2018-10 MT-KR Part 2 v02

Seoul, South Korea
October 2018
Anthony Bevilacqua, Ph.D.
Rapid Microbial Detection

Regulations, Trends, and Technology
Abstract 2
Here is a chemist's story of introducing a Rapid Microbial Detection method.

 It has always been critical for pharmaceutical producers to meet quality standards in the
life sciences. In the last 20 years, the trustworthy ability to measure chemical impurities IN
REAL TIME in a water system is routine for conductivity and TOC. This allows the
Production department to operate 24/7, and it allows the QC department to "continuously
release water", for chemical tests.
 However, there always remains the slow microbial testing, requiring at least 2-3 days to get
an initial result.
 In the meantime, the water is gone - touching product, making product, cleaning product.
 After the cost of research/development, the single greatest cost of a medicine is the final
testing. And microbial testing is by far the highest cost.
 So when a new technology comes out, here is what happens---
For internal use - Confidential

Agenda 3
1 Introduction, Water, Microbes
2 Current/Traditional Methods of Microbial Analysis
3 Current Regulations, New Regulations, Encouragement of Alternative Methods
4 Industry Drivers - PDA, OWBA and the early Adopters
5 Promotion of On-Line Measurements by Pharmacopoeia and Regulators
6 Chemical Properties of Bacteria
7 Technology in the 7000RMS
8 Case Studies and 7000RMS Features
9 Conclusion – Relate to TOC

Characteristics of Impurities in Water 4
Types of Impurities Characteristics Types of Tests
Chemical
 Organic (non-living) non-ionic, carbonaceous Total Organic Carbon
 Inorganic ionic Conductivity
Microbial
 Bacteria living, mostly organic microbial detection
 Bacterial debris dead, mostly organic bacterial endotoxin
Other
 Particulates insoluble, non-ionic filter/particle counter
 Dissolved Gases ionic (depends), inert? usually benign – not always

Bulk Water for Injection(s) - Today 5
Attribute1 USP EP JP ChP5 IP

US/EU/JP/WHO Human
Source Water drinking water Drinking water Purified Water Purified Water
consumption
Clear, colorless,
Clear and Clear, colorless, Clear, colorless,
Description odorless,
colorless liquid odorless liquid odorless liquid
tasteless
Distillation or
Distillation or Distillation or RO
Production Method RO/UF from Distillation Distillation
suitable process w/other means
Water or PW
Total Aerobic (cfu/100 mL)2 10 10 10 10 10
Conductivity (mS/cm at 25°C) 1.3 (3 stage) 3 Same as USP 2.1 offline
Same 6
as USP Same as USP Same as USP
TOC (mg/L) 0.54 0.5 0.5 0.5 0.5
Bacterial Endotoxins (EU/mL) 0.25 0.25 0.25 0.25 0.25
Nitrates (ppm) 0.2 Not detectable 0.06 0.2
Heavy Metals (ppm) Effective
0.1 2009 Not detectable 0.1 Required by PW
Acidity/Alkalinity Indicators - Indicators
pH 5.0-7.0 5.0-7.0
Calcium/Magnesium Effective Indicators
Chloride Not detectable
Effective
Required Not detectable
Sulfate NotJP 16
detectable CP 2010
Required Effective
Not detectable
Nitrite (ppm) Not detectable 0.02 IP Required
2012
Ammonium (ppm) 0.5 0.2 0.2
Oxidizable Sub (/100 mL) Not detectable Optional 5 Required
Residue on Evaporation (mg/mL) 1.0/100 1/100 Required
Note 1. All tests are maximum, unless otherwise stated.
Note 2. Microbiological testing is considered to be harmonized, with the exception noted that the EP test is written into the
Production section, and the USP test is contained in a non-compendial general information chapter 1231.
Note 3. Limits are temperature dependent. See 645 Water Conductivity
Note 4. Actual limit is Rs-Rw. See 643 Total Organic Carbon.
Note 5. WFI stored at >80°C and circulated at >70°C.
Note 6. Same as USP Water Conductivity chapter 645 Stage 2, or meets USP 645 any stage.
Bulk Purified Water - Today 6
Attribute1 USP EP JP ChP IP

US/EU/JP/WHO Human
Source Water drinking water Drinking water Potable Water Potable Water
consumption
Clear, colorless,
Clear and Clear, colorless, Clear, colorless,
Description odorless,
colorless liquid odorless liquid odorless liquid
tasteless
Distillation, RO, Distillation, RO, Distillation, RO,
Production Method Suitable process Suitable process ion-exchange, UF, ion-exchange, or ion-exchange, or
or combination suitable process suitable process
Total Aerobic (cfu/mL)2 100 100 100 100 100
Conductivity (mS/cm at 25°C) 1.3 (3 stage)3 5.1 (1 stage) 2.1 offline
Same 6
as USP Same as EP Same as USP
TOC (mg/L) 0.54 Same as USP Same as USP Same as USP Same as USP
Bacterial Endotoxins (EU/mL)
Nitrates (ppm) 0.2 (delete in 2018) Not detectable 0.06 0.2
Heavy Metals (ppm) 0.1 5 Not detectable 0.1 0.1
Acidity/Alkalinity Indicators Indicators Indicators
pH 5.0-7.0 5.0-7.0
Calcium/Magnesium Effective Effective
Indicators Indicators
Chloride Not detectable Not detectable
CP 2010 Not detectable
Sulfate JP 16
Not detectable Not detectable IP 2012
Not detectable
Nitrite (ppm) Not detectable 0.02 Not detectable
Ammonium (ppm) 0.5 0.3 0.2
Oxidizable Sub (/100 mL) Option to TOC Not detectable Option to TOC Option to TOC
Residue on Evaporation (mg/mL) 1.0/100 1/100 1.0/100
Note 1. All tests are maximum, unless otherwise stated.
Note 2. Microbiological testing is considered to be harmonized, with the exception noted that the EP test is written into the
Production section, and the USP test is contained in a non-compendial general information chapter 1231.
Note 3. Limits are temperature dependent. See 645 Water Conductivity.
Note 4. Actual limit is Rs-Rw. See 643 Total Organic Carbon.
Note 5. Not required effective Jan 1, 2009 if WFI conductivity requirements are met.
Note 6. Same as USP Water Conductivity chapter 645 Stage 2, or meets USP 645 any stage.
Agenda 7

Traditional Microbial Detection 8
The "golden method" for counting bacteria

 A colony-forming unit (CFU) is a unit used to estimate the
number of viable bacteria or fungal cells.
 Counting with CFU’s requires culturing the microbes and counts
only viable cells.
 Duration and temperature of incubation are also critical aspects of Traditional Microbial Detection
a microbiological test method.
 Classical methodologies using high-nutrient media are typically
incubated at 30–35C for 48–72 hours.
 Because of the flora in certain water systems, incubation at lower
temperatures (e.g., 20–25C) for longer periods (e.g., 5–7 days) can
recover higher microbial counts when compared to classical
methods.
 Low-nutrient media are designed for these lower temperature and
longer incubation conditions (sometimes as long as 14 and up to 21
days to maximize recovery of very slow-growing oligotrophs or
sanitant-injured microorganisms), but even high-nutrient media can
sometimes increase their recovery with these longer and cooler
incubation conditions.
Costs Associated with Microbial Sampling 9
Real-time microbial detection saves time and $

 Microbial sampling and testing has been a roadblock
for pharmaceutical manufacturing
 The costs for sampling and performing standard
bacterial plate count tests is expensive and slow -
approx. >$100/sample
 Typically, sampling frequency is done daily to a
maximum of weekly at multiple locations through out
the water system (avg. minimum of 20 points)
 Results are not obtained for 5 to 7 days and some
cultures take 21 days Current method of
bioburden detection:
 The Pharma industry estimates that over 80% of
 Costly
positive results are false-positives caused by human
 Time consuming
error
 Unreliable results from
 A Pharma facility has to decide the risk acceptance: do human errors
they use the water or wait for the test results  Delay of product
release

Agenda 10

New Chapter for Alternative Microbial Testing 11
USP General Information Chapter <1223>

Global Pharmacopeias and Regulators 12
Pharmacopeias are encouraging the validation of alternative methods
 Microbial contamination is one of

the three regulated parameters
for purified bulk waters
 USP <1223> “Validation of
Alternative Microbiological
Methods”
 Ph.EUR. 5.1.6. “Alternative
Methods for Control of
Microbiological Quality”
 FDA Guidance for Industry
defining validation criteria for
growth-based RMM
 EMA Guidance for control
strategy with rapid microbial
methods
Why On-line Microbial Monitoring? 13
Off-line method of bioburden On-line method of bioburden

detection: detection:
 Unreliable results (human errors)  Reliable, real-time results
 High risk of false-positive results  Allows real-time release of water
 Costly  Eliminates sampling error
 Time consuming  Cost-effective
 Delay of product release  No delay of product release

USP Guidelines for Microbial Monitoring 14
USP<1223> Validation of Alternative Microbiological Methods:

 Acknowledges the limitations of the use of CFU as a standard signal for microbiological
methods. " the cfu signal then is prone to underestimate the number of cells in a sample”
 Provides guidance on selection, evaluation, and use of microbiological methods as
alternatives to referee methods.
 Encourages the validation and development of alternative technologies "provided that
proper technical and scientific attention is paid to the selection, qualification, and
implementation of the method.” USP <1223> states.
 Identify suitable alternative methodology
 Development of user specifications for the equipment selection (USP<1231>)
 Demonstration of the applicability of the method as a replacement for a standard referee
method
 Qualification of the method in the laboratory
 Provides Options for demonstrating equivalence
“Alternative methods and/or procedures may be used if they provide advantages in

terms of accuracy, sensitivity, precision, selectivity, or adaptability to automation or
computerized data reduction, or in other special circumstances.” General Notices & Requirements

USP Discussion of Rapid Microbiology Methods 15
Alternative Microbiological methods will report counts that are significantly

higher – 10X, 20X, 30X or more – because it is an actual count not an
estimate

USP Discussion of Rapid Microbiology Methods 16
The viewpoint of the USP

Limitation of the CFU
The colony-forming unit (CFU) is an estimate of

viable bacterial or fungal numbers in a water, air,
soil, food or drug sample.
Unlike direct microscopic counts where all cells,

dead and living, are counted, CFU estimates viable
cells that are capable of dividing in the solid plate
count medium under the incubation conditions
employed.

European Pharmacopeia Alternative Microbial 17
Information Chapter 5.1.6.
5.1.6. ALTERNATIVE METHODS FOR CONTROL OF MICROBIOLOGICAL QUALITY

The following chapter is published for information.
1. GENERAL INTRODUCTION
The objective of this chapter is to facilitate the implementation and use of alternative microbiological
methods where this can lead to cost-effective microbiological control and improved assurance for
the quality of pharmaceutical products. These alternative methods may also find a place in
environmental monitoring.
The microbiological methods described in the European Pharmacopoeia have been used for almost
a century and these methods - for enumerating and identifying micro-organisms - still serve
microbiologists well. Over the years, these methods have been invaluable to help control and
secure the production of microbiologically-safe pharmaceutical products. Nevertheless
conventional microbiological methods are slow, and results are not available
before an incubation period of typically up to 14 days. Thus the results from the
conventional microbiological methods seldom enable proactive, corrective
action to be taken.
Alternative methods for control of microbiological quality have been introduced
in recent years, and some of these methods have shown potential for real-time
or near-real-time results with the possibility of earlier corrective action. These
new methods can also offer significant improvements in the quality of testing.

Regulators Viewpoint on Alternative Methods 18
FDA
Food and Drug Administration
 FDA encourages use of new technologies
- Rapid Microbial Methods (RMM): help meet
Quality by Design (QbD) principles.
- Regulatory mechanisms to implement  “Any alternative microbial detection
RMMs are evolving. methods may be used provided it has
 FDA Aseptic Processing Guidance (cGMPs) been demonstrated they are equivalent
or superior to the Compendia
 FDA Process Analytical Technologies-PAT
Pharmacopeia method”. FDA Senior
 FDA Strategic Plan for Regulatory Science Microbiologist
 FDA Senior Microbiologist Supports RMMs
 FDA works in partnership with USP
 FDA liaisons work with USP Committee

Agenda 19

Drivers for Alternative Methods 20
Global organizations support development of on-line bioburden analysis.
OWBA FDA
 Online Water Bioburden Analysis  FDA PAT (Process Analytical Technologies)

Workgroup Initiative
- Established as an Industry workgroup  FDA Aseptic Processing Guidance - cGMPs
to promote development and
implementation of online bioburden  FDA Strategic Plan for Regulatory Science
analyzers  FDA Microbiologists Support RMMs
- Members from Merck, Novartis,
Amgen, Fresenius, Baxter, P&G,
Roche, Sanofi and Pfizer

PDA, OWBA and ISPE 21
Pharmaceutical Industry promoting implementation and adoption of RMM
OWBA Mission
 In an effort to accelerate the implementation of

an OWBA, a collaborative workgroup was
formed, comprising representatives across the
pharmaceutical / biopharmaceutical industry.
 By leveraging lessons learned through
assessment and implementation of various
rapid microbiological methods (RMM) and
process analytical technologies (PAT) across
the healthcare and consumer products
industries, the primary goal of this workgroup
is to provide guidance regarding the
development and application of OWBA
systems that would be broadly accepted by
the industry and regulators.
Promotion of Alternative Methods by OWBA 22
OWBA Online Water Bioburden

Analysis
 Industry workgroup created to promote
development and implementation of online
bioburden analyzers
 Members from Merck, Novartis, Amgen,
Fresenius, Baxter, P&G, Biogen, Roche,
Shire, Lilly, GSK and Pfizer
Focused on reducing risk & cost by:
 Reduced Labor: Less sampling & lab-based
testing
 Better product quality/process understanding:
 Continuous monitoring-faster response to
excursion
 Fewer investigations
 Real-time release
 Better product safety
 Energy Savings:
 Reduced sanitization frequency

Agenda 23

Bacterial Detection as an At-Line Measurement 24
Bring the Microbiology Lab to the System – eliminate sampling !
 In-Line
- inside pipe, real-time
- conductivity, pressure, flow
 At-Line (Side stream)
On-Line
- attached to pipe, real-time,
flow to drain
- TOC, pH, O2, O3
- Microbiological
 Off-Line
- batch sample
- "Lab" measurements
- minutes, hours, days
- pH, chromatography
required in the Production section.
As a result of introducing non-distillation technologies into this monograph, the monograph Water,
EP Monograph – WFI Production

highly purified (1927) will be made redundant and will be deleted from the Ph. Eur.
XXXX:0169 25
WATER FOR INJECTIONS
Aqua ad iniectabile
H2 O Mr 18.02
DEFINITION
Water for the preparation of medicines for parenteral administration when water is used as vehicle
(water for injections in bulk) and for dissolving or diluting substances or preparations for parenteral
administration (sterilised water for injections).
Water for injections in bulk

PRODUCTION
Water for injections in bulk is obtained from water that complies with the regulations on water
intended for human consumption laid down by the competent authority or from purified water.
It is produced either :
– by distillation in an apparatus of which the parts in contact with the water are of neutral glass,
quartz or a suitable metal and which is fitted with an effective device to prevent the entrainment
of droplets ; The correct maintenance of the apparatus is essential. the first portion of the
distillate obtained when the apparatus begins to function is discarded and the distillate is
collected ; or
– by reverse osmosis, which may be single-pass or double-pass, coupled with other suitable
techniques such as deionisation and/or ultrafiltration.
Correct operation monitoring and maintenance of the system are essential.
In order to ensure the appropriate quality of the water, validated procedures, in-process monitoring
of the electrical conductivity, and regular total organic carbon and microbial monitoring are applied.
Water for injections in bulk is stored and distributed in conditions designed to prevent growth of
micro-organisms and to avoid any other contamination.
"Method of Manufacture" Requirements 26
The Change by EP for WFI Method of Production

 Purified Water
- USP, EP, JP, ChP, IP permits production by distillation, reverse osmosis, de-ionization,
electro-deionization, Nano, Ultra or filtration, or equivalent means.
 Water for Injection

- USP permits “distillation or a purification process that is equivalent or superior to distillation
in the removal of chemicals or microorganisms”USP27
- EP permits distillation AND RO Plus additional technology
- JP permits distillation or RO/UF when supplied PW or suitably pretreated Water
- ChP distillation only
- IP distillation only
 This change by EP has impacted the design of WFI systems.

 System Fabricators have introduced ‘Cold” WFI Systems
 The Cold WFI systems will be a stimulus for Real Time Microbial Monitoring
European Medicines Agency 27
• Questions and answers on production of water for injections

by non-distillation methods – reverse osmosis and biofilms
and control strategies.
• This set of questions and answers is intended to provide preliminary
guidance until such time the ongoing revision of Annex I of the GMP guide
is complete.
• 6. What testing should be employed during initial qualification and routine  operation sampling?  
• Testing should be conducted in line with Ph.Eur. Monograph 169 ‘Water for Injections’
• Use of rapid microbiological methods should be employed as a prerequisite to the control
strategy to aid with rapid responses to deterioration of the system.
• Article 23 of Directive 2001/83/EC states: “...the authorization holder must, in respect of the methods of manufacture and
control...take account of scientific and technical progress...”
• Quantitative microbiological test methods – in line with Ph.Eur. 5.1.6 monograph ‘Alternative Methods for control of
Microbiological Quality’.
• Due consideration should be given to employing alternate methods for the rapid quantitative determination of the
contamination levels existing within the water system. The validation of such system should be in line with the above
referenced monograph.
• Use of alternative/ rapid microbiological test methods should be employed as part of the overall
control strategy for the system.
• Taking into account the speed at which organisms can proliferate , the use of rapid microbiological test
methods and systems should be employed in order to improve or increase the probability of
early detection and allow timely action to be taken.  
WHO Encouraging On-Line Testing 28
The WHO Expert Committee

on Specifications for
Pharmaceutical Preparations
•WPU – Water for

Pharmaceutical Us
•WHO Recommendation for the

Water System is online testing
for;
• Conductivity
• TOC
• Flow
• Pressure
• Temperature
•For the Points-of-Use samples

should be taken for offline testing

Agenda 29

Exploit the Physico-Chemical Properties of Bacteria 30
Particle-Chemical duality of bacteria is measured simultaneously

 A bacteria is a particulate matter in water, with the potential to scatter light in several
physical ways
- The magnitude and direction of the scattering is informative about the type of particle
 A bacteria contain chemical metabolites – these are chromophores

- They have the ability to absorb UV light and fluoresce at distinct spectral range of
wavelengths
 These 2 properties are analyzed in real time

Principal of Operation 31
 Microorganisms (bacteria, fungi, parasites, spores) use Metabolites (NADH, Riboflavin, &
other proteins) to regulate their growth and development
 These metabolites produce intrinsic fluorescence emissions when exposed to light of
certain wavelengths
 LIF (Laser-Induced Fluorescence) is a highly sensitive technique that exploits this
phenomenon to detect microbes

Fluorescence Emission Spectra of Metabolites 32
By detection of these two metabolites we can identify bacteria and do not

need to fluoresce other metabolites
Fluorescence of metabolites
with the 405nm laser used in
7000RMS
(Hill et al, Field Ana. Chem. & Tech, 3(4-5), 221,1999)

Agenda 33

Principal of Operation 34
 7000RMS draws the sample through a flow cell into the

interrogation zone
- A UV laser light source is directed through the sample
1. Scattered light is captured and collimated within a parabolic
mirror
- The scattering of light determines the size of the particle
2. The intrinsic fluorescence is also captured - the amount of
light emitted at a longer (than 405 nm) wavelength by a
microorganism
 The two types of light (scattered/fluorescent) are optically
separated
 The technology in the software uses the combined data to
differentiate and enumerate inert particles and biological cells
- Simultaneous detection of a particle and fluorescence within
a narrow time

RMS Principle of Operation 35
Dichroic beam splitter
405nm laser

When is a particle a particle or a microbe? 36
When it scatters light and fluoresces - at the same time

 The Photodiode (PD) detects scattered light at/near 405 nm
- Plus any AS-Raman scattered light at <405 nm (very low)
 The PMT detects fluorescent light from bacteria (>405 nm)
- Plus any ASE >405 nm
 When a particle passes the interrogation (irradiated) zone and produces any light at the
PD, that signal grows and decays with a mean time of ~50 µsec
- If the amplitude and the breadth of the PD signal meets specific criteria (amplitude,
threshhold, and time), this is considered to be an INERT particle
 When a particle passes the interrogation (irradiated) zone and produces fluorescent light,
that signal grows and decays with a mean time of ~ 50 µsec
- If the amplitude and the breadth of the signal meets some specific criteria, this is a
POSSIBLE biocount
 When a particle is detected at PD and PMT at the same time (within x µsec), then this is a
BIOCOUNT!

Raw Signal at Optical Detectors in 1 sec 37
Plot of signal vs time – each channel – sample rate 100 kHz (10 µsec)

Take a closer look 38
At simultaneous detection of particle and fluorescence, this is a biocount.

How to Use Technology – PC vs QC 39
 Quality Control of Production use points

- Quality of product
Product focus - QA
- safety Uses measurement tools after the product is
- purity manufactured.
- out-of-spec investigations
- Consistency of the product
 Process Control of a Water system

- Quantity of product
- average, daily, peak
- Cost of product Engineering focus
- efficiency Uses in-process measurement tools and
- cost of production controls while the product is being
- cost of maintenance manufactured.

A Traditional Approach to a New Technology 40
A new technology cannot be used until it is approved by QUALITY 

 A new technology for manufacturing is introduced
 Advanced engineering or R&D or Operations sees a new opportunity to use in Production
to be more efficient, to increase yield, to save time, to improve product or process quality
 Water system engineering sees the potential of the technology as a means to routinely
monitor the water purification PROCESS in real time, much like TOC and conductivity
 And everyone is enthused to try the new technology (it is like a new shiny toy)
 Then what happens?

 Quality says "let's validate the technology, and if it agrees with the compendial method for
linearity, limit of detection, specificity, etc…, then it is a validated measurement"
 But--- let's look at the compendial measurement

Validation of Alternative Microbiological Methods 41
<1223> Validation Introduction

 The "gold standard" compendial method has a 0.1-1% yield
 Only large differences can be distinguished
 Chemical-based analytical quality is not possible

Optical-Based Technology for Process Control 42
Instead of prescribed treatment solution, use RMM as a dynamic solution

 Use the rapid method as a trend-based method to immediately identify upsets
- Detect excursion in real time and treat accordingly
- Situational awareness afforded by real time monitoring
 Use SPC (Statistical Process Control) as a means to identify process alarms and levels
 Facilitate Parametric Release
 “Continued Process Verification for ongoing assurance that the process remains in a state
of control” (Guidance for Industry--Process Validation; FDA, January 2011, CGMP
Revision 1)
 Lower cost, more effective, and pro-active process control

Agenda 43

Application Example: Storage/Distribution 44
Pure Water
or WFI
7000RMS can
also be
installed after
O3
the purification
unit
5000TOCi
User Objective Why the 7000RMS?

 Validate PW and WFI quality with on-line  Real time microbial detection provides
Real-time Microbial System – 7000RMS faster analysis, eliminates delays and
reduces costs
 Reduce false-positive tests from
sampling errors  Monitor and trend the water quality
thereby allowing for accurate risk
 Reduce lab testing costs and analysis
assessment
delays
 Reduce testing and insure proper and
 No delay in product release
timely sanitization For internal use - Confidential
Application Example: Point of Use Testing 45
UPW

 Rapidly validate water at point-of-use  Sample points-of-use real time
 Expand distribution monitoring to include  Reduce sample analysis time and eliminate
all Points of Use risk associated with grab samples
 Eliminate sampling errors and costs  See measurement results in seconds
 Bring the measurement to the sample

Application Example: Cold WFI Water System 46

 Rapidly validate production water  Meet PAT guidance
 Integrate the Compendial test parameters  Reduce sample analysis time and eliminate
on the water system risk associated with grab samples
 Eliminate sampling errors and delays  See microbial results in seconds
Photo credit; Bosch Packaging Technology
7000RMS Pharmaceutical User Data 47

Initial Data after Installation 48

Continuous Bioburden (Microbial) Monitoring 49
During 9 days of monitoring, no microbe count events above 10 CFU/100mL

 The 7000RMS graph below shows 432 discrete 100 mL sample results over a single 24
hour period
 During 9 days of monitoring, no microbe count events above 10 CFU/100mL
Microbe Eq
Average 0.2 3 bio count "events" above 10
Std Dev 0.9 AFU/100mL.
RSD (%) ---
Average + 3

Online Monitoring Experiment 50
Removal of the sterility filter
Significant higher Bio-Counts

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20000
40000
60000
80000
100000
120000
140000
160000
52
Lessons Learned 53
 RMS online data demonstrated the stable water quality and the state of control in the water
line
 Experiment in water line excursion alert

- Experiment:
 A sterility filter upstream in the water line from RMS installation point was removed
temporarily and re-inserted afterward
 RMS showed a corresponding response (spikes on the right hand side of preceding
slide)
 RMS’ capability of real time alerting of excursion was demonstrated

PW System–Real Time Process Control Monitoring 54
Dual Train RO systems displayed a cyclical pattern

 Dual Train RO systems displayed a cyclical pattern
 Membranes replaced, cycle continued, Reject pressure and flow rebalanced
7000RMS PW AFU/100ml Oct 11 - 14, Oct 24 - 27
800
700
600
500
400
300
200
100
0
8:47:18 20:43:59 8:40:40 20:37:21 8:34:02 20:30:43 8:27:24 20:24:04 19:36:49 7:33:30 19:30:11 7:26:52 19:23:33 7:20:14
10/11/17 10/11/17 10/12/17 10/12/17 10/13/17 10/13/17 10/14/17 10/14/17 10/24/17 10/25/17 10/25/17 10/26/17 10/26/17 10/27/17
 Discovered O-rings were cracked, replaced and cyclical pattern eliminated
7000RMS PW AFU/100ml Oct 31 - Nov 7

800
700
600
500
400
300
200
100
0
10:48:40 22:45:21 10:42:02 22:38:43 10:35:24 22:32:05 10:28:46 15:42:12 3:38:53
10/31/17 10/31/17 11/1/17 11/1/17 11/2/17 11/2/17 11/3/17 11/6/17 11/7/17

Implementation Summary 55
 For online implementation of a monitoring instrument, it’s essential to ensure everything

connected to it is clean
- Dirty tubing will give dirty reading, even when the water source is clean
 Advantages in process monitoring:
- Real time: correlate to improper operation, pin-point sources of excursion
- Trend analysis: get a real picture of aseptic operation, flag any deviation from the
normal condition

Customer Example 3: WFI loop 56
The 7000RMS provides real-time process transparency that is not possible

with an offline plate count.
Average: 236 AFU/100mL Alert Limit: 780 AFU/100mL Action Limit: 1052 AFU/100mL
Represents a single spike – that Represents an event that occurred. At this time
would not be investigated there was an increase in the demand for water & a
switch to different tank & pump.

Customer Example: WFI loop 57
The 7000RMS provides real-time process transparency on Hot WFI.

Average: 160 AFU/100mL Alert Level: 225 AFU/100mL Action Level: 258 AFU/100mL
Limit: 324 AFU/100mL
Represents a possible event that occurred. Represent single spikes – not investigated
External Bacterial Testing at BA 58
Comparison of 7000RMS Data and Traditional Plate Counting – E. coli

Comparison of 7000RMS Data and Traditional Plate Counting – B. subtilis

 Comparative test also performed at an
additional microbial testing laboratory
using the B.subtilis organism
 Plate cultures did not develop due to

organism viability issues in purified
water.
 However, the RMS results showed

detection compared to plate count
method and linear to expected inoculum
concentrations

Comparison of 7000RMS Data to projected CFU/ml – P. aeruginosa

 P. aeruginosa was consistently difficult to recover with Projected CFU (Inoculum Control) Vs. Me ler 7000
traditional plate count methods RMS
14000
y = 1.028x + 516.39
 Test was repeated with multiple replicates at varying

12000
R² = 0.99607
10000
concentrations 8000
6000
4000
 Similar results with P. aeruginosa has been observed at a 2000
facility conducting their own challenge test 0 2000 4000 6000 8000 10000 12000
14000 Membrane Filtra on Vs. Me ler 7000 RMS

14000
12000 y = 3.2997x + 3356.1

12000 R² = 0.41519
10000
8000
Projected CFU/100mL (Projected from Incoulum Control)
10000 6000
Membrane Filtration CFU/100mL 4000
8000 2000
/100mL
AFU/100mL (7000 RMS) 0

0 500 1000 1500 2000 2500 3000
6000
Membrane Filtra on Vs. Projected CFU
(Inoculum Control)
12000
4000
10000
8000
2000 6000
y = 3.3316x + 2677.5
R² = 0.44906
4000
2000
0 0
0 1 2 3 4 5 6 7 0 500 1000 1500 2000 2500 3000

Agenda 61

Adopting Technology for Real Time Water Control 62
1840 – 1991 1991 – 1996 1996-2000 2000-present

Traditional methods Verification USP Harmonize
Chemistry/Organic tests are  Thornton asked to  USP Accepted as  Adoption by

prove instrumentation Compendial Tests  EP
 Qualitative
versus wet chemistry
 Conductivity  JP
 Subject to bias
 Chemistry tests vs Instrument
 Off-line Conductivity  ChP
 TOC Instrument
- Carbon dioxide Instrument  IP
- Calcium  Oxidizable
Substances vs TOC
- Ammonia
Instrument
- Chloride
- Sulfate
- Oxidizable Substances
- Heavy Metals

Still Adopting New Technologies 63
1890 – 2015 2014-today 2018+ >2018+

Traditional Methods Verification USP/EP/JP Harmonize
Bacteria tests are  Traditional Plate Counts  Accepted by USP as  Accepted by the
vs On-Line Real Time a Compendial Test other Global
 Plate Count
Instruments Pharmacopeias
 On-Line Real Time
 Qualitative
 USP <1223> now Instruments
 Subject to bias encourages validation of
alternative microbial
 Off-line
methods
 Sampling errors  Ph.EUR. 5.1.6.
 Poor growth “Alternative Methods for
Control of Microbiological
Quality”

Mettler-Toledo Thornton 64
Thank You! 감사합니다 Terima kasih

Merci Gracias Dziękuję
Grazie Tak Tack
謝謝 σας ευχαριστώ Cảm ơn bạn
धन्यवाद Cпасибо
ありがとう
‫תודה‬ Obrigado
Danke
Go raibh maith agat

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Rapid Microbial Detection

Here is a chemist's story of introducing a Rapid Microbial Detection method.

For internal use - Confidential

1 Introduction, Water, Microbes

2 Current/Traditional Methods of Microbial Analysis

3 Current Regulations, New Regulations, Encouragement of Alternative Methods

4 Industry Drivers - PDA, OWBA and the early Adopters

5 Promotion of On-Line Measurements by Pharmacopoeia and Regulators

6 Chemical Properties of Bacteria

7 Technology in the 7000RMS

8 Case Studies and 7000RMS Features

9 Conclusion – Relate to TOC

Types of Impurities Characteristics Types of Tests

For internal use - Confidential

Attribute1 USP EP JP ChP5 IP

Attribute1 USP EP JP ChP IP

1 Introduction, Water, Microbes

2 Current/Traditional Methods of Microbial Analysis

3 Current Regulations, New Regulations, Encouragement of Alternative Methods

4 Industry Drivers - PDA, OWBA and the early Adopters

5 Promotion of On-Line Measurements by Pharmacopoeia and Regulators

6 Chemical Properties of Bacteria

7 Technology in the 7000RMS

8 Case Studies and 7000RMS Features

9 Conclusion – Relate to TOC

The "golden method" for counting bacteria

Real-time microbial detection saves time and $

For internal use - Confidential

1 Introduction, Water, Microbes

2 Current/Traditional Methods of Microbial Analysis

3 Current Regulations, New Regulations, Encouragement of Alternative Methods

4 Industry Drivers - PDA, OWBA and the early Adopters

5 Promotion of On-Line Measurements by Pharmacopoeia and Regulators

6 Chemical Properties of Bacteria

7 Technology in the 7000RMS

8 Case Studies and 7000RMS Features

9 Conclusion – Relate to TOC

USP General Information Chapter <1223>

For internal use - Confidential

Pharmacopeias are encouraging the validation of alternative methods

 Microbial contamination is one of

Off-line method of bioburden On-line method of bioburden

For internal use - Confidential

USP<1223> Validation of Alternative Microbiological Methods:

“Alternative methods and/or procedures may be used if they provide advantages in

For internal use - Confidential

Alternative Microbiological methods will report counts that are significantly

For internal use - Confidential

The viewpoint of the USP

The colony-forming unit (CFU) is an estimate of

Unlike direct microscopic counts where all cells,

For internal use - Confidential

Information Chapter 5.1.6.

5.1.6. ALTERNATIVE METHODS FOR CONTROL OF MICROBIOLOGICAL QUALITY

For internal use - Confidential

For internal use - Confidential

1 Introduction, Water, Microbes

2 Current/Traditional Methods of Microbial Analysis

3 Current Regulations, New Regulations, Encouragement of Alternative Methods

4 Industry Drivers - PDA, OWBA and the early Adopters

5 Promotion of On-Line Measurements by Pharmacopoeia and Regulators

6 Chemical Properties of Bacteria