U.S. patent application number 14/702008 was filed with the patent office on 2016-03-24 for compositions and methods to prevent aav vector aggregation.
This patent application is currently assigned to Genzyme Corporation. The applicant listed for this patent is Genzyme Corporation. Invention is credited to Guang Qu, John Fraser Wright.
Application Number | 20160083694 14/702008 |
Document ID | / |
Family ID | 35462907 |
Filed Date | 2016-03-24 |
United States Patent
Application |
20160083694 |
Kind Code |
A1 |
Wright; John Fraser ; et
al. |
March 24, 2016 |
COMPOSITIONS AND METHODS TO PREVENT AAV VECTOR AGGREGATION
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..about.500 mM) 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
6.4.times.10.sup.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.RTM. 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.
Inventors: |
Wright; John Fraser;
(Framingham, MA) ; Qu; Guang; (Framingham,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genzyme Corporation |
Framingham |
MA |
US |
|
|
Assignee: |
Genzyme Corporation
Framingham
MA
|
Family ID: |
35462907 |
Appl. No.: |
14/702008 |
Filed: |
May 1, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12661553 |
Mar 19, 2010 |
9051542 |
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14702008 |
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11141996 |
Jun 1, 2005 |
7704721 |
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12661553 |
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60575997 |
Jun 1, 2004 |
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60639222 |
Dec 22, 2004 |
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Current U.S.
Class: |
435/235.1 |
Current CPC
Class: |
C12N 2750/14111
20130101; C12N 2750/14151 20130101; C12N 7/00 20130101 |
International
Class: |
C12N 7/00 20060101
C12N007/00 |
Claims
1. A composition for the storage of purified virus particles,
comprising: purified virus particles; a pH buffer; and excipients
comprising one or more multivalent ions; wherein the ionic strength
of the composition is greater than about 200 mM.
2. The composition of claim 1, wherein the purified virus particles
are AAV virus particles.
3. The composition of claim 1, wherein one of the one or more
multivalent ions is citrate.
4. The composition of claim 1, further comprising Pluronic.RTM.
F68.
5. The composition of claim 4, wherein the Pluronic.RTM. F68 is
present at 0.001%.
6. The composition of claim 1, wherein the pH buffer is 10 mM Tris,
pH 8.0 and the excipients comprise 100 mM sodium citrate.
7. The composition of claim 1, wherein the average particle radius
(Rh) of the purified virus particles is less than about 20 nm as
measured by dynamic light scattering.
8. The composition of claim 1, wherein recovery of the purified
virus particles is at least about 90% following filtration of the
composition of virions through a 0.22 .mu.m filter.
9. A method of preventing aggregation of virions in a preparation
of virions, comprising treating said preparation of virions with
Benzonase.RTM..
10. The method of claim 9, wherein, after Benzonase.RTM. treatment,
the average particle radius (Rh) of the virions in the preparation
of virions is less than about 20 nm as measured by dynamic light
scattering.
11. The method of claim 9, wherein, after Benzonase.RTM. treatment,
recovery of the virions is at least about 90% following filtration
of the preparation of virions through a 0.22 .mu.m filter.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/661,553, filed Mar. 19, 2010, which is a continuation of
U.S. application Ser. No. 11/141,996, filed Jun. 1, 2005, now U.S.
Pat. No. 7,704,721, from which applications priority is claimed
pursuant to 35 U.S.C. .sctn.120; and claims benefit under 35 U.S.C.
.sctn.119(e) of provisional application 60/575,997, filed Jun. 1,
2004 and 60/639,222, filed Dec. 22, 2004, which applications are
hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods of
preparing and storing AAV virions that prevent aggregation.
BACKGROUND
[0003] Recombinant adeno-associated virus (rAAV) is a promising
vector for human gene transfer. 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 a member of the
Dependovirus genus of the parvoviruses. AAV serotype 2 (AAV2) is
composed of a single-strand DNA molecule of 4680 nucleotides
encoding replication (rep) and encapsidation (cap) genes flanked by
inverted terminal repeat (ITR) sequences. Berns, K. I. (1996) in
Fields Virology (B. N. Fields et. al. Eds.), pp. 2173-2197.
Lippincott-Raven Publishers, Philadelphia. The genome is packaged
by three capsid proteins (VP1, VP2 and VP3), which are
amino-terminal variants of the cap gene product. The resulting
icosahedral virus particle has a diameter of .about.26 nm. A high
resolution crystal structure of AAV2 has been reported. Xie, Q. et
al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99: 10405-10410.
[0004] The solubility of purified AAV2 virus particles is limited,
and aggregation of AAV2 particles has been described as a problem.
Croyle, M. A. et al. (2001) Gene Therapy 8: 1281-1290; Huang, J. et
al. (2000) Mol. Therapy 1: 5286; Wright, J. F. et al. (2003) Curr.
Opin. Drug Disc. Dev. 6: 174-178; Xie, Q. et al. (2004) J. Virol.
Methods 122: 17-27. In commonly used buffered-saline solutions,
significant aggregation occurs at concentrations of 10.sup.13
particles/mL, and aggregation increases at higher concentrations.
Huang and co-workers reported that AAV vectors undergo
concentration-dependent aggregation. Huang, J. et al. (2000) Mol.
Therapy 1: S286. Xie and coworkers (Xie, Q. et al. (2004)J. Virol.
Methods 122: 17-27) similarly reported that at concentrations
exceeding 0.1 mg/mL, AAV2 vectors require elevated concentrations
of salt to prevent aggregation. Aggregation of AAV2 vectors occurs
at particle concentrations exceeding 10.sup.13 particles/mL in
commonly used neutral-buffered solutions such as phosphate- and
Tris-buffered saline. This corresponds to a protein concentration
of .about.0.06 mg/mL, and emphasizes the low solubility of AAV2
under these conditions. The effective vector concentration limit
may be even lower for vectors purified using column chromatography
techniques because excess empty capsids are co-purified and
contribute to particle concentration.
[0005] Particle aggregation is a significant and not fully resolved
issue for adenovirus vectors as well. Stability of a recently
established adenovirus reference material (ARM) was recently
reported. Adadevoh, K. et al. (2002) BioProcessing 1(2): 62-69.
Aggregation of the reference material, formulated in 20 mM Tris, 25
mM NaCl, and 2.5% glycerol at pH 8.0, was assessed by dynamic light
scattering, photon correlation spectroscopy and visual appearance.
A variable level of vector aggregation following either freeze-thaw
cycling or non-frozen storage was observed, resulting in
restrictive protocols for the use of the ARM.
[0006] Aggregation can lead to losses during purification and
inconsistencies in testing of purified vector preparations. The in
vivo administration of AAV2 vectors to certain sites, such as the
central nervous system, may require small volumes of highly
concentrated vector, and the maximum achievable dose may be limited
by low vector solubility.
[0007] Vector aggregation is also likely to influence
biodistribution following in vivo administration, and cause adverse
immune responses to vectors following their administration. As has
been reported for proteins (Braun, A. et al. (1997) Pharm. Res. 14:
1472-1478), aggregation of vector may increase immunogenicity by
targeting the vector to antigen presenting cells, and inducing
enhanced immune responses to the capsid proteins and transgene
product. The reports of immune responses to AAV vectors in
pre-clinical (Chenuaud, P. et al. (2004) Blood 103: 3303-3304;
Flotte, T. R. (2004) Human Gene Ther. 15: 716-717; Gao, G. et al.
(2004) Blood 103: 3300-3302) and clinical (High, K. A. et al.
(2004) Blood 104: 121a) studies illustrate the need to address all
factors that may contribute to vector immunogenicity.
[0008] Testing protocols to characterize purified vectors are also
likely to be affected by vector aggregation. Determination of the
infectivity titer of vector was reported to be highly sensitive to
vector aggregation. Zhen, Z. et al. (2004) Human Gene Ther. 15:
709-715. An important concern is that vector aggregates may have
deleterious consequences following their in vivo administration
because their transduction efficiency, biodistribution and
immunogenicity may differ from monomeric particles. For example,
intravascular delivery of AAV vectors to hepatocytes requires that
the vectors pass through the fenestrated endothelial cell lining of
hepatic sinusoids. These fenestrations have a radius ranging from
50 to 150 nm (Meijer, K. D. F., and Molema, G. (1995) Sem. Liver
Dis. 15: 206) that is predicted to allow the passage of monomeric
AAV vectors (diameter .about.26 nm), but prevent the passage of
larger vector aggregates. In biodistribution studies in mice,
aggregated AAV2 vectors labeled with the fluorescent molecule Cy3
were sequestered in liver macrophages following vascular delivery.
Huang, J. et al. (2000) Mol. Therapy 1: 5286.
[0009] Formulation development for virus-based gene transfer
vectors is a relatively recent area of investigation, and only a
few studies have been reported describing systematic efforts to
optimize AAV vector formulation and stability. Croyle, M. A. et al.
(2001) Gene Therapy 8: 1281-1290; Wright, J. F. et al. (2003) Curr.
Opin. Drug Disc. Dev. 6: 174-178; Xie, Q. et al. (2004) J. Virol.
Methods 122: 17-27. Defining formulations compatible with
pre-clinical and clinical applications that minimize changes in
vector preparations is an important requirement to achieve
consistently high vector safety and functional characteristics. As
is well established for protein therapeutics (Chen, B. et al.
(1994) J . Pharm. Sci. 83: 1657-1661; Shire, S. J. et al. (2004) J.
Pharm. Sci. 93: 1390-1402; Wang, W. (1999) Int. J. Pharm. 185:
129-188; Won, C. M. et al. (1998) Int. J. Pharm. 167: 25-36), an
important aspect of vector stability is solubility during
preparation and storage, and vector aggregation is a problem that
needs to be fully addressed. Vector aggregation leads to losses
during vector purification, and while aggregates can be removed by
filtration, the loss in yield results in higher costs and capacity
limitations when producing vector for pre-clinical and clinical
studies. Even after filtration to remove aggregates, new aggregates
can form in concentrated preparations of AAV2 vector in
buffered-saline solutions.
[0010] The need exists for improved formulations and methods for
purification and storage of AAV vectors, such as rAAV2, that
prevent aggregation of virus particles.
SUMMARY OF THE INVENTION
[0011] These and other needs in the art are met by the present
invention, which provides high ionic strength solutions for use in
preparing and storing AAV vectors that maintain high infectivity
titer and transduction efficiency, even after freeze-thaw
cycles.
[0012] In one aspect the invention relates to methods of preventing
aggregation of virions in a preparation of virions by adding
excipients to achieve an ionic strength high enough to prevent
aggregation. In another aspect the invention relates to
compositions of virions having an ionic strength high enough to
prevent aggregation.
[0013] In some embodiments of the invention, the ionic strength is
at least about 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450
mM, 500 mM, 600 mM, 700 mM or more. In some embodiments this ionic
strength is accomplished using excipients comprising one or more
multivalent ions, for example citrate, sulfate, magnesium or
phosphate.
[0014] In additional embodiments, the osmolarity of the preparation
of virions is maintained at near isotonic levels, for example 200
mOsm, 250 mOsm, 280 mOsm, 300 mOsm, 350 mOsm or 400 mOsm, even
though the ionic strength is high enough to prevent virion
aggregation.
[0015] In some embodiments the virions are adeno-associated virus
(AAV) virions, for example AAV-2.
[0016] In other embodiments of the methods of the present invention
preparations of virions are treated with a nuclease, for example
Benzonase.RTM.. In further embodiments, nuclease treatment is
combined with addition of excipients that achieve an ionic strength
high enough to prevent aggregation.
[0017] In some embodiments of the present invention, the surfactant
Pluronic.RTM. F68 is added to a preparation of virions, for example
to 0.001%. In one embodiment, the composition comprises purified
virus particles, 10 mM Tris pH 8.0, 100 mM sodium citrate and
0.001% Pluronic.RTM. F68.
[0018] In one embodiment, AAV vectors can be stored as compositions
of the present invention at concentrations exceeding
1.times.10.sup.13 vg/mL, for example 2.times.10.sup.13,
3.times.10.sup.13, 4.times.10.sup.13, 5.times.10.sup.13 and up to
6.4.times.10.sup.13 vg/mL, without significant aggregation. In some
embodiments, AAV vectors stored using the methods and compositions
of the invention do not exhibit significant aggregation when stored
at 4.degree. C. for five days. In other embodiments, AAV vectors
that are stored as such compositions do not exhibit significant
aggregation after one, five, ten or more freeze-thaw cycles at
-20.degree. C. or at -80.degree. C.
[0019] In some embodiments, preparations of virions stored
according to the methods and compositions of the invention exhibit
an average particle radius (Rh), as measured by dynamic light
scattering, indicating that no significant aggregation of virions
has taken place. In some embodiments, preparations of virions
stored according to the methods and compositions of the invention
exhibit an average particle radius (Rh) greater than about 15 nm,
20 nm, or 30 nm.
[0020] In some embodiments, recovery of virions from preparations
of virions stored according to the methods and compositions of the
invention is greater than about 85%, 90% or 95% following
filtration through a 0.22 .mu.m filter.
[0021] In yet another aspect, the invention relates to kits
comprising the high ionic strength formulations of the invention.
In one embodiment the kit comprises a pre-mixed solution of
excipients. In another embodiment the kit comprises two or more
separate components of a high ionic strength composition of the
present invention to be mixed by a user. In some embodiments the
kit comprises sodium citrate, Tris.RTM. and Pluronic.RTM. F68. In
other embodiments, the kit further comprises instructions for
making a composition or performing a method of the present
invention.
DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A and 1B present data showing aggregation of AAV2-FIX
particles as a function of osmolarity (FIG. 1A) or ionic strength
(FIG. 1B) for various buffer compositions. AAV2-FIX vectors are
prepared by Method 2 of Example 1. Average particle radius is
measured by dynamic light scattering (DLS) following vector
dilution in varying concentrations of excipients buffered with 10
mM sodium phosphate at pH 7.5. Excipients include sodium chloride (
), sodium citrate (.smallcircle.), sodium phosphate ( ), sodium
sulfate (.quadrature.), magnesium sulfate (.tangle-solidup.), and
glycerol (.DELTA.).
[0023] FIG. 2 presents data on AAV2-FIX aggregation as a function
of the method of purification. The average particle radius is
measured by DLS following vector dilution in varying concentrations
of sodium chloride buffered with 10 mM sodium phosphate at pH 7.5.
Vectors are purified by Method 1 (double CsCl gradient)
(.smallcircle.); Method 2 (cation exchange chromatography) ( );
Method 2 plus nuclease digestion ( ); or Method 3 (chromatography
plus one CsCl gradient) (.DELTA.). Purification Methods 1-3 are
described in Example 1.
[0024] FIG. 3 presents data on transgene expression from D7/4 cells
transduced with rAAV2-AADC virions prepared and stored in high
ionic strength formulation (.quadrature.) or in a control
formulation (.smallcircle.). The concentration of AADC was measured
by ELISA (in triplicate for each data point) 72 hours
post-transduction. Error bars represent standard deviations.
DETAILED DESCRIPTION OF THE INVENTION
[0025] AAV2 vector aggregation is frequently observed in
concentrated preparations of vectors and can affect purification
recovery, and in vivo potency and safety. Hence, an important
objective for the development AAV2 vectors is to identify methods
and formulations that prevent aggregation of vectors when
concentrated stocks are prepared.
[0026] Unless otherwise indicated, the term "vector" as used herein
refers to a recombinant AAV virion, or virus particle, regardless
of the frequent use of "vector" to also refer to non-viral DNA
molecules, such as plasmids, in other contexts.
[0027] The present invention is based in part on the observation
that solution ionic strength is an important parameter in AAV
vector aggregation, implicating the involvement of ionic
interactions between virus particles in the aggregation process.
The observation that elevated ionic strength increases AAV2 vector
solubility regardless of the identity of the charged excipient
supports the hypothesis that ionic strength of solution per se,
rather than interactions involving a specific ionic species, is the
relevant physico-chemical parameter. A threshold ionic strength of
at least 200 mM is required to prevent aggregation at vector
particle concentrations examined herein.
[0028] Of practical concern, commonly used buffered saline
solutions have insufficient ionic strength to prevent AAV2 vector
aggregation at concentrations exceeding 10.sup.13 particles/mL. It
is known that high salt concentrations increase AAV2 vector
solubility (e.g. highly concentrated AAV2 vectors recovered from
gradients generally remain soluble in concentrated CsCl). However,
optimal formulations for pre-clinical and clinical studies should
be close to isotonic (280-400 mOsm), especially for in vivo
administration of vector to sites where dilution of hypertonic
solutions may be slow. In embodiments of the present invention the
exponential relationship of ionic strength with charge valency is
used to develop isotonic formulations with high ionic strengths.
Salt species with multiple charge valencies (e.g. salts of sulfate,
citrate, and phosphate) that are commonly used as excipients in
human parenteral formulations can provide the level of ionic
strength needed to prevent AAV2 vector aggregation when used at
isotonic concentrations. While isotonic (150 mM) sodium chloride
has an ionic strength of 150 mM, a value insufficient to maintain
AAV2 solubility at high vector concentrations, isotonic sodium
citrate, with an ionic strength of .about.500 mM, can support AAV2
vector concentrations of at least 6.4.times.10.sup.13 vg/mL without
aggregation.
[0029] Without intending to be limited by theory, the low
solubility of AAV2 particles may be caused by their highly
symmetrical nature in conjunction with the stabilizing effect of
complementary charged regions between neighbouring particles in
aggregates. The surface charge density based on the crystal
structure of AAV2 (Xie, Q. et al. (2002) Proc. Natl. Acad. Sci.
U.S.A. 99: 10405-10410) reveals a pattern of positive and negative
charges on the virus surface. Previous reports have shown that AAV2
vector aggregation is pH dependent, and hypothesized that amino
acids with charged side groups are involved in inter-particle
binding. Qu, G. et al. (2003) Mol. Therapy 7: 5238. These reports
hypothesized that if charged amino acid side chains are involved in
vector aggregation, high concentrations of free amino acids could
block vector particle interactions. However, we have found that
amino acids with charged side chains are not effective in
preventing AAV2 vector aggregation beyond their contribution to
ionic strength.
[0030] Vector aggregation at low ionic strength was also found to
be reduced but not prevented by efficient nuclease treatment of
purified vector particles. Digestion at an earlier stage of the
purification process (clarified HEK cell lysate) did not reduce
aggregation following vector purification. It is likely that
digestion of already purified virions is more efficient because of
a higher enzyme to nucleic acid substrate ratio. One mechanism to
explain these results is that residual nucleic acid impurities
(e.g. host cell and plasmid DNA) bound to the vector surface can
bridge to binding sites on neighbouring virus particles and thus
cause aggregation. Purified AAV2 vectors (empty capsid free) have
been reported to contain approximately 1% non-vector DNA. Smith, P.
et al. (2003) Mol. Therapy 7: 5348. While >50% of this
non-vector DNA was reported to be nuclease resistant and was
packaged within capsid particles, some impurity DNA was nuclease
resistant and appeared to be associated with the surface of
purified vector particles. The observation that efficient nuclease
treatment can reduce vector aggregation suggests that nucleic acids
associated with the vector surface at an average level not greater
than .about.25 nucleotides per vector particle can contribute to
AAV vector aggregation.
[0031] In summary, the use of high ionic strength solutions during
AAV2 vector purification and final formulation, and efficient
removal of residual vector surface DNA are two effective strategies
to achieve highly concentrated solutions of AAV2 vectors for use in
pre-clinical and clinical studies. High ionic strength solutions
and nuclease treatment can be used in combination or separately.
Although data were obtained using AAV2 vectors, the composition and
methods of the present invention may also be useful with other AAV
serotypes/variants, or other viral vectors such as adenoviruses,
lentiviruses and retroviruses.
AAV Aggregation as a Function of Excipient Concentration
[0032] Initial screening experiments are performed to elucidate the
mechanism of AAV vector aggregation and to identify classes of
excipients that can reduce/prevent aggregation. Vector aggregation
can be caused by dilution (5-fold) of vector in neutral-buffered
saline with low concentration buffer (20 mM sodium phosphate, pH
7.2). Excipients are screened using this "dilution-stress" method
to identify excipients that are able to prevent vector aggregation
when included in the diluent. For screening, aggregation is
measured by dynamic light scattering (DLS). Classes of excipients
examined included selected inorganic salts, amino acids, uncharged
carbohydrates, and surfactants. Results are presented in Table
1.
TABLE-US-00001 TABLE 1 SCREENING FOR EXCIPIENTS THAT PREVENT AAV2
VECTOR AGGREGATION USING DILUTION-STRESS METHOD Osm required to
prevent Excipient aggregation (max tested) Magnesium sulfate 180
mOsm Sodium citrate 220 mOsm Sodium chloride 320 mOsm Sodium
phosphate 220 mOsm Sodium sulfate 220 mOsm Arginine NIA (200 mOsm)
Aspartic acid 320 mOsm Glutamic acid 320 mOsm Glycine NIA (200
mOsm) Histidine NIA (200 mOsm) Lysine 300 mOsm Glycerol NIA (5%
w/v, 543 mOsm) Iodixanol NIA (5% w/v, 32 mOsm) Mannitol NIA (5%
w/v, 275 mOsm) Sorbitol NIA (5% w/v, 275 mOsm) Sucrose NIA (5% w/v,
146 mOsm) Trehalose NIA (5% w/v, 146 mOsm) Pluronic .RTM. F68 NIA
(10% w/v, 12 mOsm) Polysorbate 80 NIA (1% w/v) NIA: No inhibition
of aggregation
[0033] As illustrated in Table 1, charged excipients (inorganic
salts and amino acids) prevent aggregation when present at
sufficient concentrations. However, salt concentrations required to
prevent vector aggregation vary, ranging from 180 mOsm for
magnesium sulfate, to 320 mOsm for sodium chloride. The amino acids
arginine, aspartic acid, glutamic acid, glycine, histidine, and
lysine do not prevent aggregation at 200 mOsm, but lysine, aspartic
acid, and glutamic acid prevent aggregation at 300-320 mOsm.
Arginine, glycine and histidine were not tested at concentrations
other than 200 mOsm. Selected carbohydrates have no effect on
vector particle aggregation when present at concentrations up to 5%
w/v. For example, 5% w/v glycerol (543 mOsm) does not prevent
aggregation. The surfactants Polysorbate80 (1% w/v) and
Pluronic.RTM. F68 (10% w/v) similarly have no effect on aggregation
using the "dilution-stress" method.
AAV Aggregation as a Function of Osmolarity and Ionic Strength
[0034] FIGS. 1A and 1B show the results of a more detailed analysis
of vector aggregation as a function of the concentration of various
salts. FIG. 1A shows vector aggregation as a function of the
osmolarity of selected excipients. For charged species a
concentration-dependent inhibition of AAV2 vector aggregation is
observed. Salts with multivalent ions achieve a similar degree of
inhibition of aggregation at lower concentrations than monovalent
sodium chloride. For example, magnesium sulfate prevents
aggregation at .gtoreq.200 mOsm whereas sodium chloride requires
.gtoreq.350 mOsm to achieve a similar effect. Sodium citrate,
sodium sulfate, and sodium phosphate are intermediate in their
potency to prevent vector aggregation.
[0035] Although the results in FIG. 1A and Table 1 show no effect
of glycerol and certain sugars at concentrations up to 5% on AAV2
vector aggregation induced by low ionic strength, the data cannot
rule out improvement of AAV2 solubility at glycerol concentrations
above 5%. For example, Xie and co-workers reported that 25% (w/v)
glycerol enabled concentration of AAV2 to very high concentrations
(4.4 to 18.times.10.sup.14 particles/nil) in low ionic strength
solutions. Xie, Q. et al. (2004) J. Virol. Methods 122: 17-27.
[0036] FIG. 1B shows the data of FIG. 1 A plotted as a function of
the calculated ionic strength, rather than osmolarity, for each
excipient. FIG. 1B demonstrates that vector aggregation is
prevented when ionic strength is .about.200 mM or greater
regardless of which salt is used. These data suggested that the
ionic strength (pt) of a solution, a parameter that depends on both
solute concentration and charge valency, is the primary factor
affecting aggregation.
[0037] Ionic strengths useful to prevent aggregation in embodiments
of the present invention include, for example, 250 mM, 300 mM, 350
mM, 400 mM, 450 mM, 500 mM, 600 mM, 700 mM or higher ionic
strengths. Multivalent ions are preferred to achieve these ionic
strengths in methods and formulations of the present invention,
such as divalent, trivalent, tetravalent, pentavalent ions and ions
of even higher valency. The pH buffer in solutions and formulations
of the present invention may be phosphate, Tris, or HEPES (or other
Good's buffers), but any other suitable pH buffer may be used. In
preferred embodiments, the multivalent ions and buffer are selected
to be compatible with the target tissue for the vector being
prepared.
[0038] Use of multivalent ions in the methods and compositions of
the invention makes it possible to create compositions of high
ionic strength but relatively low osmolarity. High ionic strength
compositions of the present invention may be nearly isotonic, and
may be, for example, about 200 mOsm, 250 mOsm, 280 mOsm, 300 mOsm,
350 mOsm or 400 mOsm, although other osmolarities may be acceptable
for some uses of the compositions.
AAV Aggregation as a Function of the Method of AAV Purification
[0039] Recombinant AAV2 purified using different methods (e.g.
density gradient purification versus ion-exchange chromatography)
would be expected to have different impurity profiles. FIG. 2 shows
vector aggregation as a function of ionic strength for several
preparations of AAV differing in the purification method.
Purification methods are described in Example 1. Sodium chloride is
used to vary the ionic strength. AAV2-FIX vectors purified by
double cesium chloride gradient ultracentrifugation (Method 1), by
cation exchange column chromatography (Method 2), or by combined
column and cesium chloride gradient ultracentrifugation (Method 3)
each demonstrate similar aggregation responses as ionic strength is
decreased. In contrast, AAV2-FIX purified by the column method and
then subjected to a nuclease digestion step (Method 2+nuclease)
shows reduced aggregation at low ionic strength.
AAV Aggregation at Preparative Scale
[0040] The data in Table 1 and FIGS. 1A, 1B and 2 involve vector
aggregation at an analytical scale, employing DLS to measure
aggregation. Table 2, in contrast, shows the effects of elevated
ionic strength and nuclease treatment on AAV2 vector aggregation at
a larger scale, using methods to induce and quantify vector
aggregation that are relevant to preparative scale vector
purification. Experimental details are provided in Example 2.
Purified AAV vectors are diafiltered into solutions of various
ionic strengths, the volume is reduced to achieve high vector
concentrations, and aggregation is then assessed by measuring
vector recovery after filtration through a 0.22 .mu.m filter.
Aliquots from a single pool of AAV2-AADC vector purified by Method
1 through the second CsCl gradient centrifugation step
(1.8.times.10.sup.15 vg in 91 mL, 1.8.times.10.sup.13 vg/mL, in
.about.3M CsCl) are used as starting material in the diafiltration
experiments. Tangential flow filtration using hollow fibers is used
for diafiltration because it is scalable and yet it still enables
preparation of volumes (min. 1.4 mL), and thus AAV concentrations,
at which aggregation would be expected in neutral buffered
saline.
[0041] In Experiment 1, three hollow fiber units are used to
diafilter AAV2-AADC vector in formulations CF, TF1, or TF2, and the
volume is reduced to a target of 2.5.times.10.sup.13 vg/mL. See
Example 2. The samples are then filtered through a 0.22 .mu.m
filter. Results are shown in Table 2. Vector recovery ("Yield %")
for both elevated ionic strength formulations TF1 (95.+-.7.4%) and
TF2 (93.+-.7.4%) are significantly higher than the recovery using
the control formulation CF (77.+-.6.6%).
TABLE-US-00002 TABLE 2 AAV VECTOR RECOVERY AT PROCESS SCALE Target
Actual Yield % Experiment Formulation .mu. (mM) (vg/mL) (vg/mL)
(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)
[0042] In Experiment 2, AAV2-AADC is concentrated to a higher
target value (6.7.times.10.sup.13 vg/mL) in CF or TF2. Vector
recovery using TF2 (96.+-.4.4%) is again significantly higher than
recovery using CF (59.+-.6.0%). Within the variability of the
assays used, vector was recovered fully at both target
concentrations using TF2, indicating that aggregation was
prevented. In contrast, significant aggregation was observed at
both target concentrations using CF, and the extent of aggregation
(i.e. loss following 0.22 .mu.m filtration) was higher at the
higher target vector concentration. In an additional experiment
(not shown), 50 .mu.L samples of AAV2 vector are taken following
concentration but prior to the 0.22 .mu.m filtration step of
Experiment 2, and examined by light microscopy. Vector concentrated
in CF contains obvious amounts of visible material (not shown),
while no such material is seen in vector concentrated in TF2.
[0043] Experiment 3 examines the effect of prior nuclease digestion
of purified vector on aggregation. In the absence of nuclease
digestion recovery of AAV2-AADC in CF is 68.+-.11%, similar to the
recoveries in Experiments 1 and 2. In contrast, purified vector
treated with nuclease and then concentrated in CF gives higher
recovery (91.+-.12%). These prep scale results reflect the same
effect of nuclease digestion shown in FIG. 2 using the
"dilution-stress" (analytical scale) method.
[0044] The results presented in Table 2 demonstrate that the
methods and compositions of the present invention increase the
recovery of AAV vector recovery. For example, in various
embodiments of the present invention, recovery is improved from
less than about 80% to at least about 85%, 90%, 95% or more.
AAV Stability and Activity Following Storage or Freeze-Thaw
Cycling
[0045] Croyle and coworkers reported a significant loss of titer of
AAV and adenovirus following multiple freeze-thaw cycling in sodium
phosphate buffer, and demonstrated that the better pH buffering
provided by potassium phosphate during freeze-thaw cycling
prevented titer loss. Croyle, M. A. et al. (2001) Gene Therapy 8:
1281-1290. Results of our freeze-thaw stability study using sodium
phosphate support these findings. We find that while 150 mM sodium
phosphate provides sufficient ionic strength to prevent aggregation
during preparation and non-frozen storage of concentrated AAV2-AADC
vector, even a single freeze-thaw cycle at -20 or -80.degree. C.
results in aggregation.
[0046] AAV stability after storage or freeze-thaw (F/T) cycling is
assessed in buffers of the present invention as follows. The
concentrated vectors prepared in CF, TF1, and TF2 (Table 2,
Experiment 1) are subjected to a short stability study to
investigate whether aggregation will occur during refrigerated
storage, or following multiple freeze-thaw (F/T) cycles.
Aggregation is assessed by DLS using undiluted samples, and Rh
values >20 nm are deemed to indicate the occurrence of some
level of aggregation.
TABLE-US-00003 TABLE 3 STABILITY OF AAV2 VECTORS Particle radius -
Rh (nm) For- 4.degree. C. -20.degree. C. -80.degree. C. mulation
Pre 5 d 1 F/T 5 F/T 10 F/T 1 F/T 5 F/T 10 F/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 Pre: DLS radius measured
immediately following 0.2 .mu.m filtration. Vector concentrations
(vg/mL): CF: 1.93E13, TF1: 2.38E13, TF2: 2.33E13. TH: signal
intensity is too high to measure because of extensive
aggregation.
[0047] As shown in Table 3, AAV2-AADC vector prepared in CF shows
some aggregation after 5 days of storage at 4.degree. C., as well
as following one or more F/T cycles at -20 or -80.degree. C. For
vector prepared in TF1, no aggregation occurs after 5 days at
4.degree. C., but aggregation occurs following a single F/T cycle
at -20 or -80.degree. C. as indicated by a DLS signal intensity
that is too high to measure. Visual inspection of these samples
reveals slight cloudiness, which is consistent with aggregation.
For vector prepared in TF2, no aggregation is observed at 4.degree.
C., or following up to 10 F/T cycles at -20.degree. C. Some
aggregation is observed following 5 and 10 F/T cycles at
-80.degree. C.
[0048] AAV activity after storage or F/T cycling in TF2 is assessed
as follows. As described above, the high ionic strength, isotonic
formulation TF2 effectively prevents vector aggregation during
concentration and storage, and therefore represents a promising
candidate for further study. An important question is whether
preparation and storage of the vector in high ionic strength TF2
would adversely affect its functional activity. To assess this,
assays are performed to measure the infectious titer and the
transduction efficiency of vectors prepared and stored for an
extended period of time in TF2.
[0049] For infectivity, a highly sensitive infectivity assay
capable of detecting single infectious events is used. Zhen, Z. et
al. (2004) Human Gene Ther. 15: 709-715. AAV2-AADC is prepared in
TF2 at a concentration of 6.4.times.10.sup.13 vg/mL. After being
stored for 45 days at 4.degree. C. the preparation has a vector
genome to infectious unit ratio (vg/IU) of 13, compared to a value
of 16 vg/IU for the reference vector. This difference is not
significant given the reported variability of this assay (RSD
.about.50%).
[0050] Transduction efficiency is assessed by measuring the
expression of AADC protein by ELISA following transduction of D7/4
cells. FIG. 3 shows no significant difference between vector
prepared in TF2 and the reference control for vector input ranging
from 10 to 10.sup.5 vg/cell. Together, these data indicate that
preparation and storage of AAV2 vectors in high ionic strength TF2
does not have a deleterious effect on vector infectivity or
transduction efficiency.
Conclusion
[0051] The effect of ionic strength (.mu.) on virus particle
interactions is determined to elucidate the mechanism of vector
aggregation. The ionic strength of neutral-buffered isotonic saline
(.mu.=150 mM) is insufficient to prevent aggregation of AAV2
vectors purified by gradient ultracentrifugation or by cation
exchange chromatography at concentrations exceeding
.about.10.sup.13 particles/mL. Inclusion of sugars (sorbitol,
sucrose, mannitol, trehalose, glycerol) at concentrations up to 5%
(w/v) or of surfactants Tween80.RTM. (1%) or Pluronic.RTM. F68
(10%) does not prevent aggregation of vector particles.
[0052] In contrast, vector particles remain soluble when elevated
ionic strength solutions (.mu.>200 mM) are used during
purification and for final vector formulation. Elevated ionic
strength solutions using isotonic excipient concentrations for in
vivo administration are prepared with salts of multivalent ions,
including sodium citrate, sodium phosphate, and magnesium sulfate.
An isotonic formulation containing 10 mM Tris, 100 mM sodium
citrate, 0.001% Pluronic.RTM. F68, pH 8.0 (.mu..about.500 mM)
enables concentration of AAV2-AADC vectors to 6.4.times.10.sup.13
vg/mL with no aggregation observed during preparation and following
ten freeze-thaw cycles at -20.degree. C. See Table 3, below, and
accompanying discussion. AAV2-AADC vectors prepared and stored for
an extended period in elevated ionic strength formulation retain
high infectivity titer (13 IU/vg) and transduction efficiency.
[0053] Nuclease treatment of purified AAV2 vectors reduces the
degree of vector aggregation, implicating vector surface nucleic
acid impurities in inter-particle interactions. Hence, purification
methods to efficiently remove vector surface residual nucleic
acids, coupled with the use of elevated ionic strength isotonic
formulations, are useful methods to prevent AAV2 vector
aggregation.
EXAMPLE 1
AAV Purification Methods
[0054] AAV2 vectors expressing human coagulation factor IX (FIX) or
human amino acid decarboxylase (AADC) are produced by triple
transfection of HEK293 cells as previously described (Matsushita,
T. et al. (1998) Gene Therapy 5: 938-945), with modifications. For
the large scale preparations, cells are cultured and transfected in
850 mm.sup.2 roller bottles (Corning). Vectors are purified by one
of three methods.
[0055] In purification Method 1, modified from Matsushita,
transfected HEK293 cells in roller bottles are collected by
centrifugation (1000 g, 15 min), resuspended in 10 mM sodium
phosphate, 500 mM sodium chloride, pH 7.2, and lysed by three
freeze/thaw cycles (alternating an ethanol/dry ice bath and a
37.degree. C. water bath). The cell lysate is clarified by
centrifugation (8,000 g, 15 min). The supernatant is then diluted
to 200 mM NaCl by addition of 10 mM sodium phosphate, pH 7.2, and
digested with Benzonase.RTM. (Merck, Purity Grade 1; 200 U/mL, 1 h,
37.degree. C.). The lysate is adjusted to 25 mM CaCl.sub.2 using a
1M stock solution, and incubated at 4.degree. C. for one hour.
[0056] The mixture is centrifuged (8,000 g, 15 min), and the
supernatant containing vector is collected. To precipitate virus
from the clarified cell lysate, polyethylene glycol (PEG8000) is
added to a final concentration of 8%, the mixture incubated at
4.degree. C. for three hours, and then centrifuged (8,000 g, 15
min). The pellets containing vector are re-suspended with mixing in
0.15M NaCl, 50 mM Hepes, 25 mM EDTA, pH 8.0 and incubated at
4.degree. C. for 16 hours. The resuspended material is pooled, and
solid cesium chloride is added to a final density of 1.40 gm/ml.
Vector is then banded by ultracentrifugation (SW28, 27,000 rpm, 24
h, 20.degree. C.) using a Beckman model LE-80 centrifuge. The
centrifugation tubes are fractionated, and densities from 1.38 to
1.42 gm/mL containing vector are pooled. This material is banded a
second time by ultracentrifugation (NVT65 rotor, 65,000 rpm, 16 h,
20.degree. C.), and fractions containing purified AAV2 vectors are
pooled. To concentrate vector and to perform buffer exchange,
vectors in concentrated cesium chloride solution are subjected to
ultrafiltration/diafiltration (UF/DF) by tangential flow filtration
as described below (Example 2).
[0057] In purification Method 2, cell harvests containing AAV are
microfluidized and filtered sequentially through 0.65 and 0.22
.mu.m filters (Sartorius). Virus is purified from the clarified
cell lysates by cation exchange chromatography using Poros HS50
resin as previously described. U.S. Pat. No. 6,593,123. For the
nuclease digestion described in FIG. 2, column-purified vectors are
incubated (4 h, RT) with 100 U/mL Benzonase and 10 U/mL DNAse I
(RNAse free, Roche Diagnostics, Indianapolis, Ind.).
[0058] For purification Method 3, AAV2 vectors purified by cation
exchange chromatography are subjected to an additional cesium
chloride gradient ultracentrifugation step (SW28, 27,000 rpm, 20 h)
to remove empty capsids prior to UF/DF.
[0059] Real time quantitative PCR (Q-PCR) is used to quantify AAV
preparations as previously described. Sommer, J. M. et al. (2003)
Mol. Therapy 7: 122-128. Vectors purified by each of the three
methods are analyzed by SDS-PAGE/silver staining analysis, and in
all cases VP1, VP2 and VP3 are present in the expected ratios, with
the capsid proteins representing >95% of total proteins as
determined by scanning densitometry. However, unlike
gradient-purified AAV2 vectors purified using Methods 1 and 3,
vectors purified by Method 2 (column chromatography) contain empty
capsids, ranging from 3-10 empty capsids per vector genome.
EXAMPLE 2
Ultrafiltration and Diafiltration to Detect AAV Aggregation
[0060] Disposable hollow fiber tangential flow filtration devices
(Amersham BioSciences 8'' Midgee, 100 kDa nominal pore size) are
used to concentrate and diafilter AAV2 vectors purified by the
methods described above, and for the UF/DF experiments described in
Table 2. For all UF/DF procedures a volume of diafiltration buffer
corresponding to 10.times. the product volume is used, and it is
added in .about.1 mL increments to approximate continuous
diafiltration. Using this method, the calculated residual CsCl
after diafiltration is <0.5 mM.
[0061] The following three formulations were used for UF/DF:
Control Formulation (CF: 140 mM sodium chloride, 10 mM sodium
phosphate, 5% sorbitol, pH 7.3); Test Formulation 1 (TF1: 150 mM
sodium phosphate, pH7.5); and Test Formulation 2 (TF2: 100 mM
sodium citrate, 10 mM Tris, pH8.0). For Experiment 1 shown in Table
2, diafiltration is performed at a volume corresponding to a vector
concentration of 1.times.10.sup.13 vg/mL, and following
diafiltration the volume is reduced to a value corresponding to
2.5.times.10.sup.13 vg/mL (assuming no vector loss).
[0062] For Experiment 2, diafiltration is performed at a volume
corresponding to a 2.times.10.sup.13 vg/mL, and the volume is then
reduced to a value corresponding to 6.7.times.10.sup.13 vg/mL.
[0063] For Experiment 3 (CF.+-.Bz), AAV2-AADC (approximately
1.2.times.10.sup.14 vg) is first diafiltered into TF1 (a
formulation compatible with nuclease activity) and then passed
through a 0.22 .mu.m filter. The titer of this material is
determined, and the volume is adjusted to correspond to a
concentration of 1.times.10.sup.13 vg/mL. To 10 mL of this
material, MgCl.sub.2 is added to a concentration of 2 mM, and then
divided into two equal aliquots. One aliquot is incubated with
Benzonase (200 U/mL, 4 h, RT), and the second is mock-incubated.
Each aliquot is then diafiltered at a volume corresponding to a
vector concentration 2.times.10.sup.13 vg/mL, and then concentrated
to a 3.6.times.10.sup.13 vg/mL target. Following all UF/DF
protocols, Pluronic.RTM. F-68 (BASF Corp., Mount Olive, N.J.) from
a 1% stock is added to the vector product to a final concentration
of 0.001%, and the solution is passed through a 0.22 .mu.m syringe
filter (Sartorius). All UF/DF procedures are performed in a laminar
flow cabinet.
EXAMPLE 3
Measurement of Vector Aggregation by Dynamic Light Scattering
[0064] Purified vectors are analyzed for aggregation by dynamic
light scattering (DLS) using a Protein Solutions DynaPro 99
(.lamda.=825.4 nm). Primary data (particle radius--Rh, average
value measured over 30 cycles, 10 cycles/min) are used for all
analyses reported. A "dilution-stress" method is used to assess the
effect of varying excipients on vector aggregation. In this method,
80 .mu.L of test diluent is added to 20 .mu.L of vector solution
with mixing in the actual cuvette used for DLS measurement, and
data collection is initiated within 10 seconds of mixing. Prior to
addition of test diluents, the Rh value for AAV2 vector
preparations is measured and confirmed to be <15 nm to ensure
that the starting material is monomeric. Samples that are not 100%
monomeric are passed through a 0.22 .mu.m syringe disc filter
(Sartorius, low protein binding) to remove aggregates.
[0065] The osmolarity and ionic strength values given in FIGS. 1
and 2 are calculated using all excipients present in the mixture
(i.e. weighted: test diluent (80%) and starting vector formulation
(20%)). Osmolarity is calculated according to the equation:
Osmolarity=.SIGMA.c.sub.i, where c.sub.i is the molar concentration
of each solute species. The ionic strength (.mu.) is calculated
according to the equation: .mu.=.rho.c.sub.iz.sub.i.sup.2, where
z.sub.i is the charge on each species. In conditions that resulted
in vector aggregation (e.g. low .mu.) a progressive increase in Rh
is observed over the course of data collection. To validate the use
of the average Rh measured over the 3 minute interval following
dilution as a reliable measure of aggregation, the average rate of
increase of Rh(.DELTA.Rh/.DELTA.t) over the same time interval is
also determined (not shown). Analysis of .DELTA.Rh/.DELTA.t gives
results concordant with those obtained using the average Rh value
reported in FIGS. 1 and 2.
EXAMPLE 4
AAV Virion Infectivity
[0066] Infectivity of AAV2-AADC vectors is determined using a
highly sensitive assay as previously described. Zhen, Z. et al.
(2004) Human Gene Ther. 15: 709-715. Briefly, samples are serially
diluted (10-fold dilutions, 10 replicates/dilution) and added to
D7/4 cells (modified HeLa cells expressing AAV rep and cap) grown
in 96 well tissue culture plates (Falcon, cat. #353227) in DMEM
medium containing 10% FBS. Adenovirus (Ad-5, 100 vp/cell) is added
to each well to provide helper functions. After 48 h, replication
of AAV vector in each well is quantified by Q-PCR using
transgene-specific primers and probes, and the frequency of
infection at limiting dilution is analyzed by the Karber method to
calculate the infectivity titer. The test sample is run
concurrently with an AAV2-AADC reference previously prepared in CF
and stored at -80.degree. C.
[0067] The transduction efficiency of AAV2 vectors is quantified by
a whole cell ELISA. D7/4 cells grown in 96 well plates are infected
with 10-fold serial dilutions of the test sample and reference
vector, corresponding to 10 to 10.sup.5 vg/cell input (5
replicates/dilution). After 48 h, the culture medium is removed,
and cells are washed twice with 200 .mu.L PBS (10 mM sodium
phosphate, 140 mM sodium chloride, pH 7.2). Cells are then
permeabilized and fixed by addition of 100 .mu.L of PBS containing
0.5% Triton X-100 and 4% paraformaldehyde to each well (15 min).
The fixing solution is removed, and the cells are washed twice with
PBS containing 0.5% Triton X-100. Non-specific sites are blocked by
adding PBS containing 3% bovine serum albumin (BSA) and 0.5% Triton
X-100 (60 min).
[0068] After washing, cells are incubated for one hour with rabbit
anti-AADC IgG antibody (Chemicon, AB136), and washed. Cells are
then incubated for one hour with alkaline phosphatase-conjugated
goat anti-rabbit IgG, and washed. Antibodies are diluted 1:1000 in
PBS containing 1% BSA, 0.5% Triton X-100. Substrate (PNPP, Pierce,
cat. #34047) is then added (1 mg/mL in 1.times. diethanolamine
substrate buffer, Pierce, cat. #34064), and after incubation for 30
min the concentration of cleaved substrate is measured
spectrophotometrically (.lamda.=405 nm). Human AADC expression as a
function of vector input is fitted using a spline curve
(SigmaPlot). The AAV2-AADC reference vector is measured
concurrently with the test sample.
[0069] While preferred illustrative embodiments of the present
invention are described, it will be apparent to one skilled in the
art that various changes and modifications may be made therein
without departing from the invention, and it is intended in the
appended claims to cover all such changes and modifications that
fall within the true spirit and scope of the invention.
[0070] All publications, patents and patent applications referred
to herein are hereby incorporated by reference in their
entireties.
* * * * *