Protein Protocols Protein Protocols

Making Bispecific Antibodies 1057
Fig. 2. Chromatography profile showing the separation of parent Fab' and bispecific F(ab') 2
and F(ab') 3 products on AcA44 columns. In this case, Fab'-A(SH) and Fab'-B(mal) were mixed
at a ratio of 21 to increase the formation of bispecific F(ab') 3. The unreacted Fab' fragments
and the F(ab') 2 and F(ab') 3 products are indicated. The arrows show the points at which
protein standards eluted from the same columns.
and the eluted product monitored at 280 nm. However, we have found that F(ab')SH
rapidly reoxidizes back to F(ab') 2 while on the column. This can be overcome by alkylating
the free SH-groups by the addition of 5 µL 50 mM iodoacetamine in 1 M NTE8
to the 45-µL sample from the G25 column.
Fab' will elute from the column later than F(ab') 2 resulting in a shift in the position of
the peak on reduction. In most cases >95% of the F(ab') 2 is reduced. Following alkylation
and overnight incubation, the reaction mixture typically elutes from HPLC as a triplet,
containing a mixture of alkylated Fab∋ and Fab'(mal), which elute in a similar postion to
Fab'(SH), bispecific F(ab') 2 product, which elutes similarly to the parent F(ab') 2, and a
smaller amount of bispecific F(ab') 3, which elutes similarly to IgG.
4. Notes
1. Two SH groups may also be produced by the reduction of the heavy/light chain disulfide
bond. However, under the conditions used, this bond is not fully reduced, and any SH
groups that are produced are less likely to be available for conjugation (1,2). This procedure
relies on one maleimidated hinge SH-group remaining free for conjugation after the
intramolecular cross-linking of adjacent SH-groups with o-PDM. It follows that the Fab'
species chosen to be maleimidated must be derived from IgG with an odd number of hinge