New downstream processing strategy for the purification of monoclonal antibodies from transgenic tobacco plants

dc.contributor.author	Platis, D	en
dc.contributor.author	Drossard, J	en
dc.contributor.author	Fischer, R	en
dc.contributor.author	Ma, JK-C	en
dc.contributor.author	Labrou, NE	en
dc.date.accessioned	2014-06-06T06:48:37Z
dc.date.available	2014-06-06T06:48:37Z
dc.date.issued	2008	en
dc.identifier.issn	00219673	en
dc.identifier.uri	http://dx.doi.org/10.1016/j.chroma.2008.09.103	en
dc.identifier.uri	http://62.217.125.90/xmlui/handle/123456789/4208
dc.subject	2G12	en
dc.subject	4E10	en
dc.subject	Aqueous two phase system	en
dc.subject	Cation-exchange chromatography	en
dc.subject	Immobilized metal affinity chromatography	en
dc.subject	Molecular farming	en
dc.subject	Monoclonal antibody	en
dc.subject.other	Acids	en
dc.subject.other	Affinity chromatography	en
dc.subject.other	Agricultural products	en
dc.subject.other	Allergies	en
dc.subject.other	Antibodies	en
dc.subject.other	Chromatographic analysis	en
dc.subject.other	Chromatography	en
dc.subject.other	Colloids	en
dc.subject.other	Column chromatography	en
dc.subject.other	Diffusers (optical)	en
dc.subject.other	Electrophoresis	en
dc.subject.other	Gel permeation chromatography	en
dc.subject.other	Gelation	en
dc.subject.other	Leaching	en
dc.subject.other	Mergers and acquisitions	en
dc.subject.other	Metal recovery	en
dc.subject.other	Metal refining	en
dc.subject.other	Monoclonal antibodies	en
dc.subject.other	Network protocols	en
dc.subject.other	Phenols	en
dc.subject.other	Plants (botany)	en
dc.subject.other	Polymers	en
dc.subject.other	Positive ions	en
dc.subject.other	Sodium chloride	en
dc.subject.other	Sodium compounds	en
dc.subject.other	Surface plasmon resonance	en
dc.subject.other	Tobacco	en
dc.subject.other	Zinc	en
dc.subject.other	2G12	en
dc.subject.other	4E10	en
dc.subject.other	Aqueous two phase system	en
dc.subject.other	Cation-exchange chromatography	en
dc.subject.other	Immobilized metal affinity chromatography	en
dc.subject.other	Molecular farming	en
dc.subject.other	Purification	en
dc.subject.other	monoclonal antibody	en
dc.subject.other	monoclonal antibody 2G12	en
dc.subject.other	monoclonal antibody 4E10	en
dc.subject.other	sepharose	en
dc.subject.other	sodium chloride	en
dc.subject.other	sodium dihydrogen phosphate	en
dc.subject.other	affinity chromatography	en
dc.subject.other	animal cell	en
dc.subject.other	aqueous solution	en
dc.subject.other	article	en
dc.subject.other	controlled study	en
dc.subject.other	cost benefit analysis	en
dc.subject.other	drug purification	en
dc.subject.other	elution	en
dc.subject.other	enzyme linked immunosorbent assay	en
dc.subject.other	ion exchange chromatography	en
dc.subject.other	nonhuman	en
dc.subject.other	pH measurement	en
dc.subject.other	polyacrylamide gel electrophoresis	en
dc.subject.other	priority journal	en
dc.subject.other	purification	en
dc.subject.other	tobacco	en
dc.subject.other	Western blotting	en
dc.subject.other	Antibodies, Monoclonal	en
dc.subject.other	Chromatography, Affinity	en
dc.subject.other	Chromatography, Ion Exchange	en
dc.subject.other	HIV Antibodies	en
dc.subject.other	Humans	en
dc.subject.other	Metals	en
dc.subject.other	Plants, Genetically Modified	en
dc.subject.other	Quality Control	en
dc.subject.other	Tobacco	en
dc.subject.other	Human immunodeficiency virus	en
dc.subject.other	Nicotiana obtusifolia	en
dc.title	New downstream processing strategy for the purification of monoclonal antibodies from transgenic tobacco plants	en
heal.type	journalArticle	en
heal.identifier.primary	10.1016/j.chroma.2008.09.103	en
heal.publicationDate	2008	en
heal.abstract	Affinity chromatography on immobilized Protein A is the current method of choice for the purification of monoclonal antibodies (mAbs). Despite its widespread use it presents certain drawbacks, such as ligand instability, leaching, toxicity and high cost. In the present work, we report a new procedure for the purification of two human monoclonal anti-HIV (human immunodeficiency virus) antibodies (mAbs 2G12 and 4E10) from transgenic tobacco plants using stable and low cost chromatographic materials. The first step of the mAb 2G12 purification procedure is comprised of an aqueous two-phase partition system (ATPS) for the removal of polyphenols while providing an essential initial purification boost (2.01-fold purification). In the second step, mAb 2G12 was purified using cation-exchange chromatography (CEX) on S-Sepharose FF, by elution with 20 mM sodium phosphate buffer pH 7.5, containing 0.1 M NaCl. The eluted mAb was directly loaded onto an immobilized metal affinity chromatography column (IMAC, Zn2+-iminodiacetic acid-Sepharose 6B) and eluted by stepwise pH gradient. The proposed method offered 162-fold purification with 97.2% purity and 63% yield. Analysis of the antibody preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), enzyme immunosorbent assay (ELISA) and western blot showed that the mAb 2G12 was fully active and free of degraded variants, polyphenols and alkaloids. The effectiveness of the present purification protocol was evaluated by using a second transgenic human monoclonal anti-HIV mAb 4E10. The results showed that the same procedure can be successfully used for the purification of mAb 4E10. In the case of mAb 4E10, the proposed method offered 148-fold purification with 96.2% purity and 36% yield. Therefore, the proposed protocol may be of generic use for the purification of mAbs from transgenic tobacco plants. © 2008 Elsevier B.V. All rights reserved.	en
heal.journalName	Journal of Chromatography A	en
dc.identifier.issue	1-2	en
dc.identifier.volume	1211	en
dc.identifier.doi	10.1016/j.chroma.2008.09.103	en
dc.identifier.spage	80	en
dc.identifier.epage	89	en