Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1216, Issue 45, Pages 7851-7864Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2009.09.059
Keywords
IgM; PEG; Precipitation; Anion-exchange chromatography; Benzonase; Adsorption isotherm; Adsorption kinetics; Number of binding sites
Funding
- Biomedical Research Council of A*STAR (Agency for Science, Technology and Research), Singapore
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A two-step purification strategy comprising of polyethylene glycol (PEG) precipitation and anion-exchange chromatography was developed for a panel of monoclonal immunoglobulin M (IgM) (pI5.5-7.7) produced from hybridoma cultures. PEG precipitation was optimized with regards to concentration, pH and mixing. For anion-exchange chromatography, different resins were screened of which Fractogel EMD, a polymer grafted porous resin had the highest capacity. Despite its significantly slower mass transfer, the binding capacity was still higher compared to a convection driven resin (monolith). This purification strategy was successfully demonstrated for all 9 IgMs in the panel. In small scale most antibodies could be purified to >95% purity with the exception of two which gave a lower final purity (46% and 85%). The yield was dependent on the different antibodies ranging from 28% to 84%. Further improvement of recovery and purity was obtained by the digestion of DNA present in the hybridoma supernatant using an endonuclease, benzonase. So far this strategy has been applied for the purification of up to 21 hybridoma supernatants. (C) 2009 Elsevier B.V. All rights reserved.
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