Purification of murine IgG1 on group specific affinity sorbents.
Murine IgG1 a CD4-antibody, was produced from the cell line MAX 16 H 5. DEAE-Sepharose CL-6B and group specific sorbents based on different ligands, such as protein A-Sepharose, AvidChrom, Thiophilic Resin (T-Resin) Fractogel EMD TA 650S, Sepharose 4B-immobilized metal chelate and histidine sorbents, were compared in terms of their suitability for the large-scale purification of this IgG. Evaluation criteria were selectivity for IgG1 protein capacity and recovery, stability under cleaning-in-place (CIP) conditions and the rate of IgG1 adsorption. Both thiophilic gels performed best under these conditions, with Fractogel EMD TA 650S demonstrating slightly higher contamination with other proteins compared to the T-Resin based on 6% beaded Agarose. Higher purities were obtained with these gels compared to conventional purification techniques employing a combination of ion exchange and hydrophobic interaction chromatography. Favorable adsorption kinetics were found on DEAE-Sepharose with the effective diffusivity in the pores of the sorbent being five-times accelerated compared to the diffusion coefficient of IgG1 in solution. Multilayer adsorption of the IgG1 was experienced from adsorption isotherms on DEAE-Sepharose and Fractogel EMD TA 650S. This was linked to an early breakthrough of this protein during frontal chromatography. Co-purification of pyrogens between two-(Thiophilic Resin) and five-times (DEAE-Sepharose) of the original level was significant with all sorbents except protein A-Sepharose and AvidChrom. Thus an additional chromatographic step is required in case of pyrogen contamination of the cell culture homogenate.
Anspach
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FB
Biochemical Engineering Division, GBF-Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany.
Petsch
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Deckwer
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WD
D000818:Animals; D001709:Biotechnology; D002460:Cell Line; D002614:Chelating Agents; D002846:Chromatography, Affinity / Q000379:methods*; D004340:Drug Contamination; D005069:Evaluation Studies as Topic; D006639:Histidine; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007700:Kinetics; D008024:Ligands; D051379:Mice; D011108:Polymers; D011749:Pyrogens / Q000302:isolation & purification; D012685:Sepharose / Q000031:analogs & derivatives; D013816:Thermodynamics; D013205:Staphylococcal Protein A
D002614:Chelating Agents; D006639:Histidine; D007074:Immunoglobulin G; D008024:Ligands; D011108:Polymers; D011749:Pyrogens; D012685:Sepharose; C042030:Staphylococcal protein A-sepharose; D013205:Staphylococcal Protein A; C043982:DEAE-Sepharose CL-6B