The Importance of Accurate Microorganism Identification in Microbial Challenge Tests of Membrane Filters. Part II. The Comparison of Hydrogenophaga pseudoflava ATTC 33668 and Curvibacter sp. ATCC 700892 by Microbial Challenge Tests with Membrane Filters.

The ASTM 838-05 standard describes a bacteria challenge test procedure based on Brevundimonas diminuta (ATCC 19146) to verify a 0.2 µm rated sterilizing-grade filter. For process validation procedures a correct identification of the challenge organism is essential. The test strain ATCC 700892 repeatedly used for microbial challenge tests was incorrectly named Hydrogenophaga pseudoflava but is phylogenetically linked to the genus Curvibacter, as shown in Part I of this series. Based on these studies the misconception was consolidated that Hydrogenophaga pseudoflava, a widely isolated microorganism also found in biopharmaceutical production settings, is able to penetrate 0.2 µm rated filters. Here we show that the bacteria challenge test results of the strains Curvibacter sp. ATCC 700892 and Hydrogenophaga pseudoflava ATCC 33668 are different. In previous challenge tests analytical filter membranes were used; these do not represent the process scenarios within the sterilizing filtration in industrial processes. To represent process systems, the study data presented were determined with 10" filter cartridge elements. The strain Hydrogenophaga pseudoflava ATCC 33668 is completely retained by 0.2 µm and 0.1 µm rated filters. Depending on the different 0.2 µm filter material there are different retention rates of the strain Curvibacter sp. ATCC 700892; only the 0.1 µm rated filters showed consistent complete retention. However, up to date the genus Curvibacter seems to be of low relevance within biopharmaceutical production settings. LAY ABSTRACT: Bacteria challenge tests are used to determine the retention performance of sterilizing-grade filters. The model organism used for bacteria challenge tests and the verification of a 0.2 µm rated sterilizing-grade filter is Brevundimonas diminuta. In previous studies another proposed, model organism used for challenge tests was incorrectly labelled as Hydrogenophaga pseudoflava. Given the predefined retention characteristics, this mislabelled organism was recommended to be included in bacteria challenge studies. However, the herein presented testing demonstrated that the organism is actually phylogenetically affiliated to the genus Curvibacter and not to the strain Hydrogenophaga pseudoflava ATCC 33668. In this report, we demonstrate that the retention of the strain Hydrogenophaga pseudoflava ATCC 33668 with 0.1 µm and 0.2 µm rated filters is comparable to the retention of Brevundimonas diminuta ATCC 19146.

The importance of accurate microorganism identification in microbial challenge tests of membrane filters--part I.

Microbial challenge testing is a common procedure to determine the retention efficiency, performance, and validity of a sterilizing-grade filter. The ASTM 838-05 standard describes a bacteria challenge test procedure based on Brevundimonas diminuta (ATCC 19146), routinely used to verify a 0.2 µm rated sterilizing-grade filter. Process validation procedures most often also utilize B. diminuta (ATCC 19146), but instead of the standard procedures and fluids, process, and product parameters are employed to determine whether these parameters influence the retentivity of the filter or changes to the challenge organism, which might result in the penetration of the filter. In certain instances, the native bioburden within the drug manufacturing process is used to perform such process validation challenge tests. Filter penetrations can happen and cause concern; therefore, it is essential to identify the organism species with accuracy to avoid unnecessary confusion. This paper and its follow-up will describe such imprecision and the resulting misconceptions. It will clarify past determinations and put perspective on the findings. LAY ABSTRACT: Sterilizing-grade filters are used to remove microorganisms from biopharmaceutical solutions. To determine the retention performance of such filters, bacteria challenge tests are utilized, often with a standard challenge organism (Brevundimonas diminuta), in instances with native bioburden. The accuracy of the microorganism identification is of importance to avoid flawed results and misinterpretation of the filter's performance.

Validation of sterilizing grade filtration.

Validation consideration of sterilizing grade filters, namely 0.2 micron, changed when FDA voiced concerns about the validity of Bacterial Challenge tests performed in the past. Such validation exercises are nowadays considered to be filter qualification. Filter validation requires more thorough analysis, especially Bacterial Challenge testing with the actual drug product under process conditions. To do so, viability testing is a necessity to determine the Bacterial Challenge test methodology. Additionally to these two compulsory tests, other evaluations like extractable, adsorption and chemical compatibility tests should be considered. PDA Technical Report # 26, Sterilizing Filtration of Liquids, describes all parameters and aspects required for the comprehensive validation of filters. The report is a most helpful tool for validation of liquid filters used in the biopharmaceutical industry. It sets the cornerstones of validation requirements and other filtration considerations.

Bacterial cell size and surface charge characteristics relevant to filter validation studies.

There are two recognized mechanisms whereby organisms are retained by liquid filters; namely, sieve-retention and adsorption. The efficiency of each may be influenced by the organism, suspending milieu, and by the filtration conditions. Validations of sterilizing filtrations require the use of organism suspensions in product-specific media. However, where the product is bactericidal to the challenge organism(s), surrogate solutions may be required. The ideal surrogate solution would minimize adsorptive retention, ensuring that the sterilizing action of the filter under consideration is the consequence of sieve-retention. This review explores the impact that various physicochemical factors may have on bacterial cell size and cell surface characteristics. An understanding of interactions among challenge bacteria, suspending fluid, and filter medium is essential for the development of surrogate solutions that provide a "worst case" mileu for filter validation studies or a "placebo," non-inhibitory challenge solution.