title: Feasibility study of semi-selective protein precipitation with salt-tolerant copolymers for industrial purification of therapeutic antibodies.
abstract: We present a feasibility study for an antibody capturing process from clarified cell culture fluid using semi-selective protein precipitation with salt-tolerant copolymers. Protein precipitation is mediated by hydrophobic and electrostatic interactions with the copolymer that can be customized for the respective target. Precipitation yield with different copolymers at ionic strength of 2-22.5 mS cm⁻¹ and pH 5.0-pH 5.7 was evaluated using pure monoclonal antibody solutions. Optimized parameters were used to elucidate yield and purity of various antibodies precipitated at physiological conditions from cell culture fluid of CHO, NS0, and SP2/0 cell culture fluid. Precipitated protein was easily redissolved in small volume, enabling concentrating monoclonal antibodies (mAb) more than 40-fold and up to 100-fold, while residual polymer was removed to >98% using cationic polymer attached to silica flakes. mAb recovery of >90% and host cell protein clearance of >80% were achieved, not requiring any pre-dilution of cell culture fluid. Precipitation showed no impact on mAb binding affinity when compared to non-precipitated mAb. The obtained yield and purity were lower compared to a protein A based purification and loss of mAb was factor 1.5-3.0 higher. Yet, for high titer mAb purification processes being implemented in the future, precipitation is an attractive option due to its ease of scalability and cost-effectiveness.
authors: Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Petersenstrasse 22, 64287 Darmstadt, Germany; Merck KGaA, Darmstadt, Germany. florian.capito@external.merckgroup.com.
authors: Florian
authors: F
authors: Capito
authors: Johann
authors: J
authors: Bauer
authors: Almut
authors: A
authors: Rapp
authors: Christian
authors: C
authors: Schröter
authors: Harald
authors: H
authors: Kolmar
authors: Bernd
authors: B
authors: Stanislawski
mesh_terms: D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D001688:Biological Products / Q000302:isolation & purification*; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D005591:Chemical Fractionation / Q000379:methods*; D011232:Chemical Precipitation; D005240:Feasibility Studies; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000302:isolation & purification; D012492:Salts / Q000737:chemistry; D013678:Technology, Pharmaceutical / Q000379:methods*
chemical_list: D000906:Antibodies; D001688:Biological Products; D011994:Recombinant Proteins; D012492:Salts
keywords: 2-acrylamido-2-methylpropane sulfonic acid; 4-(acryloylamino)benzoic acid; copolymer; downstream processing; protein precipitation; protein purification