Letter to the Editor Re: "Diminutive Bacteria Useful for 0.2-μm Filter Studies, FDA's Brorson Says" 10/9/09

Letter to the Editor

Dear Editor,

With interest we read the BioPharm Bulletin, of October 4th on BioPharmInternational.com titled "Diminutive Bacteria Useful for 0.2-μm Filter Studies, FDA's Brorson Says" reporting on a presentation given by Dr. Kurt Brorson at a seminar held September 24th, 2009. A similar presentation was given at the PDA/FDA Joint Regulatory Conference on September 9th, 2008, by Jim McVey, Microbiologist TL, Office of Pharmaceutical Science, CDER, FDA, and Aquila Lee, ORISE Fellow, CDER, FDA.

The studies presented are of interest and we would like to thank Brorson and colleagues for sharing their work with industry, vendors, and the scientific community. We believe, however, that there have been misinterpretations in the BioPharm Bulletin, which we would like to clarify.

For example, the statement "Brorson shared results from studies conducted at CDER/FDA that replicated and built on results reported by Sundaram, et al., in the PDA Journal in 2001," misrepresents the work performed and published by Dr. Sundaram.^1-4 In correspondence after the 2008 PDA/FDA Meeting's presentation about the filter samples used during the challenge tests, FDA stated that the filters used in the study were obtained from "lab supply sources." Filters intended to be utilized for laboratory purposes might not and most commonly do not represent filters used in pharmaceutical and biopharmaceutical production processes. The filters used in Dr. Sundaram's experiments were filter composites, which represented filters used in production settings. These filters can have different membrane structures or filter combinations in comparison to filters intended for laboratory usage. Therefore, the work of Dr. Sundaram has not been replicated, but rather supported, to at least some extent by these studies done using laboratory filter samples.

Furthermore, following the statement in the BioPharm article that "Filter users should also understand that the 0.2-μm rating is not standardized across the industry," Brorson said, "The rating is determined from bubble point data and a mathematical equation involving several parameters that are very difficult to measure." This statement is incorrect, since there is a standard existing for 0.2-μm rated filters: ASTM F 838-05.⁵ ASTM F 838-05 evolved from a 1982 HIMA standard challenge test using B. diminuta that eliminates the uncertainties and variability from the 0.2-μm rating.⁶ The Bubble Point test is used only to verify the filter's integrity; it is not used to rate a filter.^7,8

The authors support Dr. Brorson's notion that Hydrogenophaga pseudoflava might be a good organism for any additional scientific studies to understand retention processes during different fluid and process parameters. Dr. Sundaram, though, already proved that H. pseudoflava can penetrate 0.2-μm rated filters; therefore the practical benefit of such additional studies is questionable. The presenters at the PDA/FDA meeting, as well as Dr. Brorson, underlined that the data presented do not affect the current process validation methods performed to verify the retentivity of sterilizing grade filters in production settings. We welcome and fully support this statement.

The authors believe that 0.2-μm-rated filters require appropriate process validation before their use in production processes. In addition, routine bioburden evaluation, supported by microbiological characterization and necessary controls, is essential to ensure that pre-filtration levels within the fluid streams are well controlled. We assert that such studies are absolutely necessary and that no filtration validation effort can be considered properly completed without them. Only such tests and controls can and will verify whether or not the filter chosen meets the process requirements.

Additionally, we would like to point out again, as we have in our own publications, that the penetration of 0.2-μm filters by microorganisms is neither a surprising finding, nor is it a new one. It is obvious to anyone familiar with the comparative physiology of microorganisms that a number of bacterial and viral species fall under the 0.2-μm size range. This fact only reinforces the need for proper control of bioburden accompanied by well-conducted risk analysis in the use of filters.

Scientists have understood for a century that there are microorganisms that could pass through filters, which quantitatively remove culturable bacteria and mold. In fact that appearance of mammalian pathogens and bacteriophage in filtered fluids thought to be sterile filtered contributed to the discovery of viruses. So, there is nothing new in the understanding that some microorganisms, including small bacteria, could penetrate filters generally defined as bacteria retaining.

We thank you, the editor, for your considerations and Dr. Brorson and his colleagues for their contributions. We sincerely hope that discussions among all parties interested in the utilization of filters in contamination control can ensue and that the resulting clarity benefits not only the users of filtration but the end users of biopharmaceutical and pharmaceutical products.

Theodore H. Meltzer

Russel E. Madsen, Jr.

James P. Agalloco

James E. Akers

Maik W. Jornitz

References:

1. Sundaram S, Eisenhuth J, Howard G Jr, Brandwein H. Retention of water-borne bacteria by membrane filters. Part I: Bacterial challenge tests on 0.2 and 0.22 micron rated filters. PDA J Pharm Sci Technol. 2001;55(2):65­-86.

2. Sundaram S, Mallick S, Eisenhuth J, Howard G Jr, Brandwein H. Retention of water-borne bacteria by membrane filters. Part II: Scanning electron microscopy (SEM) and fatty acid methyl ester (FAME) characterization of bacterial species recovered downstream of 0.2/0.22 micron rated filters. PDA J Pharm Sci Technol. 2001;55(2):87-113.

3. Sundaram S, Eisenhuth J, Howard G Jr, Brandwein H. Retention of water-borne bacteria by membrane filters. Part III: Bacterial challenge tests on 0.1 micron rated filters. PDA J Pharm Sci Technol. 2001:55(2):114-126.

4. Sundaram S, Lewis M, Eisenhut J, Howard G Jr, Larson B. Method for qualifying microbial removal performance of 0.1 micron rated filters. Part IV: Retention of Hydrogenophaga pseudoflava (ATCC 70090) and Ralstonia pickettii (700591) by 0.2 and 0.22 µm rated filters. PDA J Pharm Sci Technol. 2002;56(3):150-171.

5. ASTM; Committee F 838-05. Standard test method for determining bacterial retention of membrane filters utilized for liquid filtration. West Conshohocken, PA: ASTM; 2005.

6. HIMA. Microbial evaluation of filters for sterilizing liquids. Document No. 3, Volume 4. Washington, DC: Health Industry Manufacturers Association, 1982.

7. Jornitz MW, Agalloco JP, Akers JE, Madsen RE, Meltzer TH. Filter integrity testing in liquid applications, revisited. Part I. Pharm Technol. 2001; 25 (10):34-50.

8. Jornitz MW, Agalloco JP, Akers JE, Madsen RE, Meltzer TH. Filter integrity testing in liquid applications, revisited. Part 2. Pharm Technol. 2001; 25(11):24-35.

Dr. Brorson responds:

FDA is a science-based agency that conducts independent scientific studies in areas that concern the public health. We have identified bacterial penetration of 0.2-µm rated filters as an area for directed investigation. We believe that independent studies that provide a more fundamental understanding of filtration mechanisms is important from a public health standpoint and do not agree that "the practical benefit of additional studies are questionable."

Our presentation does not claim that we were solely evaluating pharmaceutical sterilizing grade filters. We stated that filter discs (0.2/0.22-µm rated) were selected and purchased based on availability from laboratory supply sources to represent multiple vendors and multiple membrane chemistries. We studied filters rated as 0.2 µm as provided; the membranes were presumed to be representative of membranes in sterilizing grade cartridges, despite potential differences in composite structure or QC testing.

The 0.2-µm filter class includes a large and diverse set of products. It includes 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, a subset of the 0.2-µm rated filters. The "0.2-µm" designation is applied to the larger and more diverse set of products. This designation is based on physical measurements (e.g., bubble point) and mathematical extrapolations, as described in our presentation. Thus, ASTM F 838-05 is not a standard for all 0.2-µm filters, and it thereby eliminates uncertainties and variability only for a subset of these filters.

The main conclusions from our studies are that Hydrogenophaga pseudoflava can consistently pass through 0.2-µm rated filters, and that this passage can be measured quantitatively. In this sense we have "replicated" Dr. Sundaram's findings, although as pointed out, "supported" might be a better word. The quantitative nature of passage lends itself to calculation of an LRV and potential uses for H. pseudoflava in R&D, process development and/or "Quality by Design" studies. We acknowledge that filtration is one part of comprehensive sterility assurance programs in place at pharmaceutical facilities. We do not state that these observations imply that passage of H. pseudoflava constitutes a significant safety issue at this time, that H. pseudoflava is a routine facility isolate, or that the current filter validation methods and sterility assurance procedures are inadequate. However, we are conducting laboratory investigations of H. pseudoflava passage, because we believe that it is prudent to build a scientific knowledgebase for risk mitigation purposes in case these or similarly diminutive bacteria ever pose a facility risk.

Penetration of 0.2-µm filters by diminutive or very thin bacteria is not a surprising finding to most filter experts. However, it is not on the radar of lab technicians and biologists, who assume that 0.2-µm filter passage is only an issue with mycoplasma. Our research studies are preliminary and ongoing and do not constitute regulatory recommendations or policy. Current recommendations and procedures for filter validation should be followed as stipulated by guidance.

David Hussong, PhD

Kurt Brorson, PhD

James Mc Vey, MS

Patrick Faustino, PhD

Scott Lute

Office of Biotechnology Products, Office of Pharmaceutical Science, and Office of Testing and Research, Center for Drug Evaluation and Research, Food and Drug Administration