Publication Date



Open access

Embargo Period


Degree Type


Degree Name

Doctor of Philosophy (PHD)


Biochemistry and Molecular Biology (Medicine)

Date of Defense


First Committee Member

Arun Malhotra

Second Committee Member

Gayathri Ratnaswamy

Third Committee Member

Thomas K. Harris

Fourth Committee Member

Sapna Deo

Fifth Committee Member

Peter Buchwald

Sixth Committee Member

Jennifer S. Chadwick


Antibody-drug conjugates (ADCs) are a newer class of targeted biologics that combine the specificity of an antibody with the cytotoxic potency of a small molecule. Only four ADCs are currently approved for market, and approximately 60 molecules are in active clinical trials in the US. Several modalities of ADCs are currently under development, one of which uses thiol-maleimide chemistry to conjugate drug-linkers at the antibody’s endogenous interchain cysteines. The most popular framework used for cysteine-linked ADCs is the IgG1 subclass, for which extensive physicochemical and structural information is available in literature. Cysteine-linked antibody-drug conjugates (ADCs) produced from IgG2 monoclonal antibodies (mAbs) are more heterogeneous than ADCs generated from IgG1 mAbs, as IgG2 ADCs are comprised of a wider distribution of molecules, typically containing 0 – 12 drug-linkers (DL) per antibody. The three disulfide isoforms (A, A/B, and B) of IgG2 antibodies confer differences in solvent accessibilities of the interchain disulfides and contribute to the structural heterogeneity of cysteine-linked ADCs. Furthermore, IgG2s are more susceptible to disulfide bond scrambling events that are favored in neutral or high pH conditions, such as those typically employed for thiol-maleimide reactions. Variably conjugated ADCs produced under either acidic or alkaline conditions using IgG2-A and IgG2-B mAbs were compared to better understand the influence of disulfide isoforms on the conjugation profiles of cysteine-linked ADCs. Higher drug-to-antibody ratios (DARs) were observed in the IgG2-A ADCs processed under acidic conditions, indicating that bond connectivity influences the rate of interchain disulfide reduction. On the other hand, both IgG2-A and IgG2-B ADCs generated using the alkaline process exhibited comparable average DARs and distributions of conjugated species, suggesting that conversion of the A to the B disulfide configuration occurs under alkaline conditions. Although the parent mAb disulfide configurations differed, the primary conjugation sites for both A and B isoforms were determined to be the hinge region cysteines for ADCs generated under both acidic and alkaline conditions. Significant efforts went into pinpointing which specific hinge cysteines are conjugated, however, further work remains to be done on this area. Several approaches were explored for this purpose, including middle-down characterization methods and novel techniques for mapping scrambled IgG2 disulfides. Additional work described in this dissertation include work initiated to solve the high-resolution structure of a full-length IgG2 antibody and its ADCs.


antibodies; antibody-drug conjugates; ADC; conjugation profile; conjugation site; bioconjugation