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.
The developed formulation could be used as an inhalation for delivering material possessing poor water solubility into the lungs.
Immunoglobulin G (IgG) consists of four subclasses in humans: IgG1, IgG2, IgG3 and IgG4, which are highly conserved but have unique differences that result in subclass-specific effector functions. Though IgG1 is the most extensively studied IgG subclass, study of other subclasses is important to understand overall immune function and for development of new therapeutics. When compared to IgG1, IgG3 exhibits a similar binding profile to Fcγ receptors and stronger activation of complement. All IgG subclasses are glycosylated at N297, which is required for Fcγ receptor and C1q complement binding as well as maintaining optimal Fc conformation. We have determined the crystal structure of homogenously glycosylated human IgG3 Fc with a GlcNAc2Man5 (Man5) high mannose glycoform at 1.8 Å resolution and compared its structural features with published structures from the other IgG subclasses. Although the overall structure of IgG3 Fc is similar to that of other subclasses, some structural perturbations based on sequence differences were revealed. For instance, the presence of R435 in IgG3 (and H435 in the other IgG subclasses) has been implicated to result in IgG3-specific properties related to binding to protein A, protein G and the neonatal Fc receptor (FcRn). The IgG3 Fc structure helps to explain some of these differences. Additionally, protein-glycan contacts observed in the crystal structure appear to correlate with IgG3 affinity for Fcγ receptors as shown by binding studies with IgG3 Fc glycoforms. Finally, this IgG3 Fc structure provides a template for further studies aimed at engineering the Fc for specific gain of function.
The use of monoclonal antibodies (mAbs) for the manufacture of innovator and biosimilar biotherapeutics has increased tremendously in recent years. From a structural perspective, mAbs have high disulfide bond content, and the correct disulfide connectivity is required for proper folding and to maintain their biological activity. Therefore, disulfide linkage mapping is an important component of mAB characterization for ensuring drug safety and efficacy. The native disulfide linkage patterns of all four subclasses of IgG antibodies have been well established since the late 1960s. Among these IgG subtypes, disulfide mediated isoforms have been identified for IgG2 and IgG4, and to a lesser extent in IgG1, which is the most studied IgG subclass. However, no studies have been carried out so far to investigate whether different IgG3 isoforms exist due to alternative disulfide connectivity. In an effort to investigate the presence of disulfide-mediated isoforms in IgG3, we employed a bottom-up mass spectrometry approach to accurately determine the disulfide bond linkages in endogenous human IgG3 monoclonal antibody and our results show that no such alternative disulfide bonds exist. While many antibody-based drugs are developed around IgG1, IgG3 represents a new, and in some cases, more desirable drug candidate. Our data represent the first demonstration that alternative disulfide bond arrangements are not present in endogenous IgG3; and therefore, they should not be present in recombinant forms used as antibody-based therapeutics.
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