WO2005066333A1

WO2005066333A1 - Stabilizing compositions for recombinant viruses

Info

Publication number: WO2005066333A1
Application number: PCT/US2004/044028
Authority: WO
Inventors: Mike Fletcher
Original assignee: Aventis Pasteur, Inc.
Priority date: 2003-12-30
Filing date: 2004-12-30
Publication date: 2005-07-21

Abstract

This invention relates to preparations of viruses, e.g. for vaccine or other pharmaceutical or research use, to their stabilization, and to processes of producing such preparations, as well as to their use, e.g. as vaccines or as virus vectors.

Description

STABILIZING COMPOSITIONS FOR RECOMBINANT VIRUSES

RELATED APPLICATIONS This application claims priority to U.S. Ser. No. 60/533,317 filed December 30, 2003.

FIELD OF THE INVENTION This invention relates to preparations of viruses, e.g. for vaccine or other pharmaceutical or research use, to their stabilization, and to processes of producing such preparations, as well as to their use, e.g. as vaccines or as virus vectors.

BACKGROUND OF THE INVENTION Numerous methods are known for producing live virus preparations for vaccine and other purposes. Compositions and methods useful in freezing, lyophilizing, or otherwise storing viable virus preparations for laboratory or vaccine use in order to preserve their activity are also known. These compositions typically contain multiple components including amino acids, peptides, proteins, carbohydrates, and buffers. Certain of these compositions are briefly described below. U.S. Pat. No. 6,258,362 provides a virus for use a vaccine in a pharmaceutical composition including polysaccharide, e.g. dextran, and/or a source of mixed aminoacids of vegetable or bacterial origin, a buffer, and a mono- or oligo-saccharide or derivative thereof. U.S. Pat. Nos. 6,225,289 and 6,514,943 describe a virus-preserving composition consisting of a liquid carrier with a polysorbate 80, L-arginine, polyvinylpyrrolidone, or trehalose stabilizing agent that can be maintained at a temperature above 0° C. for a significant period of time while maintaining a satisfactory degree of viral activity. U.S. Pat. No. 5,616,487 relates to a stabilized retrovirus composition comprising a retrovirus and an alpha-hydro-omega. -hydroxypoly(oxyethylene) poly(oxypropylene)poly(oxyefhylene) block copolymer. U.S. Pat. No. 4,338,335 and EP 0028563 describe a stabilizer for liquid vaccines containing partially hydrolyzed gelatin, a monosaccharide or disaccharide, a cell culture medium, L-glutamic acid, L-arginine and buffer to maintain pH at from about 6.0 to about 6.5. EP 0295043 describes a stabilizer for measles, mumps or rubella virus including as a stabilizing agent lactose, saccharose, D-sorbitol, sodium glutamate and gelatin hydrolysate. EP 0252059 describes stabilizers containing lactose, sorbitol, dextran, casein hydrolysate, L-glutamate, EDTA and buffer at a pH 6.7-7.2. WO 96/29096 describes production of recombinant virus vectors with at least one additive selected among arginine, glutamic acid (or sodium salt thereof), serine, glucose, inositol, lactose, mannitol, sorbitol, trehalose and xylose. U.S. Pat. No. 4,985,244 describes stabilized measles, mumps or rubella virus vaccine stabilized with lactose, saccharose, D-sorbitol, sodiumj>lutamate and relatin hydrolyzate. U.S. Pat. No. 4,622,222 describes lyophilized duck virus hepatitis vaccine lyophilized in the presence of collidone, gelatin, glucose and sucrose. U.S. Pat. No. 4,500,512 describes live yellow fever virus vaccines with stabilizers comprising phosphate buffer, calcium and magnesium ions, lactose, sorbitol and amino acid selected from histidine, alanine, valine, threonine, arginine, methionine, hydroxyproline, lysine, isoleucine, phenylalanine, serine, preferably histidine and alanine. U.S. Pat. No. 5,792,643 demonstrates the stabilization of recombinant viruses using a saccharide, high molecular weight structural additive, a buffer and water. WO 93/18790 describes the lyophilization of viral vaccines in the presence of a modified starch as a protectant. JP06234659 and U.S. Pat. No. 6,039,958 disclose stabilized live varicella virus vaccines containing a stabilizer free from calcium and magnesium ions and including gelatin, a gelatin hydrolysate, or a chelating agent such as EDTA. JP06234659-A describes a herpesvirus vaccine suspended in a solution containing NaCl, KC1, Na2HPO4, KH2PO4, sucrose, L-glutamate, gelatin, gelatin hydrolysate and EDA-3Na. WO 98/28000 describes measles, mumps, rubella, VZV or HSV vaccine formulations including a 6-carbon polyhydric alcohol, a disaccharide and a buffer. U.S. Pat. No. 3,915,794 describes stable virus preparations in a buffered aqueous solution of pH 6.5-7.5 and containing polyvinylpyrrolidone, sugar, glutamate and chelating agent. U.S. Pat. No. 4,147,772 describes a lyophilised live virus vaccine in a composition containing partially hydrolysed gelatin, a 6-carbon polyhydric alcohol, cell culture medium and acidic buffer. It remains desirable to provide further forms of stabilized virus preparations, e.g. for vaccine use. In addition, it is desirable to provide formulations for virus stabilization that are not derived from animal-based products due to concerns relating to animal-borne diseases (i.e., "mad cow" disease). It is further desirable to provide high-titer low volume formulations suitable to rapid freeze-drying treatments. Such formulations, compositions, and methods for using the same lare described herein.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Comparison of the stabilizing effects of the new stabilization buffer to former buffers.

SUMMARY OF THE INVENTION The present invention provides a method and stabilizing composition ("stabilizer") for preserving viruses, such as viral vectors. The present invention provides a method for preserving a virus comprising preparing a stabilized liquid composition comprising a virus, a liquid carrier, and at least one stabilizing agent. In one embodiment, the invention provides a stabilizing composition useful for long- term storage of viral vectors at temperatures below about 10°C. In another embodiment, a further stabilizing composition is provided for adjusting titer and/or product volume prior to freeze-drying and packaging of the virus.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides compositions for stably storing and preserving the activity of viruses, including recombinant viruses, for use as expression vectors and/or vaccines. The compositions are useful in methods of preparing and storing such viruses without a significant decrease in viral activity. A significant decrease in viral activity is considered a drop in activity of more than about 10-20%. The term "activity" is used herein with reference to viability of the virus. The present invention can be utilized to preserve (e.g., store) "active" and/or "inactive" viruses. The terms "activity", "active" and "inactive" refers to any suitable measure of the viability of a composition of a virus. Numerous measurements of virus activity, including but not limited to infectivity assays or expression analyses, are known in the art and can be used within the context of the present invention. The virus preparation is at least temporarily maintained in a composition that is in liquid form. In certain cases, it may be desirable to freeze-dry or otherwise process the virus preparation. With respect to the liquid, it is preferred that the liquid composition is a pharmaceutical composition. The pharmaceutically acceptable carrier can be a liquid carrier that contains a buffer (i.e., Tris) and a salt. Examples of suitable buffers_^ and salts, as well as other types of pharmaceutically acceptable carriers, are well known in the art. The temperature at which the liquid composition is maintained can be any suitable temperature to maintain the virus in its desired state over the time period of storage. Typically, the liquid composition is maintained at a temperature below 10 degrees C, (i.e., 5°C, -35°C). It is preferred that the stabilizing composition aid in maintaining the virus in the desired state (i.e., active, inactive) for various periods of time up to an including several weeks and/or months. The liquid composition can possess any suitable pH. A suitable pH in the context of the present invention is any pH where the virus is maintained in the liquid composition in a state capable of being later used for its intended purpose (i.e., active, inactive). The pH of the liquid composition desirably is about 6-9, 6-8.5, 6.5-8.5, 7- 8.5, 7.5-8.5, 6-8, 6.5-8, 7-8, 7.5-8, or 7- 7.5. It is generally preferred that the liquid composition have a pH somewhat above 7 but below about 8.0. The liquid composition can be placed (e.g., maintained or stored) in any suitable container. Typically, the container will comprise, consist essentially of, or consist of glass or plastic. Many different viruses may be utilized in practicing the present invention. Suitable viruses include, for example, Adeno viruses, Arbo viruses, Astro viruses, Bacteriophages, Enteroviruses, Gastroenteritis Viruses, Hantavirus, Coxsackie viruses, Hepatitis A Viruses, Hepatitis B Viruses, Hepatitis C Viruses, Herpesviruses (for example, Epstein Barr Virus (EBV), Cytomegalovirus (CMV) and Herpes Simplex Virus (HSV)), Influenza Viruses, Norwalk Viruses, Polio Viruses, Chordopoxviridae (i.e., Orthopoxvirus, vaccinia, MNA, ΝYVAC, Avipoxvirus, canarypox, ALNAC, ALNAC(2), fowlpox), Rhabdoviruses, Reoviruses Rhinoviruses, Rotavirus, Retroviruses, Baculoviridae, Caliciviridae, Caulimoviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Νodaviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Parvoviridae, Phycodnaviridae, Picomaviridae, and Togaviridae, and modified viruses originating from, based upon, or substantially similar to any of the foregoing or other suitable virus. Preferred viruses for use in practicing the present invention are poxviruses, in particular ALNAC. Other suitable viruses are known in the art as described in, for example, Fields et al., Virology (34th ed., Lippincott Williams & Wilkins (2001)). It is preferred that the stabilizer of the present invention include several components which together provide a stabilizing effect to a virus. With respect to recombinant viruses based on ALVAC, these have typically been stored as freeze- dried pellets containing sucrose, hydrolysates of casein and/or collagen, and phosphate-buffered physiological saline. These pellets are then re-hydrated in a pharmaceutically acceptable solution such as 0.4-0.9% ΝaCl. The new stabilizers, which are useful for storing viral vectors such as ALVAC recombinants, among others, includes in various combinations and amounts, a sugar having cryopreservation abilities (i.e., sucrose, sorbitol), a salt (i.e., sodium glutamate), a buffer (i.e., Tris), a dispersing agent to maintain product appearance and functionality (i.e., polyvinyl pyrrolidone), essential and non-essential amino acids to preserve viablity, and urea to provide increased thermal stability to the final product. In certain embodiments, the liquid composition may include other substances, for example other stabilizing agents, buffers, or carriers. Preferably, the pH of the stabilizer is about 7.25. In one embodiment, composition A comprising a sugar having cryopreservation abilities (i.e., sucrose, sorbitol), a salt (i.e., sodium glutamate), a buffer (i.e., Tris), a dispersing agent to maintain product appearance and functionality (i.e., polyvinyl pyrrolidone), essential and non-essential amino acids to preserve viablity, and urea to provide increased thermal stability to the final product provides the ability to stably store viral vectors at temperatures below 10°C for long periods of time (i.e., three months). In another embodiment, composition B is provided and contains approximately the same components as composition A but at about one-half the concentration as composition A. Composition B is useful for adjusting titers and/or volume prior to filling and/or freeze-drying. The new stabilizer provided herein allows for low-volume, high titer compositions of recombinant viral vectors. This allows for more efficient handling including but not limited to faster lyophilization processes. The present invention is further described in the following examples. The examples serve only to illustrate the invention and are not intended to limit the scope of the invention in any way.

EXAMPLES A novel stabilizer was developed and optimized in order to accommodate accommodate a reduction in the freeze-drying cycle of ALNAC-based expression vectors from 72 hours to 24 hours. As such, a product with the same titer parameters could be produced in a fill volume one third that of the original formulation (the fill volume being reduced from l.OmL to 0.3mL). The stabilizer is prepared as shown in Table I as follows. The amino acid mixes (essential and non-essential) are made up as solutions in WFI to the required concentrations. The remaining ingredients (which are all powders) are weighed out and added to the mixing vessel. The amino acid solutions are added with stirring. The pH is adjusted as required (approximately 7.2) and then made up to volume with WFI. The stabilizer is sterile filtered before use.

TABLE I INGREDIENT CONCENTRATION INGREDIENT CONCENTRATION

Sucrose <20g/L L-threonine 2400mg/L

Sodium Glutamate <2g/L L-tryptophan 400mg/L

Tris Buffer 14.52g/L L-valine 2350mg/L

Sorbitol <20g/L Non-essential Amino Acids in WFI <150g/L

Polyvinyl Pyrrolidone (PVP) 40 <4g/L L-alanine 890mg/L

Essential Amino Acids in WFI <150g/L L-asparagine 1500mg/L L-arginine hydrochloride 2100mg/L L-aspartic acid 1330mg/L L-cystine 1200mg/L L-glutamic acid 1470mg/L L-tyrosine 1800mg/L L-proline 1150mg/L L-histidine (free base) 800mg/L L-serine 1050mg/L L-isoleucine 2600mg/L glycine 750mg/L L-leucine 2600mg/L Urea <6g/L L-lysine hydrochloride 3650mg/L pH adjusted to 7.25±0.10 L-methionine 750mg7L ^"WFI q.s. to lOOϋmL L L-phenylalanine 1650mg/L 1 As shown in Figure 1, this stabilizer is at least as good as previously utilized stabilizers and provides the above-described advantages. All of the references cited herein, comprising patents, patent applications, and publications, are hereby incorporated in their entireties by reference. While this invention has been described with an emphasis upon preferred embodiments, it will be understood by those of ordinary skill in the art that variations of the preferred embodiments may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention comprises all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

CLAIMSWhat is claimed is:

1. A stabilizer for maintaining a virus in a desired state, the stabilizer comprising at least one sugar, at least one salt, a buffer, a dispersing agent, essential amino acids, non-essential amino acids, and urea, where the stabilizer has a pH of approximately 7.2.

2. The stabilizer of claim 1 wherein the sugar is sucrose or sorbitol.

3. The stabilizer of claim 1 wherein the salt is sodium glutamate.

4. The stabilizer of claim 1 wherein the buffer is Tris.

5. The stabilizer of claim 1 wherein the dispersing agent is polyvinyl pyrrolidone.

6. The stabilizer of claim 5 wherein the dispersing agent is polyvinyl pyrrolidone 40. 5. The stabilizer of claim 1 wherein the sugar is sucrose or sorbitol, the salt is sodium glutamate, the buffer is Tris, and the dispersing agent is polyvinyl pyrrolidone.

6. The stabilizer of claim 1 as shown in Table 1.

7. A composition comprising a stabilizer as claimed in any one of claims 1-6 and a recombinant virus.

8. The composition of claim 7 wherein the recombinant virus is a poxvirus.

9. The composition of claim 8 wherein the poxvirus is a orfhopoxvirus or an avipoxvirus.

10. The composition of claim 9 wherein the orthopoxvirus is selected from the group consisting of vaccinia, MNA, and ΝYVAC.

11. The composition of claim 10 wherein the avipoxvirus is selected from the group consisting of canarypox, fowlpox, ALVAC and ALNAC(2).

12. A lyophilized composition of any one of claims 7-11.

13. A vaccine comprising a composition of any one of claims 7-12.