Successful development of therapeutic antibodies requires high antibody stability, low off-target binding, low toxicity, low viscosity, high solubility and low levels of aggregation. Several of these properties are strongly influenced by the sequence and structural composition of antibody variable regions and especially their complementarity determining regions (CDRs). We are investigating the molecular determinants of various antibody developability properties by systematically mutating the CDRs of a panel of clinical-stage antibodies to alter their charge, polarity and hydrophobicity. We find a surprisingly strong trade-off between antibody non-specific interactions in physiological conditions and antibody self-interactions in formulation conditions. Interestingly, antibodies with strong repulsive self-interactions in formulation conditions (important for low viscosity) typically display high levels of non-specific interactions in physiological conditions (detrimental to bioavailability), and vice versa for antibodies with low levels of non-specific interactions. We find that this behavior can be largely explained by the isoelectric point of the Fv region, as highly positively charged antibodies promote repulsive interactions that promote high antibody stability at formulation conditions (pH 5-6) but strong non-specific interactions with negatively charged biomolecules at physiological pH (pH 7.4), and vice versa for antibodies with negatively charged Fv regions. Finally, we have identified intermediate ranges of Fv isoelectric point that best balance the competing demands of high antibody stability in formulation conditions and low non-specific binding in physiological conditions. We expect that these findings will improve the design and engineering of antibodies and antibody libraries with drug-like properties.

Molecular determinants of strong trade-offs between antibody formulation stability and off-target binding

ACS Fall 2021

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