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How is HPLC assessing antibody-dependent cell-mediated cytotoxicity?

6th June 2019


Fig.1. Analysis of mAb glycoforms according to their affinity to Fc Receptor/ADCC activity
Fig.2A and 2B

Regina Römling explains how HPLC is assessing antibody-dependent cell-mediated cytotoxicity

Monoclonal antibodies (mAb) are one of the fastest-growing classes of biopharmaceuticals for multiple clinical indications including cancer, cardiovascular disease, autoimmune disorders and infectious disease. An important mechanism of action (MoA) of monoclonal antibodies used in cancer treatment is the antibody-dependent cell-mediated cytotoxicity (ADCC). Selecting suitable cell lines and optimising culture conditions towards expression of antibody candidates with desired ADCC activity is an essential part of the R&D process. A fast and straightforward approach to easily access ADCC activity facilitates screening of a large number of clones or monitoring the effect of upstream process variations.

ADCC starts with the binding of the antibody to a cancer cell. Binding of the Fc domain of that antibody to Fcy receptors of effector cells triggers a process that finally kills the cancer cell. The interaction of the FcyIIIa receptor with the Fc domain is strongly influenced by the N-glycans of the antibody. As the Fcy receptor IIIa plays an key role in the ADCC process antibodies with high affinity for Fcy receptor IIIA are considered to have a high ADCC activity.

Fc receptor affinity

TSKgel FcR-IIIA-NPR is a new HPLC affinity column developed by Tosoh Corporation that is based on a recombinant version of the FcyIIIa receptor. It could be demonstrated that FcyRIIIa affinity chromatography allows fast evaluation of biologic activity and glycoform pattern of antibodies.[1] Terminal galactose residues increase affinity to FcyRIIIa while core fucose residues reduce it. This correlates with the known influence of galactose and fucose on ADCC activity. Accordingly, early eluting peaks of TSKgel FcR-IIIA-NPR represent glycoforms with low ADCC activity while late eluting peaks represent glycoforms with high ADCC activity (Fig.1).

Separation of mAb glycoforms

Fig.2 A and B demonstrate the ability of the recombinant FcyRIIIA ligand to separate antibody glycoforms depending on the structure of the oligosaccharides bound to the Fc domain of mAbs. Trastuzumab analysed with TSKgel FcR-IIIA-NPR (Fig.2A) shows a typical pattern of three peaks, corresponding with the molecule’s glycan heterogeneity. Based on the relatively high amount of fucose units the chromatogram shows a high amount of glycoforms with lower Fc receptor affinity. The corresponding analysis of a glycoengineered non-fucosylated Trastuzumab (Fig. 2B)reveals the presence of large amounts of glycoforms with high Fc receptor affinity. These non-fucosylated mAb glycoforms are supposed to show enhanced ADCC. The enhanced ADCC activity of the non-fucosylated antibody could be confirmed by ADCC reporter bioassay (data not shown).

HPLC Conditions:

Column:          TSKgel FcR-IIIA-NPR (5 µm, 4.6 mm ID x 7.5 cm L)
Mobile Phase: A: 50 mmol/L sodium citrate, pH 6.5; B: 50 mmol/L sodium citrate, pH 4.5
Flow Rate:       1 mL/min
Temp.:             25°C
Detection:        UV @ 280nm
Sample:           Trastuzumab; non-fucosylated Trastuzumab

A rapid 30-minute separation on TSKgel FcR-IIIA-NPR allows the analysis of large numbers of mAb samples to gain valuable first information on the distribution of glycoforms and expected ADCC activity. This fast and efficient method can be applied to purified samples and supernatant alike and can therefore be used in many phases of development and production, such as cell line screening in early R&D, biosimilar/originator comparison, upstream development and optimisation, monitoring of glycoengineering, or lot-to-lot comparison in QC.

References

[1]. M. Kiyoshi et al., Sci. Rep. 8: 3955 (2018) doi:10:1038/s41598-018-22199-8

Regina Römling is with Tosoh Bioscience





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