Using Retrovirus-Like Particles for Downstream Viral-Clearance Evaluation: Implications of the 2022 ICH Q5A Revision

Regulatory agencies require biopharmaceutical developers to demonstrate effective viral clearance during downstream processing. Historically, companies have outsourced clearance-validation studies to contract research organizations (CROs) that are equipped to spike samples with live pathogens, such as xenotropic murine leukemia virus–related virus (XMuLV). But as David Cetlin (senior director of MockV products from Cygnus Technologies) pointed out in a September 2023 “Ask the Expert” webinar, that strategy is cost- and time-intensive. Thus, developers often postpone viral-clearance assessment until late in process development, when it is disadvantageous to identify needed changes. Cetlin explained how MockV kits with retrovirus-like particles (RVLPs) enable developers to generate viral-clearance data quickly and economically in their own biosafety-level–1 (BSL-1) laboratories. He also highlighted how recent changes to the ICH Q5A guidance may allow for application of RVLPs during viral-clearance validation.

Leveraging RVLPs for Clearance Assessment
Historical Concerns: During the 1970s and 1980s, drug developers expressing proteins in Chinese hamster ovary (CHO) cells identified endogenous particles in process samples. Because those particles resembled retroviruses, regulatory agencies established requirements in the 1997 publication of Q5A for demonstrating endogenous-retrovirus clearance, with tests occurring before investigational new drug (IND) or biologics license application (BLA) filings.

In the 2000s, the biopharmaceutical industry began adopting XMuLV as a model virus for such studies. Meanwhile, research continued into RVLPs, with characterization efforts revealing the particles to be noninfectious. Soon after that discovery, researchers devised a method for quantifying RVLPs after protein-A and anion-exchange (AEX) chromatography steps. That prompted investigation into assessment of viral clearance by spiking samples with RVLPs rather than live XMuLV, which requires specialized work in high-BSL laboratories.

The MockV RVLP kit was designed to assist with viral-clearance evaluation, initially for process development and characterization and now also for validation studies. The kit supplies components for a spiking study, including RVLPs that are isolated from a proprietary CHO cell line and formulated in a stock solution to a concentration of 10¹⁰ RVLPs/mL. Analysis by dynamic light scattering (DLS) has confirmed that the RVLPs are the same size as XMuLVs, and transmission electron microscopy (TEM) has shown the particles to have similar morphologies, making the RVLPs suitable surrogates.

During kit application, analysts spike samples with the supplied RVLPs, then process material through a desired purification step. Purified samples are loaded onto a 96-well plate for RNA extraction, material from which is loaded onto another plate and mixed with primers and probes against a conserved region of the RVLP genome for analysis by quantitative polymerase chain reaction (qPCR). Those results enable calculation of viral-clearance capability.

Cetlin noted that the kit assay’s 96-well format enables users to analyze 23 samples (in triplicate) and requisite standards on the same plate. The assay runs over a broad dynamic range, returning log reduction values (LRVs) of 4–5 log₁₀, and it has undergone validation for accuracy and for intra- and interassay precision.

Emerging Applications
Standard Viral-Clearance Assessment: Cetlin provided several examples of Cygnus’s work with industry partners to evaluate the MockV RVLP method’s performance. One company assessed the assay’s accuracy by measuring RVLP concentrations in unspiked samples from CHO-culture supernatant. Cetlin explained that downstream teams often analyze supernatant content to establish baseline values for LRV determination over a purification process. In this case, the MockV assay and TEM returned nearly identical RVLP titers, indicating the kit method’s suitability for RVLP quantitation.

Other customers compared results from MockV RVLP spiking studies with historical data from XMuLV-based experiments to evaluate viral clearance at different downstream steps. One organization measured RVLP content in spiked samples that underwent protein A chromatography using the same resin, process parameters, and spiking loads as were applied in XMuLV studies. RVLP and XMuLV titers and associated LRVs lined up nicely (within ~0.5 log₁₀ of each other) for material collected at all process steps, including the elution fraction. Another company performed similar tests to assess a viral-filtration step, with the MockV RVLP and XMuLV spiking studies both showing full particle clearance.

Users have observed comparable results when applying the kit during AEX, cation-exchange (CEX), and hydrophobic-interaction chromatography (HIC) processes. Cetlin called attention to the HIC studies, during which XMuLV clearance was inadequate over two runs but improved in different process conditions, ultimately to full clearance. The RVLP assay returned similar values across tested conditions. Such examples, he said, indicate the MockV RVLP kit’s suitability for viral-clearance assessment and its comparability with spiking studies based on live XMuLV.

Supplementing Regulatory Justifications: Historically, MockV products have been used for research and development (R&D), process development, and process characterization. Cetlin highlighted, however, that the 2022 revision of ICH Q5A permits use of RVLPs during spiking studies for viral-clearance validation. That provision, along with permission to use prior knowledge in IND and BLA filings, could reduce drug developers’ dependence on CROs and other service providers to perform validation studies.

For instance, a developer can leverage accumulated viral-clearance data from products purified by a platform process to support filings for similar candidates that will undergo the same process. Combining historical process data with results from RVLP-spiking studies, published literature, and/or standards can help make a case to regulatory agencies about reducing the scope of — or even eliminating — requisite validation studies. As Cetlin explained, “Conducting MockV-style studies to supplement data that you have accumulated could help you to come up with the best data package possible to justify your technique.”

Cospiking Studies: Cygnus offers two MockV kits, one for spiking samples with RVLPs and another for loading noninfectious surrogates of minute virus of mice (MVM), also called mock virus particles (MVM-MVPs). Customers until recently have needed to perform distinct studies with each kit. However, Cygnus scientists have investigated the possibility of spiking samples with both particle types.

Using samples from CHO-culture supernatant bearing immunoglobulin G (IgG), the team conducted preliminary matrix-interference studies to determine whether the presence of MVM-MVPs influenced RVLP quantitation and vice versa. Regardless of the other particle type’s presence in the samples, the MockV RVLP and MVM-MVP kits accurately quantified their respective analytes. Next, supernatant samples were spiked with RVLPs only, MVM-MVPs only, or both particle types, then purified by protein G affinity chromatography. Viral-clearance data were analyzed to determine LRVs for each run type. Results from those studies showed that cospiking MVM-MVPs in the RVLP assay had no significant influence on RVLP quantitation. The same was true when cospiking RVLPs in the MVM-MVP assay.

Additional Support
Cetlin noted that Cygnus performs several services related to the MockV product line. Customers can conduct spiking studies and send samples to Cygnus for analysis. Results generally can be reported in two to three weeks. Customers also can submit process samples — e.g., from harvested CHO supernatant or protein A flowthrough — from which Cygnus scientists will isolate, concentrate, and formulate RVLP content. With that material, users can perform spiking studies with RVLPs derived from their own CHO cell lines.

Questions and Answers
Have companies already tried to supply RVLP data in place of XMuLV values for clinical or commercial filings? The Q5A revision opens up that possibility, and at recent industry events (e.g., the PDA Viral Clearance Forum in Madrid, Spain), EU regulatory representatives have supported using RVLP data for validation purposes. It might be a matter of time before companies begin submitting applications with RVLP data instead of results from new validation experiments performed with live virus.

How important is it to develop upstream-production and viral-inactivation capabilities concurrently? That strategy makes sense considering that companies often undertake process development and characterization around the same time that they optimize viral-inactivation and other post-capture steps.

Can the MVM and RVLP mock-virus kits be used to evaluate viral-inactivation capability? Cygnus discourages using the MVM-MVP kit for that step because MVM particles are extremely difficult to inactivate using low pH or chemical treatment. However, the RVLP kit can be useful for assessing inactivation processes. A working group plans to publish a study about that application early in 2024.

How can MockV kit RVLPs be distinguished from those generated by a given CHO-cell process? Generally, customers do not need to differentiate between particle types. For instance, if users are analyzing samples from after a protein-A capture step, then negligible levels of process-specific RVLPs probably remain, enabling analysts to focus on measuring spiked RVLPs. But when differentiation is necessary, one strategy is to perform preliminary analysis of sample RVLP content, then compare that titer value with one generated from running an unspiked sample on the kit. That helps to check for matrix interference and provides baseline titers for subsequent analyses of spiked samples.

What are the shelf lives of the MockV kits? The RVLP kit is stable for up to one year in 4 °C storage. The MVM kit is stable in such conditions for up to two years.

More Online
Watch the complete webinar and learn more about emerging MockV kit applications online at https://bioprocessintl.com/sponsored-content/the-use-of-retrovirus-like-particles-rvlp-to-evaluate-viral-clearance-in-downstream-processing.