Crystallization of IgG1 by mapping its liquid-liquid phase separation curves.
Monoclonal antibody therapeutics is an important and fast expanding market. While production of these molecules has been a major area of research, much less is known regarding the stabilization of these proteins for delivery as drugs. Crystallization of antibodies is one such promising route for protein stabilization at high titers, and here we took a systematic approach to initiate crystallization through nucleation in a simple PEG (polyethylene glycol), protein in water solution. A ternary mixture of globular proteins, PEG, and water will undergo a liquid-liquid phase separation (LLPS) as shown in a phase diagram or a Binodal curve. Of particular interest within the phase diagram is the position of the critical point, which is where nucleation occurs most rapidly. Detailed LLPS maps were created by increasing concentrations of PEG (from 5% to 11%) and IgG (from 1 to 20 mg/mL). By increasing the molecular weight (MW) of PEG (and hence its radius of gyration) from 1,000 to 6,000 g/mol, the temperatures of the critical point of nucleation were shown to increase. Once these curves were determined, nucleation experiments were conducted close to a chosen critical point (10.5 mg/mL IgG in 11% PEG 1000) and after 3 weeks, crystals of IgG of approximately 100 microm in size were successfully formed. This is the first example of crystallization of an antibody through systematic mapping of LLPS curves, which is a fundamental step towards the scale-up of antibody crystallization.
Jion
Adam Idu
AI
Bioprocessing Technology Institute, Biomedical Sciences Institutes, 20 Biopolis Way #06-01, Centros, Singapore.
Goh
Lin-Tang
LT
Oh
Steve K W
SK
D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D003460:Crystallization / Q000379:methods; D007074:Immunoglobulin G / Q000737:chemistry* / Q000302:isolation & purification*; D050866:Liquid Crystals; D044367:Phase Transition; D011092:Polyethylene Glycols
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011092:Polyethylene Glycols