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

  • Sebastian GiehringEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2095)


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.

Key words

Product quality Monoclonal antibodies Glycosylation Titer assay High-throughput assays Bead-based assays Bioprocess development Cell line development Lectins 


  1. 1.
    Zheng K et al (2011) The impact of glycosylation on monoclonal antibody conformation and stability. MAbs 3(6):568–576CrossRefGoogle Scholar
  2. 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):639–644CrossRefGoogle Scholar
  3. 3.
    Liu L (2018) Pharmacokinetics of monoclonal antibodies and Fc-fusion proteins. Protein Cell 9(1):15–32CrossRefGoogle Scholar
  4. 4.
    Yu M et al (2012) Production, characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans. MAbs 4(4):475–487CrossRefGoogle Scholar
  5. 5.
    Alessandri L et al (2012) Increased serum clearance of oligomannose species present on a human IgG1 molecule. MAbs 4(4):509–520CrossRefGoogle Scholar
  6. 6.
    Chung CH et al (2008) Cetuximab-induced anaphylaxis and IgE specific for Galactose-α-1,3-Galactose. N Engl J Med 358:1109–1117CrossRefGoogle Scholar
  7. 7.
    Sola RJ, Griebenow K (2010) Glycosylation of therapeutic proteins: an effective strategy to optimize efficacy. BioDrugs 24(1):9–21CrossRefGoogle Scholar
  8. 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):173–183CrossRefGoogle Scholar
  9. 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):4481–4489CrossRefGoogle Scholar
  10. 10.
    Peschke B et al (2017) Fc-Galactosylation of human immunoglobulin gamma Isotypes improves C1q binding and enhances complement-dependent cytotoxicity. Front Immunol 8:646CrossRefGoogle Scholar
  11. 11.
    Hodoniczky J et al (2005) Control of recombinant monoclonal antibody effector functions by Fc N-glycan remodeling in vitro. Biotechnol Prog 21(6):1644–1652CrossRefGoogle Scholar
  12. 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):164–177CrossRefGoogle Scholar
  13. 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):521–535CrossRefGoogle Scholar
  14. 14.
    Gramer MJ et al (2011) Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose. Biotechnol Bioeng 108(7):1591–1602CrossRefGoogle Scholar
  15. 15.
    Bruehlmann D et al (2017) Cell culture media supplemented with raffinose reproducibly enhances high mannose glycan formation. J Biotechnol 252:32–42CrossRefGoogle Scholar
  16. 16.
    Okeley NM et al (2013) Development of orally active inhibitors of protein and cellular fucosylation. PNAS 110:5404–5409CrossRefGoogle Scholar
  17. 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):816–830PubMedPubMedCentralGoogle Scholar
  18. 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):160–170CrossRefGoogle Scholar
  19. 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):175–186CrossRefGoogle Scholar
  20. 20.
    Loebrich S et al (2019) Comprehensive manipulation of glycosylation profiles across development scales. MAbs 11(2):335–349CrossRefGoogle Scholar
  21. 21.
    Wooley CF, Hayes MA (2013) Recent developments in emerging microimmunoassays. Bioanalysis 5(2):245–264CrossRefGoogle Scholar
  22. 22.
    European Medicines Agency 2012: Guideline on bioanalytical method validationGoogle Scholar
  23. 23.
    Hendrickson OD, Zherdev AV (2018) Analytical applications of lectins. Crit Rev Analyt Chem 48(4):279–292. Scholar
  24. 24.
    Lectin Frontier DataBase (LfDB), Glycoscience and Glycotechnology Research Group, National Institute of Advanced Industrial Science and Technology, Japan.
  25. 25.
    Wang L et al (2014) Cross-platform comparison of glycan microarray formats. Glycobiology 24(6):507–517CrossRefGoogle Scholar
  26. 26.
    Thompson R et al (2011) Optimization of the enzyme-linked lectin assay for enhanced glycoprotein and glycoconjugate analysis. Anal Biochem 413(2):114–122CrossRefGoogle Scholar
  27. 27.
    Geuijen KP et al (2015) Label-free glycoprofiling with multiplex surface plasmon resonance: a tool to quantify sialylation of erythropoietin. Anal Chem 87:8115–8122CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.PAIA Biotech GmbHKölnGermany

Personalised recommendations