Protein Protocols Protein Protocols

Enzymatic Digestion of MAbs 1047
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Enzymatic Digestion of Monoclonal Antibodies
Sarah M. Andrew
1. Introduction
Originally, digestion of antibodies by proteolytic enzymes was used to study their
structure. Many diverse structures can be obtained by fragmentation of the different
classes of antibody with different enzymes, or by using the same enzyme and changing
the conditions (Fig. 1). Not all the fragments obtained have significant binding activity;
for example, in several studies by this author, Fv fragments obtained by digestion
have been found to have lost their binding activity. Fragmentation of antibody is now
usually carried out to introduce required properties (e.g., a decrease in molecular size),
or to remove undesirable properties (e.g., nonspecific Fc receptor binding).
The most usual digestions carried out are:
1. Production of bivalent F(ab') 2 from mouse McAb IgG;
2. Production of univalent Fab from mouse McAb IgG;
3. Production of bivalent IgMs from mouse McAb IgM; and
4. Production of bivalent F(ab') 2µ from mouse McAb IgM.
F(ab') 2 and F(ab') 2µ are produced by digestion with pepsin and Fab is produced by
digestion with papain. One useful fragmentation uses papain that has been preactivated
with cysteine. This cleaves IgG 1 to produce F(ab') 2, and IgG 2a IgG 2b to produce Fab. It
is a very stable fragmentation in which the times of incubation are not at all critical.
The IgG-like subunit of IgM (IgMs) is the product of a mild reduction; this is most
conveniently done using cysteine, which reduces the IgM and alkylates the subunit,
thus preventing reassociation.
After digestion of the antibodies, it is necessary to purify the fragments for two
reasons: to separate the fragment from any remaining intact antibody; and to separate
the fragments of interest from other miscellaneous fragments produced by the process
of digestion. Purification of IgG fragments by protein A affinity chromatography is
possible if an intact Fc region remains after fragmentation, as it does in the case of
undigested antibody and, sometimes, in digestions with pepsin. In general this method
of purification is rough and ready and size exclusion chromatography is recommended
as an additional step.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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