1. Improving the analytical toolbox to investigate copurifying host cell proteins presence: N-(4)-(β-acetylglucosaminyl)- l-asparaginase case study
- Author
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Dominique Brault, Suzana Petrovic, Bruno Genet, Delphine Fougeron, Céline Fourneaux, Alessandro Masiero, Yann Fromentin, Bénédicte Laborderie, Jean-Michel Menet, Séverine Clavier, Shibani Mitra-Kaushik, Hagit Elmaleh, Dawid Bugnazet, Patrick Kreiss, and Francis Duffieux
- Subjects
0106 biological sciences ,0301 basic medicine ,Glycan ,host cell proteins ,medicine.drug_class ,Stability study ,In silico ,N‐(4)‐(β‐acetylglucosaminyl)‐ l‐asparaginase ,Bioengineering ,Enzyme-Linked Immunosorbent Assay ,Monoclonal antibody ,01 natural sciences ,Applied Microbiology and Biotechnology ,Article ,HCP identification and quantitation by LC–MS/MS ,L asparaginase ,03 medical and health sciences ,ARTICLES ,Tandem Mass Spectrometry ,010608 biotechnology ,medicine ,Asparaginase ,hitchhiking behavior ,Glucosamine ,biology ,Bioprocess Engineering and Supporting Technologies ,Chemistry ,Immunogenicity ,Antibodies, Monoclonal ,Proteins ,risk assessment ,Recombinant Proteins ,030104 developmental biology ,Biochemistry ,biology.protein ,HCP ELISA assay ,Immunoglobulin G1 ,Biotechnology ,Chromatography, Liquid - Abstract
Levels of host cell proteins (HCPs) in purification intermediates and drug substances (DS) of monoclonal antibodies (mAbs) must be carefully monitored for the production of safe and efficacious biotherapeutics. During the development of mAb1, an immunoglobulin G1 product, unexpected results generated with HCP Enzyme‐Linked Immunosorbent Assay (ELISA) kit triggered an investigation which led to the identification of a copurifying HCP called N‐(4)‐(β‐acetylglucosaminyl)‐l‐asparaginase (AGA, EC3.5.1.26) by liquid chromatography–tandem mass spectrometry (LC‐MS/MS). The risk assessment performed indicated a low immunogenicity risk for the copurifying HCP and an ad hoc stability study demonstrated no mAb glycan cleavage and thus no impact on product quality. Fractionation studies performed on polishing steps revealed that AGA was coeluted with the mAb. Very interestingly, the native digestion protocol implemented to go deeper in the MS–HCP profiling was found to be incompatible with correct AGA detection in last purification intermediate and DS, further suggesting a hitchhiking behavior of AGA. In silico surface characterization of AGA also supports this hypothesis. Finally, the combined support of HCP ELISA results and MS allowed process optimization and removal of this copurifying HCP., During monoclonal antibody 1 development, unexpected results generated with host cell protein (HCP) Enzyme‐Linked Immunosorbent Assay (ELISA) kit triggered an investigation which led to the identification of a copurifying HCP called N‐(4)‐(β‐acetylglucosaminyl)‐l‐asparaginase by liquid chromatography–tandem mass spectrometry (LC‐MS/MS). To mitigate the risk and improve the HCP clearance, a multidisciplinary approach was successfully applied including product and patient risk evaluation, in silico HCP surface characterization, and in‐depth understanding of this HCP behavior during downstream purification process.
- Published
- 2019