Patent Description:
AAV and adenovirus are naturally existing viruses. AAV is the only viral vector that has been widely testified not to cause cytotoxicity and immune response while adenoviruses have been found to cause immune responses. In addition, AAV shows efficient transduction and stable expression in dividing and non-dividing cells such as neuronal cells and is, therefore, considered as a most potent viral vector in both basic research and R&D of gene therapy. One example is Glybera, the first gene therapy medicine approved in European Union in <NUM>, which successfully uses AAV as vector to deliver lipoprotein lipase gene for treatment of lipoprotein lipase deficiency. Currently AAV has been used as a vector to develop gene therapy medicines in many pharmaceutical companies in the United States and around the world. Based on the advance in use, it is the urgent need to explore a convenient and efficient technology for purification of the virus.

<CIT> describes a method for the purification of a virus from a host cell, where the virus is preferably a recombinant adenovirus.

<CIT> concerns methods of adenovirus purification.

<CIT> describes an improved method for the production and purification of adenoviral vectors.

<CIT> concerns methods for generating high titer helper-free preparations of released recombinant AAV vectors.

<NPL> describes rapid and simplified purification of recombinant adeno-associated virus.

This technology provides an ideal method that yields ultra-high purity of viral production at a high recovery rate. Furthermore, the technology employs no toxic compounds and is easy to operate and readily scalable.

The invention provides a method of extracting viral particles of a recombinant adeno-associated virus (AAV) or an adenovirus (AdV) from a sample comprising cells enclosing the viral particles, comprising: lysing the cells by the addition of an alkaline lysis solution, comprising a detergent and one or more reagents, to the sample such that the sample comprises <NUM>% to <NUM>% of the detergent and has a pH of <NUM> to <NUM> for AAV, or pH of <NUM> to <NUM> for AdV, wherein the detergent is selected from the group consisting of deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, alkyl Sulfates, Polysorbate <NUM> (Tween-<NUM>), Tergitol-type NP-<NUM> and the combinations thereof, and wherein the one or more reagents is selected from the group consisting of N-cyclohexyl-<NUM>-aminopropanesulfonic acid (CAPS), glycine/sodium, glycine/potassium, sodium carbonate, potassium carbonate, sodium phosphate, potassium-phosphate and combinations thereof; precipitating host cell proteins released from the lysed cells by (a) adding a salting solution to the sample such that the sample comprises <NUM> to <NUM> of a salt and has a pH of <NUM> to <NUM>, wherein the salt is selected from the group consisting of sodium acetate, potassium acetate, sodium phosphate and combinations thereof, and (b) removing the precipitated host cell proteins to obtain a supernatant comprising the viral particles; precipitating the viral particles from the supernatant with a precipitation solution, wherein upon addition of the precipitation solution to the sample, the sample comprises <NUM>% to <NUM>% of a polyethylene glycol (PEG); and further purifying the precipitated viral particles with chromatography.

In some embodiments, the viral particles are of any one of AAV serotypes <NUM>-<NUM> or any one of AdV species A-G. In some embodiments, the detergent is deoxycholate, or is deoxycholate and sodium dodecyl sulfate (SDS).

In some embodiments, the salting solution does not include an ammonium salt.

In some embodiments, the PEG has an average molecular weight of <NUM> to <NUM>.

In some embodiments, the chromatography is silica-based chromatography, resin chromatography or ion exchange chromatography columns.

In some embodiments, the method does not include the use of an organic solvent. In some embodiments, the method does not include the use of ultracentrifugation. In some embodiments, the method does not include extraction by freeze-thaw cycles. In some embodiments, the method does not include the use of affinity chromatography.

In accordance with one embodiment of the application, provided is a kit or package comprising, in separate compartments or containers: an alkaline cell lysis solution (also referred to as an alkaline lysis solution or a lysis solution) comprising a detergent selected from the group consisting of deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, alkyl Sulfates, polysorbate <NUM> (Tween-<NUM>), Tergitol-type NP-<NUM> and a mixture thereof, and an alkali selected from the group consisting of N-cyclohexyl-<NUM>-aminopropanesulfonic acid (CAPS), glycine/sodium, glycine/potassium, sodium carbonate, potassium carbonate, sodium phosphate, potassium-phosphate and a combination thereof, at a pH of <NUM>-<NUM>; a first fractionation solution (also referred to as a salting solution or a salt solution), comprising <NUM>-<NUM> of a salt selected from the group consisting of sodium acetate, potassium acetate, sodium phosphate or the combination thereof, at a pH of <NUM>-<NUM>; a precipitation solution comprising <NUM>%-<NUM>% of a polyethylene glycol (PEG); and a solid phase chromatography column. In one embodiment, the detergent may be sodium deoxycholate (DOC).

In some embodiments, the solid phase chromatography column is selected from the group consisting of a silica-based chromatography column and a solid phase silica-based ion exchange chromatography column. In some embodiments, the solid phase chromatography column is a silica-based cation exchange chromatography column. In some embodiments, the kit or package further comprises one or more of (a) a column equilibration solution comprising <NUM>-<NUM> sodium acetate, potassium acetate, <NUM>-(N-morpholino) propanesulfonic acid (MOPS), and <NUM>-<NUM> sodium chloride at a pH of <NUM>-<NUM>, (b) a column washing solution comprising <NUM>-<NUM> sodium acetate, potassium acetate, <NUM>-(N-morpholino) propanesulfonic acid (MOPS), and <NUM>-<NUM> sodium chloride, at a pH of <NUM>-<NUM>, or (c) a column elution solution comprising <NUM>-<NUM> sodium acetate, potassium acetate, <NUM>-(N-morpholino) propanesulfonic acid (MOPS), sodium phosphate, or tris, and <NUM>-<NUM> sodium chloride or other salts, at a pH of about <NUM>-<NUM>.

In some embodiments, the solid phase chromatography column is a solid phase silica-based anion exchange chromatography column. In some embodiments, the kit or package further comprises one or more of (a) a column equilibration solution comprising <NUM>-<NUM> sodium phosphate, sodium glycine, Tris, or MOPS, and <NUM>-<NUM> sodium chloride, at a pH of <NUM>-<NUM>, (b) a column washing solution comprising about <NUM>-<NUM> sodium phosphate, sodium glycine, Tris, or MOPS, and <NUM>-<NUM> sodium chloride, at a pH of <NUM>-<NUM>, or (c) a column elution solution comprising <NUM>-<NUM> sodium acetate, sodium phosphate, Tris, or MOPS, and <NUM>-<NUM> sodium chloride, at a pH of about <NUM>-<NUM>.

In some embodiments, the kit or package further comprises a cell suspension solution comprising a serum-free Dulbecco's modified eagle medium (DMEM), a phosphate-buffered saline (PBS), or Tris-buffered saline (TBS).

In some embodiments, the kit or package further comprises a pellet suspension solution comprising <NUM>-<NUM> sodium chloride in <NUM>-<NUM> phosphate buffer or Tris buffer, at a pH of <NUM>-<NUM>, or serum-free Dulbecco's modified eagle medium (DMEM).

In some embodiments, the kit or package further comprises centrifugal filters or viral concentration sets. In some embodiments, the kit or package does not include an affinity chromatography resin or column.

As used in the present specification, the following words, phrases and symbols are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

Conventional methods for the extraction and purification of AAV and AdV particles entail the use of freeze/thaw cycles followed with density gradient ultracentrifugation. In some methods, affinity or ion exchange chromatography is not employed to improve purity of the final product. Such methods typically result in reduced infectivity of the viruses, accompanied by poor recovery. Moreover, for preclinical or clinical use of the viruses, the gradient-associated health toxicity can be a significant concern.

One embodiment of the present disclosure provides a method of extracting viral particles of a recombinant adeno-associated virus (AAV) or an adenovirus (AdV) from a sample comprising cells enclosing the viral particles, comprising: <NUM> lysing the cells by the addition of an alkaline lysis solution, comprising a detergent and one or more reagents, to the sample such that the sample comprises <NUM>% to <NUM>% of the detergent and has a pH of <NUM> to <NUM> for AAV, or <NUM> to <NUM> for AdV, wherein the detergent is selected from the group consisting of deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, alkyl Sulfates, Polysorbate <NUM> (Tween-<NUM>), Tergitol-type <NUM> NP-<NUM> and the combinations thereof, and wherein the one or more reagents is selected from the group consisting of N-cyclohexyl-<NUM>-aminopropanesulfonic acid (CAPS), glycine/sodium, glycine/potassium, sodium carbonate, potassium carbonate, sodium phosphate, potassium-phosphate and combinations thereof; precipitating host cell proteins released from the lysed cells by (a) adding a salting <NUM> solution to the sample such that the sample comprises <NUM> to <NUM> of a salt and has a pH of <NUM> to <NUM>, wherein the salt is selected from the group consisting of sodium acetate, potassium acetate, sodium phosphate and combinations thereof and (b) removing the precipitated host cell proteins to obtain a supernatant comprising the viral particles; precipitating the viral particles from the supernatant with a precipitation solution, <NUM> wherein upon addition of the precipitation solution to the sample, the sample comprises <NUM>% to <NUM>% of a polyethylene glycol (PEG); and further purifying the precipitated viral particles with chromatography. Also, in this method, the freeze/thaw cycles and density gradient ultracentrifugation are not required. The new method is easier to implement and readily scalable. As further detailed below, the method includes lysing the cells packaged with the viruses with an alkaline lysis solution followed by precipitating and removing a majority of host cell proteins with a salt solution. Furthermore, the extracted viral particles are precipitated with polyethylene glycol (PEG) and purified by chromatography. The methods disclosed herein are suitable for all different serotypes and species of AAV (e.g., adeno-associated virus serotypes <NUM>-<NUM>) or AdV (adenovirus species A-G with their respective serotypes) or other "naked" or non-naked viruses within cells after they are packaged.

"Naked viruses" refer to viruses that only have nucleocapsid, which is a protein capsid that covers the genome of the virus, and lack the viral envelop. Examples of naked viruses include, without limitation, parvoviruses, papovaviruses, adenoviruses, and reoviruses.

"Non-naked viruses" (or "enveloped viruses") refer to viruses with a lipoprotein envelope surrounding the nucleoprotein core of the virus. Examples include, without limitation, Hepatitis B viruses and Hepatitis C viruses.

In accordance with one embodiment of the disclosure, cells that enclosing AAV or AdV viruses are lysed with an alkaline lysis solution. The lysis solution comprises a detergent and one or more reagents, such that the sample comprises <NUM>% to <NUM>% of the detergent and has a pH of <NUM> to <NUM> for AAV, or <NUM> to <NUM> for AdV, wherein the detergent is selected from the group consisting of deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, alkyl Sulfates, Polysorbate <NUM> (Tween-<NUM>), Tergitol-type <NUM> NP-<NUM> and the combinations thereof, and wherein the one or more reagents is selected from the group consisting of N-cyclohexyl-<NUM>-aminopropanesulfonic acid (CAPS), glycine/sodium, glycine/potassium, sodium carbonate, potassium carbonate, sodium phosphate, potassium-phosphate and combinations thereof. The lysis solution includes a detergent which is a mild detergent or a detergent that contributes to cell lysis but does not decrease the viral activities.

An "alkaline lysis solution" or "cell lysis solution" or more generally "lysis solution", as used herein, refers to a solution that contains compound(s) or reagent(s) that maintain a pH higher than <NUM>. The alkaline lysis solution includes a detergent that is useful for breaking up cells. The detergent is a mild detergent such as deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, octyl thioglucoside, octyl glucoside, alkyl Sulfates, polysorbate <NUM> (Tween-<NUM>), Tergitol-type NP-<NUM>, or the mixtures thereof.

The pH and ingredients of the lysis solution can be determined based on the desired pH and final concentrations of the ingredients in the cell sample once the lysis solution is added to the sample. In some embodiments, the pH of the sample is from <NUM> to <NUM> for AAV, or from <NUM> to about <NUM> for AdV. The pH of the lysis solution can be achieved or adjusted with a compound or reagent, like a base, having a concentration from about <NUM> to about <NUM>, for instance.

In some embodiments, the sample has a final concentration of the detergent at <NUM>% to <NUM>%,. The final concentration of the detergent in the sample is at least <NUM>%, or alternatively at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%. The final concentration of the detergent in the sample is not greater than <NUM>%, or alternatively not greater than about <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>%. In some embodiments, the final concentration of the detergent in the sample is from about <NUM>% to about <NUM>%, from about <NUM>% to about <NUM>%, or from about <NUM>% to about <NUM>%, without limitation.

The detergent used is preferably mild unlike strong detergents such as Triton X-<NUM>. Non-limiting examples of detergents suitable for use here include deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, octyl thioglucoside, octyl glucoside, alkyl Sulfates, polysorbate <NUM> (Tween-<NUM>), and Tergitol-type NP-<NUM>. In one embodiment, the detergent used is deoxycholate, such as sodium deoxycholate (DOC).

In addition to the detergent, the lysis solution includes compound ingredients such as N-cyclohexyl-<NUM>-aminopropanesulfonic acid (CAPS), glycine/sodium, glycine/potassium, sodium carbonate, potassium carbonate, sodium phosphate, potassium-phosphate or the combinations thereof.

It is discovered herein that the cell lysis methods disclosed here, without the need of freeze/thaw cycles, can achieve fairly complete lysis of the cells while maintaining high infectivity of the viruses. Such a lysed viral sample can then be subject to a salting precipitation process which removes a majority of the contaminating host cell proteins.

The salting precipitation step of the process or also referred as first fractionation, in one embodiment, includes the addition of a salting solution that has a neutral or acidic pH (e.g., <NUM> to <NUM>) and comprises <NUM>-<NUM> of a salt selected from the group consisting of sodium acetate, potassium acetate, sodium phosphate or the combination thereof. A "salting solution" as used herein refers to a solution with the desired pH and salt at a suitable concentration as disclosed herein. Like for the lysis solution, the pH and salt concentration of the salting solution can be determined based on the desired pH and salt concentration in the viral sample and the starting condition of the sample before the salting solution is added.

In one embodiment, the pH of the viral sample, upon addition of the salting solution, is not higher than <NUM>, or alternatively not higher than <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In another embodiment, the pH of the viral sample, upon addition of the salting solution, is higher than <NUM>, or alternatively higher than <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In some embodiments, the pH of the viral sample, upon addition of the salting solution, is from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, or from about <NUM> to about <NUM>.

The salt or salts used in the salting solution are selected from one or more of the following: sodium acetate, potassium acetate, sodium phosphate, or a combination thereof. The final concentration of the salt or salts is from <NUM> to <NUM>, such as from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>.

Once the salting solution is added to the viral sample, the majority of host cell proteins can be precipitated and removed from the sample by centrifugation, while the viral particles stay in the supernatant, which can be further purified.

The salting step can remove a majority of the proteins from the sample resulting in viral particles enriched in the sample with a little protein contamination. Such a sample can be further purified as needed, just as removing the salt in the sample. According to the invention, the sample is mixed with a precipitation solution wherein upon addition of the precipitation solution to the sample, the sample comprises <NUM>% to <NUM>% of a polyethylene glycol (PEG).

The term "precipitation solution" as used herein generically refers to a solution that includes polyethylene glycol (PEG) which, when added to a viral sample, is able to precipitate viral particles. The molecular weight of the PEG is typically from about <NUM> to about <NUM>, or preferably from about <NUM> to about <NUM>. The concentration of the PEG in the precipitation solution is at least about <NUM>%. Like for the lysis solution and the salting solution, the concentration of the PEG in the precipitation solution can be determined by the desired PEG concentration in the viral sample upon addition of the precipitation solution in the viral sample.

In one embodiment, the final PEG concentration in the viral sample is <NUM>% to <NUM>%. The final PEG concentration in the viral sample is not greater than <NUM>%.

The PEG in the viral sample can precipitate the viral particles which can then be resuspended in a suitable solution.

The viral sample or resuspended viral particles are subject to chromatography. Non-limiting examples of chromatography include silica-based chromatography, resin chromatography or ion exchange chromatography columns. Conditions for using the chromatography can be set with conventional knowledge in the art.

The lysis, extraction and purification processes described here, in some embodiment, do not require (but do not exclude) certain techniques commonly used in the art. For instance, in some conventional methods, organic solvents are employed for lysing the cells or degrading proteins. No organic solvents, however, are required by the current technology.

Another commonly used method includes freeze/thaw cycles, which are also not required by the present disclosure technology. Yet another method includes ultracentrifugation, which typically employs a gradient made of cesium chloride (CsCl) or iodixanol. Ultracentrifugation or the associated gradient reagents are also not required in the present technology. Also not required is affinity chromatography, in some embodiments.

Viral particles may be prepared by the methods of the invention. It is readily appreciated that in addition to the improved efficiency of viral extraction and purification of the present technology, the end product of the technology is also superior to what can be obtained with the conventional technologies.

For instance, since the present technology may not require ultracentrifugation, the end product would necessarily not contain any contamination of the gradients, such as cesium chloride (CsCl) or iodixanol.

Also, superior to the affinity chromatography method which is not effective in removing empty viral particles from viruses, the end product of the present technology would contain a much lower amount of empty particles. The end product may contain fewer than <NUM> empty particle per <NUM>,<NUM> viral particles, or alternatively fewer than <NUM> empty particle per <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM><NUM>, <NUM><NUM>, <NUM><NUM>, <NUM><NUM>, or <NUM><NUM> viral particles.

Another difference between the product of the present process and those made by conventional technologies is that the present product has less protein contamination. The end product may contain less than about <NUM>/<NUM>,<NUM> (w/w) host cell proteins over the total amount of the viral particles, or alternatively less than about <NUM>/<NUM>,<NUM>, <NUM>/<NUM>,<NUM><NUM>/<NUM>,<NUM>, <NUM>/<NUM>,<NUM>, or <NUM>/<NUM>,<NUM>,<NUM> (w/w) host cell proteins.

Yet another advantage of the present technology is that the purified viruses retain greater infectivity as compared to what has been done in the prior art. For each <NUM><NUM> of the particles in a prepared sample, there may be at least <NUM><NUM> transducing units of active viruses. For each <NUM><NUM> of the particles in a prepared sample, there may be at least 1x10<NUM>, 2x10<NUM>, 5x10<NUM>, 2x10<NUM>, 5x10<NUM>, 1x10<NUM>, 2x10<NUM>, 5x10<NUM>, or 1x10<NUM> transducing units of viruses.

Kits and packages that include two or more items, such as solutions, columns, and filters, that are useful for viral purification, are also provided. Such kits and packages provide convenient means to practice the present technology. It is understood that the kits or packages may not need to include every single item needed for the purification process, as some of the items can be prepared on the fly or obtained from other sources.

According to the invention in some embodiments there is a kit or package comprising, in separate compartments or containers: a cell lysis solution (also referred to as an alkaline lysis solution or lysis solution) comprising a detergent selected from the group consisting of deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, alkyl Sulfates, polysorbate <NUM> (Tween-<NUM>), Tergitol-type NP-<NUM> and a mixture thereof, and an alkali selected from the group consisting of N-cyclohexyl-<NUM>-aminopropanesulfonic acid (CAPS), glycine/sodium, glycine/potassium, sodium carbonate, potassium carbonate, sodium phosphate, potassium-phosphate and a combination thereof, at a pH of <NUM>-<NUM>, a first fractionation solution (also referred to as a salting solution) comprising <NUM>-<NUM> of a salt selected from the group consisting of sodium acetate, potassium acetate, sodium phosphate or the combination thereof, at a pH of <NUM>-<NUM>, a second fractionation solution (also referred to as a precipitation solution) comprising <NUM>% to <NUM>% of a polyethylene glycol (PEG), and a solid phase chromatography column. The kit may also comprise a pellet suspension solution (also referred to as a viral suspension solution). a cell suspension solution, a column equilibration solution, a column washing solution, a column elution solution, a centrifugal filter, and suitable column adaptor and syringe or other column supportive sets or materials. Each of these items have been described with examples and are further illustrated below.

The cell suspension solution, in some embodiments, includes or consists of a serum-free Dulbecco's modified eagle medium (DMEM), a phosphate-buffered saline (PBS), or Tris-buffered saline (TBS). The cell suspension solution may have a pH of about <NUM>-<NUM>, or <NUM>-<NUM>, or <NUM>-<NUM>.

The cell lysis solution includes a detergent which is preferably a mild detergent or a detergent that contributes to cell lysis but does not decrease the viral activities. Non-limiting examples of the detergent include deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, octyl thioglucoside, octyl glucoside, alkyl Sulfates, polysorbate <NUM> (Tween-<NUM>), Tergitol-type NP-<NUM>, or the mixtures thereof. In one embodiment, the detergent used is deoxycholate, such as sodium deoxycholate (DOC).

In addition to the detergent, the lysis solution includes an alkali such as N-cyclohexyl-<NUM>-aminopropanesulfonic acid (CAPS), glycine/sodium, glycine/potassium, sodium carbonate, potassium carbonate, sodium phosphate, potassium-phosphate or the combinations thereof.

In one embodiment, cell lysis solution includes a detergent selected from the group consisting of octyl glucoside, octyl thioglucoside, polysorbate <NUM> (Tween-<NUM>), sodium deoxycholate (DOC), sodium dodecyl sulfate (SDS) and an alkali selected from the group consisting of sodium-glycine, sodium-phosphate, N-cyclohexyl-<NUM>-aminopropanesulfonic acid (CAPS) and the combination thereof.

The pH and ingredients of the lysis solution can be determined based on the desired pH and final concentrations of the ingredients in the cell sample once the lysis solution is added to the sample. In one embodiment, upon addition of the lysis solution to the cell sample, the pH of the sample is preferably at least <NUM>, or alternatively at least <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM>. In some embodiments, the pH of the sample is from about <NUM> to about <NUM>, or from about <NUM> to about <NUM>, or from about <NUM> to about <NUM>, or from about <NUM> to about <NUM>, or from about <NUM> to about <NUM>, or from about <NUM> to about <NUM>, or from about <NUM> to about <NUM>, or from about <NUM> to about <NUM>, or from about <NUM> to about <NUM>, without limitation. The pH of the lysis solution can be achieved or adjusted with a compound or reagent, like a base, having a concentration from about <NUM> to about <NUM>, for instance.

The first fractionation solution, also referred to as a salting solution, can have a neutral or acidic pH (e.g., about <NUM> to about <NUM>) and include a salt selected from the group consisting of sodium acetate, potassium acetate, sodium phosphate or the combination thereof at a concentration of <NUM>-<NUM>.

The salt or salts used in the first fractionation solution are selected from one or more of the following: sodium acetate, potassium acetate, or sodium phosphate, without limitation. Final concentration of the salt in the viral sample can be from about <NUM> and up. In one embodiment, the concentration of the salt or salts is at least <NUM>, or alternatively at least about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In one embodiment, the concentration of the salt or salts is not greater than about <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>. In some embodiments, the concentration of the salt or salts is from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, from about <NUM> to about <NUM>, without limitation.

In one embodiment, the pH of the first fractionation solution is <NUM>-<NUM>. The pH of the first fractionation solution may be from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, from <NUM> to <NUM>, or from <NUM> to <NUM>.

In one embodiment, the first fractionation solution includes <NUM>-<NUM> of a salt selected from the group consisting of sodium acetate, potassium acetate, sodium phosphate or the combination thereof, at a pH of <NUM>-<NUM>.

The second fractionation solution (also referred to as a precipitation solution) includes a polyethylene glycol (PEG). The molecular weight of the PEG is typically from about <NUM> to about <NUM>, or preferably from about <NUM> to about <NUM>. In one embodiment, the second fractionation solution includes <NUM>%-<NUM>% of a PEG having an average molecular weight of <NUM> to <NUM>.

The pellet suspension solution (also referred to as a viral suspension solution), in some embodiments, includes <NUM>-<NUM> sodium chloride in <NUM>-<NUM> phosphate buffer or Tris buffer, at a pH of about <NUM>-<NUM>. In some embodiments, the pellet suspension solution is serum-free DMEM medium.

In some embodiments, different types of solid phase chromatography columns can be included in the kits or packages. Non-limiting examples include silica-based chromatography columns and solid phase silica-based ion exchange chromatography columns. Each type of column is suitably used with optimized column equilibration solution, column washing solution and/or column elution solution.

In some embodiments, the kit or package comprises silica-based cation exchange chromatography columns and a column equilibration solution that comprises about <NUM>-<NUM> sodium acetate, potassium acetate, or <NUM>-(N-morpholino) propanesulfonic acid (MOPS), and about <NUM>-<NUM> sodium chloride, at a pH of <NUM>-<NUM>. In some embodiments, the kit or package comprises silica-based anion exchange chromatography columns and a column equilibration solution that comprises about <NUM>-<NUM> sodium phosphate, sodium glycine, Tris, or MOPS, and about <NUM>-<NUM> sodium chloride, at a pH of <NUM>-<NUM>.

In some embodiments, the kit or package comprises silica-based cation exchange chromatography columns and a column washing solution that comprises about <NUM>-<NUM> sodium acetate, potassium acetate, or <NUM>-(N-morpholino) propanesulfonic acid (MOPS), and about <NUM>-<NUM> sodium chloride, at a pH of <NUM>-<NUM>. In some embodiments, the kit or package comprises silica-based anion exchange chromatography columns and a column washing solution that comprises about <NUM>-<NUM> sodium phosphate, sodium glycine, Tris, or MOPS, and about <NUM>-<NUM> sodium chloride, at a pH of <NUM>-<NUM>.

In some embodiments, the kit or package comprises silica-based cation exchange chromatography columns and a column elution solution that comprises about <NUM>-<NUM> sodium acetate, potassium acetate, or <NUM>-(N-morpholino) propanesulfonic acid (MOPS), sodium phosphate or tris, and about <NUM>-<NUM> sodium chloride, at a pH of <NUM>-<NUM>. In some embodiments, the kit or package comprises silica-based anion exchange chromatography columns and a column elution solution that comprises about <NUM>-<NUM> sodium acetate, sodium phosphate, Tris, or MOPS, and about <NUM>-<NUM> sodium chloride, at a pH of <NUM>-<NUM>.

The kit or package may comprise column supportive sets or materials to adjust the solution flow through the columns. The kit or package may comprise column supportive materials including column top adapters and syringes. The kit or package may comprise column supportive sets that are vacuum connection units.

In some embodiments, the kit or package comprises viral concentration sets to concentrate the viral eluents from above-mentioned chromatography columns. In some embodiments, viral concentration sets are some centrifugal filters or other concentration sets.

In some embodiments, the kit or package includes at least a cell lysis solution comprising a detergent selected from the group consisting of deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, alkyl <NUM> Sulfates, polysorbate <NUM> (Tween-<NUM>), Tergitol-type NP-<NUM> and a mixture thereof, and an alkali selected from the group consisting of N-cyclohexyl-<NUM>-aminopropanesulfonic acid (CAPS), glycine/sodium, glycine/potassium, sodium carbonate, potassium carbonate, sodium phosphate, potassium-phosphate and a combination thereof, at a pH of <NUM>-<NUM>, a first fractionation solution comprising <NUM>-<NUM> of a salt selected from the group <NUM> consisting of sodium acetate, potassium acetate, sodium phosphate or the combination thereof, at a pH of <NUM>-<NUM>, a second fractionation solution comprising <NUM>% to <NUM>% of a polyethylene glycol (PEG) and a solid phase extraction-like chromatography column. In some embodiments, also included are a cell suspension solution, or a pellet suspension solution. In some embodiments, for the column, a column equilibration solution, a column washing solution, and/or a column elution solution is also provided.

Adeno-associated virus (AAV) is a small virus which infects humans and some other primate species. AAV is not currently known to cause disease. The virus causes a mild immune response, lending further support to its apparent lack of pathogenicity. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell, although in the native virus some integration of virally carried genes into the host genome does occur. These features make AAV an attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models.

The best studied AAV serotype is serotype <NUM>. Serotypes <NUM>, <NUM>, <NUM>, and <NUM> were discovered in human cells, AAV serotypes <NUM>, <NUM>, and <NUM>-<NUM> in nonhuman primate samples. There have been <NUM> AAV serotypes described. AAV capsid proteins contain <NUM> hypervariable surface regions, with most variability occurring in the threefold proximal peaks, but the parvovirus genome in general presents highly conserved replication and structural genes across serotypes. All of the known serotypes can infect cells from multiple diverse tissue types. Tissue specificity is determined by the capsid serotype and pseudotyping of AAV vectors to alter their tropism range will likely be important to their use in therapy.

Adenoviruses (AdV) are medium-sized (<NUM>-<NUM>), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double stranded DNA genome. They have a broad range of vertebrate hosts; in humans, more than <NUM> distinct adenoviral serotypes, belonging to seven species (A-G), have been found to cause a wide range of illnesses, from mild respiratory infections in young children (known as the common cold) to life-threatening multi-organ disease in people with a weakened immune system.

Methods of preparing recombinant AAV and AdV and packaging them into suitable host cells are known in the art. For AAV, viral production cells can include at least the minimum components required to generate AAV particles, where production of an AAV DNase resistant genome containing particles involves packaging an expression cassette into AAV capsids. The minimum required components include, for instance, an expression cassette to be packaged into the AAV capsids, AAV cap, and AAV rep or functional fragments thereof, and helper functions.

Suitable cells and cell lines have been described for use in production of AAV and AdV. The cells themselves may be selected from any biological organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells including insect cells, yeast cells and mammalian cells. Particularly desirable host cells are selected from among any mammalian species, including, without limitation, A549, WEHI, 3T3, 10T1/<NUM>, BHK, MDCK, COS <NUM>, COS <NUM>, BSC <NUM>, BSC <NUM>, BMT <NUM>, VERO, WI38, HeLa, HEK293, Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster.

Generally, the expression cassette is composed of, at a minimum, a <NUM>' AAV inverted terminal repeat (ITR), a nucleic acid sequence encoding a desirable therapeutic, immunogen, or antigen operably linked to regulatory sequences which direct expression thereof, and a <NUM>' AAV ITR. As disclosed herein, the <NUM>' and/or <NUM>' ITRs of AAV serotype <NUM> are used. However, <NUM>' and <NUM>' ITRs from other suitable sources may be selected. It is this expression cassette that is packaged into capsid proteins to form an AAV virion (particle).

In addition to the expression cassette, the cells contain the sequences which drive expression of AAV capsids in the cells (cap sequences) and rep sequences of the same source as the source of the AAV ITRs found in the expression cassette, or a cross-complementing source. The AAV cap and rep sequences may be independently selected from different AAV parental sequences and be introduced into the host cell in a suitable manner known to one in the art. While the full-length rep gene may be utilized, it has been found that smaller fragments thereof, i.e., the rep78/<NUM> and the rep52/<NUM> are sufficient to permit replication and packaging of the AAV.

This example tests a process for lysing, extracting and purifying AAV and AdV viral particles from their packaged cells (host cells). The example demonstrates that this process achieves higher recovery and purity of viral particles than conventional methods. In addition, this process is easy to implement and can be scaled up readily.

Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS) and cell culture dishes were purchased from Fisher Scientific. <NUM>-well and <NUM>-well cell culture plates were purchased from Santa Cruz Biotechnology. Plasmid DNA Maxiprep kit was purchased from Qiagen. Lipofectamine <NUM> Transfection Reagent was purchased from Life Technologies. DNase I, Maxima Sybr Green qPCR Master Mix (2X), Pierce BCA Protein Assay kit, SDS-PAGE minigels, Amicon Ultra-<NUM> Centrifugal Filter and all chemicals were purchased from Fisher Scientific. Silica-based chromatography columns were purchased from Agilent Technologies, Inc. and Bonna-Agela Technologies Inc.

HEK293 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) containing <NUM>% Fetal Bovine Serum (FBS) in a <NUM> incubator with <NUM>% CO<NUM> and subcultured every <NUM>-<NUM> days. Cells were split <NUM>:<NUM> in <NUM>-cm dishes for transfection for AAV packaging or for infection for AdV amplification. Cells were split onto <NUM>-well or <NUM>-well cell culture plates from infection testing.

HEK293 cells were split onto <NUM>-cm dishes and transfected on the <NUM>nd day using Lipofectamine <NUM> Transfection Reagent or calcium phosphate precipitation method. Plasmid DNA used for AAV packaging included GFP-recombinant AAV viral vector and AAV packaging plasmids. After overnight incubation, the medium was replaced with fresh growth medium and the transfected cells were incubated for additional two days before collecting cells by centrifugation at <NUM> rpm for <NUM> minutes. The AAV-packaged cells were used for viral extraction immediately or stored at -<NUM> until use. The medium was saved for PEG precipitation of viral particles within it. PEG precipitation was conducted using <NUM>-<NUM>% PEG8000 and <NUM>-hour incubation at <NUM>, followed by centrifugation at <NUM>,<NUM> rpm for <NUM> minutes. The PEG precipitates were suspended in PBS by vortexing and pipetting followed by clarifying at <NUM>×g for <NUM> minutes. The viral suspensions were used in pH, detergent and salt testing as shown in <FIG> and <FIG> panel A.

HEK293 cells were split onto a T75 flask and transfected with a GFP-recombinant adenoviral plasmid plus Lipofectamine <NUM>. The medium was replaced with fresh growth medium <NUM> hours post transfection. Cells were incubated for a week before harvesting cells. Cells were suspended in PBS and extracted by freeze/thaw <NUM> times, followed by clarifying at <NUM>×g for <NUM> minutes. One third of supernatant was used for infection of a T175 of HEK293 cells for further viral production and the rest was stored as primary stock. The infected cells were collected after a week of incubation and the viral particles were extracted by freeze/thaw <NUM> times as stock at -<NUM> until use for viral production. For testing assays, <NUM>-cm dishes of HEK293 cells were incubated for <NUM>-<NUM> days after infection with AdV virus until ><NUM>% cells were cytopathic. Cells were then collected by centrifugation at <NUM> rpm for <NUM> minutes. Cell pellets were used for viral extraction immediately or stored at -<NUM> until use. The medium was saved for PEG precipitation of viral particles within it. PEG precipitation was conducted as the same as described above for AAV and viral suspensions were used in pH, detergent and salt testing.

AAV and AdV viral solutions were mixed with an equal volume of pH buffered solutions, detergent solutions or salt solutions as indicated in figures and incubated at room temperature for <NUM> hours before used for infection in HEK293 cells. Infectivity visualized by GFP was investigated to estimate the viral activity. Determination of the ranges of pH value and amounts of detergents suitable for AAV and AdV viruses to maintain their activities is aimed to design cell lysis solutions to easily release the viral particles from their packaging cells. Determination of the viral suitability to a range of salt solutions is expected to learn the possibility to employ salting precipitation in the viral purification procedures.

Lysis of host cells is an important step that enabled rapid lysis of the viral host cells and complete release of the viral particles. Based on the results from condition testing that AAV and AdV viruses were able to maintain their activities in a wide range of pH values and certain amounts of detergents such as sodium dodecyl sulfate (SDS) and sodium deoxycholate (DOC), a serial of lysis solutions were designed, including various acidic solutions and alkaline solutions without or with addition of detergents as shown in Table <NUM> and <NUM>. After the discovery that alkaline solutions with addition of detergents such as DOC or DOC plus SDS achieved effective lysis of AAV- and AdV-packaged cells, various alkaline lysis solutions were further prepared and tested as shown in Table <NUM> and <NUM>. Suitable detergents used here could also include deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, octyl thioglucoside, octyl glucoside, alkyl Sulfates, Polysorbate <NUM> (Tween-<NUM>), Tergitol-type NP-<NUM> and their combinations. In process, AAV- or AdV-packaged cells were collected by centrifugation at <NUM> rpm for <NUM> minutes and then lysed in the above lysis solutions. Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer [<NUM> Tris-Cl, pH <NUM>-<NUM>, <NUM> NaCl, <NUM>% SDS, <NUM>% sodium deoxycholate (DOC), <NUM>% NP-<NUM> and cocktail protease inhibitors] as total protein control or extracted using <NUM> cycles of freeze in -<NUM> and thaw in a <NUM> water bath as comparative control.

As an extraction step, we first tested the possibility to use salting precipitation to remove the host cell proteins but retain the viral particles in supernatants. After testing the tolerance of AAV and AdV viruses to salt as described above in which we found that both of the viruses display wide suitability to high salt, we prepared serials of various salt solutions (Table <NUM>) for test of salting precipitation in the viral extraction following cell lysis. Salt solutions were added to cell lysates and were mixed quickly. After salting precipitation occurred, the mixed solutions were clarified by high-speed centrifugation and removed the precipitates. Supernatants were used for infection in HEK293 cells to validate viral particles remaining in solution. It was demonstrated that salting precipitation process removed the majority of impurities of host cell proteins and efficiently retained the viral particles in solution, the extracted viral solutions considerably cleaner that could be further processed with PEG fractionation or directly used for further purification by column chromatography.

PEG fractionation was then tested to desalt, enrich and further purify the extracted viral solutions following salting process. Various sizes of PEG with average molecular weights from <NUM> to <NUM> were tested by making stock solutions and adding different amounts to the viral solutions. After mixing, the mixtures were incubated on ice for <NUM>-<NUM> hours and then centrifuged at a high speed for <NUM> minutes to precipitate the viral particles. The viral particles were resuspended in PBS for infection to validate the viruses or in an appropriate buffer for further purification by column chromatography. It was found that PEG fractionation led to high-purity viral solutions that could be suitable in use for many research purposes.

The next step was column chromatography purification of the AAV and AdV particles, followed by concentration of the viral solutions with centrifugal filters, in order to yield high-titer, ultrapure viral solutions. AAV or AdV viral particles, either the extracted viral solutions from salting process or the resuspended viral solutions from PEG fractionation, were applied onto the chromatography columns that were silica-based chromatography, resin chromatography or ion exchange chromatography columns. Serials of viral suspension buffers, column washing buffers and elution buffers were prepared and optimized for different chromatography columns. The viral eluents were concentrated using Amicon Centrifugal Filters or other centrifugal filters.

Infection in HEK293 cells was performed as major means of investigation of viral activities in developing the present techniques for purification of AAV and AdV particles. Cells were split in <NUM>-well or <NUM>-well plates with <NUM>-<NUM>% confluence in DMEM containing <NUM>% FBS per well one day before use for infection. Amount of viral solutions were added to each well according to viral concentrations, ionic strength and pH value of the viral solution, ranged about <NUM>-<NUM>µl per well or diluted before used for infection. Since all viruses used are GFP-contained, the infectivity was investigated by viewing GFP cells that were photographed and counted under a fluorescence microscope. Photographs were taken under a <NUM>× lens and GFP cells were counted in fields of a <NUM>× lens. AdV infectivity was investigated in <NUM>-<NUM> days after infection while AAV infectivity was investigated in <NUM>-<NUM> days. It was important that GFP cells after infection were countable in fields of a <NUM>× lens. If numbers of GFP cells were too high or too low to count, infection was repeated with adjusted amounts of viral solutions or with new viral solutions from repeated experiments.

AAV and AdV viral solutions after chromatography column purification were titrated using quantitative real-time PCR (qPCR). The viral solutions were first treated with DNase I in <NUM>µl reaction mixture containing <NUM> units of DNase I and then titrated by means of qPCR in <NUM>µl reaction mixture containing 2x Sybr Green qPCR Master Mix and one pair of primers as listed in Table <NUM> using a standard qPCR cycling paradigm. Serials of <NUM>-fold dilutions of viral vectors were used as standards in the qPCR titration. Titers of viral solutions were calculated as viral genomic copies per milliliter (ml) using the standard curves.

Protein amounts contained in viral solutions were determined using bicinchoninic acid assay (BCA assay) following the BCA Protein Assay Kit's protocol. Briefly, the assays were conducted by adding <NUM>µl sample solutions and <NUM>µl BCA solution (mixed A and B, <NUM>:<NUM>) per well in <NUM>-well plates. The plates were incubated at <NUM> for <NUM> minutes, followed by reading at <NUM> using a plate reader. Bovine serum albumin (BSA) was used to make a standard curve with serial dilutions from a stock of <NUM>/ml. To lower influence from higher salt concentration in solutions after salting precipitation, two strategies were used: (<NUM>) The high salt samples were diluted <NUM> folds in H2O, so that <NUM>µl sample plus <NUM>µl H2O was added per well for BCA assay; (<NUM>) A similar salt solution (salt mix) was prepared and <NUM>µl of the salt mix was added to each well of those of low salt samples including standard dilutions to reduce the influence of high salt in BCA assay.

Protein amounts contained in viral solutions were visualized by means of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. Viral solutions from alkaline lysis/salting precipitation were diluted <NUM> fold with H<NUM>O and then mixed with <NUM>× SDS sample buffer. Sample solutions from freeze/thaw and RIPA lysis were mixed with <NUM>× SDS sample buffer and then diluted with <NUM>× SDS sample buffer. All samples were portioned in the same volume based on original cell suspensions and same volume of each sample was loaded onto <NUM>% SDS gels. Gels were run at <NUM> Voltages for about <NUM> hour until the dye bromophenol blue reached at the gel's bottom. After electrophoresis, gels were fixed in a fixing solution containing <NUM>% (v/v) methanol in water with <NUM>% (v/v) acetic acid before staining overnight in a Coomassie Blue Staining solution containing <NUM>% (v/v) methanol in water with <NUM>% (v/v) acetic acid and <NUM>% (w/v) Coomassie Blue R-<NUM>. Destaining was performed in a destaining solution containing <NUM>% methanol and <NUM>% acetic acid. After completely destaining, gels were kept in <NUM>% (v/v) acetic acid. Gels were scanned into image files using a scanner, of which density of gels were quantified using NIH Image J.

This example first tested the suitability of AAV and AdV to a wide range of pH values from about <NUM> to about <NUM> in order to determine pH values for development of desirable cell lysis solutions. Viral solutions were incubated with the indicated pH solutions for <NUM> hours at room temperature before used for infection in HEK293 cells. Through investigation of their GFP expression, we found that AAV maintained its infectivity at all tested pH values from pH <NUM> to pH <NUM> and AdV was stable to maintain its infectivity at the pH values from pH3. <NUM> to pH10. <NUM> but unstable over pH <NUM> (<FIG> panels A and B). In contrast, LtV was used for comparison, which has a narrow range of pH values from pH <NUM> to pH <NUM> to maintain its infectivity (<FIG> panels A and B). These results, therefore, suggest determining the desired pH values of cell lysis solutions from pH <NUM> to pH <NUM> for AAV and from pH <NUM> to pH <NUM> for AdV that mostly conduce to lysis of cells and do not obviously affect the viral activity.

To test the possibility to use detergents in cell lysis solutions for lysis of AAV- and/or AdV-packaging cells, we determined the tolerance of these viruses to detergents including sodium dodecyl sulfate (SDS), Triton X-<NUM> and sodium deoxycholate (DOC) that are commonly used for cell lysis. Viral solutions were incubated with the indicated detergents for <NUM> hours before used for infection. HEK293 cells are very sensitive to Triton X-<NUM> but not to SDS and DOC in amounts used in the testing, implicating that Triton X-<NUM> may be toxic to the viruses. SDS treatment was stringent to both viruses, which shows that AdV is more sensitive, even <NUM>% SDS abolished all viral infectivity but AAV infectivity was also dramatically decreased with increased SDS concentrations, <NUM>% down at <NUM>% SDS and <NUM>% down at <NUM>% SDS (<FIG> panels C and D). In contrast, both viruses maintain high infectivity in a range of DOC amounts from <NUM>% to <NUM>% tested (<FIG> panels C and D). It is therefore suggested from these results that DOC, but neither SDS nor Triton X-<NUM>, is the best candidate of detergent for cell lysis solutions because of its compatibility with both AAV and AdV viruses.

According to suitable pH values determined above, we prepared several acidic solutions and alkaline solutions (Table <NUM>) to test whether HEK293 cells - the viral packaging cells could be lysed under acidic and/or alkaline conditions. Cells were collected and resuspended in these solutions respectively. After mixing, we found that cells-suspended alkaline solutions were much clearer than cells-suspended acidic solutions, the former with a little of pellets out while the later with much more pellets out after centrifugation at <NUM> rpm for <NUM> minutes (Table <NUM>). It is obviously indicated that alkaline solutions with pH9. <NUM>-<NUM> partially, but not completely, lyse the viral host cells while acidic solutions with pH3. <NUM>-<NUM> did not lyse the cells. Thus, it is concluded that these alkaline solutions, although better than acidic solutions, are not capable to completely lyse the viral host cells.

As determined above, AAV and AdV were sensitive to SDS but compatible to DOC (<FIG> panels C and D). Therefore, we tested whether DOC or DOC plus SDS improves cell lysis by adding <NUM>% - <NUM>% DOC with or without <NUM>% - <NUM>% SDS to the above alkaline solutions and found that addition of the detergents led to complete lysis of cells in the pH range of pH <NUM> - <NUM> tested (Table <NUM>). Other detergents, including deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, octyl thioglucoside, octyl glucoside, alkyl Sulfates, Polysorbate <NUM> (Tween-<NUM>), Tergitol-type NP-<NUM> and their combinations, may be also suitable to conduce to cell lysis. Therefore desirable alkaline lysis solutions are suggested that contain reagents or compounds with buffering capability of pH9. <NUM>-<NUM> and appropriate amounts of detergents such as DOC and SDS.

We further investigated cell lysis with different kinds of alkaline solutions containing detergent DOC as example as listed in Table <NUM> and found all these kinds of alkaline solutions are efficient in lysis of HEK293 cells. Next, we chose <NUM> kinds of alkaline solutions as listed in Table <NUM> to lyse AAV- and AdV-packaged cells and investigated viral infectivity after lysis. It is known that freeze/thaw is most commonly used to extract AAV and AdV particles, so we extracted AAV or AdV particles from their packaged cells using freeze/thaw three times in parallel as comparative control. Viral infectivity was determined by investigation of GFP-expressed cells that were photographed (<FIG> panel A) and counted (<FIG> panel B). We found that these alkaline solutions are all effective in lysing the host cells and release both AAV and AdV viral particles that displayed higher infectivity than those extracted by freeze/thaw (<FIG> panels A and B). Consistent to the results from pH testing above, AdV infectivity was much decreased in the alkaline lysis solutions with pH11. <NUM> (<FIG> panels A and B). It is therefore concluded that alkaline solutions plus appropriate amounts of detergents such as DOC are sufficient to lyse AAV- and AdV-packaged cells and completely release the viral particles, the pH9. <NUM> to <NUM> for AAV and the pH9. <NUM> to <NUM> for AdV. This new cell lysis method can overcome the shortages of freeze/thaw method including time-consuming and viral activity-decreasing.

Massive host cell proteins exist in viral solutions as their packaged cells are lysed. How to remove these protein impurities is the major concern in these viral purifications. It is known that salting precipitation is a potent technique commonly used in protein chemistry. To test the possibility using salting precipitation to remove proteins in viral solutions, we investigated the tolerance of AAV and AdV to high salt solutions and found that both AAV and AdV maintain highly consistent infectivity after incubation with solutions of salt up to <NUM> NaCl, while LtV, in contrast, shows less tolerance to high salt solutions (<FIG>, panels A). These results implicate the possibility that salting precipitation could be used to remove protein impurities in these viral solutions.

In test of salting precipitation, we lysed AAV- and AdV-packaged cells in the <NUM> kinds of alkaline lysis solutions as listed in Table <NUM>, to these cell lysates <NUM>-<NUM> NaAc, pH <NUM> was added and mixed quickly. As expected, large precipitation occurred. The precipitates were removed by centrifugation and the supernatants were saved for infection. Results of viral infectivity show that both kinds of the viral particles perfectly remain in the supernatants after salting precipitation (<FIG> panels B and C). Importantly, we found that the new method using alkaline lysis/salting precipitation produced AAV and AdV particles <NUM>-<NUM>% higher than those extracted by freeze/thaw (<FIG> panels B and C), suggesting that alkaline lysis is more efficient than freeze/thaw to release AAV and AdV viral particles from their host cells.

To determine the effectivity of salting precipitation, we prepared two sets of samples: Set <NUM> used normal HEK293 cells and Set <NUM> used AAV-packaged HEK293 cells, and lysed these cells with alkaline lysis solutions followed by salting precipitation. After centrifugation to remove out precipitates, protein concentrations in the supernatants were measured by BCA assay (<FIG> panel A) and SDS-PAGE electrophoresis (<FIG> panel B). Both results consistently display that the majority of protein impurities in all the cell lysates was removed by salting out in comparison with whole cell lysate (RIPA buffer lysed) and extract by freeze/thaw (<FIG> panels A and B). Furthermore, a trend is observed that higher pH of alkaline lysis solutions led to more proteins removal (<FIG> panels A and B).

Investigation of salting-out effectiveness of various salt solutions discovered a variety of salts are suitable for salting precipitation to precipitate impurities out and retain the viral particles in solutions except ammonium salts that inhibit the infectivity of both AAV and AdV viruses (Table <NUM>). Besides, salt doses and pH values of salt solutions were further demonstrated that protein removal by salting precipitation is salt dose dependent and can be shifted up with lower pH (<FIG> panel C).

The next step attempted to separate the viruses from the high salt solutions either using column chromatography as described below or through fractionation of viral particles into a fraction of solution or precipitate with organic solvents or a polymer such as polyethylene glycol (PEG). In test of desalting, viral particles of both AAV and AdV were found able to be fractionated in a two-phase system including Na-HPO4/PEG or K-PO4/PEG or precipitated by organic solvents such as acetone and isopropanol. These methods, however, lead to decrease the viral infectivity (data not shown). Use of various molecular sizes of PEG from PEG3000 to PEG <NUM> showed effective fractionation of the two kinds of viruses from the high salt solutions into precipitates (data not shown). For example, as shown in <FIG> panels A and B, various amounts of PEG8000 were able to precipitate both AAV and AdV viral particles in a wide range of PEG concentrations. Results from infection further show that PEG fractionation yielded an extremely high recovery of the two kinds of viruses (<FIG> panel C) and its selectivity for precipitation of the viral particles leading to further removal of contaminant proteins (<FIG> panel D). It is, therefore, concluded that PEG fractionation is an excellent step that desalts, enriches and further purifies the viral particles following alkaline lysis/salting precipitation processes. Importantly, the viral solutions of AAV or AdV resulted from PEG fractionation are high-quality viral preparations that can be used for many research purposes.

Although the viral solutions suspended from PEG fractionation are in high quality and suitable for research purposes, further efforts were made to increase the purity of the viral particles in order to meet the requirements in use for all research purposes and/or preclinical or clinical trials. A desired choice of technique is a kind of column chromatography that is an easy, efficient and scalable technique. In testing, we found that various kinds of column chromatography are able to further purify both AAV and AdV viral particles, including silica-based chromatography columns, resin and ion exchange chromatography columns (Data not shown). As example shown in <FIG>, a kind of cation exchange columns were used to purify AAV or AdV particles from viral solutions extracted using alkaline lysis/salting precipitation as described in <FIG> (<FIG> panel A) and silica-based chromatography columns were used to purify AAV or AdV particles from viral suspensions of PEG precipitates as described in <FIG> (<FIG> panel B). After concentration of the eluted viral solutions from these columns, the resulting viral solutions were titrated using qPCR, which show high titers about <NUM><NUM> GC/ml of AAV or higher than <NUM><NUM> GC/ml of AdV (<FIG> panel C).

The whole procedure we present here for purification of AAV and AdV viral particles include <NUM> continuous steps to remove protein impurities. After cell lysis using an alkaline lysis, salting precipitation processed using a high salt solution is the first step that removes the majority of host cell proteins, about <NUM>-<NUM>% (<FIG> panels A and B). PEG fractionation is the following step that further removes contaminant proteins from the viral particles, about <NUM>-<NUM>% of remaining impurities (<FIG> panels A and B). Chromatography purification is the final step that removes remaining contaminant proteins and ensures ultrapurity of the viral solutions (<FIG> panels C and D) that can be concentrated using a centrifugal filter to yield a final high-titer viral solution (<FIG> panels E-G). The new procedures used for the two viral purifications are characterized ultrahigh purity (near <NUM>% viral particles) (<FIG> panels C, D and G), high recovery (><NUM>%) (<FIG> panel E) and high titer (up to <NUM><NUM> TU/ml) (<FIG> panel F).

A comparative study as example demonstrated the advantages of our new procedure over commercial products. Three of AAV purification kits that are marked as Kit A, Kit B and Kit C were found from the current market, commercially available from different companies. These kits were used for AAV purification from AAV-packaged cells following the product protocols, compared with our present procedure as described above (marked as C&M). Results from infection and protein assays with the resulting viral solutions are shown in <FIG>, in which we first found that C&M-purified viral solution displayed much higher infectivity ( panel A) and ><NUM>% viral recovery, while the viral recovery using Kits A, B and C was <NUM>%, <NUM>%, <NUM>% respectively (panel B); the titer of C&M-purified viral solution is near <NUM><NUM> TU/ml, <NUM>-<NUM> times over those of viral solutions purified by commercial kits (panel C), while the later contains much more contaminant proteins than the former (panels D and E). Using the viral transducing units (TU) per pg protein to estimate the viral purity, C&M-purified viral solution shows <NUM> ± <NUM> TU/pg protein, the purity <NUM>-<NUM> times over the viral solutions purified with Kits A-C (<FIG> panel F). It is, therefore, concluded that our artistic approach exhibits unparalleled advantages in yielding a high-titer, high-purity AAV solution over the current commercial kits.

In summary, this study presents a new approach for purification of AAV or AdV particles from their packaged cells, which includes a few important steps as follows (<FIG>): (<NUM>) cell lysis (cell lysates) using an alkaline solution that contains compounds with buffering capability maintaining pH value of <NUM>-<NUM>; (<NUM>) salting precipitation process by adding a salting solution (high salt) to cell lysates to remove the majority of host cell protein impurities in precipitates and retain AAV or AdV viral particles in solutions (viral extracts); (<NUM>) PEG fractionation by adding PEG to viral extracts to separate viral particles in precipitates that can be suspended in an appropriate solution (viral suspensions) and retain protein impurities in solution leading to desalting, enriching and purifying the viral particles; and (<NUM>) column chromatography by using silica-based chromatography columns to finally purify the above viral suspensions followed by concentration with centrifugal filters to produce high-titer, ultrapure viral solutions. Moreover, this is a very easy, time-saving procedure that can be finished in about <NUM> hours and is suitable for purification of all serotypes of AAV or AdV or any other species of packaged viruses retained in host cells. All the solutions used in the procedure do not contain any toxic compounds or reagents so that the resulting viral solutions can meet the quality requirements for most research purposes and/or preclinical and clinical trials.

This example tested a collection of reagents and materials, referred to as a viral purification kit, for the purification of certain viral particles. The example demonstrates that the kits can greatly improve the quality and efficiency for viral purification.

Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS) and cell culture dishes were purchased from Fisher Scientific. <NUM>-well and <NUM>-well cell culture plates were purchased from Santa Cruz Biotechnology. Plasmid DNA Maxiprep kit was purchased from Qiagen. Lipofectamine <NUM> Transfection Reagent was purchased from Life Technologies. DNase I, Maxima Sybr Green qPCR Master Mix (2X), Pierce BCA Protein Assay kit, SDS-PAGE minigels, Centrifugal Filter and all chemicals were purchased from Fisher Scientific. Silica-based chromatography columns were purchased from Agilent Technologies, Inc.

HEK293 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) containing <NUM>% Fetal Bovine Serum (FBS) in a <NUM> incubator with <NUM>% CO<NUM> and subcultured every <NUM>-<NUM> days. Cells were split <NUM>:<NUM> in <NUM>-cm dishes for transfection for AAV packaging or for infection for adenoviral amplification. Cells were split onto <NUM>-well or <NUM>-well cell culture plates from infection testing.

HEK293 cells were split onto <NUM>-cm dishes and transfected on the <NUM>nd day using Lipofectamine <NUM> Transfection Reagent or calcium phosphate precipitation method. Plasmid DNA used for AAV packaging included GFP-recombinant AAV viral vector and AAV packaging plasmids. After overnight incubation, the medium was replaced with fresh growth medium and the transfected cells were incubated for additional two days before collecting cells by centrifugation at <NUM> rpm for <NUM> minutes. The AAV-packaged cells were used for viral extraction immediately or stored at -<NUM> until use.

HEK293 cells were split onto a T75 flask and transfected with a GFP-recombinant adenoviral plasmid plus Lipofectamine <NUM>. After a week incubation to generate viruses, cells were harvested and extracted by freeze/thaw <NUM> times, followed by clarifying at <NUM>×g for <NUM> minutes. Adenovirus was then amplified in T175 flasks of HEK293 cells.

Cells were split in <NUM>-well or <NUM>-well plates with <NUM>-<NUM>% confluence in DMEM containing <NUM>% FBS per well one day before use for infection. After <NUM>-<NUM> days infection, the infectivity was investigated by viewing GFP cells that were photographed and counted under a fluorescence microscope. Photographs were taken under a <NUM>× lens and GFP cells were counted in fields of a <NUM>× lens. It was important that GFP cells after infection were countable in fields of a <NUM>× lens. If numbers of GFP cells were too high or too low to count, infection was repeated with adjusted amounts of viral solutions or with new viral solutions from repeated experiments.

Protein amounts contained in viral solutions were determined using bicinchoninic acid assay (BCA assay) following the BCA Protein Assay Kit's protocol. Briefly, samples were diluted <NUM>-<NUM> folds and the assays were then conducted by adding <NUM>µl sample solutions and <NUM>µl BCA solution (mixed A and B, <NUM>:<NUM>) per well in <NUM>-well plates. The plates were incubated at <NUM> for <NUM> minutes, followed by reading at <NUM> using a plate reader. Bovine serum albumin (BSA) was used to make a standard curve with serial dilutions from a stock of <NUM>/ml. Protein concentrations were calculated based on the standard curve.

Protein amounts contained in viral solutions were visualized by means of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis. All samples were portioned in the same volume based on original cell suspensions and same volume of each sample was loaded onto <NUM>% SDS gels. Gels were run at <NUM>-<NUM> Voltages for about <NUM> hour until the dye bromophenol blue reached at the gel's bottom. After electrophoresis, gels were fixed in a fixing solution containing <NUM>% (v/v) methanol in water with <NUM>% (v/v) acetic acid before staining overnight in a Coomassie Blue Staining solution containing <NUM>% (v/v) methanol in water with <NUM>% (v/v) acetic acid and <NUM>% (w/v) Coomassie Blue R-<NUM>. Destaining was performed in a destaining solution containing <NUM>% methanol and <NUM>% acetic acid. After completely destaining, gels were kept in <NUM>% (v/v) acetic acid. Gels were scanned into image files using a scanner.

The main components of the viral purification kits tested included the following solutions and materials:.

Cell Suspension Solution: this solution consisted of serum-free Dulbecco's Modified Eagle Medium (DMEM) or phosphate-buffered saline (PBS) or Tris buffered saline (TBS) or other buffered saline with a pH of <NUM>-<NUM>.

Cell Lysis Solution: This solution consisted of <NUM>-<NUM> sodium-glycine alkaline solution or sodium-phosphate alkaline solution or CAPS solution or other buffered solution with a pH of <NUM>-<NUM>, containing <NUM>%-<NUM>% detergents octyl glucoside or octyl thioglucoside or polysorbate <NUM> (Tween-<NUM>) or sodium deoxycholate (DOC) or sodium dodecyl sulfate (SDS) or other detergents or their combination.

<NUM>st Fractionation Solution: This solution consisted of <NUM>-<NUM> sodium acetate or potassium acetate or sodium phosphate or sodium chloride or other salt solutions with a pH of <NUM>-<NUM>.

<NUM>nd Fractionation Solution: This solution consisted of about <NUM>% to about <NUM>% of a polyethylene glycol (PEG) with an average molecular weight of about <NUM> to about <NUM>, containing sodium sulfate or sodium chloride or sodium acetate or potassium acetate or sodium phosphate or other salt solutions with a concentration of <NUM>. <NUM>-<NUM>.

Pellet Suspension Solution: This solution consisted of serum-free DMEM medium or phosphate-buffered saline (PBS) or Tris buffered saline (TBS) or other buffered saline with a pH of <NUM>-<NUM>.

Column Equilibration Solution: This solution consisted of <NUM>-<NUM> sodium acetate or potassium acetate or sodium phosphate or potassium phosphate or sodium glycine or Tris buffer or MOPS buffer with a pH of <NUM>-<NUM>, containing <NUM>-<NUM> sodium chloride or other salts, containing <NUM>-<NUM>% methanol or isopropanol or ethanol or other organic solvents.

Column Washing Solution: This solution consisted of <NUM>-<NUM> sodium acetate or potassium acetate or sodium phosphate or potassium phosphate or sodium glycine or Tris buffer or MOPS buffer with a pH of <NUM>-<NUM>, containing <NUM>-<NUM> sodium chloride or other salts, containing <NUM>-<NUM>% methanol or isopropanol or ethanol or other organic solvents.

Column Elution Solution: This solution consisted of <NUM>-<NUM> sodium acetate or potassium acetate or sodium phosphate or potassium phosphate or sodium glycine or Tris buffer or MOPS buffer with a pH of <NUM>-<NUM>, containing <NUM>-<NUM> sodium chloride or other salts.

Solid phase chromatography columns: The kits included one column for one viral purification. The chromatography columns were prefilled with medium materials such as silica or silica-based ion exchange media or other media. The prefilled columns feature high stability, high repeatability and easy to use.

Centrifugal filters or other concentration sets: The kits included one centrifugal filter or one of other concentration sets for one viral purification. For example, the centrifugal filters can efficiently reduce the volume of the viral solutions eluted from the above chromatography columns and change the buffer solution resulting in appropriate high-titer viral solutions.

Other supportive materials: The column supportive sets include column top adapters and syringes used to bring a pressure to the upper columns to flow through the solutions out or vacuum connection sets to connect to the bottom tips of the columns to give a negative pressure to the lower columns to flow through the solutions out. In addition, various sizes of lab tubes may be provided within the product kits, including <NUM>-ml microtubes, <NUM>-ml centrifuge tubes or <NUM>-ml centrifuge tubes or other tubes.

The viral purification kits can be used in a simple, efficient procedure for purification of AAV, adenovirus and other viruses from their packaging cells. The following described are certain example main steps:.

The lysis solutions were used to lyse the two viral packaged cells. In each case, viruses in cell lysates obtained from cell lysis had high viral infections, for both AAV (<FIG> panel A) and adenovirus (<FIG> panel B) in comparison with those from freeze/thaw extraction. Statistical analysis of the infection data showed higher infectivity of both viruses from the lysis than those from freeze/thaw extraction (<FIG> panel C). These data indicate that the lysis used in the products is more efficient to release viral particles from their packaged cells than freeze/thaw extraction. Freeze/thaw extraction is a commonly used method to extract AAV and adenovirus. It is a long process, taking more than <NUM> hour, that includes <NUM>-<NUM> rounds of freeze and thaw and centrifugation of clarification. In contrast, the lysis step of the kits takes <NUM>-<NUM> minutes, and is easy to carry out.

Cell lysates from the above step were applied to a first fractionation with the First Fractionation Solutions. This step is useful for removing the majority of protein impurities to precipitate by denaturation and retaining the viral particles in solution. Following addition of the first fractionation solutions to the cell lysates, most host cell proteins were denatured and precipitated. The two fractions of solution and precipitate were separated by centrifugation and the supernatants were used for infection to view the viral activities. In comparison with viral solutions from freeze/thaw extraction, the supernatants retained the viral infectivity of AAV (<FIG> panel A) and adenovirus (<FIG> panel B) to greater extents. Statistical analysis of infection data further shows that higher infectivity of the two viruses were retained in the supernatants of the first fractionation than those from freeze/thaw extraction (<FIG> panel C). The protein amounts of the supernatants were analyzed in comparison with the viral solutions from freeze/thaw extraction. Results from SDS electrophoresis (<FIG> panel A) and BCA assay (<FIG> panel B) consistently show that the protein amounts in the viral supernatants were dramatically reduced. Up to <NUM>% protein impurities were removed. This example, therefore, demonstrates that the first fractionation solutions on one hand can remove most of protein impurities to the precipitates and on other hand can efficiently maintain the viral particles in the solutions.

The second fractionation aimed to collect viral particles from the supernatants and suspend in the desired solutions for column chromatography. Following the second fractionation, the viral pellets were suspended in the Pellet Suspension Solutions and were tested for infection. Investigation of infection demonstrated that the Second Fractionation Solutions of the products efficiently recovered AAV (<FIG> panel A) and adenoviral particles (<FIG> panel B) from the viral supernatants of the first fractionation while only a small portion of viral particles were lost in the second supernatants (about <NUM>-<NUM>% (<FIG> panel C)). Apparently, the second fractionation selectively collected the viral particles into precipitates and retained protein impurities in solutions, resulting in further purification of the viral particles (<FIG>).

Noticeably, the suspensions of viral pellets from the second fractionation were already in high purity and ><NUM>% protein impurities of cell lysates were removed out (<FIG>). This purity of viruses can be used for many experimental purposes in cultured cells, especially for quick validation and testing or large-scale screening of AAV-mediated gene expression and functions.

Also included in the viral purification kits were solid phase chromatography columns such as silica-based chromatography columns and ion exchange chromatography columns or other solid phase chromatography columns and ion exchange chromatography columns. Results shown in <FIG> as examples show high viral recovery by these columns. First, a few types of commercial columns were tested to recover AAV viral particles from the supernatants of the first fractionation (<FIG> panel A, upper, Column <NUM>) and from the pellet suspensions of the second fractionation (<FIG> panel A, middle and lower; Columns <NUM> and <NUM>). These different types of solid phase chromatography columns exhibited similar effects in AAV purification, efficiently retaining the viral particles that can be eluted in the Elution Solutions. The viral infectivity of eluents showed that the viral recoveries were high, about <NUM>% (<FIG> panel A). In use of these columns for adenoviral purification, similar results were obtained in the viral purification from the pellet suspensions of the second fractionation using Column <NUM> as shown in <FIG> panel B as example.

The viral eluents from the columns were concentrated using a centrifugal filter to obtain high-titer viral solutions. The purity of the concentrated viral solutions was determined using SDS electrophoresis and BCA assay. Gel image from SDS electrophoresis exhibited clear three capsid proteins of AAV, without impurities, after the viral purification from the pellet suspensions of the second fractionation using Columns <NUM> and <NUM> while there were some impurities in the viral solution after the viral purification from the supernatants of the first fractionation using Column <NUM> (<FIG> panel A left). A clean solution of adenovirus was shown in <FIG> panel A right, which was purified from the pellet suspensions of the <NUM>nd fractionation using Column <NUM>. Results from BCA assay of the eluents from Column <NUM> demonstrated the effect of column chromatography in purification of the two viruses (<FIG> panel B), which resulted in ultrapure viral solutions (<FIG>).

The kits were compared with commercial AAV purification products. Three commercial products were used, each marked as Kit A, Kit B and Kit C. The presently developed kits are marked as "new".

Equal volumes of AAV-packaged cells were applied to each of the kits for viral purification in parallel and the resulting viral solutions were used for infection and protein analysis. Results from infection as shown in <FIG> panels A and B demonstrate the efficiency of each product in purification of AAV. The kits of the present disclosure recovered much more viral particles than the three commercial kits. The viral recovery was about <NUM>-<NUM> times higher (<FIG> panel B). Gel image from SDS electrophoresis displays a great amount of impurities in the viral solutions purified by Kits A-C but no impurities in the viral solution purified by the new kit where there are only three capsid proteins of AAV (<FIG> panel C). The protein amounts in the viral solutions were further confirmed by BCA assay, which were much higher in the commercial kit-purified viral solutions (<FIG> panel D). Taken together, these results testify that the new kits exhibited unparalleled advantages in yielding a high-titer, high-purity AAV solution over the current commercial products.

Claim 1:
A method of extracting viral particles of a recombinant adeno-associated virus (AAV) or an adenovirus (AdV) from a sample comprising cells enclosing the viral particles, comprising:
lysing the cells by the addition of an alkaline lysis solution, comprising a detergent and one or more reagents, to the sample such that the sample comprises <NUM>% to <NUM>% of the detergent and has a pH of <NUM> to <NUM> for AAV, or <NUM> to <NUM> for AdV, wherein the detergent is selected from the group consisting of deoxycholate, octyl thioglucoside, octyl glucoside, dodecyl maltoside, alkyl Sulfates, Polysorbate <NUM> (Tween-<NUM>), Tergitol-type NP-<NUM> and the combinations thereof, and wherein the one or more reagents is selected from the group consisting of N-cyclohexyl-<NUM>-aminopropanesulfonic acid (CAPS), glycine/sodium, glycine/potassium, sodium carbonate, potassium carbonate, sodium phosphate, potassium-phosphate and combinations thereof;
precipitating host cell proteins released from the lysed cells by (a) adding a salting solution to the sample such that the sample comprises <NUM> to <NUM> of a salt and has a pH of <NUM> to <NUM>, wherein the salt is selected from the group consisting of sodium acetate, potassium acetate, sodium phosphate and combinations thereof and (b) removing the precipitated host cell proteins to obtain a supernatant comprising the viral particles;
precipitating the viral particles from the supernatant with a precipitation solution, wherein upon addition of the precipitation solution to the sample, the sample comprises <NUM>% to <NUM>% of a polyethylene glycol (PEG); and
further purifying the precipitated viral particles with chromatography.