Thermal Stability of Mutant Antibody Molecules

Example 5

The thermal stability of exemplary mutant antibody molecules was determined. The thermal stability was measured by SYPRO orange. As shown in FIG. 9, FcMut008 and FcMut015 retained high melting temperature.

The impact of incorporating exemplary Fc variants on biophysical attributes was experimentally assessed. IgGs incorporating the Fc variants on motavizumab Fab were tested on SE-HPLC. All samples eluted at similar retention times as wild-type Fc, and displayed clean monomeric profile, and no aggregates were detected (FIG. 23). The IgGs were also assessed for the thermal stability of the CH2 and CH3 domains by Differential Scanning Fluorimetry (DSF). The melting temperature (T_m) of the wild type human CH2 and CH3 domain, as measured differential scanning calorimetry, is approximately 70° C. and 81.5° C., respectively (Ionescu et al., J Pharm Sci, 2008. 97(4): 1414-26). The DSF experimental results in this Example yielded similar results with a CH2 and CH3 T_M of 68.8° C. and 80.8° C., respectively. The half-life extending Fc variant YTE has been reported to decrease the T_M of the CH2 domain by 6.7° C. (Majumdar et al., MAbs, 2015. 7(1): p. 84-95.). In the experiments described in this Example, the T_M of the CH2 domain of YTE was 7.2° C. lower than WT. Additionally, mutations at 247, 257, and 308 significantly impacted the T_M of CH2. The exemplary Fc variants (FcMut183, FcMut197, FcMut213, FcMut215, FcMut228, FcMut229) were thermally stable with the T_M of the CH2 domain >64° C. (FIG. 23).