Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression

Abstract

Antibodies can undergo a variety of covalent and non-covalent degradation reactions that have adverse effects on efficacy, safety, manufacture and storage. We had identified an antibody to Angiopoietin 2 (Ang2 mAb) that neutralizes Ang2 binding to its receptor in vitro and inhibits tumor growth in vivo. Despite favorable pharmacological activity, the Ang2 mAb preparations were heterogeneous, aggregated rapidly and were poorly expressed. Here, we report the engineering of the antibody variable and constant domains to generate an antibody with reduced propensity to aggregate, enhanced homogeneity, 11°C elevated Tm, 26-fold improved level of expression and retained activity. The engineered molecule, MEDI-3617, is now compatible with the large scale material supply required for clinical trials and is currently being evaluated in Phase 1 in cancer patients. This is the first report to describe the stability engineering of a therapeutic antibody addressing non canonical cysteine residues and the design strategy reported here is generally applicable to other therapeutic antibodies and proteins.

Effects of antibody disulfide bond reduction on purification process performance and final drug substance stability

“…With a higher starting level of free thiol in the protein A product, coupled with further free thiol formation during low pH treatment, increased aggregate formation occurs. Although the exact mechanism was not investigated in this study, Buchanan and co‐workers have demonstrated through site‐directed mutagenesis that having unpaired cysteines on the surface of a mAb can lead to significantly increased rates of aggregation (Buchanan et al, 2013). Hence, a larger increase in aggregate content was observed during low pH treatment for Purification Runs 1 and 3 (which showed reduction in the HCCF), but not Purification Run 2 (no reduction in HCCF due to immediate purification after harvest).…”

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

“…Already previous studies indicated that antibodies which are more stable against thermal unfolding are able to generate clones with improved expression rates (Buchanan et al, 2013; Garber and Demarest, 2007). In our study, DSC measurements revealed that the Fv domains were the least stable and unfolded first at 52.1 ± 0.1 (2F5scFv‐Fc) and 53.5 ± 0.2 (3D6scFv‐Fc).…”

Improving biophysical properties of a bispecific antibody scaffold to aid developability

“…26 There is also the realization that development challenges can be addressed by engineering antibodies for enhanced biophysical properties. 27 This recent trend looks beyond the traditional quality by design approaches that tend to address stability and heterogeneity challenges at the formulation stage. Based on this background, we have opined that an engineered heterodimeric Fc that retains wild type antibody-like biophysical properties and consistently achieves high levels of purity is particularly important while developing a protein scaffold to be used subsequently as a platform for the design of bispecific therapeutics.…”