title: Very fast analysis of impurities in immunoglobulin concentrates using conjoint liquid chromatography on short monolithic disks.
abstract: Transferrin and albumin are often present in immunoglobulin G (IgG) concentrates and are considered as impurities. Therefore, it is important to determine their concentration in order to obtain a well-characterized biological product. Here, we describe their determination based on conjoint liquid chromatography (CLC). The established method combines two different chromatographic modes in one step: affinity and ion-exchange chromatography (IEC) combined in one column. Therefore, two CIM Protein G and one CIM quaternary amine (QA) monolithic disks were placed in series in one housing forming a CLC monolithic column. Binding conditions were optimized in a way that immunoglobulins were captured on the CIM Protein G disks, while transferrin and albumin were bound on the CIM QA disks. Subsequently, transferrin and albumin were eluted separately by a stepwise gradient with sodium chloride, whereas immunoglobulins were released from the Protein G ligands by applying low pH. A complete separation of all three proteins was achieved in less than 5 min. The method permits the quantification of albumin and transferrin in IgG concentrates and has been successfully validated.
authors.affiliation: Institute of Immunology, Inc., Zagreb, Croatia.
authors.forename: K
authors.initials: K
authors.lastname: Branovic
authors.forename: G
authors.initials: G
authors.lastname: Lattner
authors.forename: M
authors.initials: M
authors.lastname: Barut
authors.forename: A
authors.initials: A
authors.lastname: Strancar
authors.forename: Dj
authors.initials: Dj
authors.lastname: Josic
authors.forename: A
authors.initials: A
authors.lastname: Buchacher
mesh_terms: D000588:Amines / Q000737:chemistry; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D005779:Immunodiffusion; D007074:Immunoglobulin G / Q000032:analysis / Q000302:isolation & purification*; D009419:Nerve Tissue Proteins / Q000737:chemistry; D012709:Serum Albumin / Q000032:analysis*; D014168:Transferrin / Q000032:analysis*
chemical_list: D000588:Amines; C029152:G-substrate; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D012709:Serum Albumin; D014168:Transferrin