ThioMAb Drug Conjugates: Optimizing the Re-Oxidation Reaction for Conjugation

Tully, T., Genentech, Inc

ThioMAb Drug Conjugates (TDCs) are a type of therapeutic antibody to which toxins or chemotherapeutic agents are chemically linked to engineered cysteines which have been introduced into the amino acid sequence by single point mutations. Typically, two engineered cysteines are introduced per antibody on the heavy chains, light chains, or Fc. During cell culture, the engineered cysteines are capped with glutathione or cysteine available in the media through a disulfide bond. After purification of the antibody, the drug is conjugated to the engineered cysteines via a maleimide containing linker using a sequence of reduction, re-oxidation, and conjugation steps. First, the TDC is reduced sufficiently to break all interchain disulfide bonds and to releases the caps from the engineered cysteines. The reductant and free caps are removed by diafiltration. The TDC is then fully re-oxidized to reform all interchain disulfide bonds, leaving only the engineered cysteines available to be conjugated with toxin. The result is a homogeneous mixture where each antibody is conjugated with two drugs. TDCs have been demonstrated to improve the therapeutic index (maximum tolerated dose / effective dose) compared to conventional drug conjugation strategies for some antibodies.

One potential by-product of the conjugation process is mis-folded antibody due to the formation of intrachain disulfide bonds rather than interchain disulfide bonds in the hinge region of the antibody. The mis-folded antibodies may be detected under denaturing conditions (SDS-PAGE) and appear as 75 kD species containing one heavy chain and one light chain (HL) at levels of 3 to 10% of the total antibody. Formation of the mis-folded species may be minimized by optimization of the re-oxidation reaction parameters such as antibody concentration, pH, temperature, oxidizing agent level, and reaction rate. The optimized TDC conjugation process successfully minimized HL levels to ~2%.