Cleaning Validation for Biopharmaceutical Manufacturing at Genentech

ABSTRACT
Biopharmaceutical manufacturing and cleaning equipment must be designed for effective and consistent cleaning to avoid cross-contamination and the cleaning processes must be verified as effective. A cleanability study is essential before introducing a new product into the manufacturing equipment. Part 1 of this article provides background on cleaning validation and the associated regulations, cleaning methods, and the validation strategy. It also describes Genentech's approach for new product introduction using laboratory-scale and representative-scale studies. Part 2 will cover the other aspects of the cleaning validation program such as grouping strategy, validation sampling, acceptance criteria, change control, and revalidation.

Cleaning validation refers to establishing documented evidence providing a high degree of assurance that a specific cleaning process will produce consistent and reproducible cleaning results that meet a predetermined level. A cleaning program can be divided into three phases: cleaning process and cycle development, cleaning validation, and maintenance. The program should begin with equipment design evaluation and cycle and process development that includes, but is not limited to, the following: sanitary equipment design, selection of final rinse water, approved equipment specifications that address an evaluation of the compatibility of construction materials with product and cleaning solutions, sprayball design optimization, cleaning process design studies, cleaning sample assay validation, suitability of sampling, and recovery studies for assay and sampling methods. Without these design and development activities, validation could potentially lead to unnecessary troubleshooting and cleaning verification exercises.

The cleaning process should remove materials such as media, buffers, storage solutions, cell culture fluids, cell debris, non-active pharmaceutical ingredients containing placebos, and formulations and concentrations of drugs or active pharmaceutical ingredients (API). Selection of appropriate sampling to demonstrate that residues have been removed to an acceptable level is vital for the success of cleaning validation.

At Genentech, Inc., design and cleaning-cycle development is considered a prerequisite for cleaning validation. The purpose of cleaning validation at Genentech for biopharmaceuticals is to:

• Assess a new product or new equipment for cleanability before cGMP production.
• Ensure that cleaning procedures are adequate for cleaning new products or new equipment.
• Ensure that residues after cleaning of equipment are reduced to an acceptable level before the manufacture of the next run or the next product in the same equipment.
• Provide ongoing assurance that the validated cleaning procedures are in a state of control through monitoring and periodic revalidation.
• Evaluate and validate changes to cleaning processes, other manufacturing processes, and equipment to maintain these validated cleaning processes in a state of control.

REGULATORY EXPECTATIONS

Cleaning validation is driven by regulatory expectations to ensure that residues from one product will not carry over and cross-contaminate the next product. An effective cleaning program starts with appropriately designed equipment and cleaning processes, followed by validation and maintenance. The following are some good manufacturing practice (GMP) cleaning validation requirements for the biopharmaceutical industry.

US Food and Drug Administration 21 CFR Part 211: Current GMP for Finished Pharmaceuticals

§ 211.63 Equipment design, size, and location:
Equipment used in the manufacture, processing, packing, or holding of a drug product shall be of appropriate design, adequate size, and suitably located to facilitate operations for its intended use and for its cleaning and maintenance.¹

§ 211.67 Equipment cleaning and maintenance:
(a) Equipment and utensils shall be cleaned, maintained, and sanitized at appropriate intervals to prevent malfunctions or contamination that would alter the safety, identity, strength, quality, or purity of the drug product beyond the official or other established requirements. (b)(3)(5) Protection of clean equipment from contamination before use.¹

§ 211.182 Equipment cleaning and use log:
A written record of major equipment cleaning, maintenance, . . . and use shall be included in individual equipment logs that show the date, time, product, and lot number of each batch processed.¹

Basic European Union GMP Requirements for Medicinal Products
EudraLex, volume 4, Medicinal Products for Human and Veterinary Use: Good Manufacturing Practice, chapter 3, Premises and Equipment:
Principle: Premises and equipment must be located, designed, constructed, adapted, and maintained to suit the operations to be carried out. Their layout and design must aim to minimize the risk of errors and permit effective cleaning and maintenance to avoid cross-contamination, build up of dust or dirt and, in general, any adverse effect on the quality of products.²

EudraLex, volume 4, Medicinal Products for Human and Veterinary Use: Good Manufacturing Practice, annex 15, Qualification and Validation:
Cleaning Validation: Cleaning validation should be performed in order to confirm the effectiveness of a cleaning procedure. The rationale for selecting limits of carry over of product residues, cleaning agents, and microbial contamination should be logically based on the materials involved. The limits should be achievable and verifiable.²

International Conference on Harmonization Q7, Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients
The ICH Q7 was developed jointly by the European Union, Japan, and the United States for active pharmaceutical ingredient manufacturing.³ API is the drug substance before final formulation; section 12.7 contains cleaning validation requirements for APIs.

Other Guidance Documents
Additional guidance documents have been established by regulatory agencies and industry associations, such as the FDA, the Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme (PIC/S), the Canada Health Products, and the World Health Organization .^4–7

BIOPHARMACEUTICAL MANUFACTURING PROCESSES AT GENENTECH

Genentech manufactures biopharmaceutical products from E. coli and Chinese hamster ovary host cells using multiproduct, dedicated, and single-use equipment. Product manufacturing involves cell culture and bacterial fermentation processes, with the associated recovery processes (harvesting, initial and final purification), followed by formulation, aseptic filling or lyophilization, and capping.

Genentech's manufacturing processes include steps for manufacturing and purification of the API, and steps for manufacturing and packaging of the finished drug product. Steps up to and including final purification of the drug substance are considered API manufacturing; the formulation of the drug substance into finished product and the packaging of that product is considered finished drug product manufacture. This is consistent with regulatory expectations for these different categories of manufacturing, with cleaning validation requirements including predetermined acceptance criteria, which may differ for each type of manufacturing. Two separate cleaning validation master plans have been created: one for the API and one for the finished drug product.

CLEANING METHODS

Cleaning is performed to remove materials introduced into equipment during the manufacturing process. These materials may include media, buffers, storage solutions, cell debris, non-API-containing placebos, and any formulation or concentration of a given drug product or API. Manufacturing and cleaning equipment must be designed for effective and consistent cleaning. The cleaning of manufacturing equipment surfaces at Genentech uses automated, semi-automated, and manual cleaning processes. For larger, enclosed equipment, an automatic or semi-automatic clean-in-place (CIP) process is typically used. Cleaning process parameters include cleaning agent concentration, temperature, flow rates, volume, and time.

Equipment cleaning procedures use cleaning agents to aid removal of process soils. The cleaning agents may be categorized as caustic, acidic, neutral, or oxidizing. Some equipment at Genentech is cleaned with water for injection only, using no cleaning chemicals. A typical CIP process includes an initial water pre-rinse, a washing step with one or more cleaning agents, and a final rinse. Before conducting residue removal testing in cleaning validation, installation qualification and operational qualification are performed on the equipment to be cleaned and on the equipment used for the cleaning process. Manufacturing equipment is exposed to cleaning solutions by fully submerging the component (i.e., clean-out-of-place washer or manual cleaning methods); by fully flooding the product-contacting surfaces (i.e., transfer lines or manually cleaned tanks); or by directing fluids by use of spray devices such as spray balls, spray wands, and washer nozzles.

For equipment containing a spray device, qualifications include identifying and documenting the device, noting its proper orientation and alignment, and performing a spray coverage test to assure complete coverage. Spray device coverage verification testing for vessels that are cleaned in place is conducted according to an approved procedure. This procedure involves the use of a visual marker (e.g., riboflavin solution, which fluoresces under ultraviolet light) and spray devices. For glassware that is cleaned in washers, verification of coverage may be conducted with process soils, rather than riboflavin, if process soils are readily visible against translucent glass surfaces.

CLEANING VALIDATION STRATEGY

The cleaning processes for product-contact surfaces for all products manufactured in GMP equipment must be demonstrated to be effective. Product-contact surfaces are surfaces that make direct contact with product or materials introduced into equipment as part of the normal manufacturing process by their very design. Indirect-product-contact surfaces (such as buffer tanks), where there is a significant risk of residues on surfaces contaminating a subsequently manufactured product, also undergo cleaning validation.

To demonstrate the effectiveness of a cleaning process, the process is challenged. This challenge involves at least three consecutive successful cleaning process runs, after which residues are measured and results are compared to predetermined acceptance criteria.

Mock soiling is also used. Mock soiling refers to the soiling of equipment by a process other than routine manufacturing that creates a dirty equipment state equivalent to that following routine manufacturing. Mock soiling of equipment for validation purposes can be performed when equipment is not available for manufacturing soiling. Mock soiling procedures must be adequately described to simulate normal manufacturing processes.

Cleaning validation includes the establishment of dirty hold times and clean hold times. Dirty hold time is the amount of time between the end of the use of the equipment and the start of equipment cleaning. Clean hold time is the amount of time between the completion of the equipment cleaning and the next cycle of use. Cleaning processes are challenged for maximum dirty hold times during cleaning validation runs.

For clinical products, infrequently made products, or infrequently used equipment, a cleaning verification approach may be used in lieu of cleaning validation.

Single-use product-contact equipment (used once and then discarded) is excluded from cleaning validation. Single-use items include beakers, pipettes, weigh boats, silicone tubing, sample tubes, storage bags, and normal-flow filtration filters.

Product-dedicated refers to equipment that is used for a single product and then is removed as part of changeover procedures. Product-dedicated items, such as chromatography resins and tangential-flow filtration membranes, are used with one product only. The requirement for residues in dedicated equipment may differ from that for residues in equipment used for multiple products; nevertheless, the cleaning of product-contact surfaces of dedicated equipment requires cleaning validation. The validation of product-specific resin and membrane cleaning is captured in process validation protocols.

Multi-use equipment may be used to process one or more products or media components. At Genentech, the main emphasis of the equipment cleaning validation program is on multi-use equipment, because this equipment type has the highest risk of process contamination (run-to-run or product-to-product).

NEW PRODUCT INTRODUCTION

Before introducing a new product into equipment used for manufacturing a marketed product, a cleanability study is performed to determine the effectiveness of the cleaning process, using the new product on similar equipment surface types. The new product introduction (NPI) method has two purposes: to avoid cross-contamination of commercial products, and to collect development data on new products.

The cleanability study is divided into two parts: the laboratory-scale study, and the representative-scale runs. The cleanability study starts with an evaluation of the characteristics of the product and soiling at laboratory scale to determine the effectiveness of the rinse, swab, and visual inspection methods. Results of the laboratory-scale study are verified at representative scale. Representative-scale runs include three successful consecutive cleaning runs, conducted on equipment used for marketed products, which include sampling and analysis for residues, and comparison to predetermined acceptance criteria.

Laboratory-Scale Study
As part of sampling suitability testing, process residues of the new product are evaluated for recoverability by rinse and swab sampling methods. In this study (also known as recovering organic carbon by rinse and swab) total organic carbon (TOC) is analyzed to determine the sampling method that is appropriate for cleaning validation testing.

Soils from fermentation, initial purification, and final formulated bulk are used in this study. Testing is performed on each surface type (e.g., stainless steel and glass). Before spiking, soils are adequately mixed before use (by gentle inverting of the sample tube for fermentation soils or by vortexing for recovery soils). Coupons are prepared (cleaned and dried) and are spiked with protein soils at TOC concentrations similar to those in the cleaning validation acceptance criteria limit. The soiled coupons are dried for at least 24 hours, or for the specified dirty hold time, and are sampled using the rinse or the swab method. Positive and negative controls are generated, and swab and rinse water recoveries are calculated.

For highly soluble proteins, the average results of rinse and swab sampling recovery studies for fermentation, initial purification, and final bulk soils usually vary between 80% and 120%. If average recovery results for rinse or swab sampling methods is outside the acceptable range, an investigation is undertaken and a correction factor is applied for less-than-minimum recovery when reporting the equipment validation TOC results. WHO has set the following recovery levels: greater than 80% is good; greater than 50% is reasonable; and less than 50% is questionable.⁷ However, the key to recovery is consistency between samples, not just total recovery.

Representative-Scale Runs
The overall study challenges the ability of the standard cleaning procedure to remove the new product soil from representative equipment surfaces.

The cleaning process is challenged by including the maximum dirty equipment hold time.

The cleaning process may also be challenged by reducing one or more cleaning process variables—such as cleaning time, flow rate, or volume—during each run.

Sampling for residues includes rinse sampling, swab sampling, and visual inspection. An evaluation is performed to determine suitability of swab and rinse methods for validation sampling.

Results
The product residue acceptance criteria in a cleanability study are calculated using the same principles and calculations as for a validation protocol for equipment used to make marketed products. Acceptable results in the cleanability study allow a new product to be introduced and validated in equipment for marketed products; acceptable results also increase confidence in successful validation runs. Cleaning validation of the major multi-use product-contacting equipment is executed concurrent with manufacturing. Acceptable results in triplicate runs of a cleanability study in one facility constitute an acceptable basis for introducing the product into the same combination of equipment configurations and product-contact surface types in any other facility, after equivalence of equipment, cleaning methods, sampling, and acceptance criteria has been established. This equivalency should be documented in the validation protocol or in a technical report. Currently, data from full-scale cleaning validation and new product introduction methods are being generated at Genentech to determine worst-case situations and to justify reduced testing.

CONCLUSION

Cleaning validation is driven by regulatory requirements to ensure that residues from one product will not carry over and cross contaminate the next product. Appropriate design of cleaning equipment and cycle development increases success rate and reduces validation execution time. At Genentech, the cleaning program consists of equipment design and qualification, cleanability study, sampling evaluation, and meeting predetermined validation protocol acceptance criteria. Dirty and clean hold times are established during cleaning validation. Cleaning validation is supported by approved procedures and by training programs for personnel who perform the cleaning operations in the production areas and who collect validation samples. Part 2 will discuss implementation of the cleaning validation program—grouping strategy, various types of sampling and their acceptance criteria, training, change control, and revalidation.

ACKNOWLEDGEMENTS

The author is thankful to corporate quality management at Genentech, Inc., for support, and to Jenna Carlson and Ahmed Bassyouni for reviewing the manuscript and providing comments.

A. Hamid Mollah, PhD, is a senior technical manager for corporate quality and validation at Genentech, Inc., South San Francisco, CA, 650.467.1095, mollah.hamid@gene.com
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REFERENCES

1. US Food and Drug Administration. Guidance for industry. Current good manufacturing practice for finished pharmaceuticals. Rockville, MD; 2006 Apr.

2. European Commission, Enterprise Directorate General. EudraLex, vol.4, Medicinal products for human and veterinary use: Good manufacturing practice. Office for Official Publications of the European Communities: Luxembourg; 2007 Mar.

3. International Conference on Harmonization. Q7, Good manufacturing practice guide for active pharmaceutical ingredients. Geneva, Switzerland; 2000 Nov.

4. US Food and Drug Administration. Guide to inspections of validation of cleaning processes. Rockville, MD; 1993 July.

5. Pharmaceutical Inspection Convention and Pharmaceutical Inspection Co-operation Scheme. Validation master plan installation and operational qualification: Non-sterile process validation. Cleaning validation. Geneva, Switzerland; 2004 July.

6. Canada Health Products and Food Branch Inspectorate. Guidance Document. Cleaning validation guidelines: Drug and health products. Health Canada: Ottawa, Canada; 2002 Spring.

7. World Health Organization. Supplementary guidelines on good manufacturing practices: Validation. Geneva, Switzerland; 2005.

Cleaning validation, how clean is clean?

Pharmaceutical process equipment in operation.

Cleaning validation is a process to ensure that equipment cleaning procedures are removing residues to predetermined levels of acceptability. Although "equipment cleaning" is part of current Good Manufacturing Practice requirements the term "cleaning validation" was not popular until late 1980s. The need for a systematic approach to proving the effectiveness of all the cleaning procedures was achieved in 1993 with a revised Food and Drug Administration Inspection Guide on Cleaning Validation.

Pharmaceutical products and active pharmaceutical ingredients (APIs) can be contaminated by other pharmaceutical products, by cleaning agents, micro-organisms or by other material (e.g. air-borne particles, dust, lubricants). Further sources of contamination might be raw materials, intermediates, auxiliaries, etc.

In many cases, the same equipment may be used for processing different products. To avoid contamination of the following product, adequate cleaning procedures are essential.

High risk products, such as penicillins, are a major concern in the cleaning validation field. Sensitive sampling methods require development and must be applicable to each specific piece of equipment used. Due to the possibility of inter-product contamination, highly sensitive analytical methods such as Liquid Chromatography are required for trace level analysis.

These detection methods should be specific for the target analyte, sensitive for trace and ultra-trace analysis, and be sufficiently able to separate and quantify the target analyte from potential interference. Currently there are two sampling methods in use.

The direct method, incorporating a swabbing material, is favoured over the indirect rinsate analysis approach. However caution must be taken when choosing the correct swab, as factors such as recovery, background contribution and particle generation can hinder residue determinations. Therefore each should be evaluated independently and an overall correction factor applied to the swab. Swab recoveries may be determined using spiking studies incorporating coupons of equipment surfaces.

Rinsate analysis is a useful sampling tool for equipment such as blenders and reaction vessels. The theory is that by analysing an aliquot of rinse water, the total quantity of analyte residue can be estimated. This method however assumes that the residue is uniformly removed from the equipment and also presumes that if the rinsate is clean then the equipment is clean.

Probably the most important aspect of cleaning validation programmes is establishing predetermined levels of acceptability. In order to establish Acceptable Residue Limits (ARL), various product and equipment attributes are evaluated. This leads to wide variations in ARL values between different product trains and different manufacturing facilities. The most important aspect is therefore proving that the ARL values determined in the cleaning validation programme have been established using a sound scientific rationale.

Revisiting “Cleaning Verification”

This is a follow-up to my November 2009 Cleaning Memo on “continued process verification” and “continuous process verification”. You might notice that I am putting the term “cleaning verification” in quotes. The reason is that for cleaning validation purposes, “cleaning verification” has a very specific meaning. It is not just any verification that is done. For example, some people like to talk about a “revalidation” run (yearly, for example) on a cleaning process as a “verification” run. That may be appropriate (although I prefer the terminology “confirmatory” run), but it is not what I mean when I talk about “cleaning verification”. Other people like to talk about “verifying” the residues in a cleaning validation protocol. That also may be appropriate (although I prefer the terminology “measuring” the residues), but it is also not what I mean when I talk about “cleaning verification”.

So, what exactly is “cleaning verification”? I would define it something like this: “A one-time process for determining the effectiveness of a cleaning process for a specific cleaning event. “ In this sense, “cleaning verification” should be contrasted with “cleaning validation”. “Cleaning validation” is a process for determining the effectiveness and consistency of a cleaning process for defined products and equipment. If cleaning validation is considered in light of the new FDA Process Validation guidance, the validation process is (in one sense) never complete. You design the cleaning process, and then you qualify it (qualification involves what we used to call “validation runs”). Then you maintain the state of control through what the FDA refers to as “continued process verification” (not to be confused with “cleaning verification”). What I mean by cleaning validation never being complete is that the control measures for each batch following the qualification run(s) add to the supporting data for saying the cleaning process is validated.

While cleaning validation is never done, “cleaning verification” is a one-time activity. It may be repeated multiple times for an “equivalent” cleaning process, but here is what I mean by “one-time” activity. The data that is generated from a “cleaning verification” study is applicable only to that one cleaning event it is associated with. From a scientific perspective, data on one cleaning event may suggest that you will get similar data if you were to repeat that cleaning event in the future. However, from a compliance perspective, the data developed on one cleaning event does not apply to future identical cleaning events for which cleaning verification is to be done. For those of you wondering if three “cleaning verifications” on the same cleaning process constitutes “cleaning validation”, my answer is generally “No” (at least not without additional support evidence). This was discussed in my Cleaning Memo of August 2008. I believe it even more so following the redefinition of “process validation” in the November 2008 FDA Process Validation guidance.

So what is done in “cleaning verification”? Many of the same things that are done in cleaning validation protocols (or qualification protocols, if we want to adopt that terminology). You have a cleaning SOP; however, as compared to a cleaning SOP to be validated, there may not be a lot of design and development for the “cleaning verification” SOP. You establish limits for critical residues. However, in contrast to cleaning validation protocols, for “cleaning verification” protocols it is only necessary to consider the residues in light of the immediately manufactured next product. You need to have analytical methods and sampling methods. However, as compared to cleaning validation protocols, the analytical method may just be a “pass/fail” method, as opposed to a method which measures the exact amount of residue in the samples. Recovery studies are also required, but the recovery study for a pass/fail analytical method is slightly different.

In a “cleaning verification” protocol, there may be deviations in the cleaning process that occur. However, these may not be fatal to the “cleaning verification” exercise, because the key thing is whether acceptable residue data is obtained. Furthermore, if a “cleaning verification” protocol fails (that is, the residue limits are exceeded), then it is perfectly acceptable (from a compliance perspective) to clean again and tests for residues again. This process may not be desirable from a manufacturing efficiency perspective, but it is (or should be) allowed. This is one reason cleaning process SOPs associated with “cleaning verification” protocols may be “over-designed” (to the extent that they are “designed”), so that passing residue results are obtained the first time.

When can “cleaning verification” be used? It certainly can and should be used for any one-off cleaning situation, like a one-time manufacture of a clinical trial material or like cleaning after a deviation (such as a clean hold time that has been exceeded). It can also be used for any cleaning process which is done infrequently, such as cleaning after a batch manufactured only once a year.

For cleaning processes repeated frequently, it usually is an expectation that cleaning validation be done. There is an old rubric that says “If a process can be validated, it should be validated”. Now part of the reason for that is that validating a frequently repeated process usually makes good economic sense. However, there does not seem to be a good logical reason why “cleaning verification” couldn’t be done for every cleaning process that could be otherwise validated. After all, in process validation for the medical device industry, it is a well established practice that if a process cannot be 100% verified each time, then the process should be validated. The clear implication is that validation is not required if you do 100% verification. So it may be something to think about for pharmaceutical cleaning processes. I realize that this goes against “Quality by Design” principles. However, let’s not lose sight of our objective. Our objective is not to use QbD principles; our objective is to produce safe and effective products meeting quality specifications.

One last comment about “cleaning verification”. While “cleaning verification” is in contrast to cleaning validation, it still should be considered part of your cleaning validation program. In other words, it should be included in your cleaning validation master plan, cleaning validation policy, or cleaning validation quality standard (or whatever you call your high level cleaning effectiveness document). Under that high level document, you might have separate procedures for “cleaning validation” and “cleaning verification”. However, they should be tied together at the top. If you prefer to call “cleaning verification” something different in your documents, that certainly is okay provided that a clear definition is given. (I will point out that “cleaning verification” as I have described it is presented to the FDA Basic Drug School when I train that group.)

The purpose of this Cleaning Memo is to neither encourage nor discourage the utilization of “cleaning verification”. The purpose is to make sure we understand what it is, and how to utilize it appropriately.

What’s an “Equivalent” Swab?

In my training seminars (and in the recent webinar on swab sampling), I always caution against specifying a certain swab or an “equivalent”. My concern is with the “or equivalent” part. How is “equivalency” defined or determined? It is not prudent to use a certain swab from one manufacturer, and ask another manufacturer for its equivalent thinking that it will be an exact match. That second swab manufacturer may define “equivalent” as “for the same application”. For example, “this swab is equivalent because it is for the same purpose (such as for TOC swabbing)”. In such a case, are the size, shape and composition of the swab head (as well as the length and flexibility of the swab handle) the same such that performance and recoveries would be the same? For clarification, I’m not saying you can’t switch to a different swab. You can switch to a different swab, but then recovery studies should be repeated for that new swab and operator training should be required.

Ordinarily, I would not encourage a situation where you specify that either “Swab A” or “Swab B” could be used for the same residue. Certainly you can have one swab for microbial sampling and one swab for chemical residues. You might also need to have one swab for sampling with HPLC analysis and a different swab for sampling with TOC analysis. However, it is better for training purposes to only have one swab for chemical residues. Among other things, this prevents mix-ups in terms of the wrong swab being used (the “TOC swab” being used accidentally when you should be using the “HPLC swab”).

On the issue of “equivalency”, my eyes were opened even further based on some recent data obtained from a swab manufacturer. What I was really interested in was how much liquid was held by different swabs in a typical procedure where the swab is dipped in water and then the excess liquid “expressed” out by pushing it against the side of the vial. I was trying to get information that would help me understand the variability of how much liquid would be left on a surface after swabbing. The data that I saw surprised me in a different way.

What the company provided me was two sets of data using different test conditions. One condition involved dipping the swab in water for 60 seconds and then holding it out of the water for 60 seconds to allow any excess water to drip off. The second condition involved doing the same thing, except then expressing excess liquid by pressing the swab against the side of the vial.

For perspective, these were swabs which are typically used for cleaning validation, and included knit polyester and non-woven polyester, different sized swab heads, and different laundering practices. The latter refers to the fact that many swabs used for TOC analysis involve an additional laundering step to lower the “background” TOC. Also, there were no cellulosics or polyester/cellulosic blends evaluated.

Here, in a nutshell, is what the data showed. First, for most of the six swab types, there was very little difference between the amounts held by the swab with a 60-second drain time as compared to the situation where the swab head was expressed against the vial sidewall. This was not expected (at least by me). To put this in perspective, the data might be different if the water soak time was an hour before the drain step (but this is just my speculation). In other words, the rate of picking up water was no considered in this evaluation.

What was even more unexpected was the difference between what were essentially the same swabs except for the laundering step. I had expected that the amounts absorbed would increase with laundering. This was based on what I knew about cotton fabrics, that you could launder cotton to remove the natural waxes so as to make it absorb better. Data trumps theory, and the data indicated that these laundered swabs absorbed only 20% of the amount absorbed by the similar unlaundered swabs. For clarification, that is not 20% less than the amount absorbed by the unlaundered swabs; it is 20% of the amount absorbed by the unlaundered swab. This held true for two sets of swabs, one set with a relatively large swab head and one set with a relatively small swab head.

How could this be? My speculation (based on my involvement in the textile industry a long time ago) was that the unlaundered polyester had surfactants on it which were removed by the laundering process. The surfactants were part of the polyester manufacturing process (perhaps during knitting). For “TOC swabs”, the polyester is laundered to reduce the TOC value due to extractables. Was it possible that the presence of those surfactants allowed for a greater wetting, and hence greater absorbency, for the unlaundered swabs? When I suggested that explanation to the swab manufacturer, they seemed to agree that it was a reasonable explanation.

Let me also clarify that some people refer to mechanisms for holding water differently. They use absorbance for cellulosics (where the liquid is held inside the fiber) and adsorbance for polyester (where the liquid is held between the fiber bundles. For simplicity, I am referring to what is picked up by either mechanism as “absorbance.”

This limited range of these swabs should be considered when evaluating these finding. However, the surprising results (or at least what I considered surprising results) of this limited range suggest that care in specifying materials of construction of the swab is critical. And even more care needs to be used in selecting an “equivalent’ swab. This data is also only with water, and data for other solvents may be different. However, what this suggests is the need to carefully qualify swabs, as well as the need for swab manufacturers to consider adding a specification relating to “absorbance” for swabs. It also suggests that the practice of pressing the swab head against the vial to express excess liquid may not be significant.

This involves experiments that are fairly easy to perform. If anyone want to try this with their swabs (measuring absorbance under different conditions), and then wants to share the data, please send the information to me. I will try to summarize the data and share it for all to review.

Let me clarify that the point of this Cleaning Memo is not to specify certain practices. Rather it is to remind us all that for a very simple procedure, which some people like to call a “precise” sampling procedure, perhaps we still have more to learn.

Next month’s Cleaning Memo: Revisiting Cleaning Verification

© 2010, Cleaning Validation Technologies

Pharmaceutical Cleaning Validation Method References for Alconox, Inc. Detergents

A cleaning validation involves testing for acceptable residues on pharmaceutical manufacturing or medical device surfaces. The validation involves residue identification, residue detection method selection, sampling method selection, setting residue acceptance criteria, methods validation and recovery studies, and finally writing a procedure and training operators. This procedure is used to document acceptable residues 3 or more times and then a rational monitoring program to maintain a validated state is put in place. If you are changing any part of your procedure or cleaner, first clean the new way, collect data and then clean the old way before using any equipment while you are in the process of validating the new procedure.

Residue identification - in a pharmaceutical manufacturing environment involves; the cleaner, primary ingredients, excipients, decomposition products, and preservatives. This document is intended to help with the cleaner residue identification.

Residue detection method selection - for cleaners can involve specific methods for specific cleaner ingredients such as; high performance liquid chromatography (HPLC), ion selective electrodes, flame photometry, derivative UV spectroscopy, enzymatic detection and titration, or it can involve non-specific methods that detect the presence of a blend of ingredients such as: total organic carbon, pH, and conductivity. The FDA prefers specific methods, but will accept non-specific methods with adequate rationales for their use. For investigations of failures or action levels, a specific method is usually preferable. The later section of this document lists references to several methods for each cleaner brand.

Sampling method selection - for cleaners involves choosing between rinse water sampling, swabbing surfaces, coupon sampling, or placebo sampling. Rinse water sampling involves taking a sample of an equilibrated post-final rinse that has been recirculated over all surfaces. Rinse samples should be correlated to a direct measuring technique such as swabbing. Swabbing involves using wipe or swab that is moistened with high purity water (WFI) that is typically wiped over a defined area in a systematic multi-pass way always going from clean to dirty areas to avoid recontamination - ie. 10 side by side strokes vertically, 10 horizontally and 10 each with the flip side of the swab in each diagonal direction. For TOC analysis very clean low background swabs or wipes and sample vials such should be used. The Texwipe large Alpha Swab 714A or 761 have been used, these are available in kits with clean sample containers. Quartz glass fiber filter papers have been used successfully. Coupon sampling involves the use of a coupons or an actual removable piece of pipe that is dipped into high purity water to extract residues for analysis. Placebo testing involves using placebo product and analyzing for residues from the previous batch.

Setting residue acceptance criteria - in pharmaceutical and medical device manufacturing requires setting residue acceptance levels for potential residues such as the active drug, excipients, degradation products, cleaning agents, bioburden and endotoxins. These levels are determined based on potential pharmacological, safety, toxicity, stability, and contamination effects on the next product using that surface or equipment. Limits are typically set for visual, chemical, and microbiological residues.

The cleaning agent limits are generally covered under chemical criteria. Chemical limits can be expressed as a maximum concentration in the next product (ug/ml), amount per surface area (ug/cm2), amount in a swab sample (ug or ug/ml), maximum carryover in a train (mg or g), or concentration in equilabrated rinse water (ug/ml). You should have a calculated safety based acceptance limit, and you can have a lower internal action level, and a lower process control level based on actual manufacturing and measuring experience.

Cleaning agent safety based limits are typically calculated from a safety factor of an acceptable daily intake (ADI), a (1/1000 or more) reduction of an LD50 preferably by the same route of administration, or reproductive hazard levels. If the calculated limit is found to be higher than a less than 10 ppm carryover to the next batch, then the limit can be set to the more stringent 10 ppm carryover level for the safety based limit.

Calculated safety based limit in mg/cm2 or mg/ml of cleaner residue on a just cleaned equipment:

Limit (mg/cm2 or L) =	ADI carryover - see below (mg) X Smallest Next Batch (kg) Size of Shared Equipment (cm2 or L) X Biggest Daily Dose or of Next Batch (kg)
ADI carryover (mg) =	LD50 by administration route (mg/kg) X body weight (kg) X (1/ 10,000 or 1/1000*)
Comparison calculation of limit based on no more than 10 ppm carryover:
Limit (mg/cm2) =	10 mg residue on just cleaned surface X Next Batch Size(kg or L) 1 kg of L of next product X Size (cm2 or L) shared equipment
* conversion factor used to convert LD50 to acceptable daily intake, use higher number for low LD50s

Note that for many residues you can validate a visual detection limit on the order of 1-4 ug/cm2. It is possible that the visually clean criteria will be the most stringent criteria.

For example with a cleaner that has an rat oral LD50 of over 5 g/kg, the ADI calculation using a70 kg person and a safety factor of 1000 gives a result of 350mg (5 g/kg X 70 kg / 1000 ). The calculated residual acceptance limit for a 2000 kg mixer and line where there might be a next smallest batch of 1000 kg, and the area of the mixer and filling equipment which is all used in the next batch is 100,000 cm2 and the daily dose of the next product is 0.005 kg results in a calculated residual acceptance criteria of 700 mg/cm2 (350 mg X1000 kg/(100,000 cm2 X 0.005 kg). By comparison, the 10 ppm in next batch limit gives an acceptance criteria of 100 ug /cm2 (10 mg X 1000 kg/(1 kg X 100,000 cm2) X 1000ug/mg. In this case, it is likely that you will be able to show that you can visually detect down to 4 ug/cm2 and since you need to have a visually clean surface, your most stringent acceptance criteria will be the visual limit.

Note that in this example you are trying to avoid getting more than 350 mg of residue in a daily dose of the next product. In the case of small final filling equipment such as filling needles for vials or tablet punches and dies, you might need to do separate residue studies on the filling needles or punches to be sure that there was not enough residue just on that equipment to contaminate the first few bottles or tablets of the next batch with a residue of 350 mg/daily dose.

If the safety based limit in this example is set at 100 ug/cm2. Then this limit can be expressed as a rinse water concentration of 100 mg/L in a post final rinse using 100 L of recirculated to equilibrium rinse water (0.1 mg/cm2 X 100,000 cm2/100 L). This same limit could be expressed as 6.25 ug/ml or ppm total organic carbon (TOC) in a sample for a residue that is 10% TOC by weight in a 20 ml swab sample from a 25 cm2 swab area where 50% recovery has been established ((25 cm2 X 100 ug/cm2) X 50% recovery) X 10% TOC/20 ml. The same safety limit can be expressed several different ways.

The methods validation and recovery study - is the use of the sampling and detection method on known spiked surfaces at representative levels, typically spiked at 50%, 100% and 150% of the acceptable limit and at lower expected actual levels to show linearity with documented % recovery as analyzed and to determine the limit of detection and limit of quantitation. Ideally the expected values and limits should be multiples of the limits of quantitation. The % recovery is used to correlate amount detected with amount assumed to be on the surface as an acceptable residue. This is a good time to consider wipe or rinse sample storage conditions and time limits to get the sample analyzed. Rinseability profiles showing the complete rinsing of the individual detergent ingredients should be undertaken if the solubility of any detergent ingredients or the rinseability after drying is in doubt. In some cases bioburden/endotoxin levels may need to be validated. It is recommended that this process be done separately from the cleaning process so that the cleaning validation can be completed while the lengthier bioburden/endotoxin evaluation is done.

The written procedure and training of operators - involves writing out assigned responsibilities, protective clothing needs, equipment disassembly needs, monitoring procedures, documentation needs, labeling of in process and cleaned equipment with cleaning expiration date, post cleaning inspection procedures, storage conditions, and inspection required before next use. The operators then need to be trained and certified in the procedures.

Directory of cleaner residue detection methods for each Alconox detergent:

ALCONOX® :	IV A, C, D, F, and G
LIQUI-NOX® :	IV A, B, C, F, G and I
TERG-A-ZYME® :	IV A, C, D, E, F and G
ALCOJET® :	IV D, F and G
ALCOTABS® :	IV A, C, F and G
DET-O-JET® :	IV D, F, G and I
DETERGENT 8® :	IV F and J
CITRANOX® :	IV A, B, C, D, G and H
LUMINOX® :	IV F and J
CITRAJET® :	IV F and H

A. Anionic surfactant analysis methods for ALCONOX^®, LIQUI-NOX^®, TERG-A-ZYME^®, ALCOTABS^®, and CITRANOX^®. Note that the anionic surfactant is present at approximately 20% by weight in each of these detergents, except Citranox it is present at 3-5%.

1. Chemetrics Inc. water testing kit for anionic detergents, which is sensitive to 1/4 ppm. Contact Chemetrics, Inc. at 1-800-356-3072, or +540-788-9026.
2. LaMotte Chemical water testing kit for anionic detergents, which is sensitive to 1 ppm. Contact LaMotte Chemical at 1-800-344-3100, or +410-778-3100
3. Hach Company water testing method for anionic detergents, which is sensitive to 1 ppm. Contact Hach Company at 1-800-227-4224 or 303-669-3050.
4. A gradient HPLC method in "Journal of Chromatography," 302, (1984) 65-78 by Bear, Lawley and Riddle, Separation of Sulfonate and Carboxylate mixtures by ion exchange HPLC.
5. A "Synthetic Anionic Ingredient by Cationic Titration" method from ASTM D 3049-75 (reapproved 1962) which has been reported to us as having a detection limit on the order of 10 ppm using normalities of 0.004 N Hyamine. It has been suggested that using lower normality Hyamine would give lower detection limits.

B. Nonionic surfactant analysis - the detectable levels are LIQUI-NOX contains roughly 3-7% and CITRANOX contains roughly 1-5% detectable nonionic.

1. An isocratic HPLC method: Schifflet, Shapiro, Levin, and DeNisco "Validation of a Reversed-Phase HPLC Method to Determine Residual Nonoxynol-9 on Pharmaceutical Process Equipment Using a 1.5 µ Nonporous Silica (NPS) Column" to be published circa March 2000 in LC/GC.

C. Direct UV/Visible determination:

1. Direct UV/Visible determination by making a broad-spectrum scan of the detergent to determine a maximum absorbed wavelength. Make standard dilutions of the detergent you wish to analyze for, using 1ppm, 2ppm, 4ppm, 8ppm and 16ppm dilutions. Then measure their absorbence at the maximum wavelength to derive a standard curve against which you analyze the unknown sample from the rinse water or the wipe extract to determine if there is any residue. It has been reported to us that LIQUI-NOX^® has a maximum absorbence at 196-197 nm with a secondary maxima at 225-226 nm and that TERG-A-ZYME^® has a maximum absorbence at 192-193 nm. The reported detection limits were 1-2 ppm. The other detergents, ALCONOX^®, ALCOTABS^®, and CITRANOX^® should be detectable at 196-197 nm and 225-226 nm secondary wavelength.

D. Phosphate detection methods for the complex polyphosphates present in ALCONOX^®, ALCOJET^®, TERG-A-ZYME^®, DET-O-JET^® and ALCOTABS^®. Note that the content of phosphate expressed as %P is printed on the containers of the detergent. Note that these methods test for ortho-phosphate. The polyphosphates present in the detergents are acid hydrolyzable to ortho-phosphate by adding 10% of the sample volume amount of 5 N sulfuric acid and boiling gently for 30 min.

1. American Waterworks Association vol. 57 p. 917-926, 1965 by Edwards, Molof and Schneeman, Determination of Orthophosphate in Fresh and Saline Waters.
2. Hach Company phosphate analysis methods and kits. Call Hach Company at 1-800-227-4224 or 303-669-3050.

E. Protease enzyme detection method for TERG-A-ZYME^® detergent:

1. "Assay in Enzymatic Processing of Food Proteins: II. Method for Detection of Residual Proteolytic Activity" IB number 195a-GB April 1979 from Novozyme, contact them at Tel: 919-494-3000 or www.novozymes.com.

F. Total Organic Carbon (TOC) analysis has been reported to detect the organic surfactants present in ALCONOX^®(11% w/w), LIQUI-NOX^®(21% w/w), (TERG-A-ZYME® 11% w/w), ALCOJET^®(1.5% w/w), ALCOTABS^®(20% w/w), DETERGENT 8^®(38% w/w), LUMINOXtm(26% w/w) CITRANOX^®(17% w/w) and CITRAJET^® (14% w/w). You must go through the acid neutralization step or use the inorganic carbon channel on the TOC analyzer to account for inorganic carbon.

G. When rinsing with deionized water, it has been reported that conductivity has been used to detect conductive salts present in ALCONOX^®, LIQUI-NOX^®, TERG-A-ZYME^®, ALCOJET^®, ALCOTABS^®, DET-O-JET^®, and CITRANOX^®. Standard solutions of known dilution should be made up to determine the detection limits using your equipment. These limits should be reviewed to see if they are suitable for your requirements.

H. CITRANOX and CITRAJET can be detected by Citric Acid analysis both contain around 15% Citric Acid.

1. HPLC using Bio-Rad HPX-87H column, Bio-Rad Cation H Refill pre-column, 0.01 M H2S04 mobile phase, degas, 52 deg C column, 0.6 ml/min flow, 20 microliter sample loop, Waters Model 401 Refractometer detection.
2. Enzymatic detection - Taraborelli and Upton, "Enzymatic Determination of Citrate In Detergent Products" JAOCS Vol. 52, 1975 (248-251).
3. By derivatization and spectroscopy - Hartford, "Rapid spectrophotometric method for the determination of itaconic, citric aconitic and fumaric acids." Analytical Chemistry, Vol 34, No 3 1962 (426-428).

I. Ion selective electrode or flame photometry to detect potassium in Det-o-jet (appprox 13% by wt) - Standard Methods For the Examination of Water and Wastewater 20th Ed. Section 3-87.

J. Propylene glycol ether detection by GC - DETERGENT 8 and LUMINOX contains roughly 25% by weight dipropylene glycol methyl ether detectable using the the Dow Chemical analytical method DOWM-100765-ME90A June 25, 1990, contact Dow Quality/Methods at 517-636-5602.

This information is presented to help communicate our understanding of how cleaning validation has been carried out in pharmaceutical and medical device processing. The information given here is made without any representation or warrantee, as it is presented for your own investigation and verification. Request a technical bulletin for a chemical description of the ingredients in each Alconox, Inc. detergent.

To speak to a technical representative about cleaning validation, call 914-948-4040 for Malcolm McLaughlin (x160) mmclaughlin@alconox.com .

References:

1. Brewer, Rebecca Designing and Documenting Your Cleaning Validation Program to Meet FDA Requirements, Washington Group International , Philadelphia. presented at Cleaning Validation and Cleaning Processes Feb 1-2 Philadelphia, PA (2001)
2. FDA "Guide to Inspection of Cleaning Validation" (1993)
3. FDA "Guide to Inspection of Bulk Pharmaceuticals Chemicals" (1991)
4. FDA "Biotechnology Inspection Guide" (1991)
5. 21 CFR 211 and Proposed Revisions
6. Fourman and Mullen, "Determining Cleaning Validation Acceptance Limits for Pharmaceutical Manufacturing" Pharm Technol. 17 (4), 54-60 (1993)
7. Leblanc,"Establishing Scientifically Justified Acceptance Criteria for Cleaning Validation of Finished Drug Products," Pharm Technol 22 (10), 136-148 (1998)
8. Cooper, "Using Swabs for Cleaning Validation: A Review" Cleaning Validation , IVT, p 74-89 (1996)