particularly for tor is determining what species or
parameter is being measured.7 Is it
use, particularly for cleaning agent cleaning agent res- an or­ganic compound or inorganic
residues. Ref­­­er­ences are provided to compound? The next question is
guide the reader to more in-depth idues. References mea­sure­ment. How is this com­
information. are provided to pound going to be measured? Is it
Cleaning validation in the phar­ going to be swabbed from a surface
maceutical industry is of critical guide the reader to or determined from a rinse water
im­portance.1, 2, 3 There are many sample? If it is going to be swabbed
analytical techniques available that more in-depth from a surface, where will this swab­
can be used in cleaning validations. 4
information.~ bing oc­cur? Another im­portant fac­
The choice of the technique used tor in choosing an analytical tool is
in analyzing a particular sample is establishing the limits of the resi­
very important in cleaning valida­ due. The limit should always be
tion. The technique must be appropriate for measur­ established prior to selecting the analytical tool.8, 9
ing the analyte at and below the acceptance residue The limits should not be established solely based on
limit. Today’s analytical chemist has a wide vari­ detection limits of a particular method. Yet, another
ety of techniques available for use. These choices important factor in choosing an analytical tool is
in­clude specific and nonspecific methods. Many whether or not the method can be validated. If the
methods are complementary to each other. The pros method can’t be validated, then another technique

Special Edition: Cleaning Validation III 35
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

needs to be chosen. will need to be established for its analysis.
Sampling Technique Limit of Detection and Quantitation
Before choosing a method, some definitions need
The sampling technique plays a large role in to be established. The Limit of Detection (LOD) is
determining which analytical technique to use. Some the lowest amount of a compound that can be detect­
techniques are more applicable for swab samples, ed. The Limit of Quantitation (LOQ) is defined
and other techniques are more applicable for rinse as the lowest amount of a compound that can be
water sampling. The acceptable sampling techniques quantified. The LOD is usually lower than the LOQ,
include direct surface sampling (swab) and rinse but is never higher. The LOD should never be used
water samples.10 The rinse water sample is a direct to establish residue acceptance limits. The residue
measure of potential contaminants, but the analysis ac­cep­tance limit should be well above the LOQ so
should not just be a compendial test for water. Rinse that it can be accurately quantitated.
water analyses should be directed toward responses
peculiar to the possible contaminants. A questionable Specific and Nonspecific Methods
form of sampling is placebo sampling. The placebo A specific method is a method that detects a
method sampling is when the product, not contain­ unique compound in the presence of potential con­
ing the active ingredient, is processed in the specific taminants. Some examples of specific methods are
piece of equipment. This is analyzed for any active High Performance Liquid Chromatography (HPLC),
that may have been picked up from the equipment. ion chromatography, atomic absorption, inductively
A problem with placebos is the potential lack of uni­ coupled plasma, capillary electrophoresis, and other
formity. The contaminant may not be evenly distrib­ chromatographic methods. It should be noted that
uted throughout the placebo. Another problem is the HPLC is not inherently specific. What is meant is
analytical power of the tools that are used to analyze that the conditions in an HPLC measurement can
the samples. The residue levels may be extremely usually be adjusted to separate out known potential
low if in fact the contaminant is evenly distributed contaminants.
throughout the sample. The use of placebos is only Nonspecific methods are those methods that detect
acceptable if used with swab or rinse water data. any compound that produces a certain response. Some
Therefore, placebos are generally not used because examples of nonspecific methods are Total Organic
of the additional work involved. Carbon (TOC), pH, titrations, and conductivity. A
Another important factor to consider in choosing very interesting and sensitive nonspecific technique
an analytical method is the type of residue being is dynamic contact angle.11 Titrations may be specific
analyzed. Residues can be drug actives, formulation for acids or bases, but they are not specific for par­
components, cleaning agents, organic, inorganic, ticular acids or bases. There are, however, specific
water soluble, water insoluble, particulate, microbial, titrations for classes of surfactants.12
and/or endotoxins. If the residue being detected is a
drug active, and the method used for detection is the Interferences
same method that is used for quality control purposes A good nonspecific strategy that could be fol­
of the final formula, it must be established that the lowed is to first identify possible interferences. These
active has not changed its chemical nature during the interferences can be either positive or negative. The
cleaning process. That is, it must be established that nonspecific property is then measured, and the resi­
the active is still detectable and quantifiable using due is calculated as if all of the measured property
the analytical method. This can easily be established is due to that residue. For example, if the cleaning
by performing forced degradation studies. Exposing agent was the analyte and TOC was the method used,
the active to the cleaning compound at an elevated all of the TOC would be assumed to have come from
temperature and then analyzing that sample will help the cleaning agent and calculated as such. This would
determine the compatibility of the cleaner with the then provide a worst-case upper-limit value.
active. If the active has indeed changed its chemical There are many possible sources of interferences.
nature during the cleaning process, a new technique Cleaning agents and compounds can be a source of

36 Institute of Validation Technology
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

interferences, for example. Active agents and their is no need for heating or cooling the detector.
byproducts, water system components, maintenance While there are many advantages of UV detec­
materials, and the atmosphere can all be sources, as tors, there are also some significant disadvantages.
well as people, if samples are not handled properly. UV detectors cannot detect all types of compounds
The materials used to perform the analytical method and therefore are not considered to be universal.
can also be a source of interference. For example, if All compounds do not have chromophores. This is
a swab that has a high TOC value is used to sample, particularly true of surfactants that are used in the
it could increase the level of TOC detected. pharmaceutical industry. Dirty cells, air bubbles,
For specific methods, there should be no interfer­ and the use of gradients can affect baseline drift and
ence if the method is properly designed. Again, it detection capability. The limits of detection can be
should be stressed that the method must be able to fol­ higher than other detector types due to background
low the analyte after exposure to the cleaning en­viron­ interferences.
ment. It is necessary to establish that the cleaning
environment or the cleaning process does not change • Evaporative Light Scattering Detection
the analyte. For nonspecific methods (which measure a In ELSD, the compound is separated on an HPLC
nonspecific property), any compound with the prop­ column as usual, and then enters a nebulizer that is
erty that is introduced into the sample will interfere. combined with a gas stream and passed through a
For example, if the method being used is TOC, atmo­ heated column. The heated column evaporates the
spheric carbon that may enter the sample could cause mobile phase leaving the solid analyte in the column.
interference. With all nonspecific methods, there is a The solid analyte then passes through a detector that
need to identify potential sources of interference. consists of a laser or light source. The laser or light
source is scattered when it hits the solid analyte. The
n High Performance Liquid Chromatography detector then picks up this scattering.
The first technique that will be discussed is HPLC. There are many advantages associated with evap­
Almost every pharmaceutical company has an HPLC orative light scattering detectors. ELSD is claimed
instrument. HPLCs utilize a variety of detectors. to be universal. It is called universal because it can
These include ultraviolet (UV), fluorescence, elec­ detect any type of compound. ELSDs are simple,
trochemical, refractive index, conductivity, evapo­ versatile, and rugged in use. Since it is a mass
rative light scattering, and many others. The ultra­ detector, all compounds produce similar responses.
violet de­tector is by far the most common. However, Additionally, there is no baseline drift due to mobile
Evaporative Light Scattering Detection (ELSD) may phase effects.
be the most appropriate detector for cleaning agents. There are two primary disadvantages of ELSD.
We will discuss the use of both UV and ELSD detec­ First, there is a very limited choice of buffer salts
tors in depth. that can be used. Recall that the mobile phase is
evaporated or removed, leaving the analyte. Any
• Ultraviolet Detectors buffers that will not evaporate will also produce
There are many advantages of using UV detec­ solid particles that will then be detected and cause
tors. Many compounds have chromophores and interferences. The second disadvantage is that the
therefore, they can be easily detected by UV. Many nebulizer and detector must produce consistent par­
in­struments are equipped with diode array spec­ ticle sizes. This requires careful cleaning and moni­
tral capabilities. This allows for easy detection of toring of the nebulizer.
impurities or potential contaminants within peaks.
Ultraviolet detection usually requires no additional Actives and Detergent
reagents or post column or pre-column reactions. There are many types of residues that can be ana­
UV detectors are not harmful to the sample, if that is lyzed using HPLC techniques. These include both
important. They are generally inexpensive and read­ actives and detergent residues. When dealing with
ily available. Also, molar absorptivities are gener­ detergent residues, it is important to identify what
ally not affected by temperature and therefore, there is being analyzed: surfactant, builder components,

Special Edition: Cleaning Validation III 37
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

chelating agents, etc. The separation and quantita­ coating simply to make it more rugged. All common
tion of surfactants at low levels is difficult, at best. detection techniques (UV, fluorescence, etc.) can
Industry literature is full of references for surfactant be used in capillary electrophoresis detection. The
analyses using HPLC. The vast majority of tech­ capillary itself serves as the detector cell. A small
niques described in the literature are for the deter­ portion of the polyimide coating is scraped off prior
mination of surfactants in concentrated products.13,14 to use, and the bare portion of the capillary is placed
There­fore, the limits of quantitation and the limits of in the light path. This detection is different from that
detection are rather high. There are also references seen in HPLC because the detection occurs while the
for the analysis of surfactants related to the environ­ separation is taking place, rather than after separation
ment.15,16 In environmental analysis, the sample is has been completed. Using a Z-cell can increase the
pre-concentrated so that the limits of quantitation sensitivity of the technique. This is accomplished by
are very low. The pre-concentration
can be up to one thousand fold.
}The TOC is then computed by
Suggested Reading subtracting the inorganic carbon
Authors Lin, et. al., compared
the analysis of anionic, cationic, concentration from the total
and amphoteric surfactants con­ carbon concentration
taining n-dodecyl groups using
HPLC and capillary electrophore­ of the sample.~
sis.17 They found that HPLC was
best for all classes of surfactants, especially for for­ using a special accessory that bends the capillary,
mulated surfactants. Authors Carrer, et. al., utilized causing the source radiation to penetrate lengthwise
ELSD for amphoteric type surfactants.18 Amphoteric through the capillary rather than a cross-sectional
surfactants are a class of surfactants that display sampling. This, in effect, increases the path length
cationic behavior in an acidic solution and anionic of the cell. The Z-cell can be used in all types of CE
behavior in an alkaline solution. The lowest cali­ where UV detection is used.
bration standard that they utilized was 50 ppm, but CE can be used for many different types of analy­
they probably could have gone much lower. Authors ses. Surfactants can be determined quite readily using
Guerro, et. al., obtained a limit of quantitation of this technique.20,21 However, detection limits typically
0.49 ppm for alkyl polyethylene glycol ethers using are higher than with HPLC. This can be overcome by
ELSD.19 pre-concentrating the samples on the capillary itself.
A voltage is applied to the capillary in a manner that
n Capillary Electrophoresis allows the compounds to collect at one end of the
An interesting method of analysis is Capillary capillary without flowing through to the detector. An
Electrophoresis (CE). There are many different types advantage that capillary electrophoresis holds over
of CE. Capillary Zone Electrophoresis (CZE) is by HPLC is the ease with which indirect detection can
far the most common. CE instrumentation is fairly take place. Indirect detection is where a highly UV-
simple, consisting of a high voltage source, a capil­ absorbing material is included in the mobile phase.
lary, and a detector. The high voltage source is used As the analyte is eluted or travels along the capillary
to apply a potential across two solutions. One of through the detector, a negative peak is seen for the
the solutions contains the analyte, and the potential analyte. This typically is done for compounds that dis­
ap­plied to the solutions causes the analyte to migrate play low UV absorption. In addition to being useful
through the capillary, through the detector, and for the analysis of surfactants, capillary electrophore­
into the other solution. The column or capillary is sis can be used to analyze organic acids, inorganics,22
typically composed of fused silica with a polyimide and trace drug residues.23
coating. The diameter of the capillary is typically
25-75µm in diameter. The capillary has a polyimide Suggested Reading

38 Institute of Validation Technology
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

Vogt, et. al., provided a good overview of the CE of 4.0 ppm; and for HPLC, they obtained a limit
separation of cationic, anionic, and nonionic surfac­ of quantitation of 5.0 ppm.
tants using capillary electrophoresis.24 They indicated n Total Organic Carbon
that one can easily adjust the parameters of the sepa­ TOC is used widely in the pharmaceutical indus­
ration to coelute or separate oligomers. Coelution of try.31, 32, 33 The TOC is determined by the oxidation
the oligomers increased the sensitivity at the ex­pense of an organic compound into carbon dioxide. This
of increasing the potential for coeluting positive oxidation can occur through a number of mecha­
interferences. Direct UV detection could be used nisms depending on the instrument being used.
for UV-absorbing materials and indirect or non­-UV Some typical methods are persulfate, persulfate/UV
absorbing materials. oxidation, and direct combustion. The carbon diox­
Heinig, et. al., utilized micellar electrokinetic cap­ ide that is produced from these oxidations is either
illary chromatography for the separation of non-ionic measured using conductivity or infrared techniques.
alkylphenol polyoxyethylene type surfactants.25 How­ In­stru­ments generally measure the inorganic carbon
ever, the use of this method was limited because of content of a sample. The inorganic carbon consists
insufficient peak resolution and relatively low detec­ of carbon dioxide, bicarbonate, and carbonate. They
tion sensitivity. Heinig, et. al., also compared HPLC then determine the total carbon content of the sam­
and CE analyses of surfactants.26 The surfactant types ple. The TOC is then computed by subtracting the
they studied were linear alkylbenzenesulfonates, inorganic carbon concentration from the total carbon
nonylphenolpolyethoxylates, cetylpyridinium chlo­ concentration of the sample.
ride, and alkylsulfonates. For the CE analyses, they There are two primary advantages associated
utilized UV detection either in the direct or indirect with TOC. The first is that it does not take long to
modes, depending on the nature of the surfactant. develop a method. There are not a lot of variables in
For the HPLC analyses, they utilized either direct the actual analysis. The second advantage is that it is
UV detection or conductivity detection. An­ionic sur­ relatively quick. A third potential advantage (which
factant samples were pre-concentrated one thousand can also be a disadvantage) is that it will detect and
fold through the use of solid phase extraction. This analyze any compound containing carbon.
allowed for detection limits in the parts per billion As with most techniques, there are disadvantages
range to be obtained. in using TOC. A significant disadvantage is that the
Kelly, et. al., utilized CE with indirect detec­ compound or the analyte must be water soluble. This
tion to determine sodium dodecylsulfate concentra­ does not mean that the compound must be soluble in
tions.27 They also indicated that it is important to the hundreds of parts per million range but soluble in
look at the absorption of the surfactants onto filters the low parts per million range. Another disadvantage
if the samples are indeed filtered prior to analysis. is that organic solvents cannot be used. If organic
This is most important in dilute solutions. Filtering solvents were used, the TOC of the solvents would be
large volumes of sample can minimize this. Again, measured instead of the residue. There are also many
appropriate studies need to be done to determine if sources of contamination that can occur using TOC.
this indeed is a problem. These sources can include the atmosphere, the swab
Altria, et. al., examined the use of CE in the analy­ it­self, personnel, and many other sources. Methods
sis of sodium dodecylbenzenesulphonate.28 They de­veloped using TOC should be written to include
ob­tained a limit of quantitation of 0.6 ppm and a controls and blanks to identify or account for possible
0.3 ppm limit of detection. They utilized direct UV contamination. For example, a common source of con­
detection. Shamsi, et. al., utilized CE with indirect tamination is the technique used to cut the handles of
de­tection for the determination of cationic and anion­ the swabs so that they fit into the TOC vials. Many
ic surfactants.29 The authors obtained limits of detec­ times, the scissors or utensils are not clean enough
tion of 0.25 and 0.5 ppm, respectively. Heinig, et. al., for TOC use. This introduces contamination into the
also utilized CE in the analysis of cationic surfactants sampling vial when the swab is cut.
using indirect UV detection.30 They compared this
with HPLC. They obtained a limit of quantitation for Excipients

Special Edition: Cleaning Validation III 39
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

Some methods/techniques can be used in certain cleaners.39, 40, 41 Most cleaners contain sodium and/or
situations to complement each other. Examples potassium. The ion chromatography detection tech­
in­clude TOC and HPLC. Consider the case of a drug nique of suppressed conductivity is more sensitive
in the presence of excipients. The excipients are very to potassium than it is to sodium. Very low levels of
soluble in water while the drug active has ex­tremely cleaning agent can be detected using this technique.
low solubility in water. The excipients con­tribute This assumes that the rinse water used contains no
to the TOC values because they are very soluble in potassium. Ionizable organic acids are also readily
water; however, the drug active does not show up in quantitated using ion chromatography. This includes
the TOC analysis. An HPLC analysis is performed chelating agents that are often found in cleaning
to monitor the loss of the drug. The ex­ci­pients are compounds.
removed much faster from a surface during cleaning
than the drug active is removed. In this case, TOC Suggested Reading
analysis is not a good stand-alone method. It is, In determining surfactants, an excellent review
however, a good complement for the HPLC assay. concerning their analysis was done by Vogt, et. al..42
The TOC analysis enables the analyst to see what They compared the use of HPLC, CE, ion chro­
water soluble matter is left behind, if any. matography, Liquid Chromatography-Mass Spectro­
scopy (LC-MS) and Gas Chromatography-Mass
Suggested Reading Spectro­scopy (GC-MS). They also discussed pre-con­
Guazzaroni, et. al., examined the use of total centration of the samples. They compared the use of
or­ganic carbon for the analysis of detergents, endo­ solid phase extraction, super critical fluid extraction,
toxins, biological media, and polyethylene glycol.34 Soxhlet extraction, and steam distillation as means
For detergents, they were able to obtain a 0.7 ppm of pre-concentrating samples. They found, by far,
limit of quantitation. Endotoxins were found to have that the best method was solid phase extractions
a 0.2 ppm limit of quantitation. The biological media for the pre-concentration of surfactants. They also
produced a total organic carbon limit of quantitation examined the use of titrimetric methods of analysis
of 20.3 ppm; and the polyethylene glycol produced a for surfactants. For detecting anionics, substances
0.5 ppm limit of quantitation. They examined swab like methylene blue, pyridinium azo, and triphenyl­
and rinse water recoveries. They were able to obtain methane dye was used to complex the surfactants
78-101 percent recoveries utilizing swabs, and 93 prior to photometric determination. Non­ionics were
percent or better for rinse water recoveries. determined indirectly by forming a cationic complex
There are many examples in the literature that uti­ with barium. This complex was then precipitated by
lize ion chromatography as the method for analysis bismuth tetraiodide ion in acidic acid. The bismuth
of surfactants.35 The surfactants have to be charged was then quantified by potentiometric titrations.
in order to be analyzed using ion chromatography, Cationics were complexed with anionic dyes such as
that is, only anionic or cationic surfactants can be disulfine blue.
detected. Pan, et. al., recorded limits of quantitation Theile, et. al., brought up an excellent point that
down to 0.5 ppm for linear alkane sulfates and sulfo­ surfactants tend to concentrate at interfaces.43 This
nates.36 Takeda, et. al., recorded a limit of quantita­ can be a problem in extremely dilute solutions of
tion of 0.1 ppm for dodecyl alkyl sulfates.37 Nair, et. surfactants. The surfactants can collect at the surface
al., separated different sulfate, sulfonate, and cat­ of the containers that they are stored in. This may
ionic type surfactants using ion chromatography with cause errors in analysis. Proper controls in studies
suppressed conductivity detection.38 They reported should be done to determine if this is a problem.
detection limits at less than 1.0 ppm. The authors indicated that pre-concentration was
re­quired to determine very low levels of surfactant.
n Ion Chromatography Solid phase extraction was the best method for this.
In addition to its use for surfactants, ion chro­ They were also able to obtain detection limits for
matography can be used for the analysis of inor­ linear alkylbenzenesulfonates of 2.0 ppb with fluo­
ganics and other organic compounds present in rescence detection and 10.0 ppb using HPLC with

40 Institute of Validation Technology
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

UV detection after pre-concentration. phosphate (ATP) bioluminescence.50 This is based on
n Thin-Layer Chromatography the reaction of ATP with Luciferin/Luciferase. This
There are many examples in the literature for the technique is often used in biopharmaceutical facilities.
use of Thin-Layer Chromatography (TLC) for the It has extremely high sensitivity and a very high repro­
qualitative determination of surfactants.44, 45 Henrich ducibility. In many cases, the instruments can be used
described the TLC of over 150 surfactants in six at the equipment site. This technique utilizes swabs for
different TLC systems.46 This was excellent for iden­ surface analyses.
tification of the surfactants, but the author did not
attempt to quantify the surfactants. Buschmann and n Optically Stimulated Electron Emission
Kruse combined diffuse reflection infrared spectros­ In some cases, a company’s established limits of
copy and TLC, along with SIMS and TLC for sur­ residue are so low that they cannot be detected by
factant identification.47 Although these techniques conventional methods. A very sensitive method that
are tedious and time-consuming, there is no doubt may be applicable is Optically Stimulated Electron
that these methods could be developed into quantita­ Emission (OSEE).51 The instrumentation for OSEE
tive analyses. Novakovic has used high performance is fairly portable, and can be readily taken to tank
TLC for two generic drugs.48 side for analysis. The technique uses a probe that is
placed against a surface, and a UV source illuminates
Other Techniques and activates the surface. When some surfaces are
Other excellent techniques for inorganic con­ exposed to UV light at certain wavelengths, electrons
taminants, and in some cases actives, are Atomic are emitted from the surface. The instrument measures
Absorption (AA)49 and Inductively Coupled Plasma the current that is produced. If even small amounts of
(ICP) atomic emission. These techniques can detect residues are present on the surface, the current will be
inorganic contaminants down to extremely low lev­ affected. The current can be affected either in a posi­
els. Inorganic contaminants in a system are often tive or negative way depending on the nature of the
ignored. These can come from rouge that forms in residue. This is an extremely sensitive technique.
Water for Injection (WFI) systems. They can also It can be used in either a qualitative or quantitative
come from the detergent utilized in cleaning the manner.
equipment.
n Portable Mass Spectrometer
n Fourier-Transform Infrared Spectroscopy For those companies that require ultrasensitive
Fourier-Transform Infrared (FTIR) spectroscopy measurements and identification of the residues, a
is never used as a stand-alone method for analyzing technique has been developed – Lawrence Liver­
residues on equipment. This is because of the lack of more National Laboratories has developed a port­
portability of FTIR equipment and the semi-quanti­ able mass spectrometer.52 The unit consists of a gun
tative nature of the reflectance techniques used for portion of the instrument that is connected with
these types of analyses. However, it is very useful cables to vacuum pumps. The gun portion is held
in performing screening studies and in evaluating against the surface to be analyzed. A seal is formed,
po­tential cleaning agents. This is done by soiling and the surface is heated to volatilize any com­
standard coupons with the cleaning agent, allowing pounds that are present. This instrument is used not
them to dry, and performing static rinsing studies. only to measure how much of something is present,
These types of studies can indicate whether or not but also what that something is. This piece of equip­
the cleaning agent is readily removed from surfaces. ment has been utilized in the aerospace industry.
The height or area of a particular peak is measured One drawback of the portable mass spectrometer
versus the concentration of the standard coupon. is that it requires relatively flat surfaces. However,
they are currently working on adaptors to be used on
n Bioluminescence non-flat surfaces.
Bioluminescence is quite useful for biologicals.
This type of analysis usually uses Adenosine Tri­ Additional Techniques

Special Edition: Cleaning Validation III 41
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

In the biopharmaceutical industry, a wide vari­ A minimum validation requires two different
ety of techniques are utilized. These include the analysts, instruments, columns (if chromatographic
53

Enzyme-Linked Immunosorbent Assay (ELISA),54 method), days, and prepared standards and sam­
the Limulus Amoebocyte Lysate (LAL), and a wide ples.60 These are the “twos” of method validation.
variety of protein determinations. These are all con­ The point of any method validation is to show that
taminant specific assays. For example, the LAL test the method can be utilized by different analysts and/
measures the level of endotoxins present. There is or laboratories, along with the ability to produce the
also the anthrone assay that can be used to monitor same results. If a validated method is given to a lab­
the levels of carbohydrates on sur­
faces. These techniques are usually
used in combination with TOC. }For those companies that require ultra-
The nonspecific techniques of sensitive measurements and
pH, conductivity, and titrations
can be used throughout all areas identification of the residues, a
of pharmaceutical manufacturing. technique has been developed
Ob­viously, these techniques are
most often utilized in rinse water – Lawrence Livermore National
monitoring. The conductivity and Laboratories has developed a
pH of rinse water is typically moni­
tored and compared to the conduc­ portable mass spectrometer .~
tivity and pH of the water prior
to introduction to the equipment.
If acidic or alkaline materials are being measured, oratory, that laboratory must revalidate the method
titration is a very useful technique. In some cases, for their laboratory. It is not sufficient or accurate
titration can be more sensitive than performing to assume that another laboratory’s validation will
TOC analyses. The sample size can be adjusted, apply in all laboratories. For example, if a surfactant
and/or the normality of the titrant can be adjusted to is being quantitated, typically a low wavelength
increase the sensitivity of the titration. is used in a UV detector for HPLC. UV detectors
vary in their noise levels at these low wavelengths.
Method Validation A detector used in one laboratory may have sig­
nificantly less noise than a detector in another lab­
It is very important to scientifically establish the oratory. The second laboratory may not be able to
residue limit prior to choosing the method of analy­ detect at the same low level as the first laboratory.
sis. This includes the limit in the analytical sample
and the limit in the next product. This will ensure Coupons and Swabs
that the method chosen will be able to detect and Coupons can be prepared for recovery studies
quantitate the limit chosen. Once the technique for through the use of aerosol bottles available through
analysis has been chosen, it is very important to vali­ laboratory supply companies. A known weight per­
date the method used.55- 60 The validation of a meth­od cent of a solution containing the analyte can be
is very different than the validation of recovery. A sprayed fairly evenly over the surface of a coupon.
validated method is one that is rugged and robust The coupon can be swabbed using a standard tech­
enough to measure the residue limit established. nique. It does not matter how you swab the coupon,
The validation of a recovery is to determine the as long as the complete surface is covered and that the
amount that can be recovered from a surface. Again, coupons are swabbed the same way – each and every
it should be stressed that these are two completely time. The type of swabs used in recovery studies must
different validations. be the same as those used in the validation protocol. If
this is a simulated rinse procedure, then the coupons
“Twos” of Validation are rinsed and the rinse water is analyzed.

42 Institute of Validation Technology
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

For swabs, a desorption process is carried out. Detergents can be quantitated either using spe­
This can consist of simply shaking the sample vial cific or nonspecific methods; however, care must
or using an ultrasonic bath. The samples are then be taken in choosing which component is measured.
analyzed. Recovery studies are always done below For example, a detergent may contain five percent of
ac­ceptance limits in the test solution. This ensures a surfactant and 20 percent of another organic ingre­
that the limit will be (or can be) measured in the anal­ dient. Assuming equal sensitivities of the analyti­
ysis. A recovery of greater than 80 percent is good. cal methods, the limit of quantitation of the whole
If the recovery is greater than 50 percent, it may be detergent system will be lowered by a factor of four
acceptable. However, if the recovery is less than 50 if the ingredient present in the greater amount is
percent, questions arise and the source of the poor determined.
recovery should be investigated. A possible cause of If a nonspecific method (i.e., TOC) is used for
a poor re­covery can be that the residue is being too the same system, a much lower limit of quantitation
tightly held by the swab. This can be investigated could be determined simply because there would be
by spiking a swab with a known amount of residue, a tremendous amount of carbon present in the sam­
allowing it to dry, trying to desorb the res­idue, and ple. In addition, if detergent systems are combined,
following up with analysis. If the analyte is held too such as in the case of adding a detergent additive
tightly by the swab, another type of swab material to another product, the choice of a specific method
should be investigated. The recovery factor should would be made even more difficult. The question
be included in analytical calculations or in the accep­ would be, “Which detergent do I determine?” A
tance limit calculation. It should not be included in disadvantage of using a nonspecific method for the
both of the calculations. entire cleaning validation analysis is that, if there is
a failure in the future, it would not be known where
Containers the failure originally occurred. The failure could be
The choice of containers used in the analysis of due to the active, excipients, detergent system, or
samples is very important. It has been shown that, even an unknown source.
in very dilute solutions, surfactants can adsorb onto
the surfaces of sample vials. This will produce arti­ Conclusion
ficially low results in the analysis. This, however,
typically only occurs in static systems. There is no There are many different analytical techniques
need to worry about the adsorption of the surfactant available that can be used to detect residues. These
on the walls of manufacturing equipment. This is range from simple titrations to more complex LC-
because the agitation that is involved in cleaning MS. The choice of technique should be based on
removes the surfactants from the surfaces. This is what equipment is available, the type of residue,
another matter in sample vials. Appropriate spiking and the scientifically established residue limit. It is
studies should be performed to ensure that this phe­ important for an analytical chemist to keep abreast
nomenon is not occurring and will not interfere with of the literature and what techniques are available.
the analytical method. This includes both HPLC or There are techniques available that will analyze any
ion chromatography sample vials, as well as TOC residue at any level. At the end of the day, however,
sample vials. This phenomenon is not limited to sur­ it is always wise to choose the simplest technique
factants. Proteins have been shown to adsorb readily that can be used to reach the desired goal. o
onto glass surfaces. These proteins are much more
difficult to remove from surfaces than surfactants.
About the Authors
Specific Versus Nonspecific Herbert J. Kaiser, Ph.D. is Manager – Hard Surface
Products at STERIS Corporation. He has 18 years of
The choice of using a specific or nonspecific experience in cleaning and surface technologies,
method can be difficult. If a drug active is highly which includes developing products and methods
toxic, a specific method is always recommended.61 for the cleaning and analyzing of a wide variety of

Special Edition: Cleaning Validation III 43
Herbert J. Kaiser, Ph.D. & Maria Minowitz, M.L.S.

surfaces. Dr. Kaiser has developed a wide variety of Potentiometric Sensors.” Analyst. Vol. 114. 1989. pp. 1435-1441.
13. Schmitt, T. M. “HPLC Analysis of Surfactants.” Handbook of
products for the healthcare, industrial, and pharma- HPLC. Eds. Katz, E., Eksteen, R., Schoenmakers, P., Miller N.
ceutical markets. He is the sole inventor listed in Chromatography Science. Series 78. Marcel Dekker, Inc.: New
five United States Patents for various industrial treat- York. 1998. pp. 789-804.
14. McPherson, B. P., Rasmussen, H. T. “Chromatography of Cationic
ment schemes. Dr. Kaiser received his B.A. degree Surfactants: HPLC, TLC, and GLC.” Cationic Surfactants. Eds.
from St. Mary’s University in San Antonio, Texas, Cross, J., Singer, E. Surfactant Science. Series 53. Marcel Dekker,
his M.S.(R) from St. Louis University, and his Ph.D. Inc.: New York. 1994. pp. 289-326.
15. Jandera, P. “HPLC of Surfactants and Related Compounds.”
from the Un­iversity of Missouri. He is a member of Liquid Chromatography in Environmental Analysis. Ed.
the American Chemical Society and the Association Lawrence, J. Humana Press: New Jersey. 1984. pp. 115-167.
for the Ad­vancement of Medical Instrumentation. Dr. 16. Waters, J. “Analysis of Low Concentrations of Cationic Sur­factants
in Laboratory Test Liquors and Environmental Samples.” Cationic
Kaiser can be reached by phone at 314-290-4725, by Surfactants. Eds. Cross, J., Singer, E. Sur­factant Science. Series 53.
fax at 314-290-4650, or e-mail at herb_kaiser@steris. Marcel Dekker, Inc.: New York. 1994. pp. 235-256.
17. Lin, W., Lin, S., Shu, S. “Comparison of Analyses of Surfactants
com. in Cosmetics Using High-Performance Liquid Chromatography
and High Performance Capillary Electrophoresis.” Journal of
Maria Minowitz, M.L.S., Information Associate at Surfactants and Detergents. Vol. 3(1). 2000. pp. 67-72.
STERIS Corporation, has 10 years of experience in 18. Carrer, G, Faccetti, E., Valtorta, L., et. al. “An Analytical
corporate research and development librarianship. Ap­proach for the Determination of Betaines in Liquid Form­ula­
tion.” Rivista Italiana delle Sostanze Grasse. Vol. 76 (4). 1999.
She has been responsible for information manage- pp. 167-171.
ment in the disciplines of chemistry, medicine, and 19. Guerro, F., Rocca, J. L.. “RPLC Analysis of Alkyl Poly­
ethyleneglycol Ethers Using Evaporative Light Scattering De­tec­
engineering. Minowitz received her A.B. degree tion.” Chimica Oggi. Vol. 13(4-5). 1995. pp. 11-15.
from St. Louis University and an M.L.S. from the 20. Heinig, K., Vogt, C., Werner, G. “Determination of Linear
University of Missouri-Columbia. She is a member Alkylbenzenesulfonates in Industrial and Environmental Sample
by Capillary Electrophoresis.” Analyst. Vol. 123. 1998. pp. 349-
of the Special Libraries Association, Midcontinental 353.
Chapter of the Medical Library Association, and the 21. Heinig, K., Vogt, C. “Determination of Surfactants by Capillary
St. Louis Medical Librarians. Electrophoresis.” Electrophoresis. Vol. 20. 1999. pp. 3311-3328.
22. Oehrle, S. A. “Analysis of Low-Level Anions in Water Extracts
of Hard Disk Drive Heads by Capillary Electrophoresis.”
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23. Altria, K. D., Hadgett, T. A. “An Evaluation of the Use of
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3. LeBlanc, D. A. “Validated Cleaning Technologies for Pharma­ Electrophoresis.” Tenside Surfactants and Detergents. Vol.
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Pharmaceutical Technology. Vol. 22(10). 1998. pp. 136-148. 29. Shamsi, S. A., Danielson, N. D. “Individual and Simultaneous
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Pharmaceutical Technology. Vol. 22. 1998. pp. 66-74. Surfactants by Capillary Electrophoresis with Indirect
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Ionic Surfactants with Sodium Tetraphenylborate Using Simple Analysis and Fourier-Transform Infrared Spectroscopy to

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Determine Residues of Cleaning Agents on Surfaces.” Journal Integrated Approach for Validating Cleaning Procedures in
of AOAC International. Vol. 80. 1997. pp. 1078-1083. Biopharmaceutical Manufacturing Facilities.” Annals of the
33. Westman, L., Karlsson, G. “Methods for Detecting Residues of New York Academy of Sciences. Vol. 782. 1996. pp. 363-374.
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ceutical Science Technology. Vol. 54(2). 2000. pp. 365-372. Nearer the Sampling Point, Use of Simple, Rapid On-Site
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Or­ganic Carbon Analysis for Cleaning Validation in Pharma­ Environmental Release Applications.” Journal of Pharma­cy and
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41. Masters, M. B. “Use of Ion Chromatography in Surfactant 61. Segretario, J., Cook, S. C., Umbles, C. L., et. al. “Validation of
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43. Theile, B., Günther, K., Schwuger, M. “Trace Analysis of
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Detergents. Vol. 36(1). 1999. pp. 8-12, 14-18.
44. Bosdorf, V., Krüßmann, H. “Analysis of Detergents and
Cleaning Agents with Thin-Layer Chromatography.” Fourth
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Productores de Sustancias para Aplicaciones Tensioactivas.
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45. Read, H. “Surfactant Analysis Using HPTLC and the Latroscan.”
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53. Inampudi, P., Lombardo, S., Ruezinsky, G., et. al. “An

Special Edition: Cleaning Validation III 45
Control and Monitoring
of Bioburden in Biotech/
Pharmaceutical
Cleanrooms
By Raj Jaisinghani
Technovation
&
Greg Smith
Encelle, Inc.
&
Gerald Macedo
Med-Pharmex, Inc.

v

T
his paper describes results HEPA fan filter units (FFUs) was
of monitoring biotech clean­­­­­­­­ }The FDA possible. The results showed that at
rooms and a pharmaceutical the same flow rate the EEF resulted
cleanroom equipped with an Elec­ has specific in significantly lower bioburden as
trically Enhanced Fil­tration (EEF) requirements and compared to the FFUs.
system that significantly reduces
airborne bioburden in cleanrooms. guidelines for bio-
1
Background
The EEF High Ef­ficiency Part­ burden for
iculate Air (HEPA) system traps The fundamental purpose of
and kills bacteria and also im­proves various cleanrooms in the pharmaceutical,
the filtration performance of a filter pharmaceutical medical device, biotechnology, and
me­dia by two to three orders of hospital applications is to control
magnitude. In laboratory tests the operations and the amount of bioburden due to
EEF tech­nology has been shown processes.~ both internal operations and trans­
to kill Staphyl­o­coccus epidermidis port from the air. From a particu­
and Escherichia coli. These field late point of view, cleanrooms in
test results support laboratory testing and show that these industries are classified and specified accord­
basically there is no airborne bioburden in both a ing to the same cleanroom standards (e.g., Federal
Class 10 room, with terminal HEPA in addition to the Stand­ard 209E) as in other industries. It is often
EEF, and in a Class 1000 room that utilizes only the assumed that the particle (total) concentrations
EEF without any terminal HEPA filters. In the case will generally correlate to concentration of viable
of an old laboratory converted to a cleanroom, direct microorganisms. This may not always be valid.
comparison of the EEF with respect to conventional Hence, the concentration of viable organisms is also

46 Institute of Validation Technology
Raj Jaisinghani, Greg Smith, & Gerald Macedo

directly measured – both at the work surfaces (or at applied E. coli survived on the clean glass filter after
the process) and in the air. four hours of airflow, keeping in mind that E. coli is
Cleanrooms in these industries must meet separate not a hardy organism. Next about one gm of colloidal
standards for bioburden. The FDA has specific require­ kaolin was added to the E. coli solution that was to be
ments and guidelines1 for bioburden for various phar­ aerosolized. This time the recovery of E. coli was about
maceutical operations and processes. Similarly, the 104 – 105 CFU/square inch of the filter media. Similar
United States Pharmacopoeia (USP) and the European tests with S. epidermidis recovered a little more S.
Union’s GMP 2 guidelines give specific recommended epidermidisthan with E. coli even without the colloidal
limits for microbial contamination for each class of kaolin, due to the hardier nature of S. epidermidis. With
room. A cleanroom that meets the particle concentra­ 1 gm of colloidal kaolin in the 25 ml S. epidermidis
tion requirements, but does not result in the desired solution (in tryptic soy broth) the recovery of S. epider­
level of bioburden, will clearly be inadequate. midis was about 105 – 106 CFU/square inch of filter
One of the main obstacles in achieving the required media. Tests with airflow continuing for seven hours
bioburden levels is that the measurement of bioburden (following the aerosol) did not result in any significant
is time consuming. Typically, bioburden measure­ reduction in bacteria recovery. This result suggests
ment involves sampling, incubation, and counting of that, even in normal environments, bacteria can sur­
colonies. This is a time consuming pro­cess and thus vive or grow on the filters. As the trend towards using
“real” time monitoring is not possible. Thus, it is not HEPA cleanroom filters for longer periods continues,
always possible to relate higher incidents of bioburden the possibility of bacterial growth on the filter, and thus
to operating events. Recently, however, ultraviolet the rise in the airborne bioburden, also increases.
(UV) fluorescence (cf. Seaver and Eversole3, Pinnick
et al.4) technology has made it possible to achieve real EEF Technology
time monitoring of particles of biological origin. This
technology will find increasing use in the real time Jaisinghani7 has played a significant role in the
monitoring of air in hospitals, cleanrooms, and mili­ development and commercialization of EEF tech­
tary nuclear, biological and chemical (NBC) warfare nology. The most recent version (see Figure 1) of
protection systems – as a real time supplement to the this technology7 maintains the filter under an ion­
standard methods of determining bioburden. As this izing (as opposed to a simple electrostatic field)
happens, more attention will be focused on cleanroom
contamination control systems – currently mainly Figure 1
mechanical filtration. Technovation’s Ionizing
One problem with mechanical filters is that under EEF Technology
certain conditions common bacteria caught on the
Ionizing Filter
filter can start growing on the filters, grow through Wires
the media, and start shedding into the room.5,6,7 The
well-known case of the Legionnaire’s outbreak at the
Flow
veterans convention in Philadelphia has been attributed
to this phenomena. In that case the filters were sup­ Downstream
posedly in a wet state. Generally, it is accepted that Ground
bacteria is difficult to grow on clean glass fiber filter Electrode
media, used in HEPA filters, under normal humidity
conditions. However, since the function of these fil­ Control
Ionizer
Electrode
ters is to capture all particulate contamination, filters Electrode
eventually get dirty. The experiments conducted by
Jaisinghani et al.8 show that very little contaminant is
needed for growth of Staphylococcus epidermidis and H.V.
Escherichia coli on HEPA glass filters. In their experi­ Supply
ments Jaisinghani et al.8 found that very little of the

Special Edition: Cleaning Validation III 47
Raj Jaisinghani, Greg Smith, & Gerald Macedo

field. Another higher intensity ionizing field charges confirm the initial viable concentration of bacteria.
incoming particles, stabilizes the electrical fields, The rehydrated culture was then sprayed onto the filter
and increases the safety and reliability by ensuring using a Meinhard nebulizer, which was placed eight
that no spark over can occur towards the filter. This inches from the center of the filter.
method provides two fundamental benefits: A control assay was performed to determine the
amount of viable S. epidermidis on the filter, without
1. Bacteria are killed as they pass through a first application of high voltage. The bacteria were sprayed
high intensity ionizing field and then killed onto the filter as previously described, and the temper­
as they are subjected to continuous ionizing ature and humidity were monitored every 15 minutes
radiation when they are trapped on the filter. for four hours or seven hours during which the airflow
This inhibits growth of bacteria on the filter. was on. The relative humidity was held between 45-
2. The same ionizing fields enable penetration 55% using a Kaz steam vaporizer. At the end of each
re­duction by about two to three orders of mag­ control run three pieces of the filter were extracted
nitude. using a sterilized scalpel and forceps. The pieces of
filter were approximately one square inch on the face
Since the cost of the additional electrical compo­ of the filter, which when unfolded measured approxi­
nents is partially offset by the increase in filtration mately 28 square inches of filter material. Filter pieces
performance (either higher flow at the same pres­ were removed from the center of the filter, directly
sure drop and filtration efficiency or lower pressure above the center, and directly below the center.
drop at the same flow and efficiency, as compared to The samples were cut into small pieces and placed
mechanical filtration of the same size) this is a highly into 10 ml of sterile phosphate buffered saline (PBS)
cost effective method for the control of bioburden. pH 7.4 in a Nasco Whirl-Pak bag. The bags were
then processed in a Tek Mar Stomacher Lab-Blender
Laboratory Evaluation of the EEF 80 for one minute. Each sample was then serially
diluted ten-fold to 10-2, 10-3, and 10-4, then spread on
Jaisinghani et al.8 have demonstrated the bacteri­ CBA plates to determine the number of viable bacte­
cidal properties of the EEF under laboratory condi­ ria per sample filter piece.
tions. This study, conducted at Virginia Polytechnic Similar tests were then conducted by applying high
Institute, is summarized in this section. voltage to the EEF. In addition to monitoring the tem­
perature and humidity, the current was also monitored
Experimental Methods at fifteen minute intervals during the four or seven hour
S. epidermidis was grown in Tryptic Soy Broth period of airflow with the applied high voltage on.
(TSB) to a concentration of 3 x 109 colony form­
ing units (CFU)/ml. The culture was lyophilized Results and Discussion
in Wheaton vials in 5 ml aliquots – 1.5 x 1010 CFU The results are summarized in Figure 2. In the
per vial. All vials were stored in a desiccator at 4 absence of any voltage applied to the EEF unit (i.e.,
– 6ºC. control tests), viable bacteria were recovered from
Pleated 15.24cm by 15.24cm by 5cm (6” x 6” x one square inch of filter in the range of 1 x 105 CFU
2”) deep filters were first coated with colloidal kaolin to 2 x 106 CFU. Counts greater than about 3 x 106
and TSB using an Aztek airbrush. The airbrush cup CFU were too crowded to be accurately counted and
was filled with 1g kaolin suspended in 25ml TSB were considered to be too numerous to count. When
and sprayed onto a filter inside a laminar flow hood high voltage was applied for four hours, the majority
and allowed to air dry before being used. The pleated of the bacteria were killed. The kill rate increased
filters were placed in a miniature version of the EEF. with increased voltage or with the first applied field
One vial of lyophilized S. epidermidis was resuspend­ strength (applied voltage divided by the distance of
ed with 1 ml of sterile distilled water. A small aliquot the ionizer wires from the control ground electrode –
of this suspension was serially diluted ten-fold to 10-8 see Figure 1), V/d1. At a field strength (V/d1) of 4.2
and plated on Columbia Blood Agar (CBA) plates to kV/cm, there was no growth after 24 hours of incu­

48 Institute of Validation Technology
Raj Jaisinghani, Greg Smith, & Gerald Macedo

Figure 2
EEF Bactericidal Test Summary Using S. epidermidis
Filter Incubation EEF Exposure EEF Field Average Comment
Time Time Strength Colonies
Control or Hours Hours (v/d1) kv/cm #/Sq. inch
EEF
Control 24.00 0.00 0.00 1.00E+06 No additional growth
Control 24.00 0.00 0.00 1.02E+05 After 24 Hours
EEF 24.00 4.00 4.64 00.0E+00 100% Killed
EEF 24.00 4.00 3.99 3.44E+02 99.93% Killed
EEF 24.00 4.00 4.24 0.00E+00 100% Killed
EEF 24.00 4.00 4.50 0.00E+00 Some growth
EEF 24.00 4.00 4.20 0.00E+00 After 48 Hours
EEF 24.00 4.00 4.20 6.26E+03 98.75% Killed
EEF 48.00 7.00 4.20 5.44E+02 99.9% Killed
EEF 48.00 4.00 4.80 2.16E+02 99.95% Killed
EEF 48.00 4.00 4.20 3.51E+03 99.3% Killed

bation. After 48 hours, there was either no growth or Figure 3
small (in size and in number) colonies grown. These
small colonies were identified as S. epidermidis, and Model 3001 EEF Filter
were identical in biochemical profile as the isolate
used in the tests. It was concluded that four hours
at 4.2 kV/cm (V/d1) did not completely kill the S.
epidermidis. If the bacteria were not all killed, some
of them were damaged sufficiently so that no growth
or very limited growth could occur after 24 hours
incubation. When the ionizing time was increased to
seven hours, over 99% of the bacteria (as compared
to the control) were killed.
When the applied field strength, V/d1, was
increased to 4.5 kV/cm or higher, no growth occurred
on any of the filter pieces except for one experiment.
This exception may have occurred because the start­
ing dose of bacteria for this experiment was three
times higher than for the control and up to 10 times
higher than for any other experiment. Nonetheless,
there were still three to four logs of killing using an
applied field strength, V/d1 of 4.5 kV/cm or higher, ous study conducted with the EEF at the University
as compared to the control experiments. It should be of Wisconsin.
noted that, in practice, bacteria caught on the filter
are held within the ionizing field for an almost infi­ Field Results in Cleanrooms
nite amount of time, thus receiving an almost infinite
radiation dosage. Hence, in practice, the killing effi­ Model 3001B or Model 1001B BIO PLUS® EEFs
ciency should be higher even at lower field strengths. (Figure 3) manufactured by Technovation Systems,
Similar results were obtained using E. coli in a previ­ Inc., were used in the cleanroom discussed here.

Special Edition: Cleaning Validation III 49
Raj Jaisinghani, Greg Smith, & Gerald Macedo

Both models have a flow rating of about 3000 scfm flow rate utilized here (22 fpm average air velocity)
without attached ductwork. was on the lower end of flow normally used in Class
Comparison to FFU in a Converted Cleanroom 1,000 cleanrooms. This room will simply be referred
Jaisinghani et al.8 have conducted a field study com­ to, henceforth, as “the Encelle cleanroom.”
paring FFUs to an EEF in a small laboratory converted All processing and manufacturing conducted within
to a cleanroom. This will be referred to as the “older the cleanroom areas are done aseptically. Workers are
Encelle cleanroom.” (Encelle, Inc., Greenville, NC.) gowned in sterile coveralls, shoe covers, goggles, or
Encelle had four conventional HEPA fan filter units face-­masks with shields, hair nets, and sterile gloves.
(FFUs) installed in this tissue culture laboratory, prior The Class 1K cleanroom and clean Class 10K sur­
to replacing these with one Model 1001 EEF. One rounding zone are cleaned daily with a monthly rotation
model 1001 provides HEPA filtered air at about the of sterile chemicals using cleanroom equipment and
same total flow (approx. 4250 m3/h (2500 scfm) in this trained personnel. Disinfectants include Hypochlor®,
case) as the four FFUs. This allowed direct evaluation Process LpH®, Process Vesphene®, and as needed, treat­
of the effect of EEF on the bioburden in the room, ments with a spore-killing agent called SporKlenz®.
under field conditions. Airborne bioburden in the room
was reduced by as much as 75% after switching to the Sampling Methods
EEF system. The airborne bioburden in the Class 10K An environmental monitoring program has been
room was 0.021 cfu/ft3 (no process) and 0.392 cfu/ft3 designed to establish the standards and limits that
(in process) after installation of the EEF filter. are acceptable to the facility management and to
Figure 4 shows the Federal Standard 209E, USP, regulatory agencies that will audit the manufactur­
and European Union recommended airborne biobur­ ing within the cleanroom environments.
den and particulate concentration for various class Daily activities for monitoring include tempera­
cleanrooms. Clearly, from a bioburden perspective ture and pressure readings. Relative humidity is also
Encelle’s older Class 10K room performs (at rest) at a reported on sampling days.
level equivalent to a Class 100 room – without incur­ Surface samples are collected on a routine basis
ring the higher cost associated with building a Class using only sterile supplies. Surfaces monitored in­clude
100 room. Most of this benefit should be attributed to floors, equipment, walls, and ceilings. A five per­
the EEF filter system. cent sheep’s blood agar plate (three inch diameter)
is swabbed with a sterile, moist, cotton swab after
New Class 1000 Biotech Tissue sampling various surface areas. Plates are labeled and
Cul­ture Cleanroom incubated at 37ºC, with five percent carbon dioxide for
72 hours. Colony forming units that grow are counted
Facility Description and identified using standard microbial techniques.
A 1,300 square foot Class 1,000 cleanroom and Particle counting is conducted biweekly or as
900 square feet of Class 10,000 surrounding space was needed for monitoring during critical processes. A
constructed at Encelle, Inc., Greenville, NC facility. It Biotest® APC Plus particle counter measures concen­
utilized eight 3001B BIO PLUS® EEF filters. The total tration at particle sizes of 0.3, 0.5, 1.0, and 5.0µm.

Figure 4
Recommendations for Airborne Bioburden for Various Cleanroom Classes
Class Parameter 209E EU USP
10K 0.5 Um particles #/ft3 <10K <10K
10K CFU/ft3 <2.83 <2.5
1K 0.5 Um particles #/ft3 <1K <1K
1K CFU/ft3 NA NA
100 0.5 Um particles #/ft3 <100 <100
100 CFU/ft
3
<0.028 at rest; <0.283 Process <0.1 Process

50 Institute of Validation Technology
Raj Jaisinghani, Greg Smith, & Gerald Macedo

Data is collected in nearly 200 areas within the to a Class 100 cleanroom in operation. It should be
filtered-air areas. These areas are categorized by pro­ noted that the cleanroom certified as Class 100 at rest
cess and particle counts are reported to the facilities (i.e., without personnel). Similarly, the design Class
manager for evaluation and disposition if warranted. 10,000 area is functioning as a borderline Class 1,000
Data is downloaded via an RS-232 port for digital in operation. It should be noted again, that the airflow
documentation of these counts. rate used in this Class 1,000 cleanroom (22 fpm) was on
Microbial air sampling is performed in parallel the low end for a normal Class 1,000 cleanroom. The
with particle counting to provide data on airborne high performance of this room can be attributed to the
viable particulate counts and comply with Federal high degree of ceiling coverage (i.e., Airflow is highly
Standard 209E Cleanliness classes for cleanrooms distributed throughout the room) which is an inherent
and clean zones. A Biotest® Centrifugal Air Sampler feature of the Technovation BIO PLUS® EEF system.
collects 500 liters of air in each location on sterile
tryptic soy agar strips that are designed for this type Results – Airborne Microorganisms
of sampler. Strips are labeled and incubated similarly Figure 7 shows the results of airborne microor­
to the surface agar plates. Classification and identifi­ ganism monitoring in the Class 1,000 cleanroom.
cation are performed using the standards described in The results clearly indicate that, from the perspec­
the current edition of the USP. tive of airborne bioburden, the Class 1,000 area is
USP standards9 for microbial growth follow in performing at a level that is to be expected for a Class
Figure 5. 100 to Class 10 level. Note that (by comparing to the
particulate data in Figure 8) the airborne bioburden
Results – Particle Concentration does not correlate to the particulate concentration.
The particle concentration measurements are During the months of December and January, the
shown in Figure 6. sub-micron particulate concentration actually went up
From the perspective of particle counts alone, the while the airborne bioburden was reduced.
design Class 1,000 cleanroom is functioning very close Figure 8 shows the results of airborne microor­
ganism monitoring in the Class 10,000 cleanroom.
Figure 5 Note again that the bioburden does not relate to
USP Standard for Bioburden the particulate concentration. One probable reason
in Cleanrooms for this is that the bacterium are larger than the size
of the sub-micron particles being monitored. Also
Class 100 note that on the average the Class 10K area performs
Requirements between a USP Class 100 and a USP Class 10K from
Air 0.1 cfu/ft3 a bioburden perspective.
Surface 3 cfu/plate*
Gowns 5 cfu/plate Results – Surface Microorganisms
Figure 9 shows the results of surface microorgan­
Class 10,000 ism monitoring in the Class 1,000 cleanroom.
Requirements
The surface concentrations of bacteria are almost
Air 0.5 cfu/ft3 zero throughout the cleanroom suite. This can be
Surface 5 cfu/plate (10 from floor)
Gowns 10 cfu/plate
attributed to:

Class 100,000 n The cleaning protocols instituted at Encelle
Requirements n The use of the disinfecting compounds
Air 2.5 cfu/ft3
n The low airborne bioburden
Surface 20/plate
Gowns 30/plate Observations from the Encelle Cleanroom Mon­
itoring
* 2 in2 surface 1. The Encelle cleanroom operates significantly

Special Edition: Cleaning Validation III 51
Raj Jaisinghani, Greg Smith, & Gerald Macedo

Figure 6
Particle Concentration Measurements in the Encelle
Tissue Culture Laboratory
Air Particle Concentration (per ft3)
11/14/99 11/14/99 12/1/99 12/1/99 12/31/99 12/13/99 1/6/00 1/6/00 1/28/00 12/28/00
Size – > 0.3 uM 0.5 uM 0.3 uM 0.5 uM 0.3 uM 0.5 uM 0.3 uM 0.5 uM 0.3 uM 0.5 uM
Class 10,000 Design Areas Approx.
Sq. Ft.
Mechanical Corridor 235 1959 506 786 602 1163 903 801 899 852 512
Side Corridor 555 2240 642 4559 1035 3598 762 4058 687 7989 1422
Water Filtration Area 171 3649 2598 4657 2429 11406 4312 8350 4368 6378 1295
Labware Processing 212 343 102 981 719 1233 1798 243 326 618 530
Gowning Room 139 236 87 853 1063 1506 913 2874 2808 957 235
Materials Pass Through 40 257 88 450 539 2461 2463 3887 6064 1451 595
Total Square Feet – > 1312
Actual Area Classification 482 671 683 1065 1187 1859 1123 2525 1014 765
Class 1,000 Design Areas
Specialized Cleanrooom 152 158 66 223 145 350 337 313 379 754 237
Formulations Mfg. Area 142 208 24 84 52 415 228 322 307 360 126
Product Testing Area 132 28 5 9 9 43 49 19 74 19 76
Product Finishing Area 127 264 26 129 49 474 139 400 179 472 184
Product Mfg. Area 145 293 83 250 87 534 180 639 698 829 731
Refrigerator/Freezer Storage 212 262 55 233 157 832 692 427 335 659 381
Total Square Feet – > 910
Actual Area Classification 67 43 52 83 147 271 118 329 172 289

better than the design classification although the low airborne bioburden is due to the BIO
the flow rate (average room velocity = 22 fpm) PLUS® EEF filters.
used is at the low end of what is normally used 3. The surface bio contamination is almost non
in such a cleanroom. This is due to the higher existent in the Class 1K cleanroom. This may
distribution of flow rate – an inherent feature be attributed to the good cleaning practices
of the BIO PLUS® filter system. used at Encelle and due to the low airborne
2. The airborne bioburden in both the Class 1K bioburden in the suite.
and 10K areas is lower than what would be 4. The airborne bioburden seems to be lower in
expected for such rooms based on USP rec­ the winter months, although the room tempera­
ommendations. The Class 1K room has the ture is held constant at 66ºF. This may be due
bioburden of what would be expected (based to lower humidity in the winter months.
on USP) for a Class 100 room. Coupling this
observation to the laboratory studies on the New Class 10 Pharmaceutical Cleanroom
bactericidal properties of the EEF technology
and the direct comparison with respect to con­ Facility Description
ventional FFU HEPAs, it may be inferred that A 12’x20’ Class 10 cleanroom (including a

52 Institute of Validation Technology
Raj Jaisinghani, Greg Smith, & Gerald Macedo

Figure 7
Airborne Bioburden in the Encelle Class 1,000 Cleanroom
New Facility’s Microbial Air Sample Results
Collected with Biotest Plus Centrifugal Air Sampler
Design Class 1,000 11/14/99 11/30/99
Average cfu Av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr* Average cfu Av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*

Device Testing 0 0 0 0 0 0 0 0
Coating 0 0 0 0 1 0.028 0.028 0.028
Formulations 0 0 0 0 0 0 0 0
Device Manufacturing 3 0.085 0.085 0.085 2 0.057 0.057 0.057
Refrigeration 0 0 0 0 0 0 0 0
Isolation 3 0.085 0.085 0.085 15 0.425 0.425 0.425
Class 1,000 Average 1 0.028 0.028 0.028 3 0.085 0.085 0.085
12/13/99 1/28/00
Design Class 1,000 Average cfu Av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr* Average cfu Av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*
Device Testing 0 0.000 0 0 0 0.000 0 0
Coating 0 0.000 0 0 0 0.000 0 0
Formulations 0 0.000 0 0 0 0.000 0 0
Device Manufacturing 0 0.000 0 0 0 0.000 0 0
Refrigeration 0 0.000 0 0 0 0.000 0 0
Isolation 0 0.000 0 0 0 0.000 0 0
Class 1,000 Average 0 0.000 0 0 0 0.000 0 0
The time period refers to the incubation time in hours.

4’x12’ Class 10K gowning room) was constructed at hypochlorite solution.
MedPharmex in Pomona, CA using two BIO PLUS®
Model 3001B filters with eight terminal 2’x4’ HEPA Sampling Methods
filters. The Model 3001Bs were used for the 12’x16’ Air sampling was done using Tryptic Soy Agar
Class 10 inner room. The resultant average room ve­loc­ (TSA) and Sabouraud Dextrose Agar (SDA). The
ity in the Class 10 area was 24 fpm (4500 scfm). The TSA values reflect total bacterial counts while the
design specification for the room was Class 100. This SDA reflects molds and yeast, although it contains
airflow was much lower than used in a Class 100 clean­ no bacterial inhibitors. In some cases Rose Bengal
room – normally, with conventional single terminal Agar (RBA) was used. This reflects a better value for
HEPAs, at least 40 fpm average room velocity is used molds/yeast since the RBA contains bacterial growth
in a Class 100 room. However, due to the double HEPA inhibitors. Surface monitoring was done using 24-30
filter system (each Model 3001B powered Airflow cm2 RODAC plates with TSA and SDA. The TSA
through four terminal HEPAs) the cleanroom easily plates were incubated for a minimum of 48 hours at
classified as Class 10 as per Federal Standard 209E. 32.5 +/- 2.5ºC while the SDA plates were incubated
This resulted in significant energy savings. The room for a minimum of 72 hours at 22.5 +/- 2.5ºC.
was validated for bioburden initially and then has been
shut down since the facility is now being moved to Results – Airborne Microorganisms
a new location. The facility was cleaned with 0.25% The gowning room was sampled in two zones

Special Edition: Cleaning Validation III 53
Raj Jaisinghani, Greg Smith, & Gerald Macedo

Figure 8
Airborne Bioburden in the Encelle Class 10K Area
New Facility’s Microbial Air Sample Results
Collected with Biotest Plus Centrifugal Air Sampler
Design Class 10,000 11/14/99 11/30/99 Average
Average cfu Av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr* Average cfu cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*

Water Filtration Area 30 0.850 0.850 0.850 2 0.057 0.057 0.057
Side Corridor 3 0.085 0.085 0.850 3 0.058 0.085 0.085
Manufacturing Corridor 19 0.538 0.538 0.538 11 0.312 0.312 0.312
Labware Processing 5 0.142 0.142 0.142 4 0.113 0.113 0.113
Gowning Room 0 0 0 0 3 0.085 0.085 0.085
Materials Pass Through 3 0.085 0.085 0.085 11 0.312 0.312 0.312
Class 10,000 Average 10 0.283 0.283 0.283 5.67 0.160 0.160 0.172
12/13/99 1/28/00
Design Class 10,000 Average cfu Av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr* Average cfu Av. cfu/ft3 cfu/ft3/24hr* cfu/ft3/72hr*
Water Filtration Area 2 0.057 0.057 0.057 2 0.057 0.057 0.057
Side Corridor 2 0.057 0.057 0.057 0 0 0 0
Manufacturing Corridor 1 0.028 0.028 0.028 0 0 0 0
Labware Processing 0 0 0 0 0 0 0 0
Gowning Room 0 0 0 0 0 0 0 0
Materials Pass Through 2 0.057 0.057 0.057 0 0 0 0
Class 1,000 Average 1.17 0.033 0.033 0.033 0.333 0.009 0.009 0.009
The time period refers to the incubation time in hours.

while the Class 10 cleanroom was sampled in five because of the double HEPA filter system used.
zones. All plates (TSA and SDA) were negative The MedPharmex cleanroom validates as a Class
(i.e., zero counts) in all the areas. The Class 10 area 10 cleanroom, although the airflow used was lower
was also sampled using RBA and once again the than what is normally used in a Class 100 room.
results were negative – zero counts. 2. It should be noted that the MedPharmex room was
Results – Surface Microorganisms simply validated and then shut down in order to
The surface measurements were made before and move it to an adjacent facility, while the Encelle
after cleaning the newly constructed cleanroom. The room is being continuously monitored and is
results are shown in Figure 10. The 0.25% Hypo­ operational. How­ever, from the point of view
chlorite cleaning is obviously very effective in elimi­ of airborne bioburden, after the first month of
nating surface bacteria. operation the Encelle Class 1000 room operates
at an equivalent level as the MedPharmex Class
Observations From the Medpharmex Clean­room 10 room – with essentially zero airborne bacte­
Validation rial counts. The low bioburden benefit to Encelle
1. The new Encelle Class 1000 and the Med­Pharmex (this Class 1000 room is operating at essentially
Class 10 room have about the same airflow aver­ zero airborne bioburden) may be attributed to the
age velocity. From the particulate point of view the bactericidal properties of the EEF system. o
MedPharmex room operates at Class 10 simply

54 Institute of Validation Technology
Raj Jaisinghani, Greg Smith, & Gerald Macedo

Figure 9 the Un­iver­sity of Wisconsin. Jaisinghani has exten-
sive re­search experience in air and liquid filtration,
New Facility Surface colloid and aerosol science, fluid mechanics, heat
Contamination Summary transfer, and physical surface chemistry. He holds
Design Classification
10 patents and has many publications in technical
10,000 Average number of cfu/plate grown in 72 hours journals and handbooks. He can be reached by
Date – > 11/14/99 11/30/99 12/13/99 01/28/00 phone at 804-744-0604, by fax at 804-744-0677, or
by e-mail at technova@sprynet.com.
Water
Filtration Area 0 0 2 0 Greg Smith is facilities manager at Encelle, Inc.
Side Corridor 1 0 0 0 He holds a B.A. in Psychology from West Virginia
Manufacturing Corridor 0 0 0 0 University and a B.S. in Chemistry from East Carolina
Labware Processing 1 0 0 0 University. Smith has assisted in the development of
Gowning Room 0 0 0 0 medical devices and has five years experience as
Materials Pass a hospital pharmacy aseptic compounding techni-
Through 0 0 0 0 cian. He can be reached by phone at 252-355-4405
Design Classification or by e-mail at gsmith@Encelle.com.
1,000 Average number of cfu/plate grown in 72 hours
Date – > 11/14/99 11/30/99 12/13/99 01/28/00 Gerald Macedo has a B.S. degree in Pharm­acy and
Device Testing 0 0 0 0 an M.S. in Pharmaceutical Sciences. He has over
Coating 0 0 0 0 30 years experience in pharmaceutical manufactur-
Formulations 0 0 0 0 ing, with extensive experience in the manufacture
Device Manufacturing 0 0 0 0 of sterile injectables. He has ser­ved as head of
Refrigeration 0 0 0 0 man­ufactur­ing, quality control, quality assurance,
Isolation 0 0 0 0 research and development, and regulatory affairs.
Control 255 210 134 104 Macedo currently heads Med-Pharmex, Inc., a
pharmaceutical manufacturing company. He can
Figure 10 be reached by phone at 909-593-7875 or by fax at
909-593-7862.
Surface Bioburden in the
Class 10/10K Suite References
(Counts per 25 cm RODAC Plates)
2
1. FDA. “Guideline on Sterile Drug Products by Aseptic Pro­
cessing.” Rockville, MD.
Area Before Cleaning After Cleaning 2. EU. 1998. “The Rules Governing Medicinal Products in the
TSA SDA TSA SDA EU.” Good Manufacturing Processes 4. Luxembourg.
3. Seaver, M. and Eversole, J.D. 1996. “Monitoring Biological
Counts Counts Counts Counts Aerosols Using UV Fluorescence.” Proceedings 15th Annual
Gowning Meeting AAAR. October. Orlando, FL: 270.
4. Pinnick, R.G., Chen, G., and Chang, R.K. 1996. “Aerosol
Table-gowning 22 5 0 0 Analyzer for Rapid Measurements of the Fluorescence Species
Wall-gowning 3 0 0 0 of Airborne Bacteria Excited with a Conditionally Fired Pulsed
266 nm Laser.” Proceedings 15th Annual Meeting AAAR.
Class 10 October. Orlando, FL.
Tank 0 1 0 0 5. Rhodes, W.W., Rinaldi, M.G., and Gorman, G.W. 1995.
“Reduction and Growth Inhibition of Microorganisms in
Fill 21 12 0 0 Commercial and Institutional Environments.” Environmental
Filter table 9 4 0 0 Health 12 (October).
6. Tolliver, D.I. 1988. “Domestic and International Issues in
Wall 1 0 0 0 Contamination Control Technologies.” Microcontamination 6,
no. 2 (February).
7. Jaisinghani, R. A. U.S. Patent 543,383. 4 April 1995.
8. Jaisinghani, R.A., Inzana, T.J., and Glindemann, G. 1998.
About the Authors “New Bactericidal Electrically Enhanced Filtration System
for Cleanrooms.” Paper presented at the IEST 44th Annual
Rajan (Raj) Jaisinghani is a chemical engineer Technical Meeting. April. Phoenix, AZ.
with thirty years of research, product development, 9. “Microbial Evaluation of Cleanrooms and Other Controlled
Environments.” United States Pharmacopoeia, <1116>, p. 2099-
and business development experience. Jaisinghani 2106.
holds a B.S. from Banaras Hindu University, India,
and an M.S., with additional graduate work, from

Special Edition: Cleaning Validation III 55
A Cleaning Validation Program
for the ELIFA System
By LeeAnne Macaulay, Jeff Morier, Patti Hosler,
& Danuta Kierek-Jaszczuk, Ph.D.
Cangene Corporation

v

T
he Enzyme-Linked Immuno­ port individual CVPs and enforce
filtration Assay (ELIFA) }The compliance. The guidelines and pro­
provides high sensitivity of grams may cover a plethora of dif­
de­tection with rapid results. For this establishment ferent types of equipment but they
reason we developed a very sensitive, of Cleaning usually refer to equipment used in
semi-quantitative ELIFA to determine the manufacture, processing, hold­
IgA in therapeutic Win­Rho SDF™ Validation ing, filling, and packaging of raw
im­munoglobulin. In the course of the Programs (CVP) materials, inter­mediate/­final prod­
development we no­ticed that non-uni­ ucts, and associated components. The
form and unusually high background in the guidelines do not refer to equipment
(blank) re­sponses, that occurred infre­ pharmaceutical used in Research and Development
quently, greatly interfered with the (R&D), and to our understanding,
test results obtained. We hypothe­ industry is there is no regulatory requirement
sized that such background responses dictated by the for the development of CVPs for
resulted from inadequate cleaning of equipment used in these areas. The
the ELIFA apparatus. Accordingly, regulatory Easy-Titer™ ELIFA system2 is a
a cleaning program for the apparatus requirements small, micro­titer format compatible
has been devised and validated. In this apparatus developed and manufac­
paper the results supporting the hypo­ to develop tured by Pierce Chemical Company.
thesis will be presented, and the ratio­ and observe, As shown in Figure 1 (adapted from
nale and core aspects of the developed Product Instructions, Pierce Chemical
program delineated. in a fully Company) the apparatus utilizes a
documented way, nitrocellulose mem­brane (NC) sand­
Cleaning Validation wiched be­tween the sample appli­
Programs for Research effective cleaning cation plate and vacuum collection
and Development? procedures.~ chamber. Similar to the widely used
Enzyme-Linked Immunosorbent
The establishment of Cleaning Assay (ELISA), the ELIFA is an
Validation Pro­grams (CVP) in the pharmaceutical immunoassay well suited for testing of multiple sam­
industry is dictated by the regulatory requirements ples over a range of serial dilutions.3 In the ELIFA,
to develop and observe, in a fully documented way, the immunological reaction between NC immobilized
effective cleaning procedures. Regulatory guidelines ligand and ligand-specific analyte in the test sample
for validation of cleaning pro­cesses1 are meant to sup­ followed by an enzymatic reaction with a chromo­

56 Institute of Validation Technology
LeeAnne Macaulay, Jeff Morier, Patti Hosler, & Danuta Kierek-Jaszczuk, Ph.D.

Figure 1
Exploded View of the Easy-Titer™ Elifa System

Sample
Application
Thumb Plate
Screws Nitrocellulose

Clamp

Microtiter
Plate
Vacuum
Relief Valve Pump
Tubing
Gaskets
Port

Position Stops Tubing
(Acrylic Balls)

Transfer Membrane
Collection Support
Cannula Chamber Plate
Guide Pins

genic substrate gives rise to colored dots. The color ELIFA CVP – Approaches and Hallmarks
intensity of the individual dots varies proportionally
to the amount of the analyte in the samples and dots A body of experience at Cangene with valida­
produced by the samples devoid of analyte (blanks or tion5,6 or cleaning7 programs, as well as manufac­
background) are very pale or even colorless. We used turer’s cleaning instructions for the ELIFA system
the ELIFA system to research and develop a screen­ (Figure 2, adapted from Product Instructions, Pierce
ing assay for human IgA.4 The developed IgA ELIFA Chemical Company) was the foundation when devel­
will be used for testing of the licensed WinRho SDF™ oping the ELIFA CVP. Among others, the devel­
therapeutic, hence, its performance characteristics need oped program addressed the following:
to be established and validated. In pre-validation stud­
ies, however, we observed that the developed ELIFA nS  pecific design of apparatus, its individual parts
lacked reproducibility. The color of the blank dots var­ and accessories that require cleaning
ied sometimes from experiment to experiment or, even n Disassembling and re-assembling the unit
within the same experiment, from well to well. We also before and after cleaning
observed that the color of dots produced by the repli­
cated test samples occasionally varied. We postulated Figure 2
that the observed variability is a result of external con­ Cleaning of the Easy-Titer™
tamination carried over from previous experiment(s).
ELIFA System
An inadequately cleaned ELIFA apparatus would then
Clean all of the pieces to the Easy-Titer™ ELIFA
be the cause of obscured test results. We, therefore, System unit in a two percent PCC-54 solution and
decided to develop a CVP for the ELIFA apparatus then rinse with distilled water. The unit may also
before proceeding to assay validation. be soaked in the PCC-54 solution to remove stains
from the unit caused by the substrate solution.

Special Edition: Cleaning Validation III 57
LeeAnne Macaulay, Jeff Morier, Patti Hosler, & Danuta Kierek-Jaszczuk, Ph.D.

nC  leaning operations tion with two ELIFA experiments; the standard IgA
n Cleaning procedures including cleaning ves­ ELIFA and the mock ELIFA. Such a combination of
sels, agents and utensils analytical methods allowed for instantaneous moni­
n Compatibility of cleaning agents with equip­ toring of the effectiveness of the cleaning pro­cess.
ment and assay Standard IgA ELIFA method4 involved testing 96
n Decontaminating abilities of cleaning agents replicates of a sample at a high (worst-case condition)
n Sampling on cleaned equipment IgA concentration, which were applied into 96 wells of
n Analytical methods for monitoring of cleaning the ELIFA apparatus. As expected, these experiments
processes invariably produced highly colored dots (Figure 4). A
n Storage of cleaned parts tested cleaning regimen (procedure 1 or 2 in Figure
n Inspection of apparatus for cleanliness before use 3) followed by the second mock experiment was then
n Recording and documenting the cleaning pro­ executed. The mock ex­periment involved the use of
cedures the diluting buffer in lieu of a sample with high IgA
n Establishing acceptance criteria, and concentration that was also applied into 96 wells of
n Maintaining cleaning records the ELIFA apparatus. Providing that the cleaning regi­
men was effective, the mock experiment should not
Strategy for Validation of produce colored dots, as there was no specific analyte
Cleaning Procedures that could attach to the immobilized ligand to facilitate
subsequent enzymatic and color reactions. The results
Two cleaning procedures (procedure 1 and 2 in ob­tained show that whereas Procedure 1 did not
Figure 3) utilizing either enzyme or detergent-based re­move the contaminants from preceding experiments
cleaning agents were developed and tested in conjunc­ well enough (Figure 5), procedure 2 was fully effec­
Figure 3
Cleaning Procedures 1 and 2
Procedure 1 Procedure 2
Disassemble the unit by first removing the thumb Disassemble the unit by first removing the thumb screws
screws on the top of the sample application plate, then located on the top of the sample application plate, then
removing the application plate and top gasket, and removing the application plate and top gasket, and
finally unclamping the membrane support plate from finally unclamping the membrane support plate from
the collection chamber. the collection chamber.
Rinse all parts for two minutes under running Reverse Rinse all parts for two minutes under running RO water.
Osmosis (RO) water.
Immerse them into a vessel with two percent TERG-A- Immerse them into a vessel with five percent RBS10 solution
ZYME (Alconox Inc., New York, NY, U.S.A.) solution and at 50ºC and wash for five minutes by agitating the vessel.
wash for five minutes by agitating the vessel.
Rinse all parts for two minutes under RO water. Rinse all parts for two minutes under RO water.
Clean all 96 wells of the sample application plate with Clean all 96 wells of the sample application plate with
TOC swabs by dipping the swabs into the detergent TOC swabs by dipping the swabs into the detergent
solution, inserting them into wells once from top and solution, inserting them into wells once from the top and
once from the bottom, and swabbing the inner part of once from the bottom, and swabbing the inner part of
each well by turning the swab first to the right and each well by turning the swab first to the right and
then to the left. then to the left.
Clean all 96 wells of the top gasket in a similar way. Clean all 96 wells of the top gasket in a similar way.
Raise all 96 cannulas on the membrane support plate Raise all 96 cannulas on the membrane support plate and
and soak the plate for five minutes in the detergent clean them with TOC swabs by dipping the swabs into the
solution. detergent solution and swabbing the surface of individual can-
nulas and also spaces between cannulas and bottom gaskets.
Rinse each part and the spaces between the bottom Rinse each part and the spaces between the bottom
gasket and the membrane support plate for two gasket and the membrane support plate for two seconds
seconds under running RO water. under running RO water.

58 Institute of Validation Technology
LeeAnne Macaulay, Jeff Morier, Patti Hosler, & Danuta Kierek-Jaszczuk, Ph.D.

Figure 4 Figure 5
IgA ELIFA Results Obtained for a IgA ELIFA Results Obtained for a
Test Sample Containing Human Replicated Test Sample Deprived
IgA at a Concentration of 2µg/mL of Human IgA.

The experiment was performed in an apparatus
cleaned with TERG-A-ZYME (Procedure 1).
Figure 6
tive (Figure 6). Procedure 2 was then validated, in two
IgA ELIFA Results Obtained for a independent experiments performed by two analysts.
Replicated Test Sample Deprived It was shown that it invariably leads to results similar
of Human IgA. to those presented in Figure 6.

Assessment of the Effectiveness
of the Validated Procedure

The Total Organic Carbon (TOC) method is
widely utilized in industrial CVPs as it measures
low levels of carbon and is compatible with swab
sampling techniques. The standard IgA ELIFA4 fol­
lowed by the validated cleaning procedure and swab
sampling of the surface of three randomly selected
wells were, therefore, used to assess the cleanliness
of the apparatus by standard TOC. A procedure used
at Cangene8 was followed. The results obtained con­
The experiment was performed in an apparatus firm that the validated cleaning procedure was fully
cleaned with RBS (Procedure 2).
effective as the carbon concentration determined in
Figure 7
Results From Total Organic Carbon Analysis (in ppb)
Sample Sample Replica Replica Replica Average SD Percent
Number Name 1 2 3 CV
1 Well B11 277.8 271.7 268.9 272.8 4.55 1.67
2 Well D8 235.4 234.3 225.9 231.8 5.20 2.24
3 Well G2 228.4 219.1 255.3 234.3 4.73 8.03
4 Water 185.2 165.4 167.4 172.7 10.9 6.29

Special Edition: Cleaning Validation III 59
LeeAnne Macaulay, Jeff Morier, Patti Hosler, & Danuta Kierek-Jaszczuk, Ph.D.

water extracts of test samples was only slightly great­ a B.Sc. degree at the University of Winnipeg and
er than that of water used for extraction (Figure 7). received a diploma in Chemical and Bioscience
Technology from Red River College in Winnipeg.
Implementing of the ELIFA CVP She has experience in QC/QA Laboratories in the
areas of microbiology and biochemistry.
The validated ELIFA cleaning procedure will Jeff Morier is a Senior Assay Development Tech­
become part of a written Standard Operating Procedure nologist at Cangene Corporation. He received his
(SOP). Although addressing R&D instrumentation, the B.Sc. degree in Microbiology from the University
of Manitoba. He has seven years experience in the
SOP document will detail the activities that were con­
pharmaceutical industry in the areas of QC microbi-
ducted by adhering to industrial standards for cleaning
ology, QA biotechnology, and R&D experience in the
validation.1,9 The document will also advise on safety validation of immunoassays of various formats.
precautions, cleaning schedule, and assignment of
Patti Hosler is a Technician at Cangene Corporation.
responsibility for cleaning and storage of the cleaned
She completed the first year of a B.Sc. degree pro-
apparatus. The SOP document will be observed not
gram at Brandon University and received a diploma in
only when validating the performance of the IgA Chemical and Bioscience Technology from Red River
ELIFA, but also during routine use of the ELIFA sys­ College. She has seven years experience as a QA/QC
tem. It will be the subject of a periodic evaluation and, laboratory technician in the food production industry.
if deemed necessary, be updated and/or revised. Danuta W. Kierek-Jaszczuk is a Senior Research
Scientist/Assay Development Supervisor at Cangene
Conclusions Corporation. She obtained her M.S. degree in
Biology from the Nicolaus Copernicus University,
nA  comprehensive, credible CVP designed and and a Ph.D. degree in Agricultural Sciences from
developed at Cangene for the Easy-Titer™ the Polish Academy of Sciences Institute of Genetics
ELIFA system has been shown to effectively and Animal Breeding. She can be reached by
remove contaminants and residues entrapped phone at 204-275-4263, by fax at 204-269-7003,
in the apparatus after the conclusion of the and by e-mail at dkjaszcz@cangene.com.
ex­periment(s) and/or subsequent cleaning.
n The CVP has been demonstrated to vastly References
1. FDA. 1993. “Guideline to Inspection of Validation of Cleaning Pro­
re­duce the analytical background of the IgA cesses.” Office of Regulatory Affairs, USFDA, Washington, D.C.
ELIFA, improve its signal to background ratio, 2. Pierce Chemical Company. Product Instructions, Easy-Titer™
ELIFA System. Rockford, IL.
increase the quality of the test results and may, 3. Paffard, S.M., Miles, R.J., Clark, C.R., and Price, R.G. 1996.
therefore, be expected to notably support the “A Rapid and Sensitive Enzyme Linked Immunofilter Assay
(ELIFA) for Whole Bacterial Cells.” Journal of Immunological
upcoming assay validation. Methods 192, no. 1–2: 133-6.
n The CVP, by virtue of anti-viral and anti-bacte­ 4. Morier, J., Macaulay, L., and Kierek-Jaszczuk, D. “Screening for
the Presence of Human IgA in a Hyper Immune Product Using An
rial properties of the RBS10, allows for simulta­ Enzyme-Linked ImmunoFiltration Assay.” Poster Presentation at
neous decontamination and sanitization of the IBC Conference on Assay Development for Future High-Throughput
ELIFA unit, thus facilitating its safe use with Screening. 8 – 9 November 1999. Annapolis, MD.
5. Faurschou, A. 2000. General Procedure for Validation Program.
infectious samples. SOP Document # 11.001.0001.RR. Cangene Corporation.
n The CVPs generated for R&D equipment Winnipeg, MB, Canada.
6. Alejo, M. and Faurschou, A. 1998. Process Validation Qualification.
that fulfill the standards of industrial cleaning SOP Document # 11.001.0002.RR. Cangene Corp­oration.
validation not only improve the quality of the Winnipeg, MB, Canada.
7. Heise, R. and Poschner, E. 1999. Manual Cleaning and Sanitizing
as­says utilizing this equipment but may become Equipment. SOP Document # 2.010.0017.RR, Cangene Corp­
vital components of assay validation. o oration. Winnipeg, MB, Canada.
8. Shinkarik, T. 1998. Surface Sampling for Total Organic Carbon
(TOC). SOP Document # 500602.RR, Cangene Corporation.
Winnipeg, MB, Canada.
9. Chudzik, G.M. 1998. “General Guide to Recovery Studies Using
About the Authors Swab Sampling Methods For Cleaning Validation.” Journal of
Validation Technology 5, no. 1: 77-81.
LeeAnne Macaulay is a Technician at Cangene 10. Pierce Chemical Company. Product Information, RBS. Rockford, IL.
Corporation. She completed the first year towards

60 Institute of Validation Technology
A Cleaning Validation Master
Plan for Oral Solid
Dose Pharmaceutical
Manufacturing Equipment
By Julie A. Thomas
McNeil Consumer Healthcare

W
v
ith the benchmark con­ Validations of Clean­ing Processes
stantly being raised, }Often, – July 1993.” Each of these will be
many companies find discussed in greater detail in the sec­
that they are in perpetual valida­ companies have tions below.
tion mode. Often, companies have
executed validations for equipment, executed n Objective
cleaning, and processes, but the validations for n Scope
doc­­umentation no longer stands up n Introduction
to the latest in validation standards. equipment, n Responsibilities
Although these validations are gen­ n Philosophy
erally complete and on file, there cleaning, and pro- n Methodology
are many opportunities to improve n Schedule
both the supporting documenta­
cesses, but the doc-
tion and the execution. One way to umentation Objective
en­sure that your company’s policies
and procedures regarding cleaning no longer stands This section should state the pur­
validation are state-of-the-art is to pose of your cleaning master valida­
assemble a multi-disciplined team up to the latest tion plan and define whether you will
from the appropriate manufacturing validation be revalidating current procedures or
sites that can review and revise all prospectively validating new ones.
components associated with clean­ standards.~ Often, the plan will have provisions
ing validation. What follows are for both situations.
ex­cerpts from a Cleaning Validation
Master Plan (the Plan) that was painstakingly com­ Scope
posed and has now be­come the standard for planning
and executing cleaning validations at several manu­ The scope needs to list exactly which aspects of val­
facturing sites. idation will be covered in the document and to which
An outline of the Plan contains the following seven types of products and/or processes the Plan applies. For
elements, the concepts of which are taken directly example, “This document provides steps for planning,
from the FDA publication, “Guide to Inspections of executing, and maintaining equipment cleaning valida­

Special Edition: Cleaning Validation III 61
Julie A. Thomas

tions for oral solid dose products at Your Company’s for accuracy and agreement with operating
manufacturing facility in Your City, State.” practices
Introduction • Create and/or revise related SOPs and
cleaning checklists
The introduction should let the reader know • Perform cleaning processes per SOP as
what elements will be addressed in the Master referenced in the validation protocol
Validation Plan and why a formal plan is necessary. • Provide documented training for all person­
For in­stance, “This plan is intended to be a roadmap nel responsible for cleaning the equipment
clarifying the course the Company will take as it Quality Assurance
plans and executes the cleaning validations required • Review and approve protocols and reports
by current Good Manufacturing Practices (cGMP). for conformance with cGMPs and internal
This program describes and defines the various procedures
categories of cleaning validation, provides the nec­ • Provide analytical technical support
essary protocol elements, and offers guidance for • Provide documented training for all person­
un­ex­pected results. Furthermore, it includes provi­ nel responsible for sample collection and
sions for revalidation and monitoring and serves as testing
a mechanism to organize and store critical informa­ • Collect analytical samples as specified in
tion that supports the cleaning validation process.” the protocol
• Perform analytical testing using validated
Responsibilities procedures
• Label, package, and send out those samples
There are many departments and disciplines that need to be analyzed by an external
involved in planning for and executing a cleaning laboratory
validation. It is necessary to list each contributing • Review and approve analytical results
area and the associated tasks for which it is respon­ • Notify departments of test results
sible. This serves to clarify roles and to ensure that Engineering
tasks are not overlooked. Typically, representatives • Inform the affected department in advance
from Validation, Manufacturing, Quality Control, of any anticipated change to the facility or
En­gineer­ing, and Research and Development (R&D) equipment
will be needed. The following are some examples of • Include all utilities and cleaning equipment
departmental responsibilities: in the calibration and maintenance pro­
gram
Validation Specialist • Review and approve equipment drawings
• Review cleaning procedures and surface area calculations
• Assist the cleaning validation team in iden­ Research and Development
tifying equipment test sites for swab or • Provide swab and surface recovery data for
rinse samples active ingredients and cleaning agents
• Write cleaning validation protocols • Validate analytical test methods for chemi­
• Coordinate execution of the cleaning pro­ cal and cleaning agent analyses
cess with the appropriate departments and • Transfer validated methods to the site QC
laboratories laboratories and/or contract laboratories
• Prepare the sampling schedule • Provide recommended cleaning procedures
• Assemble the test data into final report for new active ingredients and/or cleaning
form for approval agents
Manufacturing
• Provide technical information for the devel­ Cleaning Validation Philosophy
opment of protocols and reports
• Review and approve protocols and reports This section discusses the considerations you

62 Institute of Validation Technology
Julie A. Thomas

have made in developing a comprehensive cleaning preparatory and includes analytical methods valida­
validation program, such as how to define equipment tion, recovery studies, surface types, degradants, and
holding time, equipment storage time, and campaign methods transfer. There is a considerable amount of
length. In general, the philosophy section presents the scientific activity that must be completed before the
Company’s position on what is being achieved by the validation can begin. These steps are explored in the
cleaning validation and how it will be demonstrated. following sections.
For instance, “Cleaning validation is required for all
manufacturing and packaging equipment that comes 1. Analytical Methods Validation
into contact with the product or product components Describe how the analytical methods will be
during production. Prior to validation, acceptance developed and validated for active ingredients, deg­
criteria will be developed for active ingredient and radants (if applicable), and cleaning agent residue.
cleaning agent residues. Verification of acceptable Validation of the method should assess reproducibil­
equipment holding time will be included as part of ity, linearity, specificity, limit of detection (LOD),
the validation. Holding time is defined as the time and swab and surface recovery. Other elements for
between the end of the last product manufactured and consideration are the instrumentation, swabbing and
the start of the cleaning process. This will demonstrate dilution solvents, dilution volume, and sample han­
that the cleaning procedure effectively removes resi­ dling and storage.2,3
due after the equipment has remained idle for a speci­
fied period of time. Additionally, holding time will be 2. Recovery Studies
evaluated to ensure storage conditions are adequate Recovery studies evaluate quantitative recovery
for a predetermined length of time. Storage time is of chemical residue from both the surface to be
defined as the time between cleaning completion and sampled and the swab material to be used for sam­
the next batch processed on the equipment. Campaign pling. The results confirm the appropriateness of
length will be determined jointly by Operations and the sampling method and material used. You should
R&D and validated with at least three iterations using determine the minimum recovery criteria for each
the maximum number of batches or maximum length surface type and state that percentage in this sec­
of time. This approach fully challenges the cleaning tion. For instance, you may want recovery values of
procedure by providing worst-case residues.” at least 70% of actual readily soluble residues, but
may choose a much lower recovery value for rela­
Cleaning Validation Methodology tively insoluble proteins.4 Most important, you must
provide data to justify the chosen value.
To ensure all of the elements are in place for a
thorough and successful validation, a chronological 3. Surface Types
methodology should be followed. One such design is Since different surface types have different affini­
illustrated through the following four phases: devel­ ties, you may want to choose a few surface materials
opment, planning, execution, and maintenance. (See to represent the many product contact surfaces used
Figure 1) In this section of the Plan, it is appropriate in manufacturing. For oral solid dose manufactur­
to include the number of sampling/testing iterations ing, you may determine that stainless steel, silicone,
required for each piece of equipment and/or each and polypropylene are the most abundant surfaces
analyte. (See Figure 2.) and that they also provide varying degrees of poros­
If you intend to reduce the number of tests ity. A matrix of all surface types and the representa­
re­quired to validate cleaning after various products tive material that will be used in recovery studies is
by using a grouping approach, it should be explained appropriate. (See Figure 3)
in this section.1
4. Degradants
Development Phase Many degradant products are more soluble in the
cleaning solvent than are the active ingredients; there­­
The initial phase of the cleaning validation plan is fore, you should determine the degree of degradant

Special Edition: Cleaning Validation III 63
Julie A. Thomas

Figure 1
Cleaning Validation Flow Diagram
Development Phase

Analytical Method
Analytical Method
Validation
Development
•D egradant
• Recovery
identification
• Surface types
• Transfer

Planning Phase

Equipment
Analyte Selection Protocol Cleaning
•S  ample site and Acceptance Development SOP
selection Criteria
•W  rite •W  rite
• Surface area
•A  ctive ingredient • Approve • Approve
calculation
• Cleaning agent • Train • Train
• Schematic

Execution Phase

Protocol Execution
•C  lean
• Sample
• Test

No
Incident
Pass?
Investigation

Yes

Validation Report
•W  rite
• Approve

Maintenance Phase

Monitoring Change Control

Revalidation

64 Institute of Validation Technology
Julie A. Thomas

Figure 2
Cleaning Iteration Summary Requirements
Sample Total Number of Iterations Conditions
Active Residue 3 1 at maximum campaign length or
maximum time period plus holding
time.
2 at maximum campaign length or
time period.
Cleaning Agent Residue 3 3 per cleaning procedure, per piece
of equipment.

testing required based on the toxicity and solubil­ 1. Equipment Information
ity of potential degradants. Likewise, active ingre­ This section should detail the methodology for
dients should be exposed to the selected cleaning providing specific equipment information. One
agent under normal usage conditions to determine option is to prepare a binder containing detailed
if degradants are formed as a result of the cleaning surface area calculations, swab sampling sites (with
process. justification), photos, and schematic diagrams for
each piece of equipment. This binder can be main­
5. Analytical Methods Transfer tained separately and used as an attachment to the
In this section, you can state how sampling and cleaning validation protocol as needed.
analytical methods will be transferred from the
R&D laboratories to the site QC laboratories and a) Sample Site Selection
how the analysts conducting validation testing will Explain how you will select sampling sites to rep­
be qualified. Reference appropriate SOPs and/or resent the product contact surface area of the equip­
De­velopment Transfer Report. ment. One of the best sources of information is the
operator who routinely cleans the equipment. He or
Planning Phase she can certainly point out the areas they find most
difficult to clean. Make the operator part of a larger
The next phase of preparation is the planning phase. team of experts to include representatives from
This is a broad category that focuses on equipment Validation, QA, and Operations, and let the team
information, analyte selection, acceptance criteria, determine the product contact surface areas that are
cleaning procedures, and protocol development. At this most difficult to clean and those that are most repre­
point, you are starting to think about what equipment sentative of the equipment. Sampling these sites will
will be included in the validation, which analytes will represent the entire equipment surface area using
be chosen, and how you will determine acceptance cri­ the assumption that residue will be evenly distrib­
teria. This leads to an in-depth review of the procedures uted over the equipment and that the most difficult
and, finally, to protocol development. to clean locations will represent the worst case for
residue removal. Include the basis for selecting each

Figure 3
Surface Recovery Matrix
Recovery Surface: 316L Stainless Steel Polyethylene Silicone
Material Used: 316L Coupon Plastic Bulk Container Hose
To Represent: 304 Stainless Teflon Rubber
Aluminum Lexan EPDM
Brass HDPE Neoprene

Special Edition: Cleaning Validation III 65
Julie A. Thomas

site. For example, sampling sites may be deemed to Figure 6
be the most difficult to clean, most difficult to dry, Kason Separator
or of different material of construction. Swab sites

Figure 4
Swab Site

can be indicated with either digital photographs or pare schematic diagrams labeled with the major sec­
suitable diagrams. (See Figure 4) tions of the equipment. (See Figure 6) The drawings
b) Surface Area Calculation do not have to be to scale, but should appropriately
An accurate surface area must be calculated for represent the equipment. If a schematic is not practical
each piece or section of equipment. This can be (i.e., a packaging line), a photograph is acceptable. The
done with manufacturer’s drawings, but should be intent is to depict the product contact surfaces that are
confirmed by field measurements. If drawings are included in the calculations. This helps to ensure that
not available, the equipment must be measured to the swab samples are taken from the intended location.
determine surface area (see Figure 5). Although not
shown here, it is advisable to include the calcula­ 2. Analyte Selection
Analyte selection is required for active, excipi­
Figure 5 ent (possibly), and cleaning agent residues. Keep
in mind that you are validating a cleaning proce­
Surface Area dure, not a manufacturing process. In the situation
Swab Number Area Swabbed where the same cleaning procedure is used for many
1 Screen/ring interface product formulas, there is an opportunity to select a
gasket representative analyte to cover multiple active ingre­
2 Discharge port – inside dients and reduce the amount of testing.
of top circular area
(top seam) a) Actives
Total contact S.A. of Kason Separator (in ) 2
3,171.2 If several active ingredients are processed in a single
piece of equipment, a marker active, or guiding sub­
Total contact S.A. of Filter Socks (in2) 15.6
stance, can be selected based on the active ingredient
solubility in water, potency, previous production expe­
tions with the schematic diagram in the equipment rience, and R&D studies. This reduces the number of
information binder mentioned above. studies required to validate the cleaning procedure.5
c) Schematic Diagram
To clearly illustrate each piece of equipment, pre­ b) Excipients

66 Institute of Validation Technology
Julie A. Thomas

The removal of excipients can either be con­ calculated using a formula such as the No Observed
firmed by visual inspection or through analytical Effect Limits (NOEL).8
testing. The approach should be stated here along 4. Cleaning Procedures
with training requirements for individuals perform­ This section should indicate that cleaning procedures
ing visual inspection. will be developed (or existing procedures reviewed)
prior to the validation. It should also list the required
c) Cleaning Agents elements for cleaning procedures, such as temperature,
Testing for cleaning agent residue is essential but pressure, water quality, cleaning agent concentration,
is often an area in which current cleaning validations spray nozzle location, etc., or it should reference where
are deficient. For most cleaning agents, a marker these requirements can be found.9 Additionally, you
compound can be selected for analysis based on the should describe the process for training the operators
recommendation of the cleaning agent manufactur­ who will be executing the validation studies.10
er. Removal of volatile cleaning agents that do not
leave a residue, such as isopropyl alcohol, may not 5. Protocol Development
need to be validated. The next step is to write a cleaning validation pro­
tocol for each cleaning procedure that you intend to
3. Acceptance Criteria validate. The protocol should describe all documenta­
The equipment must pass visual and olfac­ tion required to complete the cleaning validation. It
tory inspection, where appropriate, as defined in should also present the rationale for using a marker
the cleaning validation protocol prior to initiation active to cover validation for multiple products. For
of swabbing.6 This is a critical step in the validation ease of review, include a matrix of the products and
process that, if skipped, can lead to failed results. equipment that are covered by each validation, or
reference where this information can be found. For
a) Active Ingredient example, if there are three active ingredients processed
Acceptance criteria for active ingredients should in Fluid Bed Granulator #1, indicate which active will
be based on medical and pharmacological properties be used to represent the other two. Likewise, indicate
and scientific information. Calculations using the which pieces of equipment will be used to validate
maximum allowable carryover (MAC) and/or 10ppm
formulas can be used.7 Figure 7
To ensure that all active contact surfaces are consid­ Equipment Cleaning Matrix
ered in the carryover calculation, you may want to iden­
tify equipment trains. Acceptance criteria are calculated Active A Active B Active C Cleaning
using the surface area from the entire equipment train; Agent A
however, protocols are executed per each piece of equip­ Fluid Bed Gran 1 X – – X
ment. Equipment trains could be designated as follows: Fluid Bed Gran 2 – – – –
Starch Kettle 1 – – – X
n Granulation – granulator system through the
product container the removal of active ingredient and cleaning agent
n Compression through printing – compression, residues. (See Figure 7)
film-coating, and printing phases Execution Phase
n Packaging – product contact surfaces for each
type of packaging line When all of the supporting documentation is com­
plete, it is time to execute the plan. During the execu­
b) Cleaning Agent tion phase, you will complete the protocol, investi­
Acceptance criteria for the cleaning agent marker gate any nonconformances that may have occurred,
should be based on toxicity, limit of detection of and write a report to summarize your findings.
validated assay method, and/or data gathered dur­
ing certification studies. Acceptance criteria can be 1. Protocol Execution

Special Edition: Cleaning Validation III 67
Julie A. Thomas

Typically, three iterations of cleaning, sampling, repeatability is highly dependent on the quality and
and testing using the same procedure are required. consistency of training. Monitoring should include,
Acceptance criteria for all cleaning iterations must at a minimum, a review of changes made to the
be met for both the active ingredient and the clean­ cleaning procedure or equipment, visual inspection
ing agent. Be sure to reference the procedure where of the equipment, and direct observation of employ­
a detailed description of the chemical swab prepara­ ees executing the cleaning procedure. For some
tion and sampling methods can be found. equipment, swab samples for active ingredients may
be necessary in addition to the visual inspection
2. Incident Investigation and observation. Indicate the frequency that you
This section explains how the Company will intend to monitor the cleaning process. Reference
handle test failures and nonconformances during the appropriate SOP for detailed requirements of the
execution of the validation. Once the root cause of monitoring program.
the failure has been identified, options are to addend
the protocol or start over with a new protocol. For 2. Change Control
any incident that occurs during validation, docu­ Indicate how changes will be managed to ensure
ment the investigation along with corrective and the validated state is maintained. Any change in
preventive actions. The incident report may contain the facility, process equipment, cleaning procedure,
elements such as: cleaning agent, product formulation, or addition
of new products to the equipment train should be
n Cleaning validation protocol number documented and approved via the Change Control
n Incident report number System. The change should be reviewed by the Val­
n Equipment model and location idation Group, Operations, and QA, who will decide
n Initiator and date if revalidation is necessary. Reference appropriate
n Incident description SOPs.11
n Root cause analysis
n Corrective actions recommended/taken 3. Revalidation
n Assessment of effect on product Indicate the criteria that will be used to determine
the need for revalidation. Based on the nature of the
3. Reports change, a determination will be made as to which,
Describe the report format and content that will if any, phases of the validation must be repeated.
be used to summarize the validation. Reference Ref­erence where documentation of the revalidation
appropriate SOPs for detailed report information. will be filed.12,13
An explanation of all deviations should be included
in the validation report. Cleaning Validation Schedule

Maintenance Phase Prioritization
As is usually the case, all cleaning validations
The final phase of the Plan should specify how cannot commence at one time; therefore, it is nec­
you will maintain the conditions you have just essary to set up a priority list. Some situations to
validated. This includes periodic monitoring, using consider are:
a control of change process, and potentially, revali­
dating. n Equipment shared between products contain­
ing different active ingredients
1. Monitoring n Equipment in contact with raw material with
This section details how you will ensure that the high potential for contamination
conditions used during validation remain in con­ n Unshared primary equipment currently in use
trol during routine production. This is especially with outdated validations
important for manual cleaning procedures, where n Unshared auxiliary equipment used for pro­

68 Institute of Validation Technology
Julie A. Thomas

duction with limited potential for product References
contamination 1. Jenkins, K.M. and Vanderwielen, A.J. “Cleaning Validation: An
Tactical Schedule Overall Perspective,” Pharmaceutical Technology, April 1994,
p. 62.
A proposed schedule, including equipment pri­ 2. McCormick, P.Y. and Cullen, L.F., Pharmaceutical Process
oritization and target initiation dates, should be pre­ Validation, 2nd ed., edited by I.R. Berry and R.A. Nash, 1993,
p. 334.
sented in this section. This gives an indication that 3. Kirsch, R.B., “Validation of Analytical Methods Used in
you have contemplated the order of execution, and Pharmaceutical Cleaning Assessment and Validation,”
Pharmaceutical Technology, Analytical Validation, 1998.
it also provides a tool that can be used to track your 4. Chudzik, G.M., “General Guide to Recovery Studies Using
progress. Swab Sampling Methods for Cleaning Validation,” Journal of
Validation Technology, Vol. 5, No. 1, pp. 77-81.
5. Hall, W.E., “Your Cleaning Program: Is It Ready for the Pre-
Summary Approval Inspection?” Journal of Validation Technology, Vol.
4, No. 4, August 1998, p. 302.
6. Alvey, A.P. and Carrie, T.R., “Not Seeing is Believing – A
There are many aspects of cleaning validation Non-Traditional Approach for Cleaning Validation,” Journal of
Validation Technology, Vol. 4, No. 3, pp. 189-193.
that must be carefully planned to guarantee a suc­ 7. Fourman, G.L. and Mullen, M.V., “Determining Cleaning
cessful validation program. If you begin with a phi­ Validation Acceptance Limits for Pharmaceutical Manufact­ur
ing Operations,” Pharmaceutical Technology, 17 (4), 1993, pp.
losophy, this will set the stage for you to develop a 54-60.
structured approach. By dividing the approach into 8. Hall, W.E., “Validation of Cleaning Processes for Bulk
Pharmaceutical Chemical Processes,” Cleaning Validation An
sections, such as development, planning, execu­ Exclusive Publication, p. 4.
tion, and maintenance, it breaks down the project 9. Hall, W.E., “Proper Documentation and Written Procedures,”
into manageable segments. To complete the Plan, Journal of Validation Technology, Vol. 4, No. 3, pp. 199-201.
10. Tunner, J., “Manual Cleaning Procedure Design and Validation,”
generate a tactical schedule and begin monitoring Cleaning Validation An Exclusive Publication, p. 28.
pro­g­ress towards your new and improved cleaning 11. PDA Technical Report No. 29, “Points to Consider for Cleaning
Validation,” March 1998, p.43.
validation status. o 12. Coleman, R.C., “How Clean is Clean?” Journal of Validation
Technology, Vol. 2, No. 4, August 1996, p. 278.
13. Jenkins, K.M. and Vanderwielen, A.J., “Cleaning Validation: An
Overall Perspective,” Pharmaceutical Technology, April 1994,
About the Author p. 70.

Julie Thomas is Validation Manager at McNeil
Consumer Healthcare in Round Rock, Texas. She
has 14 years of experience in various aspects of
solid dose pharmaceutical manufacturing. Most
recently, she chaired a company-wide commit-
tee to enhance cleaning validation practices and
procedures for all McNeil facilities. She can be
reached by phone at 512-248-4470 or by e-mail at
jthomas1@mccus.jnj.com.

This article presents only one alternative for pre­
paring a Master Validation Plan. The views ex­pressed
in this article are strictly those of the author and in
no way represent the view of McNeil Con­sumer
Healthcare, Johnson & Johnson, or this publication.

© Advanstar Communications Inc. All rights reserved.

Special Edition: Cleaning Validation III 69
PROPOSED VALIDATION STANDARD
VS-3
Cleaning Validation

VALIDATION TECHNOLOGY

Journal of Validation Technology ~
Proposed Validation Standard VS-3

PROPOSED VALIDATION STANDARD VS-3
Cleaning Validation

Introduction

T his document is the third in a series of new proposed validation standards issued by the Institute of
Validation Technology Standards Committee (IVT/SC). The initial proposed standard (Process
Validation Standard VS-l: Nonaseptic Pharmaceutical Processes) was issued in February 2000, and
is intended to help practitioners worldwide who develop, implement, control, and validate processes that pro-
duce Active Pharmaceutical Ingredients (APIs) and drug products. Our second proposed validation standard
VS-2: Computer-Related System Validation was issued in May 2001. The current document (Cleaning
Proposed Validation Standard VS-3) is intended to offer more specific proposed standards for the cleaning
processes for equipment used to manufacture APIs and drug products. These proposed standards, will be used
by reviewers of manuscripts intended for publication in the lournal of Validation Technology (1VI).
Just as with the previous proposed standards, readers are encouraged to offer comments, questions, and rec-
ommendations. Such feedback will be useful to the IVT/SC and JVT editors in updating this document and in
developing future proposed standards. Technologies are continually changing, sometimes in ways that can influ-
ence the way validation is best conducted. Therefore, the IVT/SC plans to periodically update each proposed val-
idation standard, including its corresponding Preamble and reference list. In order to be dynamically responsive
to changing industrial practices and regulatory requirements, and make it easier for readers to cut and paste the
contents for their own use, all three proposed standards are available on the IVT web site at www.ivthome.com.
A fundamental need the IVT/SC intends to meet with its new proposed standards stems from the fact that most
Good Manufacturing Practice (GMP) regulations today call for numerous written procedures; for example, more
than 100 different kinds of written procedures are required to comply with current GMP regulations in the United
States. Many firms find it helpful to issue written policies in order to coordinate and reduce the number and length
of required Standard Operating Procedures (SOPs). Thus, the IVT proposed validation standards format includes
statements and definitions that can be excised and used directly or with minor editing in a firm's policies and SOPs.

Contents of the Proposed Cleaning Validation Standard
In order to be consistent with the prototype standard (Validation Standard VS-l) the Proposed Cleaning
Validation Standard VS-3 will be divided into the following five sections:

I. Policy statements - Proposed standards that indicate what is required
II. Procedural Statements - Proposed standards that describe how to meet requirements
III. Acronyms - Meaning of each acronym/abbreviation used in the document
IV. Glossary - Definition of key terms, which are highlighted and asterisked (*) when first used in the
proposed validation standard

~ Institute of Validation Technology
Proposed Validation Standard VS-3

V. Regulatory Excerpts - Regulatory language (United States, Australia, Canada, World Health Organ-
ization [WHO], Japan, and European Union) related to each Standard

T he following proposed standard is intended to reflect desirable contemporary practices, is not binding in
any way, and can be modified to suit a firm's specific needs. This proposed standard incorporates imper-
ative verbs (e.g., shall, will, must) to provide users with unambiguous quality assurance auditing tools,
and is prefaced by a Preamble that provides rationale for several of the more complex concepts. This document
is also directed toward users located at a given plant site that mayor may not be a part of a larger corporation.
Terms that are bold and asterisked (*) the first time they are used are defined in Section IV - Glossary.

I. POLICY STATEMENTS

POL 1.1
The critical cleaning processes associated with the manufacture of Active Pharmaceutical Ingredients
(API)*, critical Intermediates*, Drug Products*, or In-Process Materials* shall be validated or verified.

POL 1.2
The critical cleaning processes associated with products in the development stage of the product lifecycle
shall be verified. The administrative responsibility for such products will reside in either the appropriate
development group or in the Site Validation Steering Committee (SVSC)*. If the company decides that
responsibility for cleaning verification shall reside in the appropriate development group, then the docu-
mentation describing the verification procedure and the Cleaning Verification Protocols* must also be ap-
proved by the site Quality Authority*.

POL 1.3
During development of the new product, the manufacturing equipment, batch size, and formulation is con-
stantly changing and the cleaning procedure must be appropriate and customized for each manufacturing event.
The lifecycle for the development and validation of a new cleaning procedure consists of the following steps:

l.3.1 Determine what materials need to be cleaned from the equipment or surfaces.
1.3.2 Determine what methods should be used to evaluate the anticipated residues (from Section
1.3.1). Determine the sensitivity and reproducibility of these methods.
1.3.3 Define the Critical Product Cleaning Specifications*.
1.3.4 Define the specific equipment to be used for each development batch.
l.3.5 Define the specific formulation to be used for manufacturing each individual development batch.
1.3.6 Identify the cleaning agents to be used, if appropriate.
1.3.7 Determine what other products are manufactured in the same equipment.
1.3.8 Calculate Cleaning Verification Limits* for the specific equipment taking into account the
critical product cleaning specifications as well as the other products made in the same equip-
ment.
l.3.9 Draft a Cleaning Procedure* for the specific combination of product and manufacturing equip-
ment. Identify Critical Cleaning Process Operating Parameters* and Cleaning Agents*.
1.3.10 Prepare a cleaning verification protocol.
1.3.11 Manufacture a single product batch, clean the equipment; then test the equipment, as speci-
fied in the cleaning verification protocol.
1.3.12 Once development is complete, perform Cleaning Validation* on the first three (3) commer-
cial batches.

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Proposed Validation Standard VS-3

1.3.13 Validate analytical methods to be used for cleaning validation samples.
1.3.14 Determine recovery factors of expected residues from representative materials (stainless steel,
glass, plastics).
1.3.15 Prepare and obtain approval of a Cleaning Validation Protocol*.
1.3.16 Train and qualify operational and supervisory laboratory and production personnel in prod-
uct-specific cleaning procedures, sampling procedures, and analytical procedures.
1.3.17 Ensure that interrelated systems (automated clean-in-place, utilities, Programmable Logic
Controllers [PLCs]) are all validated.
1.3.18 Conduct Cleaning Performance Qualification (CPQ)*.
1.3.19 Assemble and document evidence that the cleaning process is acceptable and consistent.
1.3.20 Provide for retention of archived cleaning validation files for required periods following the
last commercial lot expiration date.
POL 1.4
The cleaning processes associated with products in the marketed stage of the product lifecycle shall be val-
idated for all products manufactured with a normal frequency of production. For rare instances where prod-
ucts are infrequently manufactured (e.g., one batch per year or less frequently), it may be difficult to achieve
fully validated cleaning processes and the principle of cleaning verification should be utilized. The adminis-
trative responsibility for cleaning validation and cleaning verification of products will reside in the Site
Validation Steering Committee (SVSC). The SVSC shall adjudicate cleaning validation issues and appoint
project-specific validation teams as needed that include principal(s) having experience in the cleaning
processes involved. Such SVSC responsibilities extend to cleaning processes used by contract vendors and
suppliers of the firm's drug products and/or APIs, as well as to those cleaning processes employed on-site.

POL 1.5
A written Cleaning Verification Policy (CVP) shall be used to define and describe the strategies and
approaches used to verify cleaning procedures associated with drug products, biotechnology products,
medical devices, and APIs during the development stage of the lifecycle.

POL 1.6
A written Cleaning Validation Master Plan (CVMP)* shall be used to define and coordinate validation
activities related to any cleaning process associated with the manufacture of a commercially marketed
drug product, biotechnology product, medical device, and API.

POL 1.7
Cleaning Verification Protocols shall be used to define individual cleaning verification runs.

POL 1.8
Cleaning Validation Protocols shall be used to define individual cleaning validation runs.

POL 1.9
Cleaning Verification Reports* shall be used for documenting and summarizing results of cleaning ver-
ification studies. Definite statements must be used, especially in describing the scientific rationale for the
limits chosen and whether the cleaning process was effective in meeting the limits.

POL 2.0
Cleaning Validation Reports* shall be used for documenting and summarizing results of cleaning vali-
dation studies. Definitive statements must be used, especially in describing the scientific rationale for the
limits chosen and whether the cleaning process was effective in ensuring that these limits were met.

Institute of Validation Technology
Proposed Validation Standard VS-3

POL 2.1
All Cleaning Verification Policies, Cleaning Validation Master Plans, Cleaning Verification Protocols,
Cleaning Validation Protocols, Cleaning Verification Reports and Cleaning Validation Reports must
be approved and available to the SVSc. All such cleaning documents created on-site must be approved by
the site Quality Authority and, when production is involved, also by the site Production Authority*.

POL 2.2
Relevant cleaning process verification and validation information from other divisions, departments
(including Research and Development), production sites, and outside contract services is to be gathered,
evaluated, utilized, and maintained by the SVSC.

POL 2.3
Certain cleaning processes are considered critical manufacturing steps and thus require validation (it
should be noted that not all cleaning procedures are considered critical and thus require validation). Once
the cleaning procedures are validated, they must not be altered without prior review, and any changes
should be subjected to a formal Change Control* review process prior to making the change. The site
Quality Authority must approve all changes to validated cleaning procedures.

II. PROCEDURAL STATEMENTS

PROC - 2.a [ref. POL 1.3.2]
Critical product cleaning specifications are known factors that can influence the development of the
cleaning process. These can be physical in nature such as solubility in a variety of solvents, polymorphic
crystal form, and stability. These factors could also be chemical in nature such as reactivity with water
or other solvents. They could also include medical information such as potency, toxicity, and allergenic-
ity. They could also be safety factors such as toxicity when inhaled and could require personal protec-
tion attire to protect the operator. These factors, which are normally determined during pre-formulation,
are vital information that must be known before meaningful cleaning procedures and limits can be devel-
oped.

PROC - 2.b [ref. POL 1.3.3]
During development, various types of equipment may be used in an effort to develop an optimum process
or effective product. This means that normally the specific equipment or the scale of the equipment may
vary from batch-to-batch. Because of this variability in the equipment used, the cleaning procedures may
also vary from batch-to-batch even for the same product. Therefore, the cleaning verification results apply
only to the specific cleaning event (i.e., the specific combination of equipment, processes, and materials)
used for the individual study. The cleaning verification report should contain the details of the specific
equipment (size, model number), formulation, and processes used.

PROC - 2.c [ref. POL 1.3.4]
During development, the formulation may vary from batch-to-batch in order to identify the combination
of ingredients that presents the best product performance 'in vitro' and 'in vivo'. Excipients may be var-
ied as well as the concentration of active ingredient. These combinations will present different degrees
of cleaning challenges. A given cleaning procedure may be adequate for one formulation but inadequate
for another formulation of the same active ingredient. This data will be useful for the selection of the
ultimate cleaning procedure that will be used for commercial product. It will be necessary to include the
formulation in the cleaning verification study, either by reproducing in total, or by reference to a formu-
la number.

Journal of Validation Technology
Proposed Validation Standard VS-3

PROC - 2.d [ref. POL 1.3.5]
Since it is other products made in the same equipment that will be contaminated due to inadequate clean-
ing, it is necessary to evaluate the other products made in the same equipment. Some of the factors per-
taining to other products that will be needed are:

• Batch sizes
• Normal daily doses
• Route of administration

PROC - 2.e [ref. POL 1.3.6]
In order to develop a scientific basis for cleaning verification limits, information will be needed for both
the product being cleaned as well as other products made in the same equipment. The following informa-
tion should be assembled:

• For product being cleaned
- Solubility in various solvents
- Potency
- Toxicity
- Stability (wet and dry)
- Allergenicity
- Route of administration
- Daily dosage
- Difficulty of cleaning
- Physical and chemical interaction with cleaning agent

• For other products made in same equipment
- Batch sizes
- Daily doses
- Stability
- Chemical interaction with product being cleaned
- Route of administration

The pharmacological relationships between the potential contaminating product and other products, which
could be possibly cross contaminated, may also be significant and should be considered if known. The con-
taminating product has the potential to amplify the medical activity of other products resulting in a syner-
gistic effect. The contaminant could also partially negate the medical effect of the other products by hav-
ing an antagonistic effect.

PROC - 2.f [ref. POL 1.3.7]
Just as there are critical parameters for the manufacturing process, there are critical parameters for the
cleaning process. These factors will lead to either inadequate or inconsistent cleaning if not controlled.
Critical parameters for the cleaning process must be determined and may vary from one cleaning process
to another. Some potential critical cleaning parameters (list is not all inclusive) are:

• Temperature of wash solutions
• Temperature of rinse solutions
• Amount of mixing or agitation during cleaning
• Mechanical wiping or brushing

Institute of Validation Technology
Proposed Validation Standard VS-3

• Flow rates
• Concentration of cleaning agent
• Time of washing
• Time of rinsing
• Length of time and environmental conditions (temperature, humidity) between manufacturing and
cleaning
• Nature and amounts of excipients
• Concentration or amount of residue left on equipment
• Physical properties of residues
• Chemical properties of residues
• Cleaning solvent chosen
• Soak times
• Contact time with cleaning agent
• Rinse volumes
• Order of application of cleaning solvents (acid, alkaline, and organic solvents)

PROC - 2.g [ref. 1.3.8]
Each cleaning verification protocol shall include, and is not limited to, the following:

o Statement of objective or purpose
@ Justification for cleaning verification limits, if applicable
~ Descriptions of sampling procedure(s), and locations, types, and numbers of samples to be taken
o Indications of most difficult-to-clean locations in equipment
o Experimental plan to be executed, including number of samples, and how data will be calculated
<D Descriptions of analytical methodology and sensitivity of analytical method as well as recovery factors
o Descriptions of all testing instruments to be used and specific calibration plans for each
o Complete description of acceptance criteria including visual examination (if possible) and quantitative
analytical data
o Training records of operators and analytical personnel
PROC - 2.h [ref. 1.3.11]
Prior to cleaning validation studies, analytical methods must be validated to demonstrate that they are suit-
ably sensitive to detect residues at levels below the allowable limits. Analytical Method Validation* for
cleaning validation shall include, and is not limited to, the following:

o Accuracy
@ Precision
~ Linearity
o Robustness
o Sensitivity-Limit of Detection (LOD)*, Limit of Quantitation (LOQ)*
<D Specificity

The specificity of the analytical method may not be as critical for cleaning validation as for process vali-
dation due to the fact that the levels of residue detected is very low, and often non-specific analytical meth-
ods are available that may be at least or more sensitive than specific methods. The assumption is often
made that all of the residue detected is composed of the most potent ingredient (usually the active) pre-
sent and, if this amount is still below the established limits, then the exact nature of the residue is irrele-
vant, i.e., the 'worst case' assumption was made and limits were met.

Journal of Validation Technology
Proposed Validation Standard VS-3

PROC - 2.i [ref. 1.3.12]
Following validation of the analytical method, the analytical method should be challenged concurrently
with the sampling procedure(s) to determine the percentage recovered from representative manufacturing
surfaces. The determination of recovery is important and will differ according to the composition of the sur-
face sampled (e.g., stainless steel, glass, plastics), the nature of the sampling technique, and the nature of
the residues themselves. The recovery factor must be used to correct observed analytical results to account
for portions of residue that remain on equipment even after swab and rinse sampling.

PROC - 2.j[ref. 1.3.13]
Each cleaning validation protocol shall include, and is not limited to, the following:

o Statement of objective or purpose
@ Justification for cleaning validation limits
@) Descriptions of sampling procedure(s) and diagrams of locations
o Indications of most difficult-to-clean locations in equipment
o Experimental plan to be executed, including number of cleanings to be evaluated, number of samples
from each cleaning, and how data will be calculated
<D Descriptions of analytical methodology and sensitivity of the analytical method as well as recovery factors
fi Descriptions of all testing instruments to be used and specific calibration plans for each
«l) Complete description of acceptance criteria including visual examination (if possible) and quantitative
analytical data
CD Criteria for determining when the cleaning process may be considered validated, i.e., how many suc-
cessful consecutive cleanings (normally at least three (3) are required)
@ Training records of operators and analytical personnel

PROC - 2.k [ref.1.3.14]
Prior to implementation of the cleaning validation protocol, it is important to verify the training of the pro-
duction operators who actually conduct the cleaning, sampling personnel (production, analytical, valida-
tion) who sample the equipment, analytical personnel who analyze cleaning validation samples, as well
as personnel who implement the protocol and process the documentation. If documentation does not
already exist that demonstrates each of these types of training, then the training should be done before any
actual validation runs are carried out.

PROC - 2.1 [ref. 1.3.15]
Special equipment and critical utilities such as water and steam must be qualified prior to implementation
of the cleaning validation protocol. In addition, any automated cleaning equipment such as Clean-in-
Place (CIP)* systems and their associated automated controllers must also be validated or qualified prior
to implementation of the cleaning validation protocol. In the case of CIP, Sprayball Pattern Analysis*
should be carried out to verify that cleaning solutions reach all locations in closed systems. The qualifi-
cation of equipment and utilities is normally accomplished by means of an Installation Qualification
(IQ) * and an Operational Qualification (OQ) * (see next two sections).

PROC - 2.m [ref. 1.3.15]
An Installation Qualification (IQ) must exist for all equipment that is critical to the cleaning process
including specialized cleaning aids such as Spray Devices (Sprayballs)*, equipment that delivers clean-
ing solutions, high pressure wands, water heating devices, steam generators, and utilities. The IQ is to in-
clude at least the following:

Institute of Validation Technology
Proposed Validation Standard VS-3

o List of all equipment, the operation of which has potential bearing on the quality of the cleaning process
@ As-built drawings of all specialized cleaning equipment such as pumps, high pressure delivery devices,
and hose cleaners
@) Verification that all such equipment and the installation thereof meets original intent, including applic-
able building, electrical, plumbing, and other such codes
o Preventative maintenance plans and schedules for all such equipment
PROC - 2.n [ref. 1.3.15]
An Operational Qualification (OQ) must exist for all equipment that is critical to the cleaning process and
should include at least the following:

o A list identifying each step of the cleaning process that relates to the specific equipment
@ Process operating parameters for each piece of equipment that is critical to the cleaning process
@)An OQ protocol that is designed to demonstrate via appropriate tests that the equipment operates as in-
tended throughout the cleaning process
o Report that describes the successful execution of each OQ protocol for each piece of equipment criti-
cal to the cleaning process

PROC - 2.0 [ref. 1.3.16]
At least three consecutive, successful cleanings shall be completed on the equipment used to produce the
commercial product. Normally, the cleanings follow the production of each of the batches used for the val-
idation of the manufacturing process. A Cleaning Performance Qualification (CPQ) shall be performed
when the following items are complete and commercial production has been authorized.

• The cleaning process is fully defined in writing, including identification of critical cleaning process
operating parameters
• A justification for Cleaning Validation Limits* has been prepared that takes into account the potency
and toxicity of the material, as well as the other products to be made in the same equipment
• IQ and OQ steps are complete for critical utilities and any specialized equipment used in cleaning such
as pumps, sprayballs, high pressure wand cleaners, etc.
• Operating, sampling, and analytical personnel are trained and qualified and the training is documented
• An appropriate change control procedure is in place

PROC - 2.p [ref. 1.3.17]
Once the cleaning validation protocol has been implemented on three cleanings and the sampling and test-
ing has been completed, the data must be assembled and evaluated for each cleaning event. A cleaning
validation report should be prepared that consists of:

• The cleaning validation protocol
• All data assembled in a logical format
• An analysis of the data that addresses any deviations in the protocol, explains any failures, compares
the data to the acceptance criteria, and ultimately states whether the cleaning process mayor may not
be considered validated

PROC - 2.q [ref. POL 1.5]
The Cleaning Verification Policy (CVP) can be considered to be the master plan for cleaning for a product
during the development phase of the lifecycle of the product. Since each cleaning is a unique event because
of the variability in the manufacturing equipment, formulation, and batch size between batches of the same

Journal of Validation Technology
Proposed Validation Standard VS-3

product, it is not possible to validate the cleaning process during the development phase. Still it is possible
to prepare a policy describing the testing of development equipment and what criteria will be used to deter-
mine if the equipment has been suitably cleaned. The strategy and approach to cleaning in the development
areas must be in writing and clearly explain how equipment will be sampled and tested, and how limits will
be determined, recognizing that only a single set of data will be available. Since only a single set of data is
available, it would be erroneous to refer to this situation as "validation". Thus the term "cleaning verifica-
tion" is a more appropriate description of this scenario.

PROC - 2.r [ref. POL 1.6]
The Cleaning Validation Master Plan (CVMP) may take different forms in companies around the world.
Some may have a separate independent document. Others may have a Standard Operating Procedure*
that describes in general terms how the cleaning program will operate. Still others will devote a section of
the Validation Master Plan* to cleaning. Regardless of the exact form taken, it is essential to have a writ-
ten plan describing how the cleaning program will be organized and controlled. The essential elements of
the CVMP are:

• A description of the approach and strategy to be used for controlling, verifying, and/or validating in the
various departments such as Basic Research *, Research and Development *, Scale-Up! Pilot Plant*,
Production*, Packaging*, Contract Manufacturing Facilities *, and Contract Packaging Facilities *.
• A mechanism for defining what is adequate cleaning, based on the potency, toxicity, potential aller-
genicity, potential teratogenicity, and potential carcinogenicity of the material, as well as other factors
such as route of administration and properties of the other products made in the same equipment.
• Sampling methods to be used to evaluate cleaned equipment. Examples are Swab Sampling* and Rinse
Sampling*, or a combination of these two methods depending on the nature of the equipment or product.
• Selection of sampling locations to include 'worst case' and/or most difficult-to-clean locations.
• For equipment used for manufacturing multiple products, how the Worst Case Product* for cleaning pur-
poses might be selected from a group of very similar products. Typically, a Product Matrix Approach*
is used to compare the critical cleaning properties of the products in the group. Critical cleaning proper-
ties are potency/toxicity, solubility, and the inherent difficulty of cleaning.
• Provision for how documentation will be developed, reviewed, and approved. This would include a list of
those responsible for preparing, reviewing, and approving Cleaning Verification Protocols, Cleaning
Verification Reports, Cleaning Validation Protocols, Cleaning Validation Reports, Cleaning Procedures,
Change Control Procedures, and Cleaning Monitoring Programs *.
• Criteria for Revalidation* of cleaning procedures.
• Provision for creation of a Site Validation Steering Committee (SVSC), that would serve as the group
immediately responsible for all cleaning issues. This group would normally select project teams relat-
ed to cleaning activities, e.g., for a new product.
• Training of development, pilot plant, sampling, and analytical testing personnel.
• Definition of resources required and allocated.
• Schedule of cleaning activities including cleaning validation and assignment of responsibilities.

III. ACRONYMS

API Active Pharmaceutical Ingredient
BPC Bulk Pharmaceutical Chemical
CGMPs Current Good Manufacturing Practice (U.S.)
CIP Clean-in-Place
CPQ Cleaning Performance Qualification

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Proposed Validation Standard VS-3

CVMP Cleaning Validation Master Plan
CVP Cleaning Verification Policy
IPEC International Pharmaceutical Excipients Council
IQ Installation Qualification
OQ Operational Qualification
PIC Pharmaceutical Inspection Convention
SOP Standard Operating Procedure
SVSC Site Validation Steering Committee

IV. GLOSSARY

Reference Standard
Number

POL 1.1 Active Pharmaceutical Ingredients (API) - (synonymous with drug substance). A substance
that is represented for use in a drug and, when used in the manufacturing, processing, or pack-
aging of a drug, becomes an active ingredient of a finished drug product. Such substances are
intended to furnish pharmacological activity or other direct effects in the diagnosis, cure, mit-
igation, treatment, or prevention of disease, or to affect the structure and function of the body
of humans or other animals.

Bulk Pharmaceutical Chemical (BPC) - includes active pharmaceutical ingredients (APIs) as
well as non-active excipients such as starch, lactose, rnicrocellulose, and other materials that
have no direct therapeutic effect but may indirectly affect the performance of drug dosage forms.

PROC·2.h Analytical Method Validation - documented evidence that an analytical procedure will con-
sistently detect and/or quantitate materials.

PROC-2.r Basic Research - the segment of the pharmaceutical industry that evaluates new chemical
entities for potential application to treatment of disease. This includes, but is not limited to,
basic disciplines such as biochemistry, molecular biology, toxicology, pharmacology, and
pharmacokinetics.

POL 2.3 Change Control Procedure - A procedure for:

(a.) Identifying all modifications or alterations that are potentially significant to a state of con-
trol, qualification, or validation.
(b.) Implementing corrective action, such as repair, readjustment, requalification, and/or
revalidation.
(c.) Implementing interim measures to be taken until effective corrective actions are complete.
(d.) Documenting all of the above.

POL 1.3.9 Cleaning Agents - any chemical or solvent that facilitates the cleaning of equipment by dis-
solution, hydrolysis, or other chemical or physical action.

PROC-2.r Cleaning Monitoring Program - a formal, written program describing how cleaning proce-
dures can be monitored on a regular schedule to evaluate the effectiveness and consistency of
the cleaning process.

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Proposed Validation Standard VS-3

POL 1.3.18 Cleaning Performance Qualification (CPQ) - documented evidence that a cleaning proce-
dure is consistent in removing product residue and cleaning agent from equipment.

POL 1.3.9 Cleaning Procedure - a detailed written procedure (SOP) that describes how equipment will
be disassembled, cleaned, examined, and reassembled.

POL 1.3.12 Cleaning Validation - documented evidence that a cleaning procedure is consistent in remov-
ing product residue and cleaning agents from equipment (sometimes also referred to as
Cleaning Performance Qualification [CPQ]).

POL 1.6 Cleaning Validation Master Plan (CVMP) - a comprehensive, written plan that describes the
company's strategy in ensuring that all cleaning procedures are effective and in a state of con-
trol to ensure that all products are free of contamination and of high qUality. The plan includes
or references all appropriate cleaning procedures, and SOPs describes how protocols, cleaning
validation reports, and other documentation will be assembled, provides for the testing and
analysis of data, identifies resources to be allocated, provides for training of personnel, describes
qualification of equipment, indicates the process for assigning responsibility for the various
activities, provides a criteria for revalidation of cleaning procedures, and describes a mechanism
for controlling changes to validated procedures and equipment.

PROC-2.o Cleaning Validation Limits - The maximum allowable amounts of material that can remain
on equipment after cleaning without compromising the safety of the consumer or the quality
of the product. These limits are applied during the cleaning validation study and depending on
the manufacturing circumstances, limits may be for:

• Residues of active ingredients
• Residues of excipients
• Degradation materials
• Intermediates
• Cleaning agent or by-product residuals
• Bioburden
• Endotoxin
• Other foreign materials

POL 1.3.15 Cleaning Validation Protocol - a product specific plan of sampling and testing of equipment
after at least three consecutive cleanings to establish that equipment is appropriately cleaned
after a specific product is manufactured in a development area by a specific, detailed written
cleaning procedure.

POL 2.0 Cleaning Validation Reports - a written report that summarizes results and conclusions of
the cleaning validation study and includes:

• Protocol
• Test results
• Analyses
• Conclusions
• Discussions of any deviations from procedures specified in the original protocol
• Discussion of any failures

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Proposed Validation Standard VS-3

• Indication as to whether the testing met the acceptance criteria specified in the protocol

POL 1.3.8 Cleaning Verification Limits - Maximum amount of residue that may remain on equipment
during a cleaning verification study. These limits are derived in a similar fashion to those for a
cleaning validation study, but are applied to a single cleaning event, as versus multiple clean-
ing runs (at least three) for cleaning validation studies. Just as for cleaning validation, the lim-
its may be for any of the following:

• Residues of active ingredients
• Residues of excipients
• Degradation materials
• Intermediates
• Cleaning agents or by-product residuals
• Bioburden
• Endotoxin
• Other foreign materials

POL 1.5 Cleaning Verification Policy (CVP) - a written document describing how equipment will be
verified as clean after a single manufacturing event in a development area. This is a general
document that will pertain to all cleaning in development areas.

POL 1.2 Cleaning Verification Protocol- a product specific plan of experimental sampling and test-
ing to verify that equipment is appropriately cleaned after a specific product is manufactured
in a development area.

POL 1.9 Cleaning Verification Reports - a written report that summarizes results and conclusions of
the cleaning verification study and includes:

• Protocol
• Test results
• Analyses
• Conclusions
• Discussions of any deviations in procedures from those specified in the original protocol
• Discussion of any failures
• Indication as to whether the testing met the acceptance criteria specified in the protocol

PROC-2.1 Clean-in-Place (CIP) - cleaning of equipment that is accomplished without disassembly of
the equipment but rather through the application of cleaning solutions delivered internally by
one or more internal spray devices (sprayballs) or recirculation of cleaning solution throughout
the equipment. CIP may be entirely automated or the cycle parameters may be controlled by
the operator. This type of cleaning is also known as closed system cleaning.

PROC-2.r Contract Manufacturing Facilities - facilities or companies that manufacture products for
customers on a contractual basis.

PROC-2.r Contract Packaging Facilities - facilities or companies that package products for customers
on a contractual basis.

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POL 1.3.9 Critical Cleaning Process Operating Parameter - an operating variable that is assigned a
required control range with acceptability limits, outside of which exists potential for product
or process failure. A critical process operating parameter is determined by process develop-
ment and/or investigational work.

POL 1.3.3 Critical Product Cleaning Specifications - physico-chemical properties as well as therapeutic
or medical information that are used to determine cleaning procedures and set limits for cleaning
processes. Examples are solubility, stability, hydrophobicity, therapeutic potency, and toxicity.

POL 1.1 Drug Products - a finished dosage form (e.g., tablet, capsule) that contains an API, general-
ly in association with excipients. Synonymous with finished drug product.

POL 1.1 In-Process Materials - (as applied to drug product manufacture) - any material manufac-
tured, blended, compacted, coated, granulated, encapsulated, tableted, or otherwise processed
that is produced for and used in the preparation of a drug product. (Corresponding materials
used in the preparation of APIs are referred to as intermediates.)

PROC-2.1 Installation Qualification (IQ) - documented verification that equipment, system, or a sub-
system has been properly installed, adheres to applicable codes and approved design inten-
tions, and supplier recommendations have been suitably addressed.

POL 1.1 Intermediate - a material produced during steps in the synthesis of an API that must under-
go further molecular change or processing before it becomes an API. The degree to which a
given intermediate should be rated "critical" with respect to cleaning must be determined by
a firm's experts based on such criteria as:

• Potential toxicity or other physiological activity
• Degree to which equipment used is dedicated to the process, as opposed to having multiple uses
• Ease or difficulty of removing process residuals when cleaning equipment

PROC-2.h Limit of Detection (LOD) - the lowest amount or concentration of a material that can be
detected by an analytical instrument or chemical test. Although detectable, the amount of
material in the sample cannot be determined at this level.

PROC-2.h Limit of Quantitation (LOQ) - the lowest amount or concentration of a material that can be
quantitatively determined by an analytical instrument or chemical test.

PROC-2.1 Operational Qualification (OQ) - documented verification that equipment, system, or
process performs as specified throughout representative or anticipated operating ranges.
(Note: Overlap between IQ and OQ often occurs and is considered allowable, but should be
addressed in the VMP.)

PROC-2.r Packaging - The area or department that receives bulk product and incorporates the product
in packaging that will either be sent to the customer or sent to another area for additional pack-
aging and/or labeling.

PROC-2.r Production - The unit of the company responsible for the manufacture of bulk product. This
mayor may not include the packaging function depending on the size and organization.

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POL-2.1 Production Authority - counterpart of quality authority, sometimes referred to as production
head or, in the case of FLP (Fill-Label-Pack) operations, packaging head.

PROC-2.r Product Matrix Approach - a chart that presents medical, toxicity, solubility, and other per-
tinent data so that a comparison can be made between products in the group in order that the
most risky product can be selected for cleaning validation. This 'worst case' approach obvi-
ates the need to perform cleaning validation studies on every combination of product and
equipment.

POL 1.2 Quality Authority - one or more persons who, collectively, have formal responsibilities for
specified quality-related operations, such as approval of manufacturing materials, release of
finished products, review and approval of documents, and adjudication of quality assurance
investigations. Titles of quality authority principals vary throughout the world; for example, in
the U.S., one term "the Quality Control (QC) unit," is all embracing; in the E.U. and Canada,
the head of quality control has some of the responsibilities, while a qualified person has oth-
ers; terms as responsible head (or person) and quality assurance (and/or control) department are
also used in other areas.

PROC-2.r Research and Development - The division of a company that is responsible for developing
the optimal manufacturing techniques and dosage form for a pharmaceutical product. It is also
responsible for the development of preliminary cleaning procedures for new products.

PROC-2.r Revalidation - repeating the original validation or selected portions for the purpose of
demonstrating that the process is still in a state of control and delivers acceptable product and
processes. As applied to cleaning procedures, the purpose would be to demonstrate that the
cleaning procedures are still effective in removing residues. Revalidation is a natural conse-
quence of making significant changes to equipment, manufacturing procedures, components,
cleaning procedures, and cleaning agents.

PROC-2.r Rinse Sampling - a type of sampling of cleaned equipment used in cleaning validation and
cleaning verification studies to determine if product-contact manufacturing surfaces are clean.
Controlled amounts of solvent are subjected to the equipment either under pressure or allowed
to stand in the equipment to allow dissolution of the residues. Mixing, spraying, and recircu-
lation may also be used to facilitate the detection of residues. Rinse solvents are usually
selected on the basis of residue solubility in that solvent. The rinses may be either heated or
at ambient temperature.

PROC-2.r Scale-UplPilot Plant - Functionally, this area of responsibility is between development and
full-scale production. This group is charged with scaling a formulation up from small scale to
large production scale and troubleshooting problems that arise as a result of the scale-up pro-
cess. They are also responsible for further refinements of the cleaning procedures handed over
by development.

POL 1.2 Site Validation Steering Committee (SVSC) - a standing committee with authority and
responsibilities for validation policies, practices, and adjudication of issues. Must include
quality authority and Production Authority representation, and often includes representatives
of other involved disciplines. The name of the SVSC may vary from firm-to-firm.

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PROC-2.m Spray Devices (Sprayballs) - a device that may be either permanently installed or inserted
into closed systems such as tanks specifically to provide a thorough, even coverage of the
equipment surfaces by the cleaning solutions. If fixed and spherical in shape, the device is
usually referred to as a "sprayball." Sprayballs may have fixed heads or they may rotate in
complex patterns.

PROC-2.1 Sprayball Pattern Analysis - a study that establishes that cleaning solution delivered by
sprayballs will reach all equipment surfaces, especially difficult to access or shadowed areas.
The study usually consists of coating equipment with easily detectable residue (dyes or fluo-
rescent material), activating the sprayball mechanism for a normal cycle cleaning time, and
then examining the equipment to see if any material remains on the equipment surfaces.

PROC-2.r Standard Operating Procedure (SOP) - A written document describing, in detail, a specif-
ic process or procedure. These written procedures are required by the current Good Man-
ufacturing Practice regulations for all critical processes. These procedures must be current,
detailed, controlled, and revised when necessary. All personnel must be trained in a new or
revised SOP prior to its implementation. Some companies have function specific procedures,
e.g., cleaning procedures, that take the place of SOPs.

PROC-2.r Swab Sampling - a type of sampling of cleaned equipment used in cleaning validation and
cleaning verification studies to determine if product-contact manufacturing surfaces are clean.
This type of sampling makes use of small pieces of fabric (usually polyester or other synthet-
ic material) fused to the end of a plastic strip. The swab is typically wetted with solvent
(although they can be used dry). Defined surface areas of equipment, including the most dif-
ficult-to-clean locations, are swabbed. The swab is then immersed in a vial of solvent. The
residue on the swab is dissolved in the solvent, which is subsequently analyzed for product
residues. Limits are calculated on the basis of the area swabbed.

PROC-2.r Validation Master Plan (VMP) - a comprehensive, project-oriented action plan that includes
or references all protocols, key SOPs and policies, existing Validation Task Reports *, and
other relevant materials on which the specific system or process validation effort will be
based. The plan also identifies resources to be allocated, specific personnel training, and qual-
ification requirements of relevant, organizational structure, and responsibilities of the valida-
tion team, and planned schedules. The VMP is subject to periodic revisions as defined in
change control procedures.

Validation Task Report - a written report that summarizes results and conclusions following
execution of all or any portion of a Validation Master Plan (VMP) (often referred to as a final
report if summarizing all activities of the VMP).

PROC-2.r Worst Case Product - the product selected from a group of similar products that presents the
greatest risk of carryover contamination to other products made in the same equipment by
virtue of its poor solubility, unstable chemical properties, potency, toxicity, or a combination
of these factors.

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v. SELECTED REGULATORY EXCERPTS

Regulatory Reference

FDA Proposed Amendments to current Good Manufacturing
Practice Regulations Section Ill. C (May 3, 1996)

Under CGMP, a manufacturer will set contamination limits on a substance-by-substance basis, according to both
the potency of the substance and the overall level of sensitivity to that substance.

Because other substances, such as cytotoxic agents, or other antibiotics, pose at least as great a risk of toxicity
due to cross-contamination, FDA is proposing to expand the contamination control requirements to encompass
other sources of contamination.

The Agency has refrained from establishing a list of drugs or drug products that present such an unacceptable
risk, because such a list would quickly become obsolete.

FDA Guidance for Industry: Manufacturing,Processing, or Holding
Active Pharmaceutical Ingredients Section IV.D (March, 1998)

Nondedicated equipment should be thoroughly cleaned between different products and, if necessary, after each
use to prevent contamination and cross-contamination. If cleaning a specific type of equipment is difficult, the
equipment may need to be dedicated to a particular API or intermediate.

The choice of cleaning methods, cleaning agents, and levels of cleaning should be established and justified.

FDA Guidance for Industry: Manufacturing, Processing, or Holding Active
Pharmaceutical Ingredients Section IV.E (March, 1998)

Equipment cleaning methods should be validated, where appropriate. In early synthesis steps, it may be unnec-
essary to validate cleaning methods where residues are removed by subsequent purification steps.

If various API's or intermediates are manufactured in the same equipment and the equipment is cleaned by the same
process, a worst-case API or intermediate can be selected for purposes of cleaning validation. The worst-case selec-
tion should be based on a combination of potency, toxicity, solubility, stability, and difficulty of cleaning.

The cleaning validation protocol should describe the equipment to be cleaned, methods, materials, extent of
cleaning, parameters to be monitored and controlled, and analytical methods.

Sampling should include swabbing, rinsing, or alternative methods (e.g., direct extraction), as appropriate, to
detect both insoluble and soluble residues. Swab sampling may be impractical when product contact surfaces are
not easily accessible due to equipment design and/or process limitations (e.g., inner surfaces of hoses, transfer
pipes, reactor tanks with small ports or handling toxic materials, and small intricate equipment such as microniz-
ers and microfluidizers).

Validated analytical methods sensitive enough to detect residuals or contaminants should be in place.

Residue limits should be practical, achievable, verifiable, and based on the most deleterious residue.

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FDA Guidance for Industry: Manufacturing, Processing, or Holding Active
Pharmaceutical Ingredients Section IV.F (March, 1998)

When practical, equipment in CIP systems should be disassembled during cleaning validation to facilitate in-
spection and sampling of inner product surfaces for residues or contamination, even though the equipment is not
normally disassembled during routine use.

FDA Part 211-Current Good Manufacturing Practice for Finished
Pharmaceuticals Subpart D, Section 211.67 (1990)

Equipment and utensils shall be cleaned, maintained, and sanitized at appropriate intervals to prevent contamina-
tion that would alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other
established requirements.

Written procedures shall be established and following for cleaning and maintenance of equipment, including
utensils, used in the manufacturing, processing, packing, or holding of a drug product.

FDA Guide to Inspections of Validation of Cleaning Processes, (July, 1993)

FDA expects firms to prepare specific written validation protocols in advance for the studies to be performed on
each manufacturing system or piece of equipment, which should address such issues as sampling procedures,
and analytical methods to be used, including the sensitivity of those methods.

FDA expects firms to conduct the validation studies in accordance with the protocols and to document the results
of studies.

FDA expects a final validation report which is approved by management and which states whether or not the clean-
ing process is valid. The data should support a conclusion that residues have been reduced to an "acceptable level."

Examine the design of equipment, particularly in those large systems that may employ semi-automatic or fully
automatic clean-in-place (CIP) systems since they represent significant concern. For example, sanitary type pip-
ing without ball valves should be used. When such nonsanitary ball valves are used, as is common in the bulk
drug industry, the cleaning process is more difficult.

Examine the detail and specificity of the procedure for the cleaning process being validated, and the amount of
documentation required.

When more complex cleaning procedures are required, it is important to document the critical cleaning steps (for
example certain bulk drug synthesis processes).

Determine the specificity and sensitivity of the analytical method used to detect residuals or contaminants.

The firm's rationale for the residue limits established should be logical based on the manufacturer's knowledge
of the materials involved and be practical, achievable, and verifiable.

Check the manner in which limits are established.

If a detergent or soap is used for cleaning, determine and consider the difficulty that may arise when attempting
to test for residues.

FDA Guide to Inspections of Bulk Pharmaceutical Chemicals (May, 1994)

Cross contamination is not permitted under any circumstances.

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The cleaning program should take into consideration the need for different procedures depending on what prod-
uct or intermediate was produced.

Where mUltipurpose equipment is in use, it is important to be able to determine previous usage as an aid in inves-
tigating cross-contamination or the possibility thereof.

Cleaning of multiple use equipment is an area where validation must be carried out.

Validation data should verify that the cleaning process will remove residues to an acceptable level.

There should be a written equipment cleaning procedure that provides details of what should be done and mate-
rials to be utilized.

We expect the manufacturer to establish an appropriate impurity profile for each BPC based on adequate con-
sideration of the process and test results.

PIC Document PR 1/99-2 "Cleaning Validation" Section 1.0 (April, 2000)

Cleaning procedures must strictly follow carefully established and validated methods of execution. This applies
equally to the manufacture of pharmaceutical products and bulk active ingredients.

PIC Document PR 1/99-2 "Cleaning Validation" Section 2.1 (April, 2000)

Normally only cleaning procedures for product contact surfaces need to be validated.

PIC Document PR 1/99-2 "Cleaning Validation" Section 2.2 (April, 2000)

Cleaning procedures for product changeover should be fully validated.

PIC Document PR 1/99-2 "Cleaning Validation" Section 2.6 (April, 2000)

At least three consecutive applications of the cleaning procedure should be performed and shown to be success-
ful in order to prove that the method is validated.

PIC Document PR 1/99-2 "Cleaning Validation" Section 2.8 (April, 2000)

Control of change to validated cleaning procedures is required. Revalidation should be considered under the fol-
lowing circumstances:
(a) Revalidation in cases of changes to equipment, products or processes.
(b) Periodic revalidation at defined intervals.

PIC Document PR 1/99-2 "Cleaning Validation" Section 3.1 (April, 2000)

A validation protocol is required laying down the general procedures on how cleaning processes will be validat-
ed. It should include the following:
• The objective of the validation process
• Responsibilities for performing and approving the validation study
• Description of the equipment to be used
• The interval between the end of production and the beginning of the cleaning procedures
• Cleaning procedures to be used for each product, each manufacturing system or each piece of equipment.
• Any routine monitoring requirement
• Sampling procedures, including the rationale for why a certain sampling method is used
• Data on recovery studies where appropriate

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• Analytical methods including the limit of detection and the limit of quantitation of those methods
• The acceptance criteria, including the rationale for setting specific limits
• When revalidation will be required

PIC Document PR 1/99-2 "Cleaning Validation" Section 3.3 (April, 2000)

A final validation report should be prepared. The conclusions of this report should state if the cleaning process has
been validated successfully. Limitations that apply to the use of the validated method should be defined (for exam-
ple, the analytical limit at which cleanliness can be determined). The report should be approved by management.

PIC Document PR 1/99-2 "Cleaning Validation" Section 3.4 (April, 2000)

The cleaning process should be documented in an SOP. Records should be kept of cleaning performed in such a
way that the following information is readily available:
• The area or piece of equipment cleaned
• The person who carried out the cleaning
• When the cleaning was carried out
• The SOP defining the cleaning process
• The product which was previously processed on the equipment being cleaned

PIC Document PR 1/99-2 "Cleaning Validation" Section 3.5 (April, 2000)

The cleaning record should be signed by the operator who performed the cleaning and by the person responsi-
ble for the Production and should be reviewed by Quality Assurance.

PIC Document PR 1/99-2 "Cleaning Validation" Section 4.1 (April, 2000)

Operators who perform cleaning routinely should be trained in the application of validated cleaning procedures.
Training records should be available for all training carried out.

PIC Document PR 1/99-2 "Cleaning Validation" Section 5.1 (April, 2000)

The design of the equipment should be carefully examined. Critical areas (those hardest to clean) should be iden-
tified, particularly in large systems that employ semi-automatic or fully automatic clean-in-place (CIP) systems.

PIC Document PR 1/99-2 "Cleaning Validation" Section 5.2 (April, 2000)

Dedicated equipment should be used for products, which are difficult to remove (e.g., tarry or gummy residues
in the bulk manufacturing), for equipment, which is difficult to clean (e.g., bags for fluid bed dryers), or for prod-
ucts with a high safety risk (e.g., biologicals or products of high potency which may be difficult to detect below
an acceptable limit).

PIC Document PR 1/99-2 "Cleaning Validation" Section 6.1 (April, 2000)

The existence of conditions favorable to reproduction of microorganisms (e.g., moisture, sub-strength, crevices,
and rough surfaces) and the time of storage should be considered. The aim should be to prevent excessive micro-
bial contamination.

PIC Document PR 1/99-2 "Cleaning Validation" Section 7.1 (April, 2000)

Samples should be drawn according to a written sampling plan.

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PIC Document PR 1/99-2 "Cleaning Validation" Section 7.2 (April, 2000)

There are two methods of sampling that are considered to be acceptable: direct surface sampling (swab method)
and the use of rinse solutions. A combination of the two methods is generally the most desirable, particularly in
circumstances where accessibility of equipment parts can mitigate against direct surface sampling.

PIC Document PR 1/99-2 "Cleaning Validation" Section 8.1 (April, 2000)

The efficiency of cleaning procedures for the removal of detergent residues should be evaluated. Acceptable lim-
its should be defined for levels of detergent after cleaning. Ideally, there should be no residues detected. The pos-
sibility of detergent breakdown should be considered when validating cleaning procedures.

PIC Document PR 1/99-2 "Cleaning Validation" Section 9.1 (April, 2000)

The analytical methods should be validated before the cleaning validation study is carried out.

PIC Document PR 1/99-2 "Cleaning Validation" Section 9.2 (April, 2000)

The analytical methods used to detect residuals or contaminants should be specific for the substance to be assayed
and provide a sensitivity that reflects the level of cleanliness determined to be acceptable by the company.

PIC Document PR 1/99-2 "Cleaning Validation" Section 10.1 (April, 2000)

The pharmaceutical company's rationale for selecting limits for product residues should be logically based on a con-
sideration of the materials involved and their dosage regimes. The limits should be practical, achievable, and verifiable.

PIC Document PR 1/99-2 "Cleaning Validation" Section 10.2 (April, 2000)

The approach for setting limits can be:
• Product specific cleaning validation for all products
• Grouping into product families and choosing a "worst case" product
• Grouping into groups of risk (e.g., very soluble products, similar potency, highly toxic products, difficult to detect)

PIC Document PR 1/99-2 "Cleaning Validation" Section 10.3 (April, 2000)

Carry-over of product residues should meet defined criteria, for example the most stringent of the following three criteria:
(a) No more than 0.1 % of the normal therapeutic dose of any product will appear in the maximum daily dose of
the following product.
(b) No more than 10 ppm of any product will appear in another product.
(c) No quantity of residue will be visible on the equipment after cleaning procedures are performed. Spiking
studies should determine the concentration at which most active ingredients are visible
(d) For certain allergenic ingredients, penicillins, cephalosporins, or potent steroids and cytotoxics, the limit should
be below the limit of detection by best available analytical methods. In practice, this may mean that dedicated
plants are used for these products. 0

About the Author
William E. Hall, PhD., is the President of Hall & Associates, where he provides consulting on cleaning validation,
process validation, and compliance issues for the pharmaceutical industry. Dr. Hall is internationally recognized
as an authority on the subject of cleaning validation. Dr. Hall serves on the Editorial AdviSOry Board of the Journal
of Validation Technology, and is a member of the Institute of Validation Technology Hall of Fame. Dr. Hall received
his PhD. from the University of Wisconsin, and is a former professor at the University of North Carolina. Dr. Hall
can be reached by phone at 910-458-5068, by fax at 910-458-5068, or bye-mail at CleanDoct@aol.com.

Journal of Validation Technology