Free Cysteine Thiol Groups Assessment

Creative Biolabs has established a platform to predict a series of post-translational modifications of antibody therapeutics during manufacturing. Correctly formed intra-chain disulfide bonds improve the protein tertiary structure, while inter-chain disulfide bonds contribute to the protein quaternary structure. Unexpected free cysteine residues, however, may cause problems such as protein misfolding, aggregation, increased immunogenicity or unwanted reactions with other molecules in the environment. In antibodies, free cysteines are most likely located in CDRs and cause low productivity. Besides, they can be easily chemically modified before antibody reaches its antigen in vivo and may result in an altered binding capability.

IgG4 and IgG2 are the two human IgG classes that are susceptible to hinge region disulfide scrambling. IgG2 antibodies are prone to form three main structural and functional disulfide isoforms. A major change in the antibody bioactivity and safety may be caused by the interconversion among these three isoforms. During production, the level of the formed different species should be monitored and kept within predefined limits. To minimize the variation and risk, process parameters and formulation conditions must be carefully controlled. Product candidates containing solvent exposed free cysteines or unexpected free cysteines should be avoided where possible. If the free cysteines are detected without playing an important role in function, they could be simply substituted by protein engineering.

Creative Biolabs guarantees  to search the protein candidate sequence against an internal database to locate related sequences, free cysteines and conserved disulfide bonds. We will highlight the free cysteines that do not belong to conserved regions. We will also complete a visual analysis report about possible influences from these structural features. All the antibodies are characterized in house within 2-3 weeks as customer desires.

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