Identification of a host cell protein impurity in therapeutic protein, P1.

Residual host cell proteins (HCPs) are process-related impurities present in biotherapeutics that can pose safety health risks to patients. An adequate control of HCP levels in the final product, and demonstration of HCP clearance throughout a product manufacturing process is critical for all biotherapeutic products. Developing effective downstream purification processes can be challenging as HCPs and product proteins may possess an affinity for each other or have similar physicochemical properties, resulting in co-purification. In the current study, we identified the presence of CHO-catalase subunit protein as an impurity present in purified P1 protein. This previously unreported HCP impurity, was detected in P1 protein generated in Chinese hamster ovary (CHO) cells. Purified drug substance samples contained elevated CHO HCP levels when measured using a commercial anti-CHO HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit. This finding, prompted further characterization of the HCP profile using 1D and 2D gels/ western blots using an anti-human IgG antibody as well as a commercial anti-CHO HCP antibody (Cygnus 813) for the detection of host cell proteins. The CHO-catalase protein has been characterized using a combination approach of one-dimensional (1D) and two-dimensional (2D) gels and western blotting techniques, and the identity confirmed using liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Western blot analyses using the anti-CHO HCP antibody detected a potential HCP band at ∼60 kDa and a pI of ∼8 in the purified P1 sample. The 60 kDa HCP band was excised from 1D SDS-PAGE gels and LC-MS/MS analysis identified it to be CHO-catalase subunit. The identity of catalase monomer was further confirmed by western blot analysis using a specific anti-catalase antibody.

Evaluation of automated Wes system as an analytical and characterization tool to support monoclonal antibody drug product development.

Monitoring and evaluation of critical quality attributes (cQA) in monoclonal antibodies (mAb) are a regulatory requirement in pharmaceutical industry. High molecular weight (HMW) species are of critical importance due to the potential risk associated with immunogenicity. HMW species are typically monitored by size exclusion chromatography (SEC). Although low molecular weight (LMW) species are also detected by SEC, low-resolution separation of LMW limits its capability to monitor mAb fragmentation. Recently, we have developed new methods for LMW characterization and evaluation based on the Wes instrument from ProteinSimple. The capillary western blot is based upon size-based separation in a capillary system, and detection by specific immunoprobing, following the separation. The capability of this method for characterization of mAb fragments were demonstrated. The characterization was achieved by probing two antibodies targeted to specific regions (Fc region or Fab region) of IgG1 protein. The specificity of these two antibodies was evaluated against F (ab') 2 and Fc/2 fragments generated from Ides enzyme treated IgG1 protein. The results showed the selected antibodies provide high specificity to F (ab') 2 and Fc/2 fragments. Fractions collected from SEC were used to evaluate this method. The detected fragments from SEC fractions were identified based on their estimated molecular weight and antibody detection. The result proved the capability of the capillary western blot as a characterization method for IgG1 fragments. In addition, with the specific detection to IgG1 and IgG4, the power of the capillary western blot to specifically characterize and evaluate individual IgG fragmentations in an IgG1 and IgG4 mixture was also demonstrated. When heat stressed samples were used, results showed method capability as stability indicating in IgG1 and IgG4 mixture samples. The stressed mixture samples were also evaluated by the total protein assay in which protein samples were biotinylated after separation and were labeled with HRP linked streptavidin to provide chemiluminescence detection. The results indicated total protein assay can be a useful complementary method to capillary western blot immunoassay.