Enzymatic transformation of antibodies to obtain single glycoforms
September 15-19, 2019
Introduction: Antibodies are synthesized in mammalian cell culture as heterogeneous mixtures of glycoforms. Therefore the glycan profile of monoclonal antibodies harvested from a bioreactor consists of various glycans depending upon the culture media, the control parameters and the metabolic status of the cells. Isolation of single glycoforms remains a challenge despite their value as agents of specific biological function. We report a method of sequential enzymatic-based changes to antibodies while trapped on an affinity column following harvest from a bioreactor. This method of solid phase enzymatic transformation is superior to previously reported methods because it allows a series of enzymatic steps without the need for intermediate purification of the antibody.
Results: Antibodies (camelid, Cetuximab and polyclonal human) were isolated on a solid-phase lectin column and their glycans modified by a sequential addition of enzymes for a desired transformation. Galactosylated antibodies (>90% yield) were produced by a two stage reaction. Sialylated antibodies (>90%) were produced by a 3 stage reaction involving sialidase, galactosyltransferase and finally treatment with the 2,6 sialyltransferase in the presence of CMP-NANA. Equimolar quantities of monosialylated and disialylated forms of the human antibodies (150 kDa) were produced and the results suggest that further sialylation may be limited by steric constraints within the dimeric structure. The ability to sialylate the smaller camelid antibody (80 kDa) was much greater with a high (>90%) yield of disialylated glycan structures, indicating that the steric constraints were less.
Significance: The biological activity of an antibody is highly dependent upon the glycan structure, which is described as a critical quality attribute for clinical efficacy. It is well reported that non-fucosylated or low galactosylated antibodies have high antibody-dependent cellular cytoxicity (ADCC). Sialylated antibodies have significant activity as ant-inflammatory agents. Therefore, isolating a single glycoform targeted for a specific biological activity offers considerable advantages. The method described in this abstract of enzymatic transformation during antibody purification provides a relatively simple solution to obtaining single glycoforms without the need for complex control during bioprocessing.
Michael Butler, "Enzymatic transformation of antibodies to obtain single glycoforms" in "Enzyme Engineering XXV", Huimin Zhao, University of Illinois at Urbana-Champaign, USA John Wong, Pfizer, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/enzyme_xxv/120