Purification of Fab fragments from a monoclonal antibody papain digest by Gradiflow electrophoresis

Coleman, L. and Mahler, S. M. (2003) Purification of Fab fragments from a monoclonal antibody papain digest by Gradiflow electrophoresis. Protein Expression And Purification, 32 2: 246-251. doi:10.1016/j.pep.2003.07.005

Author Coleman, L.
Mahler, S. M.
Title Purification of Fab fragments from a monoclonal antibody papain digest by Gradiflow electrophoresis
Journal name Protein Expression And Purification   Check publisher's open access policy
ISSN 1046-5928
Publication date 2003-12-01
Year available 2003
Sub-type Article (original research)
DOI 10.1016/j.pep.2003.07.005
Open Access Status
Volume 32
Issue 2
Start page 246
End page 251
Total pages 6
Place of publication Oxford
Publisher Elsevier Science
Language eng
Subject 290000 Engineering and Technology
Abstract Fab fragments isolated from papain digests of monoclonal antibodies have a wide variety of uses in analytical and in both in vivo and in vitro diagnostic applications. A novel, non-affinity method which uses the Gradiflow to purify Fab fragments from the papain digest of a mouse IgG1 anti-c-myc monoclonal antibody is described. The Gradiflow is a preparative electrophoresis instrument that uses polyacrylamide membranes of known pore size to separate proteins in solution in their native state under mild pH conditions by charge or size. The Fab and Fc fragments from the papain digestion were characterized using isoelectric focusing (IEF) and non-reducing SDS-PAGE in conjunction with IEF and Western blot. There were three Fab isoforms with pI between pH 6.5 and 7.4 while the Fc had a range of isoforms between 6.1 and 6.3. Both Fab and Fc fragments had similar M-r of 50 kDa. A charge-based purification strategy was developed to obtain a high purity Fab preparation after 10 min, confirmed by Western blot and chemiluminescence analyses. A small quantity of residual undigested IgG1 remained and was removed using a size-based separation. The efficiency of the separation despite the narrow pH range between Fab and Fe suggests that this technique may be an alternative to protein A or G affinity separation of Fc and Fab monoclonal antibody fragments from papain digests of monoclonal antibodies. (C) 2003 Elsevier Inc. All rights reserved.
Keyword Biochemical Research Methods
Biochemistry & Molecular Biology
Biotechnology & Applied Microbiology
fab antibody fragments
preparative electrophoresis
Preparative Electrophoresis
Reflux Electrophoresis
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
School of Chemical Engineering Publications
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Citation counts: TR Web of Science Citation Count  Cited 19 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 17 times in Scopus Article | Citations
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Created: Thu, 20 Sep 2007, 03:00:01 EST