A Detailed Protocol for Generation of Therapeutic Antibodies with Galactosylated Glycovariants at Laboratory Scale Using In-Vitro Glycoengineering Technology |
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Authors: | Sudhakar Voruganti Jiahui Xu Xue Li Gurusamy Balakrishnan Surinder M. Singh Sambit R. Kar Tapan K. Das |
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Affiliation: | 1. Bristol Myers Squibb, Analytical Development and Attribute Sciences, New Brunswick, NJ, USA;2. Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, United States;3. Regeneron Pharmaceuticals, Tarrytown, NY, USA |
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Abstract: | N-linked glycosylation is an important post translational modification that occurs on Asparagine 297 residue or a homologous position on the Fc portion of monoclonal antibodies (mAbs). mAb Fc glycans play important roles in antibody structure, stability, and function including effector function and pharmacokinetics. The Fc glycans are made up of a wide variety of sugars including galactose, mannose, and sialic acid. The role of galactose in mediating antibody effector functions is not well understood. Hence, there is widespread interest in the antibody research community to understand the role of galactose in antibody effector functions as galactose is a major constituent of antibody glycans. This requires generation of highly enriched galactosylated variants that has been very challenging via cell culture process. To tackle this challenge, we developed a laboratory scale biochemical process to produce highly enriched galactosylated variants. In this article, we report optimized lab-scale workflows and detailed protocols for generation of deglycosylated, hypo-galactosylated and hyper-galactosylated variants of IgG therapeutic antibodies using the in-vitro glycoengineering technology. The optimized workflows offer short turnaround time and produce highly enriched deglycosylated/hypo-galactosylated/hyper-galactosylated IgG glycovariants, with high purity & molecular integrity as demonstrated by data from an example IgG. |
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Keywords: | IgG Immunoglobulin Glycan In-vitro glycoengineering IVGE Therapeutic antibody Protocol Galactosylation Hypo-galactosylated Hyper-galactosylated Deglycosylated N-linked glycosylation Enrichment ADCC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0020" }," $$" :[{" #name" :" text" ," _" :" Antibody Dependent Cellular Cytotoxicity ADCP" },{" #name" :" keyword" ," $" :{" id" :" kwrd0030" }," $$" :[{" #name" :" text" ," _" :" Antibody Dependent Cellular Phagocytosis CD" },{" #name" :" keyword" ," $" :{" id" :" kwrd0040" }," $$" :[{" #name" :" text" ," _" :" Circular Dichroism CDC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0050" }," $$" :[{" #name" :" text" ," _" :" Complement Dependent Cytotoxicity CE-SDS" },{" #name" :" keyword" ," $" :{" id" :" kwrd0060" }," $$" :[{" #name" :" text" ," _" :" Capillary Electrophoresis Sodium Dodecyl Sulfate CGE" },{" #name" :" keyword" ," $" :{" id" :" kwrd0070" }," $$" :[{" #name" :" text" ," _" :" Capillary Gel Electrophoresis DSC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0080" }," $$" :[{" #name" :" text" ," _" :" Differential Scanning Calorimetry FLR" },{" #name" :" keyword" ," $" :{" id" :" kwrd0090" }," $$" :[{" #name" :" text" ," _" :" Fluorescence HILIC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0100" }," $$" :[{" #name" :" text" ," _" :" Hydrophilic Liquid Interaction Chromatography iCIEF" },{" #name" :" keyword" ," $" :{" id" :" kwrd0110" }," $$" :[{" #name" :" text" ," _" :" Imaged Capillary Isoelectric Focusing IVGE" },{" #name" :" keyword" ," $" :{" id" :" kwrd0120" }," $$" :[{" #name" :" text" ," _" :" In-Vitro GlycoEngineering mAb" },{" #name" :" keyword" ," $" :{" id" :" kwrd0130" }," $$" :[{" #name" :" text" ," _" :" Monoclonal Antibody LC-MS" },{" #name" :" keyword" ," $" :{" id" :" kwrd0140" }," $$" :[{" #name" :" text" ," _" :" Liquid Chromatography Mass Spectrometry SEC" },{" #name" :" keyword" ," $" :{" id" :" kwrd0150" }," $$" :[{" #name" :" text" ," _" :" Size Exclusion Chromatography UV" },{" #name" :" keyword" ," $" :{" id" :" kwrd0160" }," $$" :[{" #name" :" text" ," _" :" Ultraviolet |
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