CN101018858A - Compositions and methods to prevent AAV vector aggregation - Google Patents

Abstract

Compositions and methods are provided for preparation of concentrated stock solutions of AAV virions without aggregation. Formulations for AAV preparation and storage are high ionic strength solutions (e.g. mu~500mM) that are nonetheless isotonic with the intended target tissue. This combination of high ionic strength and modest osmolarity is achieved using salts of high valency, such as sodium citrate. AAV stock solutions up to 6.4x10<13> vg/mL are possible using the formulations of the invention, with no aggregation being observed even after ten freeze-thaw cycles. The surfactant Pluronic(R) F68 may be added at 0.001% to prevent losses of virions to surfaces during handling. Virion preparations can also be treated with nucleases to eliminate small nucleic acid strands on virions surfaces that exacerbate aggregation.

Description

Prevent the composition and the method for AAV vector aggregation

Technical field

The present invention relates to prepare and store composition and the method that prevents accumulative AAV virus particle.

Background technology

Recombinant adeno-associated virus (rAAV) is to be used for the distant view carrier that people's gene shifts.Grimm, D. and Kleinschmidt, J.A. (1999) Hum Gene Ther.10:2445-2450; High, K.A. (2001) Ann.N.Y.Acad.Sci.953:64-67; Pfeifer, A. and Verma, I.M. (2001) Ann.Rev.Genomics Hum.Genet.2:177-211.AAV is the member of Parvoviridae dependovirus.AAV serotype 2 (AAV2) is that the terminal single strand dna that oppositely repeats 4680 Nucleotide that duplicate (rep) and encapsidate (cap) gene of (ITR) sequence constitutes by the coding both sides.Bers, K.I. (1996) Fields Virology (editor such as B.N.Fields), pp.2173-2197.Lippincott-Raven Publishers，Philadelphia。Genome is by three housing albumen (VP1, VP2 and VP3) packing, and housing albumen is the N-terminal variant of cap gene product.Resulting icosahedro virion has～diameter of 26nm.Reported the crystalline structure of AAV2 high dissolution.Xie, Q etc. (2002) Proc.Natl.Acad.Sci.U.S.A.99:10405-10410.

The solubleness of purifying AAV2 virion is limited, and having described the AAV2 particle aggregation is a problem.Croyle, M.A. etc. (2001) Gene Therapy 8:1281-1290; Huang, J. etc. (2000) Mol.Therapy 1:S286; Wright, J.F. etc. (2003) Curr.Opin.Drug Disc.Dev.6:174-178; Xie, Q. etc. (2004) J.Virol.Methods 122:17-27.In buffered saline solution commonly used, 10 ¹³The concentration of particle/mL produces significant the gathering, and assembles enhancing in higher concentration.Huang and co-worker have reported that the AAV carrier stands the gathering of concentration dependent.Huang, J. etc., (2000) Mol.Therapy 1:S286.Xie and co-worker (Xie, Q. etc. (2004) J. Viral.Methods 122:17-27) have reported similarly that in the concentration that surpasses 0.1mg/ml the salt that the AAV2 carrier need improve concentration prevents to assemble.The AAV2 carrier neutral buffered solution commonly used such as phosphoric acid salt-and Tris-buffer saline middle particle concentration surpass 10 ¹³Particle/mL produces gathering.This is corresponding to the protein concn of～0.06mg/mL, and emphasizes the low solubility of AAV2 under these conditions.For the carrier that uses column chromatography technology purifying, the effective carrier concn limit even lower because co-purify excessive empty capsid and owing to granule density.

Particle aggregation is significant equally for adenovirus carrier and is the problem that does not solve fully.Reported the stability of the adenovirus of up-to-date foundation recently with reference to material (ARM).Adadevoh, K. etc. (2002) BioProcessingl (2): 62-69.Be formulated in the 20mM Tris of pH8.0, the gathering in 25mM NaCl and 2.5% glycerine by dynamic light scattering, photon correlation spectroscopy and outward appearance measurement with reference to material.Observe the variable level of three freeze-thaw circulations or non-freezing storage back vector aggregation, form the restricted embodiment of using ARM.

Gathering may cause the discordance in loss in the purge process and the test of cmy vector preparation.The AAV2 carrier is delivered medicine to privileged site in vivo,, may need the height of small volume to concentrate carrier, and maximum obtainable dosage is subjected to the restriction of low carrier solubleness as central nervous system.

As if vector aggregation also influences the bio distribution behind the vivo medicine-feeding, and causes after the administration the unfavorable immunne response to carrier.As (the Braun that has reported for protein, A. etc., (1997) Pharm.Res.14:1472-1478), by with the carrier alignment antigen presenting cell and induce the immunne response that housing albumen and transgene product are improved, the gathering of carrier has improved immunogenicity.(Chenuaud, P etc. (2004) Blood 103:3303-3304 before clinical; Flotte, T.R. (2004) Human Gene Ther.15:716-717; Gao, G etc., (2004) Blood 103:3300-3302) and clinical (High, K.A. etc., (2004) Blood 104:121a) research in reporting of AAV carrier immunne response understood need to solve immunogenic all factors of carrier that cause.

As if the test experiments scheme that characterizes cmy vector also be subjected to the influence of vector aggregation.The mensuration of having reported carrier infectious titration degree is extremely sensitive to vector aggregation.Zhen, Z. etc. (2004) Human Gene ther.15:709-715.Important concern is that vector aggregation has negative consequence after the administration in vivo, because their transduction efficiency, bio distribution and immunogenicity are different from monomer particle.For example, will be sent to the carrier that liver cell need pass hepatic sinusoid reticuloendothelial cell layer in the AAV carrier vascular.These netted layouts have 50 to 150nm radius (Meijer, K.D.F. and Molema, G (1995) Sem.Liver Dis.15:206), predict that this radius makes monomer A AV carrier (diameter～26nm) can pass through, but stop passing through than the larger vector aggregation.In the mouse body in the biodistribution research, after the vascular transmission, isolate from the Kupffer cell with the gathering AAV2 carrier of fluorescence molecule Cy3 mark.Huang, J. etc. (2000) Mol.Therapy l:S286.

For the preparation research and development based on the gene transfer vector of virus are recent relatively research fields, and the research of having only minority to describe the systems attempt of optimizing AAV carrier formulation and stability has obtained report.Croyle, M.A. etc. (2001) Gene Therapy 8:1281-1290; Wright, J.F. etc. (2003) Curr.Opin.DrugDisc.Dev.6:174-178; Xie, Q. etc. (2004) J.Virol.Methods 122:17-27.With minimize clinical that carrier formulation changes before and the suitable mutually definition preparation of clinical application be the consistent important requirement that obtains height carrier security and functional character.As for protein therapeutic known (Chen, B. etc. (1994) J. Pharm.Sci.83:1657-1661; Shire, S.J. etc. (2004) J. Pharm.Sci.93:1390-1402; Wang, W. (1999) Int.J. Pharm.185:129-188; Won, C.M. etc., (1998) Int.J.Pharm.167:25-36), the importance of carrier stability is the stability in preparation and the storage process, vector aggregation is to need the thoroughly problem of solution.Vector aggregation causes the loss in the carrier purge process, although can remove aggregation by filtering, when produce be used for clinical before during with the carrier of clinical study, production yield loss causes higher cost and production restriction.Even after removing by filter aggregation, form new aggregation in the concentrated AAV2 carrier formulation in buffer salt solution.

Existence is used for purifying and storage AAV2 carrier such as the improved formulations of rAAV2 and the needs of method to preventing the virion accumulative.

Summary of the invention

Above-mentioned these and other needs in this area have been satisfied by the present invention, the invention provides the high inonic strength solution that is used to prepare and store the AAV carrier, this AAV carrier keeps high infectious titration degree and transduction efficiency, even also is like this after freeze-thaw circulation.

In one aspect of the invention, relate to the virus particle accumulative method in the virus particle preparation that prevents, this method obtains to be enough to prevent the accumulative ionic strength by adding vehicle.In another aspect of the present invention, relate to and have the virus particle composition that is enough to prevent the accumulative ionic strength.

In some embodiments of the present invention, described ionic strength is at least about 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 600mM, 700mM or higher.In some embodiments, use and to contain one or more polyvalent ions for example Citrate trianion, vitriol, magnesium or phosphatic vehicle obtain these ionic strengths.

In some other embodiment, be maintained near isotonic water the osmolarity of virus particle preparation flat, for example, 200mOsm, 250mOsm, 280mOsm, 300mOsm, 350mOsm or 400mOsm assemble even the enough height of ionic strength prevent virus particle.

In some embodiments, virus particle is the virus particle of adeno-associated virus (AAV), for example AAV-2.

In some other embodiment of the inventive method, with nuclease Benzonase for example ^Handle the preparation of virus particle.In further embodiment, the nuclease processing is combined with the adding of vehicle, wherein the adding of this vehicle obtains to be enough to prevent the accumulative ionic strength.

In some embodiments of the present invention, with tensio-active agent Pluronic ^F68 adds in the virus particle preparation, for example to 0.001%.In one embodiment, composition comprises the virion, 10mM Tris pH8.0,100mM Trisodium Citrate of purifying and 0.001% Pluronic ^F68.

In one embodiment, the AAV carrier can be stored as composition of the present invention, concentration surpasses 1 * 10 ¹³Vg/mL, for example, 2 * 10 ¹³, 3 * 10 ¹³, 4 * 10 ¹³, 5 * 10 ¹³With up to 6.4 * 10 ¹³Vg/mL, and significantly do not assemble.In some embodiments, use the AAV carrier of method and composition storage of the present invention not present significant gathering in the time of 5 days 4 ℃ of storages.In other embodiments, after-20 ℃ or-80 ℃ one, five, ten or how freeze-thaw circulation, do not present significant gathering as the AAV carrier of such composition storage.

In some embodiments, the mean particle radius (Rh) (as recording by dynamic light scattering) that presented of virus particle preparation of the method according to this invention and composition storage shows the remarkable gathering that virus particle does not take place.In some embodiments, the virus particle preparation of the method according to this invention and composition storage presents the mean particle radius (Rh) that is higher than about 15nm, 20nm or 30nm.

In some embodiments, the virus particle that reclaims after the filtration by 0.22 μ m filter of virus particle preparation of the method according to this invention and composition storage is higher than about 85%, 90% or 95%.

More on the one hand, the present invention relates to contain the test kit of high ionic strength preparation of the present invention.In one embodiment, described test kit comprises the excipient solution that is pre-mixed.In another embodiment, described test kit comprises the part of the high ionic strength composition of the present invention that two or more separate, and mixes by the user.In some embodiments, described test kit comprises Trisodium Citrate, Tris ^And Pluronic ^F68.In other embodiments, described test kit further comprises the specification sheets for preparing composition or carry out the inventive method.

Description of drawings

The data sheet that Figure 1A and 1B present is understood the function of AAV2-FIX particle aggregation as the osmolarity (Figure 1A) or the ionic strength (Figure 1B) of various buffer compositions.Method 2 by embodiment 1 prepares the AAV2-FIX carrier.Measure mean particle radius by dynamic light scattering (DLS) behind the vehicle dilution carrier with the different concns of the sodium phosphate buffer of 10mM pH7.5.Described vehicle comprises sodium-chlor (), Trisodium Citrate (), sodium phosphate (), sodium sulfate (), sal epsom () and glycerine ().

Fig. 2 has presented the data of AAV2-FIX gathering as the function of purification process.Measure mean particle radius by dynamic light scattering (DLS) behind the different concentrations of sodium chloride dilution carrier with the 10mMpH7.5 sodium phosphate buffer.By method 1 (two CsCl gradient) (); Method 2 (cation-exchange chromatography) (); Method 2 adds nuclease digestion (); Or method 3 (chromatogram adds a CsCl gradient) is come cmy vector.Purification process 1-3 is described among the embodiment 1.

Fig. 3 has presented the data of the D7/4 cell transgene expression of the rAAV2-AADC virus particle transduction for preparing and store in high ionic strength preparation () or control formulation ().Transduce and measured the concentration (each data point triplicate) of AADC in back 72 hours by ELISA.Limit of error is represented standard deviation.

Embodiment

Usually in the concentrate formulation of carrier, observe the AAV2 vector aggregation and influence purifying and reclaim and intravital effect and security.Therefore, the free-revving engine for development AAV2 carrier is to identify that preparation prevents the method and formulation of vector aggregation when concentrating liquid storage.

Unless otherwise noted, term " carrier " refers to reorganization AAV virus particle or virion as used in this, like that also refers to non-viral DNA molecule (as plasmid) and be unlike in normally used in the other guide " carrier ".

The present invention is partly based on following observation: solution ion strength is the important parameter in the AAV vector aggregation, means to relate to the ionic interaction between the virion in the accumulation process.The solubleness that the ionic strength that raises has improved the AAV2 carrier and this observation that charged vehicle character has nothing to do have supported that solution ion strength itself interaction of specific ion material (but not relate to) is the hypothesis of relevant physical-chemical parameter.Preventing needs the limit ionic strength of 200mM at least in the gathering of the carrier granule concentration of this research.

In the practical application, the ionic strength that buffer salt solution commonly used has is not enough to prevent that concentration from surpassing 10 ¹³The gathering of the AAV2 carrier of particle/mL.Known high salt concentration improves the solubleness (for example, the height that reclaims from gradient concentrates the maintenance dissolving during concentrating CsCl usually of AAV2 carrier) of AAV2 carrier.Yet, be used for clinical before and the optimal formulation of clinical study should approach to wait and ooze (280-400mOsm), especially for the carrier vivo medicine-feeding is diluted position slowly in hypertonic solution.In embodiments of the invention, the exponential relationship of ionic strength and charge valence is used to develop have high ionic strength etc. ooze preparation.Usually the salt material with multi-charge valency (for example, vitriol, Citrate trianion and phosphoric acid salt) as vehicle in people's parenteral formulation can provide the ionic strength level that prevents that the AAV2 vector aggregation from needing when using with isotonic concentration.Although have the ionic strength of 150mM Deng oozing (150mM) sodium-chlor, this numerical value is not enough to keep AAV2 solubleness under the high carrier concn, waits to ooze Trisodium Citrate and have～ionic strength of 500mM, can support at least 6.4 * 10 ¹³The AAV2 carrier concn of vg/ml and not assembling.

Without wishing to be bound by theory, AAV2 particulate low solubility by high symmetry matter in conjunction with in the aggregation between the adjacent particles stabilising effect of complementary electric charge part cause.Disclosed the pattern of positive charge and negative charge on the virus surface based on the surface charge density (Xie, Q. etc., (2002) Proc.Natl.Acad.Sci.U.S.A.99:10405-10410) of AAV2 crystalline structure.Report before shows that the AAV2 vector aggregation is that pH is dependent, and it has supposed that the amino acid with charged side group relates to combination between particle.Qu, G etc., (2003) Mol.Therapy7:S238.If these reports have been supposed charged amino acid side chain and have related to vector aggregation that the total free aminoacids of high density can stop the interaction of carrier granule.Yet, we have found that the amino acid with charged side chain is invalid in preventing the AAV2 vector aggregation, surpass their contributions to ionic strength.

Also find to handle and reduced but do not prevent the vector aggregation of low ionic strength by cmy vector particulate effective nucleic acid enzyme.The early stage digestion (clarifying HEK cell lysate) of purge process does not reduce behind the carrier purifying to be assembled.The chances are owing to enzyme and the higher ratio of nucleic acid primer, and the digestion of purified virus particle is more effective.A kind of mechanism of explaining these results is that the remaining nucleic acid impurity (for example, host cell and plasmid DNA) in conjunction with carrier surface can bridge to and faces the binding site that connects on the virion and therefore cause gathering.The AAV2 carrier (no empty capsid) of having reported purifying contains 1% the non-carrier DNA of having an appointment.Smith, P. etc., (2003) Mol.Therapy 7:S348.Although reported this non-carrier DNA＞50%th, the nuclease resistance, and be packaged in the housing particle, some impurity DNA be the nuclease resistance and as if link to each other with the carrier particle surface of purifying.The effective nucleic acid enzyme is handled the observation that can reduce vector aggregation and is shown that the nucleic acid that links to each other with carrier surface that is not higher than each carrier granule～25 a Nucleotide mean level (ML) causes the AAV vector aggregation.

In a word, in AAV2 carrier purifying and final process for preparation effective removal of the use of high inonic strength solution and residual carrier surface DNA be obtain to be used for clinical before and the height of clinical study concentrate two kinds of available strategies of AAV2 solution.High inonic strength solution and nuclease are handled and can be used in combination or separately use.Although use the AAV2 carrier to obtain data, the compositions and methods of the invention also are useful for other AAV serotype/variants or other virus vector as adenovirus, lens virus and retrovirus.

The AAV gathering is the function of excipient concentration

Carrying out initial screening experiment illustrates the mechanism of AAV vector aggregation and identifies the classification that can reduce/prevent the accumulative vehicle.Vector aggregation can (the 20mM sodium phosphate, the dilution of carrier in neutral buffered saline pH7.2) (5 times) causes by low-concentration buffer.Use and " dilution-stress " method to screen the vehicle that can prevent vector aggregation when vehicle is included in the thinner with evaluation.In order to screen, (DLS) measures gathering by dynamic light scattering.The classification of the vehicle of studying comprises selected inorganic salt, amino acid, uncharged carbohydrate and tensio-active agent.The result is presented in the table 1.

Table 1

Use dilution-strain method to screen the vehicle that prevents the AAV2 vector aggregation

Vehicle	Prevent to assemble the Osm (maximum of being tested) of needs
		Sal epsom	180mOsm
Trisodium Citrate	220mOsm
		Sodium-chlor	320mOsm
Sodium phosphate	220mOsm
		Sodium sulfate	220mOsm
		Arginine	NIA(200mOsm)
Aspartic acid	320mOsm
		L-glutamic acid	320mOsm
Glycine	NIA(200mOsm)
		Histidine	NIA(200mOsm)
Methionin	300mOsm
Glycerine	NIA(5％w/v，543mOsm)
		Iodixanol	NIA(5％w/v，32mOsm)
N.F,USP MANNITOL	NIA(5％w/v，275mOsm)
		Sorbitol Powder	NIA(5％w/v，275mOsm)
Sucrose	NIA(5％w/v，146mOsm)
		Trehalose	NIA(5％w/v，146mOsm)
		Pluronic F68	NIA(10％w/v，12mOsm)
Polysorbate 80	NIA(1％w/v)

NIA: do not assemble and suppress

As illustrated in the table 1, when existing with enough concentration, charged vehicle (inorganic salt and amino acid) has prevented gathering.Yet, prevent that the needed salt concn of vector aggregation from being different, be 180mOsm for sal epsom, be 320mOsm for sodium-chlor.Amino acids Arginine, aspartic acid, L-glutamic acid, glycine, Histidine and Methionin do not prevent to assemble at 200mOsm, but Methionin, aspartic acid and L-glutamic acid have prevented gathering at 300-320mOsm.The not test of arginine, glycine and the Histidine concentration beyond 200mOsm.When existing with the concentration up to 5%w/v, selected carbohydrate is assembled carrier granule does not have effect.For example, the glycerine of 5%w/v (543mOsm) does not prevent to assemble.Tensio-active agent polysorbate 80 (1%w/v) and Pluronic ^F68 (10%w/v) uses " dilution-stress " method similarly gathering not to be had effect.

AAV assembles the function as osmolarity and ionic strength

Figure 1A and 1B have shown the result of vector aggregation with the more detailed analysis of various salt concn functions.Figure 1A has shown the function of vector aggregation for selected vehicle osmolarity.For charged material, observe the inhibition of AAV2 vector aggregation concentration dependent.The salt of polyvalent ion obtains similar gathering in the concentration that is lower than unit price sodium-chlor and suppresses degree.For example, the sal epsom of 200mOsm has prevented gathering, and sodium-chlor needs 350mOsm to obtain similar effect.Trisodium Citrate, sodium sulfate and sodium phosphate mediate in preventing the effectiveness of vector aggregation.

Although the result of Figure 1A and table 1 shows up to the glycerine of 5% concentration and specific sugar the AAV2 vector aggregation that low ionic strength causes is not had effect, data are not got rid of the raising that glycerol concentration is higher than 5% o'clock AAV2 solubleness.For example, Xie and co-worker have reported that the glycerine of 25% (w/v) can make the AAV2 concentration in the LISS reach very high concentration (4.4 to 18 * 10 ¹⁴Particle/mL).Xie, Q etc., (2004) J. Virol.Methods 122:17-27.

For every kind of vehicle, except osmolarity, Figure 1B has shown the curve of the data of Figure 1A as calculating ionic strength function.Figure 1B proved when ionic strength, and uses which kind of salt irrelevant.These data show that the ionic strength (μ) (it is the parameter that depends on solute concentration and charge valence) of solution is to influence the accumulative principal element.

Prevent to assemble useful ionic strength in the embodiment of the present invention and comprise, for example, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 600mM, 700mM or higher ionic strength.Preferred polyvalent ion obtains these ionic strengths in method and formulation of the present invention, as divalence, trivalent, tetravalence, pentavalent ion and even the ion of high price more.PH buffer reagent in solution of the present invention and the preparation can be phosphoric acid salt, Tris or HEPES (or other Good ' s buffer reagents), but can use any other suitable pH buffer reagent.In the preferred embodiment, select that polyvalent ion is compatible with destination organization with buffer reagent to be used for carrier to be prepared.

The use of polyvalent ion makes and can form high ionic strength but the relative low composition of osmolarity in the inventive method and the composition.High ionic strength composition of the present invention can approachingly wait and ooze, and can be, for example, about 200mOsm, 250mOsm, 280mOsm, 300mOsm, 350mOsm or 400mOsm are although other osmolarities also are acceptable for some purposes of composition.

AAV assembles the function as the AAV purification process

Expectation uses the reorganization AAV2 of different methods (for example, the density gradient purifying is to ion-exchange chromatography) purifying to have different impurity characteristics.Fig. 2 has shown that vector aggregation is as the function of ionic strength for the different several AAV preparations of purification process.Purification process is described among the embodiment 1.Use sodium-chlor to change ionic strength.By two cesium chloride gradient ultracentrifugations (method 1), by cation exchange column chromatography (method 2), or the AAV2-FIX carrier by bonded post and cesium chloride gradient ultracentrifugation (method 3) purifying has proved separately along with ionic strength reduces similar gathering response.On the contrary, the AAV2-FIX that accepts nuclease digestion step (method 2+ nuclease) then by the column method purifying shows to assemble when low ionic strength and reduces.

Preparative scale AAV assembles

Data among table 1 and Figure 1A, the 1B and 2 relate to the vector aggregation of AG, use DLS to measure gathering.On the contrary, table 2 has shown that the ionic strength that raises and nuclease handle the effect to more extensive AAV2 vector aggregation, uses and induces and the method for quantitative with preparation scale carrier purifying relevant vector aggregation.The detailed content of experiment is provided among the embodiment 2.The AAV carrier diafiltration of purifying to the solution of various ionic strengths, is reduced volume and obtains high carrier concn, and the carrier recovery of measuring then after filtering by 0.22 μ m filter is measured gathering.The sample aliquot of the single set of AAV2-AADC carrier of method 1 purifying that will be by second CsCl gradient centrifugation step is (among the 91mL 1.8 * 10 ¹⁵Vg, 1.8 * 10 ¹³Vg/mL is among～the 3MCsCl) as the starting raw material in the diafiltration experiment.Use the tangential flow filtration of hollow fiber to be used for diafiltration,, and therefore can prepare AAV concentration, be desirably in the neutral buffered saline and assemble in this concentration because it is upgradeable and still can prepares volume (minute .1.4mL).

In experiment 1, three hollow fiber devices are used for the AAV2-AADC carrier of diafiltration formulation C F, TF1 or TF2, and volume is reduced to 2.5 * 10 ¹³The target of vg/mL.Referring to embodiment 2.Then the filter of sample by 0.22 μ m filtered.The results are shown in the table 2.Be significantly higher than for the carrier recovery (" yield % ") of the preparation TF1 (95 ± 7.4%) and the TF2 (93 ± 7.4%) of rising ionic strength and use control formulation CF (77 ± 6.6%) to be reclaimed.

Table 2

Handle the AAV carrier recovery of level

Experiment	Preparation	μ(mM)	Target (vg/mL)	Actual (vg/mL)	Yield % (RSD)
						1	CF	160	2.5E13	1.93E13	77(6.6)
1	TF1	310	2.5E13	2.38E13	95(7.4)
						1	TF2	510	2.5E13	2.33E13	93(7.4)
2	CF	160	6.7E13	3.98E13	59(6.0)
						2	TF2	510	6.7E13	6.42E13	96(4.4)
3	CF(-Bz)	160	3.6E13	2.46E13	68(11)
						3	CF(+Bz)	160	3.6E13	3.29E13	91(12)

In experiment 2, AAV2-AADC is concentrated into higher target value (6.7 * 10 in CF or TF2 ¹³Vg/mL).Use the carrier recovery of TF2 (96 ± 4.4%) to be significantly higher than use (59 ± 6.0%) that CF reclaimed once more.In the mutability of used test, all reclaimed carrier at two aimed concns that use TF2, show to assemble to have obtained preventing.On the contrary, observe remarkable gathering, and accumulative degree (that is the loss after, 0.22 μ m filters) is higher in higher destination carrier concentration at two aimed concns that use CF.(not shown) in another experiment in the concentrated back of experiment 2 but the AAV2 support samples of taking out 50 μ L before 0.22 μ m filtration step, and detects by opticmicroscope.Spissated carrier contains the visible material (not shown) of obvious amount among the CF, yet does not see such material in TF2 in the spissated carrier.

The experiment 3 studied cmy vector in advance nuclease digestion to the accumulative effect.When nuclease digestion did not exist, AAV2-AADC was recovered as 68 ± 11% among the CF, and is similar with the recovery in 2 to experiment 1.On the contrary, the carrier with nuclease processing purifying concentrates in CF then, obtains higher recovery (91 ± 12%).These preparation scale result reflects the identical result of the nuclease digestion of the use shown in Fig. 2 " dilution-stress " (AG) method.

The result who presents in the table 2 has proved that method and composition of the present invention has improved the recovery of AAV carrier.For example, in the various embodiments of the present invention, will reclaim from being lower than about 80% and be increased at least about 85%, 90%, 95% or higher.

AAV stability after storage or the freeze-thaw circulation and active

Croyle and co-worker have reported the remarkable loss of repeatedly freeze-thaw circulate back AAV and adenovirus titre in sodium phosphate buffer, and have proved that the buffering of pH preferably that provides by potassiumphosphate in the freeze-thaw circulation has prevented the titre loss.Croyle, M.A. etc. (2001) Gene Therapy8:1281-1290.We use the result of the freeze-thaw stability study of sodium phosphate to support these discoveries.Although we find that the sodium phosphate of 150mM provides enough ionic strengths to prevent to concentrate the gathering of AAV2-AADC carrier in preparation and non-freezer storage process, the freeze-thaw circulation of single of-20 ℃ or-80 ℃ causes gathering.

AAV stability in following buffer reagent of the present invention after test storage or freeze-thaw (F/T) circulation.Whether the concentrated carrier for preparing among CF, TF1 and the TF2 (table 2, experiment 1) is accepted short stability study investigates in the freezer storage process or repeatedly will assemble after freeze-thaw (F/T) circulation.Use undiluted sample to measure gathering, and think that Rh value＞20nm has represented to produce the gathering of certain level by DLS.

Table 3

The stability of AAV2 carrier

	Particle radius-Rh (nm)
									Preparation	Pre	4℃	-20℃			-80℃
		5d	1F/T	5F/T	10F/T	1F/T	5F/T	10F/T
									CF	14.5	27.0	22.4	56.1	94.5	20.6	57.5	141
TF1	13.8	16.3	TH	TH	TH	TH	TH	TH
									TF2	13.8	14.4	14.2	14.0	14.1	13.8	21.3	50.9

The DLS radius of measuring immediately after Pre:0.2 μ m filters.

Carrier concn (vg/mL): CF:1.93E13, TF1:2.38E13, TF2:2.33E13.

TH: because intensive assembles, the too high so that energy measurement not of strength of signal.

As shown in table 3, the AAV2-AADC carrier for preparing among the CF demonstrates some gatherings 4 ℃ of storages after 5 days and after the one or many F/T of-20 ℃ or-80 ℃ circulation.For the carrier for preparing among the TF1, produce after following 5 days at 4 ℃ and to assemble, assemble but produce, as so that not energy measurement too high by the strength of signal shown in the DLS in the single F/T circulation back of-20 ℃ or-80 ℃.The visual inspection of these samples has shown slight muddiness, and this is consistent with gathering.For the carrier for preparing among the TF2, at 4 ℃ or do not observe gathering after up to 10F/T at-20 ℃.Under-80 ℃ 5 and 10F/T circulation after observe some gatherings.

AAV activity among the following measurement TF2 after storage or the F/T circulation.As mentioned above, the grade of high ionic strength ooze that preparation TF2 has prevented to concentrate effectively and storage process in vector aggregation, and therefore show it is to be used for the further distant view material standed for of research.Important problem is whether preparing carriers and the storage among the high ionic strength TF2 has influenced its functionally active unfriendly.In order to test this, the infection titre and the transduction efficiency that in TF2, prepare and store the carrier of time expand section have been carried out measuring.

For infectivity, used and to have detected the sensitive infectious test of height that single infects incident.Zhen, Z etc., (2004) Human Gene Ther.15:709-715.With 6.4 * 10 ¹³The concentration of vg/mL prepares AAV2-AADC in TF2., compare with the 16vg/IU of value to(for) the reference carrier after 45 days 4 ℃ of storages, the vector gene group that preparation has is 13 (vg/IU) to the ratio of infectious unit.Set the mutability (RSD～50%) of this test for report, this difference is not significant.

Measure the proteic expression of AADC by the ELISA behind the D7/4 cell transduction and test transduction efficiency.Fig. 3 has shown for 10 to 10 ⁵The carrier input of vg/ cell does not have significant difference between carrier for preparing among the TF2 and the reference contrast.Generally speaking, these data show that AAV2 preparing carriers and storage among the high ionic strength TF2 do not have disadvantageous effect to carrier infectivity or transduction efficiency.

Conclusion

Measure ionic strength (μ) to virion interactional influence illustrate the mechanism of vector aggregation.(μ=150mM) is not enough to prevent surpass～10 by gradient ultracentrifugation or the concentration by the cation-exchange chromatography purifying ionic strength of neutral buffered isotonic saline solution ¹³The gathering of the AAV2 carrier of particle/mL.Comprise sugar (Sorbitol Powder, sucrose, N.F,USP MANNITOL, trehalose, glycerine) or the tensio-active agent tween 80 of concentration up to 5% (w/v) ^(1%) or Pluronic ^F68 (10%) does not prevent the gathering of carrier granule.

On the contrary, (carrier granule keeps dissolving during μ＞200mM) to use the ionic strength solution that raises in purifying and final carrier process for preparation.The ionic strength solution of rising that use is used for the isotonic excipient concentration of vivo medicine-feeding prepares with polyvalent ion salt, comprises Trisodium Citrate, sodium phosphate and sal epsom.Contain 10mM Tris, 100mM Trisodium Citrate, 0.001%Pluronic ^F68, pH8.0 (μ～500mM) etc. ooze preparation and can make the concentration of AAV2-AADC carrier reach 6.4 * 10 ¹³Vg/mL does not observe after-20 ℃ of ten freeze-thaw circulations of preparation process neutralization and assembles.Referring to following table 3 and incidental discussion.The AAV2-AADC carrier of preparation and storage time expand section keeps high infectious titration degree (13IU/vg) and transduction efficiency in the ionic strength preparation that raises.

The nuclease of purifying AAV2 carrier is handled the degree that has reduced vector aggregation, and carrier surface nucleic acid impurity relates to the particle interphase interaction.Therefore, effectively removing the remaining purification of nucleic acids method of carrier surface and be used in combination the grade that ionic strength raises and ooze preparation, is the process useful that prevents the AAV2 vector aggregation.

Embodiment 1

The AAV purification process

(Matsushita, T. etc. (1998) Gene Therapy 5:938-945) as described above produces the AAV2 carrier of expressing human plasma thromboplastin component (FIX) or human amino acid decarboxylase (AADC) by three transfections of HEK293 cell, improves.For large-scale preparation, at 850mm ²Shake in the bottle (Corning) and cultivate and transfectional cell.Come cmy vector by a kind of in three kinds of methods.

In purification process 1, improve from Matsushita, by centrifugal (1000g, 15 minutes) collect the transfection HEK293 cell that shakes in the bottle, be resuspended to 10mM sodium phosphate, 500mM sodium-chlor, among the pH7.2, and come cracking (interchangeable ethanol/the dry ice bath and 37 ℃ of water-baths) by freezing/melt circulation for three times.By the centrifugal cell lysate (8,000g, 15 minutes) of clarifying.By adding the 10mM sodium phosphate, pH7.2 is diluted to supernatant liquor 200mM NaCl and uses Benzonase then ^(Merck, purity level 1; 200U/mL, 1h, 37 ℃) digestion.Use the 1M liquid storage that lysate is adjusted to 25mM CaCl ₂, and hatched one hour at 4 ℃.

With mixture centrifugal (8,000g, 15 minutes), and collect carrier-containing supernatant liquor.For from clarifying cell lysate precipitation virus, add the final concentration of polyoxyethylene glycol (PEG8000) to 8%, mixture was hatched three hours centrifugal then (8,000g, 15 minutes) at 4 ℃.Carrier-containing pill is resuspended to 0.15M NaCl, 50mM Hepes, 25mMEDTA by mixing, hatched 16 hours among the pH8.0 and at 4 ℃.The material that merges resuspension, and add the final densities of solid cesium chloride to 1.40gm/ml.Use then Beckman model LE-80 whizzer by ultracentrifugation with carrier layering (SW28,27,000rpm, 24h, 20 ℃).With the centrifuge tube classification, and merge carrier-containing 1.38 to 1..42gm/mL density.This material is come layering (NVT65 rotor, 65,000rpm, 16h, 20 ℃) for the second time by ultracentrifugation, and merge the fraction that contains purifying AAV2 carrier.In order to concentrate the carrier row buffering liquid exchange of going forward side by side, the carrier that concentrates in the cesium chloride solution is accepted ultrafiltration/diafiltration (UF/DF) by the tangential flow filtration of (embodiment 2) as described below.

In purification process 2, the cell harvesting thing microfluidization that will contain AAV also filters by the filter (Sartorius) of 0.65 and 0.22 μ m according to the order of sequence.The cation-exchange chromatography that passes through as described above uses Poros HS50 resin from clarifying cell lysate purified virus.U.S. patent No.6,593,123.For the nuclease digestion described in Fig. 2, with 100U/mL Benzonase and 10U/mL DNA enzyme I (no RNA enzyme, Roche Diagnostics, Indianapolis, Indiana) hatch column purification carrier (4h, RT).

For purification process 3, before UF/DF, the cesium chloride gradient ultracentrifugation step that the AAV2 carrier acceptance of cation-exchange chromatography purifying is other (SW28,27,000rpm 20h) removes empty capsid.

Use real-time quantitative PCR (Q-PCR) as described above comes quantitative AAV preparation.Sommer, J.M. etc., (2003) Mol.Therapy 7:122-128.Dye by SDS-PAGE/ silver and to analyze three kinds of methods carrier of purifying separately, and in all situations, VP1, VP2 and VP3 exist with the ratio of expection, housing albumen represents＞total protein 95%, measure as surveying close art by scanning imaging.Yet different with the gradient purifying AAV2 carrier of using method 1 and 3 purifying, the carrier by method 2 (column chromatography) purifying contains empty capsid, and each vector gene group contains 3-10 empty capsid.

Embodiment 2

Ultrafiltration and diafiltration detect AAV and assemble

Disposable hollow fiber tangential flow filtration device (Amersham BioSciences8 " Midgee, the specified pore size of 100kDa) is used for concentrating and the AAV2 carrier of diafiltration by the aforesaid method purifying, and is used for the UF/TF experiment described in the table 2.For all UF/DF programs, use diafiltration buffer volume, and add near continuous diffusion with the increment of～1mL corresponding to 10 * product volume.Use this method, the remaining CsCl＜0.5mM that is calculated after the diafiltration.

Following three kinds of preparations are used for UF/DF: control formulation (CF:140mM sodium-chlor, 10mM sodium phosphate, 5% Sorbitol Powder, pH7.3); Test formulation 1 (the TF1:150mM sodium phosphate, pH7.5); With test formulation 2 (TF2:100mM Trisodium Citrate, 10mM Tris, pH8.0).For the experiment shown in the table 21, corresponding to 1 * 10 ¹³The volume of vg/mL carrier concn carries out diafiltration, and volume is reduced to corresponding to 2.5 * 10 after the diafiltration ¹³The value of vg/mL (supposing not have the carrier loss).

For experiment 2, corresponding to 2 * 10 ¹³The vg/mL volume carries out diafiltration, and volume is reduced to corresponding to 6.7 * 10 then ¹³The value of vg/mL.

(CF ± Bz) is at first with AAV2-AADC (about 1.2 * 10 for experiment 3 ¹⁴Vg) diafiltration (preparation compatible) to TF1, the filter by 0.22 μ m then with nuclease.Measure the titre of this material, and with volume-adjustment to corresponding to 1 * 10 ¹³The concentration of vg/mL.In this material of 10mL, add MgCl ₂To the concentration of 2mM, be divided into two sample aliquot then.Sample aliquot hatches with Benzonase that (200U/mL, 4h RT), are hatched for simulation for second.Then corresponding to 2 * 10 ¹³The volume of vg/mL carrier concn is concentrated into 3.6 * 10 then with each sample aliquot diafiltration ¹³The vg/mL target.Behind all UF/DF experimental programs, will be from the Pluronic of 1% liquid storage ^F-68 (BASF Corp., Mount Olive NJ) add in the carrier product ultimate density to 0.001%, with solution by 0.22 μ m syringe filter (Sartorius).All UF/DF programs are carried out in Laminar Flow Room.

Embodiment 3

Measure vector aggregation by dynamic light scattering

(λ=825.4nm) analyzes the gathering of cmy vector to use protein soln DynaPro99 by dynamic light scattering (DLS).Raw data (particle radius-Rh, the mean value of measuring in 30 circulations, 10 cycles per minute) is used for whole analyses of being reported." dilution-stress " method is used to test the influence of various vehicle to vector aggregation.In the method, 80 μ L test thinner is added in the 20 μ L carrier solns, and be mixed in and be used for the actual cuvette that DLS measures, and begin to collect data mixing in 10 seconds.Before adding the test thinner, the Rh value and the confirmation＜15nm that measure the AAV2 carrier formulation guarantee that starting raw material is monomeric.To not that 100% monomeric sample passes 0.22 μ m syringe disc filter (Sartorius, lower protein in conjunction with) and removes aggregation.

Use all vehicle that exist in the mixture (that is, weighing: test thinner (80%) and initial vector preparation (20%)) to calculate osmolarity and the ionic strength that provides among Fig. 1 and 2.According to equation: osmolarity=c _iCalculate osmolarity, wherein c _iIt is the volumetric molar concentration of every kind of solute substance.According to equation: μ=1/2c _iz _i ²Calculate ionic strength (μ), wherein z _iIt is the electric charge on every kind of material.Under the condition that causes vector aggregation (for example, hanging down μ), observe gradual increase along with the process Rh of data gathering.In order to determine that back 3 minutes measured average Rh in interval of dilution can be used as the accumulative reliable measurements, have also measured mean rate (the Δ Rh/ Δ t) (not shown) that identical timed interval Rh increases.The analysis of Δ Rh/ Δ t has provided and those corresponding to results that use the average Rh value acquisition of being put down in writing among Fig. 1 and 2.

Embodiment 4

AAV virus particle infectivity

Use the sensitive infectivity of measuring the AAV2-AADC carrier of height as described above.Zhen, Z etc., (2004) Human Gene Ther.15:709-715.In brief, (10-doubly dilutes with the sample serial dilution, 10 replicate(determination)/dilutions) and add and to grow in 96 hole tissue culture plate (Falcn contains on cat.#353227) in the D7/4 cell in the DMEM substratum of 10%FBS (expressing the transformation Hela cell of AAV rep and cap).Adenovirus (Ad-5,100vp/ cell) added in each hole provides subsidiary function.Behind the 48h,, and calculate the infectious titration degree with the dilution infection frequency of analysis limit by the Karber method by duplicating of AAV carrier in Q-PCR use transgenosis-Auele Specific Primer and next quantitatively each hole of probe.Simultaneously with before preparation and be stored in-80 ℃ AAV2-AADC in CF with reference to moving sample.

Come the transduction efficiency of quantitative AAV2 carrier by full cell ELISA.Infect the D7/4 cell that grows in the 96 hole flat boards with the sample of 10 times of serial dilutions with reference to carrier, corresponding to 10 to 10 ⁵Vg/ cell input (5 parallel testing/extent of dilution).Behind 48h, remove substratum, (10mM sodium phosphate, 140mM sodium-chlor are pH7.2) with twice of cell washing with the PBS of 200 μ L.Adding in each hole (15 minutes) by the PBS that 100 μ L is contained 0.5% triton x-100 and 4% Paraformaldehyde 96 then changes and fixed cell thoroughly.Remove fixed solution, and with the PBS that contains 0.5% triton x-100 with twice of cell washing.The PBS that contains 3% bovine serum albumin (BSA) and 0.5% triton x-100 by adding blocks non-specific site (60 minutes).

After the washing, with rabbit anti--ADCCIgG antibody (AB136) hatched cell one hour, and washing by Chemicon.Then with alkaline phosphate link coupled goat anti--rabbit IgG is hatched cell one hour, and washing.Antibody was diluted among the PBS that contains 1%BSA, 0.5% triton x-100 with 1: 1000.Add then substrate (PNPP, Pierce, cat.#34047) (1mg/mL in the 1X diethanolamine substrate buffer solution, Pierce, cat.#34064), hatch 30 minutes after, (the λ=405nm) of the concentration by spectrophotometry division substrate.Using adaptive curve (SigmaPlot) to meet people ADCC expresses with the carrier input function.Use sample measurement AAV2-AADC with reference to carrier simultaneously.

Although described preferred exemplary embodiment of the present invention, can form that not break away from various changes and improvements of the present invention be that those skilled in the art are apparent, and determine that appended claim has contained all such changes and improvements that fall in the real spirit and scope of the present invention.

Claims (20)

1. one kind prevents virus particle accumulative method in the virus particle preparation, comprises one or more vehicle are added the ionic strength that obtains in the virus particle preparation at least about 200mM.

2. method according to claim 1, wherein said virus particle are the AAV virus particle.

3. method according to claim 1 further comprises with nuclease and handles described virus particle preparation.

4. method according to claim 3, wherein said nuclease is Benzonase ^

5. method according to claim 1, wherein said one or more vehicle comprise polyvalent ion.

6. method according to claim 5, wherein said polyvalent ion is a citrate.

7. method according to claim 1, wherein the osmolarity of virus particle preparation is not higher than about 280mOsm after adding one or more vehicle.

8. method according to claim 1, wherein after adding one or more vehicle, the virus particle mean particle radius (Rh) by dynamic light scattering measurement in the virus particle preparation is lower than about 20nm.

9. method according to claim 1, wherein after adding one or more vehicle, the virus particle after the virus particle preparation filters by 0.22 μ m filter reclaims at least about 90%.

10. one kind is used to store the purified virus grains of composition, comprising:

The virion of purifying;

The pH buffer reagent; With

The vehicle that contains one or more polyvalent ions; Wherein the ionic strength of composition is higher than about 200mM.

11. composition according to claim 10, the virion of wherein said purifying are the AAV virions.

12. composition according to claim 10, a kind of in wherein said one or more polyvalent ions is citrate.

13. composition according to claim 10 further comprises Pluronic ^F68.

14. composition according to claim 13, wherein said Pluronic ^F68 exists with 0.001%.

15. composition according to claim 10, wherein said pH buffer reagent is 10mM Tris, pH8.0, and vehicle comprises the 100mM Trisodium Citrate.

16. composition according to claim 10, wherein the purified virus particulate mean particle radius (Rh) by dynamic light scattering measurement is lower than about 20nm.

17. composition according to claim 10, wherein after the virus particle composition filtered by 0.22 μ m filter, the purified virus particulate reclaimed and is at least about 90%.

18. one kind prevents virus particle accumulative method in the virus particle preparation, comprises and use Benzonase ^Handle described virus particle preparation.

19. method according to claim 18 is wherein at Benzonase ^After the processing, the virus particle mean particle radius (Rh) by dynamic light scattering measurement in the virus particle preparation is lower than about 20nm.

20. method according to claim 18 is wherein at Benzonase ^After the processing, the virus particle after the virus particle preparation filters by 0.22 μ m filter reclaims and is at least about 90%.

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