Production and Characterization of High Coverage, Process Specific Pichia P. HCP Antibodies

The production of recombinant protein therapeutics in a number of different organisms including bacteria, yeast and mammalian cells is a rapid growing field in the pharmaceutical industry. A key requirement in this process constitutes the thorough analysis of host cell proteins (HCPs) that specifically result from growth and fermentation conditions as well as downstream purification procedures. Current guidelines call for minimum levels of HCP contaminants that may be left behind during the purification process from the expression hosts. To investigate the presence of residual contamination during the bioprocessing of a final biopharmaceutical product, a common approach is the development of polyclonal antibody reagents with maximum coverage against native HCP extracts.

Here, we describe the overall process of developing a high coverage, process-specific Pichia pastoris HCP antibodies that can be used as an analytical tool for the determination of HCPs in the production of a recombinant human protein. The overall process included characterization of HCPs by one (1D) and two dimensional (2D) SDS-PAGE at distinct early steps of bioprocessing, preparation of cascade immunization antigen to improve reactivity for low abundance/non-immunogenic proteins, continuous monitoring of immune response by 1D and 2D western blot and ELISA as well as final coverage assessment by 2D western blot analysis of purified antibody.

Extensive characterization of the resulting purified HCP antibodies by 2D western blot and ELISA guarantees comprehensive reactivity toward different HCP species that makes a reagent suitable for implementation on a high throughput enzyme-linked immunosorbent assay (ELISA) for HCP testing.