Conjugation reagents

Abstract

The invention provides compound of the general formula: ##STR00001##
in which each X independently represents a polymer chain; n represents an integer greater than 1; Q represents a linker; Y represents an amide group; and Z represents either CH.(CH.sub.2L).sub.2 or C(CH.sub.2L)(CH.sub.2), in which each L independently represents a leaving group. The compounds are useful reagents for the conjugation of polymers to proteins, the resulting conjugates being novel and also forming part of the invention.

Claims

1. A compound having the general formula: ##STR00026## in which Q is: ##STR00027## wherein each X independently represents a polymer chain; A is CO or CHOH; and Pr represents a single protein or peptide bonded via two sulfur atoms derived from a disulfide bond in said protein or peptide.

2. A compound as claimed in claim 1, in which Pr represents an IgG Fab fragment, INF-, IFN-, or consensus IFN.

3. A compound as claimed in claim 1, in which each X represents a poly(alkylene glycol), polyvinylpyrrolidone, polyacrylate, polymethacrylate, polyoxazoline, polyvinylalcohol, polyacrylamide, polymethacrylamide, HPMA copolymer, polyester, polyacetal, poly(ortho ester), polycarbonate, poly(imino carbonate), polyamide, or polysaccharide.

4. A compound as claimed in claim 3, in which each X represent poly(ethylene glycol).

5. A compound as claimed in claim 4, in which each X independently represents polyethylene glycol of the formula CH.sub.3O(CH.sub.2CH.sub.2O).sub.m in which m is the number of ethylene oxide units in X.

6. A compound as claimed in claim 1, having the formula ##STR00028## in which each X independently represents a polymer chain and Pr represents a single protein or peptide bonded at two separate points.

7. A compound as claimed in claim 6, in which each X represents a poly(alkylene glycol), polyvinylpyrrolidone, polyacrylate, polymethacrylate, polyoxazoline, polyvinylalcohol, polyacrylamide, polymethacrylamide, HPMA copolymer, polyester, polyacetal, poly(ortho ester), polycarbonate, poly(imino carbonate), polyamide, or polysaccaride.

8. A pharmaceutical composition comprising a compound as claimed in claim 1, together with a pharmaceutically acceptable carrier.

9. A pharmaceutical composition as claimed in claim 8, which contains a further active ingredient.

10. A compound as claimed in claim 7, in which each X represent poly(ethylene glycol).

11. A compound as claimed in claim 10, in which each X independently represents polyethylene glycol of the formula CH.sub.3O(CH.sub.2CH.sub.2O).sub.m in which m is the number of ethylene oxide units in X.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the SDS-PAGE gel obtained in Example 2.

(2) FIGS. 2, 3 and 4 illustrate the SDS-PAGE gels obtained in Example 3.

(3) FIG. 5 illustrates the SDS-PAGE gel obtained in Example 4.

(4) FIG. 6 illustrates the SDS-PAGE gels obtained in Example 7.

(5) The following Examples illustrate the invention.

EXAMPLE 1: PREPARATION OF PEG REAGENT 1

(6) ##STR00017##
40 (220) kDa bifurcated PEG amine 3 (MPEG being CH.sub.3O.CH.sub.2CH.sub.2O).sub.m) was purchased from NOF CORPORATION (SUNBRIGHT GL2-400 PA, lot: M7D902). 4-[2,2-bis[(p-tolylsulfonyl) methyl] acetyl]benzoic acid-NHS ester, 4 was prepared according to Brocchini et al., Nat. Protoc. 1 (2006) 2241-2252.

(7) To a single neck round-bottomed flask containing a magnetic stirrer bar, was added bifurcated PEG amine 3 (300 mg) and toluene (8 mL). The resulting homogeneous solution was evaporated under reduced pressure using a rotary evaporator for 2 h to leave a solid residue. The residue was dissolved in dichloromethane (15 mL), the flask was sealed with a septum and the mixture stirred under argon. To the solution was added activated linker 4 (27 mg), the flask was resealed with a septum and the reaction was stirred at rt overnight. The septum was removed and the volatile portion was removed via evaporation under reduced pressure using a rotary evaporator. Acetone (20 mL) was added to the residue and the solid was dissolved with gentle warming (30 C.). The resulting solution was filtered through non-absorbent cotton wool into a 50 mL Falcon tube. Cooling the solution in a dry-ice bath resulted in a thick precipitate. Centrifugation (9 C., 4000 rpm) for 30 min sedimented the precipitate. The supernatant was decanted and the pellet was again dissolved in acetone (20 mL) at 30 C. Precipitation, sedimentation and decanting were performed as previously described. A third cycle of acetone precipitation and sedimentation was performed and after decanting the supernatant, the pellet was frozen at 80 C. and then dried to constant mass under high vacuum to give PEG reagent 1 as an off-white solid (227 mg). .sup.1H NMR (CDCl.sub.3): (ppm) 2.49 (s, 6H), 3.38 (s, 6H), 3.45-3.86 (m), 4.33 (m, 1H), 7.36 (AABB, 4H), 7.64 (AABB, 2H), 7.68 (AABB, 4H), 7.83 (AABB, 2H).

EXAMPLE 2: COMPARISON OF THE REACTIVITY OF PEG REAGENTS 1 AND 2 WITH A HUMAN IGG, FAB Fragment

(8) In this example, PEG reagent 1 of Example 1 was compared with the following reagent, PEG reagent 2, in which MPEG is CH.sub.3O.(CH.sub.2CH.sub.2O).sub.m-1CH.sub.2CH.sub.2, of Cong et al., supra.:

(9) ##STR00018##

(10) A human IgG, Fab fragment solution (4.0 mg, 0.909 mL) Jackson ImmunoResearch Laboratories Inc. Cat. No. 009-000-007) was diluted to 4.95 mL with 50 mM sodium phosphate, pH 7.4 (containing 150 mM NaCl and 20 mM EDTA). To the Fab fragment solution, 1.0 M DTT (50 L) was added to give a final DTT concentration of 10 mM in order to reduce the interchain disulfide bond so that PEGylation could occur. The resulting solution was mixed gently and then stood at 4 C. for 1 h. The solution of reduced Fab was buffer exchanged into 50 mM sodium phosphate, pH 7.4 (containing 150 mM NaCl and 20 mM EDTA) using PD-10 desalting columns. The reduced Fab solution was split equally into two portions (3.5 mL, 2 mg). Two PEG reagents: PEG reagent 1 and PEG reagent 2, were dissolved in 50 mM sodium phosphate, pH 7.4 (containing 150 mM NaCl and 20 mM EDTA) at 20 mg/mL. To the first portion of Fab solution, PEG reagent 1 (75 L, 1.5 mg) was added and to the second portion of Fab solution, PEG reagent 2 (75 L, 1.5 mg) was added. Both reactions were mixed gently and then stood at 4 C. for 20 h. After 20 h the crude reaction mixtures were analysed by SDS-PAGE. The gel was stained with INSTANTBLUE and imaged using an IMAGEQUANT LAS 4010 instrument. The result is shown in FIG. 1. In FIG. 1, lane M indicates Novex Protein Standards; lane 1 indicates human IgG, Fab fragment; lane 2 indicates PEGylated product from PEG reagent 2 at 20 h; and lane 3 indicates PEGylated product from PEG reagent 1 at 20 h. From the SDS-PAGE analysis it can be seen that, while both PEG reagents 1 and 2 were successfully conjugated to the Fab fragment, the efficiency of the conjugation for PEG reagent 1 was 26%, over double of that for PEG reagent 2 (10%).

EXAMPLE 3: STABILITY COMPARISONS OF IFN -2A CONJUGATES PREPARED WITH PEG REAGENTS 1 and 2

(11) Preparation of Conjugates:

(12) A solution of IFN -2a (6.5 mg, 0.845 mg/mL) was prepared in 50 mM sodium phosphate buffer, pH 7.4 (containing 150 mM NaCl and 20 mM EDTA). The protein solution was diluted with buffer (313 L) and a 1.0 mM DTT solution in water (187.5 mL) was then added to give a final DTT concentration of 25 mM and a reaction volume of 7.5 mL. After gentle mixing, the reaction was stood at room temperature for 30 min. The reduced protein was buffer exchanged into 50 mM sodium phosphate, pH 7.4 (containing 150 mM NaCl and 20 mM EDTA) using PD-10 columns. The eluted protein solution was centrifuged (3000 g, 4 C., 5 min) and the supernatant was then quantified by UV absorbance measurements at 280 nm (0.532 mg/mL). The protein solution was diluted to 0.10 mg/mL with buffer. PEGs 1 and 2 were dissolved at 20 mg/mL in 50 mM sodium phosphate, pH 7.4 (containing 150 mM NaCl and 20 mM EDTA). Two vials were each charged with reduced IFN -2a (2.5 mg, 24.8 mL); to the first vial PEG reagent 1 (4.9 mg, 0.245 mL) was added and to the second vial PEG reagent 2 (4.9 mg, 0.245 mL) was added. The reactions were mixed gently and then stood at 4 C. for 18 h. Any reduced protein in the final reaction mixtures was oxidised by sequentially adding 5 mg/mL copper sulfate (12.18 L) and then 50:50 (mM) GSH/GSSG (0.25 mL). The reoxidation reaction was conducted at 4 C. overnight. The reaction mixtures were diluted 4 with 100 mM sodium acetate, pH 4 and then purified by cation exchange chromatography (MACROCAP SP) using a step gradient elution of 100 mM sodium acetate, pH 4 (1.0 M NaCl) with the desired conjugates eluting at 0.60-0.65 M NaCl.

(13) Stability Comparison 1. Stress Tests for IFN -2a Conjugates Prepared with PEG Reagents 1 and 2:

(14) For each of the IFN -2a samples PEGylated with 1 and 2 (in filter sterilised PBS), four vials were prepared. Each vial was loaded with 20 L of conjugate at a concentration of 200 g/mL. Two vials for each of the test samples contained 10 mM DTT. One vial with and one vial without DTT were heated to 50 C. for 1 h. The remaining vials were heated to 90 C. for 10 min. The samples (along with unconjugated protein and unstressed conjugate) were analysed by SDS-PAGEgels were stained with INSTANTBLUE and imaged using an IMAGEQUANT LAS 4010 instrument and the results are shown in FIGS. 2 and 3 for PEG 1-IFN -2a and PEG 2-IFN -2a respectively. In FIGS. 2 and 3, lane M indicates Novex Protein Standards; lane 1 indicates IFN -2a; lane 2 indicates PEG-IFN -2a; lane 3 indicates PEG-IFN -2a 50 C., 1 h; lane 4 indicates PEG-IFN -2a 50 C., DTT, 1 h; lane 5 indicates PEG-IFN -2a 90 C., DTT, min; and lane 6 indicates PEG-IFN -2a 90 C., DTT, 10 min.

(15) FIGS. 2 and 3 show that both of the conjugates tested were stable at 50 C. for 1 h. However, after heating at 50 C. for 1 h in the presence of 10 mM DTT, significantly more free protein and aggregation is observed for the conjugate prepared with PEG reagent 2. Thermal stress at 90 C. for 10 min resulted in release of free protein for the conjugate PEGylated with 2 but not for the conjugate PEGylated with 1.

(16) Stability Comparison 2. 28 Day, 40 C., Accelerated Stability Studies for IFN -2a Conjugates PEGylated with PEG Reagents 1 and 2.

(17) Solutions of the two test samples were made up in filter sterilised PBS (containing 0.01% (w/v) NaN.sub.3) at a protein concentration of 200 g/mL. For each of PEG 1-IFN -2a and PEG 2-IFN -2a four vials were loaded with 100 L of test sample. One vial for each sample was immediately frozen at 80 C. (t=0 days). The remaining three were sealed with PARAFILM and then were stored at 40 C. At 7, 14 and 28 days a sample was removed from storage and frozen at 80 C. until the completion of the study. The test samples were flash thawed in a water bath thermostated at 37 C. and analysed by SDS-PAGE (INSTANTBLUE stained and imaged using an IMAGEQUANT LAS 4010 instrument) and the result is shown in FIG. 4, in which lane M indicates Novex Protein Standards; lane 1 indicates IFN -2a (1 g); lane 2 indicates PEG 2-IFN -2a, Day 0; lane 3 indicates PEG 2-IFN -2a, Day 7; lane 4 indicates PEG 2-IFN -2a, Day 14; lane 5 indicates PEG 2-IFN -2a, day 28; lane 6 indicates PEG 1-IFN -2a, Day 0; lane 7 indicates PEG 1-IFN -2a, Day 7; lane 8 indicates PEG 1-IFN -2a, Day 14; lane 6 indicates PEG 1-IFN -2a, Day 28. In FIG. 4, it can clearly be seen that the PEG 2-IFN -2a is less stable than PEG 1-IFN -2a with more free protein and less conjugate remaining at each time point.

EXAMPLE 4: CONJUGATION OF IFN--1B WITH PEG REAGENT 1

(18) To disulfide reduced IFN--1b (9.5 mg, 0.3 mg/mL) at pH 7.3 was added a solution of PEG reagent 1 (1.7 mL, 20 mg/mL) at pH 7.3. The resulting solution was allowed to incubate at 22 C. for 4 h, whereupon the crude reaction mixture was analysed by SDS-PAGE. The gel was stained with INSTANTBLUE and imaged using an IMAGEQUANT LAS 4010 instrument. The result is shown in FIG. 5. In FIG. 5, in lane M are Novex protein standards; lane 1 is the starting IFN--1b; lane 2 is the reduced IFN--1b and lane 3 is the reaction mixture of PEG reagent 1 with IFN--1b. From the SDS-PAGE analysis it can be seen that PEGylation of IFN--1b occurred successfully with a product band visible level with the 110 kDa protein standard.

EXAMPLE 5: PREPARATION OF BRANCHED PEG REAGENT 11

(19) Step 1: Derivatisation of O-(2-aminoethyl)-O-methyl (polyethylene glycol) with Fmoc-L-aspartic acid 4-tert-butyl ester

(20) ##STR00019##

(21) To a single neck round-bottom flask was added Fmoc-L-aspartic acid 4-tert-butyl ester 5 (16.46 mg) dissolved into anhydrous dichloromethane (DCM) (5 mL). Fresh N,N-diisopropylcarbodiimide (DIPC) (6.3 L) was added and the reaction mixture was allowed to stir, at room temperature for 30 min. O-(2-aminoethyl)-O-methyl (polyethylene glycol) 10 (100 mg) was placed separately in a one neck 50 mL schlenk flask fitted with a septum and a magnetic stir bar. Azeotropic distillation was carried out using 5 mL of toluene with the aid of an oil pump fitted with an ice trap to dry the polymer prior to the coupling reaction. Anhydrous DCM (5 mL) was added to the flask under argon atmosphere to dissolve the PEG completely. 4-Dimethylaminopyridine (DMAP) (1.22 mg) was added to the PEG solution. The activated aspartic derivative was injected into the PEG solution drop-wise. The resulting solution was allowed to stir for 48 h at room temperature under argon atmosphere. After this time, volatiles were removed by roto-evaporation and the crude product was placed under vacuum for 40 min. The crude was then re-dissolved in acetone and filtered through cotton-wool into a pre-weighed centrifuge tube. The sample was placed in dry-ice for precipitation to occur. The precipitate was isolated by centrifugation (9 C., 4000 rpm, 30 min). The solid obtained was dried in a desiccator to afford a white-off product (89 mg) .sup.1H-NMR (CDCl.sub.3, 400 MHz) /ppm: 1.45 (s, 9H), 2.6-2.8 (dd, 2H), 3.38 (s, 3H), 3.5-3.8 (broad, PEG), 4.22 (t, 1H), 4.41 (d, 2H), 4.50 (m, 1H), 5.94 (br, 1H), 6.79 (br, 1H), 7.31 (t, 2H), 7.40 (t, 2H), 7.56 (d, 2H), 7.77 (d, 2H)

(22) Step 2: Removal of Fluorenylmethyloxycarbonyl from 6

(23) ##STR00020##

(24) Aspartic PEG derivative 6 (50 mg) was dissolved in dimethylformamide (DMF) (0.8 mL) in a single neck round-bottom flask (Kocienski, 1994). Under magnetic stirring, a solution of piperidine (0.2 mL) was added drop-wise. The reaction mixture was allowed to stir at room temperature for 10 min. After this time, volatiles were removed by roto-evaporation and the crude product was dried under vacuum for 1 hour. The crude was then purified by the dry-ice precipitation method described in step 1. A white product was afforded after drying in a desiccator (44 mg). .sup.1H-NMR (CDCl.sub.3, 400 MHz) /ppm: 1.36 (s, 9H), 2.76-2.49 (dd, 2H), 3.31 (s, 3H), 3.5-3.8 (broad, PEG), 4.31 (t, 1H), 6.89 (br, 1H)

(25) Step 3: Preparation of Mono-PEG Aspartic Derivative DB Reagent 8

(26) ##STR00021##

(27) To a single neck round-bottom flask was added aspartic acid 4-tert-butyl ester PEG derivative 7 (90 mg) along with 5 mL of toluene, for azeotropic distillation. After complete dryness, anhydrous DCM (5 mL) was added to the flask under argon atmosphere to dissolve 7 completely. 4-Dimethylaminopyridine (DMAP) (1.1 mg) was added to the solution. In a separate flask, the disulfide-bridging linker (15.7 mg) was added and dissolved into anhydrous DCM (5 mL). Fresh N,N-diisopropylcarbodiimide (DIPC) (4.5 mg, 5.31 L) was added and the reaction mixture was allowed to stir, at room temperature for 30 min. The activated linker was then added to the PEG solution drop-wise. After a total reaction time of 48 h, volatiles are removed by roto-evaporation and the crude product was placed under vacuum for 40 min. Purification and isolation of product was achieved by the dry-ice precipitation method described in section Step 1. The solid obtained was dried in a desiccator to afford a white-off product (83.5 mg). .sup.1H-NMR (CDCl.sub.3, 400 MHz) /ppm: 1.43 (s, 9H), 2.33 (s, 6H), 2.62-2.91 (dd, 2H), 3.14-3.22 (m, 4H), 3.36 (s, 3H), 3.5-3.8 (br, PEG), 4.89 (t, 1H), 6.97 (br, 1H), 7.05 (d, 4H), 7.12 (d, 4H), 7.56 (d, 2H), 7.73 (d, 2H)

(28) Step 4: Boc Group Removal from Mono PEG DB Aspartic Derivative 8

(29) ##STR00022##

(30) Aspartic PEG derivative 8 (50 mg) was dissolved in anhydrous DCM (0.9 mL) in a single neck round-bottom flask. Under magnetic stirring, a solution of trifluoroacetic acid (0.1 mL) was added drop-wise (Kocienski, 1994). The reaction mixture was allowed to stir at room temperature for 2 h. After this time, volatiles were removed by roto-evaporation and the crude product was dried under vacuum for 1 h. Isolation of product was achieved by the dry-ice precipitation method described previously in step 1. A white product was afforded after drying in a desiccator (39 mg). .sup.1H-NMR (CDCl.sub.3, 400 MHz) /ppm: 2.33 (s, 6H), 2.68 (m, 2H), 3.14-3.22 (m, 4H), 3.37 (s, 3H), 3.5-3.8 (broad, PEG), 4.97 (br, 1H), 7.05 (d, 4H), 7.12 (d, 4H), 7.59 (d, 2H), 7.77 (d, 2H)

(31) Step 5: Preparation of Bis-PEG Aspartic DB Reagent 10

(32) ##STR00023##

(33) Aspartic PEG 10 kDa derivative 9 (50 mg) was dissolved in toluene (5 mL) for azeotropic distillation. Fresh N,N-diisopropylcarbodiimide (DIPC) (2.4 mg, 2.79 L) was added and the reaction mixture was allowed to stir, at room temperature for 30 min. O-(2-aminoethyl)-O-methyl (polyethylene glycol) (PEG) 10 kDa (47.4 mg) was placed separately in a one neck 50 mL schlenk flask fitted with a septum and a magnetic stir bar. Azeotropic distillation was carried out using 5 mL of toluene, as previously described. Anhydrous DCM (5 mL) was added to the flask under argon atmosphere to dissolve the PEG completely. 4-Dimethylaminopyridine (DMAP) (0.58 mg) was added to the PEG solution. The activated aspartic PEG derivative was added to the PEG solution drop-wise. The resulting solution was allowed to stir for 48 h at room temperature under argon atmosphere. After this time, volatiles were removed by roto-evaporation and the crude product was placed under vacuum for 40 min. The crude was re-dissolved in acetone for the purification by the dry-ice precipitation method described in step 1. The solid obtained was dried in desiccator to afford a white-off product (79 mg). The same procedure was applied for the preparation of bis-PEG aspartic DB reagent 220 kDa. .sup.1H-NMR (CDCl.sub.3, 400 MHz) /ppm: 2.34 (s, 6H), 2.68-2.93 (m, 2H), 3.14-3.22 (m, 4H), 3.37 (s, 3H), 3.5-3.8 (broad, PEG), 4.94 (br, 1H), 7.05 (d, 4H), 7.12 (d, 4H), 7.61 (d, 2H), 7.86 (d, 2H)

(34) Step 6: Oxidation of Bis-Sulfide to Bis-Sulfone

(35) ##STR00024##

(36) Bis-PEG aspartic DB reagent 10 (20 mg) was dissolved in an aqueous solution of 50% methanol (3 mL) in a 15 mL centrifuge tube. A magnetic stirrer bar was added and while under agitation, OXONE (potassium peroxymonosulfate) was added to the solution. The reaction mixture was allowed to stir over-night, at room temperature. After this time, the solution was transferred to a 50 mL round-bottom flask and volatiles were removed by roto-evaporation. Purification of crude product was achieved by dry-ice precipitation method described in step 1. The solid obtained was dried in a desiccator to afford a white-off product (16.2 mg). .sup.1H-NMR (CDCl.sub.3, 400 MHz) /ppm: 2.46 (s, 6H), 2.68 (m, 2H), 3.14-3.22 (m, 4H), 3.37 (s, 3H), 3.5-3.8 (broad, PEG), 4.97 (br, 1H), 7.35 (d, 4H), 7.59 (d, 6H), 7.77 (d, 2H).

EXAMPLE 6: PREPARATION OF PEG REAGENT 13

(37) ##STR00025##

(38) To a single neck round-bottomed flask containing a magnetic stirrer bar, was added bifurcated PEG amine 12 (100 mg, 40 kDa, JenKem Technology, code Y-NH2-40K, lot ZZ099P120) and toluene (8 mL). The resulting homogeneous solution was evaporated under reduced pressure using a rotary evaporator for 0.5 h to leave a solid residue. The residue was dissolved in dichloromethane (3 mL), the flask was sealed with a septum and the mixture stirred under argon. To the solution was added compound 4 (8.4 mg), the flask was resealed with a septum and the reaction was stirred at room temperature for 48 h. The septum was removed and the volatile portion was removed via evaporation under reduced pressure using a rotary evaporator. Acetone (10 mL) was added to the residue and the solid was dissolved with gentle warming (30 C.). The resulting solution was filtered through non-absorbent cotton wool into a 15 mL Falcon tube. Cooling the solution in a dry-ice bath resulted in a thick precipitate. Centrifugation (9 C., 4000 rpm) for 30 min sedimented the precipitate. The supernatant was decanted and the pellet was again dissolved in acetone (10 mL) at 30 C. Precipitation, sedimentation and decanting were performed as previously described. A third cycle of acetone precipitation and sedimentation was performed and after decanting the supernatant, the pellet was frozen at 80 C. and then dried to constant mass under high vacuum to give PEG reagent 13 as a white solid (93 mg). .sup.1H NMR (CDCl.sub.3): (ppm) 2.42 (s, 6H), 3.39 (s, 6H), 3.46-3.84 (m), 4.34-4.38 (m, 1H), 7.05 (s, 1H), 7.36 (AABB, 4H), 7.64 (AABB, 2H), 7.68 (AABB, 4H), 7.83 (AABB, 2H).

EXAMPLE 7: CONJUGATION OF PEG REAGENT 13 TO A FAB

(39) A solution of a trastuzumab derived Fab (2.6 mg/mL, 2.5 mL) was buffer exchanged, using a PD-10 desalting column (GE Healthcare), into 50 mM sodium phosphate buffer, pH 7.4 (containing 150 mM NaCl and 20 mM EDTA). The resulting solution was then treated with DTT (1 M, 35 L, 1 h, 4 C.) to reduce the interchain disulfide of the Fab. The disulfide reduced Fab solution was then buffer exchanged into 50 mM sodium phosphate buffer, pH 7.4, using two PD-desalting columns. Two aliquots of the solution were then taken, each containing 1 mg of Fab. One aliquot was stored at 4 C. and the other at room temperature (RT). A solution of PEG reagent 13 was prepared in 50 mM sodium phosphate buffer, pH 7.4 (5 mg/mL) and 175 L of the solution was added to each of the reduced Fab solutions. Each of the solutions was diluted, so that final Fab concentration was 0.5 mg/mL. The reaction solutions were shaken gently before being allowed to stand for 20 h. After 20 h, the samples (along with starting Fab and reduced Fab samples) were analysed by SDS-PAGEthe gels were stained with INSTANTBLUE and imaged using an IMAGEQUANT LAS 4010 instrument. The results are shown in FIG. 6. In FIG. 6, the lane labelled M shows Novex Protein Standards; lanes 1 and 4 show the starting non-reduced Fab; lanes 2 and 5 show the reduced Fab; lane 3 shows the 4 C. reaction mixture and lane 6 shows the room temperature reaction mixture. From the SDS-PAGE analysis it can be seen that PEG reagent 13 conjugated successfully to the Fab fragment, the efficiency of the conjugation at 4 C. was 24%, and at room temperature the efficiency was 40%.