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.

Claims

1.-12. (canceled)

13. A method for disrupting influenza virions, the method comprising steps of: (i) obtaining a composition comprising influenza virions prepared from a cell culture, (ii) inactivating the influenza virions to obtain inactivated virions, and (iii) splitting the inactivated virions in the presence of a buffer having an ionic strength of from 100 mM to 800 mM, wherein the buffer comprises phosphate.

14. The method of claim 13, wherein the influenza virions are from an influenza A virus.

15. The method of claim 13, wherein the influenza virions are from an influenza A virus of H5 hemagglutinin subtype.

16. The method of claim 13, wherein the cell culture is a mammalian cell culture or an avian cell culture.

17. A method for manufacturing an influenza vaccine, the method comprising steps of: carrying out the method of claim 13, obtaining a bulk antigen preparation from the disrupted virions of step (iii), and formulating the bulk antigen preparation into an influenza vaccine.

18. The method of claim 17, wherein the influenza virions are from an influenza A virus.

19. The method of claim 17, wherein the influenza virions are from an influenza A virus of H5 hemagglutinin subtype.

20. The method of claim 17, wherein the cell culture is a mammalian cell culture or an avian cell culture.

21. The method of claim 17, wherein the influenza vaccine contains less than 10 ng host cell DNA.

22. The method of claim 17, wherein the influenza vaccine is a monovalent or trivalent influenza vaccine.

23. The method of claim 17, wherein the influenza vaccine comprises hemagglutinin from at least one B strain.

24. The method of claim 17, wherein the influenza vaccine comprises from about 3.75 μg to about 15 μg hemagglutinin, per strain, per dose.

25. The method of claim 17, wherein the step of formulating comprises combining the bulk antigen preparation with an adjuvant.

26. The method of claim 25, wherein the adjuvant is an oil-in-water emulsion.

27. The method of claim 13, wherein the buffer has an ionic strength of from 100 mM to 200 mM.

28. The method of claim 27, wherein the buffer has a pH of from 6.5 to 8.5.

29. The method of claim 13, wherein the buffer has an ionic strength of at least 200 mM and a pH of from 6.5 to 8.5.

30. The method of claim 13, wherein the buffer has a pH of from 6.5 to 8.5.

31. The method of claim 13, wherein the buffer has a pH of from 7.0 to 8.0.

32. The method of claim 13, wherein the buffer has a pH of from 7.0 to 7.8.

33. The method of claim 13, wherein the method results in a higher than 20-fold increase in antigen yield, when compared to a method for disrupting influenza virions comprising corresponding steps (i)-(iii), except that step (iii) is performed in the presence of a Tris buffer.

34. The method of claim 13, wherein the method results in an increased antigen yield, when compared to a method for disrupting influenza virions comprising corresponding steps (i)-(iii), except that the buffer in step (iii) has an ionic strength of less than 100 mM.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0190] FIGS. 1 and 2 both show SDS-PAGE analysis of five stages during antigen purification. The five lanes, from left to right, show proteins present at the following stages: (i) after β-propiolactone inactivation; (ii) after CTAB splitting; (iii) after 15 hours of CTAB removal by adsorption; (iv) after adsorption is complete; and (v) after post-adsorption sterile filtration.

MODES FOR CARRYING OUT THE INVENTION

[0191] A H5N1 strain of influenza A virus was successfully grown on MDCK cells. An initial attempt to prepare purified surface glycoproteins from this strain used a process that had previously been used successfully with a H1N1 strain, but the process performed very badly. More than 95% of viral HA had been lost after the CTAB-based splitting step, and the step used to remove CTAB also removed more than 75% of HA. Furthermore, the final HA was antigenically damaged and much of it was not detectable by SRID. Thus the H1N1 process was modified.

[0192] Virus growth and harvest were not changed. After harvest, however, ultrafiltration was used to concentrate the virions 5-fold (hollow-fiber PES membrane, 500 kDa cut-off), and the concentrated virion suspension was then diafiltered into a 10 mM phosphate buffer (pH 7.0±0.1). Virions were purified (concentrated) using a CS resin (equilibrated in the same 10 mM phosphate buffer). In a further ultrafiltration/diafiltration step a new buffer was used. Rather than use a Tris buffer, as in the H1N1 process, a phosphate buffer was used (buffer ‘A’: 50 mM phosphate, pH 7.5) for equilibration and diafiltration. Moreover, unlike the H1N1 process this buffer did not include polysorbate 80.

[0193] Purified virions were then inactivated using β-propiolactone. Unlike the H1N1 process, however, the β-propiolactone was prepared in buffer ‘A’.

[0194] Inactivated virions were then split using CTAB, but again the CTAB was prepared in buffer ‘A’. Moreover, rather than performing splitting in a low concentration of Tris buffer, it was performed in 200 mM NaCl with 50 mM phosphate, pH 7.5. Thus the ionic strength and buffer system used during splitting were both changed.

[0195] Ultracentrifugation was used to prepare surface antigens in the same way as the H1N1 process, but a subsequent step of CTAB removal (by adsorption) was altered. Whereas the CTAB adsorbent in the H1N1 process was equilibrated in a Tris buffer, for H5N1 it was equilibrated in buffer ‘A’, supplemented by 2.5 g/L of polysorbate 80. Addition of polysorbate 80 at this stage substituted for the pre-inactivation addition of polysorbate 80 in the H1N1 process.

[0196] Subsequent purification steps were the same, except again that Tris buffers were replaced by buffer ‘A’, and that the final ultrafiltration membrane was changed to reduce hydrophobicity and to decrease its size exclusion limit, with a hydrophilic cellulose acetate membrane with low intrinsic protein adsorption characteristics being selected.

[0197] FIGS. 1 and 2 show the effects of these changes on antigen purification. Whereas in FIG. 1, which shows the results of using the H1N1 process on the H5N1 virus, the amount of HA antigen is vastly decreased after splitting, the same drop is not seen in FIG. 2. Moreover, high levels of HA remain present during the subsequent purification steps.

[0198] Whereas the H1N1 process gave a final HA concentration of <10 μg/mL when performed on the H5N1 virus, the modified process provided a final concentration of 505 μg/mL i.e. a >50-fold improvement. Total HA recovery, as measured by SRID, was assessed as 36%. HA purity was very good. Residual CTAB concentration was <0.05 μg per μg of HA.

[0199] The same process is also useful for treating other influenza A viruses e.g. H1N1 strains. A modified process is also useful, in which polysorbate 80 is added after the CS chromatography step but prior to splitting, or after splitting but before CTAB removal. If the amount of polysorbate 80 added in either of these steps was less than 1.5 g/L then further polysorbate 80 is added in the subsequent purification steps, but prior to the final ultrafiltration.

[0200] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

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