Monoclonal antibodies (mAbs) are an important class of protein therapeutics. Due to their structure complexity, therapeutic mAbs need to be well characterized to ensure their structural and functional integrity, as well as safety and manufacturing consistency. Various modifications (glycosylation, oxidation, glycation, deamidation, etc.) have been described to result in altered antibody properties and activity, or even undesirable adverse biological effects. With years of experience in antibody development and characterization, Creative Biolabs offers comprehensive analytical services of mAb post-translational modifications (PTMs). Here, we focus on the analysis of cysteine (Cys)-related variants, including free sulfhydryl groups, thioether, cysteinylation, and trisulfide bond.
Disulfide bonds of Immunoglobulin G (IgG) are formed by the oxidation of 2 thiol groups within Cys residues. Intra-chain disulfide bonds are formed between two cysteines within the same antibody chain, while inter-chain disulfide bonds are formed between heavy-heavy chains or heavy-light chains. Theoretically, all cysteine residues of mAbs should be involved in the formation of either intra- or inter-chain disulfide bonds in a well-defined linkage pattern. However, several variants have been discovered, including the presence of free sulfhydryls, thioether formation, cysteinylation, and trisulfide bond.
Fig.1 Cys-related variants of IgG antibodies. (Beck, 2013)
➢ Free sulfhydryl - A disulfide bond can undergo reversible β-elimination to initially form one dehydroalanine and one persulfide on constituent chains. Continued degradation of the persulfide converts into free sulfhydryl representing a point of no return for the native disulfide. This formation of a free sulfhydryl group can increase molecular mass by 2 Da and result in size heterogeneity. Higher levels of free sulfhydryl have been associated with decreased thermal stability, the formation of covalent aggregates, and decreased potency.
➢ Thioether formation and cysteinylation - The dehydroalanine may react with the free sulfhydryl to form a non-reducible thioether bond. Alternatively, free sulfhydryls can become covalently modified by a free Cys in the solution, termed cysteinylation. The formation of thioether reduces mAb molecular weight by 32 Da due to the loss of a sulfur atom. Cysteinylation is rarely observed in mAbs but if it is observed, the effect on antibody function is significant.
➢ Trisulfide bond - Trisulfid bond is a modification formed by the insertion of a sulfur atom into an existing disulfide bond. Therefore, trisulfide bond increases mAb molecular weight by 32 Da. It is commonly observed in all IgG subtypes. Optimizing Cys feed strategies can minimize these trisulfide variants.
Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules. Incomplete formation of disulfide bonds results in heterogeneity, leading to reduced antibody stability and potency. As a result, cysteine-related variants caused by incomplete formation of disulfide bonds should be detected and quantified to ensure product quality and to lower the safety risks. Creative Biolabs offers comprehensive analytical services to help identify and quantify PTMs and physicochemical modifications of recombinant mAbs, using our advanced charge-based techniques, mass spectrometry, and colorimetric assays.
Moreover, we offer a wide spectrum of antibody function assays to help strengthen your understanding of mAb structure-function relationships. If you are interested in our services, please do not hesitate to contact us for more details.
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