Review for ADC production, quality control and functional assay

ADC drugs are essentially a mixture, which is composed of mAbs connecting different numbers of small molecule drugs. Dar represents the average number of small molecule drugs connected to each mAb. Dar directly affects the efficacy and safety of ADC drugs. In the drug development stage, the variation range of DAR value should be minimized. The coupling sites of ADC drugs are divided into amino groups on lysine residues and sulfhydryl groups on cysteine residues. The DAR coupled by lysine is often small, but there are many potential coupling sites. The coupling reaction is random and the product heterogeneity is large; There are 4 pairs of inter chain disulfide bonds used in the development of ADC drugs. The antibody converts the inter chain disulfide bond into free cysteine residues through partial reduction. The sulfhydryl group in the cysteine residues reacts with the maleimide group in the linker to form ADC. Currently, most ADCs in use or under development rely on interchain disulfide cysteines for conjugation, in which the 4 (IgG1 and IgG4) or 6 (IgG2) interchain disulfide bonds are reduced by an excess reducing agent, namely tris(2-carboxyethyl)phosphine or dithiothreitol.153 This spares disruption of intrachain disulfide bonds while freeing sulfhydryl groups from cysteine residues participating in interchain disulfide bonds (Figure 3e). The resulting product is a mixture of ADCs containing 0–8 drugs per parent IgG1 or IgG4 and 0– 12 per IgG2, with predominantly even numbered DAR (0, 2, 4, 6, 8, 10, 12) species within the ADC mixture.