Air oxidation method employed for the disulfide bond formation of natural and synthetic peptides

Abstract

Among the available protocols, chemically driven approaches to oxidize cysteine may not be required for molecules that, under the native-like conditions, naturally fold in conformations ensuring an effective pairing of the right disulfide bridge pattern. In this contest, we successfully prepared the distinctin, a natural heterodimeric peptide, and some synthetic cyclic peptides that are inhibitors of the CXCR4 receptor. In the first case, the air oxidation reaction allowed to connect two peptide chains via disulfide bridge, while in the second case allowed the cyclization of rationally designed peptides by an intramolecular disulfide bridge. Computational approaches helped to either drive de-novo design or suggest structural modifications and optimal oxidization protocols for disulfide-containing molecules. They are able to both predict and to rationalize the propensity of molecules to spontaneously fold in suitable conformations to achieve the right disulfide bridges.

Characterization of low‐abundance species in the active pharmaceutical ingredient of CIGB‐300: A clinical‐grade anticancer synthetic peptide

“…The molecular mass of this species (Table , fraction # I) is increased by 16 Da compared with the intact cyclic CIGB‐300 (Table , fraction III). This mass difference suggests the presence of an oxidation at Met 17 to a sulfoxide residue (Met 17 ox), generated mainly during synthesis and probably promoted during the cyclization step …”

“…The ESI-MS analysis (Figure 2A 17 to a sulfoxide residue (Met 17 ox), generated mainly during synthesis and probably promoted during the cyclization step. 19 The MS/MS spectra of the multiply charged ions corresponding to the intact cyclic CIGB-300 with Met 17 ox did not provide enough information to support the above-mentioned assignment (data not shown). Probably, the presence of several basic amino acids, the lack of mobile protons, [20][21][22] and the cyclic nature of this peptide (where Met 17 ox is located) impaired its efficient fragmentation by collisioninduced dissociation (CID).…”

“…Several internal fragments were also observed in the MS/MS ( Figure 8B) and assigned as A to I in has been used as a strategy to improve the biological activity of peptides. [49][50][51][52][53] In principle, dimers and other multimers (described in fraction VII) could be generated by spontaneous air oxidation during the cyclization step, 19,45 even under experimental conditions where the diluted concentration of the reduced peptide 5 did not favor the formation of intermolecular disulfide bonds. However, under our conditions after the cyclization step, RP chromatography efficiently removed all multimers including dimers, but they increased once again after lyophilization although at very low abundance levels (<0.3%).…”

On-column disulfide bond formation of monoclonal antibodies during Protein A chromatography eliminates low molecular weight species and rescues reduced antibodies

“…It is worth noting that the sulfhydryl group of cysteine is highly reactive and cysteine itself can form cystine through natural air oxidation. 27 , 46 , 47 Therefore, the stability of the redox buffer needs to be assessed to establish the expiry specification based on its reactivity. For this purpose, the redox buffer stored at room temperature and 2–8°C was used for on-column reoxidation study.…”

Optimization and kinetic modeling of interchain disulfide bond reoxidation of monoclonal antibodies in bioprocesses

“…As a post-translational modification, a disulfide bond is formed by reoxidizing two neighboring free cysteine residues. 22 – 24 Though there is a vast body of knowledge of in-vitro disulfide reoxidation for antibodies, the majority of these studies focused on the solution environment with limited investigational conditions. 8 , 18 , 19 , 25 , 26 The existing studies cannot be directly and practically implemented in manufacturing process for three reasons.…”