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1
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Wang Q, Wang T, Wu WW, Lin CY, Yang S, Yang G, Jankowska E, Hu Y, Shen RF, Betenbaugh MJ, Cipollo JF. Comprehensive N- and O-Glycoproteomic Analysis of Multiple Chinese Hamster Ovary Host Cell Lines. J Proteome Res 2022; 21:2341-2355. [PMID: 36129246 DOI: 10.1021/acs.jproteome.2c00207] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Glycoproteomic analysis of three Chinese hamster ovary (CHO) suspension host cell lines (CHO-K1, CHO-S, and CHO-Pro5) commonly utilized in biopharmaceutical settings for recombinant protein production is reported. Intracellular and secreted glycoproteins were examined. We utilized an immobilization and chemoenzymatic strategy in our analysis. Glycoproteins or glycopeptides were first immobilized through reductive amination, and the sialyl moieties were amidated for protection. The desired N- or O-glycans and glycopeptides were released from the immobilization resin by enzymatic or chemical digestion. Glycopeptides were studied by Orbitrap Liquid chromatography-mass spectrometry (LC/MS), and the released glycans were analyzed by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Differences were detected in the relative abundances of N- and O-glycopeptide types, their resident and released glycans, and their glycoprotein complexity. Ontogeny analysis revealed key differences in features, such as general metabolic and biosynthetic pathways, including glycosylation systems, as well as distributions in cellular compartments. Host cell lines and subfraction differences were observed in both N- and O-glycan and glycoprotein pools. Differences were observed in sialyl and fucosyl glycan distributions. Key differences were also observed among glycoproteins that are problematic contaminants in recombinant antibody production. The differences revealed in this study should inform the choice of cell lines best suited for a particular bioproduction application.
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Affiliation(s)
- Qiong Wang
- Laboratory of Bacterial Polysaccharides, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States.,Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21210, United States
| | - Tiexin Wang
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21210, United States
| | - Wells W Wu
- Facility for Biotechnology Resources, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States
| | - Chang-Yi Lin
- Facility for Biotechnology Resources, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States
| | - Shuang Yang
- Laboratory of Bacterial Polysaccharides, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States.,Center for Clinical Mass Spectrometry, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215123, China
| | - Ganglong Yang
- Department of Pathology, Johns Hopkins University, Baltimore, Maryland 21287, United States.,Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China
| | - Ewa Jankowska
- Laboratory of Bacterial Polysaccharides, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States
| | - Yifeng Hu
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21210, United States
| | - Rong-Fong Shen
- Facility for Biotechnology Resources, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States
| | - Michael J Betenbaugh
- Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21210, United States
| | - John F Cipollo
- Laboratory of Bacterial Polysaccharides, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland 20993, United States
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2
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Beauglehole AC, Roche Recinos D, Pegg CL, Lee YY, Turnbull V, Herrmann S, Marcellin E, Howard CB, Schulz BL. Recent advances in the production of recombinant factor IX: bioprocessing and cell engineering. Crit Rev Biotechnol 2022; 43:484-502. [PMID: 35430942 DOI: 10.1080/07388551.2022.2036691] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
Appropriate treatment of Hemophilia B is vital for patients' quality of life. Historically, the treatment used was the administration of coagulation Factor IX derived from human plasma. Advancements in recombinant technologies allowed Factor IX to be produced recombinantly. Successful recombinant production has triggered a gradual shift from the plasma derived origins of Factor IX, as it provides extended half-life and expanded production capacity. However, the complex post-translational modifications of Factor IX have made recombinant production at scale difficult. Considerable research has therefore been invested into understanding and optimizing the recombinant production of Factor IX. Here, we review the evolution of recombinant Factor IX production, focusing on recent developments in bioprocessing and cell engineering to control its post-translational modifications in its expression from Chinese Hamster Ovary (CHO) cells.
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Affiliation(s)
- Aiden C. Beauglehole
- ARC Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, QLD, Australia
- CSL Innovation, Parkville, Victoria, Australia
| | - Dinora Roche Recinos
- ARC Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, QLD, Australia
- CSL Innovation, Parkville, Victoria, Australia
| | - Cassandra L. Pegg
- ARC Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, QLD, Australia
- School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, Australia
| | | | - Victor Turnbull
- CSL Innovation, Bio21 Institute of Molecular Science and Biotechnology, Parkville, Victoria, Australia
| | - Susann Herrmann
- CSL Innovation, Bio21 Institute of Molecular Science and Biotechnology, Parkville, Victoria, Australia
| | - Esteban Marcellin
- ARC Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, QLD, Australia
| | - Christopher B. Howard
- ARC Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, QLD, Australia
| | - Benjamin L. Schulz
- ARC Training Centre for Biopharmaceutical Innovation, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, QLD, Australia
- School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, Australia
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3
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Chawla H, Jossi SE, Faustini SE, Samsudin F, Allen JD, Watanabe Y, Newby ML, Marcial-Juárez E, Lamerton RE, McLellan JS, Bond PJ, Richter AG, Cunningham AF, Crispin M. Glycosylation and Serological Reactivity of an Expression-enhanced SARS-CoV-2 Viral Spike Mimetic. J Mol Biol 2022; 434:167332. [PMID: 34717971 PMCID: PMC8550889 DOI: 10.1016/j.jmb.2021.167332] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2021] [Revised: 10/21/2021] [Accepted: 10/21/2021] [Indexed: 02/06/2023]
Abstract
Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe.
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Affiliation(s)
- Himanshi Chawla
- School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK
| | - Sian E Jossi
- Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, UK
| | - Sian E Faustini
- Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, UK
| | - Firdaus Samsudin
- Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore 138671, Singapore
| | - Joel D Allen
- School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK
| | - Yasunori Watanabe
- School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK; Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
| | - Maddy L Newby
- School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK
| | - Edith Marcial-Juárez
- Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, UK
| | - Rachel E Lamerton
- Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, UK
| | - Jason S McLellan
- Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA
| | - Peter J Bond
- Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore 138671, Singapore; Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore
| | - Alex G Richter
- Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, UK
| | - Adam F Cunningham
- Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, UK
| | - Max Crispin
- School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK.
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4
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Mota LM, Tayi VS, Butler M. Cell Free Remodeling of Glycosylation of Antibodies. Methods Mol Biol 2022; 2370:117-146. [PMID: 34611867 DOI: 10.1007/978-1-0716-1685-7_6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
The N-glycosylation profile of a monoclonal antibody (mAb) is a critical quality attribute in relation to its therapeutic application. The control of this profile during biomanufacture is difficult because of the multiple parameters that affect the glycosylation metabolism within the cell and the environment in which the cell is grown. One of the approaches that can be used to produce a preferred glycan profile or a single glycoform is through chemoenzymatic remodeling during the isolation of a mAb. Here we describe protocols that can be utilized to produce preferred glycoforms that include galactosylated, agalactosylated, or sialylated glycoforms following isolation of a mAb. Methods for analysis and assignment of structures of the samples following glycoengineering are also described. Chemoenzymatic modeling of mAb glycans has the potential for scale-up and to be introduced into biomanufacturing of mAbs with higher specific therapeutic activities.
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Affiliation(s)
- Letícia Martins Mota
- Cell Technology Group, National Institute for Bioprocessing, Research and Training (NIBRT), Dublin, Ireland
| | - Venkata S Tayi
- Department of Microbiology, University of Manitoba, Winnipeg, Canada
| | - Michael Butler
- National Institute for Bioprocessing, Research and Training (NIBRT), Dublin, Ireland.
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5
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Stevenson-Leggett P, Armstrong S, Keep S, Britton P, Bickerton E. Analysis of the avian coronavirus spike protein reveals heterogeneity in the glycans present. J Gen Virol 2021; 102. [PMID: 34424155 PMCID: PMC8513636 DOI: 10.1099/jgv.0.001642] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein’s surface. Here we used IBV propagated in embryonated hens’ eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.
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Affiliation(s)
| | - Stuart Armstrong
- Institute of Infection and Global Health, University of Liverpool, Liverpool L3 5RF, UK
| | - Sarah Keep
- The Pirbright Institute, Woking, Surrey GU24 0NF, UK
| | - Paul Britton
- The Pirbright Institute, Woking, Surrey GU24 0NF, UK
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6
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Allen JD, Chawla H, Samsudin F, Zuzic L, Shivgan AT, Watanabe Y, He WT, Callaghan S, Song G, Yong P, Brouwer PJM, Song Y, Cai Y, Duyvesteyn HME, Malinauskas T, Kint J, Pino P, Wurm MJ, Frank M, Chen B, Stuart DI, Sanders RW, Andrabi R, Burton DR, Li S, Bond PJ, Crispin M. Site-Specific Steric Control of SARS-CoV-2 Spike Glycosylation. Biochemistry 2021; 60:2153-2169. [PMID: 34213308 PMCID: PMC8262170 DOI: 10.1021/acs.biochem.1c00279] [Citation(s) in RCA: 47] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2021] [Revised: 06/18/2021] [Indexed: 02/08/2023]
Abstract
A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.
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Affiliation(s)
- Joel D. Allen
- School
of Biological Sciences, University of Southampton, Southampton SO17 1BJ, U.K.
| | - Himanshi Chawla
- School
of Biological Sciences, University of Southampton, Southampton SO17 1BJ, U.K.
| | - Firdaus Samsudin
- Bioinformatics
Institute, Agency for Science, Technology
and Research (A*STAR), Singapore 138671
| | - Lorena Zuzic
- Bioinformatics
Institute, Agency for Science, Technology
and Research (A*STAR), Singapore 138671
- Department
of Chemistry, Faculty of Science and Engineering, Manchester Institute
of Biotechnology, The University of Manchester, Manchester M1 7DN, U.K.
| | - Aishwary Tukaram Shivgan
- Bioinformatics
Institute, Agency for Science, Technology
and Research (A*STAR), Singapore 138671
- Department
of Biological Sciences, National University
of Singapore, Singapore 117543
| | - Yasunori Watanabe
- School
of Biological Sciences, University of Southampton, Southampton SO17 1BJ, U.K.
| | - Wan-ting He
- Department
of Immunology and Microbiology, The Scripps
Research Institute, La Jolla, California 92037, United States
- IAVI
Neutralizing Antibody Center, The Scripps
Research Institute, La Jolla, California 92037, United States
- Consortium
for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, California 92037, United States
| | - Sean Callaghan
- Department
of Immunology and Microbiology, The Scripps
Research Institute, La Jolla, California 92037, United States
- IAVI
Neutralizing Antibody Center, The Scripps
Research Institute, La Jolla, California 92037, United States
- Consortium
for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, California 92037, United States
| | - Ge Song
- Department
of Immunology and Microbiology, The Scripps
Research Institute, La Jolla, California 92037, United States
- IAVI
Neutralizing Antibody Center, The Scripps
Research Institute, La Jolla, California 92037, United States
- Consortium
for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, California 92037, United States
| | - Peter Yong
- Department
of Immunology and Microbiology, The Scripps
Research Institute, La Jolla, California 92037, United States
- IAVI
Neutralizing Antibody Center, The Scripps
Research Institute, La Jolla, California 92037, United States
- Consortium
for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, California 92037, United States
| | - Philip J. M. Brouwer
- Department
of Medical Microbiology, Amsterdam UMC,
University of Amsterdam, Amsterdam Infection & Immunity Institute, 1007 MB Amsterdam, The Netherlands
| | - Yutong Song
- Tsinghua-Peking
Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
- Beijing
Advanced Innovation Center for Structural Biology and Frontier Research
Center for Biological Structure, Beijing 100084, China
| | - Yongfei Cai
- Division
of Molecular Medicine, Boston Children’s
Hospital, 3 Blackfan
Street, Boston, Massachusetts 02115, United States
| | - Helen M. E. Duyvesteyn
- Division
of Structural Biology, University of Oxford,
The Wellcome Centre for Human Genetics, Headington, Oxford OX3 7BN, U.K.
| | - Tomas Malinauskas
- Division
of Structural Biology, University of Oxford,
The Wellcome Centre for Human Genetics, Headington, Oxford OX3 7BN, U.K.
| | - Joeri Kint
- ExcellGene SA, CH1870 Monthey, Switzerland
| | - Paco Pino
- ExcellGene SA, CH1870 Monthey, Switzerland
| | | | - Martin Frank
- Biognos AB, Generatorsgatan
1, 41705 Göteborg, Sweden
| | - Bing Chen
- Division
of Molecular Medicine, Boston Children’s
Hospital, 3 Blackfan
Street, Boston, Massachusetts 02115, United States
- Department
of Pediatrics, Harvard Medical School, 3 Blackfan Street, Boston, Massachusetts 02115, United States
| | - David I. Stuart
- Division
of Structural Biology, University of Oxford,
The Wellcome Centre for Human Genetics, Headington, Oxford OX3 7BN, U.K.
- Diamond Light Source Ltd., Harwell Science
& Innovation Campus, Didcot OX11 0DE, U.K.
| | - Rogier W. Sanders
- Department
of Medical Microbiology, Amsterdam UMC,
University of Amsterdam, Amsterdam Infection & Immunity Institute, 1007 MB Amsterdam, The Netherlands
- Department
of Microbiology and Immunology, Weill Medical
College of Cornell University, New York, New York 10065, United States
| | - Raiees Andrabi
- Department
of Immunology and Microbiology, The Scripps
Research Institute, La Jolla, California 92037, United States
- IAVI
Neutralizing Antibody Center, The Scripps
Research Institute, La Jolla, California 92037, United States
- Consortium
for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, California 92037, United States
| | - Dennis R. Burton
- Department
of Immunology and Microbiology, The Scripps
Research Institute, La Jolla, California 92037, United States
- IAVI
Neutralizing Antibody Center, The Scripps
Research Institute, La Jolla, California 92037, United States
- Consortium
for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, La Jolla, California 92037, United States
- Ragon Institute of Massachusetts General
Hospital, Massachusetts
Institute of Technology, and Harvard University, Cambridge, Massachusetts 02139, United States
| | - Sai Li
- Tsinghua-Peking
Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
- Beijing
Advanced Innovation Center for Structural Biology and Frontier Research
Center for Biological Structure, Beijing 100084, China
| | - Peter J. Bond
- Bioinformatics
Institute, Agency for Science, Technology
and Research (A*STAR), Singapore 138671
- Department
of Biological Sciences, National University
of Singapore, Singapore 117543
| | - Max Crispin
- School
of Biological Sciences, University of Southampton, Southampton SO17 1BJ, U.K.
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7
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Savizi ISP, Motamedian E, E Lewis N, Jimenez Del Val I, Shojaosadati SA. An integrated modular framework for modeling the effect of ammonium on the sialylation process of monoclonal antibodies produced by CHO cells. Biotechnol J 2021; 16:e2100019. [PMID: 34021707 DOI: 10.1002/biot.202100019] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2021] [Revised: 05/19/2021] [Accepted: 05/20/2021] [Indexed: 12/11/2022]
Abstract
BACKGROUND Monoclonal antibodies (mABs) have emerged as one of the most important therapeutic recombinant proteins in the pharmaceutical industry. Their immunogenicity and therapeutic efficacy are influenced by post-translational modifications, specifically the glycosylation process. Bioprocess conditions can influence the intracellular process of glycosylation. Among all the process conditions that have been recognized to affect the mAB glycoforms, the detailed mechanism underlying how ammonium could perturb glycosylation remains to be fully understood. It was shown that ammonium induces heterogeneity in protein glycosylation by altering the sialic acid content of glycoproteins. Hence, understanding this mechanism would aid pharmaceutical manufacturers to ensure consistent protein glycosylation. METHODS Three different mechanisms have been proposed to explain how ammonium influences the sialylation process. In the first, the inhibition of CMP-sialic acid transporter, which transports CMP-sialic acid (sialylation substrate) into the Golgi, by an increase in UDP-GlcNAc content that is brought about by the augmented incorporation of ammonium into glucosamine formation. In the second, ammonia diffuses into the Golgi and raises its pH, thereby decreasing the sialyltransferase enzyme activity. In the third, the reduction of sialyltransferase enzyme expression level in the presence of ammonium. We employed these mechanisms in a novel integrated modular platform to link dynamic alteration in mAB sialylation process with extracellular ammonium concentration to elucidate how ammonium alters the sialic acid content of glycoproteins. RESULTS Our results show that the sialylation reaction rate is insensitive to the first mechanism. At low ammonium concentration, the second mechanism is the controlling mechanism in mAB sialylation and by increasing the ammonium level (< 8 mM) the third mechanism becomes the controlling mechanism. At higher ammonium concentrations (> 8 mM) the second mechanism becomes predominant again. CONCLUSION The presented model in this study provides a connection between extracellular ammonium and the monoclonal antibody sialylation process. This computational tool could help scientists to develop and formulate cell culture media. The model illustrated here can assist the researchers to select culture media that ensure consistent mAB sialylation.
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Affiliation(s)
- Iman Shahidi Pour Savizi
- Faculty of Chemical Engineering, Biotechnology Department, Tarbiat Modares University, Tehran, Iran
| | - Ehsan Motamedian
- Faculty of Chemical Engineering, Biotechnology Department, Tarbiat Modares University, Tehran, Iran
| | - Nathan E Lewis
- Department of Bioengineering, University of California, La Jolla, California, USA.,School of Medicine, Novo Nordisk Foundation Center for Biosustainability at the University of California, La Jolla, California, USA.,Department of Pediatrics, School of Medicine, University of California, La Jolla, California, USA
| | | | - Seyed Abbas Shojaosadati
- Faculty of Chemical Engineering, Biotechnology Department, Tarbiat Modares University, Tehran, Iran
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8
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Modified secreted alkaline phosphatase as an improved reporter protein for N-glycosylation analysis. PLoS One 2021; 16:e0251805. [PMID: 34032812 PMCID: PMC8148361 DOI: 10.1371/journal.pone.0251805] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2021] [Accepted: 05/04/2021] [Indexed: 11/19/2022] Open
Abstract
N-glycosylation is a common posttranslational modification of proteins in eukaryotic cells. The modification is often analyzed in cells which are able to produce extracellular, glycosylated proteins. Here we report an improved method of the use of genetically modified, secreted alkaline phosphatase (SEAP) as a reporter glycoprotein which may be used for glycoanalysis. Additional N-glycosylation sites introduced by site-directed mutagenesis significantly increased secretion of the protein. An improved purification protocol of recombinant SEAP from serum or serum-free media is also proposed. The method enables fast and efficient separation of reporter glycoprotein from a relatively small amount of medium (0.5-10 ml) with a high recovery level. As a result, purified SEAP was ready for enzymatic de-glycosylation without buffer exchange, sample volume reductions or other procedures, which are usually time-consuming and may cause partial loss of the reporter glycoprotein.
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9
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Allen JD, Chawla H, Samsudin F, Zuzic L, Shivgan AT, Watanabe Y, He WT, Callaghan S, Song G, Yong P, Brouwer PJM, Song Y, Cai Y, Duyvesteyn HME, Malinauskas T, Kint J, Pino P, Wurm MJ, Frank M, Chen B, Stuart DI, Sanders RW, Andrabi R, Burton DR, Li S, Bond PJ, Crispin M. Site-specific steric control of SARS-CoV-2 spike glycosylation. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2021:2021.03.08.433764. [PMID: 33758835 PMCID: PMC7986994 DOI: 10.1101/2021.03.08.433764] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/25/2023]
Abstract
A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.
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10
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Madabhushi SR, Podtelezhnikov AA, Murgolo N, Xu S, Lin H. Understanding the effect of increased cell specific productivity on galactosylation of monoclonal antibodies produced using Chinese hamster ovary cells. J Biotechnol 2021; 329:92-103. [PMID: 33549674 DOI: 10.1016/j.jbiotec.2021.01.023] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2020] [Revised: 01/12/2021] [Accepted: 01/31/2021] [Indexed: 12/22/2022]
Abstract
Achieving optimal productivity and desired product quality of the therapeutic monoclonal antibody (mAb) is one of the primary goals of process development. Across the various mAb programs at our company, we observed that increasing the specific productivity (qp) results in a decrease in the % galactosylation (%Gal) level on the protein. In order to gain further insight into this correlation, cells were cultured under different process conditions such as pH or media osmolality or in the presence of supplements such as sodium butyrate. A range of qp and N-glycan profiles were obtained with the greatest changes observed under high pH (lower qp, higher %Gal), higher osmolality (higher qp, lower %Gal) or sodium butyrate (moderately higher qp, moderately lower %Gal) conditions. Abundance of individual glycan species highlighted different bottlenecks in the N-glycosylation pathway depending on the treatment condition. Transcriptomics analysis was performed to identify changes in gene expression profiles that correlate with the inverse relationship between qp and %Gal. Results showed downregulation of Beta-1,4-galactosyltransferase 1 (B4GalT1), UDP-GlcNAc and Mn2+ transporter (slc35a3 and slc39a8 respectively) for the high osmolality conditions. Significant downregulation of slc39a8 (Mn2+ transporter) was observed for the sodium butyrate condition. No significant differences were observed for any of the genes in the N-glycosylation pathway under the high pH condition even though this condition showed highest %Gal. Together, data suggests that different treatments have distinct complex mechanisms by which the overall glycan levels of a mAb are influenced. Further studies based on these results will help build the knowledge necessary to design strategies to obtain the desired productivity and product quality of mAbs.
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Affiliation(s)
- Sri R Madabhushi
- Biologics Upstream Process Development, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ, 07033, USA.
| | - Alexei A Podtelezhnikov
- Genetics and Pharmacogenomics, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ, 07033, USA
| | - Nicholas Murgolo
- Genetics and Pharmacogenomics, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ, 07033, USA
| | - Sen Xu
- Biologics Upstream Process Development, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ, 07033, USA
| | - Henry Lin
- Biologics Upstream Process Development, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ, 07033, USA
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11
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Song Y, Qian Y, Huang Z, Khattak SF, Li ZJ. Computational insights into O-glycosylation in a CTLA4 Fc-fusion protein linker and its impact on protein quality attributes. Comput Struct Biotechnol J 2020; 18:3925-3935. [PMID: 33335689 PMCID: PMC7734232 DOI: 10.1016/j.csbj.2020.11.037] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2020] [Revised: 11/21/2020] [Accepted: 11/23/2020] [Indexed: 12/21/2022] Open
Abstract
The hinge region of immunoglobulin G1 (IgG1) is used as a common linker for Fc-fusion therapeutic proteins. With the advances of high-resolution mass spectrometry and sample treatment strategies, unexpected O-linked glycosylation has been observed in the linker. However, the molecular mechanism involved in this unusual posttranslational modification is unknown. In this study, we applied site-direct mutagenesis, mass spectrometry, analytical chromatography, and computational modeling to investigate O-glycosylation processes in a clinically used CTLA4 Fc-fusion protein and its impacts on protein quality attributes. Surprisingly, O-glycans could be formed at new sites when an initial O-glycosylation site was eliminated, and continued to occur until all potential O-glycosylation sites were nulled. Site-preference of O-glycosylation initiation was attributed to the complex formation between the linker peptide and glycan transferase whereas the O-glycosylating efficiency and the linker flexibility were correlated using molecular modeling and simulations. As predicted, O-glycan-free CTLA4 Fc-fusion proteins were more homogenous for sialylation, and interestingly less prone to protein aggregation. Attenuating protein aggregation was a desirable effect, and could be related to the reduced presence of linker O-glycans that hindered inter-chain disulfide bond reformation. Findings from this study shed light on new therapeutic protein design and development.
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Affiliation(s)
- Yuanli Song
- Biologics Development, Bristol Myers Squibb Company, 38 Jackson Road, Devens, MA 01434, USA
| | - Yueming Qian
- Biologics Development, Bristol Myers Squibb Company, 38 Jackson Road, Devens, MA 01434, USA
| | - Zhe Huang
- Biologics Development, Bristol Myers Squibb Company, 38 Jackson Road, Devens, MA 01434, USA
| | - Sarwat F Khattak
- Biologics Development, Bristol Myers Squibb Company, 38 Jackson Road, Devens, MA 01434, USA
| | - Zheng Jian Li
- Biologics Development, Bristol Myers Squibb Company, 38 Jackson Road, Devens, MA 01434, USA
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12
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Comparison of Three Glycoproteomic Methods for the Analysis of the Secretome of CHO Cells Treated with 1,3,4- O-Bu 3ManNAc. Bioengineering (Basel) 2020; 7:bioengineering7040144. [PMID: 33182731 PMCID: PMC7712478 DOI: 10.3390/bioengineering7040144] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2020] [Revised: 10/24/2020] [Accepted: 11/06/2020] [Indexed: 01/08/2023] Open
Abstract
Comprehensive analysis of the glycoproteome is critical due to the importance of glycosylation to many aspects of protein function. The tremendous complexity of this post-translational modification, however, makes it difficult to adequately characterize the glycoproteome using any single method. To overcome this pitfall, in this report we compared three glycoproteomic analysis methods; first the recently developed N-linked glycans and glycosite-containing peptides (NGAG) chemoenzymatic method, second, solid-phase extraction of N-linked glycoproteins (SPEG), and third, hydrophilic interaction liquid chromatography (HILIC) by characterizing N-linked glycosites in the secretome of Chinese hamster ovary (CHO) cells. Interestingly, the glycosites identified by SPEG and HILIC overlapped considerably whereas NGAG identified many glycosites not observed in the other two methods. Further, utilizing enhanced intact glycopeptide identification afforded by the NGAG workflow, we found that the sugar analog 1,3,4-O-Bu3ManNAc, a "high flux" metabolic precursor for sialic acid biosynthesis, increased sialylation of secreted proteins including recombinant human erythropoietin (rhEPO).
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13
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Leiva-Carrasco MJ, Jiménez-Chávez S, Harvey DJ, Parra NC, Tavares KC, Camacho F, González A, Sánchez O, Montesino R, Toledo JR. In vivo modification of the goat mammary gland glycosylation pathway. N Biotechnol 2020; 61:11-21. [PMID: 33157282 DOI: 10.1016/j.nbt.2020.11.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2020] [Revised: 10/25/2020] [Accepted: 11/01/2020] [Indexed: 12/22/2022]
Abstract
Complex recombinant glycoproteins produced as potential biopharmaceuticals in goat's milk have an aberrant pattern of N-glycosylation due to the lack of multi-antennary structures. Overexpression of glycosyltransferases may increase oligosaccharide branching of the desired glycoproteins. Here, human erythropoietin fused to human IgG Fc (EPO-Fc) was co-expressed with N-acetyl-glucosaminyltransferase-IVa (GnT-IVa) by adenoviral transduction in goat mammary gland to evaluate the in vivo modification of N-glycosylation pattern in this tissue. Adenoviral vectors, containing the EPO-Fc and GnT-IVa sequences were assembled for in vitro and in vivo expression in mammalian cell culture or in goat mammary gland. Protein detection was assessed by gel electrophoresis and western blot, and N-glycans were identified by HPLC and mass spectrometry. GnT-IVa overexpression and its colocalization with EPO-Fc in the Golgi apparatus of SiHa cells were demonstrated. N-glycan analysis of in vitro and in vivo expression of EPO-Fc modified by GnT-IVa (EPO-Fc/GnT-IVa) showed an increase in high molecular weight structures, which corresponded to tri- and tetra-antennary N-glycans in SiHa cells and mostly tri-antennary N-glycans in goat's milk from transformed mammary tissue. The results confirmed that successful modification of the goat mammary gland secretion pathway could be achieved by co-expressing glycoenzymes together with the glycoprotein of interest. This is the first report of modification of the N-glycosylation pattern in the goat mammary gland in vivo, and constitutes a step forward for improving the use of the mammary gland as a bioreactor for the production of complex recombinant proteins.
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Affiliation(s)
- María J Leiva-Carrasco
- Biotechnology and Biopharmaceuticals Laboratory, Pathophysiology Department, School of Biological Sciences, University of Concepcion, Victor Lamas 1290, P.O. Box 160C, Concepcion, Chile; Biotechnology and Biomedicine Center SpA, Granada 168, Villumanque, Concepcion, Chile
| | - Silvana Jiménez-Chávez
- Biotechnology and Biopharmaceuticals Laboratory, Pathophysiology Department, School of Biological Sciences, University of Concepcion, Victor Lamas 1290, P.O. Box 160C, Concepcion, Chile; Biotechnology and Biomedicine Center SpA, Granada 168, Villumanque, Concepcion, Chile
| | - David J Harvey
- Oxford Glycobiology Institute, Biochemistry Department, South Parks Road, Oxford, OX1 3QU, UK
| | - Natalie C Parra
- Department of Pharmacology, School of Biological Sciences, University of Concepcion, Victor Lamas 1290, P.O. Box 160C, Concepcion, Chile
| | - Kaio C Tavares
- Molecular and Developmental Biology Laboratory, Experimental Biology Center (NUBEX), University of Fortaleza (UNIFOR), Fortaleza, CE, Brazil
| | - Frank Camacho
- Department of Pharmacology, School of Biological Sciences, University of Concepcion, Victor Lamas 1290, P.O. Box 160C, Concepcion, Chile
| | - Alain González
- Biotechnology and Biopharmaceuticals Laboratory, Pathophysiology Department, School of Biological Sciences, University of Concepcion, Victor Lamas 1290, P.O. Box 160C, Concepcion, Chile
| | - Oliberto Sánchez
- Department of Pharmacology, School of Biological Sciences, University of Concepcion, Victor Lamas 1290, P.O. Box 160C, Concepcion, Chile
| | - Raquel Montesino
- Biotechnology and Biopharmaceuticals Laboratory, Pathophysiology Department, School of Biological Sciences, University of Concepcion, Victor Lamas 1290, P.O. Box 160C, Concepcion, Chile; Biotechnology and Biomedicine Center SpA, Granada 168, Villumanque, Concepcion, Chile.
| | - Jorge R Toledo
- Biotechnology and Biopharmaceuticals Laboratory, Pathophysiology Department, School of Biological Sciences, University of Concepcion, Victor Lamas 1290, P.O. Box 160C, Concepcion, Chile; Biotechnology and Biomedicine Center SpA, Granada 168, Villumanque, Concepcion, Chile.
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14
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Grindel B, Engel BJ, Hall CG, Kelderhouse LE, Lucci A, Zacharias NM, Takahashi TT, Millward SW. Mammalian Expression and In Situ Biotinylation of Extracellular Protein Targets for Directed Evolution. ACS OMEGA 2020; 5:25440-25455. [PMID: 33043224 PMCID: PMC7542843 DOI: 10.1021/acsomega.0c03990] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/18/2020] [Accepted: 09/08/2020] [Indexed: 05/17/2023]
Abstract
Directed evolution is a powerful tool for the selection of functional ligands from molecular libraries. Extracellular domains (ECDs) of cell surface receptors are common selection targets for therapeutic and imaging agent development. Unfortunately, these proteins are often post-translationally modified and are therefore unsuitable for expression in bacterial systems. Directional immobilization of these targets is further hampered by the absence of biorthogonal groups for site-specific chemical conjugation. We have developed a nonadherent mammalian expression system for rapid, high-yield expression of biotinylated ECDs. ECDs from EGFR, HER2, and HER3 were site-specifically biotinylated in situ and recovered from the cell culture supernatant with yields of up to 10 mg/L at >90% purity. Biotinylated ECDs also contained a protease cleavage site for rapid and selective release of the ECD after immobilization on avidin/streptavidin resins and library binding. A model mRNA display selection round was carried out against the HER2 ECD with the HER2 affibody expressed as an mRNA-protein fusion. HER2 affibody-mRNA fusions were selectively released by thrombin and quantitative PCR revealed substantial improvements in the enrichment of functional affibody-mRNA fusions relative to direct PCR amplification of the resin-bound target. This methodology allows rapid purification of high-quality targets for directed evolution and selective elution of functional sequences at the conclusion of each selection round.
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Affiliation(s)
- Brian
J. Grindel
- Department
of Cancer Systems Imaging, MD Anderson Cancer
Center, Houston, Texas 77030, United States
| | - Brian J. Engel
- Department
of Cancer Systems Imaging, MD Anderson Cancer
Center, Houston, Texas 77030, United States
| | - Carolyn G. Hall
- Department
of Breast Surgical Oncology, MD Anderson
Cancer Center, Houston, Texas 77030, United States
| | - Lindsay E. Kelderhouse
- Department
of Cancer Systems Imaging, MD Anderson Cancer
Center, Houston, Texas 77030, United States
| | - Anthony Lucci
- Department
of Breast Surgical Oncology, MD Anderson
Cancer Center, Houston, Texas 77030, United States
| | - Niki M. Zacharias
- Department
of Urology, MD Anderson Cancer Center, Houston, Texas 77030, United States
| | - Terry T. Takahashi
- Department
of Chemistry, University of Southern California, Los Angeles, California 90089, United States
| | - Steven W. Millward
- Department
of Cancer Systems Imaging, MD Anderson Cancer
Center, Houston, Texas 77030, United States
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15
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Cambay F, Forest-Nault C, Dumoulin L, Seguin A, Henry O, Durocher Y, De Crescenzo G. Glycosylation of Fcγ receptors influences their interaction with various IgG1 glycoforms. Mol Immunol 2020; 121:144-158. [PMID: 32222585 DOI: 10.1016/j.molimm.2020.03.010] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2019] [Revised: 02/26/2020] [Accepted: 03/16/2020] [Indexed: 12/11/2022]
Abstract
Most of therapeutic monoclonal antibodies belong to the immunoglobulin G1 (IgG1) family; they interact with the Fcγ receptors (FcγRs) at the surface of immune cells to trigger effector functions. The IgG1-Fc N-glycans impact the interaction with FcγRs and are considered a critical quality attribute. Pioneer studies on FcγR N-glycans have unveiled an additional complexity in that the N-glycan linked on the Asn-162 of FcγRIIIa was shown to be directly involved in the strong affinity for afucosylated IgG1. The last few years have thus seen the emergence of many studies investigating the complex influence of FcγRIIIa N-glycans on the interaction with IgG1 through their glycosylation sites or their glycoprofiles. In this context, we performed site-directed mutagenesis along with glycoengineering on FcγRs (FcγRI, FcγRIIaH131/b and FcγRIIIaV158/F158) in an effort to elucidate the impact of FcγRs N-glycans on the interaction with IgG1. Furthermore, we assessed their binding to various trastuzumab glycoforms with an enhanced surface plasmon resonance assay. The FcγRIIIa N-glycans had the highest impact on the interaction with IgG1. More specifically, the N162 glycan positively influenced the affinity (15-fold) for afucosylated IgG1 while the N45 glycan presented a negative impact (2-fold) regardless of the IgG1 glycoforms. Interestingly, only the FcγRIIIa glycoprofile had an impact on the interaction with IgG1 with a 1.5-fold affinity increase when FcγRIIIa displays high-mannose glycans. These results provide invaluable insights into the complex and strong influence of N-glycosylation upon FcγRs/IgG1 binding and are instrumental to further understand the impact of FcγRs N-glycosylation in their natural forms.
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Affiliation(s)
- Florian Cambay
- Department of Chemical Engineering, Polytechnique Montréal, Montreal, Canada; Human Health Therapeutics Research Centre, National Research Council of Canada, Montreal, Canada
| | - Catherine Forest-Nault
- Department of Chemical Engineering, Polytechnique Montréal, Montreal, Canada; Human Health Therapeutics Research Centre, National Research Council of Canada, Montreal, Canada
| | - Lea Dumoulin
- Department of Chemical Engineering, Polytechnique Montréal, Montreal, Canada
| | - Alexis Seguin
- Department of Chemical Engineering, Polytechnique Montréal, Montreal, Canada
| | - Olivier Henry
- Department of Chemical Engineering, Polytechnique Montréal, Montreal, Canada
| | - Yves Durocher
- Human Health Therapeutics Research Centre, National Research Council of Canada, Montreal, Canada; Department of Biochemistry and Molecular Medicine, University of Montreal, Montreal, Canada.
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16
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Process intensification for the production of rituximab by an inducible CHO cell line. Bioprocess Biosyst Eng 2019; 42:711-725. [DOI: 10.1007/s00449-019-02075-z] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2018] [Accepted: 01/10/2019] [Indexed: 01/03/2023]
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17
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Lee JH, Kim J, Park J, Heo WD, Lee GM. Analysis of Golgi pH in Chinese hamster ovary cells using ratiometric pH‐sensitive fluorescent proteins. Biotechnol Bioeng 2019; 116:1006-1016. [DOI: 10.1002/bit.26920] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2018] [Revised: 12/18/2018] [Accepted: 01/09/2019] [Indexed: 02/06/2023]
Affiliation(s)
- Jong Hyun Lee
- Department of Biological SciencesKAISTDaejeon Republic of Korea
| | - Jihoon Kim
- Department of Biological SciencesKAISTDaejeon Republic of Korea
| | - Jong‐Ho Park
- Department of Biological SciencesKAISTDaejeon Republic of Korea
| | - Won Do Heo
- Department of Biological SciencesKAISTDaejeon Republic of Korea
| | - Gyun Min Lee
- Department of Biological SciencesKAISTDaejeon Republic of Korea
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18
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Kronimus Y, Dodel R, Galuska SP, Neumann S. IgG Fc N-glycosylation: Alterations in neurologic diseases and potential therapeutic target? J Autoimmun 2019; 96:14-23. [DOI: 10.1016/j.jaut.2018.10.006] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2018] [Revised: 10/09/2018] [Accepted: 10/11/2018] [Indexed: 12/30/2022]
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19
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Naik HM, Majewska NI, Betenbaugh MJ. Impact of nucleotide sugar metabolism on protein N-glycosylation in Chinese Hamster Ovary (CHO) cell culture. Curr Opin Chem Eng 2018. [DOI: 10.1016/j.coche.2018.10.002] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
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20
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Mellahi K, Cambay F, Brochu D, Gilbert M, Perrier M, Ansorge S, Durocher Y, Henry O. Process development for an inducible rituximab-expressing Chinese hamster ovary cell line. Biotechnol Prog 2018; 35:e2742. [PMID: 30414355 DOI: 10.1002/btpr.2742] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2018] [Revised: 10/25/2018] [Accepted: 10/31/2018] [Indexed: 12/12/2022]
Abstract
Inducible mammalian expression systems are becoming increasingly available and are not only useful for the production of cytotoxic/cytostatic products, but also confer the unique ability to uncouple the growth and production phases. In this work, we have specifically investigated how the cell culture state at the time of induction influences the cumate-inducible expression of recombinant rituximab by a GS-CHO cell line. To this end, cells grown in batch and fed-batch cultures were induced at increasing cell densities (1 to 10 × 10 6 cells/mL). In batch, the cell specific productivity and the product yield were found to reduce with increasing cell density at induction. A dynamic feeding strategy using a concentrated nutrient solution applied prior and postinduction allowed to significantly increase the integral of viable cells and led to a 3-fold increase in the volumetric productivity (1.2 g/L). The highest product yields were achieved for intermediate cell densities at induction, as cultures induced during the late exponential phase (10 × 10 6 cells/mL) were associated with a shortened production phase. The final glycosylation patterns remained however similar, irrespective of the cell density at induction. The kinetics of growth and production in a 2 L bioreactor were largely comparable to shake flasks for a similar cell density at induction. The degree of galactosylation was found to decrease over time, but the final glycan distribution at harvest was consistent to that of the shake flasks cultures. Taken together, our results provide useful insights for the rational development of fed-batch cell culture processes involving inducible CHO cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2742, 2019.
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Affiliation(s)
- Kahina Mellahi
- Dept. of Chemical Engineering, École Polytechnique de Montréal, Montréal, QC, H3C 3A7
| | - Florian Cambay
- Dept. of Chemical Engineering, École Polytechnique de Montréal, Montréal, QC, H3C 3A7
| | - Denis Brochu
- Human Health Therapeutics Research Center, National Research Council Canada, Ottawa, ON
| | - Michel Gilbert
- Human Health Therapeutics Research Center, National Research Council Canada, Ottawa, ON
| | - Michel Perrier
- Dept. of Chemical Engineering, École Polytechnique de Montréal, Montréal, QC, H3C 3A7
| | - Sven Ansorge
- Human Health Therapeutics Research Center, National Research Council Canada, Montréal, QC
| | - Yves Durocher
- Human Health Therapeutics Research Center, National Research Council Canada, Montréal, QC
| | - Olivier Henry
- Dept. of Chemical Engineering, École Polytechnique de Montréal, Montréal, QC, H3C 3A7
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21
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Loebrich S, Clark E, Ladd K, Takahashi S, Brousseau A, Kitchener S, Herbst R, Ryll T. Comprehensive manipulation of glycosylation profiles across development scales. MAbs 2018; 11:335-349. [PMID: 30252592 DOI: 10.1080/19420862.2018.1527665] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022] Open
Abstract
The extent and pattern of glycosylation on therapeutic antibodies can influence their circulatory half-life, engagement of effector functions, and immunogenicity, with direct consequences to efficacy and patient safety. Hence, controlling glycosylation patterns is central to any drug development program, yet poses a formidable challenge to the bio-manufacturing industry. Process changes, which can affect glycosylation patterns, range from manufacturing at different scales or sites, to switching production process mode, all the way to using alternative host cell lines. In the emerging space of biosimilars development, often times all of these aspects apply. Gaining a deep understanding of the direction and extent to which glycosylation quality attributes can be modulated is key for efficient fine-tuning of glycan profiles in a stage appropriate manner, but establishment of such platform knowledge is time consuming and resource intensive. Here we report an inexpensive and highly adaptable screening system for comprehensive modulation of glycans on antibodies expressed in CHO cells. We characterize 10 media additives in univariable studies and in combination, using a design of experiments approach to map the design space for tuning glycosylation profile attributes. We introduce a robust workflow that does not require automation, yet enables rapid process optimization. We demonstrate scalability across deep wells, shake flasks, AMBR-15 cell culture system, and 2 L single-use bioreactors. Further, we show that it is broadly applicable to different molecules and host cell lineages. This universal approach permits fine-tuned modulation of glycan product quality, reduces development costs, and enables agile implementation of process changes throughout the product lifecycle.
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Affiliation(s)
- Sven Loebrich
- a Cell Line and Upstream Process Development , Waltham , USA.,b Technical Operations , ImmunoGen, Waltham , USA
| | - Elisa Clark
- a Cell Line and Upstream Process Development , Waltham , USA.,b Technical Operations , ImmunoGen, Waltham , USA
| | - Kristina Ladd
- a Cell Line and Upstream Process Development , Waltham , USA.,b Technical Operations , ImmunoGen, Waltham , USA
| | - Stefani Takahashi
- b Technical Operations , ImmunoGen, Waltham , USA.,c Process Analytics , Waltham , USA
| | - Anna Brousseau
- b Technical Operations , ImmunoGen, Waltham , USA.,d High Throughput Sciences , Waltham , USA
| | - Seth Kitchener
- a Cell Line and Upstream Process Development , Waltham , USA.,b Technical Operations , ImmunoGen, Waltham , USA
| | | | - Thomas Ryll
- b Technical Operations , ImmunoGen, Waltham , USA
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22
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Continuous integrated manufacturing of therapeutic proteins. Curr Opin Biotechnol 2018; 53:76-84. [DOI: 10.1016/j.copbio.2017.12.015] [Citation(s) in RCA: 85] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2017] [Revised: 12/11/2017] [Accepted: 12/11/2017] [Indexed: 11/20/2022]
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23
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Biochemical and metabolic engineering approaches to enhance production of therapeutic proteins in animal cell cultures. Biochem Eng J 2018. [DOI: 10.1016/j.bej.2018.04.008] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
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24
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Li MY, Ebel B, Paris C, Chauchard F, Guedon E, Marc A. Real-time monitoring of antibody glycosylation site occupancy by in situ Raman spectroscopy during bioreactor CHO cell cultures. Biotechnol Prog 2018; 34:486-493. [PMID: 29314747 DOI: 10.1002/btpr.2604] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2017] [Revised: 11/14/2017] [Indexed: 12/12/2022]
Abstract
The glycosylation of therapeutic monoclonal antibodies (mAbs), a known critical quality attribute, is often greatly modified during the production process by animal cells. It is essential for biopharmaceutical industries to monitor and control this glycosylation. However, current glycosylation characterization techniques involve time- and labor-intensive analyses, often carried out at the end of the culture when the product is already synthesized. This study proposes a novel methodology for real-time monitoring of antibody glycosylation site occupancy using Raman spectroscopy. It was first observed in CHO cell batch culture that when low nutrient concentrations were reached, a decrease in mAb glycosylation was induced, which made it essential to rapidly detect this loss of product quality. By combining in situ Raman spectroscopy with chemometric tools, efficient prediction models were then developed for both glycosylated and nonglycosylated mAbs. By comparing variable importance in projection profiles of the prediction models, it was confirmed that Raman spectroscopy is a powerful method to distinguish extremely similar molecules, despite the high complexity of the culture medium. Finally, the Raman prediction models were used to monitor batch and feed-harvest cultures in situ. For the first time, it was demonstrated that the concentrations of glycosylated and nonglycosylated mAbs could be successfully and simultaneously estimated in real time with high accuracy, including their sudden variations due to medium exchanges. Raman spectroscopy can thus be considered as a promising PAT tool for feedback process control dedicated to on-line optimization of mAb quality. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:486-493, 2018.
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Affiliation(s)
- Meng-Yao Li
- Laboratoire Réactions et Génie des Procédés, CNRS-Lorraine University, UMR 7274, Vandœuvre-lès-Nancy, France
| | - Bruno Ebel
- Laboratoire Réactions et Génie des Procédés, CNRS-Lorraine University, UMR 7274, Vandœuvre-lès-Nancy, France
| | - Cédric Paris
- Platform of Structural and Metabolomics Analyses, SF4242, EFABA, Lorraine University, Vandœuvre-lès-Nancy, France
| | | | - Emmanuel Guedon
- Laboratoire Réactions et Génie des Procédés, CNRS-Lorraine University, UMR 7274, Vandœuvre-lès-Nancy, France
| | - Annie Marc
- Laboratoire Réactions et Génie des Procédés, CNRS-Lorraine University, UMR 7274, Vandœuvre-lès-Nancy, France
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25
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Mizukami A, Caron AL, Picanço-Castro V, Swiech K. Platforms for Recombinant Therapeutic Glycoprotein Production. Methods Mol Biol 2018; 1674:1-14. [PMID: 28921424 DOI: 10.1007/978-1-4939-7312-5_1] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/30/2023]
Abstract
The majority of FDA-approved biology-derived products are recombinant glycoproteins. These proteins have been used for the treatment of several diseases, with numerous products currently approved for clinical use. The choice of the expression system is a key step toward a successful functional protein production, since glycosylation influences yield, pharmacokinetics, biological activity, and immunogenicity. This chapter covers the general aspects of therapeutic recombinant glycoproteins and the platforms that are being employed for their production.
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Affiliation(s)
- Amanda Mizukami
- Center for Cell-based Therapy CTC, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil
| | - Angelo Luis Caron
- Center for Cell-based Therapy CTC, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil
- School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
| | - Virgínia Picanço-Castro
- Center for Cell-based Therapy CTC, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil
| | - Kamilla Swiech
- Center for Cell-based Therapy CTC, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil.
- School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil.
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Abstract
Fed-batch culture is the most commonly used upstream process in industry today for recombinant monoclonal antibody production using Chinese hamster ovary (CHO) cells. Developing and optimizing this process in the lab is crucial for establishing process knowledge, which enables rapid and predictable tech-transfer to manufacturing scale. In this chapter, we describe stepwise how to carry out fed-batch CHO cell culture for lab-scale antibody production.
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Affiliation(s)
- Yuzhou Fan
- Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads 223, 2800 Kgs, Lyngby, Denmark
| | - Daniel Ley
- Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads 223, 2800 Kgs, Lyngby, Denmark
| | - Mikael Rørdam Andersen
- Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads 223, 2800 Kgs, Lyngby, Denmark.
- Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet, Building 220, 2800, Kgs. Lyngby, Denmark.
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Sensitive and comprehensive analysis of O-glycosylation in biotherapeutics: a case study of novel erythropoiesis stimulating protein. Bioanalysis 2017; 9:1373-1383. [PMID: 28920453 DOI: 10.4155/bio-2017-0085] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
AIM Glycosylation of recombinant human erythropoietins (rhEPOs) is significantly associated with drug's quality and potency. Thus, comprehensive characterization of glycosylation is vital to assess the biotherapeutic quality and establish the equivalency of biosimilar rhEPOs. However, current glycan analysis mainly focuses on the N-glycans due to the absence of analytical tools to liberate O-glycans with high sensitivity. We developed selective and sensitive method to profile native O-glycans on rhEPOs. RESULTS O-glycosylation on rhEPO including O-acetylation on a sialic acid was comprehensively characterized. Details such as O-glycan structure and O-acetyl-modification site were obtained from tandem MS. CONCLUSION This method may be applied to QC and batch analysis of not only rhEPOs but also other biotherapeutics bearing multiple O-glycosylations.
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Ning T, Cui H, Sun F, Zou J. Systemic analysis of genome-wide expression profiles identified potential therapeutic targets of demethylation drugs for glioblastoma. Gene 2017; 627:387-392. [PMID: 28669930 DOI: 10.1016/j.gene.2017.06.052] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2017] [Revised: 06/26/2017] [Accepted: 06/28/2017] [Indexed: 12/31/2022]
Abstract
Glioblastoma represents one of the most aggressive malignant brain tumors with high morbidity and motility. Demethylation drugs have been developed for its treatment with little efficacy has been observed. The purpose of this study was to screen therapeutic targets of demethylation drugs or bioactive molecules for glioblastoma through systemic bioinformatics analysis. We firstly downloaded genome-wide expression profiles from the Gene Expression Omnibus (GEO) and conducted the primary analysis through R software, mainly including preprocessing of raw microarray data, transformation between probe ID and gene symbol and identification of differential expression genes (DEGs). Secondly, functional enrichment analysis was conducted via the Database for Annotation, Visualization and Integrated Discovery (DAVID) to explore biological processes involved in the development of glioblastoma. Thirdly, we constructed protein-protein interaction (PPI) network of interested genes and conducted cross analysis for multi datasets to obtain potential therapeutic targets for glioblastoma. Finally, we further confirmed the therapeutic targets through real-time RT-PCR. As a result, biological processes that related to cancer development, amino metabolism, immune response and etc. were found to be significantly enriched in genes that differential expression in glioblastoma and regulated by 5'aza-dC. Besides, network and cross analysis identified ACAT2, UFC1 and CYB5R1 as novel therapeutic targets of demethylation drugs which also confirmed by real time RT-PCR. In conclusions, our study identified several biological processes and genes that involved in the development of glioblastoma and regulated by 5'aza-dC, which would be helpful for the treatment of glioblastoma.
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Affiliation(s)
- Tongbo Ning
- Department of Neurosurgery, Weihai Central Hospital, Shandong, China
| | - Hao Cui
- Department of Neurosurgery, Weihai Central Hospital, Shandong, China
| | - Feng Sun
- Department of Neurosurgery, Weihai Central Hospital, Shandong, China
| | - Jidian Zou
- Department of Neurosurgery, Weihai Central Hospital, Shandong, China.
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29
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Hayes JM, Frostell A, Karlsson R, Müller S, Martín SM, Pauers M, Reuss F, Cosgrave EF, Anneren C, Davey GP, Rudd PM. Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab. Mol Cell Proteomics 2017; 16:1770-1788. [PMID: 28576848 DOI: 10.1074/mcp.m117.066944] [Citation(s) in RCA: 58] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2017] [Revised: 05/03/2017] [Indexed: 11/06/2022] Open
Abstract
Fc gamma receptors (FcγR) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of FcγRs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the FcγR glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fcγ receptors RI (CD64), RIIaArg131/His131 (CD32a), RIIb (CD32b) and RIIIaPhe158/Val158 (CD16a) and analyze the role of the glycans in the binding mechanism with IgG. The interactions of the monoclonal antibody rituximab with each FcγR were characterized and we discuss the CHO-FcγRIIIaPhe158/Val158 and CHO-FcγRI interactions and compare them to the equivalent interactions with human (HEK293) and murine (NS0) produced receptors. Our results reveal clear differences in the binding profiles of rituximab, which we attribute in each case to the differences in host cell-dependent FcγR glycosylation. The glycan profiles of CHO expressed FcγRI and FcγRIIIaPhe158/Val158 were compared with the glycan profiles of the receptors expressed in NS0 and HEK293 cells and we show that the glycan type and abundance differs significantly between the receptors and that these glycan differences lead to the observed differences in the respective FcγR binding patterns with rituximab. Oligomannose structures are prevalent on FcγRI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On FcγRI and FcγRIIIa large and sialylated glycans have a negative impact on rituximab binding, likely through destabilization of the interaction. In conclusion, the data show that the IgG1-FcγR binding kinetics differ depending on the glycosylation of the FcγR and further support a stabilizing role of FcγR glycans in the antibody binding interaction.
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Affiliation(s)
- Jerrard M Hayes
- From the ‡School of Biochemistry & Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Pearse St. Dublin 2, Ireland;
| | - Asa Frostell
- §GE Healthcare, Björkgatan, SE-75184 Uppsala, Sweden
| | | | - Steffen Müller
- ¶NIBRT-Glycoscience Group, NIBRT-The National Institute for Bioprocessing, Research and Training, Foster Avenue, Blackrock, County Dublin, Ireland
| | | | - Martin Pauers
- ‖Boehringer Ingelheim Pharma, Biberach/Riss, Germany
| | | | - Eoin F Cosgrave
- ¶NIBRT-Glycoscience Group, NIBRT-The National Institute for Bioprocessing, Research and Training, Foster Avenue, Blackrock, County Dublin, Ireland
| | | | - Gavin P Davey
- From the ‡School of Biochemistry & Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Pearse St. Dublin 2, Ireland
| | - Pauline M Rudd
- ¶NIBRT-Glycoscience Group, NIBRT-The National Institute for Bioprocessing, Research and Training, Foster Avenue, Blackrock, County Dublin, Ireland
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Karst DJ, Scibona E, Serra E, Bielser JM, Souquet J, Stettler M, Broly H, Soos M, Morbidelli M, Villiger TK. Modulation and modeling of monoclonal antibody N-linked glycosylation in mammalian cell perfusion reactors. Biotechnol Bioeng 2017; 114:1978-1990. [DOI: 10.1002/bit.26315] [Citation(s) in RCA: 61] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2016] [Revised: 02/24/2017] [Accepted: 04/09/2017] [Indexed: 12/23/2022]
Affiliation(s)
- Daniel J. Karst
- Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering; ETH Zurich; HCI F-129, Vladimir-Prelog-Weg 1 8093 Zurich Switzerland
| | - Ernesto Scibona
- Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering; ETH Zurich; HCI F-129, Vladimir-Prelog-Weg 1 8093 Zurich Switzerland
| | - Elisa Serra
- Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering; ETH Zurich; HCI F-129, Vladimir-Prelog-Weg 1 8093 Zurich Switzerland
| | - Jean-Marc Bielser
- Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering; ETH Zurich; HCI F-129, Vladimir-Prelog-Weg 1 8093 Zurich Switzerland
- Merck Serono SA; Biotech Process Sciences, ZI B 1809; Corsier-sur-Vevey Switzerland
| | - Jonathan Souquet
- Merck Serono SA; Biotech Process Sciences, ZI B 1809; Corsier-sur-Vevey Switzerland
| | - Matthieu Stettler
- Merck Serono SA; Biotech Process Sciences, ZI B 1809; Corsier-sur-Vevey Switzerland
| | - Hervé Broly
- Merck Serono SA; Biotech Process Sciences, ZI B 1809; Corsier-sur-Vevey Switzerland
| | - Miroslav Soos
- Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering; ETH Zurich; HCI F-129, Vladimir-Prelog-Weg 1 8093 Zurich Switzerland
- Department of Chemical Engineering; University of Chemistry and Technology; Technicka 3, 166 28 Prague Czech Republic
| | - Massimo Morbidelli
- Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering; ETH Zurich; HCI F-129, Vladimir-Prelog-Weg 1 8093 Zurich Switzerland
| | - Thomas K. Villiger
- Department of Chemistry and Applied Biosciences, Institute for Chemical and Bioengineering; ETH Zurich; HCI F-129, Vladimir-Prelog-Weg 1 8093 Zurich Switzerland
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Cha HM, Lim JH, Yeon JH, Hwang JM, Kim DI. Co-overexpression of Mgat1 and Mgat4 in CHO cells for production of highly sialylated albumin-erythropoietin. Enzyme Microb Technol 2017; 103:53-58. [PMID: 28554385 DOI: 10.1016/j.enzmictec.2017.04.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2016] [Revised: 03/30/2017] [Accepted: 04/28/2017] [Indexed: 12/22/2022]
Abstract
Terminal sialic acids on N-glycan of recombinant human erythropoietin are very important for in vivo half-life, as this glycoprotein has three N-glycosylation sites. N-acetylglucosaminyltransferases I, II, IV, and V (i.e. Mgat1, Mgat2, Mgat4, and Mgat5) catalyze the formation of a glycan antennary structure. These enzymes display different reaction kinetics for a common substrate and generally show low expression in Chinese hamster ovary (CHO) cells. Therefore, genetic control of Mgat expression is an effective method to increase sialic acid contents by enhancing glycan antennarity. To produce highly sialylated albumin-erythropoietin (Alb-EPO), we co-overexpressed the Mgat1 and Mgat4 genes in CHO cells and determined the optimal ratio of Mgat1:Mgat4 gene expression. All transfected cell lines showed increased gene expression of Mgat4, including Mgat1 overexpressing cell line. Sialic acid content of Alb-EPO was highest in co-transfected cells with excess Mgat4 gene, and these cells showed a higher tri- and tetra-antennary structure than control cells. Based on these results, we suggest that co-transfection of the Mgat1 and Mgat4 genes at a ratio of 2:8 is optimal for extension of antennary structures. Also, regulation of Mgat gene expression in the glycan biosynthesis pathway can be a novel approach to increase the terminal sialic acids of N-glycans.
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Affiliation(s)
- Hyun-Myoung Cha
- Department of Biological Engineering, Inha University, Incheon 22212, Republic of Korea
| | - Jin-Hyuk Lim
- Department of Biological Engineering, Inha University, Incheon 22212, Republic of Korea
| | - Jung-Heum Yeon
- Department of Biological Engineering, Inha University, Incheon 22212, Republic of Korea
| | - Jeong-Min Hwang
- Department of Biological Engineering, Inha University, Incheon 22212, Republic of Korea
| | - Dong-Il Kim
- Department of Biological Engineering, Inha University, Incheon 22212, Republic of Korea.
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Lee JH, Jeong YR, Kim YG, Lee GM. Understanding of decreased sialylation of Fc-fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N-glycosylation gene expression and N-linked glycan antennary profile. Biotechnol Bioeng 2017; 114:1721-1732. [PMID: 28266015 DOI: 10.1002/bit.26284] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2016] [Revised: 02/16/2017] [Accepted: 02/28/2017] [Indexed: 01/14/2023]
Abstract
To understand the effects of hyperosmolality on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing the Fc-fusion protein were cultivated in hyperosmolar medium resulting from adding NaCl (415 mOsm/kg). The hyperosmotic culture showed increased specific Fc-fusion protein productivity (qFc ) but a decreased proportion of acidic isoforms and sialic acid content of the Fc-fusion protein. The intracellular and extracellular sialidase activities in the hyperosmotic cultures were similar to those in the control culture (314 mOsm/kg), indicating that reduced sialylation of Fc-fusion protein at hyperosmolality was not due to elevated sialidase activity. Expression of 52 N-glycosylation-related genes was assessed by the NanoString nCounter system, which provides a direct digital readout using custom-designed color-coded probes. After 3 days of hyperosmotic culture, nine genes (ugp, slc35a3, slc35d2, gcs1, manea, mgat2, mgat5b, b4galt3, and b4galt4) were differentially expressed over 1.5-fold of the control, and all these genes were down-regulated. N-linked glycan analysis by anion exchange and hydrophilic interaction HPLC showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary N-linked glycans was significantly decreased upon hyperosmotic culture. Addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary N-linked glycans (P ≤ 0.05), while it increased the expression of the N-glycan branching/antennary genes (mgat2 and mgat4b). Thus, decreased expression of the genes with roles in the N-glycan biosynthesis pathway correlated with reduced sialic acid content of Fc-fusion protein caused by hyperosmolar conditions. Taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on N-glycosylation, especially sialylation, in rCHO cells. Biotechnol. Bioeng. 2017;114: 1721-1732. © 2017 Wiley Periodicals, Inc.
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Affiliation(s)
- Jong Hyun Lee
- Department of Biological Sciences, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea
| | - Yeong Ran Jeong
- Department of Biological Sciences, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea
| | - Yeon-Gu Kim
- Biotechnology Process Engineering Center, KRIBB, Ochang-eup, Cheongwon-gu, Cheongju, Republic of Korea.,Department of Bioprocess Engineering, Korea University of Science and Technology (UST), Gajeong-ro, Yuseong-gu, Daejeon, Republic of Korea
| | - Gyun Min Lee
- Department of Biological Sciences, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea
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Bora de Oliveira K, Spencer D, Barton C, Agarwal N. Site-specific monitoring of N-Glycosylation profiles of a CTLA4-Fc-fusion protein from the secretory pathway to the extracellular environment. Biotechnol Bioeng 2017; 114:1550-1560. [PMID: 28186328 DOI: 10.1002/bit.26266] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2016] [Revised: 01/31/2017] [Accepted: 02/06/2017] [Indexed: 01/18/2023]
Abstract
Glycosylation often plays a key role in the safety and efficacy of therapeutic proteins to patients, thus underlying the need for consistent control of this important post-translational modification during biologics production. In this study, we profiled the site-specific evolution of N-glycans on a CTLA4-Fc-fusion protein, from the intracellular secretory pathway to the conditioned medium (CM) in fed-batch cell culture. For this, we developed an approach that combined sub-cellular fractionation with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The study revealed that there was a significant amount of heterogeneity in the glycans displayed amongst the three distinct N-glycosylation sites. Furthermore, 54-60% of the intracellular protein was characterized by Man8 and Man9 glycans on day 10, when the cell density peaks, indicative of a significant bottleneck between the endoplasmic reticulum (ER) and the cis-Golgi. At longer culture duration, the accumulation of intracellular protein with bi-antennary-fucosylated GlcNAc-terminated residues identified the formation of another bottleneck in the medial and trans-Golgi compartments, which subsequently led to a decrease in sialylated species in the secreted protein. Glucose deprivation caused a reduction in the Man8 and Man9 glycans in favor of Man5 glycans and bi-antennary-fucosylated GlcNAc-terminated residues in the organellar pool of the Fc-fusion protein. However, transient deprivation of glucose did not lead to major differences in the glycan profile of proteins secreted into the CM. The approach developed here allows us to probe the secretory pathway and sheds light on the site-specific intracellular processing of glycans during fed-batch cell culture, thus serving as an initial step towards their rational control. Biotechnol. Bioeng. 2017;114: 1550-1560. © 2017 Wiley Periodicals, Inc.
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Affiliation(s)
| | - David Spencer
- MedImmune LLC., One MedImmune Way, Gaithersburg, Maryland 20878
| | | | - Nitin Agarwal
- MedImmune LLC., One MedImmune Way, Gaithersburg, Maryland 20878
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Fan Y, Kildegaard HF, Andersen MR. Engineer Medium and Feed for Modulating N-Glycosylation of Recombinant Protein Production in CHO Cell Culture. Methods Mol Biol 2017; 1603:209-226. [PMID: 28493133 DOI: 10.1007/978-1-4939-6972-2_14] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N-glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell. In this chapter, we will focus on how to carry out experiments for N-glycosylation modulation through medium and feed optimization. The workflow and typical methods involved in the experiment process will be presented.
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Affiliation(s)
- Yuzhou Fan
- Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
| | - Helene Faustrup Kildegaard
- The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark
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35
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Drouillard G, Hayward G, Vale J, Dutton R. A simultaneous assessment metric for MAb quantity and glycan quality. Cytotechnology 2016; 68:1987-97. [PMID: 27502107 DOI: 10.1007/s10616-016-0011-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2016] [Accepted: 07/16/2016] [Indexed: 11/28/2022] Open
Abstract
As a critical quality attribute, glycosylation represents an important consideration when analyzing the success of a glycoprotein production process. Though critical, glycosylation is not the only measure of culture success; other factors, including culture size, maintenance, and productivity, are also critical. A new metric was developed to address both product quality, as measured through glycosylation, and product quantity, as measured through product concentration. A monoclonal antibody Chinese hamster ovary cell culture model system was used to assess this metric across various media formulations. In a model test system, the metric discriminated that some media supplements had a net positive impact on productivity and glycosylation, while others had a net negative impact on productivity and glycosylation.
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36
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Liu H, Nowak C, Shao M, Ponniah G, Neill A. Impact of cell culture on recombinant monoclonal antibody product heterogeneity. Biotechnol Prog 2016; 32:1103-1112. [DOI: 10.1002/btpr.2327] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2016] [Revised: 07/19/2016] [Indexed: 12/20/2022]
Affiliation(s)
- Hongcheng Liu
- Product Characterization, Global Analytical and Pharmaceutical Development; Alexion Pharmaceuticals; CT06410 Cheshire
| | - Christine Nowak
- Product Characterization, Global Analytical and Pharmaceutical Development; Alexion Pharmaceuticals; CT06410 Cheshire
| | - Mei Shao
- Late Stage Upstream Development, Global Process Development; Alexion Pharmaceuticals; CT06410 Cheshire
| | - Gomathinayagam Ponniah
- Product Characterization, Global Analytical and Pharmaceutical Development; Alexion Pharmaceuticals; CT06410 Cheshire
| | - Alyssa Neill
- Product Characterization, Global Analytical and Pharmaceutical Development; Alexion Pharmaceuticals; CT06410 Cheshire
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Lewis AM, Croughan WD, Aranibar N, Lee AG, Warrack B, Abu-Absi NR, Patel R, Drew B, Borys MC, Reily MD, Li ZJ. Understanding and Controlling Sialylation in a CHO Fc-Fusion Process. PLoS One 2016; 11:e0157111. [PMID: 27310468 PMCID: PMC4911072 DOI: 10.1371/journal.pone.0157111] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2016] [Accepted: 05/24/2016] [Indexed: 12/13/2022] Open
Abstract
A Chinese hamster ovary (CHO) bioprocess, where the product is a sialylated Fc-fusion protein, was operated at pilot and manufacturing scale and significant variation of sialylation level was observed. In order to more tightly control glycosylation profiles, we sought to identify the cause of variability. Untargeted metabolomics and transcriptomics methods were applied to select samples from the large scale runs. Lower sialylation was correlated with elevated mannose levels, a shift in glucose metabolism, and increased oxidative stress response. Using a 5-L scale model operated with a reduced dissolved oxygen set point, we were able to reproduce the phenotypic profiles observed at manufacturing scale including lower sialylation, higher lactate and lower ammonia levels. Targeted transcriptomics and metabolomics confirmed that reduced oxygen levels resulted in increased mannose levels, a shift towards glycolysis, and increased oxidative stress response similar to the manufacturing scale. Finally, we propose a biological mechanism linking large scale operation and sialylation variation. Oxidative stress results from gas transfer limitations at large scale and the presence of oxygen dead-zones inducing upregulation of glycolysis and mannose biosynthesis, and downregulation of hexosamine biosynthesis and acetyl-CoA formation. The lower flux through the hexosamine pathway and reduced intracellular pools of acetyl-CoA led to reduced formation of N-acetylglucosamine and N-acetylneuraminic acid, both key building blocks of N-glycan structures. This study reports for the first time a link between oxidative stress and mammalian protein sialyation. In this study, process, analytical, metabolomic, and transcriptomic data at manufacturing, pilot, and laboratory scales were taken together to develop a systems level understanding of the process and identify oxygen limitation as the root cause of glycosylation variability.
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Affiliation(s)
- Amanda M. Lewis
- Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America
- * E-mail:
| | - William D. Croughan
- Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America
| | - Nelly Aranibar
- Research and Development, Bristol-Myers Squibb Company, Princeton, NJ, United States of America
| | - Alison G. Lee
- Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America
| | - Bethanne Warrack
- Research and Development, Bristol-Myers Squibb Company, Princeton, NJ, United States of America
| | - Nicholas R. Abu-Absi
- Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America
| | - Rutva Patel
- Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America
| | - Barry Drew
- Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America
| | - Michael C. Borys
- Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America
| | - Michael D. Reily
- Research and Development, Bristol-Myers Squibb Company, Princeton, NJ, United States of America
| | - Zheng Jian Li
- Biologics Development, Global Manufacturing and Supply, Bristol-Myers Squibb Company, Devens, MA, United States of America
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Meininger M, Stepath M, Hennig R, Cajic S, Rapp E, Rotering H, Wolff M, Reichl U. Sialic acid-specific affinity chromatography for the separation of erythropoietin glycoforms using serotonin as a ligand. J Chromatogr B Analyt Technol Biomed Life Sci 2016; 1012-1013:193-203. [DOI: 10.1016/j.jchromb.2016.01.005] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2015] [Revised: 12/13/2015] [Accepted: 01/05/2016] [Indexed: 10/22/2022]
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Sou SN, Sellick C, Lee K, Mason A, Kyriakopoulos S, Polizzi KM, Kontoravdi C. Understanding the impact of different bioprocess conditions on monoclonal antibody glycosylation in CHO cell cultures through experimental and computational analyses. BMC Proc 2015. [PMCID: PMC4685408 DOI: 10.1186/1753-6561-9-s9-o6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
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Vanier G, Hempel F, Chan P, Rodamer M, Vaudry D, Maier UG, Lerouge P, Bardor M. Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum. PLoS One 2015; 10:e0139282. [PMID: 26437211 PMCID: PMC4593558 DOI: 10.1371/journal.pone.0139282] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2015] [Accepted: 09/09/2015] [Indexed: 12/22/2022] Open
Abstract
Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans.
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Affiliation(s)
- Gaëtan Vanier
- Laboratoire Glycobiologie et Matrice Extracellulaire végétale Equipe d’Accueil 4358, Faculté des sciences et techniques, Université de Rouen, Normandie Université, Institut de Recherche et d’Innovation Biomédicale, Végétale Agronomie Sol Innovation, Mont-Saint-Aignan, France
| | - Franziska Hempel
- LOEWE Center for Synthetic Microbiology, Philipps-Universität Marburg, Marburg, Germany
| | - Philippe Chan
- PISSARO Proteomic Platform, Normandie Université, Institut de Recherche et d’Innovation Biomédicale, Mont-Saint-Aignan, France
| | | | - David Vaudry
- PISSARO Proteomic Platform, Normandie Université, Institut de Recherche et d’Innovation Biomédicale, Mont-Saint-Aignan, France
| | - Uwe G. Maier
- LOEWE Center for Synthetic Microbiology, Philipps-Universität Marburg, Marburg, Germany
| | - Patrice Lerouge
- Laboratoire Glycobiologie et Matrice Extracellulaire végétale Equipe d’Accueil 4358, Faculté des sciences et techniques, Université de Rouen, Normandie Université, Institut de Recherche et d’Innovation Biomédicale, Végétale Agronomie Sol Innovation, Mont-Saint-Aignan, France
| | - Muriel Bardor
- Laboratoire Glycobiologie et Matrice Extracellulaire végétale Equipe d’Accueil 4358, Faculté des sciences et techniques, Université de Rouen, Normandie Université, Institut de Recherche et d’Innovation Biomédicale, Végétale Agronomie Sol Innovation, Mont-Saint-Aignan, France
- Institut Universitaire de France, Paris, France
- * E-mail:
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Tuning a MAb glycan profile in cell culture: Supplementing N-acetylglucosamine to favour G0 glycans without compromising productivity and cell growth. J Biotechnol 2015; 214:105-12. [PMID: 26387447 DOI: 10.1016/j.jbiotec.2015.09.014] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2015] [Revised: 07/28/2015] [Accepted: 09/15/2015] [Indexed: 11/23/2022]
Abstract
Glycosylation is a critical quality attribute of many therapeutic proteins, particularly monoclonal antibodies (MAbs). Nucleotide-sugar precursors supplemented to growth medium to affect the substrate supply chain of glycosylation has yielded promising but varied results for affecting glycosylation. Glucosamine (GlcN), a precursor for N-acetylglucosamine (GlcNAc), is a major component of mammalian glycans. The supplementation of GlcN to CHO cells stably-expressing a chimeric heavy-chain monoclonal antibody, EG2-hFc, reduces the complexity of glycans to favour G0 glycoforms, while also negatively impacting cell growth. Although several researchers have examined the supplementation of glucosamine, no clear explanation of its impact on cell growth has been forthcoming. In this work, the glucosamine metabolism is examined. We identified the acetylation of GlcN to produce GlcNAc to be the most likely cause for the negative impact on growth due to the depletion of intracellular acetyl-CoA pools in the cytosol. By supplementing GlcNAc in lieu of GlcN to CHO cells producing EG2-hFc, we achieve the same shift in glycan complexity with marginal impacts on the cell growth and protein production.
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Zhu L, Guo Q, Guo H, Liu T, Zheng Y, Gu P, Chen X, Wang H, Hou S, Guo Y. Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry. MAbs 2015; 6:1474-85. [PMID: 25484062 DOI: 10.4161/mabs.36313] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
CTLA4-Ig is a highly glycosylated therapeutic fusion protein that contains multiple N- and O-glycosylation sites. Glycosylation plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. For a CTLA4-Ig biosimilar development program, comparative analytical data, especially the glycosylation data, can influence decisions about the type and amount of animal and clinical data needed to establish biosimilarity. Because of the limited clinical experience with biosimilars before approval, a comprehensive level of knowledge about the biosimilar candidates is needed to achieve subsequent development. Liquid chromatography-mass spectrometry (LC-MS) is a versatile technique for characterizing N- and O-glycosylation modification of recombinant therapeutic proteins, including 3 levels: intact protein analysis, peptide mapping analysis, and released glycans analysis. In this report, an in-depth characterization of glycosylation of a candidate biosimilar was carried out using a systematic approach: N- and O-linked glycans were identified and electron-transfer dissociation was then used to pinpoint the 4 occupied O-glycosylation sites for the first time. As the results show, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. This approach can be used to comprehensively research a candidate biosimilar Fc-fusion protein and provides a basis for future studies addressing the similarity of CTLA4-Ig biosimilars.
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Key Words
- 2-AB, 2-aminobenzamide
- CTLA-4, cytotoxic T-lymphocyte-associated antigen 4
- CTLA4-Ig fusion protein
- DTT, dithiothreitol
- EMA, European Medicines Agency
- ESI, electrospray ionization
- FA, formic acid
- FDA, Food and Drug Administration
- GFP, [Giu1]-Fibrinopeptide B
- IAM, Iodoacetamide
- LC, Liquid chromatography
- MS, mass spectrometry
- PNGase F, peptide N-glycosidase
- PTMs, post-translational modifications
- Q-Tof quadrupole-time of flight
- RA, Rheumatoid arthritis
- TIC, Total Ion Chromatography
- Tof, Time of flight
- UPLC, Ultra-performance liquid chromatography
- characterization
- glycan
- glycosylation modification
- intact protein
- mass spectrometry
- peptide mapping
- similarity
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Affiliation(s)
- Lei Zhu
- a International Joint Cancer Institute; Second Military Medical University ; Shanghai , China
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Cook MC, Kaldas SJ, Muradia G, Rosu-Myles M, Kunkel JP. Comparison of orthogonal chromatographic and lectin-affinity microarray methods for glycan profiling of a therapeutic monoclonal antibody. J Chromatogr B Analyt Technol Biomed Life Sci 2015; 997:162-78. [DOI: 10.1016/j.jchromb.2015.05.035] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2015] [Revised: 05/28/2015] [Accepted: 05/29/2015] [Indexed: 10/23/2022]
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Onitsuka M, Omasa T. Rapid evaluation of N-glycosylation status of antibodies with chemiluminescent lectin-binding assay. J Biosci Bioeng 2015; 120:107-10. [DOI: 10.1016/j.jbiosc.2014.11.015] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2014] [Revised: 11/18/2014] [Accepted: 11/18/2014] [Indexed: 01/05/2023]
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Kang S, Zhang Z, Richardson J, Shah B, Gupta S, Huang CJ, Qiu J, Le N, Lin H, Bondarenko PV. Metabolic markers associated with high mannose glycan levels of therapeutic recombinant monoclonal antibodies. J Biotechnol 2015; 203:22-31. [DOI: 10.1016/j.jbiotec.2015.03.002] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2014] [Revised: 02/28/2015] [Accepted: 03/05/2015] [Indexed: 01/21/2023]
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Li Y, Xu X, Shupe A, Yang R, Bai K, Das T, Borys MC, Li ZJ. Heterogeneous glycoform separation by process chromatography: I: Monomer purification and characterization. J Chromatogr A 2015; 1404:51-9. [PMID: 26051082 DOI: 10.1016/j.chroma.2015.05.054] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2015] [Revised: 05/18/2015] [Accepted: 05/19/2015] [Indexed: 10/23/2022]
Abstract
Fc fusion proteins with high and low sialylation were purified and separated by preparative ion-exchange and hydrophobic interaction chromatography. Heterogeneity in sialylation and glycosylation led to variation in surface charge and hydrophobicity, and resulted in multiple distinct glycoform populations in response to various purification conditions. Monomer with high sialic acid content has higher surface charge and adsorbs stronger to ion-exchange resin, while the less sialylated monomer interacts more favorably with hydrophobic resin. Extensive biophysical characterization was carried out for purified monomers at different level of sialylation. In general, different monomeric glycoforms have different surface charge and hydrophobicity, different thermal stability, and different aggregation propensity. The surface charge corresponds well with sialic acid content, as evidenced by electrophoresis, N-link domain analysis, and zeta potential results. The sialylation also contributes to minor modification of protein size, molecular mass and tertiary structure. Notably, fluorescence emission spectra and thermal transition became less distinguishable when the monomers containing low and high sialic acid were prepared in high ionic strength solution. Such finding reiterates the fact that the electrostatic forces, which are largely dependent on sialic acid content of protein, plays a dominant role in many intra- and inter-molecular interactions. Overall, the characterization data agreed well with separation behaviors and provided valuable insight to control of glycoform profile in purification process.
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Affiliation(s)
- Yi Li
- Biologics Process Development, Global Manufacturing and Supply, Bristol-Myers Squibb, Hopkinton, MA, United States.
| | - Xuankuo Xu
- Biologics Process Development, Global Manufacturing and Supply, Bristol-Myers Squibb, Hopkinton, MA, United States
| | - Alan Shupe
- Biologics Process Development, Global Manufacturing and Supply, Bristol-Myers Squibb, Hopkinton, MA, United States
| | - Rong Yang
- Mass Spectrometry and Biophysics Center of Excellence, Molecular and Analytical Development Technology, Bristol-Myers Squibb, Hopewell, NJ, United States
| | - Kevin Bai
- Mass Spectrometry and Biophysics Center of Excellence, Molecular and Analytical Development Technology, Bristol-Myers Squibb, Hopewell, NJ, United States
| | - Tapan Das
- Mass Spectrometry and Biophysics Center of Excellence, Molecular and Analytical Development Technology, Bristol-Myers Squibb, Hopewell, NJ, United States
| | - Michael C Borys
- Biologics Process Development, Global Manufacturing and Supply, Bristol-Myers Squibb, Hopkinton, MA, United States
| | - Zheng Jian Li
- Biologics Process Development, Global Manufacturing and Supply, Bristol-Myers Squibb, Hopkinton, MA, United States
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Assessment of the impact of manufacturing changes on the physicochemical properties and biological activity of Her1-ECD vaccine during product development. Vaccine 2015; 33:4292-9. [PMID: 26003492 DOI: 10.1016/j.vaccine.2015.05.018] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2014] [Revised: 05/04/2015] [Accepted: 05/07/2015] [Indexed: 11/20/2022]
Abstract
Vaccine preparations based on the extracellular domain of Her1 (Her1-ECD) have demonstrated, in vitro and in vivo, a potent antimetastatic effect on EGFR(+) Lewis lung carcinoma model, while associated side effects were absent. The Her1-ECD is a glycoprotein with a molecular weight of 105 kDa and has 11 potential sites for N-glycosylation. Currently Her1-ECD based vaccine has been evaluated in patients with hormone refractory prostate cancer. Her1-ECD molecule used for in clinical trials was obtained from culture supernatant of HEK 293 transfectomes used the protein free culture media and is purified by immunoaffinity chromatography. In order to increase the cell growth and productivity, new defined culture media have been developed (alternative culture media) in Her1-ECD vaccine production process. In this work, a comparability study was performed to evaluate the impact of process changes in the characteristics physic-chemical and biologicals of the Her1-ECD protein and the degree of similitude between both variants. Techniques such as: SDS-PAGE, SEC-HPLC, isoelectric point, peptide mapping, mass spectrometric, SCX-HPLC, oligosaccharide map, ELISA and flow cytometric were used with this aim. Results indicated that this process change decreases the degree of sialylation of the protein but does not affect its biological activity (measured as titers of Abs and recognition for A431 cell line).
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Huang CJ, Lin H, Yang JX. A robust method for increasing Fc glycan high mannose level of recombinant antibodies. Biotechnol Bioeng 2015; 112:1200-9. [DOI: 10.1002/bit.25534] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2014] [Revised: 12/15/2014] [Accepted: 12/23/2014] [Indexed: 01/03/2023]
Affiliation(s)
- Chung-Jr Huang
- Cell Science & Technology; Process and Product Development; Amgen Inc.; One Amgen Center Drive Thousand Oaks California 91320
| | - Henry Lin
- Cell Science & Technology; Process and Product Development; Amgen Inc.; One Amgen Center Drive Thousand Oaks California 91320
| | - Jerry Xiaoming Yang
- Cell Science & Technology; Process and Product Development; Amgen Inc.; One Amgen Center Drive Thousand Oaks California 91320
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Liu J, Chen X, Fan L, Deng X, Fai Poon H, Tan WS, Liu X. Monitoring sialylation levels of Fc-fusion protein using size-exclusion chromatography as a process analytical technology tool. Biotechnol Lett 2015; 37:1371-7. [DOI: 10.1007/s10529-015-1815-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2014] [Accepted: 03/12/2015] [Indexed: 11/29/2022]
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50
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Yongky A, Lee J, Le T, Mulukutla BC, Daoutidis P, Hu WS. Mechanism for multiplicity of steady states with distinct cell concentration in continuous culture of mammalian cells. Biotechnol Bioeng 2015; 112:1437-45. [PMID: 25676211 DOI: 10.1002/bit.25566] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2014] [Revised: 02/03/2015] [Accepted: 02/05/2015] [Indexed: 11/09/2022]
Abstract
Continuous culture for the production of biopharmaceutical proteins offers the possibility of steady state operations and thus more consistent product quality and increased productivity. Under some conditions, multiplicity of steady states has been observed in continuous cultures of mammalian cells, wherein with the same dilution rate and feed nutrient composition, steady states with very different cell and product concentrations may be reached. At those different steady states, cells may exhibit a high glycolysis flux with high lactate production and low cell concentration, or a low glycolysis flux with low lactate and high cell concentration. These different steady states, with different cell concentration, also have different productivity. Developing a mechanistic understanding of the occurrence of steady state multiplicity and devising a strategy to steer the culture toward the desired steady state is critical. We establish a multi-scale kinetic model that integrates a mechanistic intracellular metabolic model and cell growth model in a continuous bioreactor. We show that steady state multiplicity exists in a range of dilution rate in continuous culture as a result of the bistable behavior in glycolysis. The insights from the model were used to devise strategies to guide the culture to the desired steady state in the multiple steady state region. The model provides a guideline principle in the design of continuous culture processes of mammalian cells.
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Affiliation(s)
- Andrew Yongky
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota
| | - Jongchan Lee
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota
| | - Tung Le
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota
| | - Bhanu Chandra Mulukutla
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota
| | - Prodromos Daoutidis
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota
| | - Wei-Shou Hu
- Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota.
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