Crystallization holds the potential to be used for protein purification and low-viscosity drug substance and drug product formulations. Twenty-two different proteins (20 monoclonal antibodies and two Fc-fusions) were examined to determine the breadth of applicability of crystallization to these therapeutic proteins. Vapor diffusion technique and an evaporative screening method were used to identify crystallization conditions using around a 100 initial conditions based on reagents that are generally regarded as safe (GRAS). Of 16 IgG2 s examined, at least four formed diffraction-quality crystals and four others formed crystal-like particles. At least three of the IgG2 s that crystallized well were also crystallized under the same set of operating conditions using inexpensive GRAS reagents. The crystals were formed to high-yields in a few hours and were dissolved quickly without impacting product quality. Although only a fraction of the proteins examined crystallized, all exhibited liquid-liquid phase separation (LLPS), which could be used for their concentration or possibly purification. One of the Fc-fusions, for example, was concentrated by LLPS to a self-buffering solution at 150 g/L. Crystallization and LLPS in the salting-in region were shown to be feasible.

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Hydrophobic interaction chromatography (HIC) is a classic purification tool applied in protein and antibody, laboratory and industrial production process. It has been mainly used for the removal of both product-related impurities such as aggregates, as well as process contaminants such as host cell proteins. This review will focus on the recent development of HIC in its applications in the industrial purification processes. The process economy and requirements of high product purity and quality have driven much of the recent advancement in HIC chromatography in terms of increased throughput and enhanced selectivity or resolution. Meanwhile, high throughput screening (HTS), design of experiments (DoE) and platform approach for process development have been applied to shorten the development time. The throughput improvement has been achieved through new resins with increased binding capacity, using dual salts for load conditioning, and operating in the flow-through mode. In addition, hydrophobic interaction membrane filter chromatography technology reduces bed volumes and buffer usage and potentially improves process throughput by reducing cycle time. Selectivity and/or resolution enhancements have been achieved through optimization of operation parameters such as temperature and efforts such as application of solvent additives.

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