Study On Virus Scavenging Ability Of Monoclonal Antibody Downstream Process
Biotechnology products derived from cell lines may be at risk of virus contamination - the extent depends on a number of factors, including the source of preparation, the raw materials used, the production system, purification reagents and excipients. Studying the removal or inactivation of viruses in the manufacturing process is a key step in reducing the potential for iatrogenic transmission of pathogenic viruses and thus reducing the risk, which is essential for the safety of products. Before biological products can enter clinical trials and be marketed, their production process must be demonstrated to have the ability to remove known and presumed viruses.
01 Monoclonal antibody product virus safety assessment
The research on the virus removal ability is usually by adding the known titer indicator virus to the intermediate product at different production stages, then determining the residual virus titer in the sample treated by a specific process, and finally evaluating the log reduction value (LRV) of the virus titer. Process steps for LRV≥4log10 are generally considered to be effective virus removal steps. The process with LRV<41og10 can be considered as helpful to increase the total virus clearance in the production process. However, due to the limitations of virus verification, the process with LRV≤11og10 has little significance for virus removal, and its LRV value is not included in the total amount of the production process.
When using the method of simulated production process (reduced process), it is necessary to design a reasonable virus removal/inactivation research and test plan related to the actual production process as far as possible, especially the effective production process steps. The simulated process should be strictly consistent with the actual process in terms of test parameters and control conditions.
02 Commonly indicates viruses and verification schemes
Common indicator viruses are shown in Table 1.
At the investigational newdrug Application (IND) stage, a biotech product requires viral clearance studies for at least 2 model viruses, typically selecting one retrovirus (X-MuLV) and one parvovirus (MMV). During the biologics license application (BLA) phase, virus removal studies are typically conducted using 3-5 specific/non-specific viruses and 3-5 process steps are evaluated to demonstrate that the product has an adequate safety factor from a virus safety perspective. Due to different guidelines at home and abroad, there are some differences in the virus clearance verification steps used for declarations. The commonly used virus clearance verification schemes in mAb production process are shown in Table 2.
03 Virus removal capability of downstream processes
3.1 The ability of downstream processes to remove different viruses
As of 2009, monoclonal antibody products submitted to the FDA for IND and BLA, Protein A affinity chromatography, Low pH, anion exchange chromatography AEX (flow penetration mode), cation exchange chromatography CEX (binding - elution mode), and virus clearance validation excluding virus filtration are the most commonly used indicator virus families and the mean and range of their clearance effects are shown in Figure 1.
For retroviruses, with the exception of CEX, which has an LRV of about 3log10 (as shown in the A-bar chart in Figure 1), the other processes were robust (LRV≥4log10). Herpesviruses and retroviruses have similar clearance effects and LRV > 4log10(as shown in the B-bar chart in Figure 1). Small unenveloped viruses, such as parvovirus, are more difficult to remove effectively, are resistant to chemical treatment, and are not easily trapped by filters (shown in the C-bar chart in Figure 1). Only AEX and small virus filters were effective in removing parvoviridae (average LRV > 4log10).
The processes shown in Figure 1 are ProteinA, AEX(flow penetration mode), CEX(concomitation-elution mode), Low pH inactivation (" incomplete "versus" complete "removal), and large and small aperture devirus filtration.
3.2 Influencing factors of downstream processes on virus removal capability
The distribution of LRV values for each process is shown in Figure 2. The studies with 0 ~ 21og10 were classified as low LRV group, and those with more than 6log10 were classified as high LRV group for comparative analysis.
3.2.1 ProteinA affinity chromatography
There was no significant correlation between the LRV value of Protein A process and the balance/wash buffer, filler, eluent composition and elution pH value. The common denominator of the low LRV group is that the feedstock is quite complex, and it may be that the complex interaction of feedstock and filler negatively affects the ability of the chromatographic column to remove viruses.
3.2.2 AEX (Flow Through Mode)
Strauss et al. showed that the pH and conductivity of AEX samples can affect the LRV. However, the statistical results of Miesegaes et al showed that, similar to ProteinA, the LRV of AEX (flow penetration mode) had no significant correlation with different buffers, fillers, pH and conductivity, which may be due to the optimization of pH and conductivity in the reported case process conditions.
3.2.3 CEX(Binding - Elution Mode)
Virus removal by CEX process is not robust enough. There was no significant correlation between the LRV in the high LRV group and the buffer, experience level, or any chromatographic column properties, such as column height and resin type. However, the equilibrium/loading and elution pH values used in the high LRV study were both lower, at 5.3±0.2; In the low LRV group, the pH was 6.0±0.2. Another factor is that the salt concentration in the buffer can also cause the LRV value difference. The buffer used in the low LRV study had a higher salt concentration, with an average concentration of 290±120 mM in the loading/balancing solution and 340±70 mM in the eluent, while the concentrations of NaCl in the high LRV study were 58±27 mM and 183±31 mM respectively.
3.2.4 Low pH inactivation
In the Low pH process, pH is a key process parameter, and a pH of 3.8 is sufficient to inactivate the retrovirus by °. The low LRV group had a pH of 3.9, while the high LRV study had a pH of 3.4.
3.2.5 Removing Virus Filtering
For both small and large aperture filters, the LRV value of MuLV removal was no less than 2log10, and only 1% of the studies were below 3log10(shown in the h and i columns in Figure 2). The overall clearance of retroviruses by large aperture virus filters was lower than that by small aperture virus filters (as shown in the C-bar chart in Figure 1). The main reason affecting the result of parvovirus removal is the different filter types. In general, anti-virus filtering can reliably remove viruses.
The processes are Protein A(a), AEX penetration mode (b), CEX binding - elution mode (c), AEX binding Ⅰ elution mode (d), Low pH inactivation (" incomplete "and" complete "clearance)(e ~ g), and large pore size (h) and small pore size (i ~ j) devirus filtration (where, a ~ i: Retrovirus; j: Parvovirus).
04 Innovations and challenges in virus security assessment
The continuous development and innovation in the field of downstream biopharmaceutical processes will inevitably bring about innovations in virus clearance assessment. Advances in upstream and downstream processes-such as continuous stream bioprocesses-have made it necessary to assess and establish effective and robust virus removal processes. During continuous flow, steps such as virus inactivation and virus filtering are still expected to be performed in batch mode. Although the virus removal capability of chromatographic processes in continuous flow mode should not be substantially different from that of batch processing, it must be supported by data.
05 Outlook
From a virological safety perspective, the biopharmaceutical industry's excellent safety can largely be attributed to the industry's strict adherence to three key strategies for viral safety: adequately sourcing and testing of material and cell bank sources, documenting viral clearance assessments (virus validation studies), and in-process viral testing. The characteristics of biologic drugs produced by novel cell substrates may change with different risks, and good risk management strategies are required to ensure the safety of biologic drugs. Guidling Technology has experienced professional technical team, covering the filtration process from small trial, pilot trial, to large-scale production, our membrane and membrane filters are widely used in pre-filtration, microfiltration, ultrafiltration, nanofiltration concentration, extraction and separation; Our many product lines, from small single-use laboratory filtration to production-oriented filtration systems, sterility testing, fermentation, cell culture and more, ensure increased productivity.
Jiuling to ensure product quality as the first indicator, in the pursuit of "reduce costs, increase efficiency" on the road to explore, looking forward to working with you in the future to face the challenges of the industry and seek better development.
