Method for influenza virus protection

a technology of influenza virus and purification method, which is applied in the field of influenza virus protection, can solve the problems of high mortality rate of epidemics, significant fall in the mortality rate of high-risk populations, and high contamination of vaccines, so as to achieve simple process scale up, reduce contamination level, and maintain influenza virus potency. effect of immunogenicity and/or infectivity

Inactive Publication Date: 2010-06-24
NANOTHERAPEUTICS INC +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0022]The process of the invention is suitable for the purification of laboratory and commercially useful quantities of influenza virus, e.g. either for vaccine or as viral vector use. The invention advantageously avoids problems associated with existing methods of purifying influenza virus and relies on ultra filtration and chromatographic techniques which enable simple scale up of the process.
[0023]The process of the invention accomplishes to purify influenza virus particles from cell lysate employing optionally ultra filtration, followed by a step in which virus is bound and subsequently eluted from the chromatographic support and a final step in which virus is purified out of remaining impurities and at the same time changing the buffer in which virus was prior to the step. The present purification process greatly reduces the level of contaminants from influenza virus sample and enables the application of purified influenza virus for human use.
[0025]The process of the invention is advantageous because due to the mild conditions the potency of the influenza virus, such as immunogenicity and / or infectivity can be substantially maintained. According to a preferred embodiment of the invention, the elution buffer comprises ≦1M NaCl, preferably ≦0.8M NaCl, preferably ≦0.5M NaCl, alternatively ≦0.3M NaCl. Likewise it is possible to preserve the structural integrity of the influenza virus or its derivative when employing the process of the invention. When employing the process of the invention the influenza virus or its derivative is substantially free of DNA so that treatment with deoxyribonucleases (DNAses) can be avoided.

Problems solved by technology

Influenza A and B viruses are important respiratory pathogens, although influenza A viruses are the main cause of epidemics with high mortality rate.
Since introduction in 1940s vaccines based on virus material cultured in chicken eggs have been found to be clearly effective against influenza infection and have resulted in a significant fall in the mortality rate of high risk population.
Initially vaccines were highly contaminated by egg derived components and were highly pyrogenic and lacking in efficacy.
The patent application discloses a purification of influenza virus only from allantoic fluid and does not address virus recovery in terms of virus infectivity which remains unknown.
These properties influences down stream process significantly, since large column dimensions are needed and only a low flow rate can be used, all resulting in rather low productivity of the process.
These current methods for the purification of influenza viruses are cumbersome and do not easily allow fast large scale production of virus stocks for vaccines of requested purity.
New influenza epidemics and pandemic are likely to occur in the future and current egg-based vaccine production technology seems to be unable to respond to a pandemic crisis.
But the existing purification of influenza virus vaccine cannot follow this demand.

Method used

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  • Method for influenza virus protection
  • Method for influenza virus protection
  • Method for influenza virus protection

Examples

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example 1

[0079]This example demonstrates the preferred influenza virus purification method of the present invention using tangential flow filtration, anion exchange chromatography, size exclusion chromatography and sterile filtration followed each other in this order.

[0080]Influenza virus reassortment of A / PR / 8 / 34 Mt. Sinai with deletion in NS1 gene and IVR-116 was cultivated on Vero cells in Gibco™ OptiPRO™ SFM medium. Approximately 15 000 mL of cell lysate was clarified by centrifugation at 2400 rpm at room temperature for 10 minutes. The supernatant was collected and filtered through a Sartobran P 500 cm2 capsule filter with a cellulose acetate membrane, 0.65-0.45 μm.

[0081]The filtrate was concentrated by tangential flow filtration using Proflux system (Millipore) and hollow fibre TFF cartridge with 750 kDa nominal cut-off and 0.12 m2 area (GE Healthcare, UFP-750-E-5A). The trans-membrane pressure was 0.2 to 0.3 bar. The final volume of concentrate was 1400 ml.

[0082]The concentrate was fu...

example 2

[0090]In this example three different anion exchange functional groups are compared. The example illustrates that different anion exchange functional groups can be used for the purification of influenza virus particles.

[0091]Influenza virus reasortment of A / PR / 8 / 34 Mt. Sinai with deletion in NS1 gene and IVR-116 was cultivated on Vero cells in Gibco™ OptiPRO™ SFM medium. The cell lysate was clarified by centrifugation at 2400 rpm at room temperature for 10 minutes. The supernatant was collected and concentrated by tangential flow filtration using a Labscale TFF system (Millipore) and a Pellicone Biomax 300 kDa cut-off membrane (Millipore, PXB3 00C 50). The concentrate was used for comparison of three CIM® disks monolithic columns: CIM® QA disk, CIM® DEAE disk, and CIM® EDA disk monolithic column (BIA Separations). Following buffers were used:

[0092]Buffer A: 50 mM HEPES (pH=7.5), Buffer B: 50 mM HEPES / 0.3M NaCl (pH=7.5), Buffer C: 50 mM HEPES / 1.5M NaCl (pH=7.5).

[0093]On each column 6...

example 3

[0094]In this example three different types of chromatographic support are compared. The example illustrates that the porous particle support, membranes and monoliths can be used for the purification of influenza virus.

[0095]Influenza virus reasortment of A / PR / 8 / 34 Mt. Sinai with deletion in NS1 gene and IVR-116 was cultivated on Vero cells in Gibco™ OptiPRO™ SFM medium. The cell lysate was clarified by centrifugation at 2400 rpm at room temperature for 10 minutes. The supernatant was collected and concentrated by a tangential flow filtration using Labscale TFF system (Millipore) and a Pellicone Biomax 300 kDa cut-off membrane (Millipore, PXB3 00C 50). The concentrate was used for the comparison of three different chromatographic supports: CIM® QA disk monolithic column (BIA Separations), Q Sepharose XL virus licensed (GE Healthcare), and Mustang Q coin (Pall). The following buffers were used:

[0096]Buffer A: 50 mM HEPES (pH=7.5), Buffer B: 50 mM HEPES / 0.3M NaCl (pH=7.5), Buffer C: 5...

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Abstract

A process for the purification of influenza virus or derivative thereof comprising the steps of:providing a source having influenza virus or derivative thereof;optionally subjecting the source to a prepurification step;followed by at least one chromatographic step on chromatographic materials selected from the group consisting of porous particles having mean pore sizes of at least 20 nm, perfusion particles, gel-in-a-shell particles, tentacle like particles, membrane adsorbers, and monoliths;collecting eluting influenza virus or derivatives thereof containing fractionswith the proviso that sulfuric ester of cellulose or cross-linked polysaccharides are excluded.

Description

[0001]The invention pertains to a process for the purification of influenza virus or derivative thereof, a process for the manufacturing of influenza virus or its derivative, a fraction of influenza virus or derivative thereof, a vaccine as well as a vector comprising the influenza virus or its derivative.FIELD OF THE INVENTION[0002]The present invention relates to the purification of different quantities of influenza virus or derivative thereof. In particular, the invention provides a quick, efficient and scalable process for the purification of commercial quantities of influenza virus which can be applied for human usage e.g. prophylactic application such as vaccination.BACKGROUND OF INVENTION[0003]Influenza viruses belong to the family Orthomyxoviridae and are separated into types A, B and C according to antigenic differences. Influenza A and B viruses are important respiratory pathogens, although influenza A viruses are the main cause of epidemics with high mortality rate.[0004]...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/145C12N7/02C12N7/00
CPCC12N2760/16051C12N7/00B01D15/363B01D15/426A61K39/145B01D36/003C12N2760/16021C12N2760/16034
Inventor PETERKA, MATJAZSTRANCAR, ALESBANJAC, MARKOKRAMBERGER, PETRAROTHL, ELISABETHMUSTER, THOMAS
Owner NANOTHERAPEUTICS INC
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