Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Reexamination Certificate
2001-06-14
2003-03-25
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C435S004000, C435S005000, C210S634000, C210S638000, C210S656000, C210S660000
Reexamination Certificate
active
06537793
ABSTRACT:
The present invention relates to a new method for the purification and quantification of viral particles. More particularly, the invention relates to a method of purifying and quantifying adenoviruses by ion-exchange chromatography. The invention also relates to a method of identifying various adenovirus serotypes.
Gene therapy is currently undergoing a remarkable development and various clinical studies in humans have been in progress since the first trials conducted in 1990. Among the methods commonly used for the transfer of genes, viral vectors have proved particularly promising, and adenoviruses occupy a key position among them.
The development of adenovirus vectors in gene therapy requires access to two types of technologies which are nowadays limiting for the production of viral stocks: the first is to have a method which is rapid, is highly sensitive and is very selective for the quantification of viral particles in samples obtained from the steps of constructing and amplifying the virus considered; this point is particularly important for the optimization of the method of producing viral stocks; the second is to have a method of purification which is reliable, reproducible, simple and can be easily extrapolated on the industrial scale for the purification of virus particles.
The production of clinical batches of adenoviruses remains a long procedure because of the number of transfection and amplification steps whose productivity is not optimized. Recombinant adenoviruses are usually produced by introducing viral DNA into an encapsidation line, followed by mechanical or chemical lysis of the cells after about two or three days of culture (the kinetics of the adenoviral cycle being from 24 to 36 hours). According to another variant, the culture is continued for a longer period (8 to 12 days), and the viruses are directly harvested in the supernatant after spontaneous release by a phenomenon of autolysis of the encapsidation cells (WO 98/00524).
Generally, between 2 and 7 amplification cycles are necessary to constitute the viral stocks. A major limitation to the optimization of the method of producing viral stocks lies in the methods of titrating the viral particles. Indeed, biological methods are methods which are relatively sensitive and accurate, but are particularly long to carry out (about 4 to 15 days depending on the assay used, i.e. transgene activity (tdu) or plaque production (pfu)). Faster analytical methods have been developed but they do not have a sufficient degree of precision and sensitivity when the titrations of viral particles have to be carried out, without prior purification, in lysates, crude cellular extracts or culture supernatants. That is why successive amplification cycles are carried out with multiplicities of infection (MOI) which are estimated roughly. The result is that the amplification steps are not very reproducible, or are even sometimes longer and/or more numerous than would be necessary with an optimized method. The rapid and precise determination of the titres of adenovirus solutions would make it possible to adjust the multiplicity of infection for each step so as to optimize the entire method of producing adenovirus stocks.
The method of quantifying viral particles should satisfy several conditions. In the first place, it should be sufficiently sensitive to allow the assay of viral particles in preparations which are dilute or which have a low titre (typically<1×10
9
viral particles per ml (vp/ml)) without resorting to a prior enrichment step. It should be possible to carry out the assay of the viral particles directly in lysates or crude preparations, without the need to carry out a purification step or a prior treatment. Furthermore, this method should allow a high selectivity in order to eliminate possible interference with the numerous compounds present in the crude cellular lysates or extracts and of which the proportions may vary depending on the culture conditions.
A quantitative analytical method based on anion-exchange chromatography has been described in the literature (Huygue et al., Human Gene Ther. 6: 1403-1416, 1995; P. W. Shabram et al., Human Gene Ther. 8: 453-465, 1997). This method, which has a detection limit of the order of 1×10
8
vp/ml, is applicable to the titration of purified viral particles. However, the sensitivity of this method decreases once the analysis is carried out on lysates or crude cellular extracts. The detection limit is estimated at 2 to 5×10
9
vp/ml in such samples and this method does not make it possible to quantify the adenoviral particles in very dilute and nonpurified preparations such as the lysates of cells infected during the virus transfection and amplification steps for which the adenoviral titre is typically of the order of 1×10
8
vp/ml to 1×10
9
vp/mi. Furthermore, neither does this method make it possible to quantify the adenoviral particles from preparations obtained in certain production media free of animal proteins. Indeed, such media contain, at the end of culture, compounds of the sugar, amino acid, vitamin or phenol red type, and the like, among which some may interfere with the adenoviral particles during the quantification of the virus and which lead to the titre of the preparation being very widely overestimated. Finally, the chromatographic method reported by Shabram et al. requires a pretreatment of the sample with a nuclease with a broad activity spectrum (BENZONASE) in order to remove the nucleic acids which interfere with the detection and measurement of the particles.
As regards the preparative methods of separating adenoviruses, chromatography has been used for many years for the purification of adenoviral particles [Haruna, I., Yaosi, H., Kono, R. and Watanabe, I.
Virology
(1961) 13. 264-267; Klemperer, H. G. and Pereira, H. G.
Virology
(1959) 9, 536-545; Philipson, L.,
Virology
(1960) 10, 459-465]. Methods describing the large-scale purification of recombinant adenoviruses have been described more recently (international patent applications WO 96/27677, WO 97/08298, WO 98/00524, WO 98/22588).
Application WO 98/00524 describes in particular a method of purification using the strong anion-exchange resin Source 15Q which makes it possible to obtain, in a single chromatographic step, adenovirus preparations whose purity is at least equivalent to that obtained from preparations purified by caesium chloride gradient ultracentrifugation. This degree of purity is very high and reaches the standards required for clinical studies in humans (WHO Expert Committee on Biological Standardization, Forty-ninth Report. WHO Technical Report Series, WHO Geneva, in press).
However, when the viral titre of the preparations to be purified is low (for example in the case of an adenovirus having a low productivity, or when the purification has to be carried out using a stock obtained during an early amplification step), or alternatively when the virus production medium leads to the presence of compounds co-eluted with the adenovirus (as for example in the case of media free of calf serum), the limited performance of the chromatographic techniques previously described do not make it possible either to quantify or to purify the adenoviral particles in a single step from such a starting material.
The problem of being able to have a method of titrating viral particles from crude preparations which is rapid, sensitive and highly selective therefore exists. The problem of having a method of purification which is reliable, reproducible and makes it possible to obtain, from these same crude preparations, and preferably in a single step, viral preparations of pharmaceutical quality, also exists.
It has now been found, and this constitutes the subject of the present invention, that certain chromatography supports surprisingly exhibit quite exceptional properties for the separation of viral particles and in particular adenoviruses. These properties allow the titration and/or purification of viral particles from crude preparations, with no pr
Barbot Anne
Blanche Francis
Cameron Beatrice
Aventis Pharma S.A.
Brown Stacy S.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Park Hankyel T.
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