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Filterability and bacterial retention must be verified very early in process development to ensure successful sterilizing filtration validation.
Adjuvants are substances added to vaccine formulations to enhance, accelerate, and prolong the immune response to a vaccine. Despite these notable benefits, vaccine adjuvants also can add to concerns related to clinical efficacy, safety, and dose stability. In the manufacturing environment, adjuvants can create challenges for sterilizing membrane filter capacity and bacterial retention validation. The particulate character of some adjuvants can cause premature plugging of filter membranes, and the low surface tension of many adjuvant solutions may contribute to reduced bacterial retention efficiency and potential non-sterility. In this article, we provide some recommendations for membrane filter selection and process conditions that may enhance filter capacity and increase the probability of successful bacterial retention validation for sterilizing filtration of adjuvanted vaccines.
Immune potentiators or immunomodulators, commonly referred to as adjuvants, are gaining increased attention from vaccine manufacturers. Adjuvants are used to enhance, accelerate, and prolong the efficacy of a vaccine. They are increasingly necessary now that simple, highly purified antigens, such as recombinant proteins or DNA, which do not elicit strong immune responses on their own, are replacing more antigenic intact pathogens in vaccine preparations. Interest in adjuvants also has increased because of their dose-sparing effect, facilitating a quicker and broader response during a pandemic.1–3
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Adjuvants are a heterogeneous group of compounds (Table 1), including single compounds with intrinsic immunostimulating properties, for example, monophosphoryl lipid A (MPL) or the saponin QS21, and antigen carriers such as liposomes, microparticles, aluminum salts, but often have a combination of both immunostimulatory and carrier characteristics (e.g., emulsions and immune-stimulating complexes).3 Aluminum salts have been widely used and accepted in vaccines produced globally for several decades, whereas approval of new vaccine adjuvants has, until recently, been limited to Europe, for example, H1N1 pandemic influenza vaccines Focetria (Novartis) and Pandemrix (GSK). In the US, FDA approved the vaccine Cervarix (GSK), containing MPL, in late 2009.
Table 1. Examples of vaccine adjuvants3,6
The properties of these adjuvants or adjuvanted vaccines, in combination with specific process operating conditions, may create some challenges during sterilizing filter selection and sizing (filterability) studies and during sterilizing filter bacterial retention validation.
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.
Following the categorization of low surface tension test fluids, we analyzed events of bacterial penetration in each category and observed that the risk, qualitatively defined here as the likelihood of an occurrence of bacterial penetration during retention validation challenge, was the highest for liposome solutions, followed by lipid and lipid-like solutions and then by surfactant-containing solutions.
Figure 1. Risk of Brevundimonas diminuta penetration events with 0.2 Î¼m sterilizing grade membranes in low surface tension solutions as a function of total challenge bacteria load (CFU/cm2), relative to baseline, i.e., risk at lowest bacterial load.
With these higher penetration risk test fluids, challenge parameters associated with bacterial penetration were identified as the total Brevundimonas diminuta bacteria load (CFU/cm2 EFA) and the rate of load (CFU/min) during the B. diminuta challenge test (Figures 1 and 2). Although the minimum total load cannot be changed (because of the regulatory requirement for achieving ≥1 x 107 CFU B. diminuta/cm2 EFA during bacterial retention validation), our data suggest that B. diminuta challenge greater than 1 x 108 CFU/cm2 may significantly increase the risk of validation issues (either premature plugging or bacterial penetration) when conducting bacterial challenges of sterilizing grade membranes with these potentially higher-risk solutions. If possible, the B. diminuta bacteria challenge loading rate should be minimized.
Figure 2. Risk of Brevundimonas diminuta penetration events with 0.2 Î¼m sterilizing grade membranes in low surface tension solutions as a function of bacterial load rate (CFU/min), relative to baseline, i.e., risk at lowest bacterial load rate.
Bacterial challenge studies are conducted either under constant flow or constant inlet pressure conditions, so the categories were analyzed with regard to these challenge conditions (Figure 3). The data suggest that with higher risk solutions containing liposomes, lipids, or surfactants, a constant flow challenge condition may have a higher risk of a penetration event than constant pressure conditions. In a situation of constant flow, the pressure inevitably varies in an attempt to maintain a constant flow rate. This changing (generally increasing) pressure may result in increased potential for penetration. Similar observations have been previously reported.5
Figure 3. Risk of Brevundimonas diminuta penetration events with 0.2 Î¼m sterilizing grade membranes in categories of low surface tension solutions according to challenge method, relative to baseline, i.e., risk for surfactants solutions and constant pressure challenge method.
These categories were further compared with regard to the flux across the filter. To determine flux (flow per unit area), the maximum recorded flow rate (if available) or the average flow rate (if available) was divided by the effective filter area (EFA) of the test filter. For each category, two histograms of flux were generated; one for validated sterilizing filtrations and one for filter challenges where B. diminuta bacteria penetration was reported. The peak values of these histograms provide insight into the differences between the retentive and penetrative filtration populations, as seen in Table 2. The data suggest that increased flux (or flow rate) does not necessarily increase the risk of penetration. In fact, when all data are considered, higher flux appears to suggest a correlation with a reduced risk of penetration, and particularly in the case of lipid solutions. This however, may only be relative to the positive association between rapid plugging of the membrane and bacterial penetration.
Table 2. The peak value of flux histograms (mL/min/cm2) within a category (the shape of the histograms are skewed such that most of the test fluids have flux rates less than or equal to the peak).
A review of field and laboratory bacterial retention validation data for a variety of fluids and challenge conditions suggests that low surface tension fluids, such as many adjuvants and adjuvanted vaccines, present a higher risk of the occurrence of a bacterial penetration event during sterilizing filter validation (Table 3). Among the classified solutions examined, liposome solutions represent the highest risk, followed by lipid and finally surfactant solutions. B. diminuta bacteria loading above 1 x 108 CFU/cm2 (>10 times the minimum required challenge density) increases the chance of a penetrative event, as does a loading rate of greater than 1 x 105 CFU/min. For liposome, lipid, and surfactant solutions, bacterial challenge using constant flow may present a higher risk and we would hence recommend conducting validation studies and operating processes of low surface tension adjuvants and adjuvanted vaccines at constant pressure.
Table 3. Summary of the results of metadata analysis. Risk is qualitatively defined here as the likelihood of an occurrence of bacterial penetration during retention validation challenge.
The analysis discussed herein, along with published work by other filter manufacturers, strongly suggests that although bacterial retention by sterilizing grade filters is most commonly achieved by size exclusion, it occasionally can be strongly influenced by the nature of some feed solutions (surface tension, particle size), processing conditions (constant pressure versus constant flow, temperature, operating pressure), membrane structure, and bacterial challenge test conditions (bacterial challenge load, loading rate). Based on the reviewed data, clearly no single membrane is successful in all applications.
For the successful sterilizing filtration of adjuvants and adjuvanted vaccines, we recommend filter users consider the risk of reduced bacterial retention very early in the process, i.e., when designing the formulation, the filtration operating conditions and sequence, and when drafting the sterilizing filtration validation bacterial challenge test protocol. Several membrane candidates for bacterial retention should be tested along with filterability studies, to arrive at the candidate offering both complete bacterial retention and providing the highest filtration capacity for superior process economy. Because of the impact of operating conditions on bacterial retention, it is important to maintain process consistency during scale-up and to design the process with the end in mind.
We would like to thank our colleagues from the Pall Scientific Laboratory Services group and Vincent Guercio in particular, for their efforts in collecting and analyzing field data for this article.
ANNELIES ONRAEDT, PhD, is a global vaccine market manager, MARTHA FOLMSBEE, PhD, is a staff scientist, ANIL KUMAR is a principal R&D engineer, and JEROLD MARTIN is a senior vice president of global scientific affairs, all at Pall Life Sciences, Fribourg, Switzerland, +41 26 350 5360, email@example.com
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