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Removal of microorganisms is crucial when working with biologics. Sterile filtration offers a reliable, safe, and effective way to ensure product integrity.
In many circumstances, sterile filtration is necessary to remove particles or microorganisms that may affect the safety or efficacy of a biologic. Various processes and tests can be implemented to ensure that filtration is successful. BioPharm International spoke with Dr. Jens Meyer, product manager, filtration technologies at Sartorius Stedim Biotech; Cindy Neeley, PhD, application scientist at Thermo Fisher Scientific; and Tom Watson, global product manager at Pall Corporation, about ensuring filter validation, determining the correct filter pore size, and the importance of keeping filtration processes up to date.
The filtration processBioPharm: How can one ensure that all sterile filtration equipment and processes are up to date?
Meyer (Sartorius Stedim Biotech): Because sterilizing-grade filters consist of a very thin membrane that functions as a sterile barrier, any mechanical impact must be avoided as much as possible. In addition, wetting of a filter membrane plays a crucial role in integrity testing. Due to limitations on temperature and differential pressure, all sensors need to be routinely calibrated and checked. Obviously, the entire equipment and product-contacted surfaces have to be steam-sterilized before starting a filtration run.
Neeley (Thermo Fisher Scientific): A well-designed filtration system begins with choosing a filtration membrane appropriate for your fluid. Differences in how a filter is manufactured, the material components used, and how it performs will have a direct impact on your work. Always double-check the type of containers you entrust with valuable samples and solutions. Storage bottle and cap systems, such as the Thermo Scientific Nalgene bottles and caps, are constructed to minimize gas permeability and maintain sample integrity over time.
Watson (Pall Corporation): Given the continuing and rapid adoption of single-use systems, it is important that any filters currently being tested at benchtop or pilot scale are available in configurations that are compatible with gamma irradiation. Their performance should be validated by the filter supplier, and any process-specific filter validation studies should be performed using filters that have been sterilized by the method intended at process scale.
From an applications standpoint, newer, complex parenteral formulations using nanoparticles and liposomes tend to be harder to filter than traditional formulations. For those engaged in the manufacture of hard-to-filter formulations, it is worthwhile to identify a sterilizing-grade filter that will have good throughput capacity for these fluids. It’s also critically important to ensure that bacterial retention is confirmed early through filter validation studies.
In addition to securing a reliable sterilizing-grade filter, it can be useful to work with suppliers who can offer state-of-the-art integrity test equipment, a whole suite of complementary single-use technologies combined with systems know-how and a high level of validation expertise.
BioPharm: What methods are used to determine the pore size of a filter needed to thoroughly remove all microorganisms?
Meyer (Sartorius Stedim Biotech): According to a current American Society for Testing and Materials (ASTM) standard (F838-05), a sterilizing-grade filter must completely retain Brevundimonas diminuta microorganisms when challenged with 107 bacteria per cm² of filtration area. Standard integrity tests applied by end-users, such as the bubble point or diffusion tests, have to be correlated with the bacterial challenge tests. In principle, porometric investigations can be used to confirm a filter’s pore size distribution around 0.2 µm. For mycoplasma retention, 0.1-µm-rated membrane filters are used.
Neeley (Thermo Fisher Scientific): At Thermo Scientific, membrane bacterial retention tests are performed to determine the microbial retention capabilities of a filter with a certain pore size. Following a challenge of microorganisms of a certain level, the microorganisms are recovered from the filtrate and counted after an incubation per protocol (e.g., ASTM F838-05). The 0.2-μm filters typically used to remove bacteria are certified to retain a challenge of 1 x 107 colony-forming unit (CFU)/cm2B. diminuta.
Watson (Pall Corporation): The method used to determine whether or not a sterilizing-grade filter of a given removal rating will produce a sterile effluent in a manufacturing process is a process-specific bacterial challenge study. Ahead of such a study, it is recommended that the filter end-user begin with the selection of a filter that has been validated by its manufacturer as retentive for B. diminuta in accordance with test method ASTM F838-05 at a challenge level of 107 CFU per cm2 effective filter area. Typically, filters rated at 0.2 µm and 0.1 µm sterilizing-grade make this generic microbial retention claim.
The subsequent process-specific bacterial challenge study, performed with an appropriate challenge organism at the same challenge level, yet under ‘worst-case’ operating conditions, can confirm whether or not a selected filter will deliver a sterile final drug or vaccine product.
The use of multiple filtersBioPharm: What are the advantages and disadvantages of using multiple filters?
Meyer (Sartorius Stedim Biotech): In most cases, a single filter sufficiently ensures the sterility of a final product. For fluids containing particles, a cascade of prefilters and final filters increases filtration capability. Redundant filtration using two sterilizing-grade filters sequentially is often believed to prevent the loss of a product batch if one filter fails an integrity test. However, this approach increases safety at the expense of filtration performance, which is lowered due to loss of pressure across the first filter.
Neeley (Thermo Fisher Scientific): Manufacture certification guarantees removal of microorganisms to a certain level, even with a single filtration process. Within the manufacturer’s guidelines, single-filter filtration can be effective, economical, and environmentally friendly. Multiple filters should be used if the filtration volume or bioburden exceeds the capacity recommended by the manufacturer for a single filter.
Watson (Pall Corporation): The additive effect of multiple filters can have a positive impact on process cleanliness and safety. In the case of sterilizing-grade filters used in parenteral fill/finish applications, multiple filters (i.e., two or more identical filters positioned sequentially in a system) may be installed for different purposes. In some cases, two identical sterilizing-grade filters may be necessary because a single sterilizing-grade filter may not be able to achieve complete bacterial retention during process-specific validation studies. In such ‘serial filtration’ or ‘double filtration’ set-ups, both filters would be expected to pass an integrity test for batch release. Alternatively, a sequence of identical filters can incorporate a redundant filter. For example, in a process where a single sterilizing-grade filter has been validated to produce a sterile effluent, a redundant filter would be a duplicate of this, installed in-line.
Aside from the direct cost implications of using multiple filters, there are further considerations around the impact of extractables, adsorption, and integrity testing complexity. However, concerns over increasing costs and other factors should not detract from the economic and safety benefits that can result from more intensive filter usage. A risk-based approach with support from filter suppliers may help the filter user arrive at the appropriate filtration schematic, with the appropriate balance of safety and economy.
Compatibility testsBioPharm: What tests, if any, are conducted to determine the compatibility and interactions between the end product and filter?
Meyer (Sartorius Stedim Biotech): The chemical compatibility of the different membrane materials and of the most frequently used product ingredients is known and published by filter suppliers. If compatibility with a particular ingredient is unknown or doubtful, specific tests can reveal structural changes to the membrane, the effects on integrity test values, or the potential impact on a membrane’s bacterial retention. Process-specific filter validation allows identification of potential effects on the final product, such as by verifying the concentration of the ingredients in the first vials.
Neeley (Thermo Fisher Scientific): In general, binding tests are performed to ensure that the filter membranes do not remove any valuable components from the end product (e.g., the protein). Extractable and leachable tests of the filter membrane ensure the end product is not contaminated with the filter material. A chemical compatibility test is crucial to meet the requirements of organic solvents, or certain pH conditions. Furthermore, flow-rate tests are important for filtering fluids with high viscosity.
Environmental considerationsBioPharm: How do environmental variables within the laboratory affect the process of sterilization?
Meyer (Sartorius Stedim Biotech): General environmental parameters like room temperature and humidity will not affect the sterilization process itself. For integrity testing, all auxiliary media and equipment should be room temperature. In very rare cases, it has been shown that exposure of stainless-steel filter housings to direct sunlight might interfere with the procedure. The same could happen when hot or cold water is used for performing water intrusion tests. However, this only appears rarely and is not considered a main concern during the filtration process.
Neeley (Thermo Fisher Scientific): Sterilization by filtration is achieved when the filtrate passes through the sterilizing (usually 0.2 μm) membrane into a sterile collection vessel. Until this point, the solution is not considered sterile and does not specifically need to be handled as such, unless the work environment requires this to prevent cross-contamination. Therefore, an environment with positive air pressure (e.g., a biological safety cabinet or a clean room) is preferable.
Article DetailsBioPharm International
Vol. 28, No. 6
Pages: 24–25, 49
Citation: When referring to this article, please cite it as A. Roberts, “Sterile Filtration Techniques for Optimal Microbial Retention,” BioPharm International 28 (6) 2015.