of each step that they perform. Operators must under- stand the necessity to ensure that cleaning procedures are properly sequenced; that is, that activities are performed in the appropriate order to ensure they are not contami- nating services that have already been cleaned. General training in aseptic practices is worth its weight in gold for any facility; it provides a level of sensitivity to cross- contamination that is unparalleled.

Familiarization of employees with appropriate techniques to ensure that environmental contamination is not transferred to process and product contact surfaces is highly valuable. In particular, for manual cleaning of equipment such as automated conveyor systems or \ufb01llers, the equipment is traditionally cleaned with lint-free wipes and a bucket of a detergent solution. When cleaning this equipment it is important to ensure that the operators understand a \u201ctop-down-center-out\u201d approach to avoid contaminating already cleaned surfaces. It is not unusual to observe operators cleaning critical product-container contact surfaces and environ- mental surfaces with the same wipe and solution. It is important to help employees differentiate the surfaces and understand their role in keeping all surfaces clean.

In addition to training employees on the speci\ufb01cs of the cleaning procedures it is important to train employees in the basics of cleaning validation, especially including their role in the validation. In particular, all operators and supervisors should be aware that during a cleaning validation trial they are not themselves being judged but rather the adequacy of the SOP and that it is the techniques the SOP describes that are under challenge. Cleaning validation is also assessing the elements such as the robustness and reproducibility of the training.

Another important aspect of training is the edu- cation of inspectors. Inspectors are on the front line in helping to ensure that equipment is clean, both during the validation, and after validation is complete. Visual inspection is important to every cleaning validation program because of the assurance it provides of the baseline cleanliness of all surfaces not just those which are sampled. In addition this visual assessment can also help to ensure that excipients and all other materials not subject to analytical-speci\ufb01c analytical methods are removed. Routine visual inspection after cleaning is the one common denominator between the validation and the routine operations, ensuring that surfaces have met minimum cleanliness standards that were achieved in the original testing.

When training inspectors it is important to ensure that inspectors are made aware of appropriate inspection techniques and tools so that they do not contaminate the clean surfaces they are inspecting. It is important for them to know where to look for and how to identify residue on the surfaces. Because cleaning may be conducted either in a disassembled state or an assembled state it is important that inspectors understand whether equipment inspec- tion should be conducted assembled or disassembled. Inspectors, like cleaning personnel, should always be cognizant of wearing appropriate protective equipment such as gloves and lab coats to ensure they are not contaminating the equipment they inspect.

For routine inspection, standardized inspection tools should be used. Flashlights can be dif\ufb01cult to control unless they have rechargeable batteries and are

placed on charge frequently. Intrinsically safe, electric lights may be a better option for inspection of deep vessels or other hard to illuminate areas. Other tools such as remotely operated digital cameras or borescopes may have great utility in getting the inspection to areas that need it most. This equipment has the added bene\ufb01t of being able to capture images and saving those images as part of the cleaning or cleaning validation record.

Today\u2019s training can easily be prepared using digital images and DVDs of proper techniques. Using such tools can help ensure that a standard curriculum is applied and that the same techniques are routinely taught.

Sampling sites should be selected based on the most dif\ufb01cult to clean geometries of the equipment. These locations, however, are frequently inaccessible\u2014their very inaccessibility is what makes them dif\ufb01cult to clean! Therefore, when choosing sampling sites one must always be cognizant of the desired sampling location based both on dif\ufb01culty of cleaning and on intended sampling methods. As we will learn, sampling methods have various advantages and disadvantages that make them suitable for various geometries and locations on the equipment.

Equipment may have what we consider both hotspots and critical sites. Hotspots are locations that are likely to become dirty during the manufacturing process and are dif\ufb01cult to clean. Critical sites are those locations, which, if they were to remain dirty, would provide a disproportionate level of contamination to the next batch or portion of the next batch.

An example of a hotspot might be the bottom of an agitator or an instrument port inside a vessel that is likely to become soiled during the manufacturing process and might prove to be dif\ufb01cult to clean during the cleaning process. The use of an agitator in a mixing vessel means that any soil remaining on a surface is likely to become homogeneously distributed within the next batch. Contrast this agitator to locations such as a \ufb01lling needle, a tablet press table, or a fraction collection valve on a chromatographic skid. Each of these locations has the opportunity to affect the next dose of the product or the next portion of the batch being produced. The residue that remains on these locations will not be homogeneously mixed throughout the batch but instead will disproportio- nately contaminate a small number of doses or, worst-case, a single dose.

When selecting sample sites we must evaluate a variety of locations including hot spots and critical sites as well as some representative locations on the equip- ment. Remember to include in the process those locations which might experience recirculation or redeposition of contaminants during the cleaning process. For example, in a vessel that might use \ufb01ll-soak-and-agitate as the cleaning method, we might \ufb01nd that the agitation level falls off as the liquid is drained from the vessel. This may mean that there are signi\ufb01cant bathtub-ring risks on this type of equipment based upon the resettling of suspendedresidues on surfaces with low agitation levels.

The number of sample locations selected for any individual piece of equipment should be based on the very same considerations that were addressed in sampling location selection:

For example, in a \ufb02uidbed granulator which can be nearly two stories tall we may have dif\ufb01culties in coverage side-to-side and top to bottom. In order to ensure adequate cleaning, we may need to sample several locations on the sidewall of this equipment despite the fact that the sidewall is all of the same material of construction and not a dif\ufb01cult to clean geometry.

potential areas of weakness or locations where ill- controlled process parameters may result in variability

When determining sample size for cleaning vali- dation, we simply need to target a sample size that will provide suf\ufb01cient residue to the assay, but not collect samples so large that our recoveries may suffer. Most \ufb01rms select a convenient surface area (e.g., 100 cm2or 4 in.2), but it should be noted that it is possible to vary sample sizes slightly from sample to sample, when the sample size is accounted for in the result. Sample size variation may occur naturally based on the geometry of the equipment. For example, if a valve is 105 cm2on all product contact surfaces, it is much more appropriate to sample the full surface and account for the small overage in the equation for the results (see the Limits section) than it would be to instruct the sampling personnel to sample all of the surface except for 5 cm2.

There are a variety of sampling methods for cleaning validation. Any method can be used provided it can be demonstrated to be suitable to recover the soil reproducibly from the surface. This need for effective recovery is the reason methods validation is always coupled with an assessment of sampling method ef\ufb01ciency.

The most common sampling methods employed in cleaning validation are rinse sampling and swab sampling. Of the two, swab sampling is typically deemed by regulators to be preferable. There are clearly situations where both methods may apply, however, and it is important that any sampling method used should be based upon its strengths.

analyte. Because of the need to physically wipe the surface, swab sampling is a preferred method in locations where the surfaces are readilyaccessible to a human hand or arm. (Extension tools are also available but must also be validated for use.) The swab is typically used with a diluent (water, solvent, or a combination of the two) although the sampling may also be conducted dry. The diluent for moistening the swab must be compatible with the analytical method.

Rinse sampling, as compared to swab sampling, does not employ mechanical action on the surface other than that which is delivered by the \ufb02uid traversing the surface. Rinse sampling may be collected either as a portion of the \ufb01nal rinse of the clean-in-place process, or as a rinse applied speci\ufb01cally for the purposes of collecting a validation sample. The advantages of rinsing speci\ufb01cally to collect a sample rather than as part of the \ufb01nal rinse include the facts that:

As with swab sampling, the solvent employed for rinse sampling is selected due to its solubility and compatibility with the residue(s). Typically rinse sampling is performed with the \ufb01nal rinse water. Rinsing with alcohol or other solvents can also be performed with appropriate safety measures in place.

From the description of these two techniques it is possible to see why the swab sampling is typically preferred as opposed to rinse sampling. Regulators have long argued that the mechanical action provided by swab sampling provides bene\ufb01ts in determining whether the cleanliness of the surfaces been achieved. In 1991 when the Newark district of\ufb01ce was drafting the cleaning validation guidelines that we follow in the U.S.A. today, inspectors would frequently refer to a theory called the \u201cbaby in the bath water.\u201d They used the baby in the bath water theory to explain why they preferred swab sampling over rinse sampling. The theory was, \u201cIf you are trying to determine whether or not the baby was clean would you look at the baby? Or would you look at the bath water?\u201d The answer, of course, was that you would look at the baby!

The question asked by the regulators was if the baby represented the equipment and the bath water represented the rinse sample, why did we believe that the rinse sample would represent the cleanliness of the baby? Their concern was that if the bath water had not contacted all soiled areas, or had not had suf\ufb01cient contact time with the baby, or if the residues on the baby were poorly soluble in the rinse water, or if the residues were not homogeneously distributed in the bath water, or if the residues became too dilute in the bath water, that examining the bath water would not be an appropriate technique for establishing the baby\u2019s cleanli- ness. Therefore, they concluded that rinse would be an inappropriate technique for sampling the baby. In part

those are the very details we strive to prove with properly executed recovery studies (see the Section entitled Analysis Selection, Method Validation, Recovery Studies, and Training). If the recovery study demonstrates that the rinsing technique is adequate to demonstrate the cleanliness of the \u201cbaby,\u201d there are no further objections to the use of that rinse technique.

Direct surface sampling using FTIR or photo- electron emission techniques is on the rise in cleaning validation. With these techniques, speci\ufb01c spectra may be obtained from residues remaining on the surface, thereby directly quantitating and identifying the designated residue. These techniques are highly desirable as they directly measure the quality of the surface or the baby. The advantage of these techniques is that they represent the sampling and the analysis all in one step and there is no real \u201closs\u201d to sampling system. As with swab sampling, the direct analysis of surfaces may be limited to those areas that are accessible for inspection.

Visual techniques using the eyes or remote inspec- tion cameras are another form of direct surface sampling. These techniques are typically nonspeci\ufb01c, however, and are also nonquantitative. With the advance of digital imaging, however, before and after images, or the comparison of appearance to prior cleaning events may be accomplished, in some cases even in a side-by-side comparison in the \ufb01eld against a stored image.

Coupon analysis involves the introduction of a small soiled piece of the materials of construction of the equip- ment into the process equipment for the purpose of later removal and analysis in the laboratory. In some facilities, small portions of the equipment such as \ufb01lling needles and spool pieces may be similarly removed for the same purpose. The advantage is the opportunity to apply sampling techniques in the laboratory such as extended soaks, physical agitation or sonication, andthe application of more hazardous solvents that would be adversely indicated in the \ufb01eld. This technique also lends itself to false soiling if a worst-case soil condition is desired for sampling baked on residue, for example or for quantitative soil removal using techniques such as gravimetric analysis.

Similar \u201ccoupon\u201d approaches can also be done either by rinse or swab with small swatches of fabric or materials where testing of the surface is very dif\ufb01cult. As an example \ufb02uid bed dryers employ large bag \ufb01lters. These bags have extraordinarily large surface areas. Because they are typically considered dif\ufb01cult to clean they are frequently dedicated to an individual product. This dedication however does not exonerate the manu- facturer from the responsibility to test for cleaning agents or materials used in the cleaning process. As a result we must sample the surfaces for any residual cleaning agent. Swab sampling is typically deemed ineffectual for woven surfaces due to the complexity of the weave and the fact that residue may become trapped between the individual \ufb01bers. Rinse sampling can be very effective with woven surfaces because it provides a prolonged soak and will help to loosen or dissolve residues from the surfaces.

Because of the immense size of many of these \ufb01lter bags, however, it is impossible to provide an ef\ufb01cient sampling method. The equipment for soaking the part without using extremely large volumes of \ufb02uid is often

not available. In these cases, a small swatch of material from a bag that is to be retired, or a sample from a vendor is obtained and intentionally subjected to worst-case soiling. This coupon of fabric is subjected to the washing process with a routine \ufb01lter bag and the coupon is then returned to the laboratory for testing. Due to the small size of the swatch of fabric, the coupon can be fully immersed in a beaker for rinse recovery from that surface. Prolonged soaking of a coupon is possible to maximize recovery. Recovery studies would be necessary to con\ufb01rm the recovery associated with this technique.

Concerns exist with the coupon sampling technique when manual cleaning is performed, inasmuch as we do not want the operator to concentrate on cleaning the coupon. Although the use of this technique is fairly infrequent, it has great potential \ufb02exibility for facilities where insuf\ufb01cient product is available to soil the whole system, or where investigation into appropriate cleaning techniques is desired prior to completely soiling equip- ment. When using this technique to study cleaning processes, remember that if only the coupons are soiled, the recirculated soil load in cleaning solutions will be signi\ufb01cantly lower than when the equipment is fully soiled. This may hamper the cleaning effectiveness on a fully soiled system, especially when considering soil redeposition issues.

An additional approach that can be taken for sampling includes the placebo approach. Placebo batches are recognized as both potential cleaning tech- niques and potential sampling techniques. In the former case, a placebo material produced using all typical exci- pients but no active ingredients would be passed through a process system for the purposes of scrubbing clean the system from the prior material. The principle is that the placebo would pass along the same pathways as the product and, therefore, would have an opportunity to scrub off residual product along those pathways. Placebo sampling, on the other hand is employing a placebo and passing it through the equipment for the purpose of measuring system cleanliness.

As with rinse sampling, if a scienti\ufb01c case can be built for the use of the placebo method, it may be appropriate for either cleaning or sampling. It should be noted however, that regulators have cautioned in the U.S. cleaning validation inspection guidelines that the placebo method when used may most appropriately be used in combination with other sampling methods.

In some cases particularly in powder-based systems for safe product residues, such as those associated with topical consumer product powders, the placebo