Protein Protocols Protein Protocols

1056 French
1. Use equal amounts of F(ab') 2 from the two parent antibodies. The F(ab') 2 should be in 0.2 M
TE8 at 5–12 mg/mL in a final volume of 1–3 mL. Keep a 50 µL sample of both F(ab') 2
preparations for HPLC analysis (see Subheading 3.3.).
2. Reduce both parent F(ab') 2 preparations to Fab'(SH) using 1/10 vol F(ab') 2 reducing solution
(final concentration 20 mM 2-ME). Incubate at 30°C for 30 min and then keep on ice.
Maintain the tempterature at 0–5°C for the rest of the procedure unless stated otherwise.
3. Select the species to be maleimidated (Fab'-A[SH]) (see Note 3). Remove 2-ME by passing
through the smaller Sephadex G25 column (column 1). Collect the protein peak, which
elutes after approx 8–10 min, in a graduated glass tube in an ice bath (see Note 4). Take a
45 µL sample from the top of the peak for HPLC analysis (see Subheading 3.3.). Keep the
column running to completely elute 2-ME, which runs as a small secondary peak.
4. When the chart recorder has returned to baseline, load the second Fab'(SH) species
[Fab'-B(SH)] onto the column, and separate as for Fab'-A(SH), again taking a sample
for HPLC analysis (see Subheading 3.3.).
5. After the Fab'-B(SH) has been loaded onto the G25 column, the Fab'-A(SH) partner can
be maleimidated. Rapidly add a 1/2 vol (normally 4–5 mL) of cold o-PDM/DMF to the
Fab'-A(SH), seal the tube with Parafilm or similar, and mix by inverting two to three
times (see Note 5). Stand in an ice bath for 30 min.
6. When the Fab'-B(SH) has been collected, connect the larger Sephadex G25 column
(column 2) to the chart recorder. After the 30 min incubation, load the Fab'-A(SH)/o-
PDM/DMF mixture onto this column. Collect the Fab'-A(mal) protein peak (elutes after
8–10 min) (see Note 6).
7. Pool the Fab'-A(mal) and the Fab'-B(SH). Immediately concentrate in a stirred Amicon
concentration cell to around 5 mL, and then transfer to a tube for overnight incubation at
4°C (see Note 7).
8. During conjugation, in addition to the required BsAb, disulfide bonded homodimers may
also form. To eliminate these, after overnight incubation add 1/10 volume 1 M NTE8 to
the mixture to increase the pH, and then 1/10 vol F(ab') 2 reducing solution to reduce the
homodimer disulfide bonds. Incubate at 30°C for 30 min.
9. Alkylate to block sulphydryl groups by the addition of 1/10 vol 250 mM iodoacetamide
in 0.2 M TE8 (see Note 8). Check the composition of the mixture by HPLC (see
Subheading 3.3.).
10. Separate the products on two AcA44 columns run in series. Collect 10–15 min fractions.
A typical elution profile is shown in Fig. 2.
11. Pool the fractions containing the BsAb product. To minimize contamination, only take the
middle two-thirds of the peak. Concentrate and dialyze into appropriate buffer.
12. If required, check the final product by HPLC (see Subheading 3.3.).
3.2. Preparation of Bispecific F(ab') 3 Derivatives
This is as for the preparation of bispecific F(ab') 2 except that the ratio of Fab'(mal) to
Fab'(SH) is increased from 11 to 21 or greater. Therefore, start with at least twice as
much of the F(ab') 2 which is to provide two arms of the F(ab') 3 product.
3.3. HPLC Monitoring
For rapid analysis of products during the preparation, an HPLC system is used as
described in Subheading 2.2. This will resolve IgG, F(ab') 2, and Fab' sized molecules
in approx 20 min, and can be performed while the preparation is in progress. The parent
F(ab') 2 and the alkylated reaction mixture can be loaded directly onto the column