Methods of shifting an isoelectric profile of a protein product and uses thereof

Patent metadata

Field	Value
Publication number	US-9908932-B2
Application number	US-201514881799-A
Country	US
Kind code	B2
Filing date	Oct 13, 2015
Priority date	Oct 15, 2014
Publication date	Mar 6, 2018
Grant date	Mar 6, 2018

Abstract

Provided herein are methods shifting the isoelectric profile of a recombinant protein product and the use of such methods in the manufacture of recombinant protein products. Also provided are recombinant protein products produced by such methods.

First claim

What is claimed is:

1. A method of shifting the isoelectric profile of a recombinant protein product having seven protein subpopulations with an isoelectric point between about 5.45 and about 6.55, wherein the recombinant protein product is eculizumab and the method comprises:
(a) fed batch culturing mammalian cells comprising a nucleic acid encoding a recombinant protein in a production bioreactor under conditions sufficient to produce the product for a first period of time that consists of a growth phase or a growth phase and a stationary phase of the mammalian cells or that comprises a growth phase of the mammalian cells and terminates at a critical time point; and
(b) incubating the product under conditions sufficient to shift the isoelectric profile of the product toward a more acidic profile for a second period of time that consists of at least 6 hours of a decline phase of the mammalian cells, wherein the more acidic profile comprises an increase in the quantity of the fourth and fifth most acidic protein subpopulations of the seven protein subpopulations, and a decrease in the quantity of the first and second most basic protein subpopulations of the seven protein subpopulations, wherein the conditions sufficient to shift the isoelectric profile of the product towards a more acidic profile comprise (i) incubating in a dissolved oxygen (dO 2 ) concentration of between about 20% to about 45%; incubating in a medium having a pH of between about 7.0 and about 7.3; and/or (iii) incubating at a temperature of between about 30° C. and about 37.5° C.

2. The method of claim 1 , further comprising assaying the product to determine the isoelectric profile and repeating step (b) if the the isoelectric profile requires further shifting toward the more acidic profile when compared to a reference isoelectric profile.

3. The method of claim 1 , wherein the more acidic profile is an isoelectric profile, where:
the quantity of protein populating each of the second, third, and fourth most basic protein subpopulations of the seven protein subpopulations is ≧10% of the total mass of protein in the profile;
the quantity of protein populating each of the third and fourth most basic protein subpopulations of the seven protein subpopulations is less than the quantity of the second most basic protein subpopulation of the seven protein subpopulations;
the quantity of protein populating the most acidic protein subpopulation of the seven protein subpopulations is ≦3% of the total mass of protein in the profile;
the quantity of protein populating the second most acidic protein subpopulation of the seven protein subpopulations is ≦6% of the total mass of protein in the profile;
the quantity of protein populating the third most acidic protein subpopulation of the seven protein subpopulations is ≦9% of the total mass of protein in the profile;
the quantity of protein populating the most basic protein subpopulation of the seven protein subpopulations is ≦8% of the total mass of protein in the profile; and
there are no other protein subpopulations having a quantity of protein of ≦6% of the total mass of protein, other than the most acidic, the second most acidic, the third most acidic, and the most basic protein subpopulations of the seven protein subpopulations.

4. The method of claim 1 , wherein the dO 2 concentration is between about 20% to about 45%.

5. The method of claim 1 , wherein the dO 2 concentration is between about 30% and about 35%.

6. The method of claim 2 , wherein the assaying comprises isoelectric focusing electrophoresis.

7. The method of claim 6 , wherein the isoelectric focusing electrophoresis is capillary electrophoresis or gel electrophoresis.

8. The method of claim 1 , wherein the pH is between about 7.0 and about 7.25.

9. The method of claim 1 , wherein the pH is between about between about 7.0 and about 7.20.

10. The method of claim 1 , wherein the pH is between about 7.0 and about 7.15.

11. The method of claim 1 , wherein the temperature is between about 33° C. and about 37.5° C.

12. The method of claim 1 , wherein the conditions sufficient to shift the isoelectric profile of the product to a more acidic profile include incubating at an agitation rate of between about 200 RPM and about 400 RPM.

13. The method of claim 12 , wherein the agitation rate is between about 220 RPM and about 350 RPM, or between about 220 RPM and about 300 RPM.

14. The method of claim 1 , wherein eculizumab comprises a heavy chain comprising SEQ ID NO: 1 and light chain comprising SEQ ID NO: 2 or a heavy chain consisting of SEQ ID NO: 1 and a light chain consisting of SEQ ID NO: 2.

15. The method of claim 1 , further comprising formulating the product into a pharmaceutical composition.

16. The method of claim 2 , further comprising formulating the product into a pharmaceutical composition.

Assignees

• Alexion Pharma Inc

Inventors

• Malanson Hunter F
• Jaluria Pratik
• Alford Rachael

Classifications

• A61P7/00

  Drugs for disorders of the blood or the extracellular fluid · CPC title

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C07K16/18 Primary

  against material from animals or humans · CPC title

• G01N33/6854

  Immunoglobulins · CPC title

• C07K2317/76

  Antagonist effect on antigen, e.g. neutralization or inhibition of binding · CPC title