Trisulfide Modification Impacts the Reduction Step in Antibody–Drug Conjugation Process

Abstract

Antibody-drug conjugates (ADCs) utilizing cysteine-directed linker chemistry have cytotoxic drugs covalently bound to native heavy-heavy and heavy-light interchain disulfide bonds. The manufacture of these ADCs involves a reduction step followed by a conjugation step. When tris(2-carboxyethyl)phosphine (TCEP) is used as the reductant, the reaction stoichiometry predicts that for each molecule of TCEP added, one interchain disulfide should be reduced, generating two free thiols for drug linkage. In practice, the amount of TCEP required to achieve the desired drug-to-antibody ratio often exceeds the predicted, and is variable for different lots of monoclonal antibody starting material. We have identified the cause of this variability to be inconsistent levels of interchain trisulfide bonds in the monoclonal antibody. We propose that TCEP reacts with each trisulfide bond to form a thiophosphine and a disulfide bond, yielding no net antibody free thiols for conjugation. Antibodies with higher levels of trisulfide bonds require a greater TCEP:antibody molar ratio to achieve the targeted drug-to-antibody ratio.

In vitro and in vivo modifications of recombinant and human IgG antibodies

“…115,116 Trisulfide bonding has also been shown to affect the reduction step for the production of antibody-drug conjugation. 117 Cell culture parameters such as scale and age have a significant effect on the level of trisulfide bonds. 115,118 Trisulfide bonding can be eliminated by incubation with mild reducing reagents.…”

A high-throughput hydrophilic interaction liquid chromatography coupled with a charged aerosol detector method to assess trisulfides in IgG1 monoclonal antibodies using tris(2-carboxyethyl)phosphine reaction products: Tris(2-carboxyethyl)phosphine-oxide and tris(2-carboxyethyl)phosphine-sulfide

“…For proteins, the modification was first discovered in cysteine bridges (Cys 53 -Cys 165 and Cys 182 -Cys 189 ) of biosynthetic human growth hormone produced in Escherichia coli [1,2] and in the non-recombinant protein Cu-Zn superoxide dismutase [3]. More recently, trisulfides have been identified and characterized in recombinant monoclonal antibodies (mAbs) of all immunoglobulin G (IgG) sub-classes [4][5][6][7]. Using peptide mapping liquid chromatography mass spectrometry (LC-MS) for mAb characterization, mAb trisulfides have been found in the inter-chain disulfide bridges, predominantly in the heavy chain-light chain (HC-LC) bridges [5,[8][9][10].…”

Antibody–drug conjugates: targeted weapons against cancer

“…26 At the same time, there is a pressing need for more specific analytical techniques, useful to assess the structural effect of conjugation, 48 and this becomes even more relevant on the manufacturing side of ADCs, where further issues need to be addressed (like, for example, in the case of trisulphide modification). 49 …”