The cysteine mutant technology is a novel site specific antibody drug conjugate (ADC) technology. In this technology the mutant cysteine residues of the antibody are typically protected with cysteine or glutathione protecting groups. The conjugation protocol (total reduction & re-oxidation) for these constructs may impact the quality attributes of the resulting ADCs and in addition, involves multiple steps.<br/>A labile protecting group, 5-thio-2-nitrobenzoic acid (TNB), that can be selectively removed using tris(3-sulfophenyl) phosphine (TSPP) was discovered at the Bioprocess R&D group within Pfizer. Proof of concept using partially uncapped cysteine mutants was done to demonstrate the concept is viable. This labile TNB capping group has enabled the selective reduction for direct conjugation and therefore eliminates the harsh conditions of conventional reduction-reoxidation and buffer exchange of totally reduced antibody. As a result, the three dimensional structure of the original antibody is preserved and in addition, this protocol only involves 2 steps – selective reduction & conjugation.<br/>The BioMedicines Design department within Pfizer discovered that the Cys-capping modifications likely take place outside mammalian cells and the capping status of unpaired Cys residues can be manipulated through medium optimization. They have demonstrated that novel TNB-capping can be engineered by adding Ellman’s reagent [DTNB – 5,5’-Dithiobis-(2-nitrobenzoic acid)] into cysteine/cystine-depleted medium.<br/>Currently the Bioprocess R&D group is developing & optimizing culture conditions to maximize efficiency & homogeneity of TNB-capping and yield of protein. The progress and advantages of this alternative TNB technology for cysteine mutants in the preparation of ADCs will be presented.

Engineering TNB (5-Thio-2-NitroBenzoic acid) on antibody to enable selective reduction

255th National Meeting (2018)

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