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...

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Bibliographic Details
Published in:Journal of Chromatography A 2009-03, Vol.1216 (13), p.2676-2682
Main Authors: Tscheliessnig, Anne, Jungbauer, Alois
Format: Article
Language:English
Subjects:
High-performance monolith affinity chromatography (HPMAC)
IgG
Protein A
Quantification
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Summary: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 μg/ml. The calibration range of the method could be extended from 4 to 1000 μg/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.
ISSN:0021-9673
DOI:10.1016/j.chroma.2008.07.063