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

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 p [Formula: see text] 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 [Formula: see text] of 50kDa. A charge-based purification strategy was developed to obtain a high purity Fab preparation after 10min, 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 Fc 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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Coleman L,Mahler SM

doi

10.1016/j.pep.2003.07.005

keywords:

subject

Has Abstract

pub_date

2003-12-01 00:00:00

pages

246-51

issue

2

eissn

1046-5928

issn

1096-0279

pii

S1046592803002390

journal_volume

32

pub_type

杂志文章

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