High-performance monolith affinity chromatography for fast quantitation of immunoglobulin G.
High-performance monolith affinity chromatography employing protein A resins has been introduced previously for the fast purification of IgG from different sources. Here we describe the design and evaluation of a fast and specific method for quantitation of IgG from purified samples as well as crude supernatant from Chinese hamster ovary (CHO) cells. We used a commercially available affinity monolith with protein A as affinity ligand (CIM protein A HLD disk). Interferences of CHO host cell proteins with the quantitation of IgG from CHO supernatant were eliminated by a careful choice of the equilibration buffer. With this method developed, it is possible to quantify IgG within 5 min in a concentration range of 23-250 microg/ml. The calibration range of the method could be extended from 4 to 1000 microg/ml by adjusting the injection volume. The method was successfully validated by measuring the low limit of detection and quantification, inter- and intra-day precision and selectivity.
Tscheliessnig
Anne
A
Bioprocessing Technology Institute, Agency for Science, Technology and Research, 20 Biopolis Way, No. 06-01 Centros, Singapore 138668, Singapore. anne tscheliessnig@bti.a-star.edu.sg
Jungbauer
Alois
A
D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis*; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000737:chemistry
D007074:Immunoglobulin G; D013205:Staphylococcal Protein A