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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, size exclusion HPLC, and capillary isoelectric focusing confirmed that the glycoproteins are overall highly similar with the only major difference being glycosylation state. Binding to the activating Fc receptor, Fc?RIIIa was used to evaluate the potential biological activity of the IgG1 Fc glycoproteins. Two complementary methods using biolayer interferometry, 1 with protein G-immobilized IgG1 Fc and the other with streptavidin-immobilized Fc?RIIIa, were developed to assess Fc?RIIIa affinity in kinetic binding studies. The high-mannose IgG1 Fc and Man5-IgG1 Fc glycoforms were highly similar to one another with high affinity for Fc?RIIIa, whereas GlcNAc-Fc had weak affinity, and the nonglycosylated N297Q-Fc had no measurable affinity for Fc?RIIIa. These 4 IgG1 Fc glycoforms were also evaluated in terms of physical and chemical stability profiles and then used as a model system to mathematically assess overall biosimilarity, as described in a series of companion articles.

SUBMITTER: Okbazghi SZ

PROVIDER: S-EPMC4752726 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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

Okbazghi Solomon Z SZ More Apurva S AS White Derek R DR Duan Shaofeng S Shah Ishan S IS Joshi Sangeeta B SB Middaugh C Russell CR Volkin David B DB Tolbert Thomas J TJ

Journal of pharmaceutical sciences 20160109 2

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, size exclusion HPLC ...[more]

PMID: 26869419

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Project description:Fc gamma receptors (Fc?R) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of Fc?Rs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the Fc?R glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fc? receptors RI (CD64), RIIaArg131/His131 (CD32a), RIIb (CD32b) and RIIIaPhe158/Val158 (CD16a) and analyze the role of the glycans in the binding mechanism with IgG. The interactions of the monoclonal antibody rituximab with each Fc?R were characterized and we discuss the CHO-Fc?RIIIaPhe158/Val158 and CHO-Fc?RI interactions and compare them to the equivalent interactions with human (HEK293) and murine (NS0) produced receptors. Our results reveal clear differences in the binding profiles of rituximab, which we attribute in each case to the differences in host cell-dependent Fc?R glycosylation. The glycan profiles of CHO expressed Fc?RI and Fc?RIIIaPhe158/Val158 were compared with the glycan profiles of the receptors expressed in NS0 and HEK293 cells and we show that the glycan type and abundance differs significantly between the receptors and that these glycan differences lead to the observed differences in the respective Fc?R binding patterns with rituximab. Oligomannose structures are prevalent on Fc?RI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On Fc?RI and Fc?RIIIa large and sialylated glycans have a negative impact on rituximab binding, likely through destabilization of the interaction. In conclusion, the data show that the IgG1-Fc?R binding kinetics differ depending on the glycosylation of the Fc?R and further support a stabilizing role of Fc?R glycans in the antibody binding interaction.

Dataset Information

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

Publications

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

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