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The viral safety assurance of biological products primarily follows the basic principles of "prevention, detection, and removal." The "removal" mainly refers to the implementation of proper purification and filtration methods in the process to remove viruses. Virus removal filtration is generally based on the physical mechanism of size exclusion and is typically regarded as the most robust step in virus removal processes, especially for small viruses, achieving a removal efficacy exceeding 4 log10. Therefore, virus removal filtration has been widely applied in the purification processes of biological products, such as antibodies, recombinant proteins, and other mammalian cell expression products, biochemical extracts, coagulation factors, intravenous immunoglobulin and other blood products.
In 2021, Cobetter broke through the technical bottleneck of virus removal filtration, launching the Viruclear™ virus removal filters, which has now been over three years. Adhering to the service philosophy of providing customers with more comprehensive virus removal filtration solutions, Cobetter has been continuously engaged in research and development of virus removal membranes made from different materials to adapt to diverse application scenarios, constantly upgrading and optimizing the production processes of filters and components to enhance product filtration performance and stability.
Customer Collaboration Overview
Cobetter virus removal filter series has launched multiple models of products made from polyethersulfone (PES) and regenerated cellulose (RC) materials, allowing clients to conduct testing and selection based on the liquid condition and application scenarios. Each model has been widely applied in client sites.
Currently, over 15 clients have utilized Cobetter Viruclear™ virus filters, completing multiple batch productions with fermentation scales ranging from 2000 to 10000L. More than 10 client projects are in the clinical phase III or post-marketing change application stage, with some successful application cases accumulated. Additionally, many more client projects have completed preliminary process development/pilot research testing or are in the clinical phase I stage, with the performance of Cobetter's Viruclear™ virus filters receiving widespread recognition.
At the same time, Cobetter is continuously expanding into overseas markets, having established close communication and connections with numerous pharmaceutical clients in the field of virus removal filtration in the United States, Japan, Australia, Central and Eastern Europe, India, and other regions.
Outstanding Performance
Virus Removal Capability
Cobetter virus removal filters have demonstrated reliable parvovirus removal capabilities in numerous client testing projects, with Log reduction meeting the regulatory standard of ≥4 log10.
Capacity and Flux
Viruclear™ virus removal filters also exhibit excellent anti-clogging ability. When used in conjunction with pre-filters, they can achieve a high unit area filtration capacity, significantly reducing the cost of the virus clearance step. The testing data for intermediate products expressed in various mammalian cell projects is shown in Figure 1A, which includes McAb, double antibody, and fusion proteins, with data reflecting the quality capacity and average process flux during use. Figure 1B displays the quality capacity and average process flux of using the Viruclear™ RC H filter for filtering intermediate products with IVIG concentrations ranging from about 4.1% to 6.5% under different blood products, with testing durations varying from 6 hours to 24 hours.
The test results indicate that the filtration capacity of Cobetter Viruclear™ virus removal filters can meet client expectations.
Over the years, through product iteration and performance optimization, Cobetter Viruclear™ PES and RC virus removal filters have demonstrated filtration performance comparable to imported competitors across numerous projects at client sites, with stable supply, moderate pricing, and a high cost-performance advantage.
The following figures present experimental data obtained from head-to-head testing of Viruclear™ VF Plus and Viruclear™ RC H against competing filters.
Filtrability Testing Comparative Study (PES)
Bioprocess Laboratory head-to-head filtrability testing:
- 10.8 g/L mAb (frozen-thawed material)
- Pre-filtering with Viruclear PDT
- Samples were parallel loaded post pre-filtration
- Virus removal filtration at a constant pressure of 30 psi
Figure 2. Comparative Case Study of Filtrability Testing for Frozen-Thawed Monoclonal Antibodies with Viruclear™ VF Plus and Competing Virus Removal Filters
Filterability Testing Comparative Study (RC)
Bioprocess Laboratory head-to-head filterability testing:
- 50 g/L IVIG (frozen-thawed material)
- Separate pre-filtering treatment
- Post pre-filtering, samples were individually processed using virus removal filters at their respective recommended pressures.
In both cases mentioned above, Viruclear™ virus removal filters exhibited superior clogging resistance compared to competitors, maintaining a relatively stable filtration process flux at the backend, thereby achieving increased filtration load per unit membrane area and reduced processing time.
Membrane Structure and Strength
The outstanding filtration performance of Viruclear™ virus removal filters is attributed to their excellent membrane strength. As illustrated in the following figure, for example, the Viruclear™ RC H maintains a good linear correlation between pressure and flow rate even when the pressure is gradually increased to 60 psi, indicating that the membrane can sustain structural stability under high pressure.
Through a specialized membrane treatment process and the construction of a composite nylon support layer, the mechanical strength of the Viruclear™ virus removal membranes has been significantly enhanced, allowing them to maintain good pore morphology and internal flow channel structure even under high pressure.
Furthermore, the structure of the support layer enhances membrane strength, effectively mitigating potential damage to the raw membrane that may occur during the assembly process of the finished product. Typically, whether using silicone flat membrane bags or plastic shell membrane bags (capsule), processes such as punching, folding, edge sealing, and welding are prone to damage the unreinforced virus removal membrane, which jeopardizes product yield. However, Cobetter's construction of a support layer structure significantly reduces the complexity and operational risks associated with membrane bag production, greatly improving the production yield of finished filtration components.
The composite nylon support layer is a modified microporous nylon membrane that has been hydrophilically treated, resulting in very low protein (virus particle) adsorption levels. While the support structure provides mechanical strength, we do not expect it to contribute to the adsorption of virus particles, thereby imposing an additional, unstable virus removal mechanism.
Consequently, we conducted a retention challenge for hydrophilically modified nylon, utilizing the PP7 bacteriophage (a small virus model cited in PDA TR 41 as suitable for filter manufacturer release testing) under various buffer systems to confirm its adsorption characteristics for the bacteriophage.
The virus retention test data is summarized in the table below.
The experimental results indicate that the nylon support layer exhibits no retention efficacy for the model virus under various pH and conductivity environments.
To simulate the actual usage conditions for virus removal filtration membranes, we also employed real manufacturing processes, combining the nylon support layer with large-pore 0.22 μm PES membranes and RC membranes, and subjecting them to hydrophilic modification among other processing treatments to evaluate their removal effectiveness against the model virus in different solution environments.
Taking the PES composite membrane as an example, the LRV results for PP7 are shown in the table below:
We concurrently assessed the nylon support layer composite with the 0.22μm PES membrane and RC membrane for their virus challenge removal efficiency against Murine Minute Virus (MVM) under various solution parameters, as illustrated in the table below.
According to Annex 3 of the ICH Guideline Q5A (R2), the 95% confidence limits for detecting within-batch variation results should typically lie within ±0.5 log10 of the mean value. The results presented above all fall within this range, indicating that the nylon support layer and the nylon support-composite large-pore PES membrane exhibit no adsorption removal efficacy against the model virus.
We also investigated the robustness of virus retention of Viruclear™ PES and RC filters under different solution conditions. The physical size exclusion mechanism is generally unaffected by variations in the conditions of the loading solution. The experimental results shown in the figure below further illustrate the reliability of virus removal by membrane filtration based on size exclusion mechanisms, demonstrating that Cobetter Viruclear™ virus removal filters maintain robust virus removal efficacy across different solution environments.
Figure 5. Virus Removal Efficacy of PES Virus Removal Filters against PP7 Bacteriophage and MVM in Various Solution Environments
Figure 6: Virus removal efficiency of RC virus filtration for PP7 bacteria in different solution environments.
The combined results above reflect that the composite nylon support structure does not provide additional virus retention efficacy for virus removal filtration. The Viruclear™ virus removal filters continue to operate based on the size exclusion mechanism, relying on the narrow pore size distribution of the precise retention layer to achieve stable virus retention and removal effects under varied testing conditions.
