Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
  • C12N7/02—Recovery or purification
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
  • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
  • C12N2710/10351—Methods of production or purification of viral material
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/01—DNA viruses
  • G01N2333/075—Adenoviridae
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography

Definitions

• the invention relates to a new method for the purification and quantification of viral particles. More particularly the invention relates to a method of purification and quantification of adenovirus by ion exchange chromatography. The invention also relates to a method for identifying the various adenovirus serotypes.
• adenovirus vectors in gene therapy requires access to two types of technology which are currently limiting for the production of viral stocks: the first is to have a rapid method, with high sensitivity and very selective for the quantification of the viral particles in the samples coming from the stages of construction and amplification of the virus considered, this point is particularly important for the optimization of the production process of the viral stocks: the second is to have a purification process reliable, reproducible, simple and easily extrapolated on an industrial scale for the purification of viral particles.
• adenovirus The production of clinical batches of adenovirus remains a long process due to 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 a mechanical or chemical lysis of the cells after about two or three days of culture (the kinetics of the adenoviral cycle being 24 to 36 hours).
• the culture is continued for a longer period (8 to 12 days), and the viruses are harvested directly in the supernatant, after spontaneous release by a phenomenon of autolysis of the packaging cells (WO98 / 00524) .
• the quantification method for viral particles must satisfy several conditions. First, it must be sensitive enough to ensure the determination of viral particles in diluted preparations or with a low titer (typically ⁇ 1 x 10 ° viral particles per ml (w / ml)) without resorting to an enrichment step prior.
• the dosage of viral particles must be carried out directly in lysates or crude preparations, without it being necessary to perform a step of purification or pretreatment.
• this method must ensure high selectivity to overcome possible interference with the many compounds present in the lysates or crude cell extracts, the proportions of which can vary depending on the culture conditions.
• the detection limit is estimated at 2 to 5 x 10 ° pvml in such samples and this method does not make it possible to quantify the adenoviral particles in very diluted and unpurified preparations such as lysates of infected cells during the transfection steps and amplification of the virus for which the Adenoviral titer is typically of the order of 1 x 10 s pv / ml to 1 x 10 ° pv / ml.
• this method also does not make it possible to quantify the adenoviral particles from preparations obtained in certain production media devoid of animal proteins. Indeed, such media contain, at the end of culture, compounds of the type, sugars, amino acids, vitamins, or phenol red, etc.
• Application WO98 / 00524 describes in particular a purification method using the strong anion exchange resin Source 150 and 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 ultracentrifugation in cesium chloride gradient. 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).
• the limited performances of the chromatographic techniques described above do not allow either of quantify, nor purify adenoviral particles in a single step from such starting material.
• the problem therefore arises of being able to have a method for titrating viral particles from crude preparations which is at the same time rapid, sensitive and highly selective.
• the problem also arises of having a reliable, reproducible purification method making it possible to obtain, from these same crude preparations, and preferably in a single step, viral preparations of pharmaceutical quality.
• chromatography supports have su ⁇ renantly quite exceptional properties for the separation of viral particles and in particular of adenoviruses. These properties allow the titration and / or purification of viral particles from crude preparations, without prior treatment, with very high sensitivity and selectivity.
• the use of these supports also provides, unexpectedly, a simple and rapid analysis method for separating and identifying by chromatography, adenoviruses of different serotypes or adenoviruses modified at the fiber or hexon level.
• the present invention relates to a method of separating viral particles from a biological medium characterized in that it comprises at least one chromatography step carried out on a support comprising a matrix, groups of ion exchangers, said groups being grafted onto said matrix by means of a flexible arm.
• the matrix can be chosen from agarose. dextran, acrylamide, silica, poly [styrene-divinylbenzene], alone or in admixture.
• the matrix consists of agarose. More preferably, it is about 6% crosslinked agarose.
• the supports consisting of crosslinked agarose beads onto which flexible functionalized ion exchange arms are grafted have been developed for the preparative and industrial chromatography of biomolecules. These materials have especially been developed for the capture step (that is, the initial step of the purification process) biomolecules from crude mixtures simply released, that is to say, cleared of suspended solids constituents . Their performances have been optimized in the sense of a very high capacity for fixing the solutes on the support, a very low back pressure at high linear flow rate of liquid, a low cost as well as a very high chemical resistance cleaning agents used for regeneration.
• the flexible arm is hydrophilic in nature and consists of a polymer of synthetic or natural origin.
• polymers of synthetic origin mention may be made of polymers consisting of monomers of polyvinyl alcohols, of polyacrylamides, of polymethacrylamides or of polyvinyl ethers.
• polymers of polysaccharide nature chosen from starch, cellulose, dextran and agarose.
• the degree of polymerization of the flexible arm is approximately 30 monomeric units, more preferably, the flexible arm is a dextran with an average molecular weight of approximately 5000 Da.
• the flexible arm is functionalized by grafting a group capable of interacting with an anionic molecule.
• the group consists of an amine which can be ternary or quaternary.
• a strong anion exchanger preferably used according to the invention a chromatography support as indicated above, functionalized with quaternary amines.
• Q Sepharose® XL As a particularly preferred support for the implementation of the invention, mention may be made of Q Sepharose® XL (Amersham Pharmacia Biotech). The use of this support is mentioned in one of the examples of application WO98 / 39467.
• Purified adenoviruses are modified by treatment with polyethylene glycol (PEG). After reaction, the modified adenoviruses, the unmodified adenoviruses and the PEG are separated by passage through a column of Q Sepharose® XL. It is therefore a simple separation between starting products and end products of a chemical reaction. Those skilled in the art could not assume that this column could be successfully used for the separation of adenovirus from a complex biological medium containing various contaminating species (host DNA.
• PEG polyethylene glycol
• RNAs, proteins, lipids, lipoproteins, endotoxins such as a cell lysate of packaging.
• Q Sepharose® XL can be used for preparatory purposes because it is known that the majority of supports lose their effectiveness when large quantities of products are injected.
• the matrix consists of agarose crosslinked at 6%, it is grafted with flexible arms made of dextran and functionalized with strong anion exchange groups.
• the support has a particle size preferably between 40 and 200 ⁇ m approximately;
• the term "approximately” referring to the grain size signifies that the value to be taken into consideration is within a range of between +/- 20% relative to the value expressed. Preferably, this difference is between +/- 10% and more preferably it is between +/- 5% relative to the value expressed.
• the particle size is between
• the matrix has a dispersion such that 95% of the particles have a diameter between 0.1 and 10 times the average diameter of the particles, and preferably between 0.3 and 3 times the average diameter of the particles.
• the Q Sepharose® XL has a size distribution of beads ranging from 45 to 165 ⁇ m, centered on 90 ⁇ m. These characteristics of size and distribution of the beads make this support a preparative type chromatographic exchanger. The theory as well as the chromatographic practice indicate that such a support has very modest performances for the separation of compounds exhibiting similar chromatographic behaviors as for the interaction of ion exchange. Similarly, such a support generates wide chromatographic peaks poorly resolved, in particular due to the large size and the very wide distribution of the beads which constitute it. These expected chromatographic characteristics are verified for biomolecules in general such as proteins, which are eluted in the form of large poorly separated peaks (see Data File Pharmacia Biotech N ° 18-1 123-82).
• the adenovirus particles are eluted from this type of support in the form of an extremely fine peak. very symmetrical.
• the efficiency of a column filled with Q Sepharose® XL measured by the Height Equivalent to a Theoretical Tray (HEPT) or the number of theoretical trays per unit of column length (N / m). is 50 to 100 times higher for adenovirus (N / m: 35,000) than for proteins like bovine serum albumin (N / m: 600). See for example FIG. 1.
• this type of gel and in particular Q Sepharose® XL gel gives a chromatographic peak for the adenovirus of a fineness unmatched by the supports.
• this type of gel and in particular Q Sepharose® XL gel gives a chromatographic peak for the adenovirus of a fineness unmatched by the supports.
• the supports recommended for the separation of biomolecules mention may be made of supports whose base matrix is of the divinylbenzene polystyrene type (such as, for example, the resins Source 15Q and Source 30Q, or the resins of the type Poros HQ, Poros DE2, or Poros D ).
• supports whose base matrix is of the ethylene glycol methacrylate copolymer type, such as, for example, Toyopearl DEAE, QAE resins. and Super Q, or the Fractogel TMAE, TMAE HiCap, DMAE, or DEAE type resins, the ion exchange functional groups of which are located on linear polymer chains of polyacrylamide type grafted onto the matrix.
• supports whose base matrix is of the ethylene glycol methacrylate copolymer type, such as, for example, Toyopearl DEAE, QAE resins. and Super Q, or the Fractogel TMAE, TMAE HiCap, DMAE, or DEAE type resins, the ion exchange functional groups of which are located on linear polymer chains of polyacrylamide type grafted onto the matrix.
• the effectiveness of the supports used in the context of the present invention for the separation of adenovirus particles leads to a very high sensitivity of detection of the particles.
• the unexpected chromatographic behavior of the viral particles makes it possible to quantify the adenovirus with a detection limit much lower than the detection limit of the methods described previously.
• This detection limit is at least ten times lower, which makes it possible to reach a detection limit of the order of 1 ⁇ 10 s pv / ml in crude cell lysate type preparations and a detection limit of the order of 1 x 10 'pv / ml for purified viral preparations.
• This type of support also ensures a very high selectivity with respect to contaminants present in the samples to be analyzed such as proteins and nucleic acids.
• the proteins are in the form of very broad peaks which elute well before the viral peak.
• Nucleic acids are eluted from the column with a salt concentration much higher than the concentration necessary for the elution of the virus.
• This characteristic very different from that obtained with the chromatographic method previously described (Huygue et al., Human Gene Ther. 6: 1403-1416, 1995) makes it possible to overcome the interference of this type of compound with the viral peak.
• the preparations to be analyzed contain species co-eluted with the viral particles, the very specific form of the viral peak easily makes it possible to identify it and to carry out its quantification.
• the supports used in the context of the invention make it possible to identify and quantify very easily and with great precision the peak of the adenovirus when it is analyzed from preparations containing a large quantity of proteins and d 'nucleic acids.
• the quantitative analysis of the particles as well as the purification is also possible from preparations obtained with very varied viral production media, or media devoid of constituents of animal origin, such as albumin, whether it is d bovine, human or another origin ( Figure 2). It is also important to note that the method described in the present invention is applicable to the analysis of samples containing nucleic acids without prior treatment with a nuclease without affecting either the sensitivity or the selectivity of the method.
• another subject of the invention relates to the use of this type of support and of Q Sepharose® XL in particular, for the preparative separation or the purification of viral particles, in particular of adenovirus, from media. biological.
• a separation process can optionally comprise a prior stage of chromatography on another support such as those used in the process which is the subject of application WO98 / 00524, and in particular the Source 15Q resin.
• Such a preliminary step may prove to be advantageous in particular cases, for example if an excessive quantity of contaminants is present in the biological medium.
• Another object of the invention relates to the use of this type of support and of Q Sepharose® XL in particular, for the quantitative analysis or the titration of viral particles, in particular of adenovirus, from biological media.
• the biological medium from which is made the purification or titration of virus may be a cell supernatant of producing encapsidation of the virus or a cell lysate of encapsidation. or a prepurified solution of said virus.
• a preliminary step of ultrafiltration preferably this step is carried out by ultrafiltration tangential on membrane having a cutoff threshold between 300 and 500 kDa.
• the purification process according to the invention makes it possible to obtain viral preparations of high quality in terms of purity with high yields of particles (of the order of 75 to 80%) in one step, from a diluted stock. or / and very rich in contaminants and this, under production conditions fully compatible with industrial requirements and with the regulations concerning the production of therapeutic molecules.
• Another object of the invention relates to a method for quantifying adenovirus, characterized in that the viral particles are separated by chromatography on a support of Q Sepharose® XL type and the amount of adenovirus is determined by measuring the absorbance of the chromatography fractions.
• the method of the invention allows easier and more precise monitoring of the production kinetics, directly on homogeneous samples of supernatant, without pretreatment, which allows better reproducibility and better control of the production processes of stocks of viral particles. .
• the subject of the invention is also the use of a Sepharose® XL type Q chromatography support for the identification of different adenovirus serotypes. Indeed, and su ⁇ renantly, it has been observed that this type of support makes it possible to separate and identify in a simple and rapid manner a large variety of adenoviruses of different serotypes directly from a sample of viral production medium. by determining the retention time and the absorbance ratio at 260 nm and 280 nm from the chromatographic peak.
• the separation of viral particles for analytical or preparative purposes may be carried out by applying to the chromatography column a gradient salt elution or according to a mode isocratic, ie at constant salt concentration.
• the chromatographic support can be used in a chromatography column of conventional type or in a column suitable for high performance chromatography systems, using for example the Q Sepharose® XL support, or even in a so-called system. with "fluidized bed” or expanded, using for example the Streamline® Q XL support.
• the size of the chromatographic column is determined according to the amount of virus present in the starting material.
• the viral preparation to be purified can be applied to the support in a buffer whose conductivity is such that the virus is not retained on the support while the nucleic acids are fixed.
• the conductivity is adjusted to 45 mS / cm.
• the assay and purification and characterization methods of the different serotypes described in the present invention can be applied to different types of virus, and of adenovirus in particular, whether they are wild virus or recombinant viruses carrying a transgene of interest.
• Figure 1 elution profile of purified adenovirus and bovine albumin on Q Sepharose® XL.
• Figure 2 elution profile on Q Sepharose® XL of a viral culture supernatant obtained on a medium devoid of serum.
• Figure 3 elution profile on Q Sepharose® XL of a preparation of purified adenovirus (2 x 10 l0 pv injected).
• Figure 4 elution profile on 0 Sepharose® Fast Flow of a preparation of purified adenovirus (2 x 10 l0 pv injected).
• Figure 5 Comparison of the elution profiles of a preparation of adenovirus purified on Q Sepharose® XL and Q Sepharose® Fast Flow.
• Figure 6 elution profile on Q Sepharose® HP of a preparation of purified adenovirus (2 x 10 l0 pv injected).
• Adenoviruses are linear double-stranded DNA viruses about 36 kilobases in size. Their genome includes in particular a repeated inverted sequence (ITR) at each end, an encapsidation sequence (Psi), early genes and late genes.
• ITR inverted sequence
• Psi encapsidation sequence
• the main early genes are contained in the El. E2, E3 and E4 regions. Among these, the genes contained in the El region in particular are necessary for viral propagation.
• the main late genes are contained in regions L1 to L5.
• the genome of the Ad5 adenovirus has been fully sequenced and is accessible on the database (see in particular Genebank M73260). Likewise, parts or even all of other adenoviral genomes (Ad2, Ad7, Ad 12, etc.) have also been sequenced.
• adenovirus For their use in gene therapy, different vectors derived from adenoviruses have been prepared, incorporating different therapeutic genes. In each of these constructions, the adenovirus was modified so as to render it incapable of replication in the infected cell.
• the constructions described in the prior art are adenoviruses deleted from the E1 region essential for viral replication, at the level of which heterologous DNA sequences are inserted (Levrero et al. Gene 101 (1991) 195; Gosh-Choudhury et al. Gene 50 (1986) 161). Furthermore, to improve the properties of the vector, it has been proposed to create other deletions or modifications in the genome of the adenovirus.
• thermosensitive point mutation was introduced into the mutant ts125, making it possible to inactivate the 72 kDa DNA binding protein (DBP) (Van der Vliet et al., 1975).
• Other vectors include a deletion of another region essential for viral replication and / or spread, the E4 region.
• the E4 region is indeed involved in the regulation of the expression of late genes, in the stability of late nuclear RNA, in the extinction of the expression of cell proteins host and in the efficiency of viral DNA replication.
• Adenoviral vectors in which the E l and E4 regions are deleted therefore have very limited transcription background noise and expression of viral genes.
• Such vectors have been described by example in applications WO94 / 28152, WO95 / 02697, WO96 / 22378).
• vectors carrying a modification at the level of the IVa2 gene have also been described (WO96 / 10088).
• the recombinant adenoviruses described in the literature are produced from different serotypes of adenoviruses. There are in fact different serotypes of adenoviruses, the structure and properties of which vary somewhat, but which have a comparable genetic organization. More particularly, the recombinant adenoviruses can be of human or animal origin. As regards adenoviruses of human origin, mention may preferably be made of those classified in group C, in particular adenoviruses of type 2 (Ad2), 5 (Ad5); in group B, adenovirus type 7 (Ad7); or in group A, adenovirus type 12 (Ad 12).
• adenoviruses of animal origin mention may preferably be made of adenoviruses of canine origin, and in particular all the strains of adenoviruses CAV2 [manhattan or A26 / 61 strain (ATCC VR-800) for example].
• Other adenoviruses of animal origin are cited in particular in application WO94 26914, which is incorporated herein by reference.
• the recombinant adenovirus is a human adenovirus of group C. More preferably, it is an adenovirus Ad2 or Ad5.
• Recombinant adenoviruses are usually produced by introducing viral DNA into the packaging line, followed by lysis of the cells after approximately 2 or 3 days (the kinetics of the adenoviral cycle being 24 to 36 hours), depending on to another variant, the culture is continued until 8 to 12 days and the viral particles are released spontaneously into the culture medium by autolysis of packaging cells.
• viruses used in the context of the following examples are adenoviruses containing the lacZ marker gene from E.coli (AV, 0 CMV.lacZ). These viruses are derived from the Ad5 serotype and have the following structure:
• the culture media can vary according to the transcomplementing lines used or according to the quantities used. These environments can be the MEM, DMEM ... whether or not supplemented with calf serum and contain different concentrations of inorganic salts, sugar, amino acids, vitamins, hepes or phenol red.
• Transcomplementing cells of the E1 region such as 293 or PER.C6 cells are transfected at 60-80% confluence in a culture dish with viral DNA obtained by digestion of a plasmid carrying the adenoviral genome of interest.
• the incubation lasts from 8 to 15 days, the harvest time is judged by the observation under the microscope of the cells which round off, become more refractive and adhere more and more weakly to the culture support.
• the virus is then released from the nucleus by 3 to 6 successive thawing cycles (dry ice ethanol at -70 ° C, water bath at 37 ° C).
• the virus thus obtained is used to re-infect transcomplementing cells at a given Multiplicity of Infection (MOI) which can vary between 10 and 500 viral particles per cell, the amplified virus is obtained as previously by continuing the incubation from 40 to 72 h.
• MOI Multiplicity of Infection
• the cells are not harvested 40 to 72 hours post-infection, but the incubation is prolonged between 8 to 12 days so as to obtain total lysis of the cells without the need for proceed to freeze-thaw cycles.
• the virus is then spontaneously released into the supernatant.
• the supernatant is then clarified by filtration on filters with decreasing porosity depth (10 ⁇ m / 1.0 ⁇ m / 0.8-0.2 ⁇ m).
• the clarified supernatant is then concentrated by tangential ultrafiltration on a Millipore spiral membrane having a cutoff threshold of 300 kDa.
• the concentration factor is of the order of 20 to 100 times.
• the clarified supernatant can be used as it is for the purification of adenoviral particles by chromatography on a column of Q Sepharose® XL.
• This column is mounted on a Waters HPLC system type 626 fitted with a detection system UV / visible diode array 996 operating in the range of 200-300 nm absorbance.
• This anion exchange column is used for the separation and quantification of the viral particles.
• the column Before each analysis, the column is equilibrated at 30 ° C in a 20 mM Tris / HCl buffer, pH 7.5 at a flow rate of 1.5 ml / min.
• the sample to be analyzed containing the viral particles is injected onto the column.
• the quantity of particles injected must be less than or equal to 2x l0 12 particles / ml of support.
• the volume injected has no appreciable influence on the separation of the species, at least for an injected volume less than or equal to 50 ml per ml of gel.
• the column is rinsed with 5 volumes of the same buffer, and the fixed species are eluted with a linear gradient from 0 to 1 M NaCl in the 20 mM Tris / HCl buffer, pH 7.5 over 30 column volumes .
• the column is washed with 2 volumes of 0.5 N sodium hydroxide column before rebalancing for the next analysis.
• a standard curve at 260 nm is constructed with a preparation of adenovirus particles purified either by a CsCl gradient or by chromatography. This standard preparation was previously titrated into particles by its absorbance at 260 nm in a 0.1% SDS solution using the conversion factor of 1 ⁇ 10 10 particles per unit of absorbance at 260 nm.
• the adenovirus is eluted at the retention time of approximately 18 min and has an absorbance ratio at 260 nm compared to 280 nm of 1.30 ⁇ 0.02 (see FIG. 3).
• the suitability software of the Millennium Waters chromatographic signal acquisition and processing station automatically determines after each analysis the value of N m (calculated at mid-height) and the asymmetry (calculated at 10% of the height) of the peak.
• the value of N m on the peak adenoviral is typically 35,000 ⁇ 3,000 and the peak asymmetry factor is 1.05 ⁇ 0.05.
• the adenovirus is purified from cultures of packaging cells 293 or
• PER.C6 (WO97 / 00326).
• the virus is produced and harvested in supernatants after autolysis as described above. It is then filtered through a 0.45 ⁇ m membrane (HT Tuffryn or polysulfone) just before purification.
• the purification protocol is identical to the protocol used for the analytical separation of viral particles described above, but with a gradient of different elution. Elution is carried out with a gradient of 0.25 to 1 M NaCl over 30 column volumes. The volume of the column is adapted to the quantities of virus to be purified by considering a capacity of 1 ⁇ 10 12 particles per ml of chromatographic support. Similarly, the linear flow rate of the eluents is fixed at 300 cm / h.
• Example 1 comparison of Q type Sepharose® XL supports with Q Sepharose® Fast Flow support.
• This example illustrates the specific properties of Sepharose® XL type Q media with respect to Sepharose® Fast Flow 0 support.
• the two supports consist of balls of identical basic structure
• a purified adenovirus preparation (2 x 10 10 pv) is injected onto a column of Q Sepharose® XL (1 ml of support) and eluted with a NaCl gradient as defined in paragraph 2 of the Materials and Methods section.
• the elution profile is presented in FIG. 3.
• an identical analysis is carried out on a similar column filled with Q Sepharose® Fast Flow (FF) support.
• the elution profile is presented in FIG. 4.
• the comparison of the chromatographic performances is presented in the table below.
• Example 2 comparison of Q-type Sepharose® XL supports with Q-type Sepharose® HP support.
• This example illustrates the specific properties of Sepharose® XL type Q media with respect to Sepharose® HP 0 support.
• the two supports consist of beads of identical basic structure (cross-linked agarose at 6%).
• the Q Sepharose® XL support has a size distribution of beads ranging from 45 to 165 ⁇ m centered on 90 ⁇ m.
• the particle size of the support Q Sepharose® HP is 34 ⁇ 10 ⁇ m.
• the particle size of the Q Sepharose® HP support is finer and much less dispersed than that of the Q Sepharose® XL support.
• the Q Sepharose® HP support should therefore have chromatographic performances far superior to the performances of the Q Sepharose® XL.
• a purified adenovirus preparation (2 x 10 10 pv) is injected onto a column of Q Sepharose® XL (1 ml of support) and eluted with a NaCl gradient as defined in paragraph 2 of the Materials and Methods section.
• the elution profile is presented in FIG. 3.
• an identical analysis is carried out on a similar column filled with Q Sepharose® HP support.
• the elution profile obtained with support 0 Sepharose® HP is presented in Figure 6.
• the results presented in FIG. 6 show that the Q Sepharose® HP support has an efficiency (N / m, 15,000) substantially lower than the Q Sepharose XL support.
• the viral peak has a sensitive lag on the Q Sepharose® HP support (asymmetry, 1.6), whereas it is rigorously symmetrical on the Q Sepharose® XL support.
• the Q Sepharose® HP support does not achieve the performance of the Q Sepharose® XL support for the separation of viral particles.
• it has much higher performance for the separation of proteins, as indicated by the supplier (Amersham-Pharmacia Biotech) and as it is confirmed in our experimental conditions with bovine albumin separation experiments (table below). below).
• Example 3 comparison of Sepharose® XL type Q supports with Fractogel® TMAE (S) and Source 15Q supports.
• This example illustrates the specific properties of Sepharose® XL type Q media with respect to the Fractogel® TMAE (S) support and Source 15Q support.
• the three supports consist of balls of different structure and composition.
• Q Sepharose® XL consists of 6% cross-linked agarose.
• the Fractogel® TMAE support is a crosslinked polymethacrylate resin and the Source 15Q support consists of resin beads of the polystyrene-divinylbenzene type.
• the particle size of the Fractogel® TMAE (S) (20-40 ⁇ m) and Source 15Q (15 ⁇ m) supports is much lower and much less dispersed than that of the Q Sepharose® XL support (45-165 ⁇ m).
• the three supports present the strong exchanger groups. These are located on flexible arms fixed to the matrix in the case of Fractogel® TMAE (S) and Q Sepharose® XL, unlike Source 150 whose exchanger groups are grafted directly onto the matrix.
• a preparation of purified adenovirus (2 x 10 i0 vp) is injected onto a column of Q Sepharose XL (1 ml support) and eluted with a NaCl gradient as defined in paragraph 2 of the section Materials and Methods.
• the elution profile obtained is presented in Figure 3. Under the same conditions, an identical analysis is carried out on a similar column filled with Source 15Q support and and another analysis is carried out on a column filled with Fractogel® TMAE (S) support.
• Fractogel® TMAE does not reach the performance of the Q Sepharose® XL support for the separation of adenoviruses. On the other hand, it has much higher performance for the separation of proteins. Similarly, despite its very fine particle size and its almost monodispersed particle size distribution. Source 15Q support n 'reaches not, either, Q Sepharose XL support performance for the separation of adenoviruses.
• Table 3 comparison of chromatographic performance with Source 15Q, Q Sepharose® XL and Fractogel® TMAE (S) supports.
• Example 4 detection and quantification of wild adenovirus particles of various serotypes.
• This example illustrates a method of detection and identification of wild adenovirus particles of various serotypes based on the use of the Sepharose® XL type Q chromatographic support.
• the various wild adenoviruses were produced by infection of A549 cells cultured on DMEM medium and harvested after 3 cycles of freezing-thawing of the cells. The preparations were then filtered through an Acrodisc membrane (type HT Tuffryn 0.45 ⁇ m; GelmanSciences) before analysis.
• Acrodisc membrane type HT Tuffryn 0.45 ⁇ m; GelmanSciences
• Adenovirus 5 retention is greater (25.3 min) than the reference retention time (18 min) indicated in the Materials and Methods section.
• Table 5 correlation between the retention time of the virus and the sequence identity of the hexon.
• Example 5 detection and quantification of the recombinant adenovirus particles during the stages of production of a viral stock.
• This example illustrates the use of Sepharose® XL type Q supports for the detection and quantification of the AV, 0 CMV.lacZ recombinant adenovirus particles produced during the transfection and amplification steps with different packaging cell lines ( cells 293 or PER.C6).
• This extremely rapid analysis method provides, in a few minutes, and from a culture supernatant, the title of the adenovirus solution resulting from each step.
• This fast and sensitive method makes it possible to optimize the amplification conditions of the next step which can therefore be carried out under conditions of determined and controlled MOI.
• This analysis method was tested to control the production of recombinant adenoviruses of type AV 1 0 CMV.lacZ during the steps of transfection and amplification on 293 or PER.C6 cells.
• the adenovirus AV 1 0 CMV.lacZ was produced after transfection of 293 or PER.C6 cells with the plasmid pXL2822 digested with Pacl (Crouzet et al., Proc. Natl. Acad. Sci USA 94: 1414-1419, 1997) then infection of cells respectively 293 or PER.C6 at a determined multiplicity of infection (MOI).
• the initial transfection is carried out with 5 to 10 micrograms of viral DNA obtained by digestion of the plasmid.
• the virus was harvested by 3 freeze-thaw cycles of the cells. The preparations were then filtered through an Acrodisc membrane (of the HT Tuffryn type) of 0.45 ⁇ m before analysis.
• the various preparations are then analyzed by chromatography according to the protocol described in paragraph 2 of the Materials and Methods section.
• the titer of the adenovirus solution is determined by reference to a standard curve produced under the conditions described in paragraph 2 of the Materials and Methods section.
• Table 6 Detection and quantification of particles of recombinant adenovirus AV, 0 CMV.lacZ obtained during the production of viral stocks in two transcomplementing lines (293 or PER.C6).
• the total viral particles obtained were assayed to determine the concentration of infectious particles (pfu) and particles having the activity of the transgene (tdu).
• pfu plaque forming unit
• plaque forming unit corresponds to the infectious power of an adenovirus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 15 days, the number of plaques of infected cells.
• Table 7 relationship between the concentration of infectious particles (pfu) and particles with the activity of the transgene (tdu).
• Example 6 detection and quantification of the recombinant adenovirus particles produced during the transfection and amplification steps on an IGRP2 cell.
• This example illustrates the use of Sepharose® XL type Q supports for the detection and quantification of recombinant adenovirus particles.
• AV 30 CMV.lacZ produced during the transfection and amplification steps on an IGRP2 cell.
• the adenovirus AV 30 CMV.lacZ was produced after transfection of IGRP2 packaging cells with the plasmid pXL3005 digested with Pacl (the plasmid pXL3005 derives from the plasmid pXL281 1 described in (Crouzet et al., Proc. Natl. Acad. Sci USA 94: 1414-1419, 1997) by exchange of the RSV promoter with the CMV promoter) then infection of IGRP2 cells (WO96 / 22378) at a determined multiplicity of infection (MOI). During cell lysis, the virus was harvested by 3 freeze-thaw cycles of the cells. The preparations were then filtered through an Acrodisc membrane (of the HT Tuffryn type) of 0.45 ⁇ m before analysis.
• Acrodisc membrane of the HT Tuffryn type
• Table 8 Detection and quantification of particles of recombinant adenovirus AV? 0 CMV.lacZ obtained during the production of viral stocks.
• the total viral particles obtained were assayed to determine the concentration of particles having the transgene activity (tdu 3.2 x 10s / ml).
• a ratio vp / tdu of 43 is comparable to the ratio vp / tdu 43 and 55 obtained in Example 5 and used to correlate the physical measurement 1, 39 x 10 '° vp / ml of viral particles in biological measuring 3.2 x 10 tdu / ml of transduction units.
• Example 7 purification of the virus by chromatography on Q Sepharose® XL resin.
• the starting material consists either of the culture lysate obtained by freeze-thaw cell for producing packaging of the virus or the supernatant obtained after lysis of the cells spontaneously.
• the collected fraction is analyzed by high performance liquid chromatography (HPLC) on a Resource Q column (1 ml) in a following chromatographic system: 10 ⁇ l of the fraction purified by chromatography as described above are injected onto a Resource Q15 column ( 1 ml of gel; Pharmacia) equilibrated in 100 mM Tris / HCl buffer pH 8.0 containing 0.5 mM MgC12, (buffer B). After rinsing with 5 ml of buffer B, the adsorbed species are eluted with a linear gradient of 30 ml of NaCl (0 to 1 M) in buffer B at a flow rate of 1 ml / min. The eluted species are detected at 260 nm. After the purification step on a column of Q Sepharose XL. the fraction collected has a purity> 99% in viral particles (UV detection at 260 nm). The purification yield in viral particles is 82%.
• the electrophoresis analysis of the purified adenoviral fraction by chromatography is carried out on polyacrylamide gel (4-20%) under denaturing conditions (SDS). The protein bands are then revealed with silver nitrate. This analysis shows that the adenoviral preparation obtained by chromatography has a level of purity at least equal to that of the preparation obtained conventionally by ultracentrifugation and that it does not have any additional protein band which would indicate contamination of the preparation with non-adenoviral proteins.
• the adenoviral preparation obtained by chromatography has an absorbance ratio A 260 nm / 280 nm equal to 1.30 ⁇ 0.05. This value, which is identical to that obtained for the best preparations obtained by ultracentrifugation. indicates that the preparation is free of contaminating proteins or contaminating nucleic acids.
• the titration of the virus clearly shows the presence of infectious viral particles with a very satisfactory pv / pfu ratio (see table below) and the purified viral particles effectively have the expected infectious activity.
• Table 9 purification of AV adenovirus, 0 CMV.LacZ on Q Sepharose® XL support.
• the method described in this example therefore makes it possible to purify the adenoviral particles without affecting their infectious power, directly from a lysate of packaging cells, without prior treatment (ultrafiltration for example or treatment with a nuclease) of the material to be purified.

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