The Validation Process

Overview

Introduction

This chapter looks at the validation process. Note: This validation process is used to validate a specific computer system. It may be done on an existing computer system or on a new computer system.

Purpose

The purpose of the validation process is to provide a high degree of assurance that a specific process (or in this case computer system) will consistently produce a product (control information or data) which meets predetermined specifications and quality attributes.

The Validation Facets

The validation effort consists of 5 specific facets or processes, each alone, would not constitute a validation.  However, depending on the specifics of the application, system or process, would depend on which facets would be required.  There following facets are: ·  The Validation Master Plan (VMP) ·  The Project Plan ·  Installation Qualification (IQ) ·  Operational Qualification (OQ) ·  Performance or Process Qualification (PQ)

Types of validation

The two types of validation are: ·         Prospective validation: the validation of a new system as it is developed ·         Retrospective validation: the validation of an existing system

Validation process		The validation process and document references are shown below:
Step	Action
1	Establish Team(s)
2	Determine Validation Activities
3	Write the Validation Protocol
4	Specify the System Development Details
5	Perform Qualification Activities
6	Develop/Review Controls and Procedures
7	Certify the System
8	Review Periodically

Steps 1 to 8

Introduction

This topic provides an overview of the validation process.

Step 1: Establish team(s)

The first step in the validation process is to establish the System Validation Team and if required the System Validation Steering Team. These are the teams that will be responsible for the validation process.

Step 2: Determine validation activities

The second step in the validation process is to determine and record all of the validation activities that will be undertaken in order to validate the computer system. The validation activities are the exact details or activities that will be required for each of the steps in the validation process. The output from this activity will be the Validation Plan. Example: At step six of the validation process (Develop/Review Controls and Procedures) the exact controls and procedures that will be required to keep the computer system validated will be determined and recorded. Note: The type and number of validation activities will depend on the nature of the computer system that is being validated.

Step 3: Write the Validation Protocol

The third step in the validation process is to write the Validation Protocol. The Validation Protocol describes the procedure and the steps within the procedure that will be followed in order to validate the system. The Validation Protocol must also provide a high level description of the overall philosophy, intention and approach.

Step 4: Specify the system development details

The fourth step in the validation process is to specify the system development details. You should specify to the supplier or developer of a system that they must have: ·         a good methodology in order to develop a system ·         a formal quality management system for the development, supply and maintenance of the system You may need to specify to the supplier or developer the types of items you want to see - this could be done in the form of a Quality Plan. These items will help you ensure that the supplier or developer has a good methodology and formal quality management system in place. Examples: Items that will help you ensure a good methodology and formal quality management system include: ·         quality management procedures ·         life cycle definition ·         specifications, for example user requirements specification and functional specification ·         documentation controls and various items of documentation, for example user manuals and administrator documentation ·         testing procedures If the computer system is a new one, then the system development requirements will be identified prior to system selection/development. If the computer system is an existing one, then the system development requirements will still be identified and used as a basis against which to evaluate the system.

Step 5: Perform qualification activities

The fifth step of the validation process is to perform the qualification activities, which are comprised within the validation process. Some examples of these qualification activities include: ·         Supplier audit ·         Specification qualification ·         Design qualification ·         Installation qualification ·         Operational qualification ·        Performance qualification

Step 6: Develop / review controls and procedures

The sixth step of the validation process is to develop/review controls and procedures. If the computer system is a new one, then you will need to develop the controls and procedures, or check the suitability of existing generic procedures applicable to the site or department. If the computer system is an existing one, then you will need to review the controls and procedures and update them if required.

Step 7: Certify the system

The seventh step of the validation process is to certify the system. This step is where you certify that the validation deliverables have met the acceptance criteria that were described in the Validation Protocol. When you certify the system you should prepare a validation report. The validation report should outline the details of the validation process. Examples of details that should be outlined include: ·         what was done and the results that were obtained ·         any special considerations ·         whether the validation procedure (as described in the Validation Protocol) was followed ·         a summary of all documentation that was generated ·         the location of the validation documentation ·         the retention period for the documentation

Step 8: Review periodically

The eighth and final step of the validation process is to review the system validation periodically. The system should be reviewed periodically to provide additional assurance of validation. There should be documentation outlining the details of how the review is to be done and what the review should cover. The end result of a review should be a summary of the review and a recommendation as to what to do next.

Timing and Documentation

Introduction

This topic looks at the timing of the validation process and documentation.

Timing

Ideally, the validation process begins at the inception of the system selection or design process. It then proceeds alongside the system development and is completed prior to implementation of the system. Many aspects of computer systems validation are just "Good Informational Resources (IR) Practice" and as such should occur anyway during the implementation of a system. For many reasons, a system may not have been validated until after it has been in use for some time. The basic validation process is the same as for a new system. The timing of some of the validation activities may, however, differ. Note: Retrospective validation is becoming increasingly unacceptable to regulatory inspectors. New systems should be validated before use.

Timing for a new system

The steps in the validation process, and their associated validation activities are performed in parallel with the system development life cycle and reference the development documentation as it is produced.

Timing for an existing system

For existing systems, the validation activities will still follow the development life cycle but will reference the development documentation retrospectively.

Example

An example of the parallel between system development and validation activities is shown below. * Functional Specification can comprise mechanical, electrical and software functional specification for systems embedded in equipment ** Systems embedded in equipment with significant control and monitoring instrumentation *** Testing carried out by supplier can form part of subsequent qualification activities if adequately controlled. This can help reduce the amount of testing needed later, particularly at operational qualification.

 

Documentation

Every step in the validation process, and the activities within the steps, requires documented evidence that the steps or activities have been completed. The table below shows the documents that must be generated at each step. Note: In some cases some of these documents may not be required.

Step	Action	Documents Generated
1	Establish Validation Team(s)	·         Team Charter ·         Terms of Reference ·         Role Definition ·         Team Organization Chart
2	Determine Validation Activities	·         Validation Plan
3	Write the Validation Protocol	·         Validation Protocol
4	Specify the System Development Details	·         Systems Development Life Cycle documentation
5	Perform qualification activities	·         Supplier Audit Report ·         In-house Audit Report ·         Source Code Review Report ·         Specification Qualification Report ·         Design Qualification Report ·         Installation Qualification (IQ) Protocol ·         IQ Results ·         IQ Summary Report ·         Operational Qualification (OQ) Protocol ·         OQ Results ·         OQ Summary Report ·         Performance Qualification (PQ) Protocol ·         PQ Results ·         PQ Summary Report
6	Develop/Review Controls and Procedures	·         SOPs (Standard Operating Procedures) ·         Training procedures ·         Training records
7	Certify the System	·         Validation Report ·         Validation Certification
8	Review the System Validation Periodically	·         Periodic Review Procedure ·         Periodic Review Audit Report

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Outsourcing Critical Cleaning

Considerations for custom cleaning and packaging services

By Michael Lehtinen

The pharmaceutical industry is constantly reviewing strategies to improve profitability and process efficiency. Many technological advances have been — and continue to be — implemented to help facilitate this. However, outsourcing is an area, particularly in the current financial climate, that is providing value across the board, from R&D through to manufacturing. Regulatory compliance is influential in this shift; it is essential for risk prevention, yet can be time-consuming, dragging on internal resources and potentially impacting production run-time. Critical cleaning is one area that faces such challenges and is often under review.

Photo courtesy of EP Scientific, part of Thermo Fisher Scientific

Any pharma/biopharma company utilizing critical working environments needs to ensure that all equipment is fully validated and, where appropriate, certified ‘clean.’ This is the case for both the upstream R&D laboratory and the downstream manufacturing facility. In the manufacturing setting, depyrogenation and sterilization are a matter of paramount importance to production quality; maintaining a clean environment and utilizing certified clean vials and containers ensures the ultimate provision of microbiologically safe and uniform parenteral pharmaceuticals.

Beyond patient safety, remaining compliant to the stringent regulatory requirements is also of significant financial benefit to a pharma company in both the long and short term. A re-run of production due to contamination — or worse, a withdrawal of a product batch from the market — has massive cost implications in terms of time, materials, production and reputation. In order to gain assurance that the complex regulations associated with cleaning are consistently met and that containers for critical environment applications are processed and certified to exacting specifications, companies can outsource this time-consuming task.

What Does Critical Cleaning of Containers Involve?

Although sample tubes, containers and associated closures are not necessarily high value products, what they contain is. Consequently, ensuring these are as clean as the processes and environments in which they are used requires the understanding and adoption of proven methods to guarantee essential GMP quality assurance and traceability. An in-depth appreciation of the applications for which the containers will be used is, therefore, necessary to ensure that the best cleaning process is implemented, while keeping costs to a minimum. There are a variety of container-cleaning methodologies available, all designed to meet different cleaning requirements these include: surface modification, via silanization or siliconization, depyrogenation, sterilization and low particulate cleaning.

Following cleaning, every batch of containers must be tested and certified for cleaning validation purposes. There are a large number of potential analytical parameters necessary to enable certification. These will depend on the potential application of the container, but can include GC, GC/MS, ICP/MS, AA, and GF-AA, as well as other testing techniques such as those for endotoxin and sterility testing. Finally, to meet traceability requirements, all raw data and benchwork must be retained and samples from every lot tested should ideally be archived for 10 years.

Knowledge of appropriate safety and regulatory requirements is also essential, not only for current regulations, but also how they may change in the future. The pharmaceutical industry requires a wide selection of chemicals and processes to achieve better contamination control at lower costs. However, health and environmental concerns have resulted in restrictions on the use of certain chemicals and processes. To foresee future trends, an understanding of how the approaches of regulatory agencies may change over time is advantageous.

Why Outsource It?

It is evident that cleaning newly manufactured drug product containers to appropriate specifications is a costly process in terms of time, resources and in-house expertise. Consequently, this task has increasingly been outsourced during the last three to five years. Since outsourcing companies have the expertise to ensure that all containers are compliant with various regulations, such as those from the FDA and U.S. Pharmacopeia (USP), in-house expertise is no longer essential. Costs can, therefore, be saved on training and laboratory researchers are freed to focus on product development.

Furthermore, many pharmaceutical laboratories require a variety of different batch sizes depending on the stage in the process, ranging from discovery through to scale-up. For example, a new drug still in the discovery stages or clinical development may only require a few dozen containers to package the product under development. However, even though such experiments are only being performed on a small scale in the laboratory, the protocols adopted should be similar to those used in final production to ensure that results are not compromised by laboratory grade containers. As data is accumulated the number of experiments and tests increases, driving the need to a few hundred containers. However, these volumes are still very small when compared to the equipment used to process the final product. The process of loading and setting up a high speed line, which is likely to be installed in a large pharmaceutical company, is far greater than that of actually running the product through the process. That is, if the setup time on the processing equipment is one hour and the machine is capable of producing 10,000 vials per hour, the inefficiency is evident if only a few hundred containers are required. The ability to outsource the supply of flexible quantities is invaluable. This ensures the provision of smaller quantities of product until the need for the item reaches a point of efficiency, and the production of final product on large scale equipment, which may exceed 100,000 vials per day, can move “in house”.

Outsourcing of custom cleaning and packaging, therefore, has some clear advantages for the pharmaceutical industry:

• Access to critical cleaning expertise
• Production of containers to exacting regulatory specifications is not a distraction from the company’s product expertise
• Economy of scale
• Accommodation of flexible demands and customization for new applications
• Testing, full documentation and ‘cradle to grave’ traceability

Assessing Your Needs

Each of the above is a sufficient reason for looking to outsource critical cleaning of drug product containers and packaging, but how should the appropriate supplier be chosen? There is no single answer. However, one good starting point would be reduction in risk, both in terms of safety and financially. The various cleaning options should then be weighted according to their influence on risk to the whole business. Requirements to be considered may include the following:

How ‘clean’ is your process?

USP describes limits and methods of analysis for a variety of contaminants. The key regulations relating to the cleaning of containers are: USP 788 for particulate matter in injections, USP 643 regulating Total Organic Carbon (TOC) as an indirect measure of organic molecules, USP 85 relating to acceptable levels of bacterial endotoxins, and USP 71 regarding sterility testing. Will the cleaning processes available ensure that all critical cleaning requirements are met, or even exceeded, cost effectively?

What cleaning methodologies do I need?

Table 1 provides a breakdown of the types of cleaning options available for differing applications.

Table 1: Types of cleaning options available for different cleanroom process applications

Advice and assistance required?

Many API (Active Pharmaceutical Ingredient) manufacturing companies are comfortable with meeting regulatory requirements and will simply outsource for purposes of flexibility of volume. However, there may be companies that are required to supply an intermediate pharmaceutical compound. Although the product itself is not pharmaceutical grade, it is required to meet the expectations of the API manufacturer. This means that the product can be manufactured in an industrial environment, but should have final packaging that meets the strict requirements set by the API manufacturer. These may be guidelines on particulate matter or trace compounds. This is where an expert custom cleaning company can assist by providing the correct level of cleaning to match the customer’s price with their needs.

Help! I need greater throughput now!

The following example demonstrates how this can be rapidly addressed. A contract pharmaceutical manufacturer that fills a product, such as the influenza vaccine, has an established production line capable of meeting a desired throughput. When the swine flu pandemic struck in the fall of 2009, the demand for flu vaccines dramatically increased. While the production of the liquid vaccine was scaled up to meet the demand, the ability of the equipment to process the containers was insufficient. Outsourcing container processing was an opportunity to supplement the supply of the packaging to ensure adequate quantities could be produced to rapidly meet the temporary peak in demand.

I need a customized product

The development of a new application may result in the need for a new and innovative solution, particularly due to the growing need for higher standards of purity demanded by today’s ultra-sensitive analytical instrumentation. Frequently, pharma and biopharma customers require expertise in resolving the difficult challenge of sourcing containers as clean as the facilities in which they will be used. Here the key questions to be asked are:

• Will you send your own materials or do you require them to be supplied?
• What cleaning process(es) do you need?
• What packaging configuration is required? – e.g. Cleanroom bag, autoclave bag, foil wrap, etc.
• What are your certification requirements? – e.g. Certificate of Analysis, Certificate of Processing, selected testing by USP methods, Certificate of Sterility, etc.

Finding the best supplier for you

Once you have decided upon your key requirements, you now need to decide on the best supplier to service these. It is probable that the basic requirements for supply of a clean container can be met by any of several companies in the industry. However, confidence in the quality of containers supplied for use within critical areas is key, along with the ability to understand, advise on and meet very specific needs.

Figure 1: Flow diagram demonstrating how utilizing external resources for custom cleaning and packaging services frees up valuable internal resources

Trust & reputation

Evidence of experience and continuous performance in meeting critical cleaning regulations should be a crucial element in the decision making process. Furthermore, a company should be able to demonstrate that it has never had any instances of product that has caused illness. A good reputation must be earned by building up trust with customers, and an open-door audit policy is an excellent way of developing this, whilst also ensuring that manufacturing companies can satisfy regulatory needs without compromise.

GMP quality compliance

Outsourcing to a reputable company can significantly decrease the amount of in-house compliance work that need be undertaken. If the outsourced company can offer ‘cradle to grave’ traceability through batch record tracking, this ensures that, should the FDA have any questions at any time, the necessary paperwork will always be available to provide an immediate answer. In addition, manufacturing processes and cleanroom facilities that ensure products are handled in Class 100/10 environments can only further secure product quality.

Flexibility

The ability to provide varying quantities of customized end products through processing, packaging and certification to meet the exact requirements of a customer is highly advantageous, particularly if the customer has its own specific containers that need custom cleaning. Furthermore, a supplier that can interact at the customer’s convenience is also an ideal scenario. For example, the ability to visit a customer’s facility and undertake all protocols and validation on its behalf could be seen as ideal.

Return on investment

Selecting a contract cleaning services company that can provide flexibility, confidence and efficiency is no mean feat and there are many considerations to take into account. We have looked at the required expertise, capabilities and services, however, it is ultimately the bottom line — and potential for savings that can be demonstrated by such a company — that is often the critical factor in the decision making process. Figure 1 highlights where outsourcing an expert critical cleaning company can certainly deliver return on investment (ROI). Indeed, value is added since most discovered compounds do not make it to commercialization. This is because internal resources only have an ROI if the product is a success; the truth is that fewer than one in 10,000 are successful.

Validating Sterile Filtration: Overcome the Fear of Failure 4

“I need an absolute 0.1- or 0.2-µm-rated filter.”
 
 As a former FDA authority, since retired, once observed, “The word ‘absolute’ should be used only in conjunction with vodka.” Absoluteness implies a complete independence from conditions, an inherent ability to retain particles larger that than the filter’s pore size rating, regardless of any other considerations. Without a complete knowledge of the properties of the particles and filter pores at our disposal, the statement is devoid of technical significance or guidance. It may, perhaps, be used in ignorance (although cynics may suspect that its utility derives from marketing efforts, a practice not unknown in the competitive world of sales.)
Control vs. Fear
As Sandman elucidated, human beings like to be in control, and, if this status cannot be achieved, may move rapidly to fear. Unfortunately, when sterile filtration is concerned, fear can result in the installation of wasteful, unnecessary safety nets that can create more problems than they solve.
Being in control is the desired state, and such control can only come from process validation studies. Their authority is at least as old as Lord Kelvin’s basic scientific principle, “When you can measure what you are speaking about, and can express it in numbers, you know something about it.”
It speaks to validation. In sterile filtration, as in most areas of pharmaceutical manufacturing, science-based validation is the best cure for fear.

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References
1. Hessler, A., Sandman, P.M. Squeaky Clean? Not Even Close. http://www.nytimes.com/2004/01/28/dining/squeaky-clean-not-even-close.html?sec=health?pagewanted=1
2. FDA. Guideline on General Principles of Process Validation, FDA CDER, 1987.
3. Agalloco, J.P. “Compliance Risk Management Using a Top-Down Validation Approach,” Pharmaceutical Technology, July 2008.
4. PDA Technical Report 26 (2008), Sterilizing Filtration of Liquids, Parenteral Drug Association, Bethesda, MD.
5. Ridgway, H.F., Rigby, M.G., and Argo, D.G. “Adhesion of a Mycobacterium to Cellulose Diacetate Membranes Used in Reverse Osmosis.” Applied and Environmental Microbiology 47, 1984, pp. 61-67.
6. Tolliver, D.L. and Schroeder, H.G. “Particle Control in Semiconductor Process Streams.” Microcontamination (l), 1983, pp. 34-43 and 78.
7. Bowman, F.W, Calhoun, M.P. and White, M. “Microbiological Methods for Quality Control of Membrane Filters.” J. Pharm. Sci., 56/2, 1967, pp. 453-459.
8. Leahy, T.J., Sullivan, M.J. “Validation of Bacterial Retention Capabilities of Membrane Filters.” Pharmaceutical Technology 2(11), 1978, pp. 64-75.
9. FDA. Guideline on Sterile Drug Products Produced by Aseptic Processing, FDA CDER, 1987.
10. Sundaram, S., Eisenhuth, J., Howard Jr., G.H., and Brandwein, H. “Part 1: Bacterial Challenge Tests on 0.2 and 0.22 Micron Rated Filters.” PDA Journal of Pharmaceutical Science and Technology, 55 (2), 1984, pp. 65-86.
11. Sundaram, S., Auriemma, M., Howard Jr., G.H., Brandwein, H., and Leo, F. “An Application of Membrane Filtration for Removal of Diminutive Bioburden Organisms in Pharmaceutical Products and Processes,” PDA Jour. Pharm. Sci. and Technol. 53 (4), 1999, pp. 186-201.
12. Krygier, V. Rating of Fine Membrane Filters Used in the Semiconductor Industry, Transcripts of Fifth Annual Semiconductor Pure Water Conference, (1986), pp. 232-251, San Francisco, CA
13. PDA/FDA Special Scientific Forum, Bethesda, MD; Validation of Microbial Retention of Sterilizing Filters, July 12-13, 1995.
14. Mittleman, M.W., Jornitz, M.W., Meltzer, T.H., “Bacterial Cell Size and Surface Charge Characteristics Relevant to Filter Validation Studies,” PDA Jour. of Pharm. Sci. and Technol. 52 (1), 1998, pp. 37-42.
15. Agalloco, J., Letter to the Editor—re: “It just doesn’t matter, It just doesn’t matter, It just doesn’t matter.” PDA Journal of Science and Technology. Vol 52, No. 3, pp. 149-150.

Validating Sterile Filtration: Overcome the Fear of Failure 3

Based on available data, long term filtrations may best be handled by 0.1-µm-rated filters, subject to validations being performed. However, in other cases, substituting 0.1-µm-rated for 0.2-µm-rated membranes may be unnecessary, and could result in significant penalties, including:
 Slower flow and processing rates, resulting in longer term operations.
 Higher costs for larger EFAs
 More leaching and extractables
 Higher product losses, due to adsorptive bonding to the ultimately greater filter area used.
A responsible choice requires that both the 0.1 µm-rated membranes and the 0.2 µm-rated membranes be validated.
If both types of filter prove appropriate, the higher pore size rating should be used to avoid the penalties of reduced flows. If, however, the validation data do not permit a clear resolution, the 0.1 µm-rated membranes should be used, since retention is more critical than flow rate or flux.
Below, we address some of the common sterile filtration concerns, requirements or practices that appear to be motivated by fear and can best be resolved by careful process validation.
“0.2-µm filters are penetrated by organisms. The industry is, therefore, required to switch to 0.1-µm-rated filters.”
In certain specific processes, 0.2-µm–rated filter can be penetrated by organisms, or by organisms which would normally be retained by such filters. In such cases, the flltrative removal of the organisms may well require the use of 0.1 µm–rated filters. Such instances are not new. Their occurrences have been considered by regulators for years, at least since the PDA and FDA held a special forum on this topic in 1995 [13].
   
Certain organisms, such as Burkholderia pickettii, Burkholderia cepacia, and Pseudomonas aerugenosa. shrink as a result of their immersion in fluid media that are only minimally nutritious for them [14]. Their reduction in size renders as invalid validations that use B. diminuta as a model. Brevundimonas diminuta can undergo shape alterations in minimally nutritious media but is not listed as undergoing size alterations occasioned by contacts with process fluids.
The fact that some microbes require 0.1-µm–rated filters to arrest them does not signify that all organisms are so disposed. The necessitated switch from 0.2-µm-rated to 0.1-µm–rated happens in only roughly 0.005% – 0.01 % of sterilizing grade filtration applications.
A mandated switch is therefore scientifically and statistically unfounded. Its promulgation may be shunned and process validation activities and data used as performance verification. Sole reliance on pore size ratings have been found obsolete anyway.
“Increasingly there are detectable but non-culturable organisms or L-forms or nano-bacteria in our processes.”
Conclusions cannot be made regarding the sterile filtration of microorganisms unless the methods of quantifying them by culturing and counting are available. Organisms such as the L-forms, nanobacteria, and “viable but non-culturable” entities may not be amenable to such analyses. Concerns about their presence may be justified, but without the means to cultivate and count them, it is impossible to attest to their complete absence.
It follows that a sterilizing filter can be judged only by its performance in the removal of identifiable and culturable organisms known to be present in the drug preparation [15]. The complex of influences governing the outcome of an intended sterilizing filtration necessitates a careful validation of the process, including that of the filter [4]. The very drug preparation of interest, the exact membrane type, the precise filtration conditions, and the specific organism type(s) of concern must be employed in the necessary validation.
“Redundant 0.2-µm filtration is necessary and should be used.”
Not necessarily. Again, proper process validation will disclose whether a single filter will do the job or not. However, there are some specific applications which traditionally, for whatever reason, utilize a second (redundant) filter as an “insurance filter,” i.e. if the first filter fails, the second may compensate. This holds, however, only when each filter has been validated to show specified retentivity.
Even so, the wisdom of the exercise deserves careful evaluation, as it assumes added costs for membrane EFA, increased leachables and extractables. The loss of drug product may needlessly be incurred by the filter’s heightened product hold-up, and unspecified adsorption.
    
“The maximum bioburden in front of a sterilizing filter should be 10 cfu per 100 mL of fluid.”
This is true if one wishes to accord with EMA regulations, and especially if one wishes to export product to Europe. The FDA makes no such stipulation, but bases its approval on process validation.
Seemingly in conflict, the two views arise from the same premise. The EMA regulation tries to establish the same sterility assurance level (SAL) for filtration as for thermal sterilization. EMA recognizes that, the greater the number of challenges, the more likely that at least one will succeed.
The FDA seems to agree, in that if the filter can sustain the removal of organism burdens far above those liable to be encountered in real life situations, it can assuredly withstand lesser insults. If, as the authors see it, the FDA’s massive challenge fails to breach the filter’s pores, it is needless to compel bioburden assessment in front of the filter. Filter validation would gainfully serve the intended purpose. Process validation, effectively conducted, would reliably demonstrate the filter action.

Validating Sterile Filtration: Overcome the Fear of Failure 2

From Tolliver and Schroeder (1983) courtesy of Microcontamination
B. diminuta should not be viewed as a universal model organism, as some native bioburden may be a better alternative as challenge organism, being close to the actual process settings. Unfortunately, rare penetrations of sterilizing grade filters have caused an exaggerated doubt in the reliability of filtration. Appropriate process validation though should render such doubts and be trusted by even the most critical reviewer of sterile filtration..
Sources of Variability: Size and Shape
B. diminuta varies in size and shape, depending on how it is cultivated. Back in 1967, Bowman and colleagues described the B. diminuta size as 0.3 × 1.0 μm [7]. However, in 1978, Leahy and Sullivan found [8] that the organism grown at 30 °C and incubated for 24 hours in saline lactose broth, a minimally nutritional medium for that microbe, yielded cocci-like cells approximately 0.3 × 1.0 μm (Figure 1). Similar considerations have to be accounted for when challenge tests are performed with native bioburden forms.
B. diminuta are typically cultivated to develop as spherical a form as possible, since spheres are least amenable to retention. Thus, Leahy and Sullivan proposed, back in 1978, that it be used as the model organism for 0.2/0.22-μm-rated membranes, partly because of its size relative to the 0.2-μm dimension [8].
Subsequently, the FDA designated it for that very purpose [9], defining a sterilizing filter as one that retains a minimum of 1 x 107 cfu of Brevundimonas diminuta ATCC 19146 / per cm2 of effective filtration area (EFA).
Although it isn’t the smallest organism known, B. diminuta was considered diminutive enough to represent whatever smaller organisms were likely to be present in pharmaceutical preparations. The smaller the test organism, goes the logic, the more likely that its removal by a filter would assure the sieve retention of larger organisms.
However, ease and safety of cultivation and handling are also important considerations. In 2001, Sundaram and colleagues found an increasing number of cases where filtration in 0.2/0.22-µm-rated membranes failed to yield sterile effluent [10]. Experimental studies showed that penetrating organisms had shrunken because they had been cultivated in broths that were nutritionally inadequate. In such cases, the physicochemistry of the suspending fluid may serve to alter the size of the suspended organisms as expressed by the Donnan equilibrium consequent to ionic strengths.
Organism Shrinkage During Processing
Sundaram’s team [11] also found that organisms underwent size changes after exposure to certain drugs. In cases with 0.2-µm-rated membranes, the researchers found, the larger pore size could only provide sterile effluent and/or a high titer reduction with regard to certain organisms for various lengths of time, before penetration occurred.
Penetration times varied from 24 to 96 hours, and the cumulative challenge at which penetration was first observed ranged from 1.2 x 107 to 1.1 x 108 cfu/cm2. Two 0.2-µm rated Nylon-66 filters in series were unable to fully retain Ralstonia pickettii (now Burkholderia pickettii) with penetration observed at 72 hours, corresponding to a cumulative challenge of 2.4 x 107 cfu/cm2. The more extensive penetration of the Nylon-66 membranes, compared with the PVDFs, is in keeping with their greater degree of openness, as Krygier and colleagues showed in 1986 [12].
As a result, it has been suggested that 0.1-µm-rated membranes be substituted for their 0.2-µm-rated counterparts.
Sundaram’s team evaluated five 0.1-µm-rated membranes and found that they yielded sterile effluent over the entire duration of the test (120-196 hours), up to challenge levels of 5.7 x 107 to 2.0 x 108 cfu/cm2. Similar results were obtained with the PVDF filters tested; no B. pickettii were detected at challenge levels of 5.9 x 107-6.0 x 108 cfu/cm2.
In addition, all 0.1-µm-rated filters tested provided consistent and complete retention of B. pickettii for the entire duration of the test (120-192 hours), suggesting that the smaller pores would ensure sterile product at conditions where penetration could occur through conventional 0.2- and 0.22-µm-rated sterilizing grade filters. Proponents argue that using 0.1-μm-rated membranes would permit longer term formulation and filtration operations. In fact, 0.1 µm-rated filters may be the best choice for long-term filtrations.
However, penetration has also been found in 0.1 µm-rated filters. In 1999, Sundaram’s team found that B. pickettii, when its size was so affected, could be retained by certain 0.1-µm-rated filters. But, in a similar situation, they found that only four of seven commercially available 0.1-μm-rated membranes could remove a particular organism. Just because one type of membrane so classified may provide proper retention, does not mean that any other 0.1-µm-rated membrane can also be depended upon for a like result.
It is important to remember that today, there are no industry standards by which 0.1 filters can be judged. In addition, more research clearly needs to be done into the kinetics behind the organism’s size changes, evaluating different organisms in different fluids.