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    • 专利摘要:
    METHOD FOR PURIFYING ADENOVIRUS PARTICLES FROM A CELL SUSPENSION. The invention provides a method for large scale purification of adenoviruses from high cell density suspensions using host cell DNA fragmentation and/or precipitation followed by a clarification step with tangential flow filtration.
    公开号:BR112012008507B1
    申请号:R112012008507-7
    申请日:2010-10-14
    公开日:2021-05-04
    发明作者:Marcel Leo De Vocht；Marloes Veenstra
    申请人:Janssen Vaccines & Prevention B.V.；
    IPC主号:
    专利说明:

    [0001] The invention concerns the field of virus production. More particularly, it concerns improved methods for purifying adenovirus particles from a cell suspension. Fundamentals of Invention
    [0002] Recent developments in the field of vaccine production have created the need for large-scale manufacturing. Robust, high-throughput processes are needed to support the crowd with sufficient quantities of (recombinant) vaccines to fight infectious diseases.
    [0003] Vaccines against infectious diseases can be based on recombinant adenovirus particles. For this reason, huge efforts are being put into optimizing cell-based processes for adenovirus production. Cells are being grown at increasing densities and subsequently infected in order to obtain higher virus yields. Such high cell density processes are being disclosed for example in WO 2010/060719 by Crucell Holland BV, and in Yuk et al. (2004). A process for producing large concentrations of recombinant adenoviruses has been described therein. This optimized process has the ability to infect cultures at high cell density (eg higher than 5 x 106 cells/ml) while preserving a high viral productivity per cell. Together, it offers a method to obtain a solution of collected virus with high virus concentration in a single bioreactor. The viral particle yields (VP) of said processes are about 1.5 to 2.5 x 10-2 VP/ml.
    [0004] Processes in which cells are cultured at high densities are prone to accumulation of high amounts of cell fragments and host cell DNA. These contaminants have to be disposed of further down the purification process, which is an inconvenient operation. A method for discarding host cell DNA from a harvested cell culture was previously disclosed in US7326555. The method consists of selectively precipitating host cell DNA out of the cell culture. A selectable precipitating agent can specifically bind to host cell DNA and leave adenovirus particles unprecipitated. The method in this reference however has only been described for cell cultures with low cell density, in which cell fragments and host cell DNA are present in low amounts.
    [0005] It has not been known until now that said process could be applied in a culture containing high cell densities. In contrast, from the prior art a strong suggestion can be deduced that a precipitating agent as used in said method would not selectively precipitate host cell DNA out of the culture and would precipitate viral particles when used in high concentrations (Goerke et al. 2004) .
    Cell culture harvests containing adenovirus are generally further processed to obtain purified adenovirus. A clarification step using for example deep filtration and/or tangential flow filtration (TFF) is usually included in said purification process. The use of TFF requires a relatively clean harvest, i.e. containing limited amounts of cell fragments or other impurities such as for example host cell DNA. An excess of said impurities would possibly block the filters. As a consequence, clarification by TFF is usually used further down in the purification process for example as a third or fourth step of the process.
    [0007] Separation of adenoviruses from an adenovirus-containing cell suspension directly after harvesting using tangential flow filtration has been previously described for example in EP1371723. However, adenovirus was grown in adherent cells, which remained in the bioreactor after harvest. Therefore, the suspension containing virus that was processed still contained very low concentrations of cell fragments and host cell DNA. WO2006/052302 also describes the use of TFF directly after harvesting. However, the cell densities from the virus-containing harvest used at this point were much lower than 5 x 106 cells/ml. As disclosed herein, the use of TFF in the clarification step directly after harvesting is not practicable for cell cultures containing high cell densities.
    [0008] As cell culture processes are being increased and cells are being cultured at increasing densities, there is a need in the industry for downstream processes that would enable the treatment of high density cell suspensions. This applies in particular to the field of adenovirus production. Invention Summary
    [0009] The present invention relates to methods of purifying adenovirus particles from a cell lysate from a cell suspension, in particular from a high density cell suspension.
    [00010] Attempts to purify adenoviruses from a high density cell suspension with existing processes resulted in very low virus recovery as exemplified here (Example 1). The impurity concentrations in said high density cell suspension obtained after harvesting were in general too high to allow direct adenovirus purification. Downstream processing of high density cell suspensions using known processes would typically require a large number of steps. A first filtration step would consist of a coarse filtration to remove large precipitates and cell debris. Subsequently, one or two more selective filtration steps would be required to obtain a sufficiently purified adenovirus suspension.
    [00011] We surprisingly found and disclosed here that directly subsequent to the preparation of an adenovirus-containing cell lysate, the consecutive use of fragmentation and/or precipitation of the host cell DNA followed by a clarification step comprising tangential flow filtration (TFF ) resulted in a highly purified adenovirus suspension.
    [00012] Surprisingly, cell lysate containing adenovirus, large amounts of cell fragments, host cell DNA and other impurities would be efficiently processed to purified adenovirus with the present invention. With this, the present invention provides a new process adapted for the removal of host cell DNA in large-scale adenovirus purification processes.
    [00013] With the incorporation of DNA fragmentation or precipitation within the purification process, a single clarification step was sufficient to purify adenoviruses from a high density cell suspension, using TFF for clarification.
    [00014] The invention provides a method for purifying adenovirus particles from a cell suspension having a cell density ranging from 5 x 106 to 150 x 106 cells per ml, said method comprising: a) lysing the cells within the said cell suspension; b) fragmenting and/or precipitating host cell DNA within said cell suspension; and c) subjecting the cell suspension obtained from step b) to clarification by tangential flow filtration.
    [00015] In some embodiments said adenovirus is purified from a cell suspension having a cell density ranging from 5 x 106 to 150 x 106 cells per ml, for example 5 x 106 to 50 x 106 cells per ml or 10 x 106 to 30 x 106 cells per ml.
    [00016] In another embodiment said precipitation in step b) is performed by selectively precipitating the host cell DNA away from the adenovirus particles by adding a selective precipitating agent.
    [00017] In a preferred embodiment, the tangential flow filtration is performed with membrane having a pore size ranging from 0.1 to 0.65 µm.
    [00018] In other preferred embodiments, the tangential flow filtration is performed with a hollow fiber. In yet another preferred embodiment, said tangential flow filtration is performed with an ATF system. Brief description of figures
    [00019] FIG. 1. Recovery of adenovirus and precipitated host cell DNA, plotted against the concentration of domifen bromide in low (2.5 x 106 to 3.5 x 106 vc/ml) and high (20 x 106 to) cell suspensions 30 x 106 vc/ml).
    [00020] FIG. 2. Recovery of adenovirus and precipitated host cell DNA, plotted against domifen bromide concentration in low (2.5 x 106 to 3.5 x 106 vc/ml) and high (18 x 106 to 25) cell suspensions x 106 vc/ml). Detailed description of the invention
    [00021] The present invention relates to methods of purifying adenovirus particles from a high density cell suspension. According to the invention, high density cell suspensions are obtained by culturing cells to high cell densities. Such cultivation for example can be carried out in batch, fed-batch or perfusion mode. Methods to grow cells to high cell densities are known to the person skilled in the art. Specific methods for obtaining high cell density cultures are disclosed for example in WO2004/099396, WO2005/095578, WO2008/006494, WO 2010/060719.
    [00022] According to the present invention, a high density cell suspension contains between about 5 x 106 and 150 x 106 cells/ml, for example between about 8 x 106 and 120 x 106 cells/ml, for example between about 12 x 106 and 100 x 106 cells/ml, for example between about 20 x 106 and 80 x 106 cells/ml.
    [00023] In a preferred embodiment of the present invention, the cell density in said high density cell suspension ranges between about 10 x 106 and 50 x 106 cells/ml, for example at least about 15 x 106 cells µg/ml, for example at least about 20 x 106 cells/ml, for example at least about 25 x 106, for example up to about 30 x 106 cells/ml, for example up to about 35 x 106 cells/ml, for example up to about 40 x 106 cells/ml, for example up to about 45 x 106 cells/ml.
    [00024] According to the invention, high cell density cultures are infected with adenovirus particles in order to allow said adenovirus to propagate in the cell suspension. With this, high density cell suspensions are obtained that contain high concentrations of adenoviruses, in a single bioreactor. Methods for infecting high cell density cultures are also known to the person skilled in the art. Specific methods for obtaining said high cell density cultures with high virus concentration are disclosed for example in EP08168181.9, Cortin et al. 2004 and Yuk et al. 2004. These references describe processes for producing large quantities of recombinant adenoviruses. These processes have the ability to infect cultures at high cell density while preserving high adenovirus productivity per cell. Together, it offers a method to obtain a high density cell suspension with high adenovirus concentrations in a single bioreactor. Typical yields of standard procedures for example for recombinant adenovirus 35 (rAd35) are about 1.5 to 2.5 x 1012 VP/ml. Since the adenovirus has propagated in the cell culture, killing most of the cells, the adenovirus particles are, according to the present invention, purified from the high density cell suspension.
    [00025] The method of the present invention as a first step includes lysing the cells contained in the high density cell suspension. Lysing high-density cell suspensions, which have been infected with adenovirus particles, causes large amounts of cell fragments and host cell DNA to accumulate in the cell suspension. these accumulations make subsequent downstream processing of the cell suspension inconvenient.
    [00026] The present invention provides a method adapted to purify adenovirus particles from the cell lysate of high density cell suspensions. Large amounts of host cell DNA can be selectively precipitated away from adenovirus particles within the high density cell suspension by adding a selective precipitating agent to the cell lysate such that at least about 80% of the host cell DNA are precipitated away from the high density cell suspension containing the adenovirus particles. As disclosed herein, the precipitation step allows precipitation of contaminating host cell DNA, with at least an 80% reduction in host cell DNA, preferably 90% and even more preferably, as exemplified herein, a reduction of about 95%. % in host cell DNA following clarification with TFF. Lyse
    [00027] The first step of the process includes lysing the cells within the cell suspension. This first step, in which cell membranes are lysed, allows the harvesting of both associated (intracellular) and non-associated (extracellular) adenoviruses with the cell from the infected high density cell suspension. Detergent lysis of the host cell, while the preferred method of lysing virus-containing host cells, can be substituted for non-mechanical lysis methods (such as enzymatic treatment) and/or mechanical shear methods (such as fiber ultrafiltration hollow) to release maximum amounts of adenovirus. Methods which can be used for active cell lysis are known to the person skilled in the art, and for example discussed in WO 98/22588, p. 28-35. Methods useful in this regard are, for example, freeze-thaw, solid shear, hypertonic and/or hypotonic lysis, liquid shear, sonication, high pressure extrusion, detergent lysis, combinations of the above, and the like. In one embodiment of the invention, cells are lysed using at least one detergent. The use of a lysing detergent has the advantage that it is an easy method, and that it is easily expandable.
    [00028] The detergents that can be used, and the ways they are used, are generally known to the person skilled in the art. Several examples are for example discussed in WO 98/22588, p. 29-33. Detergents, as used herein, may include but are not limited to anionic, cationic, zwitterionic, and nonionic detergents. Examples of detergents are for example Triton and/or Polysorbate-80. In one embodiment, the detergent used is Triton X-100. Furthermore, a solvent such as TNBP can be added to the lysate or clarified lysate at low concentration to complement these detergents in their ability to inactivate enveloped viruses. Also, autolysis of adenovirus-infected host cells therein can provide substantial release of intracellular adenoviruses and can be used in the methods of the invention. Therefore, any form of host cell lysis that is known in the art can be used to release intracellular viruses into the host cell culture medium for eventual harvesting by the methods disclosed herein. It is evident to the person skilled in the art that the optimum concentration of detergent can vary, for example within the range of about 0.1% to 1% (w/w). Selectable fragmentation and precipitation
    [00029] Following lysis, host cell DNA can be fragmented or precipitated out of the virus-containing cell suspension.
    [00030] In a preferred embodiment, host cell DNA is precipitated by the addition of a selective precipitating agent (SPA) solution. This step allows for selectable precipitation of host cell DNA while leaving the viral particles unchanged in the liquid phase. As exemplified herein, this early stage precipitation step results in about at least 90% reduction in host cell DNA following clarification.
    [00031] SPAs that may be useful in the practice of the present invention include, but are not limited to, amine copolymers, quaternary ammonium compounds, and any of their respective mixtures. More specifically, the many forms of polyethylene (PEI) are very efficient at neutralizing excess anionic charge (DNA impurities). A list of possible SPAs that can be used appropriately in the present invention is given in US7326555 (column 12, lines 56-67 and column 13, lines 1-28), incorporated herein by reference. Suitable SPAs for use in the present invention include but are not limited to the following classes and examples of commercially available products: monoalkyltrimethyl ammonium salts (examples of commercially available products include cetyltrimethyl ammonium bromide or chloride such as CTAB, tetradecyltrimethyl bromide or chloride ammonium (TTA), alkyltrimethyl ammonium, alkylaryltrimethyl ammonium chloride, dodecyltrimethyl ammonium bromide or chloride, dodecyldimethyl-2-phenoxyethyl ammonium bromide, hexadecylamine: chloride or bromide salt, dodecyl amine or chloride salt, and cetyldimethylethyl bromide or chloride ammonium), monoalkyldimethylbenzyl ammonium salts (examples include alkyldimethylbenzyl ammonium chlorides and benzethonium chloride such as BTC), dialkyldimethyl ammonium salts (commercial products include domiphen bromide (DB), didecyldimethyl ammonium halides, and octyldodecyldimethyl chloride or bromide ammonium), heteroaromatic ammonium salts (the commercial products s include cetylpyridium halides (CPC or bromide salt and hexadecylpyridinium bromide or chloride), cis isomer of 1-[3-chloroallyl]-3,5,7-triaza-1-azoniaadamantane, alkyl-isoquinolinium bromide, and chloride of alkyldimethylnaphthylmethyl ammonium (BTC 1110). Polysubstituted quaternary ammonium salts, (commercially available products include, but are not limited to alkyldimethylbenzyl ammonium saccharate and alkyldimethylethylbenzyl ammonium cyclohexylsulfamate), bis-quaternary ammonium salts (product examples include 1,10-bis(chloride) 2-methyl-4-aminoquinolinium)-decane, 1,6-bis[1-methyl-3-(2,2,6-trimethylcyclohexyl)-propyldimethylammonium chloride]hexane or triclobisonium chloride, and the bis-quat referred to as CDQ by Buckman Brochures), and polymeric quaternary ammonium salts (include polyionenes such as poly[oxyethylene(dimethyliminium)ethylene(dimethyliminium) ethylene] dichloride, poly[N-3-dimethylammonium)propyl]-N-[3-dichloride ethylene-oxyethylenedimethylammonium)propyl]urea, and alpha-4-[1-tris(2-hydroxyethylene)ammonium chloride). As the skilled technician will understand from US7326555, in which several of these have been shown to work and in which it has been shown that the skilled person can routinely find the appropriate concentrations for these compounds to selectively precipitate DNA, these are examples of SPAs, and based on disclosure in this and the disclosure of the present invention it is clear that these will also be suitable in the present invention.
    [00032] In a preferred embodiment, cationic detergents are used in the present invention. In an even more preferred embodiment, dialkyldimethyl ammonium salts such as domifen bromide (DB) are used in the present invention. Although a large number of potential SPAs can be used to practice the present invention, domifen bromide is of particular interest primarily due to its availability as a GMP grade raw material and current use in other products intended for human use. More specifically, since domifen bromide is extensively used as an active ingredient in oral care products as well as topical antibiotic creams, this molecule is produced in large quantities and released under cGMP conditions.
    [00033] The optimal SPA concentration that is used in high density cell suspensions to precipitate host cell DNA out of the cell suspension has been determined here. Although it was predicted, based on the prior art, that adenovirus particles would immediately precipitate when brought into contact with high concentrations of SPA, unexpectedly, the adenovirus particles remained unprecipitated. In fact, it has been shown in the prior art, for example in US7326555, that in low density cell suspensions (up to 1 x 106 cells/ml), adenovirus particles precipitate when the concentration of cationic detergent is increased.
    The suspension as produced by the lysis of high cell density cultures as disclosed herein will contain vastly increased amounts of host cell DNA and other impurities and therefore will require increased amounts of cationic detergent (eg increased by a factor 2). It was thus expected, based on the extrapolation of the results in low cell density, that this increase in the concentration of cationic detergent would lead to precipitation of all adenovirus particles present in the suspension.
    [00035] However, surprisingly, at high concentrations of SPA, selectable removal of contaminating host cell DNA from a high density cell suspension containing viral particles was still possible. In a preferred embodiment of the present invention, p SPA, preferably DB, is added at a concentration ranging from 1.2 to 5 mM. In an even more preferred embodiment the SPA, preferably DB, is added at a concentration ranging from 1.3 to 2.2 mM, for example from 1.4 to 2 mM, for example from 1.4 to 1.8 mM, for example from 1.5 to 1.6 mM. Based on the present disclosure, it is clear that the person skilled in the art knows how to determine the appropriate SPA concentration windows for a given cell density at harvest.
    [00036] The appropriate concentration of DB to treat a high density cell suspension containing adenovirus which comprises a cell density ranging between 10 x 106 and 150 x 106 cells/ml ranges between about 1.2 mM and 5 mM. The appropriate concentration of DB to treat a high density cell suspension containing adenovirus which comprises a cell density ranging from 10 x 106 to 50 x 106 cells/ml ranges from about 1.3 mM to 2.2 mM. The appropriate concentration of DB to treat a high density cell suspension harvest containing adenovirus which comprises a cell density ranging between 10 x 106 and 30 x 106 cells/ml ranges between about 1.3 and 2 mM, for example between about 1.4 and 1.9 mM, for example between about 1.4 and 1.8 mM, for example between about 1.4 and 1.7 mM, for example between about 1.45 and 1, 65 mM, for example from about 1.5 to 1.55 mM.
    [00037] It will be within the purview of the person skilled in the art to test potential substitutes for the SPAs disclosed herein to identify a compound that effectively precipitates nucleic acid molecules and other cellular fragments out of adenovirus particles as exemplified herein for domifen bromide (DB ). Therefore, this present invention relates in part to methods of purifying adenovirus particles from a high density cell suspension. Said methods comprise selectively precipitating host cell nucleic acid molecules out of adenovirus particles within the high density cell suspension after lysis by adding a selectable precipitating agent to the host cell culture medium after lysis.
    [00038] Although the preferred method to remove host cell DNA from the cell suspension is selective precipitation, the invention is not limited thereto. Any other method of removing host cell DNA is also included in the present invention.
    [00039] Therefore, in one embodiment, the host cell's DNA is fragmented, ie: cut into pieces. According to the present invention, fragmentation of host cell DNA following lysis can be accomplished by adding a nuclease into the cell suspension. Exemplary nucleases suitable for use in the present invention include Benzonase®, Pulmozyme®, or any other DNase and/or RNase commonly used in the art. In preferred embodiments of the invention, the nuclease is Benzonase®, which rapidly hydrolyzes nucleic acids by hydrolyzing internal phosphodiether bonds between specific nucleotides, thereby reducing the viscosity of the cell lysate. Benzonase® can be obtained commercially, for example from Merck KGaA (code W214950).
    [00040] The concentration of nuclease that is required for proper fragmentation depends on the host cell density, temperature and reaction time. The person skilled in the art knows how to determine and optimize the required concentration of nuclease for successful fragmentation of host cell DNA. Clarification
    [00041] The SPA-treated cell lysate obtained from the previous steps is subsequently clarified to remove precipitated host cell DNA, cell fragments and other impurities.
    [00042] Attempts to directly purify adenoviruses from a high density cell suspension with a stepped clarification process resulted in low virus recovery, as exemplified here (Example 1). The impurity concentrations in said high density cell suspension obtained after harvesting were in general too high to allow virus purification. Typically, combinations of at least two consecutive filters, as often used and recommended in the art and as exemplified herein, are necessary for proper clarification.
    [00043] We surprisingly found and disclosed here that combining DNA precipitation with a clarification step using TFF allows virus purification from a high density cell suspension with high recovery. A single tangential flow filtration step was sufficient to remove cell fragments and nucleic acid precipitates from the virus-containing suspension. Host cell DNA was precipitated by more than 95% and viral recovery was higher than 80%. Consequently, according to the present invention, it is possible to use TFF as a single clarification step. The clarified virus-containing suspension obtained with the method of the present invention is substantially reduced in host cell DNA and other impurities compared to the original lysate (obtained in step a) of the present method).
    [00044] According to the present invention, the filters used in a TFF configuration are for example GE Healthcare hollow fiber filters or JM separations; other alternatives are flat screen filters from Millipore or Sartorius stedim biotech. In said TFF configuration, viral particles are collected in the permeate while cell fragments, host cell DNA precipitate and other impurities remain in the retentate. It was shown here that hollow fiber filters were very suitable for processing high density cell suspensions. Therefore, in a preferred embodiment of the present invention, TFF is performed with a hollow fiber filter.
    [00045] According to the present invention, the pore size of said filters is preferably ranging from 0.1 to 1 µm. In a preferred embodiment of the present invention the pore size is ranging from 0.2 to 0.65 µm. Said pore sizes allow viral particles to pass the membrane and cell debris, precipitated host cell DNA and other impurities are retained by the filter. filter modules are preferably pre-wetted with water following the manufacturer's instructions. Liquid is recirculating through the modules using tubing and a peristaltic pump.
    [00046] In certain embodiments of the present invention TFF is used in the form of alternating tangential filtration (ATF). ATF is a form of tangential flow filtration and it was found here that clarification using ATF was particularly well suited for viral recovery from suspensions with high concentrations of cell fragments, host cell DNA and other impurities. The flow Tangential flow can be obtained according to methods known to the person skilled in the art and as described for example in US 6,544,424. The advantage of using an ATF system (eg ATF System, Refine Technology, Co., East Hanover, NJ) is that the feed and hold currents change direction with each cycle of the ATF pump (the cycle consists of one mode of pressurization and exhaust). This creates a reverse flow cleaning action and a transmembrane pressure (TMP) that continuously changes across the membrane. During exhaustion a vacuum is created during which some material is back-flushed from the permeate side to the retentate side, which results in cleaning of the membrane. The use of ATF will result in less membrane clogging which results in a higher viral recovery yield. The person skilled in the art can determine the optimal ATF and permeate flow rates for maximum yield.
    [00047] According to the present invention, the filters used in said ATF system are, for example, GE healthcare hollow fiber filters.
    [00048] An additional advantage of using an ATF system is that the array that is used for clarification can be used initially to culture cells in perfusion mode. In fact, a bioreactor connected to an ATF system can be used in the first part to grow cells to high cell densities. Very high cell densities of more than 100 x 106 viable cells/ml can be obtained using an ATF perfusion system, for example with PER.C6 cells (see for example Yallop et al, 2005 and WO 2005/095578 ). Once the cells have reached a high cell density, they can be infected in order to obtain a cell suspension containing highly concentrated virus. The ATF system that remains connected to the bioreactor throughout the process can subsequently be used to purify the virus from said high density cell suspension.
    [00049] The combination of selectable precipitation with clarification by TFF removes at least 70%, preferably at least 80% and even more preferably at least 90% of the host cell DNA contained in the high density cell lysate obtained after harvest. Additional purification methods
    [00050] In certain embodiments, the harvested viral particles are further purified. Further purification of the virus can be carried out in several steps comprising concentration, ultrafiltration, diafiltration or chromatographic separation as described for example in WO 2005080556, incorporated herein by reference. Other steps such as anion exchange membrane chromatography, sterile filtration, reverse phase absorption, hydroxyapatite chromatography can also be used. These steps are for example disclosed in US 7326555, incorporated herein in its entirety by reference. The person skilled in the art knows how to find the optimal conditions for each purification step. Also WO 98/22588, incorporated herein in its entirety by reference, describes methods for the production and purification of viral particles.
    [00051] In certain embodiments according to the invention, the clarified adenovirus particle suspension can be treated by ultrafiltration. Ultrafiltration is used to concentrate the viral suspension. The suspension can be concentrated 5 to 20 times and possibly treated with nuclease (as mentioned above). Another aspect of the invention is the subsequent introduction of an exchange buffer via diafiltration. Diafiltration, or buffer exchange, using ultrafilters is a way for the removal and exchange of salts, sugars and the like. The person skilled in the art knows under what conditions the buffer exchange must take place and which buffers are appropriate for this step.
    [00052] The particular ultrafiltration membrane selected will be of a size small enough to retain adenovirus particles but large enough to effectively clean impurities. Depending on the manufacturer and type of membrane, nominal molecular weight cutoffs between 10 and 1000 kDa may be appropriate. Ultrafiltration using the tangential flow mode is preferred. In said mode, the step can be controlled by setting a fixed cross flow with or without back pressure on tissue return, setting a fixed transmembrane pressure, or setting both cross flow and permeate flow.
    [00053] According to the invention, a next step can be an anion exchange chromatography step. During said step the adenovirus particles are attached to a positively charged material, for example a membrane, cartridge or column. Subsequent elution allows separating the viral particles from the remaining host cell impurities and DNA.
    [00054] For adenovirus purification with a Mustang Q membrane absorber, the NaCl concentration for loading and washing would presumably be anywhere from 0.3 to 0.4 M at pH 7.5 and would change on alternating pH. Most preferably the NaCl concentration is 0.35 M. The pH of the buffers needs to be high enough for the adenovirus to bind (greater than approximately 6.5). In addition, the pH of the buffer system must also be low enough to avoid viral instability. The precise maximum pH that is usable will depend on the specific stability profile of the adenovirus and the buffer components, and can be easily determined by the person skilled in the art for this particular application. As a guideline and certainly not a limitation, the pH would potentially range from about 5 to 10.
    [00055] The presence of 0.1% PS-80 in the buffers is highly preferred to achieve low residual DNA levels in the product because this attenuates adenovirus/DNA association and adenovirus aggregation. It will be within the domain of routine experimentation for the person skilled in the art to establish higher or lower detergent concentrations or alternative detergents that would be useful to promote dissociation of adenovirus particles away from other adenoviruses as well as various cell contaminants. It is also within this same domain of experimentation that the person skilled in the art can choose an alternative detergent for the process buffer. Examples for such alternative detergents can be found in US 7326555. Anion exchange membrane chromatography products such as those produced by Pall (eg Mustang® series) and Sartorius (eg Sartobind series) are suitable for viral purification according to the present invention. US Patent 6,485,958 or WO 05/080556 describe the use of anion exchange chromatography for the purification of recombinant adenoviruses.
    [00056] The binding capacity for the virus in a membrane absorber such as Mustang Q (Pall Corporation) is extremely high, and on the order of 7 x 1013 VP/ml. Other membrane absorbers and resins which are suitable for adenovirus purification in this process include but are by no means limited to Source 15Q and Source 30Q (GE life sciences), Q-Sepharose XL (GE life sciences), Fractogel TMAE (EM industries), Sartobind Q (Sartorius), Adsept Q (Natrix separations), CIM QA (BIA separations). Adenovirus elution would preferably be performed using a buffer containing NaCl. The skilled person knows how to optimize NaCl concentration.
    [00057] In certain embodiments, it is preferred to use at least one anion exchange chromatography step. After the anion exchange chromatography step, the adenovirus can be sufficiently pure. In certain embodiments however a size exclusion chromatography step is further performed to increase the robustness of the process. This step can be either before or after the anion exchange chromatography step. Obviously, other purification steps can also be suitably combined with an anion exchange chromatography step. The use of anion exchange chromatography for the purification of adenoviruses has been extensively described, and this aspect is therefore well within the reach of the person skilled in the art. Many different chromatography matrices have been used for adenovirus purification and are suitable, and the person skilled in the art can easily find the optimal anion exchange material to purify the adenovirus.
    [00058] In any particular embodiment of the present invention, the anion exchange product can be diafiltered into the formulation buffer and sterile filtered. Alternatively, an additional chromatography step (eg cation exchange) can be added before or after diafiltration with the potential to improve the robustness of impurity and/or virus/prion clearance.
    [00059] An additional ultrafiltration step may also be possible at this stage. Tangential flow ultrafiltration is useful in removing residual protein and nucleic acid and exchanging adenovirus in a formulation buffer. The choice of membranes between 300 kDa and 500 kDa is dictated by tradeoffs between yield and improved impurity clearance. Other membrane configurations (such as a hollow fiber) are acceptable substitutes. The selected ultrafiltration membrane will be of a size small enough to retain adenovirus particles but large enough to effectively purge impurities. Depending on the manufacturer and type of membrane, nominal molecular weight cutoffs between 100 and 1000 kDa may be appropriate.
    [00060] A sterile filtration step can be included in the process, which is helpful in eliminating the bioburden. The product can be filtered through a modified 0.22 micron polyvinylidene fluoride (PVDF) membrane (eg Millipore, Millipak).
    [00061] Optional downstream processing steps can be added into the process. These for example would include a size exclusion chromatography step, a reversed phase absorption step and/or a hydroxyapatite chromatography step. More details on each of these steps can be found for example in US 7326555, WO 03/097797, WO 02/44348.
    [00062] The international application WO 97/08298 describes the purification of adenoviruses using certain chromatographic matrices to prevent virus damage, including anion exchange and size exclusion steps.
    [00063] Certain ultrafiltration methods are also very suitable for the purification of adenoviruses, as disclosed in WO 2006/108707. Such steps can be performed in addition to or instead of certain chromatographic purification steps. Scale of cell culture systems and downstream processing systems
    [00064] The processes of the present invention are scalable. The cell cultures used in the present invention range from small scale cultures (eg 1 to 10 liter runs) to medium scale cultures (eg 20 to 1000 L runs) to large scale commercial preparations such as rounds of yield from 1000 to 50,000 L. The initial steps of the process (lysis, deep filtration and ultrafiltration) increase with the culture volume while anion exchange chromatography and subsequent steps increase with the entry of adenoviral particle. Therefore, the size of the last steps will be based on a bioreactor productivity estimate of at least 1 x 1012 adenovirus particles per ml (vp/ml). These high adenovirus yields for example can be obtained by infecting high cell density cultures (as described for example in EP08168181.9). Further purification of these high density cell suspensions containing high concentrations of adenovirus particles is made possible with the present invention. The possibility to process these suspensions, which contain high amounts of cell fragments and host cell DNA, allow the purification of high amounts of adenovirus particles per suspension volume. It is the merit of this invention to provide a method for processing cell culture batches with high cell densities, containing high concentrations of adenovirus particles and thus allowing very high yields per volume processed. The present method, while applicable for large scale cell cultures will allow cells to be grown on a smaller scale, albeit for higher cell densities and still achieve high yields of adenoviruses that can still be efficiently processed. This method offers the possibility to process highly concentrated batches of adenoviruses so that it will have a huge impact on the entire adenovirus purification industry. Adenovirus and producer cells
    [00065] The invention concerns the purification of adenoviruses. An adenovirus according to this invention can be any wild-type, modified, mutated and/or recombinant adenoviral vector adenovirus. Of specific interest in gene vaccination and/or gene therapy applications is the use of a 1st or 2nd generation replication incompetent adenovirus mutilated by E1 or other deletions, including gutless adenoviral vectors. The adenovirus genome is generally associated with benign pathologies in humans. The genome is receptive to manipulation, depending on the strategy used to construct the respective vector. A replication incompetent virus, such as recombinant adenovirus vector 35 (rAd35) or 26 (rAd26) (as exemplified herein) requires a producer cell line that complements the deletions.
    [00066] A producer cell (sometimes also referred to in the art and here as 'packaging cell' or 'complementary cell' or 'host cell') can be any producer cell in which a desired adenovirus can be propagated. For example, propagation of recombinant adenoviral vectors is done in producer cells that complement adenovirus deficiencies. Such producer cells preferably have in their genome at least one adenovirus E1 sequence, and thus are capable of complementing recombinant adenoviruses with a deletion in the E1 region. Furthermore, adenovirus may have a deletion in the E3 region, which is dispensable from the Ad genome, and therefore such a deletion does not have to be complemented. Any producer cell that complements E1 can be used, such as E1 immortalized human retina cells, for example 911 or PER.C6 cells (see US Patent 5,994,128), E1 transformed amniocytes (See EP patent 1230354), E1 transformed A549 cells (see e.g. WO 98/39411, US Patent 5,891,690), GH329: HeLa (Gao et al, 2000, Human Gene Therapy 11: 213-219), 293, and the like. In certain embodiments, the producer cells are for example HEK293 cells, or PER.C6 cells, or 911 cells, or IT293SF cells, and the like. Preferably PER.C6 cells (ECACC deposit no. 96022940, filed February 29, 1996 at ECACC, CAMR, Porton Down, Salisbury SP4 OJG, United Kingdom; see US Patent 5,994,128), or cells derived therefrom are used as cells producers.
    [00067] The replication-deficient adenoviral vector can be generated by using any species, strain, subtype, or mixture of species, strains, or subtypes, of an adenovirus or a chimeric adenovirus as the source of the vector DNA (see for example WO 96/26281, WO 00/03029), which for example can provide the adenoviral vector with the ability to infect certain desired cell types. In a preferred embodiment of the present invention, rAd35 or rAd26 is used as an adenovirus.
    [00068] The person skilled in the art will be aware of the possibilities to propagate adenoviral vectors of different serotypes in specific host cells, using methods such as for example disclosed in US Patent 6,492,169 or in WO 03/104467, and references therein. For example, for E1-deficient rAd35 propagation, specific producer cells expressing Ad35 E1B-55K can be constructed, for example based on existing producer cells expressing Ad5 E1A and E1B such as PER.C6 or HEK293 cells (see , for example US 6,492,169), as known to the authorized person. Alternatively and preferably, existing complementary (Ad5-) cell lines such as for example PER.C6 or HEK293 can be used without modification of the cells for propagating E1-deficient rAd35 or rAd26 by including the E4-orf6 coding sequence of Ad5 within the rAd35 or rAd26 vector, as extensively disclosed for example in WO 03/104467, incorporated herein in its entirety by reference. Thus, propagation of adenoviral vectors of any serotype can be done in producer cells using means and methods well known to the person skilled in the art. Adenoviral vectors, methods for their construction and methods for their propagation, are well known in the art and are described, for example, in U.S. Pats. US Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, and 6,113,913, and Thomas Shenk , "Adenoviridae and their Replication", MS Horwitz, "Adenoviruses", Chapters 67 and 68, respectively, in Virology, BN Fields et al., eds., 3rd ed., Raven Press, Ltd., New York (1996), and other references mentioned herein.
    [00069] The invention is further explained in the following examples. The examples do not limit the invention in any way. They merely serve to clarify the invention. EXAMPLES Example 1: Direct TFF clarification does not work for high cell density harvests
    [00070] PER.C6 cells were grown in a bioreactor at 37°C and infected with Ad35 in serum-free culture medium for 3 days. Cells were harvested at a cell density between 20 and 30 x 106 cells/ml and viral titers from 1 to 1.5 x 1012 VP/ml. Viruses were released from the cells by adding the nonionic detergents Triton X-100 and Tween-80 to final concentrations of 0.1 and 0.05% respectively. The lysis time was between 2 and 24 hours. Subsequent to lysis, the virus-containing crop was clarified by tangential flow filtration (TFF).
    [00071] Clarification was performed using a 0.2 g hollow fiber filter (GE Healthcare, model CFP-2-E-4MA, 0.042 m2) or a 0.65 g (GE Healthcare, model CFP-6- D-4MA, 0.046 m2). One liter of lysed harvest was concentrated by at least a factor of 3. Following clarification a flow was performed using three times the volume of retentate, in a bleed and feed operation. The filtration experiments were carried out in a permeate flux ranging between 15 and 40 LMH. Viral recovery at the clarification step was determined in the following table. Table 1

    [00072] There was no viral recovery after clarification. This was somewhat unexpected, as for example EP1371723 and WO2006/052302 describe the use of TFF for the clarification of successful adenovirus harvests. Apparently, the high cell densities of the current process impede this step and make the TFF unsuitable for direct clarification. Example 2: Selectable Precipitation of Host Cell DNA in High Density Cell Suspensions
    Selectable host cell DNA precipitation has been demonstrated in the prior art (Goerke et al (2005), US7326555) for cell densities of up to 1 x 106 cells/ml. It was shown there that (at low cell densities) at least 80% of the host cell DNA was precipitated out of the cell suspension with a 90% recovery of the viral particles. However, it has until now been completely unknown whether such selectable precipitation would be feasible at high cell density, as such cell suspensions would contain very high amounts of host cell DNA and cell fragments, and therefore much larger amounts of precipitating agent would be expected. of DNA would be required, whereas extrapolation of prior art data would suggest that such higher concentrations of DNA precipitating agent would also precipitate adenovirus.
    [00074] In order to explore the possibility of DNA precipitation at high cell densities, host cell DNA precipitation was tested in small scale test tubes containing cell densities up to 30 x 106 cells/ml. The small-scale test tube template was used as a rapid screening tool to test whether selectable DNA precipitation still occurs at high cell densities.
    [00075] PER.C6 cells were cultured in a bioreactor and infected with Adenovirus 35 (Ad35) and cultured at 37°C in serum-free culture medium for 3 days. Cells were harvested at a cell density between 2 and 30 x 106 cells/ml and viral titers ranging from 8 x 1010 to 1.5 x 1012 VP/ml. Cell lysis was performed over a period of 2 to 24 hours (hours), by adding the nonionic detergents Triton X-100 and Tween-80 to final concentrations of 0.1% and 0.05% respectively. Increased concentrations of domifen bromide (DB) in 40 mM NaCl were added to 3.5 ml of lysed harvest, followed by immediate vortexing for 1 minute. Precipitated material was removed with syringe filters containing 0.45 g of polyvinylidene fluoride (PVDF). The filtrates were analyzed for concentrations of Ad35 and host cell DNA using an HPLC-AEX and Q-PCR assay respectively.
    [00076] Fig. 1 shows viral recovery and precipitated DNA of the host cell, plotted against the concentration of domifen bromide. Curves plotted in triangles are obtained from cell culture harvests having cell densities ranging from 2.5 x 106 to 3.5 x 106 cells/ml. The curves depicted in the circles are obtained from cell culture harvests having cell densities ranging from 20 x 106 to 30 x 106 cells/ml. The C* (Concentration of Domifen Bromide that shows 90% viral recovery) at low and high cell densities and the related percentage of precipitated host cell DNA is highlighted in the graphs.
    [00077] The DB concentration that is required to precipitate more than 90% of the host cell DNA at cell densities ranging between 20 x 106 and 30 x 106 cells/ml is increased by a factor of at least 2.5 times compared to the DB concentration required at cell densities which are 10 times lower. Surprisingly, the increased DB concentration did not precipitate the viral particles, as would be expected from extrapolating the curves obtained at lower cell density densities.
    [00078] The experiment was repeated with cell culture harvests having cell densities ranging between 18 x 106 and 25 x 106 cells/ml. Fig. 2 shows viral recovery and precipitated host cell DNA, plotted against the concentration of domifen bromide. Curves plotted in triangles are obtained from cell culture harvests having cell densities ranging from 2.5 x 106 to 3.5 x 106 cells/ml. Curves plotted in circles are obtained from cell culture harvests having densities ranging from 18 x 106 to 25 x 106 cells/ml. The C* (Concentration of Domifen Bromide that gives 90% viral recovery) at low and high cell densities and the related percentage of precipitated host cell DNA are highlighted in the graphs.
    [00079] As can be mentioned from the graphs in Figs 1 and 2, the DB concentration giving 90% viral recovery (C*) for the high density cell suspensions may differ slightly between individual experiments, and this is part of the normal variation. However, the graphs consistently demonstrate that selectable DNA precipitation is possible also at high cell densities, and that the adequate concentration of SPA (here DB) is significantly higher than for low cell density cultures, but much higher. lower than would be expected based on extrapolation. Thus, the skilled person will recognize that there is a range rather than a fixed point of suitable concentrations for the selective precipitation agent, and based on the disclosure here, the appropriate range can be found. For example, appropriate DB concentrations to treat an adenovirus containing high density cell suspension harvest comprising a cell density ranging from about 10 x 106 to 30 x 106 cells/ml range from about 1.3 to 2 mM.
    [00080] Based on this result, the person skilled in the art will now be aware that DNA precipitation can be extrapolated to adenovirus-containing suspensions even at higher cell densities, for example around 40 x 106 cells/ml, per example of about 50 x 106 cells/ml, for example up to about 100 x 106 cells/ml, for example up to about 150 x 106 cells/ml, and that adenovirus from such high density cell suspensions can be purified with the process of the present invention.
    [00081] Thus, it is possible according to the present invention to use selectable DNA precipitation in the purification of adenovirus particles from high cell density suspensions. Example 3: Selectable Precipitation of Host Cell DNA in Larger Scale High Density Cell Suspensions
    [00082] DNA precipitation was tested at scales ranging from 0.5 L to 20 L. DB concentrations used for DNA precipitation were based on previous experimental results (Fig. 2). About 80% of the C* concentration as determined in the small scale test tube model was used.
    [00083] In perfusion mode, the perfusion which was performed with an ATF system, was started 4 days after inoculation at a cell density of approximately 2.5 x 106 total cells/ml. After 14 days of perfusion the cell suspension was diluted with fresh serum free medium in the bioreactor to a cell density of about 13 x 106 cells/ml. Subsequently the bioreactor was infected with Ad35 virus. The ATF system was started 5 hours after infection at a relief rate of half 2 vessel volumes per day. After 3 days (after infection) cells were harvested. The cell density at harvest (CDAH) is given in Table 2.
    [00084] Subsequent to harvesting, cells were lysed within a period of 2 to 24 hours by adding the nonionic detergents Triton X-100 and Tween-80 to final concentrations of respectively 0.1 and 0.05%. Domifen bromide was added to the lysed harvest to final concentrations of 0.72 and 1.52 mM in 40 mM NaCl. The precipitated lysate was clarified using two consecutive loaded deep filters with different pore sizes. The estimated pore sizes of both filters were varied between ~10 to ~5 μm (Millistak + CE20) and ~1 to ~0.2 μm (Cuno Zeta plus 50CP) respectively. The first filter was used to remove large cell debris and impurities. After said pre-filtration, the second filter was used to remove remaining impurities and precipitated DNA from the virus-containing suspension. Clarification was carried out at a constant flow of 100 LMH (liter per square meter per hour) until the pressure reached 5 psi (34.5 kPa).
    [00085] Table 2 shows the process parameters and results of the purification process. Lysis, DNA precipitation (ptt DNA) and clarification were performed using 8 different harvests, which differed in volume, cell density at harvest (CDAH) and viral titer. Crops were collected from 2L or 10L bioreactors. The percentage of precipitated host cell DNA (HC-DNA) and the viral recovery in the precipitation step were determined. Table 2

    [00086] It is concluded that the actionable precipitation of host cell DNA is possible at high cell density. In fact, although the DB concentration was increased (by a factor of 2), the viral particles remained unprecipitated (see recovery higher than 70%) and the HC-DNA reduction was higher than 98%.
    [00087] This process offers a method of discarding host cell DNA from harvesting high cell density cultures without compromising viral recovery. With this it allows for the processing of large volumes of high density cell suspensions, which is necessary in industrial processes.
    [00088] It should be mentioned that for practical reasons, a single concentration of DB (1.52 mM) was used for the selectable precipitation of DNA in experiments 4 to 8. Said experiments show that adenovirus-containing suspensions having a wide range ( 9.1 x 106 to 25.8 x 106 vc/ml) of cell densities can be treated with 1.52 mM DB. This is consistent with the above notion that the relationship between adequate selective precipitating agent concentrations and cell density is not a very fixed relationship, but rather provides variation so that a range of precipitating agent concentrations is suitable for a given cell density.
    [00089] The appropriate concentration of DB to treat a high density cell suspension containing adenovirus comprising a cell density ranging between 10 x 106 and 50 x 106 cells/ml ranges between about 1.3 mM and 2.2 mM . The appropriate concentration of DB to treat a high density cell suspension harvest containing adenovirus which comprises a cell density ranging from 10 x 106 to 30 x 106 cells/ml ranges from about 1.3 to 2 mM.
    [00090] It is mentioned that for some experiments at high cell densities (experiments # 6 and 7), reduced viral recovery and filtration capacity in the 2nd filter, when compared to the low cell density harvests, were observed. Furthermore, the use of two separate filters in a row makes the process laborious and expensive.
    [00091] Example 4: Single-step clarification of high cell density adenovirus preparations by selectable precipitation of host cell DNA followed by TFF
    [00092] PER.C6 cells were cultured in a perfusion bioreactor and infected with Ad35 and cultured at 36°C in serum-free culture medium for 3 days. Cells were harvested at a cell density between 20 and 30 x 106 cells/ml and viral titers of 1 to 1.5 x 1012 VP/ml. Viruses were released from the cells by adding the nonionic detergents Triton X-100 and Tween-80 to final concentrations of 0.1 and 0.05% respectively. The lysis time was between 2 and 24 hours. Subsequent to cell lysis, a DNA precipitation step was performed followed by clarification.
    [00093] DNA precipitation was carried out using 0.063 % to 0.077 % domifen bromide (DB) in 40 mM NaCl. DNA precipitation was carried out for 3 hours with an addition time of 2 hours DB at a stirring speed of 0.17 to 1.52 m/s-1. Clarification was performed using tangential flow filtration with a 0.65 µm hollow fiber filter (GE Healthcare, model CFP-6-D-4MA, 0.046 m2). One liter of lysed harvest and precipitated DNA was concentrated by at least a factor of 3. Following clarification a water flow was carried out using three times the volume of retentate, in a bleed and feed operation. The filtration experiments were carried out in a permeate flux ranging between 15 and 40 LMH. Lysis followed by DNA precipitation and clarification were performed using 3 different harvests. The percentage of precipitated host cell DNA, concentration of host cell DNA per 1x 1011 viral particles, and viral recovery after selectable precipitation and clarification were determined in Table 3. Table 3

    [00094] Similar to the previous example, it was unexpectedly shown here that selective DNA precipitation was possible at high cell density. In fact, although the DB concentration was increased, the viral particles remained unprecipitated (see recovery higher than 80%) and the HC-DNA reduction was higher than 99%.
    [00095] Despite the flaw in example 1, it is shown in this example that tangential flow filtration, when used in combination with selectable HC-DNA precipitation allowed the high density cell suspensions to be clarified.
    [00096] The use of tangential flow filtration instead of a set of two distinct depth filters (as exemplified previously) offers the possibility to process a high density cell suspension to a clarified adenovirus suspension in a single filtration step.
    [00097] The combination of selectable HC-DNA precipitation with the use of tangential flow filtration allows removal of host cell DNA from the high density cell suspension harvest, and unexpectedly does so without compromising adenovirus recovery. In fact, overall adenovirus recovery using this process can be even higher than using the classic two-step filtration clarification processes using deep filters. Furthermore, it makes the purification process less labor intensive and expensive when compared to the process with deep filters. With this it allows for the processing of large volumes of high density cell suspensions, which are required in industrial processes. References Cortin V, Thibault J, Jacob D, Gamier A. High-Titer Adenovirus Vector Production in 293S Cell Perfusion Culture. Biotechnol. Program 2004. Goerke A, To B, Lee A, Sagar S, Konz K. Development of a Novel Adenovirus Purification Process Utilizing Selective Precipitation of Cellular DNA. Biotechnology and bioengineering, Vol. 91, No. 1, July 5, 2005. Yuk IHY, Olsen MM, Geyer S, Forestell SP. Perfusion Cultures of Human Tumor Cells: A Scalable Production Platform for Oncolytic Adenoviral Vectors. Biotechnol. Bioengine. 86: 637-641 (2004).
    权利要求:
    Claims (9)
    [0001]
    1. Method for purifying adenovirus particles from a cell suspension having a cell density ranging from 5 x 106 to 150 x 106 cells per ml, said method characterized in that it comprises the steps of: a) cultivating a host cell suspension having a cell density between 5 x 106 and 150 x 106 cells per ml and infecting the cell suspension; b) lysing the cells within said cell suspension with a detergent; c) selectively precipitating host cell DNA within said cell suspension by adding a selective precipitating agent to the cell suspension, wherein the selective precipitating agent is selected from the group consisting of quaternary ammonium compounds, amine copolymer and mixtures of the same; or fragment host cell DNA within said cell suspension by adding a nuclease; d) subjecting the cell suspension obtained from step b) to clarification by tangential flow filtration, to obtain a suspension of purified adenovirus,
    [0002]
    2. Method for purifying adenovirus particles according to claim 1, characterized in that said precipitation in step b) is carried out by selectively precipitating host cell DNA out of the viral particles by adding a selective precipitation agent .
    [0003]
    3. Method according to claim 2, characterized in that said selective precipitation agent is domifen bromide (DB).
    [0004]
    4. Method according to claim 3, characterized in that the concentration of domifen bromide (DB) is ranging from 1.2 to 5 mM.
    [0005]
    5. Method according to claim 3, characterized in that the cell density is ranging from about 10 x 106 to 30 x 106 cells per ml and the concentration of domifen bromide (DB) is ranging from 1.3 to 2 mM.
    [0006]
    6. Method according to any one of claims 1 to 5, characterized in that said tangential flow filtration is performed with a membrane having a pore size ranging from 0.1 to 0.65 µm.
    [0007]
    7. Method according to any one of claims 1 to 6, characterized in that said tangential flow filtration is performed with a hollow fiber.
    [0008]
    8. Method according to any one of claims 1 to 7, characterized in that the tangential flow day filtration is performed with an ATF system.
    [0009]
    9. Method according to any one of claims 1 to 8, characterized in that at least 80% of the host cell DNA was removed in the suspension of purified adenovirus.
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    法律状态:
    2018-06-19| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2018-08-21| B25D| Requested change of name of applicant approved|Owner name: JANSSEN VACCINES AND PREVENTION B.V (NL) |
    2018-08-21| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-10-15| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|
    2019-12-31| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-06-09| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2020-12-29| B25G| Requested change of headquarter approved|Owner name: JANSSEN VACCINES AND PREVENTION B.V. (NL) |
    2021-03-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-05-04| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/10/2010, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME MEDIDA CAUTELAR DE 07/04/2021 - ADI 5.529/DF |
    2021-05-25| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/10/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |
    优先权:
    申请号 | 申请日 | 专利标题
    US27906009P| true| 2009-10-15|2009-10-15|
    US61/279,060|2009-10-15|
    EP09173119|2009-10-15|
    EP09173119.0|2009-10-15|
    PCT/EP2010/065436|WO2011045381A1|2009-10-15|2010-10-14|Process for adenovirus purification from high cell density cultures|
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