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Last Updated: January 28, 2022

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Claims for Patent: 10,266,606

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Summary for Patent: 10,266,606
Title:Method for purifying Cys-linked antibody-drug conjugates
Abstract: The present invention relates to a method for purifying a mixture of cysteine-linked antibody-drug conjugates, wherein the amount of non-conjugated antibody is in the range of 0-40% by weight, using hydrophobic interaction chromatography (HIC). The mixture is loaded onto a preparative HIC column using a 0.2-1.5 M aqueous salt solution, in which non-conjugated antibody is collected in a flow-through fraction, followed by elution of a purified mixture of cysteine-linked antibody-drug conjugates using a 0-100 mM aqueous salt solution.
Inventor(s): De Roo; Guy (Nijmegen, NL), Verstegen; Ruud Martin (Nijmegen, NL), Coumans; Rudy Gerardus Elisabeth (Nijmegen, NL)
Assignee: Synthon Biopharmaceuticals B.V. (Nijmegen, NL)
Application Number:15/110,169
Patent Claims:1. A method for obtaining a purified mixture of cysteine-linked antibody-drug conjugates, which comprises: a. providing a cysteine-linked antibody-drug conjugates mixture in a 0.2-1.5 M aqueous salt solution, wherein said mixture of cysteine-linked antibody-drug conjugates is of formula (I) ##STR00010## wherein Ab is an antibody, L is a linking group selected from ##STR00011## V.sup.1 is a conditionally-cleavable dipeptide of natural and/or unnatural amino acids, CL is a cyclization linker selected from ##STR00012## wherein n is an integer of from 1 to 16, R is selected from H, CH.sub.3, CH.sub.2CH.sub.3, OCH.sub.3, OCH.sub.2CH.sub.3, CF.sub.3, OCF.sub.3, Cl, F, q ranges from 0 to 8, and DB is a DNA binding moiety selected from ##STR00013## ##STR00014## ##STR00015## and wherein the amount of non-conjugated antibody in said mixture is in the range of 10-40% by weight; b. loading said solution onto a preparative hydrophobic interaction chromatography column; c. collecting a flow-through fraction that contains non-conjugated antibody; d. washing said column with a 0.2-1.5 M aqueous salt solution while collecting the flow-through fraction; and e. eluting said column with a 0-100 mM aqueous salt solution to obtain a purified mixture of cysteine-linked antibody-drug conjugates; wherein no organic solvent is added in steps a, b, d, and e.

2. The method according to claim 1, wherein said column is packed with Fractogel EMD propyl, Fractrogel EMD phenyl, Butyl-S sepharose, Octyl Sepharose, Capto Octyl, Capto Butyl, Capto Phenyl ImpRes, Capto Butyl ImpRes, Toyopearl PPG-600M, Toyopearl Hexyl-650, Toyopearl Butyl-650, Toyopearl Phenyl-650, Toyopearl Ether-650, Macroprep t-Butyl, Macroprep phenyl, Cellufine Butyl, Cellufine Phenyl or Poros HP2.

3. The method according to claim 1, wherein said column has a diameter in the range of 4.0-2,000 mm.

4. The method according to claim 1, wherein the column loading is in the range of 5-50 g/L of column packing material.

5. The method according to claim 1, wherein the column contains column packing material that has an average particle size in the range of 30-180 .mu.m.

6. The method according to claim 1, wherein the salt of the aqueous salt solution in each of steps (a), (d), and (e) is selected from the group consisting of potassium thiocyanate, sodium chloride, potassium chloride, ammonium chloride, sodium sulphate, potassium sulphate and ammonium sulphate.

7. The method according to claim 1, wherein the aqueous salt solution in each of steps (a), (d) and (e) further contains a buffer.

8. The method according to claim 7, wherein the buffer in each solution is selected from the group consisting of sodium phosphate, potassium phosphate, ammonium phosphate, sodium acetate, potassium acetate, sodium citrate, potassium citrate, ammonium citrate and mixtures thereof.

9. The method according to claim 7, wherein each of said aqueous salt solution is buffered to a pH of from about 4 to about 8.

10. The method according to claim 1, wherein the elution in step e is performed in a reverse mode.

11. The method according to claim 1, wherein the Ab is selected from the group consisting of an anti-CD19 antibody, an anti-CD22antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD74 antibody, an anti-CD138 antibody, an anti-CLL-1 antibody, an anti-5T4 antibody, an anti-CD303 antibody, an anti-Tag 72antibody, an anti-Lewis A like carbohydrate antibody, an anti-EphB3 antibody, an anti-HMW-MAA antibody, an anti-CD38 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-MN antibody, an anti-HER2 antibody, an anti-PSMA antibody, an anti-EGFR antibody, an anti-CD203c antibody, an anti-SLC44A4 antibody, an anti-Nectin-4antibody, an anti-mesothelin antibody, an anti-CD44 antibody, an anti-CD79antibody, an anti-FcRL5 antibody, an anti-MUC16 antibody, an anti-NaPi2b antibody, an anti-STEAP-1 antibody, an anti-ETBR antibody, an anti-TF antibody, an anti-MUC1 antibody, an anti-HGFR antibody, an anti-CD37 antibody, an anti-FOLR1 antibody, an anti-CEACAM antibody, an anti-TROP2 antibody, an anti-GCC antibody, an anti-Lewis Y antibody, an anti-LIV1 antibody, an anti-DLL3antibody, and an anti-EPCAM antibody.

12. The method according to claim 1, wherein the mixture of cysteine-linked antibody-drug conjugates of step (a) is of the formula (II) ##STR00016## wherein Ab is trastuzumab and q ranges from 0 to 8.

13. The method according to claim 12, wherein the purified mixture of said cysteine-linked antibody-drug conjugates of the formula (II) has an average drug-to-antibody ratio (DAR) of from 2.6 to 2.9.

14. The method according to claim 13, wherein the average DAR is 2.80.

15. The method according to claim 3, wherein said column has a diameter in the range of 15-2,000 mm.

16. The method according to claim 4, wherein the column loading is in the range of 5-40 g/L of column packing material.

17. The method according to claim 6, wherein the salt is sodium chloride or ammonium sulphate.

18. The method according to claim 8, wherein the buffer is sodium phosphate or sodium acetate.

19. The method according to claim 13, wherein the average DAR is from 2.7 to 2.9.

20. The purified mixture of cysteine-linked antibody-drug conjugates as obtained according to claim 1.

21. The purified mixture according to claim 20, wherein the purified mixture of cysteine-linked antibody-drug conjugates has an average DAR of from 2.6 to 2.9.

22. The purified mixture according to claim 21, wherein the average DAR is from 2.7 to 2.9.

23. The purified mixture according to claim 22, wherein the average DAR is 2.80.

24. The purified mixture of cysteine-linked antibody-drug conjugates of the formula (II) as obtained according to claim 12.

25. The purified mixture according to claim 24, wherein the purified mixture of said cysteine-linked antibody-drug conjugates of the formula (II) has an average drug-to-antibody ratio (DAR) of from 2.6 to 2.9.

26. The purified mixture according to claim 25, wherein the average DAR is from 2.7 to 2.9.

27. The purified mixture according to claim 26, wherein the average DAR is 2.80.

28. The method according to claim 1, wherein said purified mixture of cysteine-linked antibody-drug conjugates has an average drug-to-antibody ratio of 2 to 3.

Details for Patent 10,266,606

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Genentech, Inc. HERCEPTIN trastuzumab For Injection 103792 1998-09-25 ⤷  Sign up for a Free Trial 2034-01-10
Genentech, Inc. HERCEPTIN trastuzumab For Injection 103792 2017-02-10 ⤷  Sign up for a Free Trial 2034-01-10
Genentech, Inc. HERCEPTIN HYLECTA trastuzumab and hyaluronidase-oysk Injection 761106 2019-02-28 ⤷  Sign up for a Free Trial 2034-01-10
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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