Design, synthesis and evaluation of pyridine-based chromatographic adsorbents for antibody purification

• Published in: Journal of Chromatography A Vol. 1355; pp. 15 - 25
• Main Authors: Mountford, Simon J., Daly, Rachel, Robinson, Andrea J., Hearn, Milton T.W.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 15-08-2014; Elsevier
• Subjects: Adsorbents; Adsorption; Affinity; Affinity chromatography; Analytical, structural and metabolic biochemistry; Animals; Antibodies; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Binding; Biological and medical sciences; Biotechnology; CHO Cells; Chromatography; Chromatography, Affinity - methods; Cricetinae; Cricetulus; Design analysis; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; General pharmacology; IgG; Immunoglobulin G - chemistry; Immunoglobulin G - immunology; Ligands; Medical sciences; Methods. Procedures. Technologies; Monoclonal antibodies; Others; Pharmaceutical technology. Pharmaceutical industry; Pharmacology. Drug treatments; Protein purification; Proteins; Pyridine ligands; Pyridines - chemistry; Sepharose - chemistry; Various methods and equipments; Monoclonal antibodies; IgG; Pyridine ligands; Affinity chromatography; Protein purification; Purification; Glycoprotein; Monoclonal antibody; Agarose; Protein; Pyridine derivatives; Affinity adsorbent; Separation method; Pyridine
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2014.06.006

•New adsorbents for monoclonal antibody purification have been developed.•Structure-retention characteristics of the immobilised ligands have been delineated.•Conditions to achieve high capacities and recoveries have been established.•Applications with crude cell culture broths for mAb purification demonstrated.
The structure-based design and synthesis of four series of adsorbents for antibody purification by affinity chromatography has been investigated. The structures of 10 ligands were based on pyridine compounds that possessed thioalkyl substituents containing a primary amine, which was required for immobilisation of the ligands onto an epoxy-activated matrix (epoxy-Sepharose Fast Flow®). These new adsorbents were screened in monoclonal antibody binding assays in order to determine optimal buffer conditions for capture and elution under static and dynamic adsorption conditions. From batch binding measurements, the binding affinities, KD's, were found to be in the range of 3–5μM and the maximum capacities, qm's were between 12 and 30mgmAb/mL resin, depending on the substitution pattern of the thioalkylamine in the N-heterocyclic ring structure of the ligands. The amount of monoclonal antibody bound and eluted under overload conditions was influenced by the concentration of the sample loaded, the flow rate at which the sample was applied and the loading/volume. Further, the ability of these new adsorbents to selectively capture monoclonal antibodies of the class IgG1 from supernatants derived from genetically engineered CHO cells cultured in chemically defined media was investigated, documenting efficient capture and recovery of the mAb.