Gd x

Example 1

[Figure (not displayed)]

Under an argon atmosphere, in a 10 mL Schlenk tube were placed HO-U-SUC-TOB (82.2 mg, 66.0 μmol) and MeOC(O)-TOB (100 mg, 106 μmol) and they were dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (37.8 mg, 197 μmol) and U-CE phosphoramidite (150 mg, 197 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1 hr. To the reaction solution was added 2,2,2-trifluoroethanol (70.3 μL, 983 μmol), and the mixture was stirred at room temperature for 15 min. After stirring, PADS (178 mg, 590 μmol) was added and the mixture was stirred at room temperature for 2 hr. 5-Methoxyindole (193 mg, 1.31 mmol) and trifluoroacetic acid (45.2 μL, 590 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (85.5 μL, 649 μmol) was added, acetonitrile (10 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (2′-O-methyl-uridine-3′-[O-(2-cyanoethyl)]phosphorothionyl 2′-O-methyl-uridin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (197 mg, yield 95%).

(1) Preparation of Reaction Solution

Under an argon atmosphere, HO-dT-SUC-TOB (100 mg, 80.8 μmol) and MeOC(O)-TOB (100 mg, 106 μmol) were dissolved in dehydrated dichloromethane (4.0 mL). To this solution was added a mixture of dG-CE phosphoramidite (204 mg, 242 μmol) and 5-benzylthio-1H-tetrazole (46.5 mg, 242 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 30 min. Completion of the reaction was confirmed by thin layer chromatography (dichloromethane/methanol=10/1 (volume ratio)), a quencher in the kind and amount shown in Table 1 was added and the mixture was stirred at room temperature for 30 min. Then, an oxidant shown in Table 1 (254 μmol, 1.05 molar equivalents relative to phosphoramidite monomer) was added and the mixture was stirred at room temperature for 1 hr to prepare a reaction solution.

(2) Preparation of Test Solution (Pre-Treatment for Analysis)

The obtained reaction solution (50 μL) was dispensed to a 1.5 mL vial, diluted with tetrahydrofuran (450 μL), to which DBU (20 μL) was added and the mixture was stirred for 30 sec to prepare a test solution.

(3) Analysis

The obtained test solution was measured by mass spectrometry using LC-TOF MS (Agilent6230). The amount of the byproduct was calculated by the following formula based on the abundance of each compound observed (object compound and byproduct).
amount(%) of branch product=(abundance of branch product/abundance of object compound)×100amount(%) of phosphoric acid triester cleavage product=(abundance of phosphoric acid triester cleavage product/abundance of object compound)×100

As used herein, the object compound refers to the object oligonucleotide contained in the reaction solution, the branch product refers to a byproduct produced by falling off of an amino-protecting group of nucleic acid base of the object compound and binding of the amino group and a monomer, and the phosphoric acid triester cleavage product refers to a byproduct produced by cleavage of phosphoric acid triester of the object compound. The results are shown in Table 1.

As shown in Table 1, when CSO was used as an oxidant and morpholine, tetrahydrofurfuryl alcohol, diethylene glycol or ethylene glycol was used as a quencher, production of a branch product and a phosphoric acid triester cleavage product was confirmed to have been effectively suppressed.

(1) Synthesis of Dimer

Under an argon atmosphere, in a 300 mL four-necked flask were placed HO-dT-SUC-TOB (619 mg, 500 μmol) and Ac-TOB (619 mg, 648 μmol), and they were dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (288 mg, 1.50 mmol) and dT-CE phosphoramidite (1.12 g, 1.50 mmol) prepared by dissolving in dehydrated acetonitrile (3.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added methanol (608 μL, 15.0 mmol), and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (340 mg, 1.65 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.47 g, 10.0 mmol) and trifluoroacetic acid (345 μL, 4.50 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (654 μL, 4.95 mmol) was added, acetonitrile (150 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (762 mg, yield 95%).

(2) Synthesis of Trimer

Under an argon atmosphere, in a 300 mL four-necked flask was placed the dimer (762 mg, 473 μmol) obtained in the above-mentioned (1), and it was dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (273 mg, 1.42 mmol) and dA-CE phosphoramidite (1.22 g, 1.42 mmol) prepared by dissolving in dehydrated acetonitrile (3.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added methanol (575 μL, 14.2 mmol), and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (321 mg, 1.56 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.39 g, 9.46 mmol) and trifluoroacetic acid (326 μL, 4.26 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution.

Since Reference Example 1 includes steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, it is one embodiment of the present invention.

Example 2

(1) Synthesis of Phosphorothioate Dimer Wherein 3′-Hydroxy Group is Protected by Anchor

[Figure (not displayed)]

Under an argon atmosphere, in a 200 mL four-necked flask were placed HO-dT-SUC-TOB (619 mg, 0.500 mmol) and AcO-TOB (773 mg, 0.808 mmol) and they were dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (288 mg, 1.50 mmol) and dT-CE phosphoramidite (1.12 g, 1.50 mmol) prepared by dissolving in dehydrated acetonitrile (2.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added 2,2,2-trifluoroethanol (1.07 mL, 15.0 mmol), and the mixture was stirred at room temperature for 15 min. After stirring, a mixture of acetic acid (172 μL, 3.00 mmol) and 2,4,6-trimethylpyridine (594 μL, 4.50 mmol) was further added, and the mixture was stirred for 15 min at room temperature. DDTT (340 mg, 1.65 mmol) was added, and the mixture was stirred at room temperature for 30 min. After stirring, 5-methoxyindole (1.47 g, 10.0 mmol) and trifluoroacetic acid (689 μL, 9.00 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (1.31 mL, 9.90 mmol) was added, acetonitrile (150 mL) was added to the reaction solution and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (789 mg, yield 98%).

(2) Synthesis of Phosphorothioate 19-Mer Wherein 3′-Hydroxy Group is Protected by Anchor

An operation similar to that in the above-mentioned (1) was further repeated 18 times to give a 19-mer (deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-benzoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-benzoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl 3,4,5-tris(octadecyloxy)benzyl succinate) (2.78 g).

(3) Synthesis of Phosphorothioate 20-Mer Wherein 3′-Hydroxy Group is Protected by Anchor and 5′-Hydroxy Group is Protected by DMTr Group

Under an argon atmosphere, in a 200 mL four-necked flask was placed the 19-mer (2.78 g) obtained in the above-mentioned (2), and it was dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (287 mg, 1.49 mmol) and dT-CE phosphoramidite (1.11 g, 1.49 mmol) prepared by dissolving in dehydrated acetonitrile (2.0 mL), and the mixture was stirred at room temperature for 1.5 hr. DDTT (338 mg, 1.64 mmol) was added, and the mixture was stirred at room temperature for 30 min. After stirring, acetonitrile (150 mL) was added to the reaction solution and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a 20-mer (5′-O-(4,4′-dimethoxytrityl)-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-acetyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-acetyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-acetyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl 3,4,5-tris(octadecyloxy)benzyl succinate) as a white solid (2.79 g).

(4) Removal of DMTr Group

Under an argon atmosphere, in a 100 mL two-necked flask was placed the 20-mer (1.00 g) obtained in the above-mentioned (3) and it was dissolved in dehydrated dichloromethane (10 m). 5-Methoxyindole (245 mg, 1.66 mmol) and trifluoroacetic acid (100 μL, 1.31 mmol) were added and the mixture was stirred at room temperature for 1.5 hr, and neutralized with 2,4,6-trimethylpyridine (190 μL, 1.44 mmol). Acetonitrile (150 mL) was added, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a 20-mer wherein the DMTr group was removed (deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-acetyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-acetyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-acetyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl 3,4,5-tris(octadecyloxy)benzyl succinate) as a white solid (867 mg).

(5) Deprotection

A mixture of the 20-mer (20 mg) obtained in the above-mentioned (4) and 30 wt % aqueous ammonia (5.00 mL) was placed in an autoclave and heated at 55° C. for 16 hr and cooled to room temperature. Insoluble material in the reaction solution was removed by a syringe filter (Whatman 25 mm GD/X PVDF 0.45 μm) and the filtrate was freeze-dried to give the object deoxythymidine-3′-phosphorothionyl-deoxycytidine-3′-phosphorothionyl-deoxycytidine-3′-phosphorothionyl-deoxycytidine-3′-phosphorothionyl-deoxyguanosine-3′-phosphorothionyl-deoxycytidine-3′-phosphorothionyl-deoxycytidine-3′-phosphorothionyl-deoxythymidine-3′-phosphorothionyl-deoxyguanosine-3′-phosphorothionyl-deoxythymidine-3′-phosphorothionyl-deoxyguanosine-3′-phosphorothionyl-deoxyadenosine-3′-phosphorothionyl-deoxycytidine-3′-phosphorothionyl-deoxyadenosine-3′-phosphorothionyl-deoxythymidine-3′-phosphorothionyl-deoxyguanosine-3′-phosphorothionyl-deoxycytidine-3′-phosphorothionyl-deoxyadenosine-3′-phosphorothionyl-deoxythymidine-3′-phosphorothionyl-deoxythymidine.

HPLC (WATERS XBridge™ C18 2.5 μm 4.6×75 mm column, flow rate 1.0 mL/min, 8 mM TEA+100 mM HFIP, MeOH, gradient: 0-10 min; 5 to 60%, λ=260 nm):Rt=6.87 min (83.3 area %); TOF/MS: 6646.05

(1) Preparation of Reaction Solution

Under an argon atmosphere, HO-dT-SUC-TOB (100 mg, 80.8 mol) was dissolved in dehydrated dichloromethane (4.0 mL), a mixed solution of rA_OMe(Bz)-CE phosphoramidite (215 mg, 242 mol) and 5-benzylthio-1H-tetrazole (46.5 mg, 242 μmol) in dehydrated acetonitrile (0.5 mL) was added, and the mixture was stirred at room temperature for 1 hr. Completion of the reaction was confirmed by thin layer chromatography (dichloromethane/methanol=10/1 (volume ratio)), a quencher in the kind and amount shown in Table 2 was added and the mixture was stirred at room temperature for 30 min. Then, CSO (58.2 mg, 254 μmol) was added and the mixture was stirred at room temperature for 1 hr. Furthermore, to the reaction solution were added 5-methoxyindole (238 mg, 1.62 mmol) and trifluoroacetic acid (92.5 mL, 1.21 mmol) and the mixture was stirred at room temperature for 18 hr to prepare a reaction solution.

(2) Preparation and Analysis of Test Solution

In the same manner as in Experimental Example 1, the prepared test solution was measured by mass spectrometry and the amount of the byproduct was calculated by the following formula based on the abundance of each compound observed (object compound and byproduct).
amount(%) of+1monomer product=(abundance of+1monomer product/abundance of object compound)×100amount(%) of base-part deprotected product=(abundance of base-part deprotected product/abundance of object compound)×100

As used herein, the +1 monomer product refers to a byproduct produced by binding of one redundant monomer to the object compound, and the base-part deprotected product refers to a byproduct produced by falling off of an amino-protecting group of nucleic acid base of the object compound. The results are shown in Table 2.

As shown in Table 2, when tetrahydrofurfuryl alcohol, diethylene glycol or ethylene glycol was used as a quencher, production of a +1 monomer product and a base-part deprotected product was confirmed to have been effectively suppressed.

(1) Synthesis of Dimer

Under an argon atmosphere, in a 10 mL Schlenk tube were placed HO-dT-SUC-TOB (80.4 mg, 64.9 mol) and Ac-TOB (99.6 mg, 104 μmol) and they were dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (37.4 mg, 195 μmol) and dT-CE phosphoramidite (145 mg, 195 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr was stirred. To the reaction solution was added methanol (39.5 μL, 975 μmol), a mixture of acetic acid (22.3 μL, 390 μmol) was added, and 2,4,6-trimethylpyridine (77.0 μL, 585 μmol), and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (44.0 mg, 215 μmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.47 g, 10.0 mmol) and trifluoroacetic acid (89.6 μL, 1.17 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (170 μL, 1.29 mmol) was added, acetonitrile (10 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (104 mg, yield 99%).

(2) Synthesis of Trimer

Under an argon atmosphere, in a 300 mL four-necked flask was placed the dimer (104 mg, 64.2 μmol) obtained in the above-mentioned (1) and it was dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (37.5 mg, 195 μmol) and dA-CE phosphoramidite (165 mg, 195 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added methanol (39.5 μL, 975 μmol), a mixture of acetic acid (22.3 μL, 390 μmol) and 2,4,6-trimethylpyridine (77.0 μL, 585 μmol) was added, and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (44.0 mg, 214 μmol) was added and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (158 mg, 1.07 mmol) and trifluoroacetic acid (74.7 μL, 975 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution.

Since Reference Example 2 includes steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, it is one embodiment of the present invention.

Example 3

(1) Synthesis of Phosphorothioate Dimer Wherein 3′-Hydroxy Group is Protected by Anchor

Under an argon atmosphere, in a 10 mL Schlenk tube were placed HO-dT-SUC-TOB (100 mg, 81 μmol) and MeOC(O)-TOB (100 mg, 106 μmol), and they were dissolved in dehydrated dichloromethane (4.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (46.6 mg, 242 μmol) and dT-CE phosphoramidite (181 mg, 242 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added 2,2,2-trifluoroethanol (86.6 μL, 1.21 mmol), and the mixture was stirred at room temperature for 15 min. Furthermore, a mixture of acetic acid (13.9 μL, 242 μmol) and 2,4,6-trimethylpyridine (31.9 μL, 242 μmol), and DDTT (54.7 mg, 267 μmol) were added, and the mixture was stirred at room temperature for 30 min. After stirring, 5-methoxyindole (238 mg, 1.62 mmol) and trifluoroacetic acid (55.7 μL, 727 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added acetonitrile (10 mL), and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO while washing with acetonitrile (20 mL) and dried under reduced pressure to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (222 mg, yield 96%).

(2) Synthesis of Phosphorothioate Trimer Wherein 3′-Hydroxy Group is Protected by Anchor

An operation similar to that in the above-mentioned (1) was further repeated once to give a trimer (N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl3,4,5-tris(octadecyloxy)benzyl succinate) (251 mg, yield 89%).

(1) Preparation of Reaction Solution

As shown in the below-mentioned Reference Examples 1 to 11, the reaction solution was prepared.

(2) Preparation and Analysis of Test Solution

In the same manner as in Experimental Example 2, the prepared test solution was measured by mass spectrometry, and the amount of the +1 monomer product and the amount of the base-part deprotected product were calculated. The results are shown in Table 3.

As shown in Table 3, when methanol, t-butanol, 2,2,2-trifluoroethanol or 2-propanol was used as a quencher, production of a +1 monomer product was confirmed to have been suppressed. Furthermore, when t-butanol, 2,2,2-trifluoroethanol or 2-propanol was used, production of a base-part deprotected product was also confirmed to have been suppressed. In addition, when a neutralized salt of acetic acid and 2,4,6-trimethylpyridine was copresent together with alcohol confirmed to have a production suppressive effect on a +1 monomer product and a base-part deprotected product, production of a byproduct was confirmed to have been suppressed.

(1) Synthesis of Dimer

Under an argon atmosphere, in a 300 mL four-necked flask were placed HO-dT-SUC-TOB (618 mg, 499 μmol) and Ac-TOB (618 mg, 646 μmol), and they were dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (288 mg, 1.50 mmol) and dT-CE phosphoramidite (1.11 g, 1.50 mmol) prepared by dissolving in dehydrated acetonitrile (3.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added a mixture of acetic acid (857 μL, 15.0 mmol) and pyridine (1.82 mL, 22.4 mmol), and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (338 mg, 1.65 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.47 g, 10.0 mmol) and trifluoroacetic acid (344 μL, 4.49 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (653 μL, 4.94 mmol) was added, acetonitrile (150 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (753 mg, yield 94%).

(2) Synthesis of Trimer

Under an argon atmosphere, in a 300 mL four-necked flask was placed the dimer (753 mg, 468 μmol) obtained in the above-mentioned (1), and it was dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (270 mg, 1.40 mmol) and dA-CE phosphoramidite (1.20 g, 1.40 mmol) prepared by dissolving in dehydrated acetonitrile (3.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added a mixture of acetic acid (402 μL, 7.01 mmol) and pyridine (567 μL, 7.01 mmol), and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (317 mg, 1.54 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.38 g, 9.35 mmol) and trifluoroacetic acid (322 μL, 4.21 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution.

Since Reference Example 3 includes steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, it is one embodiment of the present invention.

Example 4

[Figure (not displayed)]

Under an argon atmosphere, in a 10 mL Schlenk tube were placed HO-dT-SUC-TOB (100 mg, 81 μmol) and MeOC(O)-TOB (100 mg, 106 μmol), and they were dissolved in dehydrated dichloromethane (5.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (46.6 mg, 242 μmol) and dT-CE phosphoramidite (181 mg, 242 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added 2,2,2-trifluoroethanol (86.6 μL, 1.21 mmol), and the mixture was stirred at room temperature for 15 min. Furthermore, DPTT (28.0 mg, 73.0 μmol) was added, and the mixture was stirred at room temperature for 30 min. After stirring, 5-methoxyindole (234 mg, 1.62 mmol) and trifluoroacetic acid (55.7 μL, 727 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. The reaction solution was neutralized with 2,4,6-trimethylpyridine (105 μL, 800 μmol), triethyl phosphite (8.6 μL, 73.0 μmol) was added and the mixture was stirred at room temperature for 15 min. After stirring, acetonitrile (10 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (222 mg, yield 88%).

(1) Preparation of Reaction Solution

As shown in the below-mentioned Reference Examples 12 to 15, a reaction solution was prepared.

(2) Preparation of Test Solution

To the reaction solutions obtained in Reference Examples 12 to 15 were added a quencher and a neutralized salt in the kind and amount shown in Table 4. The reaction solution (50 μL) was dispensed to a 1.5 mL vial, DDTT (2.5 mg, 12 μmol) was added and the mixture was shaken for 30 sec. The mixture was diluted with tetrahydrofuran (450 μL), DBU (20 μL) was added and the mixture was stirred for 30 sec to give a test solution.

(3) Analysis

The obtained test solution was measured by mass spectrometry using LC-TOF MS (Agilent6230). The amount of the unreacted material was calculated by the following formula based on the abundance (m/z=2) of each compound observed (object compound and unreacted material).
amount(%) of unreacted material=(abundance of unreacted material/abundance of object compound)×100

As used herein, the unreacted material refers to a phosphoramidite monomer used for preparing the reaction solution.

As shown in Table 4, when t-butanol or 2,2,2-trifluoroethanol and a neutralized salt of acetic acid and 2,4,6-trimethylpyridine were copresent, condensation reaction was confirmed to have proceeded effectively without leaving an unreacted material during condensation reaction.

(1) Synthesis of Dimer

Under an argon atmosphere, in a 300 mL four-necked flask were placed HO-dT-SUC-TOB (619 mg, 500 μmol) and Ac-TOB (770 mg, 807 μmol), and they were dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (288 mg, 1.50 mmol) and dT-CE phosphoramidite (1.12 g, 1.50 mmol) prepared by dissolving in dehydrated acetonitrile (3.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added a mixture of acetic acid (172 μL, 3.00 mmol) and N-methylimidazole (178 μL, 2.25 mmol), and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (339 mg, 1.65 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.47 g, 10.0 mmol) and trifluoroacetic acid (574 μL, 7.50 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, N-methylimidazole (653 μL, 8.25 mmol) was added, acetonitrile (150 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (792 mg, 98%).

(2) Synthesis of Trimer

Under an argon atmosphere, in a 300 mL four-necked flask was placed the dimer (792 mg, 492 μmol) obtained in the above-mentioned (1), and it was dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (284 mg, 1.48 mmol) and dA-CE phosphoramidite (1.27 g, 1.48 mmol) prepared by dissolving in dehydrated acetonitrile (3.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added a mixture of acetic acid (169 μL, 2.95 mmol) and N-methylimidazole (175 μL, 2.21 mmol), and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (333 mg, 1.62 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.45 g, 9.83 mmol) and trifluoroacetic acid (565 μL, 7.37 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution.

Since Reference Example 4 includes steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, it is one embodiment of the present invention.

Example 5

[Figure (not displayed)]

Under an argon atmosphere, in a 10 mL Schlenk tube were placed HO-dT-SUC-TOB (80.3 mg, 65.0 μmol) and MeOC(O)-TOB (99.0 mg, 105 μmol) and they were dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (37.4 mg, 195 μmol) and dT-CE phosphoramidite (145 mg, 195 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added 2,2,2-trifluoroethanol (69.5 μmL, 973 μmol), and the mixture was stirred at room temperature for 15 min. Furthermore, PADS (177 mg, 584 μmol) was added, and the mixture was stirred at room temperature for 30 min. After stirring, 5-methoxyindole (191 mg, 1.30 mmol) and trifluoroacetic acid (44.7 μL, 584 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. The reaction solution was neutralized with 2,4,6-trimethylpyridine (84.6 μL, 642 μml). Acetonitrile (10 mL) was added, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (73.3 mg, 85%).

TOF/MS: 1557.0116

The phosphorothioate 10-mer obtained in the below-mentioned Reference Examples 16-20 were analyzed by HPLC and the purity of the obtained 10-mer (area %) was calculated. The results of the purity (area %) of quencher, neutralized salt and 10-mer used are shown in Table 5.

As shown in Table 5, when 2,2,2-trifluoroethanol or t-butanol, and a neutralized salt of acetic acid and 2,4,6-trimethylpyridine was used, the purity of 10-mer was confirmed to be high.

(1) Synthesis of Dimer

Under an argon atmosphere, in a 300 mL four-necked flask were placed HO-dT-SUC-TOB (619 mg, 500 μmol) and Ac-TOB (770 mg, 807 μmol), and they were dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (288 mg, 1.50 mmol) and dT-CE phosphoramidite (1.12 g, 1.50 mmol) prepared by dissolving in dehydrated acetonitrile (3.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added methanol (608 μL, 15.0 mmol), a mixture of acetic acid (172 μL, 3.00 mmol) and N-methylimidazole (356 μL, 4.50 mmol) was added, and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (339 mg, 1.65 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.47 g, 10.0 mmol) and trifluoroacetic acid (689 μL, 9.00 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, N-methylimidazole (784 μL, 9.90 mmol) was added, acetonitrile (150 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (779 mg, yield 97%).

(2) Synthesis of Trimer

Under an argon atmosphere, in a 300 mL four-necked flask was placed the dimer (779 mg, 483 μmol) obtained in the above-mentioned (1), and it was dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (279 mg, 1.45 mmol) and dA-CE phosphoramidite (1.24 g, 1.45 mmol) prepared by dissolving in dehydrated acetonitrile (3.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added methanol (588 μL, 14.5 mmol), a mixture of acetic acid (166 μL, 2.90 mmol) and N-methylimidazole (344 μL, 4.35 mmol) was added, and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (328 mg, 1.60 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.42 g, 9.67 mmol) and trifluoroacetic acid (666 μL, 8.70 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution.

Since Reference Example 5 includes steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, it is one embodiment of the present invention.

Example 6

[Figure (not displayed)]

Under an argon atmosphere, in a 10 mL Schlenk tube were placed deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)cyclohexyl-1-methyl]succinate (500 mg, 0.402 mmol) and methyl 3,4,5-tris(octadecyloxy)cyclohexyl-1-carboxylate (500 mg, 0.528 mmol), and they were dissolved in dehydrated dichloromethane (4.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (116 mg, 0.603 mmol) and dT-CE phosphoramidite (449 mg, 0.603 mmol) prepared by dissolving in dehydrated acetonitrile (0.4 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added 2,2,2-trifluoroethanol (129 μL, 1.81 mmol), and the mixture was stirred at room temperature for 15 min. Furthermore, DPTT (54.1 mg, 0.141 mmol) was added, and the mixture was stirred at room temperature for 30 min. After stirring, 5-methoxyindole (1.18 g, 8.04 mmol) and trifluoroacetic acid (115 μL, 1.51 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. The reaction solution was neutralized with 2,4,6-trimethylpyridine (218 μL, 1.66 mmol), acetonitrile (20 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)cyclohexyl-1-methyl]succinate) as a white solid (1.15 g, yield 100%).

(1) Synthesis of Dimer

Under an argon atmosphere, in a 10 mL Schlenk tube were placed HO-dT-SUC-TOB (80.3 mg, 65.0 μmol) and Ac-TOB (100 mg, 105 μmol) and they were dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (37.4 mg, 195 μmol) and dT-CE phosphoramidite (145 mg, 195 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added t-butanol (186 μL, 1.95 mmol), and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (44.0 mg, 214 μmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (94.6 mg, 642 μmol) and trifluoroacetic acid (44.7 μL, 584 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (84.6 μL, 642 μmol) was added, acetonitrile (10 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (104 mg, yield 100%).

(2) Synthesis of Trimer

Under an argon atmosphere, in a 300 mL four-necked flask was placed the dimer (104 mg, 65.0 μmol) obtained in the above-mentioned (1) and it was dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (37.3 mg, 194 μmol) and dA-CE phosphoramidite (167 mg, 194 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution. The reaction solution obtained at this time point was used as the reaction solution of Reference Example 13 in the above-mentioned Experimental Example 4.

To the reaction solution obtained as mentioned above was added t-butanol (186 μL, 1.94 mmol), and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (43.9 mg, 214 μmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (94.3 mg, 641 μmol) and trifluoroacetic acid (44.6 μL, 583 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution. The reaction solution obtained at this time point was used as the reaction solution of Reference Example 6 in the above-mentioned Experimental Example 3.

Since Reference Example 6 and Reference Example 13 include steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, they are each one embodiment of the present invention.

Example 7

[Figure (not displayed)]

Under an argon atmosphere, in a 10 mL Schlenk tube were placed deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzoylpiperazine]succinate (100 mg, 76.0 μmmol) and methyl 3,4,5-tris(octadecyloxy)cyclohexyl-1-carboxylate (100 mg, 0.106 mmol) and they were dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (43.7 mg, 227 μmop and 2′-OMe-rA(Bz)-CE phosphoramidite (202 mg, 0.227 mmol) prepared by dissolving in dehydrated acetonitrile (0.3 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added morpholine (99.0 μL, 1.14 mmol), and the mixture was stirred at room temperature for 15 min. Furthermore, (2R,8aS)-(+)-(camphorylsulfonyl)oxaziridine (54.7 mg, 0.239 mmol) was added, and the mixture was stirred at room temperature for 1 hr. 5-Methoxyindole (223 mg, 1.52 mmol) and trifluoroacetic acid (34.8 μt, 0.455 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. The reaction solution was neutralized with 2,4,6-trimethylpyridine (65.9 μL, 500 μmol), acetonitrile (10 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (N⁶-benzoyl-2′-O-methyl-adenosine-3′-[O-(2-cyanoethyl)]phosphoryl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzoylpiperazine]succinate) as a white solid (214 mg, yield 89%).

(1) Synthesis of Dimer

Under an argon atmosphere, in a 300 mL four-necked flask were placed HO-dT-SUC-TOB (619 mg, 500 μmol) and Ac-TOB (772 mg, 808 mol), and they were dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (288 mg, 1.50 mmol) and dT-CE phosphoramidite (1.12 g, 1.50 mmol) prepared by dissolving in dehydrated acetonitrile (3.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added t-butanol (1.43 mL, 15.0 mmol), a mixture of acetic acid (173 μL, 3.00 mmol) and 2,4,6-trimethylpyridine (594 μL, 4.50 mmol) was added, and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (339 mg, 1.65 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.47 g, 10.0 mmol) and trifluoroacetic acid (689 μL, 8.99 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (1.31 mL, 9.89 mmol) was added, acetonitrile (150 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (798 mg, yield 99%).

(2) Synthesis of Trimer

Under an argon atmosphere, in a 300 mL four-necked flask was placed the dimer (798 mg, 495 μmol) obtained in the above-mentioned (1), and it was dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (286 mg, 1.49 mmol) and dA-CE phosphoramidite, (1.27 g, 1.49 mmol) prepared by dissolving in dehydrated acetonitrile (3.0 mL), and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution. The reaction solution obtained at this time point was used as the reaction solution of Reference Example 14 in the above-mentioned Experimental Example 4.

To the reaction solution obtained as mentioned above was added t-butanol (1.42 mL, 14.8 mmol), a mixture of acetic acid (171 μL, 2.97 mmol) and 2,4,6-trimethylpyridine (589 μL, 4.46 mmol) was added, and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (335 mg, 1.63 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.46 g, 9.90 mmol) and trifluoroacetic acid (569 μL, 7.43 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution. The reaction solution obtained at this time point was used as the reaction solution of Reference Example 7 in the above-mentioned Experimental Example 3.

Since Reference Example 7 and Reference Example 14 include steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, they are each one embodiment of the present invention.

Example 8

[Figure (not displayed)]

Under an argon atmosphere, in a 10 mL Schlenk tube were placed 3,4,5-tris(octadecyloxy)benzyl]succinate (100 mg, 81.0 μmmol) and methyl 3,4,5-tris(octadecyloxy)phenyl-1-carboxylate (100 mg, 0.106 mmol), and they were dissolved in dehydrated dichloromethane (5.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (46.6 mg, 242 μmol) and dG-CE phosphoramidite (204 mg, 0.242 mmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added morpholine (106 μL, 1.21 mmol), and the mixture was stirred at room temperature for 15 min. Furthermore, a mixed solution of iodine (64.6 mg, 0.254 mmol), 2,4,6-trimethylpyridine (83.8 μL, 0.636 mmol) and water (6.60 μL, 0.364 mmol) was added, and the mixture was stirred at room temperature for 1 hr. 5-Methoxyindole (238 mg, 1.62 mmol) and trifluoroacetic acid (55.7 μL, 0.727 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. The reaction solution was neutralized with 2,4,6-trimethylpyridine (105 μL, 800 μmol), acetonitrile (10 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give dinucleotide (N²-isobutyryl-deoxyadenosine-3′-[O-(2-cyanoethyl)]phosphoryl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (209 mg, yield 88%).

(1) Synthesis of Dimer

Under an argon atmosphere, in a 10 mL Schlenk tube was placed HO-dT-SUC-TOB (80.3 g, 65.0 μmol) and it was dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (37.4 mg, 195 μmol) and dT-CE phosphoramidite (145 mg, 195 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added 2-propanol (74.8 μL, 974 μmol), a mixture of acetic acid (22.2 μL, 390 μmol) and 2,4,6-trimethylpyridine (77.1 μL, 585 μmol) was added, and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (44.0 mg, 214 μmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (94.6 mg, 643 μmol) and trifluoroacetic acid (44.7 μL, 584 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (84.6 μL, 643 μmol) was added, acetonitrile (10 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (101 mg, yield 98%).

(2) Synthesis of Trimer

Under an argon atmosphere, in a 300 mL four-necked flask was placed the dimer (101 mg, 63.0 μmol) obtained in the above-mentioned (1) and it was dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (36.3 mg, 189 μmol) and dA-CE phosphoramidite (162 mg, 189 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added 2-propanol (72.5 μL, 944 μmol), a mixture of acetic acid (10.8 μL, 189 μmol) and 2,4,6-trimethylpyridine (37.3 μL, 283 μmol) was added, and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (42.6 mg, 208 μmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (91.7 mg, 623 μmol) and trifluoroacetic acid (43.4 μL, 566 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution.

Since Reference Example 8 includes steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, it is one embodiment of the present invention.

Example 9

(1) Synthesis of Dimer

Under an argon atmosphere, in a 300 mL four-necked flask was placed HO-dT-SUC-TOB (180 mg, 64.9 μmol) and it was dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (37.4 mg, 195 μmol) and dT-CE phosphoramidite (145 mg, 195 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added 2,2,2-trifluoroethanol (140 μL, 1.95 mmol), a mixture of acetic acid (22.3 μL, 390 μmol) and 2,4,6-trimethylpyridine (77.0 μL, 585 μmol) was added, and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (44.0 mg, 215 μmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (189 mg, 1.17 mmol) and trifluoroacetic acid (89.6 μL, 1.17 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (170 μL, 1.29 mmol) was added, acetonitrile (10 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (104 mg, yield 99%).

(2) Synthesis of Trimer

Under an argon atmosphere, in a 300 mL four-necked flask was placed the dimer (101 mg, 62.5 μmol) obtained in the above-mentioned (1) and it was dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (36.1 mg, 188 μmol) and dA-CE phosphoramidite (161 mg, 188 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution. The reaction solution obtained at this time point was used as the reaction solution of Reference Example 15 in the above-mentioned Experimental Example 4.

To the reaction solution obtained as mentioned above was added 2,2,2-trifluoroethanol (134 μL, 1.88 mmol), a mixture of acetic acid (21.5 μL, 376 μmol) and 2,4,6-trimethylpyridine (74.3 μL, 564 μmol) was added, and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (42.5 mg, 207 μmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (147 mg, 1.00 mmol) and trifluoroacetic acid (72.0 μL, 910 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution. The reaction solution obtained at this time point was used as the reaction solution of Reference Example 9 in the above-mentioned Experimental Example 3.

Since Reference Example 9 and Reference Example 15 include steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, they are each one embodiment of the present invention.

Example 10

Under an argon atmosphere, in a 10 mL Schlenk tube was placed HO-dA-SUC-TOB (182 mg, 60.5 μmol) and it was dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (35.0 mg, 182 μmol) and dT-CE phosphoramidite (135 mg, 182 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added 2,2,2-trifluoroethanol (65.3 μL, 910 μmol), and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (41.1 mg, 200 μmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (88.4 mg, 600 μmol) and trifluoroacetic acid (41.8 μL, 546 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution.

Example 11

Under an argon atmosphere, in a 10 mL Schlenk tube was placed HO-dA-SUC-TOB (182 mg, 60.5 μmol) and it was dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (35.0 mg, 182 μmol) and dT-CE phosphoramidite (135 mg, 182 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added t-butanol (87.0 μL, 910 μmol), and the mixture was stirred at room temperature for min. After stirring, DDTT (41.1 mg, 200 μmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (88.4 mg, 600 μmol) and trifluoroacetic acid (41.8 μL, 546 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution.

Example 12

(1) Synthesis of Dimer

Under an argon atmosphere, in a 10 mL Schlenk tube were placed HO-dT-SUC-TOB (80.3 mg, 65.0 μmol) and Ac-TOB (100 mg, 105 μmol) and they were dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (37.4 mg, 195 μmol) and dT-CE phosphoramidite (145 mg, 195 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added 2,2,2-trifluoroethanol (69.6 μL, 973 μmol), DDTT (44.0 mg, 214 μmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (94.6 mg, 642 μmol) and trifluoroacetic acid (44.7 μL, 584 μmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (84.6 μL, 642 μmol) was added, acetonitrile (10 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (98.3 mg, yield 94%).

(2) Synthesis of Trimer

Under an argon atmosphere, in a 300 mL four-necked flask was placed the dimer (98.3 mg, 61.0 μmol) obtained in the above-mentioned (1) and it was dissolved in dehydrated dichloromethane (3.0 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (35.2 mg, 183 μmol) and dA-CE phosphoramidite (157 mg, 183 μmol) prepared by dissolving in dehydrated acetonitrile (0.5 mL), and the mixture was stirred at room temperature for 1.5 hr to prepare a reaction solution.

Since Reference Example 12 includes steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, it is one embodiment of the present invention.

Example 16

(1) Synthesis of Phosphorothioate Dimer Wherein 3′-Hydroxy Group is Protected by Anchor

Under an argon atmosphere, in a 300 mL four-necked flask were placed HO-dT-SUC-TOB (619 mg, 500 mol) and Ac-TOB (619 mg, 648 μmol), and they were dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (288 mg, 1.50 mmol) and dT-CE phosphoramidite (1.12 g, 1.50 mmol) prepared by dissolving in dehydrated acetonitrile (3.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added methanol (608 μL, 15.0 mmol), and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (340 mg, 1.65 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.47 g, 10.0 mmol) and trifluoroacetic acid (345 μL, 4.50 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (654 μL, 4.95 mmol) was added, acetonitrile (150 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (762 mg, yield 95%).

(2) Synthesis of Phosphorothioate 9-Mer Wherein 3′-Hydroxy Group is Protected by Anchor

An operation similar to that in the above-mentioned (1) was further repeated 7 times to give a 9-mer (N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl 3,4,5-tris(octadecyloxy)benzyl succinate) (2.03 g).

(3) Synthesis of Phosphorothioate 10-Mer Wherein 3′-Hydroxy Group is Protected by Anchor and 5′-Hydroxy Group is Protected by DMTr Group

Under an argon atmosphere, in a 300 mL four-necked flask was placed the 9-mer (2.03 g) of the above-mentioned (2), and it was dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (228 mg, 1.19 mmol) and dG-CE phosphoramidite (1.00 g, 1.19 mmol) prepared by dissolving in dehydrated acetonitrile (2.0 mL), and the mixture was stirred at room temperature for 1.5 hr. DDTT (338 mg, 1.64 mmol) was added, and the mixture was stirred at room temperature for 30 min. After stirring, acetonitrile (150 mL) was added to the reaction solution and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a 10-mer (5′-O-(4,4′-dimethoxytrityl)-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl 3,4,5-tris(octadecyloxy)benzyl succinate) as a white solid (2.28 g).

(4) Deprotection

A mixture of the 10-mer (20 mg) obtained in the above-mentioned (3) and 30 wt % aqueous ammonia (5.00 mL) was placed in an autoclave, heated at 55° C. for 16 hr and cooled to room temperature. Insoluble material in the reaction solution was removed by a syringe filter (Whatman 25 mm GD/X PVDF 0.45 μm), and the filtrate was freeze-dried to give the object deoxyguanidyl-[3′→5′]-deoxyadenylyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxyadenylyl-[3′→5′ ]-deoxythymidinyl-[3′→5′]-deoxyguanidyl-[3′→5′ ]-deoxycytidinyl-[3′→5′ ]-deoxyadenylyl-[3′→5′]-deoxythymidinyl-[3′→5′ ]-deoxythymidine.

HPLC (WATERS XBridge™ C18 2.5 μm 4.6×75 mm column, flow rate 1.0 mL/min, 8 mM TEA+100 mM HFIP, MeOH, gradient: 0-10 min; 5 to 60%, λ=260 nm):Rt=6.61, 6.75 min (41.3+41.3%);

TOF/MS: 3471.489

Since Reference Example 16 includes steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, it is one embodiment of the present invention.

Example 17

(1) Synthesis of Phosphorothioate Dimer Wherein 3′-Hydroxy Group is Protected by Anchor

Under an argon atmosphere, in a 300 mL four-necked flask were placed HO-dT-SUC-TOB (619 mg, 500 μmol) and Ac-TOB (619 mg, 648 μmol), and they were dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (288 mg, 1.50 mmol) and dT-CE phosphoramidite (1.12 g, 1.50 mmol) prepared by dissolving in dehydrated acetonitrile (2.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added methanol (608 μL, 15.0 mmol), and the mixture was stirred at room temperature for 15 min. Thereafter, a separately prepared mixture of acetic acid (172 μL, 3.00 mmol) and 2,4,6-trimethylpyridine (595 μL, 4.50 mmol) was added, DDTT (339 mg, 1.65 mmol) was further added and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.47 g, 10.0 mmol) and trifluoroacetic acid (689 μL, 9.00 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. The mixture was neutralized with 2,4,6-trimethylpyridine (654 μL, 4.95 mmol), acetonitrile (150 mL) was added, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (765 mg, yield 95%).

(2) Synthesis of Phosphorothioate 9-Mer Wherein 3′-Hydroxy Group is Protected by Anchor

An operation similar to that in the above-mentioned (1) was further repeated 7 times to give a 9-mer (N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[0-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl 3,4,5-tris(octadecyloxy)benzyl succinate) (2.18 g).

(3) Synthesis of Phosphorothioate 10-Mer Wherein 3′-Hydroxy Group is Protected by Anchor and 5′-Hydroxy Group is Protected by DMTr Group

Under an argon atmosphere, in a 300 mL four-necked flask was placed the 9-mer (2.18 g) obtained in the above-mentioned (2), and it was dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (272 mg, 1.42 mmol) and dG-CE phosphoramidite (1.19 g, 1.42 mmol) prepared by dissolving in dehydrated acetonitrile (2.0 mL), and the mixture was stirred at room temperature for 1.5 hr. DDTT (320 mg, 1.56 mmol) was added, and the mixture was stirred at room temperature for 30 min. After stirring, acetonitrile (150 mL) was added to the reaction solution and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a 10-mer (5′-O-(4,4′-dimethoxytrityl)-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl3,4,5-tris(octadecyloxy)benzyl succinate) as a white solid (2.39 g).

(4) Deprotection

A mixture of 10-mer (20 mg) obtained in the above-mentioned (3) and 30 wt % aqueous ammonia (5.00 mL) was placed in an autoclave, heated at 55° C. for 16 hr and cooled to room temperature. Insoluble material in the reaction solution was removed by a syringe filter (Whatman 25 mm GD/X PVDF 0.45 μm), and the filtrate was freeze-dried to give the object deoxyguanidyl-[3′→5′]-deoxyadenylyl-[3′→5′]-deoxycytidinyl-[3′→5′ ]-deoxyadenylyl-[3′→5′ ]-deoxythymidinyl-[3′→5′ ]-deoxyguanidyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxyadenylyl-[3′→5′]-deoxythymidinyl-[3′→5′ ]-deoxythymidine.

HPLC (WATERS XBridge™ C18 2.5 μm 4.6×75 mm column, flow rate 1.0 mL/min, 8 mM TEA+100 mM HFIP, MeOH, gradient: 0-10 min; 5 to 60%, λ=260 nm):Rt=7.06, 7.19 min (41.1+42.3%);

TOF/MS: 3471.49

Since Reference Example 17 includes steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, it is one embodiment of the present invention.

Example 18

(1) Synthesis of Phosphorothioate Dimer Wherein 3′-Hydroxy Group is Protected by Anchor

Under an argon atmosphere, in a 300 mL four-necked flask were placed HO-dT-SUC-TOB (617.5 mg, 499 μmol) and Ac-TOB (619 mg, 648 μmol), and they were dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (288 mg, 1.50 mmol) and dT-CE phosphoramidite (1.12 g, 1.50 mmol) prepared by dissolving in dehydrated acetonitrile (3.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added a separately prepared mixture of acetic acid (857 μL, 15.0 mmol) and dehydrated pyridine (1.82 mL, 22.5 mmol), and the mixture was stirred at room temperature for 15 min. After stirring, DDTT (338 mg, 1.65 mmol) was added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.47 g, 10.0 mmol) and trifluoroacetic acid (345 μL, 4.50 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (653 μL, 4.94 mmol) was added, acetonitrile (150 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (753 mg, yield 94%).

(2) Synthesis of Phosphorothioate 9-Mer Wherein 3′-Hydroxy Group is Protected by Anchor

An operation similar to that in the above-mentioned (1) was further repeated 7 times to give a 9-mer (N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[0-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl 3,4,5-tris(octadecyloxy)benzyl succinate) (1.41 g).

(3) Synthesis of Phosphorothioate 10-Mer Wherein 3′-Hydroxy Group is Protected by Anchor and 5′-Hydroxy Group is Protected by DMTr Group

Under an argon atmosphere, in a 300 mL four-necked flask was placed the 9-mer (1.41 g) obtained in the above-mentioned (2), and it was dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (159 mg, 829 μmol) and dG-CE phosphoramidite (696 mg, 829 μmol) prepared by dissolving in dehydrated acetonitrile (2.0 mL), and the mixture was stirred at room temperature for 1.5 hr. DDTT (187 mg, 912 μmol) was added, and the mixture was stirred at room temperature for 30 min. After stirring, acetonitrile (150 mL) was added to the reaction solution and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a 10-mer (5′-O-(4,4′-dimethoxytrityl)-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[0-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl 3,4,5-tris(octadecyloxy)benzyl succinate) as a white solid (1.51 g).

(4) Deprotection

A mixture of the 10-mer (20 mg) obtained in the above-mentioned (3) and 30 wt % aqueous ammonia (5.00 mL) was placed in an autoclave, heated at 55° C. for 16 hr and cooled to room temperature. Insoluble material in the reaction solution was removed by a syringe filter (Whatman 25 mm GD/X PVDF 0.45 μm), and the filtrate was freeze-dried to give the object deoxyguanidyl-[3′→5′]-deoxyadenylyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxyadenylyl-[3′→5′]-deoxythymidinyl-[3′→5′]-deoxyguanidyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxyadenylyl-[3′→5′ ]-deoxythymidinyl-[3′→5′ ]-deoxythymidine.

HPLC (WATERS XBridge™ C18 2.5 μm 4.6×75 mm column, flow rate 1.0 mL/min, 8 mM TEA+100 mM HFIP, MeOH, gradient: 0-10 min; 5 to 60%, λ=260 nm):Rt=7.32, 7.47 min (40.3 area %+39.7 area %); TOF/MS: 3471.493

Since Reference Example 18 includes steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, it is one embodiment of the present invention.

Example 19

(1) Synthesis of Phosphorothioate Dimer Wherein 3′-Hydroxy Group is Protected by Anchor

Under an argon atmosphere, in a 300 mL four-necked flask were placed HO-dT-SUC-TOB (619 mg, 500 μmol) and Ac-TOB (619 mg, 648 μmol), and they were dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (288 mg, 1.50 mmol) and dT-CE phosphoramidite (1.12 g, 1.50 mmol) prepared by dissolving in dehydrated acetonitrile (2.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added a mixture of 2,2,2-trifluoroethanol (1.09 mL, 15.0 mmol), and the mixture was stirred at room temperature for 15 min. Thereafter, a separately prepared mixture of acetic acid (172 μL, 3.00 mmol) and 2,4,6-trimethylpyridine (595 μL, 4.50 mmol) was added, DDTT (339 mg, 1.65 mmol) was further added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.47 g, 10.0 mmol) and trifluoroacetic acid (345 μL, 4.50 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (653 μL, 4.94 mmol) was added, acetonitrile (150 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (788 mg, yield 98%).

(2) Synthesis of Phosphorothioate 9-Mer Wherein 3′-Hydroxy Group is Protected by Anchor

An operation similar to that in the above-mentioned (1) was further repeated 7 times to give a 9-mer (N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[0-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl 3,4,5-tris(octadecyloxy)benzyl succinate) (1.97 g).

(3) Synthesis of Phosphorothioate 10-Mer Wherein 3′-Hydroxy Group is Protected by Anchor and 5′-Hydroxy Group is Protected by DMTr Group

Under an argon atmosphere, in a 300 mL four-necked flask was placed the compound (1.97 g) of the above-mentioned (2), and it was dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (222 mg, 1.16 mmol) and dG-CE phosphoramidite (972 mg, 1.16 mmol) prepared by dissolving in dehydrated acetonitrile (2.0 mL), and the mixture was stirred at room temperature for 1.5 hr. DDTT (261 mg, 1.27 mmol) was added, and the mixture was stirred at room temperature for 30 min. After stirring, acetonitrile (150 mL) was added to the reaction solution and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a 10-mer (5′-O-(4,4′-dimethoxytrityl)-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[0-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl 3,4,5-tris(octadecyloxy)benzyl succinate) as a white solid (2.21 g).

(4) Deprotection

A mixture of the 10-mer (20 mg) obtained in the above-mentioned (3) and 30 wt % aqueous ammonia (5.00 mL) was placed in an autoclave, heated at 55° C. for 16 hr and cooled to room temperature. Insoluble material in the reaction solution was removed by a syringe filter (Whatman 25 mm GD/X PVDF 0.45 μm), and the filtrate was freeze-dried to give the object deoxyguanidyl-[3′→5′]-deoxyadenylyl-[3′→5′]-deoxycytidinyl-[3′→5′ ]-deoxyadenylyl-[3′→5′ ]-deoxythymidinyl-[3′→5′ ]-deoxyguanidyl-[3′→5′ ]-deoxycytidinyl-[3′→5′ ]-deoxyadenylyl-[3′→5′ ]-deoxythymidinyl-[3′→5′]-deoxythymidine.

HPLC (WATERS XBridge™ C18 2.5 μm 4.6×75 mm column, flow rate 1.0 mL/min, 8 mM TEA+100 mM HFIP, MeOH, gradient: 0-10 min; 5 to 60%, λ=260 nm):Rt=7.36, 7.48 min (48.8 area %+44.8 area %); TOF/MS: 3471.495

Since Reference Example 19 includes steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, it is one embodiment of the present invention.

Example 20

(1) Synthesis of Phosphorothioate Dimer Wherein 3′-Hydroxy Group is Protected by Anchor

Under an argon atmosphere, in a 300 mL four-necked flask were placed HO-dT-SUC-TOB (619 mg, 500 μmol) and Ac-TOB (619 mg, 648 μmol), and they were dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (288 mg, 1.50 mmol) and dT-CE phosphoramidite (1.12 g, 1.50 mmol) prepared by dissolving in dehydrated acetonitrile (2.0 mL), and the mixture was stirred at room temperature for 1.5 hr. To the reaction solution was added t-butanol (1.43 mL, 15.0 mmol), and the mixture was stirred at room temperature for 15 min. Thereafter, a separately prepared mixture of acetic acid (173 μL, 3.00 mmol) and 2,4,6-trimethylpyridine (594 μL, 4.50 mmol) was added, DDTT (339 mg, 1.65 mmol) was further added, and the mixture was stirred at room temperature for 1 hr. Thereafter, 5-methoxyindole (1.47 g, 10.0 mmol) and trifluoroacetic acid (689 L, 8.99 mmol) were successively added, and the mixture was stirred at room temperature for 1.5 hr. Furthermore, 2,4,6-trimethylpyridine (1.31 mL, 9.89 mmol) was added, acetonitrile (150 mL) was added to the reaction solution, and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give a dimer (deoxythymidine-3′-[O-(2-cyanoethyl)]phosphorothionyl deoxythymidin-3′-yl-[3,4,5-tris(octadecyloxy)benzyl]succinate) as a white solid (798 mg, yield 99%).

(2) Synthesis of Phosphorothioate 9-Mer Wherein 3′-Hydroxy Group is Protected by Anchor

An operation similar to that in the above-mentioned (1) was further repeated 7 times to give a 9-mer (N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[0-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl 3,4,5-tris(octadecyloxy)benzyl succinate) (1.93 g).

(3) Synthesis of Phosphorothioate 10-Mer Wherein 3′-Hydroxy Group is Protected by Anchor and 5′-Hydroxy Group is Protected by DMTr Group

Under an argon atmosphere, in a 300 mL four-necked flask was placed the 9-mer (1.93 g) obtained in the above-mentioned (2), and it was dissolved in dehydrated dichloromethane (25 mL). To the obtained solution was added a mixed solution of 5-benzylthio-1H-tetrazole (218 mg, 1.13 mmol) and dG-CE phosphoramidite (950 mg, 1.13 mmol) prepared by dissolving in dehydrated acetonitrile (2.0 mL), and the mixture was stirred at room temperature for 1.5 hr. DDTT (256 mg, 1.23 mmol) was added, and the mixture was stirred at room temperature for 30 min. After stirring, acetonitrile (150 mL) was added to the reaction solution and the precipitated solid was collected by suction filtration using a KIRIYAMA ROHTO and dried to give 10-mer (5′-O-(4,4′-dimethoxytrityl)-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N²-isobutyryl-deoxyguanosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-N⁴-(2-hexyl)decanoyl-deoxycytidine 3-[O-(2-cyanoethyl)]phosphorothionyl-N⁶-benzoyl-deoxyadenosine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidine 3′-[O-(2-cyanoethyl)]phosphorothionyl-deoxythymidin-3′-yl3,4,5-tris(octadecyloxy)benzyl succinate) as a white solid (2.14 g).

(4) Deprotection

A mixture of the 10-mer (20 mg) obtained in the above-mentioned (3) and 30 wt % aqueous ammonia (5.00 mL) was placed in an autoclave, heated at 55° C. for 16 hr and cooled to room temperature. Insoluble material in the reaction solution was removed by a syringe filter (Whatman 25 mm GD/X PVDF 0.45 μm), and the filtrate was freeze-dried to give the object deoxyguanidyl-[3′→5′]-deoxyadenylyl-[3′→5′]-deoxycytidinyl-[3′→5′′]-deoxyadenylyl-[3′→5′]-deoxythymidinyl-[3′→5′ ]-deoxyguanidyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxyadenylyl-[3′→5′]-deoxythymidinyl-[3′→5′]-deoxythymidine.

HPLC (WATERS XBridge™ C18 2.5 μm 4.6×75 mm column, flow rate 1.0 mL/min, 8 mM TEA+100 mM HFIP, MeOH, gradient: 0-10 min; 5 to 60%, λ=260 nm):Rt=6.95, 7.08 min (44.3 area %+42.7 area %); TOF/MS: 3471.493

Since Reference Example 20 includes steps (1), (3), (4) and (6) in the synthesis of the above-mentioned dimer, it is one embodiment of the present invention.

Example 1

A. Materials and Methods

1. Screening of Precipitation Recovery at 4° C.

To measure monoclonal antibody purity and yield, the monoclonal antibody-containing supernatant was diluted 1:5 with HQ-H₂O before 96% (v/v) ethanol was added to a final concentration of 33% (v/v). The solution was incubated overnight in the cold room (4° C.) under slight stirring (400 rpm). Aliquots of 10 ml were collected and filtered using either a planar filter (Millex GV, Sartorius, Bedford, USA) or a depth filter (GD/X, Whatman, Little Chalfont, UK). The planar filter uses a PVDF Durapore membrane with a cut-off of 0.22 μm while the depth filter uses four layers of different filtration material (layer 1+2: glass microfiber 10 to 1 μm; layer 3: glass microfiber prefilter 0.7 μm; layer 4: PDVDF 0.2 μm). Following a factorial design plan, the precipitates were then washed or not washed, dissolved immediately or after a delay in histidine buffer (20 mM histidine, 100 mM NaCl, pH 6.0), and the flow direction for dissolving of the precipitate was identical or opposing to the flow direction of precipitate collection. All dissolved precipitates were analyzed by analytical protein A chromatography. The purity was calculated as area of IgG peak divided by the sum of the areas of the flow-through and the IgG peak.

2. Evaluation of the Purification Strategies for Monoclonal Antibody-containing Supernatant A and B.

The respective supernatant was transferred into the reactor vessels of an Integrity 10 (Thermo Fisher Scientific, Rochford, UK) and equilibrated at 20° C. (˜room temperature). For all experiments, the starting volumes were selected so that the last precipitation step started with 10 ml of adjusted cell culture supernatant. Three different methodologies were used: (1) initial precipitation of the impurities such as DNA and protein impurities; (2) selective precipitation of IgG; and, (3) precipitation out of clarified cell culture supernatant or only diluted clarified cell culture supernatant. For the initial precipitation of impurities, two or three precipitation steps may be required. The first precipitation steps (1 and/or 2) aim at the removal of the impurities by precipitation. In this case, the precipitates are discarded and the remaining precipitation supernatant is used to proceed. The precipitate was only collected using depth filters (GD/X, Whatman) after the final step. It is then washed using a tempered ethanol solution of respective concentration and dissolved in histidine buffer (20 mM histidine, 100 mM NaCl, pH 6.0). For the selective precipitation of IgG, only one precipitation step may be required. The precipitate may then be collected, washed and dissolved. For the precipitation of IgG from the clarified cell culture supernatant and diluted (1:4 with HQ-H₂O) cell culture supernatant no adjustment besides temperature control is required. Again the precipitates are collected, washed and dissolved as described before. Adjustments were performed as follows: (a) adjustment of pH using HCl or NaOH; (b) adjustment of the conductivity using either saturated NaCl or CaCl₂; (c) control and if required further adjustment of pH; (d) simultaneous adjustment of temperature and ethanol addition. The ethanol was pre-cooled at −10° C. and added at 2-4 μl/min. After addition of ethanol the suspension was incubated for at least two hours. For each precipitation step, the required adjustments were performed in this given sequence. All handling was performed at room temperature unless otherwise indicated. Syringes and filters were also stored at room temperature unless otherwise indicated. The dissolved precipitates were analysed for IgG concentration (analytical protein A chromatography), protein impurity concentration (Bradford assay, excluding IgG) and DNA concentration (Picogreen assay). Additionally the dissolved precipitates were analysed by SEC. Screening of precipitation recovery at −10° C. Aliquots of 5 ml of cell culture supernatant of monoclonal antibody A were transferred into the reactor vessels of the Integrity 10 (Thermo Fisher Scientific) and tempered to 4° C. Upon addition of 96% (v/v) ethanol to a final concentration of 40% (v/v) over 30 minutes while the temperature was linearly decreased to −10° C. The suspension was then mixed at 400 rpm or 1000 rpm and after two hours collected by filtration using a depth filter (GD/X, Whatman). The filters and syringes used were tempered at room temperature, 4° C. in the laboratory fridge or ˜−20° C. in the laboratory freezer. The precipitates were washed using a tempered ethanol solution (−10° C., 40% (v/v) ethanol) and then dissolved in 10 ml histidine buffer (20 mM histidine, 100 mM NaCl, pH 6.0). All handling was performed at room temperature unless otherwise indicated. The precipitation supernatants, the washing solution and the dissolved precipitates were collected and analysed by analytical protein A chromatography. The respective purity was calculated again as area of IgG peak divided by the sum of the areas of the flow-through and the IgG peak. A detailed description of the experimental set-up is provided below. All experiments were performed in triplicates unless otherwise indicated.

3. Elution Profile of Dissolved Precipitate—Recovery and Purity

The same experimental set-up as for the screening of precipitation recovery at −10° C. was used for aliquots of 5 ml of monoclonal antibody-containing cell culture supernatant. The obtained precipitate was not washed. The dissolved precipitate was collected in aliquots of 1 ml and each aliquot analysed by analytical protein A chromatography. The respective purity was calculated again as area of IgG peak divided by the sum of the areas of the flow-through and the IgG peak.

4. Comparison of Different Alcohols for Precipitation

The alcohols used were methanol (Methanol LCMS Chromasolv, Fluka), ethanol (Ethanol 96% Emprove exp, Merck), isopropanol (Isorpopanol LiChrosolv, Merck), acetone (Aceton p.a., Merck) or 1,2-propandiol (Acros Organics). Aliquots of 10 ml of the cell culture supernatant C were transferred into the reactor vessels of an Integrity 10 (Thermo Fisher Scientific) and tempered to 4° C. During addition of the respective alcohol to a final concentration of either 10% (v/v) or 30% (v/v) over 1 h the temperature was linearly decreased to either 0° C. or −5° C. No precipitation was observed for acetone or 1,2-propandiol. The precipitates were collected by depth filtration (GD/X, Whatman) and dissolved without prior washing using 10 ml of histidine buffer (20 mM histidine, 100 mM NaCl, pH 6.0). All experiments were performed in triplicates. The concentration of IgG in the precipitation supernatant and the analysis of the composition of the dissolved precipitates were performed using SEC (Bio-SEC3, Agilent).

B. Isolation of Proteins

1. Influence of Filter Type and Conditions on Recovery and Purity after Precipitation at 4° C.

Additional parameters of precipitate collection and dissolution were also evaluated (Table 1). All experiments were performed with the clarified culture monoclonal antibody-containing supernatant (e.g., supernatant B). The “planar filter” corresponds to the Millex GV 0.22 μm. It uses a PVDF Durapore membrane with a cut-off of 0.22 nm and a filtration area of 3.9 cm². The “depth filter” corresponds to the GD/X 0.2 μm from Whatman. Its filtration material consists of 4 layers: layer 1+layer 2 consist of glass microfiber with a cut-off of 10 to 1 μm; layer 3 consists of a glass microfiber with a cut-off of 0.7 μm; layer 4 consists of a PVDF membrane with 0.2 μm cut-off. The advantage of the “depth filter” is the easier filterability of the precipitate particles (1-30 μm). Compared to a “planar filter” larger volumes of precipitation suspension can be filtrated before clogging of the membrane is observed. However, dissolution of the collected precipitate is not so trivial. The higher hold-up volume (1 ml or 250 μl with air purge) compared to the hold-up volume of the “planar filter” (100 μl) results in sample dilution and even incomplete recovery. Additionally other post-filtration steps such as washing or drying of the pellet might have an effect on purity and/or yield. Washing of the precipitate using a pure precipitant solution of correct concentration and temperature can help in increasing purity. However, washing puts additional stress on the particle and might result in non-specific wash-out, in particular if the particles are small. The impact of drying is of interest to evaluate if the precipitate could be stored in the syringe filter or if it should be dissolved and collected immediately.

The direction of the flow during dissolution of the pellet may also have an effect on the efficiency of dissolution. Therefore, the pellet was dissolved using the same flow direction as for filtration (“in-flow direction of dissolution”) or a flow direction contrary to the flow for filtration (“against-flow direction of dissolution”). Undissolved particles were retained in the filter using “in-flow direction of dissolution” but washed out and collected together with the dissolved particles using against-flow direction of dissolution. This might result in a decreased purity but also a higher yield. Using the factors given in Table 1 a four-factorial design plan, similar to the design plans described previously, was set up:

The clarified cell culture supernatant monoclonal was diluted 1:5 with RO-water and ethanol added to a final concentration of ˜33% (v/v) and incubated the suspension o/n at 4° C. under mild stirring (400 rpm). The taken samples were then processed as given in Table 2. Precipitation yield was low (40-50%) due to the low antibody concentration in the starting material.

The IgG concentration of all samples was analysed by analytical protein A chromatography. Purity was calculated using the relation of the area of the flowthrough peak to the area of the IgG peak as determined by analytical protein A chromatography. This served as a sufficient and fast approximation when only differences within a sample set need to be evaluated. The effect on yield, mass balance and purity were calculated to isolate the main factors and interacting factors with significant impact (Table 3). As shown therein, factor D as well as the interacting factors AB and CD had significant impact on yield and mass balance, while for purity only factor C had a significant impact.

The plots of the main factors and selected interacting factors are provided in FIGS. 2 and 3. FIG. 2 illustrates the main and the interacting plots with the mass balance as the response function. On average, the mass balance was about 86%±6%. Considering the rather small scale (10 ml) of the reactions, this is not unexpected. Filter type and flow direction also displayed a slight impact. This correlates well with the minute percent contribution observed (Table 3). Washing of the precipitate had no effect on the mass balance. The most significant impact among the main factors is the factor of “drying of the precipitate in the filter” (“D”). It was determined that storage of the precipitate in the filter over longer periods (=“drying”) may not be advisable as it may lead to a decrease in yield (only 83% as compared to 89%).

The selection of the flow direction for dissolution (B) of the pellet should be selected depending on the filter (A) used (FIG. 2). While the planar filter resulted in only slightly better results when operated against flow, the depth filter is typically only applicable if the pellet is dissolved in flow. This was probably due to the difference in their structure. The planar filter is a symmetric filter while the depth filter is an asymmetric filter. Also, the evaluation of the interacting factors showed that drying of the precipitate when not washed leads to a significant decrease in mass balance (FIG. 2) while for the washed pellet no influence of drying was found. The mass balance for the unwashed pellet is typically higher than that of the washed pellet. As the mass balance and the yield are correlated, the same observations as for the mass balance are also true for the yield (FIG. 3). As mentioned previously, the yield may be low (40-50%) due to the low concentration of the antibody in the diluted cell culture supernatant. The washed, dissolved precipitate has a significantly higher purity (80%±2%) than the unwashed, dissolved precipitate (65%±3%).

Based on this data, a depth filter was tested for filtration as it allows a fast collection of the precipitate. The dilution during dissolution of the pellet due to the washing-out effect of the depth filter is at this scale acceptable. As given before, the depth filter is an asymmetric filter and the same flow direction need to be used throughout the experiment. Despite the loss of material, the effect of washing on purity is significant (˜20% higher purity). A delay in dissolution of the precipitate results in material loss and will therefore also be avoided. Any precipitate recovered should typically be immediately dissolved after washing.

2. Comparison of Different Alcohols for Precipitation

The use of various alcohols to precipitate antibodies from cell culture supernatants was also tested. A list of alcohols tested in these experiments are shown in Table 4.

FIG. 28 illustrates the solubility curves of monoclonal antibody following precipitation using methanol, ethanol or isopropanol at 0° C. or −5° C. At −5° C., the solubility of monoclonal antibody A was very similar to precipitation at 0° C., although differences in slope and intercept were observed. Three data points are presented for each alcohol at 0° C. The data suggest that the behavior of the different alcohols is, at least at low temperatures, similar and that ethanol could be replaced by either methanol or isopropanol.

3. Optimization of CaCl₂ Precipitation

For the monoclonal antibody-containing cell culture supernatants A and C, CaCl₂ precipitation with varying concentrations of CaCl₂ (5-100 mM) and a subsequent ethanol precipitation (25% (v/v), −10° C.) were tested. As supernatant A has a higher phosphate concentration (˜3.1 mM) than supernatant C (0.2 mM), CaCl₂ precipitation was expected to be more pronounced for supernatant A. CaCl₂ precipitation was performed at room temperature and pH 8.5 (adjusted using NaOH). The CaCl₂ precipitate was removed from the supernatant by centrifugation (4000 g, 15 min) and the pH of the supernatant brought to pH 6.5 using HCl. As shown in FIG. 29, the impurity (HCP) concentration of supernatant A is lower than for supernatant C, which is in agreement with the initial HCP concentration in the supernatants (supernatant A: 54212 ppm; supernatant C: 180099 ppm). FIG. 29 also shows that the CaCl₂ concentration in the first step has a significant influence on the final HCP level for supernatant A and a marginal influence for supernatant C. The IgG concentration is almost independent of CaCl₂ concentration used in the first step for both of the tested supernatants. A larger standard deviation can be found at lower CaCl₂ concentrations for supernatant A.

Higher CaCl₂ concentrations were also test (500 mM; FIG. 30). As shown in FIG. 2-11B, impurity (HCP) concentration decreases further for supernatant A to 250 mM CaCl₂ while it remains almost constant for supernatant C. The increase of HCP concentration for supernatant A at 500 mM CaCl₂ is due to the significant loss of IgG. The optimum CaCl₂ concentration for HCP removal was therefore determined to be about 150-250 mM.

As shown below, phosphate concentration may improve isolation of monoclonal antibodies from cell culture supernatant. The phosphate concentration of supernatant A is higher than for supernatant C, which could result in a stronger influence of CaCl₂ on HCP removal. The isolation of monoclonal antibody from supernatant C by CaCl₂ precipitation and cold ethanol precipitation (CEP) in the presence of elevated phosphate concentrations was therefore tested. Phosphate was added to a final concentration of 4.0 mM to the supernatant, and then CaCl₂ precipitation and CEP were carried out. The results were compared with supernatant containing the “original” phosphate concentration of about 0.2 mM phosphate. As shown in FIG. 31, the addition of phosphate did not significantly change the results.

To determine if the effect on HCP reduction is due to CaCl₂-phosphate precipitation or the high conductivity caused by CaCl₂, the following purification strategies were compared for supernatant A. As the supernatant contains the IgG in a rather complex matrix, the first CaCl₂ precipitation and CEP aimed to obtain a relatively pure IgG in a defined matrix (20 mM histidine, 150 mM NaCl, pH 6.0). Therefore, impurities were first removed by addition of CaCl₂ to a final concentration of 250 mM. The supernatant was subsequently subjected to CEP using 25% (v/v) ethanol (final concentration) (−10° C., 2 h). The dissolved precipitate was then divided into three parts. To one part, first 300 mM Na₂HPO₄ was added to a final concentration of 4 mM phosphate followed by 4000 mM CaCl₂ to a final concentration of 250 mM CaCl₂. The other two parts were mixed with CaCl₂ to a final concentration of 250 mM or NaCl to a final concentration of 500 mM. NaCl required a higher concentration to obtain a similar conductivity as for 250 mM CaCl₂ (˜50 mS/cm). As shown in FIG. 32, reduction of impurities is more effective when repeating CaCl₂ precipitation and CEP (FIG. 32(A)) than by increasing the conductivity of the second CEP step (FIG. 32(C,D)). And when (only) salt is added to increase the conductivity before the second CEP step, NaCl may be more efficient (˜2500 ppm) than CaCl₂ (˜4000 ppm). While it is possible to increase conductivity to improve HCP reduction during CEP, it may be more advisable to perform a second CaCl₂ precipitation.

4. Isolation of Monoclonal Antibodies from Monoclonal Antibody-containing Cell Culture “Supernatant A”.

Six different purification strategies were compared (Table 5) to determine the optimal conditions for purification of monoclonal antibody from monoclonal antibody-containing supernatant A (“supernatant A” containing monoclonal antibody having a pI˜9.2) from cell-free culture supernatant.

Strategy “D” uses NaCl instead of CaCl₂ and aims at keeping the impurities soluble while the antibody is selectively precipitated. The two remaining precipitation strategies, “E” and “F”, are used as reference methods where the clarified cell culture supernatant is used without further adjustments except a 1:4 dilution in case of strategy “F”. For simplicity we analysed only the dissolved precipitates (Table 6).

Comparison of the analytical SEC chromatograms of the different purification strategies are given in FIG. 5. As shown therein, strategies “A”, “B” and “C” result in higher purity than strategies “D”, “E”, and “F”. These strategies provided for the removal of high molecular weight impurities, presumably consisting mainly of DNA. Low yields may be improved using depth filters. Strategy “A” may be replaced by strategy “B”, with no significant loss in purity or recovery.

5. Isolation of Monoclonal Antibodies from Monoclonal Antibody-containing Cell Culture “Supernatant B”.

Five different purification strategies were evaluated to determine the optimal conditions for purifying monoclonal antibody from monoclonal antibody-containing cell culture supernatant B (“supernatant B” containing the same antibody as A, but produced using a different cell line; pI˜9.2), as summarized in Table 7.

A modified Strategy C was also used to isolation monoclonal antibody from supernatant B (Tables 9 and 10). It was surprisingly found that the solubility of the resulting monoclonal antibody was different from that isolated from supernatant A using the same procedures (e.g., as the monoclonal antibodies in A and B differ only in that each is prepared using a different cell line).

In Table 2-9, purity is calculated as IgG content by protein A chromatography and Bradford assay. As shown in Table 2-9, strategy C provided a monoclonal antibody B product with high solubility. Strategy G provided significant reduction in DNA but also a significant loss of IgG. The overall yield was between 22 and 99%. Modification of the culture supernatant (e.g, Strategies J and K) provided higher yields. SEC chromatograms are provided in FIG. 8. This data demonstrates that the highest purity and yield result from Strategy C.

6. Isolation of Monoclonal Antibodies from Monoclonal Antibody-containing Cell Culture “Supernatant C”.

Five different purification strategies were evaluated to determine the optimal conditions for purifying monoclonal antibody from monoclonal antibody-containing cell culture supernatant C (“supernatant C” containing monoclonal antibody having a pI˜6.8), as summarized in Table 12.

The modified Strategy C (conductivity of 29 mS/cm) was also tested on supernatant C (Table 14). It was not expected that Strategy C would provide a sufficiently pure “C” preparation considering the predicted low solubility thereof. One would have predicted that DNA would co-precipitate and the monoclonal antibody yield would be low. Table 15 provides an overview of the composition of the various dissolved pellets. As shown therein, despite the addition of phosphate prior to calcium chloride, DNA removal was not complete.

Based on the results presented in Table 2-13 and FIGS. 2-7B, strategies C and P (1:4 dilution of supernatant in H₂O followed by the addition of 15% (v/v) ethanol) are optimal. According to the SEC data, Strategy M may provide the highest purity but the yield provided by Strategy C is significantly higher.

7. Further Modified Strategy C (Addition of Phosphate)

Supernatants A, B and C were separately evaluated using a modified strategy involving the addition of phosphate. The method included an initial CaCl₂ precipitation followed by a first ethanol precipitation. The pellet from the first ethanol precipitation was then dissolved in 20 mM histidine, 100 mM NaCl, pH 6.5. Sodium phosphate was then added to a final concentration of 4 mM to assist in the second CaCl₂ precipitation. All experiments were performed in quadruplets and the precipitates recovered by centrifugation. This purification procedure is illustrated in FIG. 10.

Table 16 provides the mass balance for the small-scale purification of IgG from supernatant B using this further modified strategy C. The required purity (100-fold HCP reduction) is achieved after the second ethanol precipitation. However, additional precipitation steps resulted in a significant loss of IgG over the course of the purification. Each step provided a yield of 64-77%, resulting in an overall yield of 26%. This is evident from the SEC chromatograms (FIG. 11; note: “C3” results not represented in Table 16). SEC chromatograms for the ethanol and CaCl₂ precipitates were compared. As shown in FIG. 12, the impurities eluting at about 12 minutes in the precipitates of step 1 are removed in the precipitates of step 2 (except C3).

This process was also used to purify antibody from supernatant C. The results are summarized in Table 17. The overall HCP reduction was about 80-fold. Recovery data is presented in FIG. 13. As shown in FIG. 14, the impurities eluting at about 12 minutes (e.g., typically between about ten to 12 minutes) in the precipitates of step 1 are removed in the precipitates of step 2 (except C3).

Mass balance data resulting from purification of monoclonal antibody from supernatant A, B, C, or D using modified strategy C (FIG. 17) is presented in Tables 18, 19, 20, and 21:

8. Large-scale Isolation Procedures

In some smaller scale embodiments, the use of 50 mM CaCl₂ for the CaCl₂ precipitation step provided a yield of only about 18% and a HCP reduction of only about 43-fold. At higher CaCl₂ concentrations (added up to 29 mS/cm), significantly higher HCP reduction (159-fold) and yield (26%) were observed. In the large-scale isolation procedures described here, 250 mM CaCl₂ (˜50 mS/cm) was selected as providing high HCP reduction and good yield. The CEP step was maintained at −10° C., 2 h, and 25% (v/v) ethanol. The CaCl₂ precipitation was performed at room temperature using a beaker and a magnetic stirrer. For adjustment of pH, CaCl₂ and phosphate concentrations a 25% HCl, 10.0 M NaOH, 5.0 M CaCl₂ and a 0.3 M N₂HPO₄ solution were used. For CEP, the solutions were transferred to an EasyMax reactor. FIG. 18 describes the temperature profile, the IR probe response, and the ethanol concentration of the first (A) and second (B) CEP precipitation in the EasyMax reactor. Ethanol was added over 4 h while simultaneously decreasing the temperature from 4° C. to −10° C. where it was then kept for 2 h to reach precipitation equilibrium. As shown in FIG. 18, the first and second CEP precipitation begins at about three hours (−6° C., >20% (v/v) ethanol). The turbidity reaches quickly its equilibrium, suggesting that the precipitation is a rather fast reaction. During the purification, samples were drawn after each precipitation step and analysed by analytical protein A chromatography, SEC, and CHO HCP ELISA (Table 22). As shown therein, the yield increased to 64% and a high HCP reduction (83-fold) was observed. As shown in FIG. 19, the final dissolved precipitate is of high purity and the IgG obtained is highly monomeric.

In another embodiment, the precipitate was isolated after the second CaCl₂ precipitation using a syringe filter (Whatman, 0.2 μm) instead of centrifugation. The samples were drawn after each precipitation step and analysed by analytical protein A chromatography, SEC and CHO HCP ELISA (Table 23). As shown therein, yield increased to 71% and HCP reduction remained high (128-fold). However, due to the longer exposure to room temperature, the precipitate collected by filtration (2^nd CEP) partially dissolved and the soluble IgG fraction found in the supernatant was higher (167 mg/l) than expected (64 mg/l) resulting in a lower step yield for the 2^nd CEP (84%) than for the 1^st CEP (94%). Assuming a similar concentration of the soluble fraction of the 2^nd CEP as for the 1^st CEP, the step yield as well as the overall yield would have been higher (step yield ˜95%; overall yield ˜80.5%). As seen in FIG. 20, the final dissolved precipitate is of high purity and the IgG obtained is highly monomeric. This procedure was repeated with another supernatant sample and similar results were observed (91-fold HCP reduction; Table 24, FIG. 21). As shown in FIG. 21, the resulting precipitate is of high purity and the IgG obtained is highly monomeric.

This procedure was also carried out using supernatant C (Table 25). As shown therein, the yield increased to 91% but the HCP reduction was only 23-fold. As can also be seen in FIG. 22, the final dissolved precipitate is of high purity but contained a larger amount of IgG aggregates.

This procedure was also carried out on Supernatant D. The precipitation was performed as previously and the samples drawn after each precipitation step analysed by analytical protein A chromatography, SEC and CHO HCP ELISA (Table 26). The yield increased to 76% but the HCP reduction was only 48-fold. As shown in FIG. 23, the final dissolved precipitate is high purity IgG.

9. Modified Method Including a Single CaCl₂ Precipitation

It was concluded that the first ethanol precipitation step had the most significant impact on impurity (HPC) removal. In some cases, the first CaCl₂ precipitation was observed to only slightly influence impurity removal and result in significant IgG loss (e.g., 25%). A modified strategy in which the first CaCl₂ precipitation step was removed while the first and second ethanol precipitation steps and the second CaCl₂ precipitation maintained was therefore developed.

To optimize the first ethanol precipitation step, crude supernatant A was mixed with ethanol to a final ethanol concentration of 10%, 15%, 20%, 25%, or 30%. The solubility of IgG in each preparation is shown in FIG. 15(A). As shown therein, IgG is not precipitated in the presence of 10% ethanol but begins to precipitate with 15% ethanol and reaches equilibrium at 25% ethanol. FIG. 15(B) shows no difference between IgG and DNA for 20%, 25%, and 30% but a significant difference in HCP (30% ethanol significantly higher). A final concentration of 25% was therefore selected as optimal for the first ethanol precipitation step.

The effect of washing after the first ethanol precipitation step was also examined. The precipitate from ethanol precipitation was collected by centrifugation and immediately dissolved in histidine buffer (20 mM histidine, 100 mM NaCl, pH 6.0) or washed three times with 25% ethanol before being dissolved in the histidine buffer. As shown in Table 27, washing resulted in a significantly reduced yield (60%) without a significant improvement in HCP reduction (from 2.9-fold without wash to 3.6-fold with wash).

As mentioned above, the CaCl₂ precipitation step preceding the first ethanol precipitation step was excluded. However, the CaCl₂ precipitation following the first ethanol precipitation was retained and optimized as described herein. A variety of conditions were evaluated as shown in Table 28:

The second ethanol precipitation step, which followed the first ethanol precipitation step (−10° C., 25% ethanol) and CaCl₂ precipitation steps (20° C., pH 8.5, 4 mM phosphate, 50 mM CaCl₂) described above, was also optimized. Ethanol was added to a final concentration of 10%, 15%, 20%, 25%, or 30%. After incubation for two hours at −10° C., the precipitate was collected by centrifugation and dissolved in histidine buffer (20 mM histidine, 100 mM NaCl, pH 6.0). The soluble and precipitated fractions were then analyzed, as presented in FIG. 2-7I. As shown therein, no significant difference between these conditions was observed. Twenty-five percent ethanol was therefore selected as optimal.

The HCP concentration in the dissolved pellet resulting from this modified method was significantly higher than that produced by the original method (e.g., FIGS. 1, 10). While the presence of CaCl₂ appeared not to influence HCP concentration, the presence of CaCl₂ improved HCP removal in the subsequent ethanol precipitation (Table 29).

As shown herein, variations in the ethanol precipitation steps did not typically provide improvements in HCP content or yield although 25% ethanol and −10° C. were determined to be optimal. Washing had a beneficial effect on purity but also resulted in lower yield. Typically, then, the washing steps may not be necessary. And, while the CaCl₂ step may have little effect on HCP removal, it was found to be an important (e.g., critical) precursor step to the ethanol precipitation step. The use of 50 mM CaCl₂ was also found to be optimal (e.g., as compared to 120-150 mM CaCl₂ in the original method). While yield was typically comparable, lower concentrations of CaCl₂ typically led to greater HCP reduction.

10. Influence of Filtration Conditions on Recovery and Purity after Precipitation at −10° C.

The yields observed for the purification strategies evaluated were sometimes significantly lower than expected. Comparing the experimental set-up of the filter screening to the conditions of the real purifications, significant differences resulted from changes in stirrer speed and temperature. The filter screening was performed in the cold room at 4° C. in a 400 ml vessel with a stirrer speed of 400 rpm. The purifications were performed at ambient temperature. Only the precipitate and the washing solution were at the required temperature (e.g., −10° C.). The stirrer speed may be significantly higher (1000 rpm for a volume of 10 ml). Three parameters were selected for screening: stirrer speed, washing conditions, and the storage temperature of syringes and filters. As the lab reactor used for the precipitation method screening is at ambient temperature, all handling had to be performed at that temperature. In order to evaluate if these parameters resulted in the low recovery, a second filter screening was performed, as summarized in Table 30.

The concentration of IgG in the precipitation supernatant, the washing fraction, and the dissolved precipitate was determined by analytical protein A chromatography. The IgG present in the precipitation supernatant and the washing fraction was below the lower limit of quantification of the method used. It was therefore concluded that the yield is only negligible different from the mass balance. For a first estimation of the impact of the three main factors the average yield was calculated (FIG. 24). The stirrer speed had no significant impact on the average yield (˜70%) but the standard deviation of the yield is for a stirrer speed of 400 rpm significantly higher than for 1000 rpm (likely due to differences in the particle size and density). The energy input, in our case due to the mechanical stirring, is an important parameter for the particle size and density: In general, particle size decreases and particle density increases with energy input. Also, a certain energy input is required to form homogenous particles. It was assumed that the particles obtained at a stirrer speed of 400 rpm are large, not that dense and of inhomogeneous size. Therefore, significant differences in yield can be observed. On the other hand, the particles obtained at a stirrer speed of 1000 rpm are smaller but denser and should be of a homogenous size distribution. It was determined that washing results in a loss of about 10% IgG as compared to no washing. The standard deviation is similar for either case. It was observed that storage at room temperature results in a lower yield (60%) as compared to storage at 4° C. (80%) or −20° C. (70%).

A more detailed analysis of the influence of stirrer speed and washing is provided in FIG. 25. The precipitates obtained at a stirrer speed of 400 rpm show a similar average yield when washed or when not washed. Only the standard deviation of the samples which were washed is larger than the samples which were not washed. In case of the higher stirrer speed, the standard deviation of the washed precipitates and the unwashed precipitates is similar but the average of the washed precipitates is lower. It was assumed that the smaller particles formed at 1000 rpm were more easily dissolved than the particles at 400 rpm. Therefore, washing of particles formed at 1000 rpm has more impact. In contrast the particles formed at 400 rpm are not homogenous, therefore the large standard deviation is observed in case of the washed precipitate. Depending on the sample a part of the precipitate can be easily dissolved during washing. A stirrer speed of about 600 rpm was found optimal in these experiments. Lower speed may lead to non-homogeneous particle size and varying yield. A higher stirring speed will result in small particles which can easily dissolve during washing. The syringes and filters may also be stored at 4° C., which should also significantly reduce loss of precipitate during collection. With these conditions, the evaluation of the precipitation strategies will be repeated. After up-scaling, collection may be performed by centrifugation.

11. Elution Profile of Dissolved Precipitate—Recovery and Purity

The wash-out effect of the depth filters used for recovery collection and dissolution was also studied. Monoclonal antibody A supernatant was precipitated using 40% (v/v) ethanol and dissolved with aliquots of 1 ml histidine buffer (20 mM histidine, 100 mM NaCl, pH 6.0). The effluent was collected and analyzed by analytical protein A chromatography. The purity of the effluent fractions was also determined by comparison of the area of the IgG peak and the total peak areas. This method was found sufficient for monitoring the purity trend of the effluent fraction. In FIG. 26, the concentration and purity of the effluent fraction is depicted. As shown therein, the first fraction is of low purity due to the precipitation supernatant still present in the filter. Purity increases in the second fraction and also a concentration spike is observed. In the following fractions, no trend regarding purity was observed (although it varied between 40-50%). This is not too high and may be ascribed to unspecific precipitation conditions and the rather simple method of purity determination. The concentration was stable in the first five fractions before an exponential wash-out was observed. From the calculated yield of each fraction, and the cumulative yield, a dissolution volume of approximately 10 ml was estimated to be sufficient for obtaining a suitable, though not complete, recovery of precipitated IgG. Precipitation collection by filtration is typically most useful at smaller scale (<20 ml). With up-scaling, centrifugation may be used as it allows faster and easier handling of larger volumes.

C. Conclusions

The comparison of the purification strategies for the monoclonal antibody-containing supernatants A, B and C surprisingly showed that the purification strategies presented here may be used to selectively separate IgG from protein impurities and DNA directly from cell culture supernatant with exceptional purity and yield. For example, for supernatant A, purification strategies B and C may be optimal. For supernatant B, strategies C and K may be optimal. And for supernatant C, purification strategies C, M and O may be optimal. Purification strategy C provides good results for all supernatants and may therefore be the most generally optimal strategy. In addition, in some instances, it may be optimal to perform two precipitations to obtain a satisfactory purity (HCP). Modified strategy C (e.g., FIG. 17) may also be optimal and generally applicable to the isolation of many different monoclonal antibodies (e.g., of different pIs) from different sources (e.g., supernatant of various types of cells). Decreased yield may result from the method of precipitate recovery (e.g., washing and dissolution). In particular, reducing the temperature difference between the precipitate and the syringes and filters by using tempered syringes and filters (4° C.) provides increased yield. Comparison of the different alcohols for their effect on antibody precipitation led to the conclusion that ethanol could be replaced by methanol or isopropanol with no obvious change in the precipitation behaviour of the antibody.

While the present invention has been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the invention as claimed.

Example 1

DNA Sequencing of the BASB041 Gene from Two N. meningitidis Strains.

A: BASB041 in N. meningitidis serogroup B strain ATCC13090.

The BASB041 gene of N. meningitidis strain ATCC 13090 is shown in SEQ ID NO:1 The translation of the BASB041 polynucleotide sequence, shown in SEQ ID NO:2, shows significant similarity (28% identity in a 130 amino acids overlap) to a hypothetical protein of Aquifex aeolicus. The BASB041 polypeptide contains a signal sequence characteristic of a lipoprotein, and could thus be inserted into the outer membrane of the bacterium.

The sequence of the BASB041 gene was further confirmed as follows. For this purpose, genomic DNA was extracted from 10¹⁰ cells of the N. meningitidis cells (strain ATCC 13090) using the QIAGEN genomic DNA extraction kit (Qiagen Gmbh), and 1 μg of this material was submitted to Polymerase Chain Reaction DNA amplification using primers lip5-01 (5′-AAT GAA AAC CGT TTC CAC CGC-3′) [SEQ ID NO:7] and lip5-02 (5′-TCA TTT CTC CTT AAC GGT-3′) [SEQ ID NO:8]. This PCR product was gel-purified and subjected to DNA sequencing using the Big Dye Cycle Sequencing kit (Perkin-Elmer) and an ABI 373A/PRISM DNA sequencer. DNA sequencing was performed on both strands with a redundancy of 2 and the full-length sequence was assembled using the SeqMan program from the DNASTAR Lasergene software package. The resulting DNA sequence and deduced polypeptide sequence are shown as SEQ ID NO:3 and SEQ ID NO:4 respectively. It should be noticed that the DNA sequence of SEQ ID NO:3 has an additional nucleotide at position 616 relative to SEQ ID NO:1.

B: BASB041 in N. meningitidis Serogroup B Strain H44/76.

The sequence of the BASB041 gene was also determined in another N. meningitidis serogroup B strain, the strain H44/76. For this purpose, genomic DNA was extracted from the N. meningitidis strain H44/76 using the experimental conditions presented in the previous paragraph. This material (1 kg) was then submitted to Polymerase Chain Reaction DNA amplification using primers LipS-01 and Lip5-02 specific for the BASB041 gene. The PCR amplicon was then submitted to DNA sequencing using the Big Dyes kit (Applied biosystems) and analyzed on a ABI 373/A DNA sequencer in the conditions described by the supplier. As a result, the polynucleotide and deduced polypeptide sequences, referred to as SEQ ID NO:5 and SEQ ID NO:6 respectively, were obtained. It should be noticed that the DNA sequence of SEQ ID NO:5 has an additional nucleotide at position 616 relative to SEQ ID NO:1. Using the MegAlign program in the DNASTAR Lasergene package, an alignment of the polynucleotide sequences of SEQ ID NO:1, 3 and 5 was performed, and is displayed in FIG. 1; a pairwise comparison of identities is summarized in Table 1, showing that the three BASB041 polynucleotide gene sequences are all similar at identity level greater than 99.0%. Using the same MegAlign program, an alignment of the polypeptide sequences of SEQ ID NO:2, 4 and 6 was performed, and is displayed in FIG. 2. A pairwise comparison of identities is summarized in Table 2, showing that SEQ ID NO:4 and 6 are 100% identical; their dissimilarity with SEQ ID NO:2 is entirely contained in the last 18 residues, and is due to the missing nucleotide in SEQ ID NO:1 relative to SEQ ID NO:3 and 5.

Taken together, these data inditate strong sequence conservation of the BASB041 gene among the two N. meningitidis serogroup B strains.

The NdeI and SalI restriction sites engineered into the forward Lip5-Fm/p (5′-AGG CAG AGG CAT ATG AAA ACC GTT TCC ACC GCC GTT GTC CTT GC-3′) ([SEQ ID NO:9]) and reverse Lip5-RCf/p (5′-AGG CAG AGG GTC GAC TTT CTC CTT AAC GGT TGG GTT GCC ATG CGC-3′)([SEQ ID NO:10]) amplification primers, respectively, permitted directional cloning of a BASB041 PCR product into the commercially available E. coli expression plasmid pET24b (Novagen, USA, kanamycin resistant) such that a mature BASB041 protein could be expressed as a fusion protein containing a (His)6 affinity chromatography tag at the C-terminus. The BASB041 PCR product was purified from the amplification reaction using silica gel-based spin columns (QiaGen) according to the manufacturers instructions. To produce the required NdeI and Sail termini necessary for cloning, purified PCR product was sequentially digested to completion with NdeI and Sail restriction enzymes as recommended by the manufacturer (Life Technologies). Following the first restriction digestion, the PCR product was purified via spin column as above to remove salts and eluted in sterile water prior to the second enzyme digestion. The digested DNA fragment was again purified using silica gel-based spin columns prior to ligation with the pET24b plasmid.

B: Production of Expression Vector.

To prepare the expression plasmid pET24b for ligation, it was similarly digested to completion with both NdeI and SalI and then treated with calf intestinal phosphatase (CIP, ˜0.02 units/pmole of 5′ end, Life Technologies) as directed by the manufacturer to prevent self-ligation. An approximately 5-fold molar excess of the digested fragment to the prepared vector was used to program the ligation reaction. A standard ˜20 μl ligation reaction (˜16° C., ˜16 hours), using methods well known in the art, was performed using T4 DNA ligase (˜2.0 units/reaction, Life Technologies). An aliquot of the ligation (˜5 μl) was used to transform electro-competent BL21 DE3 cells according to methods well known in the art. Following a ˜2-3 hour outgrowth period at 37° C. in ˜1.0 ml of LB broth, transformed cells were plated on LB agar plates containing kanamycin (50 μg/ml. The antibiotic was included in the selection media to ensure that all transformed cells carried the pET24b plasmid (KnR). Plates were incubated overnight at 37° C. for ˜16 hours. Individual KnR colonies were picked with sterile toothpicks and used to “patch” inoculate fresh LB KnR plates as well as a ˜1.0 ml LB KnR broth culture. Both the patch plates and the broth culture were incubated overnight at 37° C. in either a standard incubator (plates) or a shaking water bath.

A whole cell-based PCR analysis was employed to verify that transformants contained the BASB041 DNA insert. Here, the ˜1.0 ml overnight LB Kn broth culture was transferred to a 1.5 ml polypropylene tube and the cells collected by centrifugation in a Beckman microcentrifuge (˜3 min., room temperature, ˜12,000×g). The cell pellet was suspended in ˜200 μl of sterile water and a ˜10 μl aliquot used to program a ˜50 μl final volume PCR reaction containing both BASB041 forward and reverse amplification primers. Final concentrations of the PCR reaction components were essentially the same as those specified in example 2 except ˜5.0 units of Taq polymerase was used. The initial 95° C. denaturation step was increased to 3 minutes to ensure thermal disruption of the bacterial cells and liberation of plasmid DNA. An ABI Model 9700 thermal cycler and a 32 cycle, three-step thermal amplification profile, i.e. 95° C., 45 sec; 55-58° C., 45 sec, 72° C., 1 min., were used to amplify the BASB041 PCR fragment from the lysed transformant samples. Following thermal amplification, a ˜20 μl aliquot of the reaction was analyzed by agarose gel electrophoresis (0.8% agarose in a Tris-acetate-EDTA (TAE) buffer). DNA fragments were visualized by UV illumination after gel electrophoresis and ethidium bromide staining. A DNA molecular size standard (1 Kb ladder, Life Technologies) was electrophoresed in parallel with the test samples and was used to estimate the size of the PCR products. Transformants that produced the expected PCR product were identified as strains containing a BASB041 expression construct. Expression plasmid containing strains were then analyzed for the inducible expression of recombinant BASB041.

C: Expression Analysis of PCR-Positive Transformants.

For each PCR-positive transformant identified above, ˜5.0 ml of LB broth containing kanamycin (50 μg/ml) was inoculated with cells from the patch plate and grown overnight at 37° C. with shaking (˜250 rpm). An aliquot of the overnight seed culture (˜1.0 ml) was inoculated into a 125 ml erlenmeyer flask containing ˜25 ml of LB Kn broth and grown at 37° C. with shaking (˜250 rpm) until the culture turbidity reached O.D.600 of ˜0.5, i.e. mid-log phase (usually about 1.5-2.0 hours). At this time approximately half of the culture (˜12.5 ml) was transferred to a second 125 ml flask and expression of recombinant BASB041 protein induced by the addition of IPTG (1.0 M stock prepared in sterile water, Sigma) to a final concentration of 1.0 mM. Incubation of both the IPTG-induced and non-induced cultures continued for an additional ˜4 hours at 37° C. with shaking. Samples (˜1.0 ml) of both induced and non-induced cultures were removed after the induction period and the cells collected by centrifugation in a microcentrifuge at room temperature for ˜3 minutes. Individual cell pellets were suspended in ˜50 μl of sterile water, then mixed with an equal volume of 2× Laemelli SDS-PAGE sample buffer containing 2-mercaptoethanol, and placed in boiling water bath for ˜3 min to denature protein. Equal volumes (˜15 μl) of both the crude IPTG-induced and the non-induced cell lysates were loaded onto duplicate 12% Tris/glycine polyacrylamide gel (1 mm thick Mini-gels, Novex). The induced and non-induced lysate samples were electrophoresed together with prestained molecular weight markers (See Blue, Novex) under conventional conditions using a standard SDS/Tris/glycine running buffer (BioRad). Following electrophoresis, one gel was stained with commassie brilliant blue R250 (BioRad) and then destained to visualize novel BASB041 IPTG-inducible protein(s). The second gel was electroblotted onto a PVDF membrane (0.45 micron pore size, Novex) for ˜2 hrs at 4° C. using a BioRad Mini-Protean II blotting apparatus and Towbin's methanol (20%) transfer buffer. Blocking of the membrane and antibody incubations were performed according to methods well known in the art. A monoclonal anti-RGS (His) antibody, followed by a second rabbit anti-mouse antibody conjugated to HRP (QiaGen), was used to confirm the expression and identity of the BASB041 recombinant protein. Visualization of the anti-His antibody reactive pattern was achieved using either an ABT insoluble substrate or using Hyperfilm with the Amersham ECL chemiluminescence system.

Production of Recombinant BASB041

Bacterial Strain

A recombinant expression strain of E. coli BL21 DE3 containing a pET24b plasmid encoding BASB041 from N. meningitidis was used to produce cell mass for purification of recombinant protein. The expression strain was cultivated on LB agar plates containing 50 μg/ml kanamycin (“Kn”) to ensure plasmid maintenance. For cryopreservation at −80° C., the strain was propagated in LB broth containing the same concentration of antibiotic then mixed with an equal volume of LB broth containing 30% (w/v) glycerol.

Media

The fermentation medium used for the production of recombinant protein consisted of 2×YT broth (Difco) containing 50 μg/ml Kn. Antifoam was added to medium for the fermentor at 0.25 ml/L (Antifoam 204, Sigma). To induce expression of the BASB041 recombinant protein, IPTG (Isopropyl β-D-Thiogalactopyranoside) was added to the fermentor (1 mM, final).

Fermentation

A 500-ml erlenmeyer seed flask, containing 50 ml working volume, was inoculated with 0.3 ml of rapidly thawed frozen culture, or several colonies from a selective agar plate culture, and incubated for approximately 12 hours at 37±1° C. on a shaking platform at 150 rpm (Innova 2100, New Brunswick Scientific). This seed culture was then used to inoculate a 5-L working volume fermentor containing 2×YT broth and both Kn antibiotic. The fermentor (Bioflo 3000, New Brunswick Scientific) was operated at 37+1° C., 0.2-0.4 VVM air sparge, 250 rpm in Rushton impellers. The pH was not controlled in either the flask seed culture or the fermentor. During fermentation, the pH ranged 6.5 to 7.3 in the fermentor. IPTG (1.0 M stock, prepared in sterile water) was added to the fermentor when the culture reached mid-log of growth (˜0.7 O.D.600 units). Cells were induced for 2-4 hours then harvested by centrifugation using either a 28RS Heraeus (Sepatech) or RC5C superspeed centrifuge (Sorvall Instruments). Cell paste was stored at −20 C until processed.

Purification

Imidazole and biotechnology grade or better reagents were all obtained from Ameresco Chemical, Solon, Ohio. Triton X-100 (t-Octylphenoxypolyethoxy-ethanol), Triton X-114, sodium phosphate, monobasic, and urea were reagent grade or better and obtained from Sigma Chemical Company, St. Louis, Mo. Dulbecco's Phosphate Buffered Saline (1×PBS) was obtained from Quality Biological, Inc., Gaithersburg, Md. Dulbecco's Phosphate Buffered Saline (10×PBS) was obtained from BioWhittaker, Walkersville, Md. Penta-His Antibody, BSA free was obtained from QiaGen, Valencia, Calif. Peroxidase-conjugated AffiniPure Goat Anti-mouse IgG was obtained from Jackson Immuno Research, West Grove, Penn. All other chemicals were reagent grade or better.

Ni-chelatin Sepharose Fast Flow resin was obtained from Pharmacia, Sweden. Precast Tris-Glycine 4-20% and 10-20% polyacrylamide gels, all running buffers and solutions, SeeBlue Pre-Stained Standards, MultiMark Multi-Colored Standards and PVDF transfer membranes were obtained from Novex, San Diego, Calif. SDS-PAGE Silver Stain kits were obtained from Daiichi Pure Chemicals Company Limited, Tokyo, Japan. Coomassie Stain Solution was obtained from Bio-Rad Laboratories, Hercules, Calif. Acrodisc® PF 0.2 m syringe filters were obtained from Pall Gelman Sciences, Ann Arbor, Mich. GD/X 25 mm disposable syringe filters were obtained from Whatman Inc., Clifton, N.J. Dialysis tubing 8,000 MWCO was obtained from BioDesign Inc. Od New York, Carmal New York. BCA Protein Assay Reagents and Snake Skin dialysis tubing 3,500 MWCO were obtained from Pierce Chemical Co., Rockford, Ill.

Extraction Protocol

Cell paste was thawed at room temperature for 30 to 60 minutes. Five to six grams of material was weighed out into a 50-ml disposable centrifuge tube. Recombinant BASB041 antigen was purified by extraction of cell membranes with 1.0% Triton X114, and allowing phase partitioning based on Triton X114 cloud point at 37° C. The Triton X114 phase was diluted with 50 mM Tris-HCl containing 10% glycerol, 5% ethylene glycol and 0.5% Triton X100. This was applied to nickel-chelating Sepharose Fast Flow. The protein is afterwards eluted with 200 mM imidazole to affinity purify the histidine-tagged protein and yielded greater than 90% pure protein.

Binding of BASB041 to Nickel Affinity Resin

After extraction, the mixture was incubated to the nickel-chelating Sepharose Fast Flow and placed at room temperature with gentle agitation for one hour. After one hour, the nickel-chelating Sepharose Fast Flow is packed into an XK16 Pharmacia column, and eluted afterwards with 500 mM imidazole buffer to affinity purify the histidine tagged protein. This fraction was then dialyzed against 25 mM Phosphate buffer (pH7.0) containing 0.1% of Triton. This sample was then applied to a TOYOPEARL BUTYL_—650M column which was equilibrated with 25 mM Phosphate buffer (pH7.0) containing 2M sodium chloride and 0.1% of Triton. Elution was performed in the following buffer with 25 mM Phosphate buffer (pH7.0) containing 1M sodium chloride and 0.1% of Triton. This fraction was further applied to a DEAE-sepharose-FF in presence of 50 mM tris(pH7.5) containing 2 mMEDTA, 10 mMsodium chloride and 0.005% Triton X100. The DEAE flowthrough was dialyzed against PBS (pH7.4) containing 0.1% of Triton and stored at −70 at a concentration of 490 μg/ml.

Final Formulation

BASB041 was formulated by dialysis overnight against, three changes of 0.1% Triton X-100 and 1×PBS, pH 7.4. The purified protein was characterized and used to produce antibodies as described below.

Biochemical Characterizations: SDS-PAGE and Western Blot Analysis

The recombinant purified protein was resolved on 4-20% polyacrylamide gels and electrophoretically transferred to PVDF membranes at 100 V for 1 hour as previously described (Thebaine et al. 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354). The PVDF membranes were then pretreated with 25 ml of Dulbecco's phosphate buffered saline containing 5% non-fat dry milk. All subsequent incubations were carried out using this pretreatment buffer.

PVDF membranes were incubated with a dilution of anti-His tail antibodies for 1 hour at room temperature. PVDF membranes were then washed twice with wash buffer (20 mM Tris buffer, pH 7.5, containing 150 mM sodium chloride and 0.05% Tween-20). PVDF membranes were incubated with 25 ml of a 1:5000 dilution of peroxidase-labeled species specific conjugate for 30 minutes at room temperature. PVDF membranes were then washed 4 times with wash buffer, and were developed with 3-amino-9-ethylcarbazole and urea peroxide as supplied by Zymed (San Francisco, Calif.) for 10 minutes each.

The results of an SDS-PAGE (FIG. 3) show a protein about 31 kDa purified to greater than 90% and that is reactive to an anti-RGS (His) antibody by western blots (FIG. 3) of the SDS-PAGE.

Immunization of Mice with Recombinant BASB041

Partially purified recombinant BASB041 protein expressed in E. coli has been injected three times in Balb/C mice on days 0, 14 and 28 (10 animals/group). Animals were injected by the subcutaneous route with around 5 μg of antigen in two different formulations: either adsorbed on 100 μg AlPO₄ or formulated in SBAS2 emulsion (SB62 emulsion containing 5 μg MPL and 5 μg QS21 per dose). A negative control group consisting of mice immunized with the SBAS2 emulsion only has also been added in the experiment. Mice were bled on days 28 (14 days Post II) and 35 (7 days Post III) in order to detect specific anti-BASB041 antibodies. Specific anti-BASB041 antibodies were measured by western-blotting on pooled sera (from 10 mice/group) from both formulations (on day 7 Post III only), using recombinant protein (part of the gel) and Neisseria meningitidis B strains.

Recognition of the BASB041 epitopes on different Neisseria meningitidis Serogroup B strains by western-blotting

In this test, immunized mice sera (pooled) have been tested by western-blotting for recognition of the BASB041 epitopes on seven different Neisseria meningitidis B strains: H44/76 (B:15:P1.7, 16, lineage ET-5), M97 250687 (B:4:P1.15), BZ10 (B:2b:P1.2, lineage A4), BZ198 (B:NT*: -, lineage 3), EG328 (B:NT*, lineage ST-18), NGP165 (B:2a:P1.2, ET 37 cluster) and the ATCC 13090 (B:15:P1.15) Neisseria meningitidis B strains, as well as on partially purified recombinant BASB041 protein. (*: NT: Not Typed).

Briefly, 10 μl (>10⁸ cells/lane) of each sample treated with sample buffer (10 min at 95° C.) are put into a SDS-PAGE gradient gel (Tris-glycine 4-20%, Novex, code no EC60252). Electrophoretic migration occurs at 125 volts for 90 min. Afterwards, proteins are transferred to nitrocellulose sheet (0.45 μm, Bio-rad code no 162-0114) at 100 volts for 1 hour using a Bio-rad Trans-blot system (code no 170-3930). Filter was blocked with PBS-0.05% Tween 20 overnight at room temperature, before incubation with the mice sera containing the anti-BASB041 antibodies from both AlPO₄ and SBAS2 formulations. These sera are diluted 100 times in PBS-0.05% Tween 20, and incubated on the nitrocellulose sheet for two hours at room temperature with gentle shaking, using a mini-blotter system (Miniprotean, Bio-rad code no 170-4017). After three repeated washing steps in PBS-0.05% Tween 20 for 5 min., the nitrocellulose sheet is incubated at room temperature for 1 hour under gentle shaking with the appropriate conjugate (biotinylated anti-mouse Ig antibodies from sheep, Amersham code no RPN1001) diluted at 1/500 in the same washing buffer. The membrane is washed three times as previously, and incubated for 30 min with agitation using the streptavidin-peroxidase complex (Amersham code no 1051) diluted at 1/1000 in the washing buffer. After the last three repeated washing steps, the revelation occurs during the 20 min incubation time in a 50 ml solution containing 30 mg 4-chloro-1-naphtol (Sigma), 10 ml methanol, 40 ml PBS, and 30 μl of H₂O₂. The staining is stopped while washing the membrane several times in distillated water.

Results illustrated in FIGS. 4 and 5 show that all strains tested present the expected bands around 25-30 kDa (major) and 50 kDa (minor), which are recognized at the same level in all of the Neisseria meningitidis B strains tested. This means that the BASB041 protein is expressed in probably all Neisseria meningitidis B strains. In both figures, the recombinant BASB041 protein is also clearly recognized by mice sera at the same MW (second lane after the MW). Another band at around 20 kDa is known to be non-specific. This BASB041 protein is not recognized anymore in E. coli preparation.

Presence of Anti-BASB041 Antibodies in Sera from Convalescent Patients.

In this test, several convalescent sera have been tested by western-blotting for recognition of the purified recombinant BASB041 protein.

Briefly, 5 μg of partially purified BASB041 Neisseria meningitidis B protein are put into a SDS-PAGE gradient gel (4-20%, Novex, code no EC60252) for electrophoretic migration. Proteins are transferred to nitrocellulose sheet (0.45 μm, Bio-rad code no 162-0114) at 100 volts for 1 hour using a Bio-rad Trans-blot system (code no 170-3930). Afterwards, filter is blocked with PBS-0.05% Tween 20 overnight at room temperature, before incubation with the human sera. The following convalescent sera were tested: patients #262068, 261732, 262117, 261659, 261469, 261979, and 261324. These sera are diluted 100 times in PBS-0.05% Tween 20, and incubated on the nitrocellulose sheet for two hours at room temperature with gentle shaking, using a mini-blotter system (Miniprotean, Bio-rad code no 170-4017). After three repeated washing steps in PBS-0.05% Tween 20 for 5 min., the nitrocellulose sheet is incubated at room temperature for 1 hour under gentle shaking with the appropriate conjugate (biotinylated anti-human Ig antibodies, from sheep, Amersham code no RPN1003) diluted at 1/500 in the same washing buffer. The membrane is washed three times as previously, and incubated for 30 min with agitation using the streptavidin-peroxidase complex (Amersham code no 1051) diluted at 1/1000 in the washing buffer. After the last three repeated washing steps, the revelation occurs during the 20 min incubation time in a 50 ml solution containing 30 mg 4-chloro-1-naphtol (Sigma), 10 ml methanol, 40 ml of ultra-pure water, and 30 μl of H₂O₂. The staining is stopped while washing the membrane several times in distillated water. Results illustrated in FIGS. 6 and 7 show that all the 7 convalescents react against the major band of recombinant BASB041 protein at around 25-30 kDa. All of them react with around the same intensity, with slightly lower reactivity with patients 261979. In the right part of the western-blot, the reaction against the same 25-30 kD band is observed with the immunized mice sera, plus the band recognized at around 50 kDa.

Example 2

DNA Sequencing of the BASB043 Gene from Two N. meningitidis Strains.

A: BASB043 in N. meningitidis Serogroup B Strain ATCC13090.

The BASB043 gene of N. meningitidis strain ATCC 13090 is shown in SEQ ID NO:11. The translation of the BASB043 polynucleotide sequence, shown in SEQ ID NO:12, did not show any significant similarity to any known protein. The BASB043 polypeptide contains however a signal sequence characteristic of a lipoprotein, and could thus be inserted into the outer membrane of the bacterium.

The sequence of the BASB043 gene was further confirmed as follows. For this purpose, genomic DNA was extracted from 10¹⁰ cells of the N. meningitidis cells (strain ATCC 13090) using the QIAGEN genomic DNA extraction kit (Qiagen Gmbh), and 1 μg of this material was submitted to Polymerase Chain Reaction DNA amplification using primers lip7-01 (5′-ATG AAA AAA TAC CTT ATC CCT CTT TCC-3′) [SEQ ID NO:13] and lip7-02 (5′-TCA TTT CAA GGG CTG CAT-3′) [SEQ ID NO:14]. This PCR product was gel-purified and subjected to DNA sequencing using the Big Dye Cycle Sequencing kit (Perkin-Elmer) and an ABI 373A/PRISM DNA sequencer. DNA sequencing was performed on both strands with a redundancy of 2 and the full-length sequence was assembled using the SeqMan program from the DNASTAR Lasergene software package. The resulting DNA sequence turned out to be 100% identical to SEQ ID NO:11.

B: BASB043 in N. meningitidis Serogroup B Strain H44/76.

The sequence of the BASB043 gene was also determined in another N. meningitidis serogroup B strain, the strain H44/76. For this purpose, genomic DNA was extracted from the N. meningitidis strain H44/76 using the experimental conditions presented in the previous paragraph. This material (1 μg) was then submitted to Polymerase Chain Reaction DNA amplification using primers Lip7-01 and Lip7-02 specific for the BASB043 gene. The PCR amplicon was then submitted to DNA sequencing using the Big Dyes kit (Applied biosystems) and analyzed on a ABI 373/A DNA sequencer in the conditions described by the supplier. As a result, the polynucleotide sequence turned out to be 100% identical to SEQ ID NO:11.

Taken together, these data indicate strong sequence conservation of the BASB043 gene among the two N. meningitidis serogroup B strains.

Construction of Plasmid to Express Recombinant BASB043

A: Cloning of BASB043.

The NdeI and XhoI restriction sites engineered into the forward Lip7-Fm/p (5′-AGG CAG AGG CAT ATG AAA AAA TAC CTT ATC CCT CTT TCC ATT GCC-3′) ([SEQ ID NO:15]) and reverse Lip7-RCf/p (5′-AGG CAG AGG CTC GAG TTT CAA GGG CTG CAT CTT CAT CAC TTC-3′) ([SEQ ID NO:16]) amplification primers, respectively, permitted directional cloning of a BASB043 PCR product into the commercially available E. coli expression plasmid pET24b (Novagen, USA, kanamycin resistant) such that a mature BASB043 protein could be expressed as a fusion protein containing a (His)6 affinity chromatography tag at the C-terminus. The BASB043 PCR product was purified from the amplification reaction using silica gel-based spin columns (QiaGen) according to the manufacturers instructions. To produce the required NdeI and XhoI termini necessary for cloning, purified PCR product was sequentially digested to completion with NdeI and XhoI restriction enzymes as recommended by the manufacturer (Life Technologies). Following the first restriction digestion, the PCR product was purified via spin column as above to remove salts and eluted in sterile water prior to the second enzyme digestion. The digested DNA fragment was again purified using silica gel-based spin columns prior to ligation with the pET24b plasmid.

B: Production of Expression Vector.

To prepare the expression plasmid pET24b for ligation, it was similarly digested to completion with both NdeI and XhoI and then treated with calf intestinal phosphatase (CIP, ˜0.02 units/pmole of 5′ end, Life Technologies) as directed by the manufacturer to prevent self-ligation. An approximately 5-fold molar excess of the digested fragment to the prepared vector was used to program the ligation reaction. A standard ˜20 μl ligation reaction (˜16° C., ˜16 hours), using methods well known in the art, was performed using T4 DNA ligase (˜2.0 units/reaction, Life Technologies). An aliquot of the ligation (˜5 μl) was used to transform electro-competent BL21 DE3 cells according to methods well known in the art. Following a ˜2-3 hour outgrowth period at 37° C. in ˜1.0 ml of LB broth, transformed cells were plated on LB agar plates containing kanamycin (50 μg/ml. The antibiotic was included in the selection media to ensure that all transformed cells carried the pET24b plasmid (KnR). Plates were incubated overnight at 37° C. for ˜16 hours. Individual KnR colonies were picked with sterile toothpicks and used to “patch” inoculate fresh LB KnR plates as well as a ˜1.0 ml LB KnR broth culture. Both the patch plates and the broth culture were incubated overnight at 37° C. in either a standard incubator (plates) or a shaking water bath.

A whole cell-based PCR analysis was employed to verify that transformants contained the BASB043 DNA insert. Here, the ˜1.0 ml overnight LB Kn broth culture was transferred to a 1.5 ml polypropylene tube and the cells collected by centrifugation in a Beckman microcentrifuge (˜3 min., room temperature, ˜12,000×g). The cell pellet was suspended in ˜200 μl of sterile water and a ˜1011 aliquot used to program a −50 μl final volume PCR reaction containing both BASB043 forward and reverse amplification primers. Final concentrations of the PCR reaction components were essentially the same as those specified in example 2 except ˜5.0 units of Taq polymerase was used. The initial 95° C. denaturation step was increased to 3 minutes to ensure thermal disruption of the bacterial cells and liberation of plasmid DNA. An ABI Model 9700 thermal cycler and a 32 cycle, three-step thermal amplification profile, i.e. 95° C., 45 sec; 55-58° C., 45 sec, 72° C., 1 min., were used to amplify the BASB043 PCR fragment from the lysed transformant samples. Following thermal amplification, a ˜20 μl aliquot of the reaction was analyzed by agarose gel electrophoresis (0.8% agarose in a Tris-acetate-EDTA (TAE) buffer). DNA fragments were visualized by UV illumination after gel electrophoresis and ethidium bromide staining. A DNA molecular size standard (1 Kb ladder, Life Technologies) was electrophoresed in parallel with the test samples and was used to estimate the size of the PCR products. Transformants that produced the expected PCR product were identified as strains containing a BASB043 expression construct. Expression plasmid containing strains were then analyzed for the inducible expression of recombinant BASB043.

C: Expression Analysis of PCR-Positive Transformants.

For each PCR-positive transformant identified above, ˜5.0 ml of LB broth containing kanamycin (50 μg/ml) was inoculated with cells from the patch plate and grown overnight at 37° C. with shaking (˜250 rpm). An aliquot of the overnight seed culture (˜1.0 ml) was inoculated into a 125 ml erlenmeyer flask containing ˜25 ml of LB Kn broth and grown at 37° C. with shaking (˜250 rpm) until the culture turbidity reached O.D.600 of ˜0.5, i.e. mid-log phase (usually about 1.5-2.0 hours). At this time approximately half of the culture (˜12.5 ml) was transferred to a second 125 ml flask and expression of recombinant BASB043 protein induced by the addition of IPTG (1.0 M stock prepared in sterile water, Sigma) to a final concentration of 1.0 mM. Incubation of both the IPTG-induced and non-induced cultures continued for an additional ˜4 hours at 37° C. with shaking. Samples (˜1.0 ml) of both induced and non-induced cultures were removed after the induction period and the cells collected by centrifugation in a microcentrifuge at room temperature for ˜3 minutes. Individual cell pellets were suspended in ˜50 μl of sterile water, then mixed with an equal volume of 2× Laemelli SDS-PAGE sample buffer containing 2-mercaptoethanol, and placed in boiling water bath for ˜3 min to denature protein. Equal volumes (˜15 μl) of both the crude IPTG-induced and the non-induced cell lysates were loaded onto duplicate 12% Tris/glycine polyacrylamide gel (1 mm thick Mini-gels, Novex). The induced and non-induced lysate samples were electrophoresed together with prestained molecular weight markers (SeeBlue, Novex) under conventional conditions using a standard SDS/Tris/glycine running buffer (BioRad). Following electrophoresis, one gel was stained with commassie brilliant blue R250 (BioRad) and then destained to visualize novel BASB043 IPTG-inducible protein(s). The second gel was electroblotted onto a PVDF membrane (0.45 micron pore size, Novex) for ˜2 hrs at 4° C. using a BioRad Mini-Protean II blotting apparatus and Towbin's methanol (20%) transfer buffer. Blocking of the membrane and antibody incubations were performed according to methods well known in the art. A monoclonal anti-RGS (His) antibody, followed by a second rabbit anti-mouse antibody conjugated to HRP (QiaGen), was used to confirm the expression and identity of the BASB043 recombinant protein. Visualization of the anti-His antibody reactive pattern was achieved using either an ABT insoluble substrate or using Hyperfilm with the Amersham ECL chemiluminescence system.

Production of Recombinant BASB043

Bacterial Strain

A recombinant expression strain of E. coli BL21 DE3 containing a pET24b plasmid encoding BASB043 from N. meningitidis. was used to produce cell mass for purification of recombinant protein. The expression strain was cultivated on LB agar plates containing 50 μg/ml kanamycin (“Kn”) to ensure plasmid maintenance. For cryopreservation at −80° C., the strain was propagated in LB broth containing the same concentration of antibiotic then mixed with an equal volume of LB broth containing 30% (w/v) glycerol.

Media

The fermentation medium used for the production of recombinant protein consisted of 2×YT broth (Difco) containing 50 μg/ml Kn. Antifoam was added to medium for the fermentor at 0.25 ml/L (Antifoam 204, Sigma). To induce expression of the BASB043 recombinant protein, IPTG (Isopropyl β-D-Thiogalactopyranoside) was added to the fermentor (1 mM, final).

Fermentation

A 500-ml erlenmeyer seed flask, containing 50 ml working volume, was inoculated with 0.3 ml of rapidly thawed frozen culture, or several colonies from a selective agar plate culture, and incubated for approximately 12 hours at 37±1° C. on a shaking platform at 150 rpm (Innova 2100, New Brunswick Scientific). This seed culture was then used to inoculate a 5-L working volume fermentor containing 2×YT broth and both Kn antibiotic. The fermentor (Bioflo 3000, New Brunswick Scientific) was operated at 37±1° C., 0.2-0.4 VVM air sparge, 250 rpm in Rushton impellers. The pH was not controlled in either the flask seed culture or the fermentor. During fermentation, the pH ranged 6.5 to 7.3 in the fermentor. IPTG (1.0 M stock, prepared in sterile water) was added to the fermentor when the culture reached mid-log of growth (˜0.7 O.D.600 units). Cells were induced for 2-4 hours then harvested by centrifugation using either a 28RS Heraeus (Sepatech) or RC5C superspeed centrifuge (Sorvall Instruments). Cell paste was stored at −20 C until processed.

Purification

Imidazole and biotechnology grade or better reagents were all obtained from Ameresco Chemical, Solon, Ohio. Triton X-100 (t-Octylphenoxypolyethoxy-ethanol), Triton X-114, sodium phosphate, monobasic, and urea were reagent grade or better and obtained from Sigma Chemical Company, St. Louis, Mo. Dulbecco's Phosphate Buffered XhoIne (1×PBS) was obtained from Quality Biological, Inc., Gaithersburg, Md. Dulbecco's Phosphate Buffered XhoIne (10×PBS) was obtained from BioWhittaker, Walkersville, Md. Penta-His Antibody, BSA free was obtained from QiaGen, Valencia, Calif. Peroxidase-conjugated AffiniPure Goat Anti-mouse IgG was obtained from Jackson Immuno Research, West Grove, Penn. All other chemicals were reagent grade or better.

Ni-chelatin Sepharose Fast Flow resin was obtained from Pharmacia, Sweden. Precast Tris-Glycine 4-20% and 10-20% polyacrylamide gels, all running buffers and solutions, SeeBlue Pre-Stained Standards, MultiMark Multi-Colored Standards and PVDF transfer membranes were obtained from Novex, San Diego, Calif. SDS-PAGE Silver Stain kits were obtained from Daiichi Pure Chemicals Company Limited, Tokyo, Japan. Coomassie Stain Solution was obtained from Bio-Rad Laboratories, Hercules, Calif. Acrodisc® PF 0.2 m syringe filters were obtained from Pall Gelman Sciences, Ann Arbor, Mich. GD/X 25 mm disposable syringe filters were obtained from Whatman Inc., Clifton, N.J. Dialysis tubing 8,000 MWCO was obtained from BioDesign Inc. Od New York, Carmal New York. BCA Protein Assay Reagents and Snake Skin dialysis tubing 3,500 MWCO were obtained from Pierce Chemical Co., Rockford, Ill.

Extraction Protocol

Cell paste was thawed at room temperature for 30 to 60 minutes. Five to six grams of material was weighed out into a 50-ml disposable centrifuge tube. Recombinant BASB043 antigen was purified by extraction of cell membranes with 25 mM Tris-HCl containing 4M guanidine-HCl. The supernatent was applied to nickel-chelating Sepharose Fast Flow. The protein is afterwards eluted with 200 mM imidazole to affinity purify the histidine-tagged protein and yielded greater than 90% pure protein.

Binding of BASB043 to Nickel Affinity Resin

After extraction, the mixture was incubated to the nickel-chelating Sepharose Fast Flow and placed at room temperature with gentle agitation for one hour. After one hour, the nickel-chelating Sepharose Fast Flow is packed into an XK16 Pharmacia column, and eluted afterwards with 200 mM imidazole buffer to affinity purify the histidine tagged protein and yielded greater than 90% pure protein. The fraction was dialyzed against PBS (pH7.4) containing 0.1% of Triton and stored at −70 at a concentration of 500 μg/ml.

Final Formulation

BASB043 was formulated by dialysis overnight against, three changes of 0.1% Triton X-100 and 1×PBS, pH 7.4. The purified protein was characterized and used to produce antibodies as described below.

Biochemical Characterizations: SDS-PAGE and Western Blot Analysis

The recombinant purified protein was resolved on 4-20% polyacrylamide gels and electrophoretically transferred to PVDF membranes at 100 V for 1 hour as previously described (Thebaine et al. 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354). The PVDF membranes were then pretreated with 25 ml of Dulbecco's phosphate buffered XhoIne containing 5% non-fat dry milk. All subsequent incubations were carried out using this pretreatment buffer.

PVDF membranes were incubated with a dilution of anti-His tail antibodies for 1 hour at room temperature. PVDF membranes were then washed twice with wash buffer (20 mM Tris buffer, pH 7.5, containing 150 mM sodium chloride and 0.05% Tween-20). PVDF membranes were incubated with 25 ml of a 1:5000 dilution of peroxidase-labeled species specific conjugate for 30 minutes at room temperature. PVDF membranes were then washed 4 times with wash buffer, and were developed with 3-amino-9-ethylcarbazole and urea peroxide as supplied by Zymed (San Francisco, Calif.) for 10 minutes each.

The results of an SDS-PAGE (FIG. 8) show a protein about 16 kDa purified to greater than 90% and that is reactive to an anti-RGS (His) antibody by western blots (FIG. 8) of the SDS-PAGE.

Immunization of Mice with Recombinant BASB043

Partially purified recombinant BASB043 protein expressed in E. coli has been injected three times in Balb/C mice on days 0, 14 and 28 (10 animals/group). Animals were injected by the subcutaneous route with around 5 μg of antigen in two different formulations: either adsorbed on 100 μg AlPO₄ or formulated in SBAS2 emulsion (SB62 emulsion containing 5 μg MPL and 5 μg QS21 per dose). A negative control group consisting of mice immunized with the SBAS2 emulsion only has also been added in the experiment. Mice were bled on days 28 (14 days Post II) and 35 (7 days Post III) in order to detect specific anti-BASB043 antibodies. Specific anti-BASB043 antibodies were measured by western-blotting on pooled sera (from 10 mice/group) from both formulations (on day 7 Post III only), using recombinant protein (part of the gel) and Neisseria meningitidis B strains.

Recognition of the BASB043 epitopes on different Neisseria meningitidis B strains by western-blotting

In this test, immunized mice sera (pooled) have been tested by western-blotting for recognition of the BASB043 epitopes on seven different Neisseria meningitidis B strains: H44/76 (B:15:P1.7, 16, lineage ET-5), M97 250687 (B:4:P1.15), BZ10 (B:2b:P1.2, lineage A4), BZ198 (B:NT*: -, lineage 3), EG328 (B:NT*, lineage ST-18), NGP165 (B:2a:P1.2, ET 37 cluster) and the ATCC 13090 (B:15:P1.15) Neisseria meningitidis B strains, as well as on partially purified recombinant BASB043 protein. (*: NT: Not Typed).

Briefly, 10 μl (>10⁸ cells/lane) of each sample treated with sample buffer (10 min at 95° C.) are put into a SDS-PAGE gradient gel (Tris-glycine 4-20%, Novex, code no EC60252). Electrophoretic migration occurs at 125 volts for 90 min. Afterwards, proteins are transferred to nitrocellulose sheet (0.45 μm, Bio-rad code no 162-0114) at 100 volts for 1 hour using a Bio-rad Trans-blot system (code no 170-3930). Filter was blocked with PBS-0.05% Tween 20 overnight at room temperature, before incubation with the mice sera containing the anti-BASB043 antibodies from both AlPO₄ and SBAS2 formulations. These sera are diluted 100 times in PBS-0.05% Tween 20, and incubated on the nitrocellulose sheet for two hours at room temperature with gentle shaking, using a mini-blotter system (Miniprotean, Bio-rad code no 170-4017). After three repeated washing steps in PBS-0.05% Tween 20 for 5 min., the nitrocellulose sheet is incubated at room temperature for 1 hour under gentle shaking with the appropriate conjugate (biotinylated anti-mouse Ig antibodies from sheep, Amersham code no RPN1001) diluted at 1/500 in the same washing buffer. The membrane is washed three times as previously, and incubated for 30 min with agitation using the streptavidin-peroxidase complex (Amersham code no 1051) diluted at 1/1000 in the washing buffer. After the last three repeated washing steps, the revelation occurs during the 20 min incubation time in a 50 ml solution containing 30 mg 4-chloro-1-naphtol (Sigma), 10 ml methanol, 40 ml PBS, and 30 μl of H₂O₂. The staining is stopped while washing the membrane several times in distillated water.

Results illustrated in FIGS. 9 and 10 show that all strains tested present the expected bands around 20 kDa (major), 25 and 35-40 kDa (minors), which are recognized at the same level in all of the Neisseria meningitidis B strains tested. This means that the BASB043 protein is expressed in probably all Neisseria meningitidis B strains. In both figures, the recombinant BASB043 protein is also clearly recognized by mice sera at the same MW (second lane after the MW). This BASB043 protein is not recognized anymore on E. coli preparation.

Presence of Anti-BASB043 Antibodies in Sera from Convalescent Patients.

In this test, several convalescent sera have been tested by western-blotting for recognition of the purified recombinant BASB043 protein.

Briefly, 5 μg of partially purified BASB043 Neisseria meningitidis B protein are put into a SDS-PAGE gradient gel (4-20%, Novex, code no EC60252) for electrophoretic migration. Proteins are transferred to nitrocellulose sheet (0.45 μm, Bio-rad code no 162-0114) at 100 volts for 1 hour using a Bio-rad Trans-blot system (code no 170-3930). Afterwards, filter is blocked with PBS-0.05% Tween 20 overnight at room temperature, before incubation with the human sera. The following convalescent sera were tested: patients #261469, 261979, 261324, 261732, 262117 and 261659. These sera are diluted 100 times in PBS-0.05% Tween 20, and incubated on the nitrocellulose sheet for two hours at room temperature with gentle shaking, using a mini-blotter system (Miniprotean, Bio-rad code no 170-4017). After three repeated washing steps in PBS-0.05% Tween 20 for 5 min., the nitrocellulose sheet is incubated at room temperature for 1 hour under gentle shaking with the appropriate conjugate (biotinylated anti-human Ig antibodies, from sheep, Amersham code no RPN1003) diluted at 1/500 in the same washing buffer. The membrane is washed three times as previously, and incubated for 30 min with agitation using the streptavidin-peroxidase complex (Amersham code no 1051) diluted at 1/1000 in the washing buffer. After the last three repeated washing steps, the revelation occurs during the 20 min incubation time in a 50 ml solution containing 30 mg 4-chloro-1-naphtol (Sigma), 10 ml methanol, 40 ml of ultra-pure water, and 30 μl of H₂O₂. The staining is stopped while washing the membrane several times in distillated water.

Results illustrated in FIGS. 11 and 12 show that all the 6 convalescents react against the major band of recombinant BASB043 protein at around 20 kDa. Human sera recognize only this band while mice sera recognize the three major bands of BASB043. All of these human convalescent react with a very high intensity, except for convalescent no 262117 who shows a lower reactivity. In the right part of the western-blot, the reaction against the same 25-30 kD band is observed with the immunized mice sera, plus the band recognized at around 50 kDa. Few bands at a lower MW are also observed. Few leaks also are visible in FIG. 11.