Sterilization of parenterals by filtration
In the sixth of a series of 10 articles encompassing asceptic
processing for parenterals, Monisha Narke, Director, Klenzaids GMP Academy
focuses on sterilization by filtration
is a preferred method of sterilizing drug product solutions. A sterilizing grade
filter should be validated to reproducibly remove viable microorganisms from
the process steam. Whatever filter or combination of filters is used, validation
should include microbiological challenges to simulate worse case production
conditions for the material to be filtered and integrity test results of the
filters used for the study. The microorganism Brevundimonas - ATCC 19146 - when
properly grown, harvested and used, is a common challenge microorganism for
0.2 m rated filters because of its small size - 0.3 m mean diameter.
Membrane filters are thin, uniform porous sheets that trap all particles larger
in size than the pores in the membrane or larger than their interstices. Particle
entrapment by sieving alone is an excessively simplistic interpretation of what
happens to particles during membrane filtration.
In reality, by spatial sequestration, membrane filters are also capable of retaining
particles that are dimensionally smaller than their pores. These mechanisms
a ) Spatial entrapment apply equally to liquid and gas filtration; they include
inertial impaction to the walls or surfaces of the pores and lodgment in crevices
b ) Physicochemical interactions with the filter medium can be very complex
and differ according to the types of particles, the fluid in which they are
suspended, and the dynamics of the filtration system.
c ) Electrostatic forces are of more importance in gas filtration than in liquid
d ) Adsorption of surfactants from liquid suspensions to both particles and
pore surfaces may result in mutually repulsive ionic forces that encourage the
passage of particles.
e ) Liquid suspensions through covalent bonding, and through attraction by van
der Walls forces when zeta potentials of both particles and pore surfaces are
low - usually less than 30 mV.
Pore size rating
Pore size ratings of commercially available membranes are not indices of maximum
pore opening diameters obtained from direct microscopic measurement or by particle
passage methods. Ratings are almost always determined by indirect means based
on theoretical considerations and on non-destructive test technology.
Factually, one can expect to find membranes with specified pore size ratings
to have a portion of pores with larger diameters than the rating would suggest,
yet quite capable of retaining microorganisms physically smaller than these
pores. Electron microscope studies of commercially available membranes that
had pore size ratings of 0.22 m and that had been shown to be capable of meeting
the FDA's microbiological particle passage criterion revealed pores with diameters
larger than 0.3 m on their surfaces and throughout their depths.
Microbial particle passage
Pore size is clearly not the only factor influencing the retention of microorganisms
on or in membrane filters. The FDA's microbiological particle passage standard,
that a sterilizing filter should be capable of retaining a challenge of at least
107 microorganisms of a particular type per cm2, is an end-result criterion
that does not by itself help to quantify measurable characteristic of membranes
or membrane types that contribute to retention.
The most discriminating experimental conditions exist when all pores are being
challenged by potential penetrates, i.e., when the challenge is sufficient to
cover the membrane surface completely but below the clogging concentration.
Scanning electron micrographs of this situation show a double layer of cells
of Ps. diminuta from a challenge concentration of 108 per cm2 on the surface
of 0.22 m pore size rated membranes. When membranes become clogged, excessively
high differential pressures are required to maintain a constant flow rate, and
furthermore the proportion of the challenge population penetrating the membrane
Applications of sterilizing filtration
There are two main occasions for the use of filtration in the sterilization
of fluids. The first is when fluids are damaged or destroyed by exposure to
other sterilization processes; the second is when the scale of sterilization
is too large to be addressed through other sterilization processes.
Aseptic processing of liquid pharmaceutical formulation implies
the use of sterile filtration. Filtration should not be the method of choice
ahead of thermal sterilization, but it is not out of the question; some heat-stable
pharmaceutical liquid products are currently being filter-sterilized and aseptically
processed. Sterilizing membranes for this purpose should have a pore size rating
no greater than 0.22 m. It is normal to filter from a clean compounding area
through an intact physical barrier into the aseptic filling room where the filling
machines are located. There may be intermediate vessels, or filtration may be
directly to the filling machine. It is good practice to mount a further sterilizing
filter at or close to the point of fill.
Fig 1 : Schematic representation of a single disc filter mounting assembly
Construction of sterilizing filters
For all but the most unusual circumstances, there are two types of sterilizing
filters available, disc and cartridge filters. With disc filters, the necessary
surface area for filtration is achieved through a large flat circular membrane.
With cartridge filters, the membrane is folded and pleated into a more compact
design. Regardless of these differences, there are commonalities in housing
designs made necessary by the requirement to avoid microbiological contamination
arising from processes intended to remove or reduce microbiological contamination.
All filter housings should be made from smooth polished materials, and where
possible clamps should be used rather than screw threads where contaminants
can increase in numbers. Large disc filters are hardly ever seen nowadays for
production purposes. Cartridge filters predominate; the reason for this has
probably more to do with marketing than with science or economics, but it is
correct to say that large disc filters take longer to change, clean, and replace
than cartridge filters, particularly with stacked disc combinations.
1. Disc Filters
Traditional types of large disc filter holders, if used at all for production
purposes, are usually about 300 mm in diameter. The direction of fluid flow
is from above the filter to below. The membrane is sandwiched between metal
inlet and outlet plates equipped with the sanitary connections necessary to
operate the filter. Because of the fragility of disc-type membranes, there must
always be a support plate directly beneath the membrane. Support plates must
be porous, often photoetched, chemically inert, and have minimal effects on
flow rate. This often means an uneasy compromise. Prevention of flow restriction
requires extensive void space; this acts against the plate's mechanical strength.
Usually there is also a loose mesh drain plate beneath the support plate and
resting on the outlet plate. When serially stacked disc filters are used, each
membrane requires its own support plate but there will only be one drain plate
as depicted in Fig 1.
Small disc filters and small single-use stacked disc filters in plastic housings
are available and have the appearance of cartride filters but consist of a series
of separate mebranes rather than of one continuous pleated membrane. This added
complexity may make sterilization by saturated steam more difficult. Deep vacuums
intended to ensure effective air removal from the filter media may raise the
membranes from their support screens, thus imposing an undesirable strain on
the adhesion between the two. This may lead to loss of integrity.
2. Cartridge Filters
There are two major components of cartridge filters, the cartridge and the housing.
The operational part of the cartridge is the membrane, pleated to provide a
significantly large surface area in a compact presentation. A cartridge filter
of 5 cm diameter and 25 cm length may contain upto about 6500 cm2 of pleated
Filtration of liquids
The general case for pharmaceutical products includes sterile filtration of
small-volume parenterals either in aseptic manufacture or prior to terminal
sterilization; sterile filtration of ophthalmic products; and filtration of
large volume parenterals prior to terminal sterilization.
For sterile filtration of ophthalmics and small-volume parenteral products it
is not unusual to find several filters mounted in series. For instance a compounded
bulk product may be filtered through the wall from a clean area into an aseptic
filling room. In these cases, there are usually two filters mounted in series,
one on either side of the wall. The filtrate may be fed directly to a filling
machine, alternatively it may be collected in an intermediate vessel, held for
a while, and then filled out. Intermediate vessels should be equipped with sterile
vent filters to prevent pressure increases leading to blow-backs.
Most large-volume parenterals are terminally sterilized, but regardless of this
it is quite usual to find them being passed through a sterilizing filter prior
to autoclaving. This is because of the risk of endotoxic shock from parenteral
infusion of large volumes containing only small concentrations of non-viable
but still pyrogenic microbial material.
Filtration of gases
Cartridge-type hydrophobic membrane filtration has largely replaced depth filtration
as a means of sterilization of gases. Collection of particles from a gas stream
by membrane filtration is, as with liquid filtration, a function of both sieving
and other means of retention. Adsorption and electrostatic attraction are far
more important to retention particles in gas filtration than in liquid filtration.
Because there are more mechanisms and interactions between pore surfaces and
particles, removal of particles is more easily accomplished from gas streams
than from liquids. Gases are quite satisfactorily sterilized using 0.45 m pore
size rated membranes.
Sterile filtration of gases has three main applications :
- First, when sterile air is required as an ingredient
gas in fermentation process;
- Second, when gasses are used for service purposes
in sterile manufacture, for instance to actuate valves and to stabilize head
space contents ; and,
- Third, to protect the venting of sterile enclosures
such as storage tanks.
In-process verification of the sterility of filtered gases may be done by constantly
bleeding off a trickle of gas through a pressure reducer on the downstream side
of the sterilizing filter. The bleed may be filtered through a gelatin membrane,
which should be removed daily or at other suitable intervals for incubation
and examination for evidence of microbiological contamination.
Membrane filters and their housings should be chemically and biological inert.
Filter manufacturers are therefore obliged to provide evidence that minimal
amounts of chemical substances are released from the materials going into their
Since the membrane usually presents the largest surface area of material in
commercial filters, it is from membrane contamination during manufacture that
most extractables arise. Dust particles, chemical pore formers, and solvents
may be left on membranes. Wetting agents may be released from hydrophobic membranes.
Indeed, Triton X-100 was at one time widely used as a wetting agent for sterilizing
membranes, until it was found to be cytotoxic.
It is not common for filter users to perform microbial retention tests. In principle,
microbial retention testing is quite simple; the test organism is Pseudomonas
diminuta ATCC 19146 and the challenge is 107 bacteria per cm2 of effective filtration
area. The entire filtrate that has passed through the filter under test is collected
on an analytical membrane; this is then incubated on an appropriate medium,
and any bacteria that have passed through the test membrane are counted as colonies.
The ability of the test filter to retain the challenge organism is expressed
as the log reduction value - LRV - of the test filter. The LRV is defined as
the log10 of the ratio of the number of organisms in the challenge to the number
of organisms in the filtrate. Most commercial filters tested by this method
yield a sterile filtrate; in these circumstances 1 is substituted in the denominator
of the equation required to calculate the LRV, and the results are reported
as greater than the LRV calculated.
Ps. diminuta is an inherently small, motile, gram-negative bacterium that was
originally isolated as a contaminant of filter sterilized fluids. Its size,
however, is not independent of nutritional and physiological factors that may
be encountered in its cultivation. Smallest cell sizes occur only in the stationary
phase of the organism's growth cycle. Regrettably for microbial retention testing,
microbial cultures in this phase of growth also contain significant numbers
of dead cells and debris. These particles that are not discernable as part of
the viable microbial challenge and are capable of clogging the pores of the
filters under test and effectively reducing the challenge. In other phases of
the growth cycle, the size of Ps. diminuta is larger than it's quoted 0.3 m.
Filtration flow rate
In some cases the time taken to filter a specified volume of product under constant
pressure may be taken as an index of filter integrity. Unduly fast filtration
may be indicative of a major loss of physical integrity. Filtration flow rate
is not sufficiently sensitive to detect defects in filters, which although physically
quite small could contribute significantly to loss of sterility.
Bubble point test
The classic laboratory bubble point test looks crude from a technological standpoint
but is in fact very sensitive for determining the bubble point for a sample
of membrane. A wetted filter element is held between support screens, and the
differential pressure across the filter is gradually increased by manually adjusting
a supply of compressed air. The pressure at which the first bubble appears downstream
of the filter is read off from an upstream pressure gauge. Everything is dependent
upon successful visual detection of the first bubble.
Filter integrity may be evaluated using the fact that gas is able to flow through
wetted filters by diffusion at pressures lower than the bubble point pressure.
In practice, diffusion tests are automated, equipment is available to measure
flow rate across filters by upstream or downstream flow meters or by upstream
pressure gauges. Measurement by downstream flow meter is termed forward flow
a term origianally used to describe their own variant of diffusion testing,
now widely used to describe any type of diffusion testing regardless of location
of the measuring device. Diffusion testing is done in-place, in-process, and
Process hold - pressure decay - test
Pressure hold tests require a wetted filter to be pressurized with gas. Further
supply of gas is then shut-off. The pressure decay in the filter is monitored
over time and compared to pre-determined standards.
The pressure hold test can be set up as a variant of the bubble point test by
using a pressure slightly below the estimated bubble point. It can be set up
as a variant of the diffusion test with the pressure around 80 percent of the
bubble point pressure, or it can be set up at lower pressures around 5 - 10
percent of the bubble point pressure. The test can be done over a very short
period of time or over a protracted hold time. Pressure decay tests are mainly
used as prefiltration integrity tests for cartridge filters.
Water penetration test
Hydrophobic filters are mainly tested by bubble point, diffusion, or pressure
decay methods based on wetting with solvents with lower surface tensions than
those of the membranes. This is because the void space of the membrane will
not be completely penetrated by water to allow these tests to be applicable.
The main alternative solvents used in association with hydrophobic filters are
aqueous solutions of isopropanol. This practice, although perfectly legitimate,
introduces other problems, namely the problem of removing all of the solvent,
to ensure that product coming into contact with filtered gas or air is not to
be adulterated, and the problem of flammability of solvents. The water penetration
- water intrusion - test is an alternative method applicable only to hydrophobic
filters, which by using water rather than solvents avoid the problems described
Microbiological methods of monitoring the effluent from a sterilizing filter
are too insensitive to discern any but the most serious failures of the filtration
system. Such failures are better detected by the non-microbiological in-process
integrity tests described above. It is, however, normal in the application of
sterile filtration to ensure that the microbiological challenge to the filter
is well within the filter's sterilizing capability. Microbiological limits should
be set therefore for the non-sterile challenge material; if measured numbers
are high or exceed justifiable limits, prefiltration or some other means of
reducing the microbial challenge should be introduced.
Reasons for filtration failure
In certain circumstances sterilizing filters may allow the passage of microorganisms.
It may be of value for preventing this to summarize the main reasons for the
occurrence of non-sterile effluents.
o Incorrect assembly
Sterilizing filters must be assembled aseptically. It is difficult to legislate
this except by thorough training and good supervision. The number and frequency
of aseptic connections should be minimized as far as practicality dictates.
o Passage through defects
Filters may be flawed or defective. The defects may be within the membrane,
or in the housing, or between the membrane and housings. Routine integrity testing
should be capable of revealing these types of defects through considerations
of all data from bubble point, diffusion, and pressure hold tests.
o Passage through the membrane
The mechanism of particle retention by membrane filters is not confined to sieving.
It is not therefore out of the question that there may be a coincidence of a
microorganism at the bottom end of its size range and a pore at the top end
of its size range. Part of the answer to this possible failure mode is for filter
manfuacturers to control their pore size within bands of only narrow variation.
From the user's point of view, the challenge should be kept well within the
capability of the filter.
Microorganisms retained on a wet membrane for an extended period of time at
ambient temperatures may be able to reproduce and grow through to the downstream
side of the filter. This is probably more likely with air filters than when
liquid filters are kept in place. In part it is due to the likelyhood of moisture
being carried in the airstream. It can be avoided by prefiltration and by controlling
the duration of use.
The separate and independent components of an effective filtration
system manufacture overlap rather than about against one another. Momentary
failure of any one system does not necessarily compromise product sterility.
Nonetheless, good assurance of avoidance of contamination can only be obtained
from knowledge that each system is capable of performing as it ought to perform
validation and that it continues to operate effectively in routine use routine
( The author is Director, Klenzaids GMP Academy.She can
be contacted at firstname.lastname@example.org)