Engineering of a two-step purification strategy for a panel of monoclonal immunoglobulin M directed against undifferentiated human embryonic stem cells
Tscheliessnig, A. Ong, D. Lee, J. Pan, S. Satianegara, G. Schriebl, K. CHOO BOON HWA,ANDRE Jungbauer, A.
Tscheliessnig, A.
Ong, D.
Lee, J.
Pan, S.
Satianegara, G.
Schriebl, K.
Jungbauer, A.
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Alternative Title
Abstract
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) (pI 5.5-7.7) 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 2 l hybridoma supernatants. © 2009 Elsevier B.V. All rights reserved.
Keywords
Adsorption isotherm, Adsorption kinetics, Anion-exchange chromatography, Benzonase, IgM, Number of binding sites, PEG, Precipitation
Source Title
Journal of Chromatography A
Publisher
Series/Report No.
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Date
2009-11-06
DOI
10.1016/j.chroma.2009.09.059
Type
Article