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Consideration on Sterilizing Grade Liquid Filtration – Pore Size and Filtration Sterilization -

M.W. Jornitz, K. Kawamura, PhD and T. H. Meltzer, PhD

Introduction

There has been an evolution of our understanding of membranes and of membrane

filtration over the half-century of their use. Their early successes in producing sterile liquid

filtrates led to the optimistic belief that such membranes were absolute; and that they

unquestionably removed from pharmaceutical preparations the organisms commonly suspended

therein. The filtrative action was seen to result from sieve retention, the mechanism whereby

particles (organisms) larger in size than the pores become spatially restrained from passage

through the filters. Nevertheless recent findings show that adsorptive sequestration plays a role in

filtrative sterilization, which in itself can be influenced by the drug product properties to be

filtered. Appropriate process and filter validation is required to determine the performance of a

filter and its function (PDA Technical Report 26).

Microbial Retention and Brevundimonas diminuta

B. diminuta (ATCC 19146), previously classified as Pseudimonas diminuta, came to

serve as the model organism for pharmaceutical filtration. These microbes, suspended in a

penicillinase solution were found to penetrate 0.45 µm-rated membranes, the “sterilizing

membranes” at the time, but were restrained by the tighter 0.2 µm filters devised for that very

purpose. However, the invoking of the sieve retention mechanism was called into question

because the 0.45 µm-rated membrane did remove these organisms from aqueous suspensions

absent penicillinase (Bowman et al. 1967). The rationalization was that the organisms were

removed by adsorptive arrests to the filter surfaces (as well as by sieving) unless protein

competitively pre-empted the adsorptive sites. As a consequence, the adsorptive sequestration

mechanism came to be recognized.

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Adsorption is governed by filtration conditions, such as the challenge density, the ionic

strength of the solution, possibly the temperature/viscosity, and most importantly by the applied

differential pressure (Mittleman et al. 1998). Dependence upon such various influences, by

definition, signifies the non-absoluteness of filters and of filtration (Tanny et al. 1979).

Absoluteness would mean freedom from such dependencies. However, so reliable is the

sterilizing removal of B. diminuta by 0.2 (0.22) µm-rated membranes that these membranes have

come to be designated as the “sterilizing filters”, whereby the use of a 0.45 µm rated membrane

can also result in a sterile filtrate, depending on the process conditions. Therefore only

appropriate process validation enables to determine the required performance of a filter

independent of its pore size designation. Pore size designation becomes a mere logistical

necessity or an indicator for the filter manufacturer and user.

Sterilizing Grade Filter Rating

Filter manufacturers designate their filters 0.2 (0.22) µm-rated membrane products if they

are capable of withstanding challenges of 1 x 107 B. diminuta organisms per square centimeters

of membrane surface (HIMA 1982, ASTM 1988). The challenge is normally performed using a

suspension of a suitable concentration of B. diminuta in from 2 to 20 liters (usually) of saline

lactose broth, employing about 2 bar (30 psi) differential pressure for the filtration. Withstanding

the challenge test does not necessarily mean that such filter membranes are of 0.2 µm rating. As

in Figure 1. membranes designated 0.2 µm can be of larger pore size and still result in a sterile

effluent, when challenged as stated above. Adsorptive capture may be the effective in this case.

Given the possibility of adsorptive sequestration and of the resulting influences of the several

conditions of the filtration on the outcome of the organism/membrane confrontation, the FDA

insists that users of membranes designated by their manufacturers as 0.2 µm or “sterilizing

filters”, experimentally demonstrate the sterilizing proclivities under “worse case conditions”,

under the severest conditions of the processing operation (PDA/FDA 1995).

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This FDA requirement, thus, in itself recognizes that a membrane that sterilizes under

one set of circumstances may not so perform under another even when the same organism is

involved. As stated, the model organism is the B. diminuta grown under stipulated conditions

(Leahy and Sullivan 1978, Fennington and Howard 1997). The present definition of “sterilizing

filter” is, then, referenced in terms of this organisms size and its adsorptive proclivities.

Pore Size Distribution 0.2 micron

Different Filter Manufacturers

min. average max.0

0,2

0,4

0,6

0,8Pore Size (µm)

F1 F2 F3 F4 F5 F6

Figure 1.: Pore size distribution measured with Coulter Porometer of 0.2 µm rated filter

membranes

Bubble Point Correlations and Retention Ratings

Efforts have been made to match the B. diminuta size to the 0.2 µm-rated pore size; a

relationship expressive of the sieve retention mechanism. The results are clouded if only because

pore-size ratings are innocent of any measurement standards. However, correlations have been

developed between organism retention levels and bubble point integrity test values that are

indicative of a filters largest pores (Johnston and Meltzer 1979) (See Figure 2).

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On the basis of this correlation, a particular filter can be designated as a “sterilizing filter” given

a sufficiency of B. diminuta retention. The bubble point will differ for each polymeric type, but

for any type the label 0.2 (0.22) applies to membrane that completely retains the B. diminuta

challenge. The numerical value should not be entertained seriously. It is a vestigial sign only.

There is, then, an identity between the bubble point measurement and the affixing of the 0.2

(0.22) µm label based on B. diminuta retention.

Again, it is evident that the appellation “sterilizing filter” is defined in terms of B.

diminuta removals. Its arrest by filters depends upon its particle size/pore size relationship and

upon its capability to adsorb to the membrane polymer under the selected filtration conditions. It

is hardly likely to serve as a universal model for all other microbes, of whatever size, and under

all other filtration conditions.

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Still there is often surprise when filters with the 0.2 or 0.22 µm designations fail to retain

organisms. Improper imputations of poor filter quality may be made, as if the action of the

“sterilizing filter” were absolute and independent of the organisms involved, of the nature of the

suspending fluid, and of the filtration conditions. Additionally the pore size distribution in itself

shows that larger pores are involved. This again underlines the necessity of filter validation

under process conditions, instead of overall statements that a smaller pore size designation would

retain with “absolute” efficiency.

Smaller Organisms Concerns

There is a concern that organisms smaller than B. diminuta may be present in certain

pharmaceutical preparations. The 0.2 µm-rated membranes may not perform as sterilizing filters

for the smaller organisms. No surprise, but used, by distinguished filter manufacturers, to move

the industry to utilize a filter of different pore size designations, organism penetration of 0.2 µm

rated filters has been experienced on specific occasions, especially hostile process and product

conditions. Organisms present in Water for Injection may, on account of the poor nutritional

environment, be reduced in size (Gould 1997, Collentro 1999). Known for years are P. cepacia

and P. fluorescens, which are able to adapt to a low nutritious environment by reducing the

surface/volume ratio. These smaller organisms, which are mainly waterborne organisms may be

able to penetrate 0.2 µm rated filters (Carter and Levy, 1998). L-form organisms, devoid of their

more rigid outer membranes, may be capable of negotiating the tortuous pore paths of filters

(Thomas et al. 1991). They are not retained by 0.2 µm-rated filters (Hargreaves 1993), as are

Mycoplasma not. In these instances 0.1 µm rated or tighter filters have to be used. Heresay

mentioned that “nanobacteria” have been found in sera. This issue has been debated at a special

PDA discussion at the PDA Annual Meeting 1999 without conclusion, whether such organisms

are existing or not. To elevate the scope of discussions, there is the threat of the unknown posed

by viable but non-culturable microbes (Colwell 1996), which are difficult to determine.

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Substitution of 0.1 for 0.2 Membranes

It has been suggested that such smaller organisms may be better restrained from passage

by 0.1 µm-rated filters. It is advanced, therefore, the 0.1 µm-rated membrane should replace the

0.2 µm-rated as the sterilizing filter. Unfortunately such statement is utilized in a generalistic

way, missing the scope and importance of process validation, suggesting again absoluteness.

As an alternative to the use of 0.1 µm-rated membranes, the use of two 0.2 µm-rated

filters in series is also suggested. Jornitz et al. (1999) indicate, however, that double 0.2 µm-

rated membranes, aside from augmenting adsorption effects, are not likely to be as efficient as

the 0.1 µm-rated membrane.

It is advocated that substitution of 0.1 µm-rated filters for their 0.2 rated counterparts,

however prudently intended, should not be implemented unless the need is experimentally

indicated. Such substitutions may involve filtration problems and would most likely compel

revalidations.

Whether the interchange of 0.1 and 0.2 µm-rated filters requires revalidation deserves

consideration. The finer pored membrane structures may be more promotive of adsorptive

sequestration since they offer more pore surface area. Viscosity effects may become enhanced;

flow rates being reduced. Borderline incompatibilities may become exaggerated, added

solid/liquid interfaces manifesting themselves. Additionally, the narrower pores may undergo

wetting with greater reluctance, almost all the newer membrane polymeric materials of

construction being borderline in their hydrophilicity. This could occasion an increase in (false)

integrity test failures, usually caused by incomplete wetting of the pore surfaces. Steam-in-place

failures could increase. These could eventuate from the greater impediment to the penetration of

narrower apertures by steam (Young et al., 1994), and by its more ready condensation upon

increased surface areas (Steere and Meltzer 1998).

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Above all, the improved, hoped for organism retention would require documented

experimental confirmation. As Lindenblatt (1999) indicated, there are different retention

performances by 0.1 µm rated filters from different manufacturers. Some of those were able to

retain mycoplasma, others not. Due to the lack of an appropriate challenge standard for 0.1 µm

rated filters, the user either relies on the manufacturers data or performs proper validation

studies. Such validation studies would also reveal, whether or not a 0.1 µm rated filter is

absolutely necessary or not. It may well be that a 0.2 µm rated filter would have sufficient

retention capabilities. Therefore validation overcomes the generalistic term as mentioned

previously.

Flow-Rate Differences

1Differential Pressure (bar)

0

5

10

15

20

25Flow Rate (ml/cm²/min)

0.1 µm 0.2 µm 0.45 µm

Figure 3.: Flow rate differences of various pore-size ratings

There are, as stated, applications for which the 0.1 µm-rated filters are clearly indicated.

Risk assessments for other applications are appropriate. There are, however, penalties in flow

rates and potentially in throughputs to be considered. Figure 3., illustrates the dramatic decrease

in flux, for at least one type of membrane, that results when a 0.45 µm-rated filter is substituted

for by a 0.2-and then by a 0.1 µm-rated membrane. The flux decreases by 75 % from a 0.2 µm

filter rating to a 0.1 µm.

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Some of this loss, but hardly all, can perhaps be compensated for by improved filter

design. However, tighter filter design will ineluctably result in flow rate diminution. Reliance

upon thinner membranes should be made with caution. They are more prone to imperfections.

Additionally, the use of prefilters to minimize increased flow-blocking particle accretions on the

tighter membranes could be necessitated. The application of 0.1 µm-rated membranes has its

uses. The exercise should not, however, receive cavalier endorsements; it has its costs.

Validation is required and revalidation when changes are made to validated and approved

processes, under all circumstances.

Validation - Product Wet Integrity Testing

Filter manufacturers ascertain the proper water bubble point value for their type of filter

that accords with the required 1 x 107 cfu retention per cm2 of filter surface. In filter

manufacture it is common to provide a safety margin by furnishing customers with membranes

of higher-than-minimum water bubble points. The penalty imposed on the filter user by this

insurance is a somewhat lower rate of flow. The classic situation involves a filter

manufacturers determining the minimum bubble point for water, its translation to the product

bubble point, and the testing of the filter for integrity. The integrity tests ascertain the ratio of the

water/product bubble points.

This is useful in determining whether the product bubble point meets the minimum

allowable test value or not (PDA Technical Report 26). Three filter specimens are used in the

integrity testing. At least one should have a bubble point value close to the minimum. Otherwise,

the level set will reflect an unnecessarily high value. This, in turn, means that the filter user will

not be entitled to employ filters with lower integrity values. Following such an evaluation, the

filter purveyor will be enjoined from selling for that validated application filters having bubble

points between the minimum and chose of the tested value.

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Filter Manufactures Choice

The minimum accepted bubble point is that which correlates to the required organism

retention level. It would seem to be in the filters manufacturers interest to provide membranes

with as close a value to the minimum bubble point as possible so as not to exclude from use

filters still possessed of a sufficiency of retention but having bubble points lower than that tested

in the validation. However, there is a problem. Many filter manufacturers have until now elected

to produce membrane with higher than minimum bubble points, both as a safety factor for users

and perhaps in connection with minimizing scrap rates. Filter manufacturers, therefore, must

make a choice. One option lies with furnishing membranes having lower but acceptable bubble

points consonant with the minimum water bubble point and its matching organism retention

abilities. This would avoid precluding the use of membranes whose water bubble point

automatically sets the product bubble point as that of the filter used in the validation.

A second possibility is for the filter manufacturer to designate a new minimum bubble

point specification for the membrane he now regularly produces. To be sure, this would forego

offering filters of lower bubble points but still above those representing the correlation to the

required organism retentions. The safety factor of higher values would automatically inhere. The

cost to the filter manufacturer would be minimal if his product is consistent in its bubble point

value. The scrap rate, too, would reflect the constancy of manufacture, but the cutoff level

would be that of the (higher) new minimum setting. Implications that this change in catalogue

specifications implies a corrective to previous lower bubble point values would be inappropriate.

Implications of Higher Minimum Bubble Points

How the filter producers address their problem, essentially a business decision, need not

concern the user. Most prefer to offer membrane having bubble points closer to the minimum

value stipulated in their product descriptions. At least one filter manufacturer, however, has

elected to specify a new minimum water-wet bubble point for his product. The new range,

routinely manufactured, is sanctioned by its being above the filter manufacturers minimum

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value, the level at which that filter type retains 1 x 107 cfu/cm2 of filter surface. Redefining the

minimum bubble point of the membrane being offered to the drug industry, in effect a change in

the catalogue listing, in no way modifies the adequacy of the value actually determined by the

filter manufacturer, if the value changed is higher than the previously stated value. Even then

one should not take such changed and new value for granted. In filter production, membranes

are produced within a specified bubble point range, an upper (maximum) and lower (minimum)

bubble point value. An upward shift of this specified band may excessively elevate the upper

limit, thereby changing filter performance. One is called upon to supply evidence that the filters

performance did not change, i.e. beside the achievement of a sterile effluent, certain other

properties have to be evaluated, as for example described in PDA Technical Report 26. The

adsorptivity should be evaluated; same for flow rates, pressure conditions, extractable levels, etc.

When the bubble point is changed now to a higher level, one has to ask whether this will

influence the pore size and pore size distribution, and with it the flow rate and, therefore, contact

time or time to filter the required batch volume. If, because of increased contact time between

the solution and the filter, the adsorptive effect could be heightened, the product could change in

respect to its activity, potency and strength. Changes in the process, of equipment settings or

parameters have to be evaluated in respect to their potential adverse effect on the process and/or

drug product. Therefore, a change of integrity test value of a sterilizing grade filter, even to a

higher assurance level, requires testing to achieve documented evidence that it will not have

adverse effects.

Either way, filter users will have at their disposable microporous membranes dedicated to

sterile filtrations from which minimum product bubble point can be arrived at with confidence.

Summation

One can clearly state that there is no absoluteness in filtration. The question is, is absoluteness

required. Aseptic processing in biopharmaceutical processes require assured sterile filtrates, but

not absoluteness. Thorough filter and process validation, properly performed, will assure sterile

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filtrates. The filter user has to know the process and microbial circumstances and according to

these parameters validate a filter, which sterilizes the liquid product reliably. Therefore the need

is a sterilizing filter, independently from the pore size. A sterilizing grade filter can be a 0.45 µm

pore size filter is certain applications, in other instances 0.2 µm or 0.1 µm rated filters could be

an improved alternative. Appropriate filter validation performed by the filter user, supported by

the filter manufacturer, will determine which of the above ratings will be the best choice for the

specific process and not overall statements or unnecessary fears.

Reference:

American Society for Testing and Materials (ASTM), Standard F838-83, Standard Test Method

for Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration. 1983,

Revised 1988

Bowman, F. W., Calhoun, M.P., and White, M., (1967). Microbiological methods for quality

control of membrane filters, J. Pharm. Sci., 56: 222-225.

Carter, J.R. and Levy, R.V., (1998). Microbial Retention Testing in the Validation of Sterilizing

Filtration". in Filtration in the Biopharmaceutical Industry. Meltzer, T.H. and Jornitz, M.W.,

Eds., Marcel Dekker, New York.

Collentro, W. V. (1999), “The Use of 0.1-µm Membrane Filters in USP Purified Water Systems

– Case Histories, Part I”, Pharm. Tech.: 176-192

Colwell, R.R. and Huq, A., (1995)."Viable but Non-Culturable Bacteria and Their Implications

for Water Purification", Ultrapure Water 12(3), pp. 67-74

Fennington, G.J. and Howard, G., Jr., (1997). Preparation and evaluation of bacterial

stock for filter validation, PDA J. Pharm. Sci. Technol. 51(4): 153-155.

Hargreaves, P., (1993)."Paul Hargreaves Speaks Out on Pharmaceutical Manufacturing" PDA

Letter 10-13.

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HIMA, (1982). Microbial Evaluation of Filters for Sterilizing Liquids, Document No. 3,

Volume 4. Health Industry Manufacturers Association, Washington, D.C.

Jornitz, M. W. and Meltzer, T. H., (1999). Addressing Uncertainties In Sterile Filtrations;

Substituting Double Filters and 0. 1 for 0.2 µm Rating.

Leahy, T.J.; Sullivan, M.J., (1978). "Validation of Bacterial Retention Capabilities of Membrane

Filters", Pharmaceutical Technology 2(11) pp. 64-75.

Lindenblatt, J. (1999). “0.1 µm versus 0.2 µm Final Filtration in BFS Operations”, Minutes

from AGM, BFS News, Autum Edition 1999: 44-48

Mittleman, M.W., Jornitz, M.W. and Meltzer, T.H., (1998). "Bacterial Cell Size and Surface

Charge Characteristics Relevant to Filter Validation Studies," PDA J. Pharm. Sci. Tech. 52 (1):

37-42.

PDA Special Scientific Forum, Bethesda, MD; Validation of Microbial Retention of Sterilizing

Filters, July 12 – 13, 1995

Steere, W.C. and Meltzer, T.H., (1998). "Operational Considerations in the Steam Sterilization

of Cartridge Filters," Pharm. Tech., 17 (9): 98-110.

Tanny, G.B., Strong, D.K, Presswood, W.G., and Meltzer, T.H., (1979). "The Adsorptive

Retention of Pseudomonas diminuta by Membrane Filters," J. Parenteral Drug Assoc., 33(l),

40-51.

Technical Report No. 26, Sterilizing Filtration of Liquids. PDA Journal of Pharmaceutical

Science and Technology, Vol. 52 No. S1, 1998

Thomas, A.J., Durkheim, H.H. Alpark, M.J. and Evers, P., (1991). "Dection of L-forms of

Pseudomonas aeruginosa during Microbiological Validation of Filters". Pharm. Tech. 15 (1):

74-80.

Young, J.H., Ferko, B.L. and Gaber, R.P., (1994). "Parameters Covering Steam Sterilization of

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Deadlegs", J, Pharm Sci & Tech 48(3), pp. 140-147.