Validation of Viral Safety of Recombinant Proteins

Presented by Franz Nothelfer

Franz Nothelfer is associate director of protein science at Boehringer Ingelheim Pharma, Germany.

Cheap, effective, and highly focused tools for the elimination of viruses from recombinant cells are always much in demand because of their wide spread use in the production of biologic drugs. In this presentation on viral safety aspects in downstream development of recombinant proteins, Nothelfer introduced the topic by discussing some general rules for comprehensive evaluation of this complex issue. He stated that validation of virus reduction should be performed before the onset of clinical trials. At least two orthogonal steps should be assessed. The reproducibility of an effective virus reduction step should be demonstrated by at least two independent experiments. Virus reduction studies should include both an enveloped and a small nonenveloped virus (preferably a parvovirus). If cells containing endogenous retroviruses or retrovirus-like particles are used in the protocol, then a specific model virus (such as a murine leukemia virus) should be used to validate the inactivation/removal of viruses to demonstrate full clearance of particles present in the unprocessed bulk.

In this context the term orthogonal, as it relates to process development, is generally understood to mean that multistep purification procedures should use separation mechanisms that are distinct from one another.

Nothelfer discussed his team’s work on different orthogonal steps used in the downstream process for removing viruses. One approach is the inactivation of enveloped viruses at low pH value or through the use of detergents such as Triton X100. An alternative line of attack for the removal of small viruses is the use of a filter with a 20-nm pore size.

Figure 1: Platform processes for monoclonal antibodies (MAbs); AEX = anion-exchange chromatography; CEX = cation-exchange chromatography; UF/DF = ultra/diafiltration

Low pH treatment was found to be extremely effective for removing enveloped viruses in a model system. It was determined that sodium acetate buffer inactivates MuLV (murine leukemia virus) completely at pH values <pH 3.9 and in sodium citrate buffer at a pH value of 3.3. However, inactivation at higher pH values was more difficult to predict for the citrate buffer system.

Nothelfer noted the following guidelines for recombinant protein purification: The downstream process for a recombinant protein should be able to eliminate more virus than is estimated to be present in a single dose equivalent of unprocessed bulk material. And low pH treatment, virus filtration, and anionexchange chromatography are favored because they represent robust virus removal steps in a MAb purification process.

It should be recognized that the downstream treatment for a recombinant protein should be able to eliminate substantially more virus than is estimated to be present in a single-dose equivalent of unprocessed bulk. Before the development of a downstream process of a recombinant protein, the process steps have to be evaluated as potential virus removal steps.

It is essential that these guidelines be followed precisely, because an inadequate viral safety program may cause a substantial delay of clinical studies and approval of a marketable product. For blockbuster biologic drugs, that could result in losses running to millions of euros per day.