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Verifying Membrane Filter–Based Bacterial Removal Using Titer Reduction

Medical Device & Diagnostic Industry Magazine MDDI Article Index An MD&DI July 1999 Column When relying on filters for bacterial removal, it's necessary to know which membranes to use to reach the desired results.

Medical Device & Diagnostic Industry Magazine
MDDI Article Index

An MD&DI July 1999 Column

When relying on filters for bacterial removal, it's necessary to know which membranes to use to reach the desired results.

The need to control microbial bioburden has led to point-of-use bacterial sterilization using filter membranes to remove particles and bacteria from critical fluids.1,2 The microbial rating of a membrane filter assembly is determined by its ability to retain particle sizes represented by specified strains of microorganisms.3 Bacterial challenges are typically performed during validation to evaluate membrane filter performance. Verification of bacterial removal is typically performed by challenging a membrane filter assembly with a suspension of a known organism. The titer reduction is the ratio of influent colony forming units (CFU) to effluent bacteria colonies found downstream of the membrane.4

For example, an upstream challenge of 1013 ending in an effluent count of 104 results in a titer reduction of 1 x 109. This measurement means that the probability of a cell passing through the filter is 0.0000001%. Another way of viewing this is that there is a 99.9999999% probability of bacterial retention.

When no colonies are detected downstream of a filter assembly, the titer reduction is expressed as greater than the total number of CFU influent to the membrane filter assembly.5 (For an upstream challenge of 1013 with a resulting sterile effluent count of 0, the titer reduction is >1 x 1013. In other words, the probability of a cell making it downstream of the filter is <0.00000000001%, and the probability of retention is >99.99999999999%.1

The correlation between nondestructive integrity and bacterial retention test results is important to ensure that the membranes used will clarify the critical fluid as required for the process in question. This correlation is obtained empirically in a validation process according to the test parameters used. Nondestructive integrity tests accepted by the industry are the forward flow, pressure hold, bubble point, and water intrusion tests. These tests will be discussed under "Filter Integrity Tests." As always, it is necessary to perform enough test runs to ensure a statistically valid sample size.

MEMBRANE AND FILTER ASSEMBLY RATING: MICROBIAL CHALLENGE TESTS

Industry and regulatory agencies have specified test organisms for identification of filter ratings. The influent concentration of CFU of the specified organism per membrane surface area is specified, along with the test pressure.

Filter membranes rated at 0.2 µm remove a majority of contaminating microorganisms and must provide a sterile effluent when challenged with 107 microorganisms of Brevundimonas (Pseudomonas) diminuta (ATCC19146) per cm2 of membrane surface under a pressure > 30 psi (Figure 1).6 According to FDA guidelines, a filter membrane that produces effluent free of Brevundimonas diminuta, such as the 0.2 µm filter, is referred to as a sterlizing filter membrane. However, these filters are not absolute; they can remove bacteria, but not viruses.

Figure 1. The rod-shaped Brevundimonas (Pseudomonas) diminuta at 3000x magnification. This microorganism is 0.3 µm in diameter and 0.6 to 0.8 µm long.4

Filter membranes rated at 0.1 µm can be tested with a strain of Mycoplasma, which lack a rigid cell wall. A common choice is Acholeplasma laidlawii (ATCC 23206) tested at a pressure of 7 psi. The spherical organisms vary in size from less than 0.1 µm to approximately 0.4 µm in diameter. These filter membranes usually can retain a culture of 107 CFU/cm2 of membrane surface.8

Some analytical filter membranes, rated at 0.45 µm, retain larger organisms, such as a culture of 107 CFU/cm2 of membrane surface, under the pressure specified. However, standards for retention are less rigorous than those used for the bacterial sterilizing filters. Serratia marcescens (ATCC 14756) is often used to evaluate membranes at test pressures as low as 5 psi.8 This organism is 0.45-µm wide and 0.8-µm long.

DESCRIPTION OF MICROBIAL CHALLENGE TO MEMBRANE FILTER ASSEMBLY

To determine the bacterial retention characteristics of a membrane filter, a specific liquid concentration of bacteria is passed through a known area of the filter in a test assembly (Figures 2 and 3).

Figure 2. Disc scale test setup for bacteria challenge test (from personal communication with G. Fennington).

The test equipment is cleaned with isopropyl alcohol, and the membrane (or filter assembly) is sterilized via autoclave. The appropriate microorganism is removed from stock, stirred, sonicated, diluted with carrier fluid (e.g., saline solution) and restirred. The challenge organisms include those discussed earlier.

Figure 3. Large-scale test setup for bacteria challenge test.9

At an appropriate flow rate, the bacterial challenge suspension is passed through the test filter assembly and the analysis membrane to achieve a challenge level of >1 x 107 CFU/cm2 of membrane surface. Filter effluent is collected and evaluated for microbial contamination. The differential pressure is recorded, and the titer reduction is calculated.

TYPICAL RESULTS OF MEMBRANE AND FILTER ASSEMBLY EVALUATIONS

Microbial challenge tests were designed to approximate an extreme bioburden under aqueous process conditions with the filter on-line for six to eight hours. The microbial grades of many membranes have a high efficiency of bacterial removal when used in liquid filter assemblies (Tables I-IV).

 

 

 

 

 

Membrane
Polymer
Contaminant Diameter
(µm)
Membrane Area
(sq ft)
Titer
Reduction
P
(psid)
Sterile
Filter
Modified nylon667 Bacteriophage T1 0.05 0.015 2.29 x 107 NAa
Modified nylon667 Acholeplasma laidlawii 0.1–0.4 8.6 >1 x 1013 NAa
Modified nylon667 B. diminuta 0.3 8.6 >1 x 1012 Yes
a Not applicable. Sterility has been defined using a test challenge of Brevundimonas (Pseudomonas) diminuta.



Table I. 0.04-µm-rated membrane (filter assembly) efficiency data at a challenge level of >1 x 107 CFU/cm2 of membrane surface.

 

 

 

 

 

 

 

 

Membrane
Polymer
Contaminant Diameter
(µm)
Membrane Area
(sq ft)
Titer
Reduction
P
(psid)
Sterile
Filter
Nylon669 Acholeplasma laidlawii 0.1–0.4 7.5 >1 x 1010 75 NAa
Modified nylon667 Acholeplasma laidlawii 0.1–0.4 8.6 >1 x 1012 1 NAa
Polyethersulphone10 Acholeplasma laidlawii 0.1–0.4 0.5 >1 x 1010 30 Yes
Nylon669 B. diminuta 0.3 7.5 >30 Yes
Modified nylon667 B. diminuta 0.3 7.5 >1 x 1013 Yes
Modified PVDF
hydrophilic11
B. diminuta 0.3 5.4 >1.1 x 1010 65 Yes
a Not applicable. Sterility has been defined using a test challenge of Brevundimonas (Pseudomonas) diminuta.



Table II. 0.1-µm-rated membrane (filter assembly) efficiency data at a challenge level of >1 x 107 CFU/cm2 of membrane surface.

 

 

 

 

 

 

 

 

 

 

 

Membrane
Polymer
Contaminant Diameter
(µm)
Membrane Area
(sq ft)
Titer
Reduction
P
(psid)
Sterile
Filter
Nylon669 Acholeplasma laidlawii 0.1–0.4 7.5 >1 x 1011 40 NAa
Modified nylon667 Acholeplasma laidlawii 0.1–0.4 7.5 >1 x 1012 1 NAa
Nylon669 B. diminuta 0.3 5.0 >1 x 1013 >38 Yes
Modified nylon667 B. diminuta 0.3 8.5 >1 x 1013 >38 Yes
Modified PVDF
hydrophobic13
B. diminuta 0.3 5.4 >1.1 x 1010 Yes
Modified PVDF
hydrophilic12
B. diminuta 0.3 >8 x 1011 Yes
Polyamide hydroxyl
modified14
B. diminuta 0.3 5.0 >7 x 1011 40 Yes
Polyethersulphone15 B. diminuta 0.3 0.5 >1 x 1010 30 Yes
PTFE16 B. diminuta 0.3 >1 x 1011 Yes
a Not applicable. Sterility has been defined using a test challenge of Brevundimonas (Pseudomonas) diminuta.



Table III. 0.2-µm-rated membrane (filter assembly) efficiency data at a challenge level of >1 x 107 CFU/cm2 of membrane surface.

 

 

 

 

 

 

 

 

Membrane
Polymer
Contaminant Diameter
(µm)
Membrane Area
(sq ft)
Titer
Reduction
P
(psid)
Sterile
Filter
Nylon669 Acholeplasma laidlawii 0.1–0.4 7.5 >1 x 1010 75 NAa
Modified nylon667 Acholeplasma laidlawii 0.1–0.4 8.8 >1 x 1012 1 NAa
Nylon669 B. diminuta 0.3 8.5 >1 x 1012 Yes
Modified nylon667 B. diminuta 0.3 8.8 >1 x 1013 40 Yes
Nylon669 Serratia marcescens 0.45 8.8 >1 x 1012 NAa
Modified nylon667 Serratia marcescens 0.45 8.8 >1 x 1012 NAa
a Not applicable. Sterility has been defined using a test challenge of Brevundimonas (Pseudomonas) diminuta.



Table IV. 0.45-µm-rated membrane (filter assembly) efficiency data at a challenge level of >1 x 107 CFU/cm2 of membrane surface.

FILTER INTEGRITY TESTS

The application of a predetermined air pressure to a wetted membrane to quantitatively measure the sum of diffusive air flow and the bulk flow through any larger pores is the forward-flow test (Figure 4).

Figure 4. Test setup for the forward-flow integrity test.11

The pressure hold test is run by pressurizing the filter housing that contains the membrane to a predetermined setting, isolating it from the pressure source, and measuring the quantitative decay in pressure over time caused by the diffusion of air through the wetted membrane. These data can be used to determine the bubble point (Figure 5).

Figure 5. Pressure-hold test results as related to the bubble point.17

Bubble point tests entail pressurizing one side of a wetted membrane with a layer of the wetting fluid on the other side, and slowly increasing the pressure, thereby displacing liquid from the largest pore and noting the bulk air flow (where the majority of the air is going, which is toward the largest pore). A manual bubble point test apparatus is described by Brantley and Martin (Figure 6).17

Figure 6. Test setup for the manual bubble-point test.17

The water-intrusion test involves filling the upstream side of a test assembly (Figure 7) with water, pressurizing that side of the hydrophobic membrane, and slowly increasing the pressure. An integrity device is used to measure the flow of gas and air pressure. The water intrusion pressure is the pressure at which air is displaced from the largest pore and the water flow is visible at the filter outlet.

Figure 7. Test setup for water-intrusion test.18

Empirically correlating the results of any of the above four tests with the bacterial challenge test results in validation data applicable to the test methods and test parameters.

CONCLUSION

Membrane filter assemblies used in laboratory devices to remove particles and bacteria from critical fluids should be rated to determine their ability to retain small biological test particles of a size represented by specified strains of microorganisms. Verification of bacterial removal is typically performed by challenging the filter assembly with a suspension of a known organism and can be reported in terms of titer reduction, the ratio of the number of influent colony forming units to effluent bacteria colonies found downstream of the membrane. Although the principle behind filters is simple—the separation of bacteria from fluid, in this instance—the membranes used need to withstand high pressure yet still perform at high levels. The correlation between nondestructive integrity test results and bacterial retention test results is important to ensure that the membranes used in critical fluid processes will clarify the critical fluid and provide adequate microbial control. Such a correlation is obtained empirically in a validation process applicable to the test parameters used.

REFERENCES

1. JP Heggers, "Nosocomial Infections of Today, Tomorrow," Advanced Laboratory, July (1998): 76-82.

2. JA O'Brien, "Meeting TB Regulations in the Clinical Lab," Advanced Laboratory, July (1998): 89-92.

3. O Masako, N Yamada, and M Toya, "Bacterial Retention Mechanisms of Membrane Filters," Pharmaceutical Technology Japan 7, No.11 (1991).

4. DB Pall and E Kirnbauer, "Bacterial Removal Prediction in Membrane Filters" (paper presented at the 52nd Colloidal and Surface Science Symposium, University of Tennessee, Knoxville, TN, 1978).

5. Validation Guide for Pall 0.2 µm Ultipor N66 and N66 Posidyne Membrane Cartridge TR-680c (East Hills, NY: Pall Ultrafine Filtration Company, 1997).

6. FDA Guidelines on Sterile Drug Products Produced by Aseptic Processing. (Rockville, MD: Food & Drug Administration, June 1987).

7. 'N66' Posidyne Filter Guide: Nylon 66 Positive Zeta Potential High Area Filter Elements for Microbial Removal and Particulate Removal; Filtration from 5 Micrometers to Molecular Levels USD950g (East Hills, NY: Pall Process Filtration, 1994).

8. The United States Pharmacopeia, USP 23, and The National Formulary, NF 18. (1994). United States Pharmacopeial Convention, Inc., Rockville, MD, Chapter 1211.

9. Validation Guide for Pall 0.1 µm Nylon 66 Membrane Cartridge TR-690 (East Hills, NY: Pall Ultrafine Filtration Company, 1997).

10. SuporFlow 100 P-Grade and Supor DCF 0.1µm Validation Guide PN32756 (Ann Arbor, MI: Gelman Sciences, 1996).

11. Validation Guide for Pall 0.1 µm Fluorodyne II DJPL Filters Fluoro-DJLP-VG (East Hills, NY: Pall BioPharmaceuticals, 1998).

12. Validation Guide for Pall 0.2 µm Fluorodyne II DFLP Filters TR1195 (East Hills, NY: Pall Ultrafine Filtration Company, 1995).

13. Validation Guide for Pall Emflon II Membrane Cartridges for Air and Gas Filtration TR870B (East Hills, NY: Pall Process Filtration Company Pall Ultrafine Filtration Company, 1994).

14. Validation Guide for Pall Bio-Inert 0.2 µm Membrane Filter Cartridges TR890 (East Hills, NY: Pall Ultrafine Filtration Company, 1998).

15. SuporFlow 200 P-Grade and Supor DCF 0.2 µm Validation Guide PN31202 (Ann Arbor, MI: Gelman Sciences, 1996).

16. Sol-Vent II Validation Guide PN31664 (Ann Arbor, MI: Gelman Sciences, 1996).

17. JD Brantley and JM Martin, "Integrity Testing of Membrane Filters to Assure Production of Sterile Effluent," Genetic Engineering News 17, No. 10 (1997): 24.

18. The Pall Water Intrusion Test for Hydrophobic Sterile Gas Filters PBB-STR-29 (East Hills, NY: Pall Ultrafine Filtration Company, 1996).

Jane J. Janas, PhD, is a senior staff scientist with Pall Corp., (Port Washington, NY).

 


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