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High-Throughput Quantification and Glycosylation Analysis of Antibodies Using Bead-Based Assays

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Animal Cell Biotechnology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2095))

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Abstract

A novel version of bead -based assays with fluorescence detection enables the high-throughput analysis of antibodies and proteins. The protocols are carried out in special 384-well plates, require very few manual interventions, and are easy to automate. Here we describe how the technology can be used to determine antibody titers and screen for product glycosylation, a critical quality attribute, early in cell line and bioprocess development.

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References

  1. Zheng K et al (2011) The impact of glycosylation on monoclonal antibody conformation and stability. MAbs 3(6):568–576

    Article  Google Scholar 

  2. Onitsuka M et al (2014) Glycosylation analysis of an aggregated antibody produced by Chinese hamster ovary cells in bioreactor culture. J Biosci Bioeng 117(5):639–644

    Article  CAS  Google Scholar 

  3. Liu L (2018) Pharmacokinetics of monoclonal antibodies and Fc-fusion proteins. Protein Cell 9(1):15–32

    Article  CAS  Google Scholar 

  4. Yu M et al (2012) Production, characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans. MAbs 4(4):475–487

    Article  Google Scholar 

  5. Alessandri L et al (2012) Increased serum clearance of oligomannose species present on a human IgG1 molecule. MAbs 4(4):509–520

    Article  Google Scholar 

  6. Chung CH et al (2008) Cetuximab-induced anaphylaxis and IgE specific for Galactose-α-1,3-Galactose. N Engl J Med 358:1109–1117

    Article  CAS  Google Scholar 

  7. Sola RJ, Griebenow K (2010) Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs 24(1):9–21

    Article  CAS  Google Scholar 

  8. Liu SD et al (2015) Afucosylated antibodies increase activation of FcγRIIIa-dependent signaling components to intensify processes promoting ADCC. Cancer Imunol Res 3(2):173–183

    Article  CAS  Google Scholar 

  9. Junttila TT et al (2010) Superior in vivo efficacy of Afucosylated Trastuzumab in the treatment of HER2-amplified breast cancer. Cancer Res 70(11):4481–4489

    Article  CAS  Google Scholar 

  10. Peschke B et al (2017) Fc-Galactosylation of human immunoglobulin gamma Isotypes improves C1q binding and enhances complement-dependent cytotoxicity. Front Immunol 8:646

    Article  Google Scholar 

  11. Hodoniczky J et al (2005) Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog 21(6):1644–1652

    Article  CAS  Google Scholar 

  12. Wong D et al (2005) Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures. Biotechnol Bioeng 89(2):164–177

    Article  CAS  Google Scholar 

  13. Fan Y et al (2015) Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation. Biotechnol Bioeng 112(3):521–535

    Article  CAS  Google Scholar 

  14. Gramer MJ et al (2011) Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose. Biotechnol Bioeng 108(7):1591–1602

    Article  CAS  Google Scholar 

  15. Bruehlmann D et al (2017) Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation. J Biotechnol 252:32–42

    Article  Google Scholar 

  16. Okeley NM et al (2013) Development of orally active inhibitors of protein and cellular fucosylation. PNAS 110:5404–5409

    Article  CAS  Google Scholar 

  17. Ehret J et al (2019) Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells. Biotechnol Bioeng 116(4):816–830

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Rouiller Y et al (2016) Screening and assessment of performance and molecule quality attributes of industrial cell lines across different fed-batch systems. Biotechnol Prog 32(1):160–170

    Article  CAS  Google Scholar 

  19. Mora A et al (2018) Sustaining an efficient and effective CHO cell line development platform by incorporation of 24-deep well plate screening and multivariate analysis. Biotechnol Prog 34(1):175–186

    Article  CAS  Google Scholar 

  20. Loebrich S et al (2019) Comprehensive manipulation of glycosylation profiles across development scales. MAbs 11(2):335–349

    Article  CAS  Google Scholar 

  21. Wooley CF, Hayes MA (2013) Recent developments in emerging microimmunoassays. Bioanalysis 5(2):245–264

    Article  Google Scholar 

  22. European Medicines Agency 2012: Guideline on bioanalytical method validation

    Google Scholar 

  23. Hendrickson OD, Zherdev AV (2018) Analytical applications of lectins. Crit Rev Analyt Chem 48(4):279–292. https://doi.org/10.1080/10408347.2017.1422965

    Article  CAS  Google Scholar 

  24. Lectin Frontier DataBase (LfDB), Glycoscience and Glycotechnology Research Group, National Institute of Advanced Industrial Science and Technology, Japan. https://acgg.asia/lfdb2/index

  25. Wang L et al (2014) Cross-platform comparison of glycan microarray formats. Glycobiology 24(6):507–517

    Article  CAS  Google Scholar 

  26. Thompson R et al (2011) Optimization of the enzyme-linked lectin assay for enhanced glycoprotein and glycoconjugate analysis. Anal Biochem 413(2):114–122

    Article  CAS  Google Scholar 

  27. Geuijen KP et al (2015) Label-free glycoprofiling with multiplex surface plasmon resonance: a tool to quantify sialylation of erythropoietin. Anal Chem 87:8115–8122

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. PAIA Biotech GmbH, Köln, Germany

    Sebastian Giehring

Authors
  1. Sebastian Giehring
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Correspondence to Sebastian Giehring .

Editor information

Editors and Affiliations

  1. Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology (TUHH), Hamburg, Germany

    Ralf Pörtner

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Giehring, S. (2020). High-Throughput Quantification and Glycosylation Analysis of Antibodies Using Bead-Based Assays. In: Pörtner, R. (eds) Animal Cell Biotechnology. Methods in Molecular Biology, vol 2095. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0191-4_15

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  • DOI: https://doi.org/10.1007/978-1-0716-0191-4_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0190-7

  • Online ISBN: 978-1-0716-0191-4

  • eBook Packages: Springer Protocols

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