title: Crystallization and liquid-liquid phase separation of monoclonal antibodies and fc-fusion proteins: screening results.
abstract: 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.
authors.affiliation: Dept. of Purification Process Development, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA.
authors.forename: Egor
authors.initials: E
authors.lastname: Trilisky
authors.forename: Ronald
authors.initials: R
authors.lastname: Gillespie
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Osslund
authors.forename: Suresh
authors.initials: S
authors.lastname: Vunnum
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins