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A technical rountable featuring Sartorius Stedim Biotech, Pall Life Sciences, 3M Purification, Asahi Kasei Bioprocess, and Bio-Rad Laboratories.
Adjuvants are becoming more common in vaccine and other drug formulations to increase therapeutic response. Some of these substances, however, are close enough in size to bacteria that they are unable to pass through sterilizing-grade filters. Others have low surface tension that can reduce a filter's bacterial retention. As a result, adjuvants can cause premature plugging of filter membranes and reduce filter capacity. BioPharm International spoke to several industry experts to gain insight on resolving these technical challenges.
BioPharm: Novel adjuvants are often based on emulsions or liposomes, which are suspensions of small particles made up of surfactant or lipid particles. Because these formulations have a relatively high viscosity and because the typical particle size of the micelles or liposomes is close to the size of the smallest bacteria to retain, they result in a difficult separation process. In addition, these fluid streams often contain high particle loads which can cause premature plugging of sterilizing-grade filters. How can pharmaceutical or filter manufacturers reduce such filter plugging or pore blockage?
(PHOTO CREDIT: SARTORIUS STEDIM)
Bromm (Sartorius Stedim): One possibility for filter manufactures to deal with these challenges is to develop sterilizing-grade filters that specifically address these needs. According to our experience at Sartorius Stedim, highly asymmetric membranes, such as polyethersulfone (PES) membranes provide higher flow rate and capacity for such type of formulations compared with symmetric membranes. According to practical experiences, the use of a heterogeneous double-layer membrane construction provides total throughput advantages compared with single layer membrane filters. The prefilter (i.e., upstream layer) protects the final membrane (i.e., downstream layer) from premature plugging. Of high importance is to find the optimal graduation between two membranes. Studies with model solutions and test results with actual formulations in field tests have demonstrated that the combination of a finer prefilter membrane with the final 0.2 µm membrane achieves better results compared with combinations with a coarser prefilter membrane for adjuvants applications.
Pharmaceutical manufacturers should carry out filtration studies to compare the performance of different membrane materials and construction principals of filters to find out the optimal solution for their specific formulation. Furthermore, the use of prefilters should be considered in such studies to protect final sterilizing-grade filters effectively and to reduce costs and filtration time. These studies can be used to determine the optimal parameters for the filtration process, such as differential pressure or temperature. Increasing the temperature can enhance filterability depending on the stability of the solution at higher temperatures. The same filter-selection process may be applied for other protein therapeutics or vaccines.
Martin (Pall): Pharmaceutical manufacturers can reduce filter plugging by optimizing formulation and process conditions for desired filter life, along with selection of appropriate filters with suitable capacity. Filter manufacturers can provide technical support for this process by conducting feasibility (filterability) trials, selecting appropriate filter-media grades, sizing of filter cartridges or capsules, as well as ultimately applying that knowledge to the development of new filters capable of providing greater capacity.
Process parameters such as pressure, temperature, and flux (i.e., flow per unit area) can have a large impact on filter throughput and capacity. For example, with complex plugging biological fluids, performing the filtration in a constant flow mode, increasing pressure differential to maintain flux rather than operating under a constant pressure mode can often have a positive impact on filtration throughput (capacity). Process temperature can also have an impact but is product-dependent and needs feasibility (filterability) tests to determine whether an improvement can be achieved through modification. Optimizing these performance variables is an acceptable (and recommended) technique to reduce the risk of premature blockage for vaccines or protein therapeutics.
Koklitis (3M): The plugging of membrane filter systems by adjuvants is particularly undesirable when the process step has been validated to provide sterility assurance. The risk of filter plugging can be reduced by careful control of the filtration operating conditions, such as inlet pressure and optimum flux. The lifetime of the sterilizing-grade filter membrane will be greatly determined by the particle load in the process feedstream and the capacity may be extended with a prefiltration stage. A prefilter rated at 0.45 μm will remove larger emulsion micelles or liposomes which might ordinarily plug a sterilizing 0.2 μm membrane. Another option is to consider a 0.2 μm-rated bioburden reduction membrane as a prefilter. This can be of the same material as the final sterilizing membrane to simplify validation and may be effective for removing larger particle sizes from the process stream as a result of its pore size distribution. The prefiltration system selected should be sized appropriately to meet the demands of the process stream to minimize the expense associated with the final sterilizing membrane stage. When emulsions are used, the pharmaceutical manufacturer could investigate an adjuvant formulation with a sufficiently small particle size to make it filter-sterilizable.
Some studies with oil-in-water emulsions have shown that increasing the pressure drop across the membrane can increase filter capacity. The coating of bacteria on the membrane with emulsion has been considered to contribute to bacterial penetration. In such instances, higher bacterial retention may be achieved by increasing the temperature if cold conditions are currently used. However, the reasons for adopting cold filtration (e.g., to maintain protein stability) may present an obstacle to implementing a change.
Powell (Asahi): This is rather hard to answer because the blocking can occur due to a wide range of issues related to product use and conditions such as pH, conductivity, protein concentration, viscosity, temperature, membrane incompatibility with what is in the adjuvant, and so forth. The best solution would be to better characterize the adjuvant, the product, and the combination to find the most stable and best filter condition possible, where material is not precipitating, too viscous, too high a concentration, and/or at the early stage or "edge" of aggregation and the filter type where the adjuvant's oil, if present, does not bind to or change/damage the membrane itself.
There are really two choices: the brute force method, where one throws more membrane at the problem, or the better method, which would be to choose the right adjuvant for the job and choose conditions that fit into a high stability window of operation for the API. Another more sophisticated solution to these kinds of clogging problems is to use a cascade of filters that end in the final desired porosity. The upstream filter(s) can act as prefilters to increase final filter capacity.
BioPharm: Low surface tension of some adjuvant solutions can reduce the efficiency of filters' bacterial retention. How can this problem be mitigated?
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Bromm (Sartorius Stedim): It is advisable and required by regulators to carry out a comprehensive filter validation study, including bacteria-retention testing, simulating worst-case process parameters with actual product formulation using process related (i.e., pleated) scale-down filter devices. The design of the filtration system should consider reducing filtration time and differential pressure because these two parameters, among others, may increase the risk for bacterial breakthrough. During a filter evaluation study, the impact of different inlet pressure filtration conditions should be assessed, including constant flow or constant pressure conditions. Constant flow conditions may increase the risk of bacterial breakthrough, because of the increased differential pressure required to keep the flow constant during the filtration process and increased filter blocking.
The use of filters specifically designed for adjuvant filtration as explained above is highly recommended because those filters will keep the process parameters at a moderate level. It is recommended to carry out a bacterial-retention study early in the filter-selection process to find the optimal solution based on retention efficiency and highest filtration capacity.
Martin (Pall): Statistical and empirical studies at Pall Corporation have identified low surface tension of some adjuvant solutions as a risk factor for reduced bacterial retention efficiency of most sterilizing grade 0.2 μm rated membrane filters. The mechanism by which bacterial retention is reduced under lower surface tension in these fluids is not yet fully elucidated. Some mitigating factors appear to be membrane structure and layering of multilayer media, operating conditions, as well as reduction of bacterial bioburden or challenge levels and reducing challenge duration. Fluid surface tension affects the interactions between the bacteria and the membrane flow-path surfaces, but detailed mechanisms are not well known and specific surface tension thresholds cannot be determined.
Membrane surface chemistry is also an element that may mitigate the negative impact of fluid surface tension. Determining how and to what extent membrane-surface chemistry can enhance retention requires extensive studies. Filters with positive zeta potential, which provide enhanced adsorptive removal properties for bacteria in aqueous ionic solutions, have been used in the past for such purposes. This was also one of the capability advantages of asbestos-containing filters, although these are no longer used because of asbestos safety concerns.
Koklitis (3M): Such reduced filter efficiency can be related to the mechanisms involved in bacterial retention, which can be based not only on sieving but also on entrapment and electrostatic attraction. The adsorption of bacteria to the membrane polymer surface can be caused by any combination of forces, including hydrogen bonding, charge-induced, and Van der Waals interactions. The presence of liposomes, oils, or surfactants in a process stream can disrupt these adsorptive interactions and consequently reduce retention of bacteria within the membrane structure.
When there may be a high risk of bacterial penetration, it should be identified and considered in the planning of a filter validation study. The required minimum bacteria challenge (1 × 107 colony forming units of Brevundimonas diminuta per cm2 effective filter area) must apply, although an upper challenge level can be considered and restricted to one log higher. In a full-scale production process, the bacterial challenge to the final filter membrane may be controlled by introducing a prefiltration stage that has been demonstrated to be effective for bioburden reduction. The careful management and control of the operating conditions during process filtration will also help mitigate the risk of bacterial penetration, with attention to flow rate and filter area sizing to avoid high pressure drop.
Powell (Asahi): This issue is typically not applicable to Asahi products, but with some filters, the lower surface tension can change the effective porosity rating of the membrane, allowing larger particles to slip through the membrane's holes. These low viscosity adjuvants effect the thickness of the boundary layer (where flow velocities at the membrane surface are at or close to zero) which, in turn, alters the effective pore size under those conditions. It can also affect how the API and contaminants build up around the membrane's pores hence altering the effective pore size. One can screen different membrane types, porosities, and brands of filters, and work closely with the membrane filter supplier to choose the best filter for the application.
BioPharm: Can certain types of adjuvants cause fewer problems with regard to filters' bacterial retention?
Bromm (Sartorius Stedim): A review of validation studies and field tests for a broader variety of fluid formulations indicates that low surface tension formulations, such as many adjuvants or adjuvanted vaccines, present a higher risk for bacterial penetration of sterilizing-grade membrane filters. Among such formulations, according to the data analyzed, liposome formulations present a higher risk than surfactant containing solutions. Therefore, the use of such formulations may be a suitable alternative to replace more critical formulations where applicable.
Martin (Pall): It is possible that certain adjuvants and related low surface tension fluids may be intrinsically less likely than others to cause reduced retention efficiency by membrane filters. However, there is insufficient data at this time to draw firm conclusions and make recommendations. In addition, awareness among vaccine producers that selection of surfactant-containing adjuvants and processing conditions can influence bacterial retention efficiency of sterilizing filters is not yet widespread. Until then, filter manufacturers must continue to work with vaccine developers to define appropriate membranes and optimize reasonable processing conditions to sterilize any vaccine formulation. Certainly, elaboration of an optimum adjuvant with such a goal would require an extensive amount of work and a very close partnership between filter manufacturers and vaccine producers.
Koklitis (3M): The choice of adjuvant is dependent on meeting the requirements of the process under consideration. The pros and cons of using a particular type of adjuvant must be considered and compared. When liposomes are selected as adjuvants their role as antigen carriers is utilized along with their immunological enhancement effect.
Powell (Asahi): These issues should be discussed with the membrane supplier's technical support teams and if they can't help, the filters must be screened to choose the best solution for the filter application. The answer depends on the membrane chemistry, but for large porosity filters, surfactant-containing solutions are typically not that large of a problem. Smaller porosity filters can be dramatically impacted in a negative way.
BioPharm: How can manufacturers maintain product flow g during adjuvant filtration?
Bromm (Sartorius Stedim): For the manufacturer of the adjuvants, it is important to study and understand the process variables involved in making the adjuvant. The process variables identified to have a significant impact on the filterability of the formulation should be controlled carefully and kept within a narrow operating window. This will enable constant performance of the filtration process within established process parameters.
Martin (Pall): Filter plugging may or may not be an inherent part of a filtration process, depending on the particulate nature of the influent solution. An efficient filter is designed to retain bacteria and therefore tends to retain any particulate of a similar and larger size (e.g., micelles, liposomes). The ideal filter, with an extremely narrow pore-size distribution, a very high porosity, free of pinholes or other defects, and with sufficient area, will present the best compromise between bacterial retention and filtration capacity.
If a specific flow rate is desired over the duration of a filtration operation where the potential for plugging exists, the filtration operation should be performed under constant flow mode using an appropriately sized filtration area. Product flow can be maintained by increasing the inlet pressure as needed. Throughput of complex biological fluids often benefits from operation in his constant flow mode, as opposed to operating at high initial pressure and allowing flux to decay as the filter plugs.
With adjuvanated vaccines, or similar products at risk for reduced bacterial retention efficiency, preliminary filterability trial performed at the initial stages of process developments can identify filters providing the highest level of sterility assurance for further formulation or process optimization, perhaps including limited microbial challenges to confirm initial suitability. Further filterability studies can then focus on optimizing process time and economy under operating parameters known to further increase bacterial retention likelihood with these highest assurance filters. This will maximize both retention and throughput to provide for successful sterilizing filtration, validation, and processing.
Koklitis (3M): As mentioned, the careful management and control of the operating conditions during process filtration is usually advantageous for achieving consistency and robustness. In addition, the choice of filter membrane type can can contribute to maintaining a consistent flow. An asymmetric membrane structure, with a more open upstream zone, can provide a relatively higher initial flux, for example, which results in higher filter capacity for some process streams.
A higher filter surface area can be obtained per cartridge cylinder by selecting products that use advanced pleat technologies, thus enabling higher throughput without increasing filter-system size. This approach may help with filtering highly viscous process streams, such as emulsions.
Powell (Asahi): Large areas of membrane is the brainless solution, but working with filter vendors and doing DOE-based filter screening under the desired, "high stability" API conditions is the better choice.
Just like in horse racing, where some horses perform better than others on different courses, choosing the right filter type or perhaps a cascade of filters can solve the problem and provide a balanced solution to your filtration problem. If your feed contaminant is primarily a slowly precipitating molecule of some sort, a relatively small coarse filter such as a 1 μm or 5 μm might be able to trap it and allow a medium-sized sterile grade filter handle the higher flow rate and process larger volumes.
Depth filters often provide significantly higher capacity than membrane filters so placing them upstream of a sterile grade filter is often a good idea when possible. As with any filter, but especially depth filters, a study of undesired reduction (by binding) in solution components should be considered. Find a balanced approach to this cascade of filters, with each filter sized appropriately to deal with and control the specific contaminant that causes the processing roadblock.
Featured in the roundtable: Holger Bromm, director of marketing and product management filtration technologies at Sartorius Stedim Biotech; Jerold Martin, senior vice-president of Global Scientific Affairs at Pall Life Sciences; Peter Koklitis, a technical filtration specialist at 3M Purification in the United Kingdom; and Jim Powell, business development manager of Asahi Kasei Bioprocess.