Purification process monitoring in monoclonal antibody preparation: Contamination with viruses, DNA and peptide growth factors

Abstract

Administration in vivo of monoclonal antibodies to humans is challenged by considerations regarding their safety. Contamination with viruses, potentially oncogenic nucleic acids and biologically active components like growth factors and hormones forms a serious point of concern in this respect. We have investigated the potential risk of viral contamination by measuring the reduction of 12 different viruses (after spiking) in the standard downstream purification process of ascitic fluid. Depending on the type of virus added and the purification step employed, the reduction of infectious virus particles varies considerably. The overall reduction ranges from about 10 3, observed for a member of the family of Papovaviridae, to more than 10 12 for members of the families of Herpesviridae and Orthomyxoviridae. Using hybridization analysis with a mouse (genomic) DNA probe, we show that the amount of residual DNA in ascitic fluids may also vary considerably, ranging from 75 ng/ml to 1 μg/ml. In crude preparations produced in cell culture, much lower DNA concentrations are found (0·3 ng/ml). When standard downstream purification procedures are applied to ascitic fluid, a significant reduction of residual DNA levels is observed in the purified monoclonal antibody preparations and in intermediate fractions. The overall reduction factors vary from about 10 3 to 10 4, which is also confirmed by spiking experiments with either purified DNA or crude chromatin-like DNA. Using in-vitro cellular assays, we further show that peptide growth factors like PDGF and TGF β are present in considerable amounts in ascitic fluids. The observed biological activities, however, are completely eliminated during the purification steps applied.