PREPARING ANTIBODIES FROM CHO CELL CULTURES FOR CONJUGATION
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.
1 . 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 detectable level in step (b), performing a purification step to remove the enzyme to a non-detectable level, wherein the purification step is selected from a step of (i) loading the preparation on a protein A column with a salt wash, (ii) performing depth filtration, (iii) using anion exchange with a quaternary ammonium ion column, and (iv) performing phenyl membrane filtration;
(d) if the enzyme is not detected at a detectable level in step (b) or (c), proceeding to step (d); and
(e) conjugating the at least partially purified antibody via one or more sulfhydryl groups to a drug to produce the conjugated antibody.
2 . The method of claim 1 , wherein the CHO cell sulfhydryl oxidizing enzyme is QSOX1.
3 . The method of claim 1 , wherein the testing comprises identifying a band of 65-75 kDa on a gel.
4 . The method of claim 3 , wherein the band is identified by western blot or silver stain.
5 . The method of claim 1 , wherein the testing comprises a functional test for QSOX1 activity.
6 . The method of claim 5 , wherein the functional test produces hydrogen peroxide as an indicator of the activity.
7 . The method of claim 1 , wherein the drug is selected from anti-tubulin agents, DNA minor groove binding agents, DNA replication inhibitors, chemotherapy sensitizers and pyrrolobenzodiazepine dimer.
8 . The method of claim 1 , wherein the antibody is an antibody produced in CHO cells.
9 . 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.
10 . The method of claim 1 , wherein the salt wash has a concentration of 150-500 mM NaCl.
11 . The method of claim 1 , wherein the depth filtration is performed at 230 L/m 2 /hr and uses at least 15 L/m 2 of an equilibration buffer with a pH of 7.5-8 and NaCl concentration of 50-100 mM.
12 . The method of claim 1 , wherein the anion exchange with a quaternary ammonium ion column is performed with a buffer having a pH of 7.5-8 and a conductivity of less than or equal to 11 mS/cm.
13 . The method of claim 1 , wherein the phenyl membrane filtration is performed using a buffer having a pH of 6-8 and 0.3-0.4M sodium citrate.
14 . The method of claim 1 , wherein the non-detectable level is a level less than 0.5 μg/ml or 33 ppm.
15 . A method of producing a conjugated antibody, comprising purifying an antibody from a culture of CHO cells by loading the antibody on a protein A column with a salt wash, 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.
16 . The method of claim 15 , wherein the salt wash has a concentration of 150-500 mM NaCl.
17 . A method of producing a conjugated antibody, comprising purifying an antibody from a culture of CHO cells by performing depth filtration, 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.
18 . The method of claim 17 , wherein the depth filtration is performed at 230 L/m 2 /hr and uses at least 15 L/m 2 of an equilibration buffer with a pH of 7.5-8 and NaCl concentration of 50-100 mM.
19 . 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, 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.
20 . An antibody preparation, wherein an amount of CHO cell sulfhydryl oxidizing enzyme in the preparation is present at a level less than 0.5 μg/ml or 33 ppm, and wherein the 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).