title: An innovative approach for the characterization of the isoforms of a monoclonal antibody product.
abstract: Protein biopharmaceuticals, such as monoclonal antibodies (mAbs) are widely used for the prevention and treatment of various diseases. The complex and lengthy upstream and downstream production methods of the antibodies make them susceptible to physical and chemical modifications. Several IgG1 immunoglobulins are used as medical agents for the treatment of colon, breast, and head and neck cancers, and at least four to eight isoforms exist in the products. The regulatory agencies understand the complex nature of the antibody molecules and allow the manufactures to set their own specifications for lot release, provided the safety and efficacy of the products are established in animal models prior to clinical trials. During the manufacture of a mAb product, we observed lot-to-lot variability in the isoform content and, although the variability is within the set specifications for lot release, made attempts to gain mechanistic insight by isolating and characterizing the individual isoforms. Matrix-assisted laser desorption/ionization (MALDI) and liquid chromatography (LC)/mass spectrometry (MS)/MS analyses of the isolated isoforms indicate that this variability is caused by sialic acid content, as well as truncation of C-terminal lysine of the individual isoforms. Sialidase and carboxypeptidase treatment of the product confirm the observations made by MALDI and LC/MS/MS.
authors: Bioanalytical Science, ImClone Systems, Branchburg, NJ, USA.
authors: Shanmuuga
authors: S
authors: Sundaram
authors: Alice
authors: A
authors: Matathia
authors: Jun
authors: J
authors: Qian
authors: Jingming
authors: J
authors: Zhang
authors: Ming-Ching
authors: MC
authors: Hsieh
authors: Tun
authors: T
authors: Liu
authors: Richard
authors: R
authors: Crowley
authors: Babita
authors: B
authors: Parekh
authors: Qinwei
authors: Q
authors: Zhou
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D001709:Biotechnology / Q000379:methods; D002268:Carboxypeptidases / Q000378:metabolism; D002853:Chromatography, Liquid; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000032:analysis; D013058:Mass Spectrometry; D019158:N-Acetylneuraminic Acid / Q000032:analysis; D009439:Neuraminidase / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D020033:Protein Isoforms; D009439:Neuraminidase; D002268:Carboxypeptidases; D019158:N-Acetylneuraminic Acid; D008239:Lysine