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Russell E. Madsen is the former senior vice-president of science and technology at PDA, and is a current member of Pharmaceutical Technology's editorial advisory board.
Theodore H. Meltzer is principle of Capitola Consultancy.
James E. Akers is the president of Akers Kennedy & Associates, PO Box 22562, Kansas City, MO 64113, firstname.lastname@example.org.
Model organisms are useful when validating sterile filtration, but successful retention of the model organism does not always guarantee that effluent is sterile. The authors explore the various factors that influence sterile filtration.
There are advantages and disadvantages to using model microorganisms in the validation of sterilizing filtration. Although filtration validation studies may demonstrate the removal of the challenge organisms (whatever they may be), those studies can only imply that any particular filter used to filter a batch of drug product will yield a sterile filtrate. Successfully passing a bubble-point test (1) (or multipoint diffusive-flow integrity test), the results of which have been correlated to successful microbial challenge testing and validation studies, provides additional assurance that a sterile filtrate has been obtained. The filter manufacturer's quality-management system and production-control program further support the validity of the sterilizing filtration process. All three elements must be present to predict the successful outcome of a particular sterile filtration event (2).
Perhaps the best method of determining whether a filter can yield sterile effluent is to subject the drug product to a proper microbial challenge and to analyze the filtrate for its microorganism content. This method of direct measurement would seem to be the most reliable way of characterizing a filter's sterilizing ability. Even though an individual filter can be characterized by a direct microbial challenge, however, the tested filter may not then be used in a processing operation because it has been contaminated by the test organisms.
In practice, the measurement of a filter's sterilizing capability is derived from a correlation of its degree of bacterial retention with a characterization of its largest pores, essentially in terms of their width. The latter is obtained by means of the bubble-point integrity test method (3). It is necessary to confirm this correlative relationship because to assume it exists could prove a mistake. For instance, two qualities or properties of a filter may so constantly occur together as to be assumed to be correlated. Actually, they may in fact be two independent but parallel events that are not correlated (1). For example, single-point air diffusion rates of a filter measured at 5 psi had formerly been thought to correlate with a bubble point of some 50 psi for that filter, but it was discovered that it was necessary to determine the bubble point by measurements made at the actual bubble-point pressure. It did not necessarily correlate with the extension of the 5 psi single-point air diffusion test reading in all cases. So what had seemed to be a correlation turned out to be a case of parallel events. As a result, the use of single-point diffusive airflows measured at 5 psi was abandoned. In the present instance, extensive examination by many investigators has established the validity of correlating the bubble point with microbial retention (4).
An inherent assumption
A filter can be characterized by direct microbial challenge as being capable of producing a sterile effluent. Extrapolating from the properties of the tested filter to other "identical" filters would, however, involve assumptions related to the uniformity of the filters' manufacture. To investigate this aspect of the filtration picture to a statistically meaningful level would involve a large number of correlated studies. Moreover, it might well necessitate the disclosure of proprietary information in a competitive market. This would prove highly impractical. From this one consideration alone, it becomes evident that a fundamental assumption must be made, namely, that the filter manufacturers can be depended upon to control their manufacturing processes so that "identical" filters achieve the same level of retention performance.
Factors affecting pore-size ratings
Reliance upon the filter manufacturer is one necessary factor in this action. Identifying the requirements does not ensure the attainment of sterile effluent.
The sterilizing filter concept
Until rather recently, it was believed that the sterilization of liquids could be achieved by their filtration through a sterilizing membrane whose proper and pertinent identity was confirmed by its pore-size rating, which was itself determined by integrity testing. The pore-size rating, plotted against organism size, indicated an inverse correlation. However arranged, organism removal by a filter was seen as resulting from the mechanism of size exclusion, also known as sieve retention, wherein the organism is retained because it is larger in size and shape than the filter's pores. The sizes of various organisms are available from listings in the literature, and the size of a filter's largest pores derives from its bubble-point value. Therefore, it was possible to accommodate the size of the restraining pore to the size of the organism to be retained. In the interest of obtaining a maximum flow rate consonant with complete organism removal, the 0.2/0.22-μm-rated membranes were widely accepted as being sterilizing filters.
The sterilizing filter was defined in 1987 by the US Food and Drug Administration on the basis of its retaining a minimum of 1 × 107 colony-forming units (cfu) of Brevundimonas diminuta (at that time taxonomically identified as Pseudomonas diminuta) per square centimeter of effective filtration area (5).
The situation is complicated in its application by the absence of pore-size rating standards. The pore sizes may not be assumed to have been rated in the same fashion by the various filter manufacturers. Thus, while filters may bear the same pore-size designation, they may not be identical in this regard. Nevertheless, such filters may prove interchangeable in filtration processing operations. Manipulative adaptations often make it possible to substitute one such filter for another.
Although the individual rating methods differ, bubble-point measurements performed in as similar a manner as possible enable comparisons to be made among the various types of filters. Given the importance of gauging the sizes of the largest pores, the integrity testing should be as accurate and reproducible as possible. Accordingly, using automated integrity test instruments is recommended, because their use eliminates the subjectivity inherent in manual integrity testing (6). For the same reason, the bubble point should be identified by the straight line plotted from multipoint diffusion measurements extended through and beyond it to a robust flow of air. Single-point diffusive airflow measurements are not sufficiently reliable for this purpose (7).
Limitations of the B. diminuta model
Advantage of the model organism
There are numerous types and sizes of microorganisms. To explore the sterilizing capabilities of the available filters with each of these different organisms would not be practical and might not even be meaningful from the standpoint of assessing patient risk. Given the large number of organism types and their differences in size, it would be advantageous to select for testing an organism that could serve as a model for all the others, or at least for those commonly encountered in pharmaceutical operations. The smaller the test organism, the more its removal by a filter would ensure the sieve retention of larger organisms. Properties other than size, however, also are important in the selection of the model microbe. Ease and safety in cultivation and handling are real considerations. The likelihood of the selected organisms' being encountered in pharmaceutical operations is another influence on its selection.
In the mid-1960s, Frances Bowman of FDA identified an organism that would penetrate the 0.45-μm-rated membranes that typically were used for sterilizing filtration at that time (8). The organism, currently known as B. diminuta, American Type Culture Collection 19146, had the ability to distinguish between 0.22- and 0.45-μm-rated membranes (it readily penetrated the latter) and, at concentrations higher than about 1 × 107 cfu/cm2, it penetrated 0.22-μm-rated membranes, demonstrating that because penetration was concentration-dependent, mechanisms other than sieve retention were active. Although not the smallest organism known (smaller organisms had been known for decades), B. diminuta generally was considered small enough to represent whatever smaller organisms were likely to be present in pharmaceutical preparations. ASTM standard F 838, "Standard Test Method for Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration," is based on the use of B. diminuta as the challenge organism. The standard was originally approved in 1983 and was designed to determine the bacterial retention characteristics of membrane filters for liquid filtration.
Bioburden organisms as models
Since then, however, some 25 cases have been noted wherein the 0.2/0.22-μm-rated membranes qualified by withstanding the requisite B. diminuta challenge did not yield sterile effluent (9, 10). Obviously, B. diminuta does not serve as a universal model for all organisms. This finding has led some to advocate that the choice of the model organism whereby a filter will be qualified for use as a sterilizing filter should be an organism native to the drug preparation. In this view, B. diminuta suffers from the demerit of not being part of the drug preparation's bioburden. Be that as it may, selecting a component of the bioburden as the model organism is not enough. The test organism must be amenable to identification and cultivation. Its life stages must allow amply for its management in the necessary test manipulations. Above all, its susceptibility to size diminution by contact with a given drug preparation will have to be investigated, and the kinetics and direction of the morphological changes, if any, will require elucidation.
Insufficiency of the model organism
As previously stated, it had been believed that a sterilizing filter could be defined by way of its pore-size designation as identified by integrity testing. Developments in filtration practices showed this belief to be too simplistic. What had once seemed simple now is recognized as being quite complex. It was discovered that the conclusions based on pore-size ratings were subject to modification by the physicochemical specificity of the organism-suspending fluid, the individuality of the organism type in its size-changing response to the fluid, the possible change in pore size induced by the fluid, and the adsorptive qualities of the filter resulting from its particular polymeric composition, all influenced by the filtration conditions in their numerous varieties, but especially by the transmembrane pressure (11, 12).
A filter may not sterilize the same preparation under different filtration conditions, especially under dissimilar differential pressures (13). A given membrane may or may not retain a particular organism type suspended in a different drug vehicle (8). The organism type need not remain constant in size, but may alter in response to its suspending fluid (14–16). The effect of the vehicle upon the polymeric membrane may cause a change in its pore size (17).
The certainty of obtaining sterile effluent requires far more than the identification of a sterilizing filter by a pore-size rating. The variety of influences governing the outcome of an intended sterilizing filtration necessitates a careful validation of the process, including the filter. The very drug preparation of interest, the exact membrane type, the precise filtration conditions, and the prefiltration bioburden, including specific organism type(s) and number, should be used in the necessary validation.
Limitations on the use of the B. diminuta model
Given the complexity of the organism removal operation, it is doubtful whether a universal sterilizing filter can be devised. Certainly, there is no known absolute filter, one that will retain all organisms under all conditions, especially if viruses are included. Therefore, the successful attainment of a sterile filtration with regard to specified organisms of interest must in every individual filtration sterilization be corroborated by the documented experimental evidence that constitutes validation. The specifics of such operations are discussed in the Parenteral Drug Association's "Technical Report No. 26." Adherence to the teachings of this report is a second factor that is essential to attaining sterile effluent.
Obviously, the many organisms whose presence in pharmaceutical contexts deserves consideration may differ from one another so greatly as to compel individual techniques for their identification and cultivation. Conclusions cannot be made regarding the sterile filtration of microorganisms unless methods of quantifying them by culturing and counting are available. Organisms such as the L-forms, nanobacteria, and "viable but nonculturable" 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 (18).
Model organisms, although perhaps necessitated at least as an opening gambit, will not truly serve their intended, more general use as surrogates for most other organisms. In the present circumstance, however, B. diminuta can serve as a model as well as any bioburden organism. Albeit with surprise to some and consternation to others, B. diminuta did fail to serve as a filtration model in some specific circumstances. Nevertheless, the failures were relatively few. Moreover, they were the result of organism size reductions occasioned for particular organisms by contact with particular drug preparations. Many involved in pharmaceutical filtration were unaware that an organism's morphology could reflect its manner of cultivation, as well as the nutritional value of its suspending fluid, but those facts are known to microbiologists.
It was not, nor is it as yet, sufficiently recognized within the pharmaceutical industry that organisms penetrating the 0.2/0.22-μm-rated membranes were likely to have undergone size reductions occasioned by exposures to particular drug preparations (Ralstonia pickettii, Burkholderia cepacia, and B. diminuta in particular). The dependability of the exclusion mechanism was predicated on the stability of the organism and pore sizes as measured in certain specific situations. It does require modification when organisms or pore sizes do not remain constant. Each may undergo change upon contact with particular drugs. At present, little is known about the kinetics of these size alterations.
Summary and conclusion
Bacterial challenge studies should be conducted under process conditions by means of inoculating the product to be evaluated with B. diminuta to achieve a challenge level of 1 × 107 cfu/cm2 and evaluating the filtrate for the presence of the challenge organism. In the event that B. diminuta is not viable in the product formulation, precondition the filter by recirculating product through it to simulate process conditions and follow this with a microbial challenge by modifying the process to ensure the viability of the challenge organism (e.g., change temperature), modifying the formulation to ensure the viability of the challenge organism (e.g., adjust pH, remove bactericidal component), reducing the exposure time to ensure the challenge organism remains viable, or change from B. diminuta to a microorganism that has been isolated from the formulation. It is important to use the product formulation if possible, because there have been instances in which B. diminuta has penetrated a sterilizing filter in contact with the product formulation but has been retained by the same filter when inoculated into a surrogate fluid.
The ability to predict the successful outcome of a particular sterile filtration event requires a filter manufacturing enterprise that is highly capable and consistent, a dependable and accurate method to conduct integrity testing, and possession of the understanding and ability to implement effective filtration validation strategies.
Russell E. Madsen* is president of the Williamsburg Group, LLC, 18907 Lindenhouse Rd., Gaithersburg, MD 20879, tel. 301.869. 5016, email@example.com James E. Akers is president of Akers Kennedy and Associates. Maik W. Jornitz is group vice-president of global product management, bioprocess of Sartorius North America. Theodore H. Meltzer is a consultant for Capitola Consulting. Madsen, Akers, and Meltzer also are members of Pharmaceutical Technology's editorial advisory board.
*To whom all correspondence should be addressed.
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