Use of Hydrogenophaga pseudoflava Penetration To Quantitatively Assess the Impact of Filtration Parameters for 0.2-Micrometer-Pore-Size Filters

Abstract

Filters rated as having a 0.2-m pore size (0.2-m-rated filters) are used in laboratory and manufacturing settings for diverse applications of bacterial and particle removal from process fluids, analytical test articles, and gasses. Using Hydrogenophaga pseudoflava, a diminutive bacterium with an unusual geometry (i.e., it is very thin), we evaluated passage through 0.2-m-rated filters and the impact of filtration process parameters and bacterial challenge density. We show that consistent H. pseudoflava passage occurs through 0.2-m-rated filters. This is in contrast to an absence of significant passage of nutritionally challenged bacteria that are of similar size (i.e., hydrodynamic diameter) but dissimilar geometry.The 0.2-m-pore-size filter class (0.2-m-rated filters) includes a large and diverse set of products (22). They include air filters, particle reduction filters, filters used for bioburden reduction, lab-grade filters, and "sterilizing-grade" filters used in sterile-dosage-form manufacture. ASTM F 838-05, the Brevundimonas diminuta challenge test, is a standard for the "sterilizing-grade" filters (4), a subset of the 0.2-m-rated filters. The "0.2-m" designation is applied to the larger and more diverse set of products. This designation is based on physical measurements (e.g., the bubble point, the force necessary to extrude air through the capillary network of a wet filter) and mathematical extrapolations (5,14,29).The current filter validation approach for parenteral pharmaceuticals involves a demonstration of removal of 7 log 10 CFU/cm 2 of nutritionally starved B. diminuta from bulk drug product liquids (4,8,11,29). B. diminuta can penetrate 0.2-m-rated filters, but only sporadically and at low levels (12, 21). Larger bacteria (Listeria monocytogenes) have been demonstrated to be able to penetrate 0.2-m filters after long-term exposure (27). Recently, a species of small waterborne bacteria, Hydrogenophaga pseudoflava, has been shown to penetrate 0.2-m-rated filters (31-36) to a greater extent than the abovedescribed bacteria. None of these bacteria are actually physically smaller than 0.2 m, even H. pseudoflava (25,37,38).Because H. pseudoflava penetrates 0.2-m-rated filters in a potentially quantifiable manner, it can be used to study filtration efficiency. In this report, we evaluate the impact of filtration process parameters and bacterial challenge density on passage. We benchmark H. pseudoflava passage against that of nutritionally challenged bacteria which are of similar size (i.e., hydrodynamic diameter) but dissimilar geometry. MATERIALS AND METHODSBacteria. Hydrogenophaga (38) (formerly Pseudomonas) pseudoflava (ATCC 700892), Brevundimonas (37) (also formerly Pseudomonas) diminuta (ATCC 19146), Serratia marcescens (10) (ATCC 13880 and ATCC 8100), and Ralstonia pickettii (39) (ATCC 700590 and ATCC 49129) were purchased from the American Type Culture Collection (Manassas, VA) or from Fisher Scientific (Waltham, MA).Growth conditions. An H. pseudoflava stock was prepared by seeding with a 1:10...

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