Membrane Technology And Vaccine Clarification (Ⅱ)
In the previous article, we had some preliminary introduction to vaccines and vaccine clarification strategies, and we will continue to explore them in the rest of this article. Following on from the above, we will continue to share vaccine clarifications and related membrane tissue applications.
2.2.2 Influence of physical and chemical properties of virus
After considering the production system and the methods to remove the relevant contaminants in the clarification step, it is important to take into account the characteristics of the virus and focus on maximizing the virus yield.
2.2.2.1 Easy virus adsorption
Positively charged materials and filter aids (such as diatomite) have been developed to improve deep filtration effects. Although the positive charge increases the capture of nucleic acids and HCP, diatomite is known to bind cell debris and colloids. However, these materials may also retain the virus through the adsorption mechanism. Since the virus is usually negatively charged in solution, electrostatic interactions with the positively charged filter may occur.
Viruses may also bind by their hydrophobic or nonspecific interactions with certain filter materials (such as diatomite or glass fibers). Enveloped viruses, due to their lipid envelope, are more susceptible to this adsorption. If the virus is adsorbed to the filter through electrostatic interactions, and the virus particles are deattached due to salt competition, rinsing the filter with a highly conductive buffer can partially recover the virus. However, this may also elute contaminants such as HCP or nucleic acids. Therefore, the use of an alternative filter material, such as the more inert polypropylene, is preferred.
Adenovirus is easily adsorbed, but different results have been confirmed. Using positively charged diatomite and deep filters. Borosilicate glass fiber filter material is also very well recovered. On the other hand, a patent proposed by Weggeman involving clarification of 20 – 40% adenovirus losses at PER, et al. Cell cultures were prepared with similarly positively charged deep filters containing diatomite. In this case, the nominal polypropylene filter showed a very high viral recovery rate (> 90%).
It is well known that influenza viruses are prone to adsorption loss during clarification. Therefore, the use of a no-charge filter, the polypropylene-based filter, is suitable for clarifying the influenza collection. Thompson et al reported the use of a nominal rated 1.2 μ m polypropylene filter followed by 0.45 μm PVDF membrane to clarify cell-based influenza virus produced by MDCK cells. A total of nine purification tests were performed at the 20L scale, loading 111 L / m2 for 1.2 μ m polypropylene filter and 105 L / m2 for 0.45 μm PVDF filter. The results showed that the majority of the running viruses recovered well (78-154%). They also reported up to 58% removal of hcDNA, but no significant HCP removal.
2.2.2.2 Clipping sensitive viruses
Some viruses (encapsulated or non-encapsulated) exhibit low mechanical resistance and may be destroyed by shear exposure during centrifugation and membrane filtration steps. Shearing forces generated during purification steps involving filtration or chromatography may cause the viral envelope to fall off, thus affecting infectivity. Depending on the size, thickness, and geometry of the capsid, the viral capsid may be brittle or, conversely, resilient to high pressures. Some enveloped viruses, such as influenza viruses, are elastic to mechanical stress and can withstand large deformation. On the other hand, shear force may cause the envelope of less resistant viruses, such as retroviruses, to fall off, thus affecting the infectivity of the virus.
Extracellular generated envelope VLPs are also very vulnerable. High shear rates are generated in the centrifugal process, mainly at the inlet and outlet parts (high shear rates are generated at the gas-liquid interface). When the virus was purified by gradient centrifugation, the transduction ability of some retroviruses was significantly weakened. When designing centrifugal separation, the relative instability of viral particles to shear forces must be considered. Centrifugal force is not the only source of shear shock, more important is the equipment design, especially at the import and export also have significant shear shock. Differences in the design of different scales may lead to differences in the yield and recovery of shear-sensitive viruses at different scales.
Shear-sensitive viruses should be carefully designed because the magnitude of the shear stress and the time of exposure to the stress (due to recirculation) can be high. For shear-sensitive viruses, open circuit devices (hollow fiber or open plate devices) are preferred to reduce turbulence and shear forces in the feed channel.
The choice of operating parameters should also minimize damage to the virus particles: low cross flow, medium transmembrane pressure (TMP), and short processing time.
Membrane contamination under high pressure leads to loss of viral infectivity, possibly due to the forces that shear action may act on the viral envelope. Membrane based separation is based on size, and the accumulation of large molecular weight viral inhibitors and viral particles can reduce the infectivity of viral vectors.
Degradation of shear-sensitive viruses during deep filtration is not widely documented. Loss of viruses in deep filtration is most often attributed to trapping, adsorption, or time - and temperature-dependent viral degradation of the product. In fact, even though mechanical stress may occur in NFF systems, the exposure time for NFF products to shear is very short compared to other technologies because of the rapid single pass experienced in NFF products.
2.2.2.3 Intercept according to the aperture size
Viruses over 100 nm can be retained by removing mycoplasma or sterile grade membranes (0.22 μm and below). In this case, special attention should be paid to the selection of filters. For microfiltration TFF steps, 0.45μm or 0.65μm membranes are preferred for good product channels. For NFF multi-step filtration, the densest layer is ≥ 0.45μm. Care should be taken when choosing a deep filter, as some deep filter devices may contain a layer of film, which can result in product loss from retention drive. Virus aggregation negatively affects virus production and enhances virus retention due to virus size.
According to a patent by Andre and Champluvier, homogenization can prevent or limit filter clogging by reducing the size of the aggregate, providing a higher yield. Homogenization also improved the filtration capacity of the harvest, which increased by 2.4-3 times.
Too much impurity may interfere with virus recovery. Impurities tend to clog the filter, and clogged membrane pores may result in reduced virus passage rates. In a patent by de Vocht and Veenstra, it is mentioned that the direct clarification of high cell density Per. Collection with TFF(0.65 or 0.2 μm membrane) resulted in adenovirus-free virus recovery. Recovery can be achieved by selective precipitation removal of host cell DNA prior to the 0.65 μm TFF step. 70% of adenoviruses.
2.3 Case study: Optimization of viral vaccine clarification
At the 2011 International Conference on Biological Processes, Sanofi Pasteur presented a rational approach to screening filters for the development of new clarified sequences for candidate viral vaccines. The research aims to overcome the problems faced in optimizing cell and virus culture processes. Modifications to the upstream process resulted in a 20% yield loss and premature filter contamination during the clarification step, resulting in no scale-up. In order to establish a robust and scalable clarification step, a complete re-development of the filter sequence was required, with viral recovery rates higher than 85%.
Based on internal experience and scientific publications, the team selected 27 filters for an initial screening study. Small scale virus adsorption tests were performed on various filter media (polypropylene, nylon, cellulose ester, glass fiber, charged adsorption filter) and structures (pleated or deep filter). The virus yield was measured by ELISA and the clarifying efficiency of the preselected filtrate was compared by checking the reduction of turbidity. Preliminary screening studies showed that nylon and charged filters retained viral particles and virus recovery. Ten percent. The virus recovery rate of polypropylene and polyether sulfone filter was >. 80%. The recovery rate of cellulose ester and glass fiber filters depends on the evaluation of the filter (20% or 90%).
As a second step, Sanofi Pasteur evaluated several combinations (phase 2 or phase 3 sequences) of the seven filters pre-selected in the screening study. The constant flow classification test was carried out with a small filter. In addition, this experiment used a higher yield than the screening study. Based on the results of viral recovery and filter capacity, the team chose the two best combinations for further study.
- Sequence 1(Stage 2):30 μm nominal rated folded polypropylene prefilter, followed by a composite cellulose ester and glass fiber multilayer filter (1/0.5 μm porosity)
- Sequence 2(stage 3): the same prefilter (30 μm nominal rated polypropylene filter), followed by an intermediate multilayer polypropylene filter and finally an asymmetric polyether sulfone film.
The robustness of these two clarified sequences has been challenged by repeated constant flow sizing experiments with different harvest batches. While both potential sequences demonstrated enhanced capabilities compared to the reference sequence, only sequence 1 achieved virus recovery objectives (> 85%), as shown in Figure 1.
Figure 1 Average virus recovery at each filtering step. Stability studies were evaluated for three filtration sequences, of which only sequence 1 using a 30 μm nominal rated folded polypropylene and a 1.0/0.5 μm cellulose ester and glass fiber filter met the global recovery target.
Centrifugation was also evaluated as the primary clarification step, followed by final filtration of 0.45 μm. Several speed/duration pairs were tested. Although the filtration rate of 0.45 μm was increased by two times, the final yield was lower than the target of 85%. As a result, centrifugation has not been studied further.
Finally, the performance of the polypropylene and glass fiber filtration sequences was evaluated on a larger scale (160 L bioreactor size). The filter sequence is shown in Figure 2.
Figure 2 Clarifies the filter combination and a graphical representation of the step yield of the string. Train A is the traditional process and train B is the optimized process. The optimized sequence B can reduce the pre-filtration area by 3 times, cancel the intermediate filtration step, and reduce the final filtration area by 10 times, thus increasing the global virus recovery by 3%.
Several batches were successfully clarified with no signs of filter blockage, process time in line with production limits, and virus yield of > Eighty-five percent. Clarification step optimization had no effect on downstream steps and key quality attributes of the vaccine. Therefore, the selected clarification sequence was used in the vaccine production process (1000 L sized bioreactor) and the performance was successfully confirmed.
03 Clarification of bacterial vaccines
3.1 Considerations for clarification of bacterial vaccines
According to the Medical Thesaurus (2015), a bacterial vaccine is defined as a suspension of diluted or killed bacteria or their antigenic derivatives that is used to induce an immune response to prevent or treat bacterial diseases. More generally, bacterial vaccines can be divided into four sub-categories based on the type of active antigen. This agent can be:
- Kills or weakens the whole living bacteria. Also known as the BCG vaccine.
- Purification of antigenic determinants (subunit vaccines). Anthrax vaccine or acellular pertussis vaccine.
- Bacterial toxins (toxoids). Diphtheria and tetanus toxoids.
- Plasmid (pDNA).
Due to the wide heterogeneity of the family's products, upstream and downstream process challenges largely depend on the type of vaccine being produced. Therefore, after the initial fermentation step can be purified or not purified, so that the clarification step can be carried out.
3.2 Bacterial vaccine clarification strategy
3.2.1 Toxoid
The two most common toxoids produced for vaccine use are diphtheria and tetanus, which are produced by Corynebacterium diphtheriae and Clostridium tetani, respectively. Production of both vaccines is subject to strict regulatory requirements. The WHO technical report and its annexes make clear recommendations to ensure the quality, safety and effectiveness of tetanus and diphtheria vaccines. General Good Manufacturing Practices apply to the production of both vaccines, and employees must be properly trained and receive booster immunization against both diseases.
GMP strictly requires that the purity and quality of the final product must be demonstrated. According to WHO and EP, the efficacy of a final tetanus vaccine must be determined by comparison in vivo or with any other proven method with an appropriate reference substance calibrated in international units according to the International Standard for tetanus toxoid. Updated requirements for efficacy were published in 2011 and may vary depending on the method of assessment. The safety (toxin-free and restorative toxicity) of each batch of vaccine must also be demonstrated. Finally, the stability of vaccines, especially in real time, must be addressed.
3.2.2 Plasmid DNA vaccine
Plasmid DNA vaccines are used for animal health purposes, and several plasmid DNA vaccines for human use are in various stages of development and clinical evaluation. After E. coli fermentation, the bacteria are collected and cleaved to release plasmid DNA.
Removal of cell debris is usually accomplished by centrifugation or filtration. The subject has been covered extensively in recent publications. In this publication, current upstream, downstream, and formulation pDNA processes and challenges.
The authors also provide insight into the gaps at each step of the typical pDNA manufacturing process and potential future innovations and/or current technology gaps that could lead to further process optimization.
Plasmid DNA vaccines are prepared in two steps. First, the bacterial cells are removed from the culture medium, and second, the cell debris after the cell lysis is removed. Depending on the scale, the cells are collected using centrifugation or TFF microfiltration. The disc-pile centrifuge is ejected intermittently at high speed, and the yield of supercoiled plasmids is poor due to shear damage during discharge. If centrifugation must be used, a solid bowl centrifuge is best. Open channel, flat TFF devices with 0.1 or 0.2 μm microfiltration membranes or hollow fiber devices can work well.
Because hollow fiber devices have a high solid load capacity, they are sometimes given priority. Typically these processes operate at 3-5 times the concentration, followed by 3-5 volumes of transfiltration. In order to reduce shear and better control membrane polarization, penetration control operations are highly recommended. Although centrifuges are more cost-effective in large-scale commercial operations, smaller scale processes tend to use filtration because of portability and ease of operation.
Flocculants have been used to facilitate processing, but it can lead to product loss. Some also recommend using inert diatomaceous earth particles, followed by bag filtration.
Cell lysis produces viscous products, including large particles, cell fragments, soluble impurities, fine colloidal particles, and pDNA. Due to the complexity of the material, removing such fine solids is a difficult separation. Gradient density deep filter or open hole structure (> 0.45m) membrane filters are good at removing cell debris. Due to the strong clogging of cell debris, low flow or low pressure filtration is preferred. Tff-based microfilters have been used for this step and industrial-scale bag filters. Static (in a mixing vessel) and continuous (using an in-line static mixer) cracking requires different filters.
3.3 Case study: Comparison of the efficacy of centrifugation, NFF and TFF methods for clarifying tetanus toxins
Muniandi et al. compared three different methods for clarifying tetanus toxins and toxoids from fermentation fluids, namely centrifugation, deep filtration (NFF), and TFF. The test material was produced in a 400L fermenter using a modified Miller (MMM) medium. In the centrifugation study, the cells were separated from the heart at 4000 RPM in a 6 × 1L container for 60min. Samples of supernatant were taken to detect toxoid recovery. Deep filtration uses 0.45μm and 0.22μm deep filters containing diatomaceous earth and cellulose to clarify the fermentation broth. The process is carried out at a temperature of 35℃ and 12psi.
An open flat panel TFF module is thermally bonded to a 0.22μm PVDF membrane in the TFF method. The TFF-based clarification process was performed at a cross flow rate of 2000 L/h at 23℃, and the clarified filtrate was concentrated at a cross flow rate of 1000 L/h at 25℃ using a conventional TFF sandwich 30kD PES membrane. The clarified meat liquid (about 6L) is concentrated 10 times in this ultrafiltration process. Tetanus toxoid tests were performed on concentrated retainer samples to assess product recovery.
Deep filtration resulted in a product recovery rate of approximately 89%, with TFF units resulting in a product recovery rate of over 97%. Microfiltration and ultrafiltration processes consistently yield higher product recoveries than the NFF process. These results are based on flocculation tests (Lf).
04 Clarification of polysaccharide vaccines
4.1 Consideration of polysaccharide vaccine clarification
The production process of both uncoupled/free polysaccharide vaccines and coupled polysaccharide vaccines begins with the culture of the host bacteria in a fermenter. At the end of fermentation, bacteria can be treated with cleaning agents such as DOC(sodium deoxycholate), Triton®X-100, or other suitable reagents to destroy the bacteria and promote the release of polysaccharides. Due to the large battery capacity, collection directly via NFF is not economically feasible as throughput can be very low. Therefore, the ideal choice is to use a centrifuge to separate the cell clumps. The TFF microfiltration range can also be used. The cell-free center/penetrant containing the polysaccharide of interest is further clarified by an NFF deep filtration system, followed by biobattered filtration, and then proceeded to downstream treatment for further purification.
4.2 Polysaccharide vaccine clarification strategy
4.2.1 First Clarification Procedure
Centrifugation is one of the most common techniques for separating cells from fermentation fluids. Depending on the scale, continuous centrifugation or batch centrifugation can be selected. It is important to note that proper optimization of centrifugation conditions and their operation is essential for successful downstream purification. When selecting a specific TFF membrane and pore size, it is important to keep in mind the molecular weight of the polysaccharides, which are often large and structurally complex, with molecular weights ranging from approximately 500kDa to more than 1000kDa. Due to the large open pore size, the use of 0.22μm, 0.45μm, 0.65μm MF membranes can ensure the successful recovery of PS molecules in the osmotic solution.
4.2.2 Secondary Clarification Procedure
The clarity/turbidity of the cell-free fermentation solution that reaches the secondary clarification step depends on the specific bacteria, cleavage type, individual serum type, and the technique used for the primary clarification step. The turbidity of the center can range from about 50 to 150 NTU. A positively charged fractional density deep filter made of impregnated diatomite with filled cellulose fibers can be used to clarify and reduce its turbidity to < 5NTU.
The volume throughput of this deep filter can range from approximately 150 L/m3 to 250 L/m3. Typically, the clarified product solution is filtered through a subsequent 0.45 μm biosupported reduction grade or 0.22 μm sterilized grade membrane to remove any remaining cell particles, colloids, and potential microorganisms.
4.3 Case study: Clarification of the center of Streptococcus pneumoniae fermentation broth after centrifugation
The cells were separated by adding 0.1% (v/v) type 8 Streptococcus pneumoniae fermentation broth (20 L) by continuous centrifugation. The center of the collection is filtered through two separate positively charged and diatomaceous earth deep filters containing cellulose fibers. The individual deep filter filtrate was then filtered through a bioloaded reduction grade PVDF 0.45μm membrane. All filtration tests were performed in constant flow mode with peristaltic pumps. Filtration tests using a charged deep filter and Streptococcus pneumoniae serotype 8 fermentation broth resulted in turbidity dropping from approximately 120 NTU to 3 NTU. The tests were carried out at a flow rate of 140,150 L/m2 /hr and an endpoint pressure difference of 20-25 psi, with a volume throughput of approximately 180-200 L/m2.
Similar filtration tests were performed on the fermentation broth of Streptococcus pneumoniae serotype 19A. The centrifuged 19A liquid is clarified through a charged deep filter, which reduces the turbidity from about 40 NTU to 3 NTU. The tests were performed at a constant flow rate of about 140-160 L/ m2 /hr, and a volumetric throughput of 200-230L/ m2 was achieved at an endpoint pressure of about 15 psid. HLPC analysis of product samples collected during filtration evaluation tests showed no significant yield loss for deep filtration or 0.45 μm (or 0.22 μm) membranes.
05 Conclusion
The development of clarification processes requires the integration of several unit processes such as centrifugation, TFF-MF, deep filtration and aseptic filtration. The optimization of the clarification process requires an understanding of how different unit operations affect each other. The challenge is to select technologies and tools (equipment and fixtures) to meet the increasingly complex requirements of process fluids produced by today's more efficient bioreactors. The increase in upstream productivity (viral titer, cell density, etc.), cell debris, and cell lysis products increases the difficulty of the clarification process and confuses the choice of separation and filtration equipment.
Equipment design, ease of use and cleanliness should be considered when making process scale selection. This will ensure efficient conversion and operator safety when dealing with discarded filters. In order to develop the clarification process, strong integration of the clarification steps is important to ensure that the processing of the upstream harvest is cost-effective. A range of filtration units are readily available to facilitate laboratory testing, pilot production, and full-size processing. By implementing a well-designed scale-up work plan that evaluates multiple clarification options, one can confidently select and size clarification filters to protect downstream unit operations while reducing operating costs.
Vaccine clarification presents several challenges. Typically, the filtration process needs to be tailored to the production system, inactivation or lysis agent, and antigen presentation, not necessarily vaccines. Traditional vaccine processes typically use centrifugation to initially clarify the vaccine. Modern vaccines with multiple technology platforms and smaller processing volumes make vaccines more suitable for clarification by membrane-based technologies. Newly developed vaccines using modern cell lines and expression systems and using more defined cell culture conditions make many vaccine processes more conducive to filtration.
However, heterogeneity in the antigenic component or "target antigen" of vaccine products increases the complexity of filtration clarification. Antigens vary in size, surface chemistry, and charge. These characteristics affect the yield and recovery of antigens. Vaccines present unique challenges for clarification, mainly due to the size of their macromolecules. This, combined with capability issues around clarification, increases the need for guidance on process development strategies.
The size and scale of a commercial-scale operation for vaccine production has a significant impact on the choice of clarification technology. Being located upstream of the process, proper clarification optimization is critical to the success of downstream unit operations, maximizing the yield, recovery and robustness of the process. While centrifugation remains a viable technical option for primary clarification, open channel microfiltration units (TFF) for primary clarification and fine deep filters or membrane filters for secondary clarification are gaining acceptance in the vaccine industry. This change is driven by the need for faster processing, rapid process development, portable processes, and single-use implementations. NFF offers an economical process suitable for small to large scale single-use options. Due to changing regulatory needs, the availability of gamma irradiation pre-aseptic devices or modules designed for autoclaving promotes faster adaptation of NFF or TFF based technologies.
Many classic vaccine processes involve the evolution of clarification unit operations, largely due to regulatory constraints and the associated high costs of revalidation and resubmission or clinical trials. The platform process using the filtration based clarification scheme has been widely used in several biological agents with a high degree of success. The examples and cases outlined in this document show that vaccine manufacturers have the potential to achieve this level of stability, economic viability, and single-use utility by following the template approach.
Other advantages of filtration over centrifugation are shear sensitive viruses or viruses that tend to accumulate at the air interface. As device manufacturers bring new products to market, vaccine manufacturers will continue to be better equipped for the clarification process.
As the first company in the localization circuit, Guidling Technology has accumulated sufficient relevant experience in vaccine clarification. Guidling Technology is a development and production company focusing on biopharmaceutical and cell culture, purification and separation. Products are widely used in biomedicine, diagnosis, industrial fluid filtration, detection, clarification, purification and concentration process; Guidling has successfully developed ultrafiltration centrifuge tube, ultrafiltration/microfiltration membrane cassette, virus removal filter, tangential flow filter device, deep membrane stack, etc., which fully meet the application scenarios of biopharmaceutical and cell culture.
Our membranes and membrane filters are widely used in concentration, extraction and separation of pre-filtration, microfiltration, ultrafiltration and nanofiltration. Our wide range of product lines, from small single-use laboratory filtration to production-type filtration systems, sterility testing, fermentation, cell culture and more, can meet the needs of testing and production.
