Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis

Abstract

Four different well-defined IgG1 Fc glycoforms are proposed as a model system to examine important biological and physicochemical features for protein drug biosimilar analyses. The IgG1 Fc glycoforms were produced by yeast expression combined with in vitro enzymatic synthesis as a series of sequentially truncated, high mannose IgG1 Fc glycoforms with an anticipated range of biological activity and structural stability. Initial characterization with mass spectrometry, SDS-PAGE, SEC, and cIEF confirmed the glycoproteins are overall highly similar with the only major difference being glycosylation state. Binding to the activating Fc receptor FcγRIIIa was used to evaluate the potential biological activity of the IgG1 Fc glycoproteins. Two complementary methods utilizing biolayer interferometry (BLI), one with protein G immobilized IgG1 Fc and the other with streptavidin immobilized FcγRIIIa, were developed to assess FcγRIIIa affinity in kinetic binding studies. The HM-Fc and Man5-Fc were highly similar to one another with high affinity for FcγRIIIa, while GlcNAc-Fc had weak affinity, and the non-glycosylated N297Q-Fc had no measurable affinity for FcγRIIIa. These four IgG1 Fc glycoforms were also evaluated in terms of physical and chemical stability profiles, and then used as model system to mathematically assess overall biosimilarity, as described in a series of companion papers.

Structural characterization of the Man5 glycoform of human IgG3 Fc

“…The weaker affinity of the GlcNAc-IgG3 Fc was driven by a faster dissociation rate, with the association rate remaining largely unaffected. A similar trend in FcγRIIIA binding was also seen in IgG1 Fc, as previously reported by our group (Okbazghi et al, 2016) and others (Subedi et al, 2014). These results indicate that protein-glycan interactions of the core pentasaccharide (GlcNAc 2 Man 3 ) were necessary in maintaining high affinity FcγRIIIA binding, and that the IgG3 Fc interactions with GlcNAc1 are essential for FcγRIIIA binding.…”

“…The present construct was modified with mutation N392K to delete a potential glycosylation site N 392 TT. The protein was expressed as previously described in glycosylation-deficient Pichia pastoris (SMD1168) yeast with the OCH1 and PNO1 gene deletions (Okbazghi et al, 2016). The strain was further modified by deleting the BMT1 and BMT2 genes to reduce β-mannosylation (Hopkins et al, 2011).…”

“…α-1,2 mannosidase (Bt3990) (Cuskin et al, 2015) and endomannosidase (supplementary information) to convert high mannose glycosylation on the expressed protein to the homogenous GlcNAc 2 Man 5 (Man5) glycoform. Man5-IgG3 Fc was characterized by SDS-PAGE and intact protein mass spectrometry, as per previously described method (Okbazghi et al, 2016). …”

“…The high affinity polymorph (V158) of FcγRIIIA was cloned and expressed in yeast P. pastoris and purified using Ni 2+ -NTA affinity and phenyl sepharose chromatographies, as previously described (Okbazghi et al, 2016; Xiao et al, 2009). Different IgG3 Fc glycoforms were assessed for FcγRIIIA binding with BioLayer Interferometry (BLI) using BLItz.…”

The Botanical Drug Substance Crofelemer as a Model System for Comparative Characterization of Complex Mixture Drugs

“…This approach has recently been demonstrated in proof-of-concept studies from various analytical data sets from four different well-defined IgG1-Fc glycoforms. 14–17 Multiple lots of a drug product are usually necessary to determine the critical quality tests and their acceptable limits, i.e. the critical quality attributes (CQA).…”