Science Pool

Improving Virus Clearing Studies in Recombinant Protein Production

Key Takeaways:

  • Non-infectious, purified RVLPs can be used in place of model viruses to predict process performance for viral clearance.
  • Initial screenings show that clearance also trends similarly between bench screenings and higher throughput plate screenings.
  • Plate-based screening of RVLPs in-process can examine up to 24 different run conditions simultaneously and uses less viral surrogate compared to bench scale runs, allowing for greater evaluation and confidence going into formal viral clearance studies.

Improving virus clearing studies in recombinant protein production

Chinese hamster ovary (CHO) cells are the most frequently used mammalian host cells for the industrial manufacturing of recombinant protein therapeutics. They can produce recombinant proteins on the scale of up to 10 gram per liter of culture. However, they are also known to contain type‐C endogenous retrovirus (ERV) sequences in their genome and to release retroviral‐like particles. Although evidence for their infectivity is missing, this has raised safety concerns, and regulatory agencies require demonstration that the purification process removes or inactivates viruses.

Viral clearance validation is assessed through “spiking studies”, whereby model mammalian viruses are introduced into process material which then undergoes the purification technique to be tested. Viral quantity before and after processing is determined through infectivity or qPCR assay. As these studies use live viruses, they require specialized Biological Safety Level laboratories (BSL) and experienced personnel and can create a substantial bottleneck because typically only 3rd party facilities are qualified to perform these studies.

As an alternative, Just – Evotec Biologics is in the early stages of establishing a high-throughput process using commercially available purified retrovirus-like particles from Cygnus Technologies LLC. These particles are non-infectious and mimic the physicochemical properties of live infectious viruses. By using these particles as spiking agents, the retroviral clearance capability of downstream unit operations can be studied, assessed, and quantified by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Usually, this is performed at bench scale using chromatography columns.

In a poster presented at this year’s ACS spring conference entitled High throughput optimization of chromatography steps for viral clearance using retrovirus-like particles (RVLPs), researchers from Just-Evotec Biologics detailed the high-throughput workflow for the analysis of RVLP content for rapid analysis of in-process samples.

The research team compared common bench scale chromatography runs with a plate-based screen using resin-loaded filter plates and a liquid handling robot. While at bench scale, only a single set of run conditions can be tested at a time, the plate-based screening can examine up to 24 different run conditions simultaneously. It also uses less RVLP stock solution. The researchers expect that plate-based screening of RVLPs will not only save time and costs, but also allows for better evaluation and confidence before formal viral clearance studies.