Removal of residual cell culture impurities

Patent metadata

Field	Value
Publication number	US-2016287693-A1
Application number	US-201415035302-A
Country	US
Kind code	A1
Filing date	Nov 6, 2014
Priority date	Nov 15, 2013
Publication date	Oct 6, 2016
Grant date	—

Abstract

The present application discloses methods for removing residual impurities from protein preparations. Such methods include addition of an anionic detergent to a solution comprising proteins of interest and cellular contaminants under non-precipitating conditions and passing the solution through an ion exchange column.

First claim

1 . A method for removing residual cellular DNA from a sample comprising a protein of interest, the method comprising the steps of:
a. adding an anionic detergent to a solution comprising a protein of interest under non-precipitating conditions; and, b. passing the solution through an ion exchange matrix, whereby the residual cellular DNA is bound to the ion exchange resin, so as to obtain an eluate comprising the protein of interest, wherein the eluate is substantially free of cellular DNA.

2 . A method for removing residual cellular DNA from a sample comprising viral protein, the method comprising the steps of:
a. providing a sample comprising virus grown in a host cell system, wherein the virus expresses viral protein of interest; b. splitting the virus; c. adding an anionic detergent to a solution comprising the viral proteins under non-precipitating conditions; and, d. passing the solution through an ion exchange matrix, whereby residual cellular DNA is bound to the ion exchange resin, so as to obtain an eluate comprising the viral protein of interest.

3 . A method for preparing an influenza vaccine composition comprising immunogenic proteins derived from a virus propagated on a cell culture, the method comprising the steps of:
a. growing a virus in cell culture, b. adding a fatty acid detergent to a solution comprising the immunogenic protein under non-precipitating conditions, and c. processing the immunogenic protein on an ion exchange matrix.

4 . A method for removing Influenza Nuclear Protein (NP) from a preparation comprising virus proteins of interest, the method comprising the steps of:
a. splitting a virus preparation derived from cell culture or eggs, b. adding an anionic detergent to the virus preparation under non-precipitating conditions, and c. processing the virus preparation through an ion exchange matrix, whereby the nuclear protein is bound to the ion exchange matrix.

5 . The method of claim 1 further comprising a clarification step.

6 . The method of claim 1 further comprising a concentration step.

7 . The method of claim 1 further comprising a depth filtration step.

8 . The method of claim 3 further comprising a splitting step prior to the detergent step (b).

9 . The method of claim 1 further comprising an inactivation step prior to the ion exchange step.

10 . The method of claim 8 wherein the splitting step comprises treatment with cetyltrimethylammonium bromide.

11 . The method of claim 1 , wherein the anionic detergent is not the same as other detergents used in the process.

12 . The method of claim 1 wherein the anionic detergent is a fatty acid detergent.

13 . The method of claim 1 wherein the anionic detergent is carboxylic detergent which comprises 4-10 carbons in length.

14 . The method of claim 13 wherein the carboxylic acid detergent is selected from the group consisting of caprylic(8), valeric(5), caproic(6), enanthic(7), pelargonic(9), capric(10) acid under conditions in which no precipitation of the viral proteins occurs.

15 . The method of claim 1 wherein the anionic detergent is present at a concentration of 25 mM-500 mM.

16 . The method of claim 9 , wherein the inactivating step comprises inactivation with betapropiolactone.

17 . The method of claim 1 , wherein the ion exchange resin is Sartobind Q.

18 . The method of claim 1 , wherein the ion exchange resin is Fractogel TMAE.

19 . (canceled)

20 . The method of claim 1 wherein the amount of the protein of interest recovered is at least 90%.

21 . An influenza vaccine comprising less than 5 ng residual DNA per dose at a fragment size of less than 200 bases, and less than 0.5 μg NP per 10 μg of HA.

22 . The influenza vaccine of claim 21 , further comprising an adjuvant.

23 . The influenza vaccine of claim 22 , wherein the adjuvant is selected from the group consisting of: alum adjuvants, oil-in-water adjuvants, and Toll-like receptor (TLR) agonists.

24 . A method for producing an influenza vaccine, the method comprising the steps of:
a. splitting a virus grown in host cells, b. adding an anionic detergent to a solution comprising viral protein of interest from the virus under non-precipitating conditions, c. passing the solution through an ion exchange matrix, whereby the residual cellular DNA is bound to the ion exchange resin, so as to obtain an eluate comprising the viral protein of interest; d. optionally further processing the eluate to provide a preparation comprising the viral protein of interest; and, e. carrying out sterile filtration of the preparation, and optionally filling and packaging.

Assignees

• Novartis Ag

Inventors

• Norman Carnley
• Suda Eric
• Dowless Kayla
• Astigarraga Ruiz
• Bastek Patrick
• Yannone Vaishali

Classifications

• A61K2039/53

  DNA (RNA) vaccination · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• A61K39/145 Primary

  Orthomyxoviridae, e.g. influenza virus · CPC title

• A61K39/12 Primary

  Viral antigens · CPC title

• C07K1/18 Primary

  Ion-exchange chromatography · CPC title