The impact of trisulfide modification of antibodies on the properties of antibody-drug conjugates manufactured using thiol chemistry

Abstract

Antibody-drug conjugates (ADCs) are promising biotherapeutic agents for the treatment of cancer. The careful monitoring of critical quality attributes is important for ADCs' development, manufacturing and production. In this work, the effect of the presence of a trisulfide bond in the monoclonal antibody (mAb) conjugated to DM4 cytotoxic payload through a disulfide-bond linker sulfo-SPDB (sSPDB) was investigated. Three lots of antibody containing variable levels of trisulfide bonds were used. The identity and levels of trisulfide bonds were determined by liquid chromatography/ mass spectrometry (MS)/MS analysis. The antibodies were conjugated to sSPDB-DM4 to generate ADCs. Further analysis indicated that the drug-to-antibody ratio (DAR) value, a critical quality attribute, slightly increased for the conjugates made from antibody containing higher levels of trisulfide bond. Also, higher fragmentation levels were observed in the conjugates with more trisulfide bond. Detailed characterization by MS revealed that a small amount of DM4 payload was directly attached to inter-chain cysteine residues by disulfide or trisulfide bonds. Overall, our investigation indicated that the trisulfide bond present in the mAb could react with DM4 during the conjugation process. Therefore, the presence of trisulfide bonds in the antibody moiety should be carefully monitored and well controlled during the development of a maytansinoid ADC.

Fluctuations in dissolved oxygen concentration during a CHO cell culture process affects monoclonal antibody productivity and the sulfhydryl‐drug conjugation process

“…For interchain cysteine conjugated ADCs, control of the reduction step is critical for achieving desired DAR). The average DAR value is considered to be a critical quality attribute and must be well controlled . Most major structural modifications to the antibody are monitored as critical material attributes (CMAs) as these modifications can affect the reduction step and consequently, the final DAR of the ADC.…”

“…The average DAR value is considered to be a critical quality attribute and must be well controlled. 19 Most major structural modifications to the antibody are monitored as critical material attributes (CMAs) as these modifications can affect the reduction step and consequently, the final DAR of the ADC. Trisulfide modifications are difficult to assay and are typically not listed as a CMA; though, trisulfide modifications have been reported in multiple subtypes of mAbs 20 and have been shown to impact the reduction step in the ADC process when linkage occurs through native interchain disulfide bonds.…”

Simplified identification of disulfide, trisulfide, and thioether pairs with 213 nm UVPD

“…44 The increased bond length between cysteine residues results in higher reactivity, as is well documented in a previous study examining trisulfides in mAbs. 45…”

Antibody‐Incorporated Nanomedicines for Cancer Therapy

“…[133] The free thiol group of cysteine has been utilized as another functional group commonly adopted for antibody conjugation. [134,135] Via the reduction of inter-chain disulfide bonds by tris(2-carboxyethyl) phosphine (TCEP), the free cysteines can provide 0, 2, 4, 6, or 8 sites for conjugation per antibody molecule. [134] TCEP can also be utilized for the mild reduction of C-terminal cysteines of antibodies for further conjugation with nanoparticles.…”