This study underscored the importance of monitoring and controlling the trisulfide bonds during the process development of cysteine-linked ADCs. Pharmaceutical companies continue to develop linkers and cytotoxic agents to expand their arsenal of ADCs. the antibody moiety should be carefully HJ1 monitored and well controlled during the development of a maytansinoid ADC. KEYWORDS:Antibody-drug conjugates (ADC), drug-to-antibody ratio (DAR), LC-MS, sSPDB-DM4, PCI-27483 trisulfide bond == Abbreviations == monoclonal antibodies Antibody-drug conjugate crucial PCI-27483 quality contributes electrospray ionization mass spectrometry drug-to-antibody ratios succinimidyltrans-4-(maleimidylmethyl) cyclohexane-1-carboxylate N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate N(2)-deacetyl-N(2)-(4-mercapto-4-methyl-1-oxopentyl)-maytansine capillary electrophoresis == Introduction == Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies (mAbs) coupled to potent cytotoxic brokers through various linker technologies. These molecules take advantage of the biologic specificity of mAbs and the high potency of chemotherapeutic brokers. The results of such combinations are more targeted and efficacious novel biotherapeutic products.1-6By combining the target-specific capabilities of antibodies with the tumor-killing ability of cytotoxic molecules, ADCs allow sensitive discrimination between healthy and tumor tissue and work as magic bullets for targeted cancer therapy.4So far, 3 ADCs have been approved by the US Food and Drug Administration, and 2 of these are currently marketed: brentuximab vedotin7(Adcetris) and ado-trastuzumab emtansine8,9(T-DM1, Kadcyla). Many more ADCs are currently under development and in clinical trials, representing a new wave of cancer drug development. A well-selected linker between the antibody and cytotoxic agent (payload) is usually a crucial aspect for the success of an ADC.10-14Appropriate linker technology will ensure the general stability of ADCs duringin vivocirculation and efficient drug release once internalized.4,10,15Linkers are generally categorized into 2 groups: cleavable and non-cleavable. PCI-27483 For example, ado-trastuzumab emtansine contains a non-cleavable linker, succinimidyltrans-4-(maleimidylmethyl) cyclohexane-1-carboxylate (SMCC). The maleimide moiety of SMCC links to the free sulfhydryl group of DM1, the cytotoxic agent, through a non-cleavable thioether bond.8In contrast, brentuximab vedotin contains a peptide linker that can be cleaved by proteases.7,16,17Other cleavable linkers include acid-labile linkers and thiol chemistry linkers, which form a disulfide bond between linker and cytotoxic agent.4,18The mechanism of release of the payloads is dependent on linker chemistry. For ado-trastuzumab emtansine, the cytotoxic drug is usually released after degradation of the ADC in lysosomes,19,20while for brentuximab vedotin, the cytotoxic drug is usually released by cathepsin.7,17Both cleavable and non-cleavable linkers should be stable in the blood stream becausein vivoinstability of an ADC may affect the off-target toxicity. Conventionally, conjugation of the linker to the mAb was achieved through reactive amino acid groups such as lysine (e.g., ado-trastuzumab emtansine) or free thiols of cysteines from reduction of inter-chain disulfide bonds (e.g., brentuximab vedotin). In the latter case, the inter-chain cysteine was first reduced by tris(2-carboxyethyl)phosphine (TCEP), and the free cysteine therefore could conjugate to linker to generate the ADC. However, both methods will generate heterogeneous ADCs. Lysine-based conjugation generally results in 0 to 8 or more payloads per molecule, while the conjugation sites can be more than 20 per half antibody.21Cysteine conjugation, which occurs through reduction of inter-chain disulfide, results in 0, 2, 4, 6, to 8 linker-payloads per molecule, although the number of sites of conjugation is limited to 8. Therefore, the drug-to-antibody ratio (DAR) value is usually calculated as the average number of payload per antibody molecule. The average DAR value is usually a critical quality attribute (CQA) for ADCs, and it should be well controlled during the conjugation process and must be consistent between batches.22,23 It is very important to identify and monitor CQAs during the ADC process development, manufacturing and lot release. Disulfide bonds are critical for antibody structures, stability and biologic function. Each subclass of IgG molecules has well-defined homogenous disulfide bond structures; however, free cysteine residues, scrambled disulfide bonds and the presence of trisulfide bonds and thioether bonds have also been widely reported.24-26More recently, trisulfide bonds have been reported in all 4 subclasses of human IgG molecules.27-31In a recent study of ADC development using inter-chain cysteine-directed linker chemistry as described above, trisulfide bonds have PCI-27483 been reported to affect the reduction step by TCEP during the.