Characterization of Human IgG1 Fc Region Stability and Aggregation Propensity

Abstract

Monoclonal therapeutic antibodies (MAb) are highly valued for their efficacy in treating ailments ranging from arthritis to cancer. Unrivaled specificity and infrequent adverse effects benefit patients in ways traditional small molecule therapeutics cannot. However, formation of aggregates during expression, purification, or storage as a drug substance can lead to product loss and increased immunogenicity. Glycans present on the antibodies also contribute to both aggregation propensity and antibody effector functions. Understanding the importance of glycans in aggregation and protein function is important in limiting costs and maximizing therapeutic efficacy. Antibodies are comprised of two heavy chains and two light chains, and regions are alternatively classified into Fab (fragment antigen binding) and Fc (fragment crystallizable) regions. Only heavy chain subunits exist in the Fc region and remain constant between MAbs of the same isotype (commonly human IgG1). Uniformity of Fc regions between antibodies allows for creation of fusion proteins containing an Fc region that facilitates affinity purification using a standardized protocol. The Fc region is N-glycosylated at Asn-297, and glycoform identities drive effector functions via interactions with specific Fcγ receptors (FcγRs). We have utilized mammalian cells to express the human IgG1 Fc region and will present initial studies on efforts to purify and characterize the aggregation propensity and stability of this antibody fragment under controlled stress conditions (e.g. thermal, mechanical, etc.). Data collected from these conditions has potential applications in building and modifying protein stability models and could improve the rational design of future recombinant therapeutic antibodies.