The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and long-term stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability.
Submitter: Marie Ary
Submission Date: Nov. 29, 2018, 5:01 p.m.
|Number of data points||69|
|Proteins||Anti-LOX-1 (lectin-type optimized LDL receptor 1) mAb1 ; Oxidized low-density lipoprotein receptor 1 (LOX-1)|
|Assays/Quantities/Protocols||Experimental Assay: Tm (DSC) ; Experimental Assay: Tm (tryptophan fluorescence) ; Experimental Assay: F350/F330 ; Experimental Assay: T_onset RALS ; Experimental Assay: Tm2 (2nd unfolding transition) (tryptophan fluorescence) ; Experimental Assay: Titer ; Experimental Assay: Kd ; Experimental Assay: Monomer portion, day 0 ; Experimental Assay: Monomer portion, day 17 ; Experimental Assay: Monomer portion, day 81 ; Experimental Assay: Monomer portion, day 126|
|Libraries||Biophysical characterization of mAb1*_WT and variants (Table 2) ; Expression levels of mAb1/mAb1*_WT and variants (Table 1) ; Kd for mAb1*_WT and mAb1 variants (Table 3) ; Long-term stability (monomer portion) after storage at 40 C (Fig 4)|