title: Influence of glycosylation pattern on the molecular properties of monoclonal antibodies.
abstract: Glycosylation is an important post-translational modification during protein production in eukaryotic cells, and it is essential for protein structure, stability, half-life, and biological functions. In this study, we produced various monoclonal antibody (mAb) glycoforms from Chinese hamster ovary (CHO) cells, including the natively glycosylated antibody, the enriched G0 form, the deglycosylated form, the afucosylated form, and the high mannose form, and we compared their intrinsic properties, side-by-side, through biophysical and biochemical approaches. Spectroscopic analysis indicates no measureable secondary or tertiary structural changes after in vitro or in vivo modification of the glycosylation pattern. Thermal unfolding experiments show that the high mannose and deglycosylated forms have reduced thermal stability of the CH2 domain compared with the other tested glycoforms. We also observed that the individual domain's thermal stability could be pH dependent. Proteolysis analysis indicates that glycosylation plays an important role in stabilizing mAbs against proteases. The stability of antibody glycoforms at the storage condition (2-8 °C) and at accelerated conditions (30 and 40 °C) was evaluated, and the results indicate that glycosylation patterns do not substantially affect the storage stability of the antibody we studied.
authors.affiliation: Late Stage Pharmaceutical Development; Genentech Inc.; South San Francisco, CA USA; Formerly at Oceanside Pharma Technical Development; Genentech Inc.; Oceanside, CA USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Zheng
authors.affiliation: Formerly at Oceanside Pharma Technical Development; Genentech Inc.; Oceanside, CA USA; Department of Cancer Biology; Perelman School of Medicine; University of Pennsylvania; Philadelphia, PA USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Yarmarkovich
authors.affiliation: Formerly at Oceanside Pharma Technical Development; Genentech Inc.; Oceanside, CA USA; Department of Chemistry Development; Illumina Inc.; San Diego, CA USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Bantog
authors.affiliation: Formerly at Oceanside Pharma Technical Development; Genentech Inc.; Oceanside, CA USA; Biotherapeutics Development Unit; Novartis Inc.; San Diego, CA USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Bayer
authors.affiliation: Early Stage Pharmaceutical Development; Genentech Inc.; South San Francisco, CA USA.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Patapoff
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D055592:Biophysical Phenomena; D016466:CHO Cells; D002152:Calorimetry, Differential Scanning; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006358:Hot Temperature; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D010447:Peptide Hydrolases; D011487:Protein Conformation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D010447:Peptide Hydrolases
keywords: Fourier transform infrared spectroscopy; differential scanning calorimetry; glycosylation; intrinsic fluorescence; liquid chromatography–mass spectroscopy; monoclonal antibody; size-exclusion chromatography; stability