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Abstract
Protein aggregation remains a major area of focus in the production of monoclonal antibodies. Improving the intrinsic properties of antibodies can improve manufacturability, attrition rates, safety, formulation, titers, immunogenicity, and solubility. Here, we explore the potential of predicting and reducing the aggregation propensity of monoclonal antibodies, based on the identification of aggregation-prone regions and their contribution to the thermodynamic stability of the protein. Although aggregation-prone regions are thought to occur in the antigen binding region to drive hydrophobic binding with antigen, we were able to rationally design variants that display a marked decrease in aggregation propensity while retaining antigen binding through the introduction of artificial aggregation gatekeeper residues. The reduction in aggregation propensity was accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process. The data presented show that this approach can significantly reduce liabilities in novel therapeutic antibodies and proteins, leading to a more efficient path to clinical studies.
Highlights
Protein particles are assemblies built up of native and/or denatured proteins [1] that generally have a negative impact on manufacturability, stability, safety, titers, immunogenicity, and solubility of biologics in general [2,3,4,5,6]
In a Stretch-plot, each aggregation-prone region (APR) is represented by a single point, of which the ordinate is determined by the local aggregation propensity of the sequence segment and the abscissa by its contribution to the free energy of folding (ΔGcontrib)
The analysis clearly revealed that APRs in the framework region (FR) are, in large majority, thermodynamically stable and well protected from aggregation (Fig. 1c)
Summary
Protein particles are assemblies built up of native and/or denatured proteins [1] that generally have a negative impact on manufacturability, stability, safety, titers, immunogenicity, and solubility of biologics in general [2,3,4,5,6]. We investigate the impact of β-aggregation-prone sequences on protein particle formation and assess our ability to predict and suppress antibody particle formation based on this structural mechanism alone. Common methods of aggregation prediction are geared toward the identification of APRs in the primary sequence [2,3,4,5,8,9]. These prediction methods establish the theoretical aggregation potential of the protein in the unfolded state, called the “intrinsic aggregation propensity”.
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