EP-4736870-A2 - IMPROVEMENTS IN PREPARATION OF INFLUENZA VIRUS VACCINE ANTIGENS

Abstract

A number of improvements for preparing vaccine antigens from disintegrated influenza viruses are disclosed. A splitting step can be followed by detergent exchange. Splitting can take place in the presence of a buffer with a higher ionic strength and/or in the presence of phosphate buffer.

Inventors

• HAUSSMANN, CHRISTOPH
• HAUSCHILD, FRANK
• JOBST, BJOERN

Assignees

• Seqirus UK Limited

Dates

Publication Date

20260506

Application Date

20090318

Claims (15)

1. A process for preparing an influenza vaccine containing an adjuvant, wherein: (a) a hemagglutinin-containing antigen preparation is obtained from a virus grown in an MDCK cell line by a process in which influenza virions are disrupted, wherein the process for disrupting influenza virions, comprises steps of: (i) obtaining a composition comprising influenza virions in the absence of detergent; (ii) performing ultrafiltration or diafiltration on the virion-containing composition; (iii) adding a detergent to the filtered virion-containing composition without splitting the virions, wherein the amount of detergent added is less than 1.5g/L; (iv) inactivating the influenza virions; and (v) splitting the inactivated virions with a detergent; wherein further quantities of the same detergent which was used in step (iii) are added after step (v); and (b) the hemagglutinin-containing antigen preparation is used to prepare the vaccine.
2. The process of claim 1, wherein the detergent used in step (v) is different from the detergent used in step (iii).
3. The process of claim 1, wherein the further addition takes place prior to a further ultrafiltration/diafiltration step.
4. The process of any preceding claim, wherein the detergent added in step (v) is an ionic detergent, e.g. CTAB.
5. The process of any preceding claim, wherein the detergent added in step (iii) is polysorbate 80.
6. The process of any preceding claim, wherein the influenza virions are from an influenza A virus.
7. The process of any preceding claim, wherein the influenza virions are from an influenza A virus of (a) H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16 hemagglutinin subtype, (b) H2, H5, H7 or H9 hemagglutinin subtype, or (c) H5 hemagglutinin subtype.
8. The process of any preceding claim, wherein the influenza virions are prepared from MDCK cells grown in serum-free culture media and/or protein-free media.
9. The process of any preceding claim, wherein the vaccine includes antigen from 1, 2, 3, 4 or more influenza strains, including influenza A virus and/or influenza B virus.
10. The process of claim 9, wherein the vaccine includes two influenza A strains (H1N1 and H3N2) and one influenza B strain.
11. The process of claim 10, wherein the vaccine is a trivalent vaccine.
12. The process of any preceding claim, wherein the vaccine contains hemagglutinin at 7.5µg per strain or 15µg per strain.
13. The process of any preceding claim, wherein the adjuvant comprises an oil-in-water emulsion.
14. The process of claim 13, wherein the adjuvant is a submicron emulsion of squalene, Tween 80 and Span 85, for example, wherein the adjuvant is MF59.
15. The process of any preceding claim, wherein the vaccine is a purified surface antigen vaccine.

Description

This application claims priority from US provisional application 61/069,868 (filed March 18th 2008), the complete contents of which are incorporated herein by reference. TECHNICAL FIELD This invention is in the field of influenza vaccine preparation. BACKGROUND ART Influenza vaccines currently in general use are described in chapters 17 & 18 of reference 1. They are based on live virus or inactivated virus, and inactivated vaccines can be based on whole virus, 'split' virus or on purified surface antigens (including haemagglutinin and also, usually, neuraminidase). A variety of different procedures are known for preparing both split and surface antigen vaccines. For example, various splitting procedures are disclosed in references 2-8, and different methods for preparing surface antigen vaccines are disclosed in references 9-14 It is an object of the invention to provide further and improved methods for preparing split and surface antigen vaccines. DISCLOSURE OF THE INVENTION The inventor has devised a number of improvements to existing processes for preparing vaccine antigens from disintegrated influenza viruses. Modified timing of detergent use In an existing purification process, influenza virions are exposed to a first detergent (e.g. a polysorbate, such as polysorbate 80) prior to splitting by a second detergent (e.g. CTAB). The first detergent is used prior to virus inactivation, whereas the second detergent is added after inactivation. The second detergent is removed later in the process, but the first detergent remains, thereby facilitating antigen solubility. In contrast, according to a first aspect of the present invention the first detergent is used later in the process, and in particular after the second detergent has already been used for splitting virions. This change in timing and order has been associated with a >20-fold increase in antigen yield, in particular with a H5 subtype influenza A virus. Thus the invention provides a process for disrupting influenza virions, comprising steps of: (i) obtaining a composition comprising influenza virions in the absence of detergent; (ii) inactivating the influenza virions in the absence of detergent; (iii) splitting the inactivated virions with a reagent comprising a first detergent; and (iv) exchanging the first detergent for a second detergent. The detergent exchange step may occur any time after the splitting step, but ideally occurs prior to a step of virion capture e.g. by a process selected from affinity capture, pseudo-affinity capture, chromatography or adsorption (see below). Thus the second detergent may be added after splitting but before virion capture. Further quantities of the second detergent may be added after virion capture, particularly if the amount of second detergent previously added was less than 1.5g/L. Disrupted virions obtained by this process can be used for the preparation of influenza vaccines. The invention also provides, in a process for disrupting influenza virions, the improvements consisting of: inactivating influenza virions in the absence of detergent; splitting the inactivated virions with a reagent comprising a first detergent; and exchanging the first detergent for a second detergent after splitting. In alternative embodiments of the first aspect, the second detergent is added after inactivation but before splitting. Thus the invention provides a process for disrupting influenza virions, comprising steps of: (i) obtaining a composition comprising influenza virions in the absence of detergent; (ii) inactivating the influenza virions in the absence of detergent; (iii) adding a second detergent without splitting the inactivated virions; (iv) splitting the inactivated virions in the presence of the second detergent with a reagent comprising a first detergent; and (v) removing the first detergent while retaining the second detergent. Disrupted virions obtained by this process can be used for the preparation of influenza vaccines. The invention also provides, in a process for disrupting influenza virions, the improvements consisting of: inactivating influenza virions in the absence of detergent; splitting the inactivated virions with a reagent comprising a first detergent, in the presence of a second detergent; and removing the first detergent after splitting while retaining the second detergent. In a further alternative embodiment with modified timing of detergent use, a detergent is added prior to virion inactivation, but after an ultrafiltration/diafiltration step performed on virions. Thus the invention provides a process for disrupting influenza virions, comprising steps of: (i) obtaining a composition comprising influenza virions in the absence of detergent; (ii) performing ultrafiltration/diafiltration on the virion-containing composition; (iii) adding a detergent to the filtered virion-containing composition without splitting the virions; and (iv) inactivating the influenza virions. The process may include a fur