**12. Conclusions**

TSKgel FcR-IIIA-NPR affinity chromatography column is a unique tool separating monoclonal antibodies into three peaks based on their glycosylation profile. Selectivity of the genetically engineered FcγRIIIa ligand is very specific to the mAb based on its glycan composition on highly conserved Asn-297 residue. IgG1, IgG3 and IgG4 subclasses bind to the FcR column whereas IgG2 subclass does not have affinity. IgA and IgM also don't bind to this column. Non-glycosylated mAb also does not bind to the column. Importantly, this column can be used for fast evaluation of antibody's ADCC effector function since the peak profile correlates well with the ADCC activity. Longer the retention time, higher is the ADCC activity.

The generally accepted workflow for mAb characterization, based on its glycan content typically follows the three different pathways (**Figure 31**, panel A). These are reporter bioassay for monitoring ADCC activity, Surface Plasmon Resonance (SPR) for measuring FcR affinity and U/HPLC-MS analysis for characterization of the glycan structure. Characterization of mAb on TSKgel FcR-IIIA-NPR column can combine these three pathways to one workflow (**Figure 31**, Panel B) in most circumstances.

The column is expected to be useful in several application areas (**Figure 32**) including (a) early screening of ADCC activity, (b) upstream (cell culture) optimization, (c) quality control of the mAb lot-to-lot consistency and (d) comparison between innovator and biosimilar products. Overall, this novel FcR affinity column is anticipated to be useful in research and development, characterization, manufacturing and quality control.

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*Analytical Characterization of Monoclonal Antibodies with Novel Fc Receptor-Based…*

Chromatogram showing separation of mAb glycoforms using preparative TSKgel FcR-IIIA-5PW column (**Figure 30**, Panel B) was kindly provided by Scott L. Melideo, Ph.D., Tosoh Bioscience LLC. We sincerely thank Oscar Yamasaki and Hiroshi Tomizawa (Tosoh Bioscience, Tokyo, Japan) for their valuable comments

All the authors are current employees of Tosoh Bioscience, part of the Tosoh Corporation, that markets the TSKgel FcR-IIIA-NPR affinity column. Beyond this, the authors are not aware of any affiliations, memberships, funding, or financial

, Egbert Müller2

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Toru Tanaka3

holding that might be perceived as affecting the objectivity of this work.

\*, Jukka Kervinen1

1 Tosoh Bioscience LLC, King of Prussia, PA, USA

2 Tosoh Bioscience GmbH, Darmstadt, Germany

3 Bioscience Division, Tosoh Corporation, Japan

provided the original work is properly cited.

\*Address all correspondence to: atis.chakrabarti@tosoh.com

*DOI: http://dx.doi.org/10.5772/intechopen.95356*

**Acknowledgements**

**Figure 32.**

**Conflict of interest**

**Author details**

Atis Chakrabarti1

and Kazuaki Muranaka<sup>3</sup>

and suggestions to the manuscript.

*Application areas of analytical and preparative FcR columns.*

**Figure 31.** *Workflow for the characterization of mAb.*

*Analytical Characterization of Monoclonal Antibodies with Novel Fc Receptor-Based… DOI: http://dx.doi.org/10.5772/intechopen.95356*

**Figure 32.** *Application areas of analytical and preparative FcR columns.*
