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Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product

Coronavirus Disease 2019 (COVID-19) is a major public health problem worldwide with 5–10% hospitalization and 2–3% global mortality rates at the time of this publication. The disease is caused by a betacoronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The receptor-bind...

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Autores principales: Szabo, Miklos, Sarkozy, Daniel, Szigeti, Marton, Farsang, Robert, Kardos, Zsofia, Kozma, Adam, Csanky, Eszter, Chung, Doo Soo, Szekanecz, Zoltan, Guttman, Andras
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8957997/
https://www.ncbi.nlm.nih.gov/pubmed/35350184
http://dx.doi.org/10.3389/fbioe.2022.839374
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author Szabo, Miklos
Sarkozy, Daniel
Szigeti, Marton
Farsang, Robert
Kardos, Zsofia
Kozma, Adam
Csanky, Eszter
Chung, Doo Soo
Szekanecz, Zoltan
Guttman, Andras
author_facet Szabo, Miklos
Sarkozy, Daniel
Szigeti, Marton
Farsang, Robert
Kardos, Zsofia
Kozma, Adam
Csanky, Eszter
Chung, Doo Soo
Szekanecz, Zoltan
Guttman, Andras
author_sort Szabo, Miklos
collection PubMed
description Coronavirus Disease 2019 (COVID-19) is a major public health problem worldwide with 5–10% hospitalization and 2–3% global mortality rates at the time of this publication. The disease is caused by a betacoronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The receptor-binding domain (RBD) of the Spike protein expressed on the surface of the virus plays a key role in the viral entry into the host cell via the angiotensin-converting enzyme 2 receptor. Neutralizing monoclonal antibodies having the RBD as a target have the ability to inhibit angiotensin-converting enzyme 2 (ACE2) receptor binding, therefore, prevent SARS-CoV-2 infection, represent a promising pharmacological strategy. Bamlanivimab is the first anti-spike neutralizing monoclonal antibody, which got an emergency use authorization from the FDA for COVID-19 treatment. Albeit, bamlanivimab is primarily a neutralizing mAb, some of its effector function related activity was also emphasized. The effector function of antibody therapeutics is greatly affected by their N-linked carbohydrates at the conserved Fc region, possibly influenced by the manufacturing process. Various capillary gel electrophoresis methods are widely accepted in the biopharmaceutical industry for the characterization of therapeutic antibodies. In this paper we introduce a capillary gel electrophoresis based workflow for 1) size heterogeneity analysis to determine the presence/absence of the non-glycosylated heavy chain (NGHC) fragment (SDS-CGE); 2) capillary gel isoelectric focusing for possible N-glycosylation mediated charge heterogeneity determination, e.g., for excess sialylation and finally, 3) capillary gel electrophoresis for N-glycosylation profiling and sequencing. Our results have shown the presence of negligible amount of non-glycosylated heavy chain (NGHC) while 25% acidic charge variants were detected. Comprehensive N-glycosylation characterization revealed the occurrence of approximately 8.2% core-afucosylated complex and 17% galactosylated N-linked oligosaccharides, suggesting the possible existence of antibody dependent cell mediated cytotoxicity (ADCC) effector function in addition to the generally considered neutralizing effect of this particular therapeutic antibody molecule.
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spelling pubmed-89579972022-03-28 Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product Szabo, Miklos Sarkozy, Daniel Szigeti, Marton Farsang, Robert Kardos, Zsofia Kozma, Adam Csanky, Eszter Chung, Doo Soo Szekanecz, Zoltan Guttman, Andras Front Bioeng Biotechnol Bioengineering and Biotechnology Coronavirus Disease 2019 (COVID-19) is a major public health problem worldwide with 5–10% hospitalization and 2–3% global mortality rates at the time of this publication. The disease is caused by a betacoronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The receptor-binding domain (RBD) of the Spike protein expressed on the surface of the virus plays a key role in the viral entry into the host cell via the angiotensin-converting enzyme 2 receptor. Neutralizing monoclonal antibodies having the RBD as a target have the ability to inhibit angiotensin-converting enzyme 2 (ACE2) receptor binding, therefore, prevent SARS-CoV-2 infection, represent a promising pharmacological strategy. Bamlanivimab is the first anti-spike neutralizing monoclonal antibody, which got an emergency use authorization from the FDA for COVID-19 treatment. Albeit, bamlanivimab is primarily a neutralizing mAb, some of its effector function related activity was also emphasized. The effector function of antibody therapeutics is greatly affected by their N-linked carbohydrates at the conserved Fc region, possibly influenced by the manufacturing process. Various capillary gel electrophoresis methods are widely accepted in the biopharmaceutical industry for the characterization of therapeutic antibodies. In this paper we introduce a capillary gel electrophoresis based workflow for 1) size heterogeneity analysis to determine the presence/absence of the non-glycosylated heavy chain (NGHC) fragment (SDS-CGE); 2) capillary gel isoelectric focusing for possible N-glycosylation mediated charge heterogeneity determination, e.g., for excess sialylation and finally, 3) capillary gel electrophoresis for N-glycosylation profiling and sequencing. Our results have shown the presence of negligible amount of non-glycosylated heavy chain (NGHC) while 25% acidic charge variants were detected. Comprehensive N-glycosylation characterization revealed the occurrence of approximately 8.2% core-afucosylated complex and 17% galactosylated N-linked oligosaccharides, suggesting the possible existence of antibody dependent cell mediated cytotoxicity (ADCC) effector function in addition to the generally considered neutralizing effect of this particular therapeutic antibody molecule. Frontiers Media S.A. 2022-03-08 /pmc/articles/PMC8957997/ /pubmed/35350184 http://dx.doi.org/10.3389/fbioe.2022.839374 Text en Copyright © 2022 Szabo, Sarkozy, Szigeti, Farsang, Kardos, Kozma, Csanky, Chung, Szekanecz and Guttman. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Szabo, Miklos
Sarkozy, Daniel
Szigeti, Marton
Farsang, Robert
Kardos, Zsofia
Kozma, Adam
Csanky, Eszter
Chung, Doo Soo
Szekanecz, Zoltan
Guttman, Andras
Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product
title Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product
title_full Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product
title_fullStr Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product
title_full_unstemmed Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product
title_short Introduction of a Capillary Gel Electrophoresis-Based Workflow for Biotherapeutics Characterization: Size, Charge, and N-Glycosylation Variant Analysis of Bamlanivimab, an Anti-SARS-CoV-2 Product
title_sort introduction of a capillary gel electrophoresis-based workflow for biotherapeutics characterization: size, charge, and n-glycosylation variant analysis of bamlanivimab, an anti-sars-cov-2 product
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8957997/
https://www.ncbi.nlm.nih.gov/pubmed/35350184
http://dx.doi.org/10.3389/fbioe.2022.839374
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