Professional Documents
Culture Documents
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).
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.
1
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
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).
2
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.
0,6
0,4
0,2
0
min. average max.
F1 F2 F3 F4 F5 F6
Figure 1.: Pore size distribution measured with Coulter Porometer of 0.2 µm rated filter
membranes
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).
3
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
4
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.
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.
5
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).
6
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
Flow Rate (ml/cm²/min)
25
20
15
10
5
0
1
Differential Pressure (bar)
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.
7
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.
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.
8
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.
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
9
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
10
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
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.,
Collentro, W. V. (1999), “The Use of 0.1-µm Membrane Filters in USP Purified Water Systems
Colwell, R.R. and Huq, A., (1995)."Viable but Non-Culturable Bacteria and Their Implications
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.
Letter 10-13.
11
HIMA, (1982). Microbial Evaluation of Filters for Sterilizing Liquids, Document No. 3,
Leahy, T.J.; Sullivan, M.J., (1978). "Validation of Bacterial Retention Capabilities of Membrane
Lindenblatt, J. (1999). “0.1 µm versus 0.2 µm Final Filtration in BFS Operations”, Minutes
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
Steere, W.C. and Meltzer, T.H., (1998). "Operational Considerations in the Steam Sterilization
Tanny, G.B., Strong, D.K, Presswood, W.G., and Meltzer, T.H., (1979). "The Adsorptive
40-51.
Technical Report No. 26, Sterilizing Filtration of Liquids. PDA Journal of Pharmaceutical
Thomas, A.J., Durkheim, H.H. Alpark, M.J. and Evers, P., (1991). "Dection of L-forms of
74-80.
Young, J.H., Ferko, B.L. and Gaber, R.P., (1994). "Parameters Covering Steam Sterilization of
12
Deadlegs", J, Pharm Sci & Tech 48(3), pp. 140-147.
13