**Acknowledgements**

*Monoclonal Antibodies*

**12. Conclusions**

**Figure 30.**

turing and quality control.

*Workflow for the characterization of mAb.*

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, manufac-

*Separation of mAb glycoforms using (A) analytical FcR column and (B) preparative FcR column.*

**102**

**Figure 31.**

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 and suggestions to the manuscript.
