An explorative study towards the chemical synthesis of the immunoglobulin G1 Fc CH3 domain

Abstract

Monoclonal antibodies, fusion proteins including the immunoglobulin fragment c (Ig Fc) CH2‐CH3 domains, and engineered antibodies are prominent representatives of an important class of drugs and drug candidates, which are referred to as biotherapeutics or biopharmaceuticals. These recombinant proteins are highly heterogeneous due to their glycosylation pattern. In addition, enzyme‐independent reactions, like deamidation, dehydration, and oxidation of sensitive side chains, may contribute to their heterogeneity in a minor amount. To investigate the biological impact of a spontaneous chemical modification, especially if found to be recurrent in a biotherapeutic, it would be necessary to reproduce it in a homogeneous manner. Herein, we undertook an explorative study towards the chemical synthesis of the IgG1 Fc CH3 domain, which has been shown to undergo spontaneous changes like succinimide formation and methionine oxidation. We used Fmoc‐solid‐phase peptide synthesis (SPPS) and native chemical ligation (NCL) to test the accessibility of large fragments of the IgG1 Fc CH3 domain. In general, the incorporation of pseudoproline dipeptides improved the quality of the crude peptide precursors; however, sequences larger than 44 residues could not be achieved by standard stepwise elongation with Fmoc‐SPPS. In contrast, the application of NCL with cysteine residues, which were either native or introduced ad hoc, allowed the assembly of the C‐terminal IgG1 Fc CH3 sequence 371 to 450. The syntheses reported here show advantages and limitations of the chemical approaches chosen for the preparation of the synthetic IgG1 Fc CH3 domain and will guide future plans towards the synthesis of both the native and selectively modified full‐length domain.

Susceptibility of protein therapeutics to spontaneous chemical modifications by oxidation, cyclization, and elimination reactions

“…As a matter of fact, a lot of efforts are made to develop analytical tools and chemical protocols for the detection of spontaneous modifications in proteins and biopharmaceuticals (Alcock et al 2018;Beck et al 2013;Berkowitz et al 2012;Forstenlehner et al 2015;Grassi et al 2017;Kettenhofen and Wood 2010;Regl et al 2017;Schweida et al 2019). Moreover, (semi)synthetic approaches are applied to reproduce proteins containing the spontaneous modification of interest at selected positions, to evaluate its impact on the protein structure and biology: for example, an explorative study has been conducted to assess the synthetic accessibility of the Fc CH3 IgG1 region containing a modified residue, i.e., Met(O), at the desired position, to avoid the postproduction treatment with strong oxidants (Grassi et al 2018). Furthermore, the semisynthetic approach has been used to prepare the two site-selective glycated proteins Hsp27 (Matveenko et al 2016) and Tau (Ellmer et al 2019): glycation refers to a class of non-enzymatic modifications of biomolecules containing nitrogen nucleophiles that react with the carbonyl group of ketoses or aldoses to build a Schiff base.…”