This protocol describes the production of bispecific F(ab')₂ antibody derivatives (BsAbs) by the linking of two Fab∋ fragments via their hinge region SH groups using the bifunctional crosslinker o-phenylenedimaleimide (o-PDM) as described.

1.Materials

1.1 Reagents

1. 2 M TE8: 2 M Tris-HCl, pH 8.0, 100 mM ethylenediaminetetra-acetic acid (EDTA). Prepare 0.2 M TE8 from 2 M stock.

2. F(ab')₂ reducing solution: 220 mM 2-ME, 1 mM EDTA. Make up 10 mL. Use a fume cupboard.

3. Sephadex G25 (Pharmacia, Uppsala, Sweden) and Ultragel AcA44 (Biosepra S. A.,Villeneuve la Garenne, France) gel filtration media.

4. G25 column buffer (50 mM AE); 3.35 g sodium acetate, 526 µL glacial acetic acid, 0.186 g EDTA, made up to 1 L. Degas before use under vacuum or using nitrogen.

5. High performance liquid chromatography (HPLC) buffer (0.2 M phosphate, pH 7.0): Add 0.2 M Na₂HPO₄ to 0.2 M NaH₂PO₄ to obtain the required pH.
6. o-PDM/DMF for Fab'(SH) alkylation: 12 mM o-PDM in dimethylformamide. Make up just prior to use. Chill in a methylated spirit/ice bath. Caution: o-PDM is toxic and should be handled with care.

7. NTE8, 1 M: 1 M NaCl, 0.2 M Tris-HCl, pH 8.0, 10 mM EDTA.

8. Iodoacetamide: 250 mM in 0.2 M TE8 and 50 mM in 1 M NTE8.



1.2 Chromatography Equipment

1. Two chromatography columns packed with Sephadex G25 and equilibrated and run in 50 mM AE are required. The first (column 1) should be 1.6 cm in diameter,packed to a height of 25 cm with gel, and pumped at approx 60 mL/h. The second (column 2) should be 2.6 cm in diameter, packed to a height of 20 cm with gel, and pumped at approx 200 mL/h. The columns must be fitted with two end-flow adaptors and water jackets to allow chilling throughout the procedure. Pharmacia K Series columns are ideal.

2. Two larger columns packed with polyacrylamide agarose gel and run in 0.2 M TE8 are used for the size exclusion chromatography of the BsAb products. These should be 2.6 cm in diameter, and packed to a height of 80 cm with gel. The two columns should be joined in series using Teflon capillary tubing and pumped at approx 30 mL/h. Chilling is not required at this stage of the preparation, and the columns can be run at room temperature.

3. Two peristaltic pumps capable of rates between 15 and 200 mL/h for column chromatography.

4. Chiller/circulator to cool columns. A polystyrene box containing water and crushed ice and a submersible garden pond pump (rate approx 10 L/min) can be used as an alternative to a commercial chiller.

5. UV monitor, chart recorder, and fraction collector.

6. Amicon stirred concentration cell (Series 8000, 50 or 200 mL) with a 10,000 Mr cutoff filter for concentration of products.

7. HPLC system fitted with Zorbex Bio series GF250 column (Du Pont Company,Wilmington, DE) or equivalent gel-permeation column capable of fractionation up to approx 250,000 M_r.

2.Method

2.1 Preparation of Bispecific F(ab')₂ Derivatives

The method described here is for the preparation of F(ab')₂ BsAb starting with 5–20 mg of each parent F(ab')2 to obtain 1–8 mg of BsAb product.

1. Use equal amounts of F(ab')₂ from the two parent antibodies. The F(ab')₂ should be in 0.2 MTE8 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.

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]). 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. Take a 45 µL sample from the top of the peak for HPLC analysis. 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.

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. 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)/oPDM/DMF mixture onto this column. Collect the Fab'-A(mal) protein peak (elutes after 8–10 min).

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.

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. Check the composition of the mixture by HPLC.

10. Separate the products on two AcA44 columns run in series. Collect 10–15 min fractions.A typical elution profile is shown in Fig.

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.


2.2 Preparation of Bispecific F(ab')₃ Derivativess



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.


2.3 HPLC Monitoring



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 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.

3.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. 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 region disulfide bonds. Of the mouse IgG subclasses, IgG1 and IgG2a (three bonds), and IgG3 (one bond) qualify, whereas IgG2b (four bonds) does not. F(ab')₂ derived from rabbit Ig (one bond) and rat IgG1 (three bonds)can also be employed. However, rat IgG2a and IgG2c both have two, and rat IgG2b has four and so cannot be used as the maleimidated partner.

2. If F(ab')₃ derivatives are required, the number of SH-groups at the hinge of the unmaleimidated partner should be at least two and preferably three, because this determines the number of Fab'(mal) arms that can be conjugated.

3. We have found that a few antibodies give consistently low yields of BsAb when used as the maleimidated partner. If large quantities of a derivative are required, it is worthwhile performing small scale pilot preparations to determine which maleimidated partner gives the optimal yield.

4. It is very important to avoid contamination of the Fab'-A(SH) with 2-ME. In order to minimize the risk, stop collecting when the recorder has returned two-thirds of the way back to the baseline. In order to ensure that Fab'-B(SH) is not contaminated with 2-ME left over from the first run, make sure that it is not loaded until the chart recorder has returned to the baseline.

5. Sometimes the mixture becomes slightly cloudy.

6. To avoid contamination with o-PDM/DMF, which elutes as a second large peak, stop collecting when the chart recorder has returned halfway to baseline.

7. To avoid loss of product, slightly over concentrate, then wash the cell with a small volume of chilled buffer.

8. It is very important to add excess iodoacetamide at this stage; otherwise the BsAb derivative can precipitate.