Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
2001-10-05
2004-05-11
Priebe, Scott D. (Department: 1635)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S320100
Reexamination Certificate
active
06734008
ABSTRACT:
The present invention relates to the preservation of adenoviruses in a stabilized and storable form and to the liquid or frozen compositions intended for this preservation.
The preservation of adenoviruses in stable compositions is a known problem, which has been the subject of many studies, without as a result giving truly satisfactory results up until now. Recombinant viruses can in fact be used only if they can be stored successfully without degradation and without loss of their infectivity.
The physical state of the adenoviral particles, in solution, undergoes two types of impairments simultaneously and rapidly over time: on the one hand, aggregation (coagulation) of the particles in the form of lumps or filaments, which is an extremely serious phenomenon because it is irreversible, and, on the other hand, disintegration of the icosahedral structure of the capsids themselves (by lysis of the capsids and release of the capsomers into the medium).
In International Application WO 98/02522, there has been described a method of preserving infectious recombinant viruses in the form of a suspension in an aqueous solution comprising sucrose. The recommended compositions do not contain glycerol and contain, according to a preferred aspect, at least one divalent cation or monovalent alkali metal cation salt.
However, it can be shown that sucrose does not systematically provide stabilization at +4° C., and depending on the cases, stabilization at this temperature never exceeds a period of 2 weeks, or is at best less than 2 months.
In J. Virology, 70(11), 7498-7509 (1996), there have been described adenovirus preservations in the form of buffer solutions based on Tris buffer and glycerol, but systematically containing magnesium chloride. These preparations require preservation at very low temperatures, of the order of −70° C. to avoid a reduction in their infectivity.
In Human Gene Therapy, 7, 1693-99 (1996), there is also used a formulation for storage based on Tris buffer and glycerol, and comprising magnesium chloride. This formulation is preserved at −80° C.
More generally, the Tris-based formulations described in the literature up until now systematically contain magnesium chloride (1 mM in general) and/or a salt at physiological concentration (generally NaCl at 150 mM). There may be mentioned, for example by way of illustration, [Cell, 68, 143-155 (1992); Nature Genetics, 3, 229-234 (1993); Human Gene Therapy, 6, 5-11 (1995); Human Gene Therapy, 6, 145-153 (1995); Human Gene Therapy, 6, 277-287 (1995); Human Gene Therapy, 6, 643-666 (1995); Human Gene Therapy, 6, 1039-1044 (1995); Human Gene Therapy, 6, 1317-1322 (1995); Human Gene Therapy, 6, 1587-1593 (1995); Human Gene Therapy, 7, 2177-2184 (1996); J. Virology, 73, 1601-1608 (1999)]. When specified by the authors, the preservation of the virus is systematically carried out at −70° C./−80° C.
It has since been shown that the variation in the viral structure (aggregation and lysis) is a phenomenon which is highly dependent on factors such as the concentration of certain cationic counterions added to the solution and also such as the adenovirus concentration. Above concentrations of about 1E12 vp/ml, the aggregation and disintegration of the capsids (and therefore the loss of infectivity) are observed within a few hours to a few days, depending on the pharmaceutical compositions used, whereas these phenomena become slower at low concentration. Because of this, it is particularly difficult to preserve for a prolonged period compositions with high concentrations of viral particles.
It has thus been found, and this is what constitutes the subject of the present invention, that infectious recombinant adenoviruses could be preserved in aqueous medium, at temperatures of between +4 and +20° C., in the form of a suspension in a buffer solution capable of maintaining the pH of the medium at slightly alkaline vales, supplemented with glycerol and without addition of divalent metal cations or of alkali metal cations.
More specifically, the pH of the medium is maintained between 8.0 and 9.6.
The liquid or frozen compositions containing the adenoviral particles in a buffer solution supplemented with glycerol also fall within the scope of the present invention. These compositions have the considerable advantage of a good stability, but also of being particularly suited to the preservation of high concentrations of viral particles for a prolonged period.
According to the invention, the buffer solution capable of maintaining the pH of the medium between 8.0 and 9.6 consists either of an acid/base system comprising Tris [tris(hydroxymethyl)-aminomethane], or lysine and an acid chosen from a strong acid (hydrochloric acid for example) or a weak acid (maleic acid, malic acid or acetic acid for example), or of an acid/base system comprising Hepes [2-(4-(2-hydroxyethylpiperazin)-1-yl)ethanesulphonic acid] and a strong base (sodium hydroxide for example).
Preferably, the pH is maintained between 8.4 and 8.8 and still more particularly at 8.4 (value measured at 25° C.).
The concentration of the buffer solution is determined so as to exert the buffering effect within a limit and in a volume where the pH value is not affected. The molar concentration of acid+base may vary from 10 to 500 mM, preferably from 20 to 100 mM, and more particularly it is maintained at 20 mM. Among the buffer systems according to the invention, the Tris/HCl buffer solution at a concentration of 20 mM gives particularly satisfactory results.
According to the invention, the composition contains 10 to 50% glycerol, and preferably between 20 and 25% (volume for volume).
The compositions according to the invention may, in addition, optionally contain other adjuvants. The latter may be chosen from polymers (polyethylene glycols, for example PEG 400, PEG 8000), the pluronics (Pluronic F68 for example), in particular in an amount of about 1 to 20% by weight, the polysorbates (in particular Tween-20, for example at 0.01 to 1% by weight), or chosen from sugars such as for example sucrose, mannitol or dextrose (in particular in an amount of about 5 to 10%) or alternatively from alcohols (in particular ethanol) in an amount of 1 to 10% by volume.
The compositions according to the invention are particularly suited to the preservation of adenoviral preparations of high concentration. Indeed, the compositions thus stabilized make it possible to maintain the viral particles in a liquid aqueous suspension (in particular between +4 and +20° C.) while preserving the infectivity of the virus, this being given at very high viral concentrations (from values of 1E8 vp/ml up to 1E12 vp/ml or up to 1E13 vp/ml and which may even be as high as 5E13 vp/ml).
According to another alternative of the invention, the virus may also be frozen at −20° C., thus preserved for several months or for a longer period, and then thawed in this formulation with no harm either to its structure or its infectivity.
According to the invention, the compositions may be prepared by suspending, in the buffer solution, adenoviral particles initially obtained in aqueous solution, and then purified, followed by the addition of glycerol and optionally by the addition of an adjuvant as cited above.
The infectious recombinant adenovirus may be obtained according to the usual methods of production in cells of encapsidation transcomplementing lines, for example the cells of the 293 line or of the PER-C6 line. The viral particles may then be purified by caesium chloride gradient centrifugation as described, for example, in Journal of General Virology, 34, 19-35 (1977). More preferably, the viral particles are purified by liquid chromatography in anion-exchange mode, gel filtration, in hydrophobic mode or by metal chelation. The purification by anion exchange is particularly advantageous since it makes it possible to obtain, in a single chromatographic step, a pure viral preparation, free of the proteins, the nucleic acids and other impurities and m
Blanche Francis
Shih Shian-Jiun
Finnegan Henderson Farabow Garrett & Dunner LLP
Gencell S.A.
Priebe Scott D.
Whiteman Brian
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