title: High productivity purification of immunoglobulin G monoclonal antibodies on starch-coated magnetic nanoparticles by steric exclusion of polyethylene glycol.
abstract: We achieved exceptionally high capacity capture of monoclonal IgG by adding 200 nm starch-coated magnetic particles as nucleation centers, adding polyethylene glycol (PEG), then collecting the particle-associated antibody in a magnetic field. Experimental data suggest that accretion of IgG begins on particle surfaces then continues with fusion of particle-centric accretions up to about 1mm in a process that closely parallels PEG precipitation. An embedded nanoparticle mass of 1.3% of the IgG mass is adequate to enable efficient magnetic collection of the associated IgG. Recovery of purified IgG averaged 98% up to loads of 78 mg of IgG per mg of particles. Converted to an equivalent volume of settled particles, this represents about 58 g IgG per mL of nanoparticles, which is roughly 1000 times higher than the average capacity of commercial protein A porous particles packed in columns. When applied to cell culture harvest clarified by centrifugation and microfiltration, performing the nanoparticle technique under physiological conditions permitted only a 10-fold reduction of host cell protein (HCP) contamination and IgG recovery less than 50%. Application of a more capable clarification method and operating the nanoparticle method at 0.5-1.0M NaCl supported more than 99% HCP reduction and 87% IgG recovery. The high salt concentration also dramatically diminished the influence of operating pH on selectivity. The nanoparticle step was followed by sample application without buffer exchange to a column packed with multimodal electropositive-hydrophobic particles that reduced HCP to 2 ppm. Aggregate content was reduced from 4.9 to 3.6% at the nanoparticle step, then to less than 0.05% at the multimodal step. The multimodal step also removed residual PEG. Overall IgG recovery was 69%. The ability of the system to achieve purity similar to protein A, but dramatically higher productivity than packed columns, suggests that the technique could evolve as a credible option for industrial purification of monoclonal antibodies.
authors: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore 138668, Singapore. Electronic address: petegagnon@bti.a-star.edu.sg.
authors: Pete
authors: P
authors: Gagnon
authors: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore 138668, Singapore.
authors: Phyllicia
authors: P
authors: Toh
authors: Bioprocessing Technology Institute, 20 Biopolis Way, Centros #06-01, Singapore 138668, Singapore.
authors: Jeremy
authors: J
authors: Lee
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000302:isolation & purification*; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D008970:Molecular Weight; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry*; D013213:Starch / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D058185:Magnetite Nanoparticles; D011092:Polyethylene Glycols; D013213:Starch
keywords: Antibody purification; Magnetic nanoparticles; Nucleation centers; Polyethylene glycol; Productivity; Steric exclusion