Preparing antibodies from CHO cell cultures for conjugation

Patent metadata

Field	Value
Publication number	US-10457976-B2
Application number	US-201415039179-A
Country	US
Kind code	B2
Filing date	Nov 21, 2014
Priority date	Nov 25, 2013
Publication date	Oct 29, 2019
Grant date	Oct 29, 2019

Abstract

The invention is based in part on the observation that a CHO cell oxidizing enzyme, particularly QSOX1, can survive a seemingly rigorous antibody purification process to reduce subsequent conjugation efficiency of the antibody to a drug. Whether the oxidizing enzyme survives the purification procedure depends on which purification techniques are employed which can vary from one antibody to another. With knowledge that contamination with a CHO cell oxidizing enzyme is a potential problem for subsequent conjugation, a suitable purification scheme can be devised for any antibody that eliminates or at least reduces CHO oxidizing enzyme(s) to an acceptable level.

First claim

What is claimed is:

1. A method of producing a conjugated antibody, comprising:
(a) performing at least one purification step of a purification scheme to obtain at least a partially purified preparation of an antibody from a culture of CHO cells expressing the antibody;
(b) testing the preparation for presence of a CHO cell sulfhydryl oxidizing enzyme;
(c) if the enzyme is detected at a level greater than 0.5 μg/ml or 33 ppm in the preparation of step (b), repeating steps (a) and (b);
(d) if the enzyme is detected at a level less than 0.5 μg/ml or 33 ppm in the preparation of step (b), performing the at least one purification step to obtain at least a partially purified preparation of antibody, wherein the at least one purification step of the purification scheme comprises at least one of a step of (i) washing a protein A column with a salt wash at a concentration of 150-500 mM NaCl, (ii) performing depth filtration at 230 L/m 2 /hr and using at least 15 L/m 2 of an equilibration buffer with a pH of 7.5-8 and NaCl concentration of 50-100 mM, (iii) using anion exchange with a quaternary ammonium ion column with a buffer having a pH of 7.5-8 and a conductivity of less than or equal to 11 mS/cm, and (iv) performing phenyl membrane filtration using a buffer having a pH of 6-8 and 0.3-0.4M sodium citrate; and
(e) conjugating the at least partially purified antibody via one or more sulfhydryl groups to a cytotoxic drug to produce the conjugated antibody.

2. The method of claim 1 , wherein the CHO cell sulfhydryl oxidizing enzyme is selected from at least one of quiescin Q6 sulfhydryl oxidase 1 (QSOX1), Q6 sulfhydryl oxidase 2 (QSOX2), and Augmenter of Liver Regeneration (ALR).

3. The method of claim 2 , wherein the CHO cell sulfhydryl oxidizing enzyme is QSOX1.

4. The method of claim 1 , wherein steps (d) and (e) are performed multiple times on different cultures of the antibody over a period of at least a year.

5. The method of claim 1 , wherein the testing comprises identifying a band of 65-75 kDa on a gel.

6. The method of claim 5 , wherein the band is identified by western blot or silver stain.

7. The method of claim 1 , wherein the testing comprises a functional test for QSOX1 activity.

8. The method of claim 7 , wherein the functional test produces hydrogen peroxide as an indicator of the activity.

9. The method of claim 8 , wherein the activity is inhibited by zinc ions, which inhibition is reversed by EDTA.

10. The method of claim 1 , wherein the antibody is not brentuximab.

11. The method of claim 1 , wherein the cytotoxic drug is selected from anti-tubulin agents, DNA minor groove binding agents, DNA replication inhibitors, chemotherapy sensitizers and pyrrolobenzodiazepine dimer.

12. A method of producing a conjugated antibody, comprising:
purifying an antibody from a culture of CHO cells by anion exchange with a quaternary ammonium ion column using a buffer having a pH of 7.5-8 and a conductivity less than or equal to 11 mS/cm, wherein the antibody is separated from QSOX1 enzyme in the culture; and conjugating the purified antibody via one or more sulfhydryl groups to a cytotoxic drug to produce the conjugated antibody.

13. A method of producing a conjugated antibody, comprising:
(a) performing at least one purification step of a purification scheme to obtain at least a partially purified preparation of an antibody from a culture of CHO cells expressing the antibody;
(b) conjugating the at least partially purified antibody via one or more sulfhydryl groups to a cytotoxic drug under conditions to produce the conjugated antibody; wherein an unacceptably low level of conjugated antibody is produced;
(c) testing the purified preparation of step (a) for presence of a CHO cell sulfhydryl oxidizing enzyme selected from at least one of quiescin Q6 sulfhydryl oxidase 1 (QSOX1), Q6 sulfhydryl oxidase 2 (QSOX2), and Augmenter of Liver Regeneration (ALR);
(d) if the enzyme is detected at a level greater than 0.5 μg/ml or 33 ppm in the preparation of step (c), performing steps (a) and (c) with a different purification step until the enzyme is detected at a level less than 0.5 μg/ml or 33 ppm in the preparation of step (c);
(e) performing the at least one purification step resulting in detection of the enzyme at a level less than 0.5 μg/ml or 33 ppm on a second culture of CHO cells expressing the antibody to obtain purified antibody, wherein the at least one purification step of the purification scheme resulting in detection of the enzyme at a level less than 0.5 μg/ml or 33 ppm comprises at least one of a step of (i) washing a protein A column with a salt wash at a concentration of 150-500 mM NaCl, (ii) performing depth filtration at 230 L/m 2 /hr and using at least 15 L/m 2 of an equilibration buffer with a pH of 7.5-8 and NaCl concentration of 50-100 mM, (iii) using anion exchange with a quaternary ammonium ion column with a buffer having a pH of 7.5-8 and a conductivity of less than or equal to 11 mS/cm, and (iv) performing phenyl membrane filtration using a buffer having a pH of 6-8 and 0.3-0.4M sodium citrate; and
(f) conjugating the purified antibody via one or more sulfhydryl groups to a cytotoxic drug to produce the conjugated antibody.

14. A method of producing a conjugated antibody, comprising:
(a) obtaining a preparation of antibody;
(b) testing the preparation for the presence of a CHO cell sulfhydryl oxidizing enzyme selected from at least one of quiescin Q6 sulfhydryl oxidase 1 (QSOX1), Q6 sulfhydryl oxidase 2 (QSOX2), and Augmenter of Liver Regeneration (ALR);
(c) if the enzyme is detected at a level greater than 0.5 μg/ml or 33 ppm in the preparation of step (b) performing a purification step to remove the enzyme to a level less than 0.5 μg/ml or 33 ppm, wherein the purification step is selected from a step of (i) washing a protein A column with a salt wash at a concentration of 150-500 mM NaCl, (ii) performing depth filtration at 230 L/m 2 /hr and using at least 15 L/m 2 of an equilibration buffer with a pH of 7.5-8 and NaCl concentration of 50-100 mM, (iii) using anion exchange with a quaternary ammonium ion column with a buffer having a pH of 7.5-8 and a conductivity of less than or equal to 11 mS/cm, and (iv) performing phenyl membrane filtration using a buffer having a pH of 6-8 and 0.3-0.4M sodium citrate; and
(d) conjugating the at least partially purified antibody via one or more sulfhydryl groups to a drug to produce the conjugated antibody.

15. The method claim 1 , wherein the antibody is an antibody produced in CHO cells.

16. The method claim 1 , wherein the antibody preparation has already been subjected to at least one purification step.

17. The method of claim 16 wherein the purification step is a chromatography purification step.

18. The method of claim 1 , wherein the testing involves monitoring the reaction between free thiol groups and DTNB in a control sample and a test sample and comparing the two.

19. The method of claim 1 , wherein the CHO cell sulfhydryl oxidizing enzyme comprises an amino acid sequence having 90% or higher sequence identity to SEQ ID NO: 2 and retaining sulfhydryl oxidizing ability.

20. The method of claim 1 , wherein the CHO cell sulfhydryl oxidizing enzyme comprises an amino acid sequence having amino acids 94 to 571 of SEQ ID NO: 2 and retaining sulfhydryl oxidizing ability.

Assignees

• Seattle Genetics Inc

Inventors

• Beam Kevin
• Meyer Damon
• Hayes Bradley
• Lyon Robert
• Valliere-Douglass John

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• A61K47/68 Primary

  the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment · CPC title

• C12N9/0051

  acting on a sulfur group of donors (1.8) · CPC title