The Challenges of Filter Qualification Filterability
The particulate size of some adjuvants (aluminum salts, liposomes, and microparticles) may be too large to pass through the
pores of a sterilizing grade filter membrane (with a typical mean pore diameter ~0.2 μm, i.e., 200 nm) and aseptic preparation
of an adjuvant vaccine formulation may have to be carried out in lieu of final sterilizing filtration. The membrane pore rating
and material of construction also may influence the integrity of the liposomes or microparticles or reduce the mean particle
size of the emulsion.4,5 Typical sizes of emulsions and liposome solutions can range from <100 nm up to 600 nm (0.6 μm), as measured using dynamic
light scattering (DLS). For example, the squalene oil-in-water emulsion MF59 (Table 1) has a particle size of ~165 nm, while
emulsion SB62 has a particle size of ~155 nm, both with a broad particle size distribution.6
It has been observed that a complete transmission of liposomes can be achieved through 0.2 μm rated membranes, including liposomes
that were estimated to be larger in size than the mean pore rating.7 A comparison of liposome size distributions by DLS and transmission electron microscopy (TEM) imaging, however, has revealed
that DLS may not be very accurate in predicting liposome size relative to filterability. Specifically, liposomes examined
by TEM were found to be ~3 times smaller than those obtained by DLS, suggesting that liposome size measurements using dynamic
light scattering may, at times, overestimate the effective liposome size in regard to filtration.8 This could potentially explain the observations of large liposome transmission through 0.2 μm rated membranes.7
Even when sterilizing filtration is an option based on antigen size, adjuvants may cause premature plugging of the filter
membrane, reducing filter capacity. This can be linked to the solution viscosity or to the particulate character of the adjuvant
in the formulation, as described above. For a model oil-in-water emulsion, it has been reported that membrane capacity is
increased with decreasing adjuvant particle size.5,9 This suggests a known mechanism where membrane capacity may be overchallenged with particles similar in size to the filter's
effective pore size rating. The flux decays seen with such adjuvants or adjuvanted vaccines may be a function of the membrane
structure, its effective pore size distribution, and total porosity.
Prefiltration can be a useful procedure for avoiding overchallenging a membrane with particles larger than, or in the same
size range as the effective mean pore size of the membrane. Prefiltration typically helps in improving throughput by removing
larger emulsion droplets or liposomes that otherwise plug the sterilizing membrane. Prefiltration or bioburden reduction membranes
rated at 0.2 μm (mean effective pore size) can represent a fairly wide range of pore size distributions and retention efficiencies
for nm or sub-micron–sized particles. Hence, the decision of whether to use coarser or finer 0.2 μm rated prefilters, is dependent
on feed characteristics as well as overall process economics.
Another challenge that adjuvanted solutions pose for sterilizing grade membranes is a potential reduction in bacterial retention
efficiency. During the process-specific bacterial retention validation of a sterilizing grade filter membrane, worst-case
conditions are simulated. In rare cases, under the high bacterial challenge levels imposed, poor filtration properties and
reduced bacterial retention may be observed. Investigations are currently in progress to identify key process parameters that
may affect bacterial retention in the presence of adjuvants. Some parameters that might reduce bacterial retention have been
described for a model oil-in-water emulsion.5,9 Amongst them were the plugging behavior of the model emulsion, the membrane properties, temperature, and operating pressure.
For this model oil-in-water emulsion, and for a selected sterilizing grade membrane filter, performing filtration at ambient
temperature (versus at cold temperature), lowering operating pressure, and using a prefiltration step before sterilizing grade
filtration may improve bacterial retention and the probability of demonstrating a sterile effluent.
Given that more controlled studies are not yet available, we evaluated our extensive database of field studies and sterilizing
filtration validation studies to identify process parameters that may affect bacterial retention. We offer our observations
as preliminary recommendations to minimize the risk of bacterial penetration during validation studies and thus reduce efforts
and costs to repeat studies, as well as to avoid delays in time-to-market.
In general, adjuvants and adjuvanted vaccines are most often solutions containing oils or surfactants, formulated as such,
in emulsions or as liposomes with a reduced surface tension (Table 1). For example, squalene-based emulsions like SB62 or
MF59 typically have a surface tension of ~33 dynes/cm2.6
In the process of validating numerous sterilizing filtration steps through various 0.2 μm rated filters, under a multitude
of conditions, we have observed that test fluids with a "low" surface tension (<68 dynes/cm2) present a higher risk of reducing bacterial retention of sterilizing grade filters than fluids with a "high" surface tension
(~70 dynes/cm2, comparable to that of water). This categorization was based on surface tension data when available or rational assumptions
based on the chemical composition of the test fluid solution when surface tension data were not available (for example, the
presence of a surfactant or lipid suggested the likelihood of reduced surface tension in the test fluid).
We further analyzed formulation data of low surface tension test fluids and categorized them (where information was available)
as 1) liposome solutions, 2) lipid and lipid-like solutions (i.e. emulsions, proteins with long chain fatty acids, sterols,
etc.), and 3) surfactant solutions. In the interest of a broad analysis, we didn't limit ourselves to adjuvants or adjuvanted
vaccines, but considered all low surface tension solutions to better understand what parameters might increase the risk of
bacterial penetration during validation challenges of sterilizing grade membranes.