Method of producing an immunogenic composition

Patent metadata

Field	Value
Publication number	US-11957746-B2
Application number	US-201916980785-A
Country	US
Kind code	B2
Filing date	Mar 25, 2019
Priority date	Mar 26, 2018
Publication date	Apr 16, 2024
Grant date	Apr 16, 2024

Abstract

The present invention in particular relates to a method of producing an immunogenic composition exhibiting reduced virucidal activity, as well as to the immunogenic composition and uses thereof, wherein the method in particular comprises the steps of: (a) providing a mixture with a first liquid and a recombinant protein, (b) concentrating the recombinant protein in the mixture by removing a portion of the first liquid from the mixture, and (c) processing the solution resulting from step (b) by continuous diafiltration.

First claim

The invention claimed is:

1. A method of producing an immunogenic composition comprising a recombinant PCV2 ORF2 protein, wherein the method comprises the steps of:
(a)(i) providing a mixture containing
a first liquid,
recombinant PCV2 ORF2 protein and/or virus-like particles comprising a plurality of a recombinant PCV2 ORF2 protein, and
a vector comprising a nucleic acid sequence encoding said recombinant protein;
(ii) inactivating the vector by adding an inactivating agent to the mixture of step (i);
(iii) neutralizing the inactivating agent by adding a neutralizing agent to the mixture resulting from step (ii), wherein the neutralization is carried out in a first container, and wherein the mixture resulting from neutralizing the inactivating agent is transferred from the first container to a second container connected with a filter system, and wherein after transferring the mixture from the first to the second container a valve between the first container and the second container is closed, and the empty first container is filled with a second liquid different from the first liquid;

(b) concentrating the recombinant PCV2 ORF2 protein and/or said virus-like particles in the mixture resulting from step (a)(iii) by removing a portion of the first liquid from the mixture by circulating the mixture through the second container and the filter system until the concentrating is completed, and
(c) processing the solution resulting from step (b) by continuous diafiltration such that the concentration of the neutralized inactivating agent and/or the concentration of the neutralizing agent is decreased in the process solution, wherein said continuous diafiltration comprises:
feeding the solution into the filter system containing at least one filter, wherein the at least one filter comprises a filter membrane having a membrane pore size allowing the neutralized inactivating agent and/or the neutralizing agent to pass through while retaining the recombinant PCV2 ORF2 protein and/or said virus-like particles in the bulk flow,
discharging the permeate comprising the neutralized inactivating agent and/or the neutralizing agent,
adding the second liquid to the bulk flow at a rate equal to the permeate flow, wherein the valve between the first and the second container is opened and the second liquid is continuously led from the first container to the second container while the mixture is circulated through the filter system and the second container.

2. The method of claim 1 , wherein in step (c) the at least one filter comprises at least one flat sheet or at least one hollow fiber filter.

3. The method of claim 2 , wherein
the at least one flat sheet filter is at least one cassette filter.

4. The method of claim 1 ,
wherein the at least one filter comprises 2-8 filters or 5-7 filters,
or wherein each of the at least one filter has a total filter area of about 16-26 m 2 or of about 20-22 m 2 ,
or wherein the filter membrane has an average pore size that is smaller than the recombinant PCV2 ORF2 protein and/or said virus-like particles, or wherein the filter membrane has a molecular weight cut off of between about 200 kDa and about 500 kDa,
or wherein the filter membrane comprises a material selected from the group consisting of polyethersulfone, cellulose hydrate, regenerated cellulose, stabilized cellulose, cross-linked cellulose, cross-linked cellulose hydrate, cellulose acetate, polyamide, polyurethane, polypropylene, polysulfone, polycarbonate, nylon, polyimide, and combinations thereof,
or wherein the filter membrane comprises polyethersulfone, or wherein the filter membrane is a stabilized cellulose based membrane.

5. The method of claim 1 , wherein in step (b) and in step (c) the same filter system is utilized.

6. The method of claim 1 , wherein said first liquid comprises a portion of cell culture medium or consists of cell culture medium, and wherein the cell culture medium is insect cell culture medium,
or wherein the volume of the mixture resulting from step (a) is from 1000 L to 10000 L, or from 2000 L to 8000 L, or from 3000 L to 5000 L,
or wherein in step (b) said recombinant PCV2 ORF2 protein and/or said virus-like particles is finally concentrated at least 5×, preferably at least 8×, or 9× to 11×, in comparison to the volume of the mixture resulting from step (a).

7. The method of claim 1 , wherein the second liquid is a buffer solution or P-Saline or phosphate buffered saline (PBS),
wherein said P-saline comprises a buffer solution composed of 0.8-0.9% (w/v) NaCl dissolved in water and pH adjusted to 6.8-7.0,
or wherein in step (c) the total volume of the second liquid added to the bulk flow is at least 5×, at least 7× or at least 9×, the volume of the solution resulting from step (b), or wherein the total volume of the second liquid added to the bulk flow is about the volume of the mixture resulting from step (a).

8. The method of claim 1 , wherein said method further comprises the step of
(d) admixing the mixture remaining after step (c) with a further component selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and combinations thereof, and wherein the concentration of the recombinant PCV2 ORF2 protein and/or the concentration of said virus-like particles structures in the solution resulting from said admixing is about the concentration of the PCV2 ORF2 recombinant protein and/or said virus-like particles in the mixture resulting from step (a).

9. The method of claim 8 , wherein said recombinant PCV2 ORF2 protein comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.5%, or 100% sequence identity with the sequence of SEQ ID NO:1 or SEQ ID NO:2.

10. The method of claim 1 , wherein the vector is a recombinant virus or baculovirus, or wherein the nucleic acid sequence is a DNA sequence,
or wherein the inactivating agent is an aziridine compound or binary ethylenimine (BEI), or wherein the inactivating agent is added in a molar excess in relation to the vector,
or wherein the neutralizing agent is sodium thiosulfate or wherein the neutralizing agent is added in a molar excess in relation to the inactivating agent.

11. The method according to claim 1 , wherein the virucidal activity of the immunogenic composition resulting from said method is reduced by at least 20% as compared to an immunogenic composition mixture that has not undergone the concentrating of step (b) and the continuous diafiltration of step (c) of said method, or wherein the immunogenic composition produced by said method causes a loss of less than 1 log TCID 50 per mL of a live virus or of less than 0.7 log TCID 50 per mL of a live virus, when the live virus is mixed with the immunogenic composition for four or more hours at room temperature.

12. The method according to claim 8 , wherein the method further comprises the step of combining the mixture remaining after step (c) and/or step (d) with at least one additional antigen.

13. The method according to claim 12 , wherein the at least one additional antigen is Porcine Reproductive and Respiratory Syndrome (PRRS) virus.

14. An immunogenic composition obtained by the method according to claim 12 .

15. The immunogenic composition according to claim 14 , for use in a method of
inducing a protective immune response against at least one pathogen or
reducing one or more clinical signs of at least one pathogen infection
in an animal, wherein the at least one pathogen is selected

Assignees

• Boehringer Ingelheim Animal Health Usa Inc

Inventors

• Wilmes Leigh
• Coons Matthew
• Brown Amanda
• Gassel Michael Johannes
• Orveillon Francois-Xavier
• Toepfer Katharina Hedwig
• Bautista Elida
• Schlesinger Kathy

Classifications

• C12N2710/14034

  Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein · CPC title

• C12N2710/14063

  by chemical treatment · CPC title

• C12N2710/14043

  viral genome or elements thereof as genetic vectore · CPC title

• C12N2750/10034

  Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein · CPC title

• A61K2039/5256

  expressing foreign proteins · CPC title