Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design

Abstract

While the concept of Quality-by-Design is addressed at the upstream and downstream process development stages, we questioned whether there are advantages to addressing the issues of biologics quality early in the design of the molecule based on fundamental biophysical characterization, and thereby reduce complexities in the product development stages. Although limited number of bispecific therapeutics are in clinic, these developments have been plagued with difficulty in producing materials of sufficient quality and quantity for both preclinical and clinical studies. The engineered heterodimeric Fc is an industry-wide favorite scaffold for the design of bispecific protein therapeutics because of its structural, and potentially pharmacokinetic, similarity to the natural antibody. Development of molecules based on this concept, however, is challenged by the presence of potential homodimer contamination and stability loss relative to the natural Fc. We engineered a heterodimeric Fc with high heterodimeric specificity that also retains natural Fc-like biophysical properties, and demonstrate here that use of engineered Fc domains that mirror the natural system translates into an efficient and robust upstream stable cell line selection process as a first step toward a more developable therapeutic.

Keywords: Fc engineering; LC-MS of antibody; antibody engineering; antibody stability; biophysical characterization of antibody; bispecific antibody; heterodimeric antibody; quality by design.

Figure 1.(A) The figure illustrates the total number of variants that were experimentally tested and the progress in achieving > 95% heterodimer purity and wild type Fc stability over the three design iterations. The heterodimer specificity was measured by LC-MS and the thermal stability is expressed by the melting temperature (Tm) of the CH3 domain, measured by differential scanning calorimetry. The detailed design process is described in the Methods and Supplemental Material. (B) Selected variants from the 3 iterations of the design process in (A), including the final lead variant ZW1. (C) Superposition of the ZW1 Fc crystal structure and a wild type Fc crystal structure (PDB ID: 2WAH) to illustrate the equivalence of the heterodimeric ZW1 and the homodimeric wild type CH3 domain structure. The close-up shows a stereo representation of the CH3 domain interface.

Figure 2.(A) Differential scanning calorimetry (DSC) of ZW1 in comparison to wild type Fc and the Knob-into-hole (KiH) control. The melting temperature of the CH2 and CH3 domains are highlighted in green and blue respectively. (B) Forced degradation study of purified ZW1. Presence of high molecular weight (HMW) and low molecular weight (LMW) contaminants was determined by HPL-SEC. (C) Pre-formulation aggregation / turbidity screening of ZW1. The susceptibility of ZW1 to form visibly aggregates (precipitate) was evaluated under different buffer conditions by thermal melting and detection of the sample turbidity (see Supplementary Material and Methods for evaluation of pH conditions). (D) Analysis of purified ZW1 and trastuzumab at 100mg/ml by size exclusion chromatography.

Figure 3. (A) Heterodimer purity of ZW1 in comparison to the knob-into-hole control. In order to estimate the robustness of heterodimer formation, heterodimers were produced by transient co-expression (three plasmids for light chain, heavy chain A and heavy chain B) using three different DNA ratios of the two heavy chains A and B (e.g., ratios A:B = 1:1.5; 1:1; 1.5:1). The heterodimer purity of the ProteinA purified product was determined by LC-MS following EndoS deglycosylation as described in detail in the Methods. As illustrated by the LC-MS spectra, ZW1 forms exclusively heterodimers over the tested expression range, while the KiH control forms close to 10% homodimers if chain A is slightly higher expressed than chain B. (B)-(C) Stable cell line generation of ZW1 and determination of heterodimer purity by LC-MS. The figure in (B) illustrates the generation of stable pools using different ratios of the light and heavy chains for co-transfection. The panel on the right shows the heterodimer purity of the different pools as determined by LC-MS. (C) LC-MS heterodimer purity of two stable clones. Two stable clones (1 and 2) were evaluated under different culture conditions in fed-batch cultures (conditions A-D) and the heterodimer purity of the ProteinA purified product was evaluated by LC-MS.