Methods of producing immunoconjugates
The present invention provides methods of preparing active immunoconjugates, including anti-CD22 immunoconjugates. Suitably, the methods include a fed-batch refolding process and/or column stripping process that result in an increase in yield of the immunoconjugate over other processes that do not utilize the methods.
1. A method of preparing an active immunoconjugate, wherein said immunoconjugate is composed of two polypeptide chains joined by a disulfide bond, the method comprising refolding said immunoconjugate in a fed-batch refolding process in a refold buffer having a pH of 9.5 or less, and purifying the refolded immunoconjugate using a two cycle elution on an ion exchange column, wherein the column is stripped between a first elution and a second elution with a stripping buffer comprising arginine, urea and dithiothreitol (DTT), wherein the stripping buffer comprises 0.25M to about 0.75 M arginine, 7M to about 9 M urea and 9 mM to about 11 mM DTT.
2. The method of claim 1 , wherein an amount of the immunoconjugate recovered from the method of preparation is at least three-hundred % (300%) greater than an amount of the immunoconjugate recovered utilizing a method that does not comprise a fed-batch refolding process and/or purification on an ion exchange column that has been stripped using the stripping buffer.
3. The method of claim 2 , wherein the polypeptide or immunoconjugate comprises an antibody or antigen binding fragment thereof.
4. The method of claim 3 , wherein the antibody or antigen binding fragment comprises a Fab, a Fab′, a F(ab′)2, a Fd, a single chain Fv or scFv, a disulfide linked Fv, a V-NAR domain, an IgNar, an intrabody, an IgGΔCH2, a minibody, a F(ab′)3 a tetrabody, a triabody, a diabody, a single-domain antibody, DVD-Ig, Fcab, mAb 2 , a (scFv)2, or a scFv-Fc.
5. The method of claim 2 , wherein the polypeptide or immunoconjugate comprises a toxin.
6. The method of claim 5 , wherein the toxin is a Pseudomonas exotoxin, or variant thereof.
7. The method of claim 6 , wherein said Pseudomonas exotoxin, or variant thereof has an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-22.
8. The method of claim 7 , wherein said Pseudomonas exotoxin, or variant thereof has the amino acid sequence of SEQ ID NO:22.
9. The method of claim 3 , wherein said antibody or antigen binding fragment thereof comprises a VH and a VL sequence.
10. The method of claim 9 , wherein said VH sequence is selected from the group consisting of SEQ ID NOs: 6-11.
11. The method of claim 9 , wherein said VL sequence is selected from the group consisting of SEQ ID NOs: 2, and 12-15.
12. The method of claim 2 , wherein the immunoconjugate comprises an anti-CD22 antibody or antigen binding fragment thereof and a PE or variant thereof.
13. The method of claim 12 , wherein the immunoconjugate is the Moxetumomab pasudotox immunotoxin comprising the VH-PE38 subunit of SEQ ID NO: 1 and the VL subunit of SEQ ID NO:2.
14. The method of claim 2 , wherein the refold buffer has a pH of 9.4.
15. The method of claim 2 , wherein the fed batch process uses an addition rate of about 52 mL of solubilized inclusion bodies per L of refold buffer per hour to about 13 mL solubilized inclusion bodies per L refold buffer per hour.
16. The method of claim 15 , wherein the fed batch process uses an addition rate of about 35 mL of solubilized inclusion bodies per L of refold buffer per hour to about 17 mL solubilized inclusion bodies per L refold buffer per hour.
17. The method of claim 16 , wherein the fed batch process uses an addition rate of about 30 mL of solubilized inclusion bodies per L of refold buffer per hour to about 18 mL solubilized inclusion bodies per L refold buffer per hour.
18. The method of claim 17 , wherein the fed batch process uses an addition rate of about 26 mL of solubilized inclusion bodies per L of refold buffer per hour.
19. The method of claim 15 , wherein the fed batch process occurs over a period of about 2 to about 8 hours.