Development of affinity ligands for antibody glycoform separations based on the Fc-gamma receptor

Biopharmaceuticals continue to grow as a fraction of pharmaceutical sales, the search for new products with increased efficacy often means the detailed glycobiology of the product and its interactions with the immune system are critical. Current products including antibodies and erythropoietin are heterogeneous in their glycosylation state, even though it is known that for example non-fucosylated antibodies are more potent in raising antibody-dependent cell cytotoxicity (ADCC) [1] than fucosylated glycoforms. Current products using CHO cell lines express high levels of fucosylation. Processes capable of homogeneous glycosylation have therefore generated considerable interest and investment. These approaches have generally been upstream focused e.g. to knock out fut8 in CHO [2] and create a fucosylation deficient host cell or engineer human glycosylation in yeast [3].

Here we propose to take a downstream processing based solution by designing affinity ligands capable of glycoform recognition for use in chromatography. This concept is based on the naturally occurring interaction between antibodies and immune system which is mediated by the Fc gamma receptor. It has been used in affinity chromatography using the wild-type protein as the ligand to perform preparative separations [4] and characterise the strength of the Fc gamma/Ab interactions [5]. To be a useful basis for manufacturing such ligands need to be stable and robust in their separation properties. The project will therefore examine rational protein engineering of the Fc gamma ligand for this purpose a strategy that has been effective in improving the protein A ligand that is the default technology for mAb manufacturing.

The wild-type Fc gamma 3a ligand has been shown to have selectivity between fucosylated and non-fucosylated antibody glycoforms hence it is of clinical and commercial interest. However it has five glycosylation sites all of which exhibit a considerable diversity in the structure. Using this as a starting point the project will:
-Design a cloning, expression and purification strategy for the Fc gamma affinity ligands.
-To generate components for model separation mixtures using an enzyme based strategy to remodel glycosylation on antibodies to create homogeneous preparations e.g. non-fucosylated structures.
-A similar strategy to the above will be employed to create Fc gamma 3a ligands of defined glycan structure.
-The ligands will then be immobilised onto agarose using site specific chemistry and their performance in affinity chromatography tested using the model antibody mixtures.
-The chromatography will then be applied to both the separation of antibody glycoforms in CHO expression systems and in fractionated blood.

References
[1] Enhanced Effector Functions Due to Antibody Defucosylation Depend on the Effector Cell Fcy Receptor Profile. Bruggeman, CW., Dekkers, G., et al. The Journal of Immunology. 2017, 199:204-211.
[2] Functional knockout of FUT8 in Chinese hamster ovary cells using CRISPR/Cas9 to produce a defucosylated antibody. Tao, S., Chaodong, L., Lei, H., et al. Eng. Life. Sci. 2015, 15: 660-666.
[3] GlycoFi's technology to control the glycosylation of recombinant therapeutic proteins. Beck A, Cochet O, Wurch T. Expert Opin Drug Discov. 2010, 5(1):95-111.
[4] Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered Fc?Receptor IIIa-Immobilized Column. Scientific Reports. 2018, 8:3955
[5] In Vitro Glycoengineering of IgG1 and Its Effect on Fc Receptor Binding and ADCC Activity. Thomann, M., Schlothauer, T., Dashivets, T., et al. Plos One. 2015, 10(8):e0134949.