Biophysical investigations into the impact of crystallization conditions on monoclonal antibody properties and purification.

Crystallization of monoclonal antibodies (mAbs) is a challenging problem given their large size and inherent structural complexity. Thousands of conditions need to be screened to arrive at a promising crystallization hit. Historically, crystallization has been successfully applied for preparative-scale purification and manufacturing of small molecules but rarely to produce mAbs, large globular proteins and peptides other than insulins. There is tremendous interest in the biopharmaceutical industry in exploring and implementing alternative strategies for the purification of biologics. One such non-platform, unit operation is crystallization. However, translatability across scales, robustness and amenability for large-scale processing are key challenges that need to be overcome to realize the success of crystallization as a unit operation in biomanufacturing. In this work, we take a biophysical characterization- driven approach to 1) elucidate differences between two unique crystallization conditions, previously identified via high-throughput screening, for a IgG4 mAb, and 2) demonstrate that the fundamental understanding generated from this biophysical characterization can enable a rational selection of crystallization conditions for bioprocessing applications. Results show that the crystallization conditions, both of which crystallize the mAb in two unique crystal structures, can impact the mAb structure and solution-state biophysical properties quite differently leading to significantly different outcomes in thermal and colloidal stability of the protein as well as in the purification of the mAb via crystallization as a unit operation. Second, this work highlights that co-crystallization of a large macromolecule like a mAb with a small molecule like caffeine can dramatically influence solution stability as well as impurity rejection. Third, and related to above we demonstrate that not all crystallization conditions that lead to structure elucidation are amenable for bioprocessing, as certain solution conditions can negatively impact protein solution stability and conformation, impurity co-precipitation thereby leading to lower impurity clearance from complex bioprocess feeds. For bioprocessing applications it is therefore critical to understand the impact of a condition on not just the pure mAb but also on the mAb in complex solutions alongside impurities.