Title

Systematic Investigation of Factors Affecting the Viscosity and Self-Association Issues in High Concentration Monoclonal Antibody Solutions

Date of Completion

January 2010

Keywords

Chemistry, Biochemistry|Health Sciences, Pharmacy|Biophysics, General

Degree

Ph.D.

Abstract

High concentration subcutaneous (SC) dosing regimens are desirable for monoclonal antibody (MAb) therapeutics to ensure patient convenience, manufacturing ease as well as product marketability. High protein concentration often results in a dramatic increase in solution viscosity and self-association issues, which remain critical concerns in formulation development. ^ The effect of different variables, pertaining to both inherent molecular and solution properties, on the viscosity of different MAbs was systematically analyzed. The high concentration viscosity behavior could not be explained based on the net charge, effective molecular size and/or excluded volume effects. The nature of intermolecular interactions governed the high concentration behavior, wherein stronger intermolecular attraction led to a higher solution viscosity. To elucidate the influence of solution conditions, the viscosity of a number of MAbs and bovine serum Albumin (BSA) was studied as a function of pH and ionic strength. Except for MAb-1, a higher viscosity was observed near the isoelectric point (pI) of the molecules. At the pI, the net molecular charge induced repulsion is minimal, whereas the charge distribution becomes most conspicuous. The resulting dominance of non-specific attractive interaction leads to a higher solution viscosity at the pI. ^ MAb-1 showed a high viscosity nearly two units below the measured pI, which could not be explained based on the electroviscous effects or the non-specific electrostatic interactions. Intermolecular attractions, conducive to self-association and high viscosity, persisted in MAb-1 at pH 6.0, despite a net positive charge. These intermolecular attractions at pH 6.0 were invoked specifically from a number of charged residues in the complementarily determining regions (CDR). ^ Further, several mutants were designed where the charged residues in MAb-1 were swapped with those present in MAb-2 at their respective positions and vice versa. The replacement of these charged residues in the CDR of MAb-1 resulted in the loss of self-association and solution viscosity, confirming that the exposed charged residues were mainly responsible for the highly viscous behavior of MAb-1. ^ These results demonstrated that at high protein concentrations, the charge distribution and the local details of the molecule are more critical in governing the viscosity and self-associating behavior than the overall net charge. ^

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