US20230047531A1 - Method for determining the loading state of an aav particle by nuclear magnetic resonance relaxometry - Google Patents

Method for determining the loading state of an aav particle by nuclear magnetic resonance relaxometry Download PDF

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US20230047531A1
US20230047531A1 US17/886,357 US202217886357A US2023047531A1 US 20230047531 A1 US20230047531 A1 US 20230047531A1 US 202217886357 A US202217886357 A US 202217886357A US 2023047531 A1 US2023047531 A1 US 2023047531A1
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dna
aav particles
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Maximilian Hartl
Dinah Funke
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Hoffmann La Roche Inc
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
    • G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
    • G01N24/087—Structure determination of a chemical compound, e.g. of a biomolecule such as a protein
    • G—PHYSICS
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    • G01N30/02—Column chromatography
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    • C—CHEMISTRY; METALLURGY
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
    • G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
    • G01N24/085—Analysis of materials for the purpose of controlling industrial production systems
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/14—Preparation by elimination of some components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/448—Relaxometry, i.e. quantification of relaxation times or spin density
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/46—NMR spectroscopy
    • G01R33/465—NMR spectroscopy applied to biological material, e.g. in vitro testing
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material
    • G—PHYSICS
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins

Definitions

  • the current invention is in the field of gene therapy.
  • a method for the determination of the loading state of a viral particle that is the ratio between full and empty viral particles. To achieve this aim nuclear magnetic resonance is used.
  • Gene therapy refers broadly to the therapeutic administration of genetic material to modify gene expression of living cells and thereby alter their biological properties. After decades of research, gene therapies have progressed to the market and are expected to become increasingly important. In general, gene therapy can be divided into either in vivo or ex vivo approaches.
  • AAV adeno-associated viral
  • An AAV is a small, naturally occurring, non-pathogenic parvovirus, which is composed of a non-enveloped icosahedral capsid. It contains a linear, single stranded DNA genome of approximately 4.7 kb.
  • the genome of wild-type AAV vectors carries two genes, rep and cap, which are flanked by inverted terminal repeats (ITRs). ITRs are necessary in cis for viral replication and packaging.
  • the rep gene encodes for four different proteins, whose expression is driven by two alternative promoters, P5 and P19. Additionally different forms are generated by alternative splicing.
  • the Rep proteins have multiple functions, such as, e.g., DNA binding, endonuclease and helicase activity. They play a role in gene regulation, site-specific integration, excision, replication and packaging.
  • the cap gene codes for three capsid proteins and one assembly-activating protein. Differential expression of these proteins is accomplished by alternative splicing and alternative start codon usage and driven by a single promoter, P40, which is located in the coding region of the rep gene.
  • the viral genes are replaced with a transgene expression cassette, which remains flanked by the viral ITRs, but encodes a gene of interest under the control of a promoter of choice.
  • the engineered rAAV vector does not undergo site-specific integration into the host genome, remaining predominantly episomal in the nucleus of transduced cells.
  • helper genes are provided in nature by co-infected helper viruses, such as, e.g., adenovirus or herpes simplex virus.
  • helper viruses such as, e.g., adenovirus or herpes simplex virus.
  • helper viruses such as, e.g., adenovirus or herpes simplex virus.
  • helper viruses such as, e.g., adenovirus or herpes simplex virus.
  • E1A, E1B, E2A, E4 and VA five adenoviral genes, i.e. E1A, E1B, E2A, E4 and VA, are known to be essential for AAV replication.
  • VA is a small RNA gene.
  • the therapeutic DNA carrying the transgene flanked by ITRs is introduced into a packaging host cell line, which also comprise rep and cap genes as well as the required helper genes to produce rAAV particles.
  • a packaging host cell line which also comprise rep and cap genes as well as the required helper genes to produce rAAV particles.
  • the packaging efficiency i.e. the efficiency of introducing the DNA into the viral capsid during the rAAV particle generation inside the packaging cell line, is less than 100% rAAV
  • the obtained rAAV particle preparations are mixtures of full, i.e. DNA containing, and empty, i.e. without DNA, viral capsids.
  • the composition of the mixture has a direct effect.
  • Nuclear magnetic resonance is a technique that has been used in a wide range of applications to study biomolecules.
  • Conclusions regarding different aspects of biomolecules in a sample may be based on NMR signals of the biomolecules, e.g. proteins or peptides, themselves or on NMR signals derived from the solution comprising the biomolecule.
  • Shigemitsu et al. (Anal. Biochem. 498 (2016) 59-67) describe the measurement of NMR signals of amyloid beta peptide monomers and dimers.
  • Metz and Mader Int. J. Pharmaceut. 364 (2008)170-175) show NMR experiments for the characterization of emulsions and lipid ingredients that may specifically be used in the pharmaceutical field, in particular, in drug delivery.
  • the current invention is based, at least in part, on the finding that the transverse nuclear magnetic spin relaxation time T 2 and the transverse nuclear magnetic spin relaxation rate R 2 , respectively, of protons of water molecules in an aqueous solution comprising viral particles depends on the loading status of the viral particle.
  • one aspect of the current invention is a method for determining the ratio/fraction/concentration of loaded viral particles in a sample
  • the NMR parameter is indicative of the transverse nuclear magnetic spin relaxation of water in the solution or fraction thereof, specifically of the protons of the water molecules in the aqueous solution.
  • the NMR parameter is the transverse nuclear magnetic spin relaxation time T 2 or the transverse nuclear magnetic spin relaxation rate R 2 of the protons of the water molecules in the aqueous solution.
  • the isolated, (monomeric) viral particle has a molecular weight in the range of 5,000 kDa to 6,000 kDa. In one preferred embodiment, the isolated, (monomeric) viral particle has a molecular weight of 5,000 kDa to 5,250 kDa.
  • the viral particle is a virus, a virus-like particle (VLP), or a liposome. In a further embodiment, the viral particle is a virus or a VLP. In another embodiment, the viral particle is a non-enveloped virus or a VLP of a non-enveloped virus. In one preferred embodiment, the viral particle is an adeno-associated virus particle (AAV particle).
  • AAV particle adeno-associated virus particle
  • the loaded viral particles are DNA-containing viral particles. In one preferred embodiment, the loaded viral particles are DNA-containing AAV particles.
  • the total concentration of the viral particles in the aqueous solution is at least 1*10 12 vp/mL (viral particles per mL), in one embodiment at least 5*10 12 vp/mL, in one preferred embodiment at least 1*10 13 vp/mL, in one embodiment at least 2*10 13 vp/mL.
  • the aqueous solution is a phosphate buffered saline solution. In one preferred embodiment, the aqueous solution is a phosphate buffered saline solution comprising a detergent and optionally citrate.
  • the method is for determining the ratio/fraction/concentration of loaded viral particles in a sample, wherein the sample comprises up to 80% of DNA-containing viral particles, in one preferred embodiment up to 80% of DNA-containing AAV particles, and the calibration function is a linear function.
  • the calibration function is a second order polynomial function.
  • the method is for determining the ratio/fraction/concentration of loaded viral particles in a sample, wherein the sample comprises up to 100% of DNA-containing viral particles, in one preferred embodiment up to 100% of DNA-containing AAV particles, and the calibration function is a second order polynomial function.
  • the calibration function is based on the same NMR parameter determined for at least two (calibration) samples with different ratios of DNA-containing and empty AAV particles.
  • the method according to the current invention is in certain embodiments an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to producing an aqueous solution comprising (a mixture of) DNA-containing and empty AAV particles, and/or the steps indicated elsewhere herein. Moreover, one or more of said steps may be performed by automated equipment.
  • the present invention also relates to a use of the method according to the present invention, for a purpose selected from the group consisting of producing a recombinant AAV particle and release of recombinant AAV particle preparations for therapeutic application, especially real-time release.
  • one aspect of the current invention is the use of the transverse nuclear magnetic spin relaxation time T 2 for the determination of the loading status of viral particles, in one preferred embodiment of AAV particles.
  • the loading status is the ratio of DNA-containing viral particles to empty viral particles.
  • the loading status is the fraction of DNA-containing virsal particles of the total viral particles.
  • the use is an in vitro use.
  • the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and claimed herein.
  • the particular features presented herein, especially presented as aspects or embodiments can be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter includes any suitable combination of the features disclosed herein.
  • the description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.
  • a method for determining the ratio/fraction/concentration of DNA-containing AAV particles in a sample comprises the steps of determining the water proton transverse relaxation time (T2) or the water proton transverse relaxation rate (R2), of the protons of the water molecules in an aqueous solution comprising a mixture of DNA-containing and empty AAV particles, by applying an NMR measurement to the solution and thereafter determining the ratio/fraction/concentration of DNA-containing AAV particles with the determined nuclear magnetic spin relaxation based on a calibration function.
  • T2 water proton transverse relaxation time
  • R2 water proton transverse relaxation rate
  • the current invention is based, at least in part, on the finding that the transverse nuclear magnetic spin relaxation time T 2 and the transverse nuclear magnetic spin relaxation rate R 2 , respectively, of protons of water molecules in an aqueous solution comprising higher order protein structures, such as DNA-containing AAV particles, is affected by the internal composition of the higher order protein structure.
  • recombinant DNA technology enables the generation of derivatives of a nucleic acid.
  • Such derivatives can, for example, be modified in individual or several nucleotide positions by substitution, alteration, exchange, deletion or insertion.
  • the modification or derivatization can, for example, be carried out by means of site directed mutagenesis.
  • Such modifications can easily be carried out by a person skilled in the art (see e.g. Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B. D., and Higgins, S. G., Nucleic acid hybridization—a practical approach (1985) IRL Press, Oxford, England).
  • standard conditions if not otherwise noted, relates to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e. preferably, a temperature of 25° C. and an absolute pressure of 100 kPa; also preferably, standard conditions include a pH value of 7.
  • SATP ambient temperature and pressure
  • aqueous solution relates to any liquid preparation wherein the concentration of water (H 2 O) in the solvent is at least 50% (w/v), in an embodiment at least 75% (w/v), in a further embodiment at least 90% (w/v), in a further embodiment at least 95% (w/v), in a further embodiment at least 98% (w/v), in a further embodiment at least 99% (w/v), in a further embodiment is at least 99.5% (w/v).
  • the term aqueous solution encompasses liquid preparations comprising up to 50% (w/v) D20 or PEG (poly ethylene glycol).
  • aqueous solution indicates that at least a fraction of the compounds (solutes) in the solution is dissolved in the solvent.
  • Methods for preparing solutions are known in the art.
  • the term aqueous solution in an embodiment, relates to a liquid preparation comprising viral particles, such as AAV particles, which are, at least partially, dissolved in a solvent comprising, in an embodiment consisting of, water.
  • the aqueous solution is a phosphate buffered saline solution.
  • the aqueous solution is a phosphate buffered saline solution comprising about 0.001% (w/v) of a detergent and optionally about 100 mM citrate.
  • the detergent is selected from poloxamers and polysorbates. Poloxamers are commercially available under the tradenames Pluronic® or Kolliphor® or Synperonic®. Polysorbates are commercially available under the tradenames Tween®, Scattics®, Alkest® and Canarcel®.
  • the detergent is poloxamer 188 (tradename: Pluronic F-68) and the optional citrate is sodium citrate.
  • Pluronic F-68 tradename: Pluronic F-68
  • a phosphate buffered saline solution comprises about 137 mM sodium chloride, about 2.7 mM potassium chloride and about 12 mM phosphate as a mixture of hydrogen phosphate and dihydrogen phosphate, adjusted to a pH value of 7.4.
  • determining is used as understood by the skilled person and relates to determining a value of a relevant parameter, in particular an NMR parameter with a suitable method.
  • the determination is a continuous in-line determination.
  • the aliquots are virtual aliquots.
  • the aliquots are generated as a continuous stream of liquid.
  • the concentration, the NMR parameter and/or any other parameters(s) optionally determined is/are determined in a flow-through cell, in particular, in case of the concentration, in a flow-through cuvette.
  • the parameters may be determined simultaneously or sequentially, in specific embodiments, the parameters are determined sequentially.
  • empty capsid refers to a viral particle, such as an AAV particle that includes a viral protein shell but that lacks in whole or part a nucleic acid that encodes a protein or is transcribed into a transcript of interest. Accordingly, the empty capsid does not function to transfer a nucleic acid that encodes a protein or is transcribed into a transcript of interest into the host cell.
  • endogenous denotes that something is naturally occurring within a cell; naturally produced by a cell; endogenous gene locus/cell-endogenous gene locus: naturally occurring locus in a cell.
  • an exogenous nucleotide sequence indicates that a nucleotide sequence does not originate from a specific cell and is introduced into said cell by DNA delivery methods, e.g., by transfection, electroporation, or transduction by viral vectors.
  • an exogenous nucleotide sequence is an artificial sequence wherein the artificiality can originate, e.g., from the combination of subsequences of different origin (e.g. a combination of a recombinase recognition sequence with an SV40 promoter and a coding sequence of green fluorescent protein is an artificial nucleic acid) or from the deletion of parts of a sequence (e.g.
  • endogenous refers to a nucleotide sequence originating from a cell.
  • An “exogenous” nucleotide sequence can have an “endogenous” counterpart that is identical in base compositions, but where the “exogenous” sequence is introduced into the cell, e.g., via recombinant DNA technology.
  • impurities denotes all compounds decreasing purity of a viral particle preparation, such as an AAV particle preparation, especially including empty viral particles.
  • impurities are AAV particle-specific by-products, especially empty AAV particles, and host cell protein by-products.
  • nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • isolated polypeptide or antibody refers to a polypeptide molecule or antibody molecule that has been separated from a component of its natural environment.
  • the NMR parameter comprises or is a relaxation time or a relaxation rate determined in an 1 H 2 O NMR measurement.
  • the NMR parameter comprises or is at least one of a transverse nuclear magnetic spin relaxation time T 2 and a transverse nuclear magnetic spin relaxation rate R 2 of water in an aqueous solution or aliquot thereof.
  • the NMR parameter is indicative of a transverse nuclear magnetic spin relaxation of water in an aqueous solution or aliquot thereof, specifically of protons in water in solution or aliquot thereof.
  • the magnetic field strength during the determination is of from 0.1 Tesla (T) to 24 T, in certain embodiments of from 0.2 T to 10 T, in further embodiments of from 0.3 T to 5 T, in further embodiments of from 0.4 T to 2 T. In one embodiment, the magnetic field strength is in the range of 0.45 to 0.50 T. In one preferred embodiment, the magnetic field strength is about 0.47 T.
  • the resonance frequency is of from 5 MHz to 500 MHz, in further embodiments of from 7.5 MHz to 200 MHz, in further embodiments of from 10 MHz to 100 MHz, in further embodiments of from 15 MHz to 50 MHz. In one preferred embodiment, the resonance frequency is about 20 MHz.
  • the NMR parameter is corrected for changes not caused by viral particles, such as AAV particles, in particular for solute effects, which may in certain embodiments be based on pH value and/or ion concentration.
  • such correction is provided by performing a pre-run using a sample with known viral particle content and otherwise identical composition as the samples to be tested.
  • nuclear magnetic resonance measurement device and “NMR measurement device”, as used herein, equally include any and all devices configured for determining an NMR parameter by applying an NMR measurement to an aqueous solution or an aliquot thereof.
  • the NMR measurement device is configured to perform measurement of a nuclear magnetic spin relaxation value, in certain embodiments of the water proton nuclear magnetic spin relaxation value.
  • the NMR measurement device is configured for in-line measurement, in further embodiments for continuous in-line measurement. Suitable devices are known in the art, e.g. from Metz & Mader (Int. J. Pharmaceut. 364 (2008) 170) and from Taraban et al. (Anal. Chem. 89 (2017) 5494; 7th Annual PANIC Conference, 2019, Poster presentation P35: “Water Flow-NMR—A Prospective Contact-Free In-Line Analytical Tool for Continuous Biomanufacturing”).
  • a “recombinant AAV vector” is derived from the wild-type genome of a virus, such as AAV by using molecular methods to remove the wild type genome from the virus (e.g., AAV), and replacing it with a non-native nucleic acid, such as a nucleic acid transcribed into a transcript or that encodes a protein. Typically, for AAV one or both inverted terminal repeat (ITR) sequences of AAV genome are retained in the AAV vector.
  • ITR inverted terminal repeat
  • a “recombinant” AAV vector is distinguished from a naturally occurring viral AAV genome, since all or a part of the viral genome has been replaced with a non-native (i.e., heterologous) sequence with respect to the natural viral genomic nucleic acid. Incorporation of a non-native sequence therefore defines the viral vector (e.g., AAV) as a “recombinant” vector, which in the case of AAV can be referred to as a “rAAV vector.”
  • a recombinant viral vector sequence (e.g., an AAV vector sequence) can be packaged —referred to herein as a “particle”—for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo.
  • a recombinant vector sequence is encapsulated or packaged into an AAV particle
  • the particle can also be referred to as an “AAV particle” or a “rAAV particle”.
  • Such particles include proteins that encapsulated or package the vector genome.
  • Particular examples include viral envelope proteins, and in the case of AAV, capsid proteins, such as AAV VP1, VP2 and VP3.
  • AAV variants including capsid variants may not be serologically distinct from a reference AAV or other AAV serotype, they differ by at least one nucleotide or amino acid residue compared to the reference or other AAV serotype.
  • a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest.
  • the new virus e.g., AAV
  • this new virus e.g., AAV
  • serology testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype according to the traditional definition of serotype.
  • serotype broadly refers to both serologically distinct viruses (e.g., AAV) as well as viruses (e.g., AAV) that are not serologically distinct that may be within a subgroup or a variant of a given serotype.
  • transduce and “transfect” refer to introduction of a molecule such as a nucleic acid (plasmid) into a cell.
  • a cell has been “transduced” or “transfected” when exogenous nucleic acid has been introduced inside the cell membrane.
  • a “transduced cell” is a cell into which a “nucleic acid” or “polynucleotide” has been introduced, or a progeny thereof in which an exogenous nucleic acid has been introduced.
  • a “transduced” cell (e.g., in a mammal, such as a cell or tissue or organ cell) is a genetic change in a cell following incorporation of an exogenous molecule, for example, a nucleic acid (e.g., a transgene).
  • a “transduced” cell(s) can be propagated and the introduced nucleic acid transcribed and/or protein expressed.
  • the nucleic acid in a “transduced” or “transfected” cell, may or may not be integrated into genomic nucleic acid. If an introduced nucleic acid becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in that cell or organism and further passed on to or inherited by progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may exist in the recipient cell or host organism extrachromosomally, or only transiently. A number of techniques are known, see, e.g., Graham et al. (1973) Virology, 52:456; Sambrook et al.
  • plasmid backbone This non-vector portion of the recombinant plasmid is referred to as the “plasmid backbone”, which is important for cloning and amplification of the plasmid, a process that is needed for propagation and recombinant virus production, but is not itself packaged or encapsulated into virus (e.g., AAV) particles.
  • a “vector” refers to the nucleic acid that is packaged or encapsulated by a virus particle (e.g., AAV).
  • the current invention is based, at least in part, on the finding that the transverse nuclear magnetic spin relaxation time T 2 and the transverse nuclear magnetic spin relaxation rate R 2 , respectively, of protons of water molecules in an aqueous solution comprising viral particles depends on the loading status (full vs. empty) of the viral particle.
  • one aspect of the current invention is a method for determining the ratio/fraction/concentration of loaded viral particles in a sample
  • the NMR parameter is indicative of a transverse nuclear magnetic spin relaxation of water in the solution or fraction thereof, specifically of protons in water in solution or fraction thereof.
  • the NMR parameter is the transverse nuclear magnetic spin relaxation time T 2 or the transverse nuclear magnetic spin relaxation rate R 2 of the protons of the water molecules in the aqueous solution.
  • the viral particle is a virus, a virus-like particle (VLP), or a liposome. In a further embodiment, the viral particle is a virus or a VLP. In another embodiment, the viral particle is a non-enveloped virus or a VLP of a non-enveloped virus. In one preferred embodiment, the viral particle is an adeno-associated virus particle (AAV particle).
  • AAV particle adeno-associated virus particle
  • the loaded viral particles are DNA-containing viral particles. In one preferred embodiment, the loaded viral particles are DNA-containing AAV particles.
  • the aqueous solution is a phosphate buffered saline solution.
  • the aqueous solution is a phosphate buffered saline solution comprising a detergent and optionally citrate, especially about 0.001% (w/v) detergent and optionally about 100 mM citrate.
  • the method is for determining the ratio/fraction/concentration of loaded viral particles in a sample, wherein the sample comprises up to 100% of DNA-containing viral particles, in one preferred embodiment up to 100% of DNA-containing AAV particles, and the calibration function is a linear function.
  • the method is for determining the ratio/fraction/concentration of loaded viral particles in a sample, wherein the sample comprises between 50% and 100% of DNA-containing viral particles, in one preferred embodiment between 50% and 100% of DNA-containing AAV particles, and the calibration function is a linear function.
  • the calibration function is a second order polynomial function.
  • the method is for determining the ratio/fraction/concentration of loaded viral particles in a sample, wherein the sample comprises up to 100% of DNA-containing viral particles, in one preferred embodiment up to 100% of DNA-containing AAV particles, and the calibration function is a second order polynomial function.
  • the method is for determining the ratio/fraction/concentration of loaded viral particles in a sample, wherein the sample comprises between 50% and 100% of DNA-containing viral particles, in one preferred embodiment between 50% and 100% of DNA-containing AAV particles, and the calibration function is a second order polynomial function.
  • the calibration function is based on the same NMR parameter determined for at least two samples with different ratios of DNA-containing and empty AAV particles.
  • the method according to the current invention is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to producing an aqueous solution comprising (a mixture of) DNA-containing and empty AAV particles, and/or the steps indicated elsewhere herein. Moreover, one or more of said steps may be performed by automated equipment.
  • the present invention also relates to a use of the method according to the present invention, for a purpose selected from the group consisting of producing an AAV particle, purifying an AAV particle, removing impurities from a preparation of an AAV particle, and release of recombinant AAV particle preparations for therapeutic application, especially real-time release.
  • the current invention is further based, at least in part, on the finding that the transverse nuclear magnetic spin relaxation time T 2 and the transverse nuclear magnetic spin relaxation rate R 2 , respectively, of protons of water molecules in an aqueous solution comprising AAV particles depends on the loading status (full vs. empty) of the AAV particle.
  • the current invention is based, at least in part, that the transverse nuclear magnetic spin relaxation time T 2 can be used for the determination of the loading status of AAV particles independent of the serotype of the AAV particle, thus is independent of the serotype of the AAV particle.
  • the fraction/ratio/percentage/concentration of DNA-containing AAV particles can be obtained by determining the transverse nuclear magnetic spin relaxation time T 2 in an aqueous solution comprising DNA-containing and empty AAV particles by water proton NMR ( 1 H 2 O NMR) and correlating said time with standard/calibration values. That is, it has been found that the water proton transverse spin relaxation time T 2 correlates with the fraction/ratio/percentage/concentration of DNA-containing AAV particles in an aqueous sample.
  • FIG. 2 shows the dependency of T 2 on the DNA-loading of AAV particles of the serotype 6 (AAV6), i.e. the dependency of T 2 on the concentration of DNA-containing AAV6 particles in an aqueous sample.
  • AAV6 serotype 6
  • FIG. 3 shows the dependency of T 2 on the DNA-loading of AAV particles of the serotype 8 (AAV8), i.e. the dependency of T 2 on the concentration of DNA-containing AAV8 particles in an aqueous sample. It can be seen that the absolute difference in the relaxation time T 2 between empty AAV8 particles (0% full) and completely DNA-containing AAV8 particles (100% full) at a concentration of 2*10 13 particles/mL is 206.9 ms and the relative difference is 14%.
  • the data points can be fitted with a second order polynomial function with an R 2 value of 0.9633.
  • one aspect of the current invention is a method for determining the ratio of DNA-containing AAV particles to empty AAV particles in a sample.
  • one aspect of the current invention is a method for determining the fraction of DNA-containing AAV particles with respect to all AAV particles in a sample.
  • one aspect of the current invention is a method for determining the concentration of DNA-containing AAV particles in a sample.
  • the method according to these three aspects of the invention comprises the following steps:
  • the NMR parameter is indicative of a transverse nuclear magnetic spin relaxation of water in the solution or aliquot thereof, specifically of protons in water in solution or aliquot thereof.
  • the NMR parameter is the transverse nuclear magnetic spin relaxation time T 2 or the transverse nuclear magnetic spin relaxation rate R 2 of the protons of the water molecules in the aqueous solution.
  • the NMR parameter is the water proton transverse relaxation time (T 2 ) of the protons of the water molecules in the aqueous solution.
  • the method according to the current invention is performed at conditions wherein the relative difference of the transverse nuclear magnetic spin relaxation time T 2 determined with 100% empty AAV particles and determined with 100% DNA-containing AAV particles is at least 5%. In one embodiment, the method is performed at conditions wherein the relative difference of the transverse nuclear magnetic spin relaxation time T 2 determined with 100% empty AAV particles and determined with 100% DNA-containing AAV particles is at least 8%. In one preferred embodiment, the method is performed at conditions wherein the relative difference of the transverse nuclear magnetic spin relaxation time T 2 determined with 100% empty AAV particles and determined with 100% DNA-containing AAV particles is at least 10%.
  • the conditions are a temperature, a total concentration of AAV particles in the aqueous solution, a magnetic field strength, a resonance frequency, a buffer condition, optionally a detergent concentration, and optionally a salt concentration.
  • the smaller of the two transverse nuclear magnetic spin relaxation times T 2 is set to 100% for the calculation of the relative difference. The calculation is done according to the following equation:
  • the magnetic field strength during the determination of the NMR parameter is of from 0.1 T to 24 T, in certain embodiments of from 0.2 T to 10 T, in further embodiments of from 0.3 T to 5 T, in further embodiments of from 0.4 T to 2 T. In one embodiment, the magnetic field strength is in the range of 0.45 T to 0.50 T. In one preferred embodiment, the magnetic field strength is about 0.47 T.
  • the resonance frequency during the determination of the NMR parameter is of from 5 MHz to 500 MHz, in further embodiments of from 7.5 MHz to 200 MHz, in further embodiments of from 10 MHz to 100 MHz, in further embodiments of from 15 MHz to 50 MHz. In one preferred embodiment, the resonance frequency is about 20 MHz.
  • the method is performed at about room temperature. In one embodiment, the method is performed at a temperature in the range from 18° C. to 25° C. In one embodiment, the method is performed at a temperature in the range from 19° C. to 22° C. In one preferred embodiment, the method is performed at a temperature of about 20° C.
  • the aqueous solution is a phosphate buffered saline solution.
  • the aqueous solution is a phosphate buffered saline solution comprising a detergent.
  • the detergent is selected from the groups of poloxamers and polysorbates.
  • the detergent is selected from the group of detergents consisting of poloxamer 188, poloxamer 407, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 and polysorbate 100.
  • the detergent is present at a concentration of about 0.001% (w/v).
  • the detergent is poloxamer 188.
  • the aqueous solution further comprises a citrate (salt of citric acid).
  • the citrate is present at a concentration of 100 mM.
  • the citrate is sodium citrate.
  • a phosphate buffered saline solution comprises about 137 mM sodium chloride, about 2.7 mM potassium chloride and about 12 mM phosphate as a mixture of hydrogen phosphate and dihydrogen phosphate, adjusted to a pH value of 7.4.
  • the AAV particle has a serotype selected from the group of AAV serotypes consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and mixtures as well as chimeras thereof.
  • the AAV particle has a serotype selected from the group of AAV serotypes consisting of AAV2, AAV4, AAV6, AAV8 and AAV9.
  • the AAV particle is of the AAV2 serotype, or the AAV6 serotype, or the AAV8 serotype.
  • the AAV particle is an AAV8 particle and the aqueous solution is a phosphate buffered saline solution additionally comprising about 0.001% (w/v) poloxamer 188.
  • the AAV particle is an AAV2 particle and the aqueous solution is a phosphate buffered saline solution additionally comprising about 0.001% (w/v) poloxamer 188 and about 100 mM sodium citrate.
  • the AAV particle is an AAV6 particle and the aqueous solution is a phosphate buffered saline solution additionally comprising about 0.001% (w/v) poloxamer 188 and about 100 mM sodium citrate.
  • the method comprises as first step the step of
  • the method comprises as second step the step of
  • the calibration function is based on the same NMR parameter determined for at least two samples with different known concentrations and/or ratios of DNA-containing and empty AAV particles.
  • the calibration function is a linear function.
  • the method is for determining the ratio/fraction/concentration of loaded viral particles in a sample, wherein the sample comprises up to 80% of DNA-containing viral particles, in one preferred embodiment up to 80% of DNA-containing AAV particles, and the calibration function is a linear function.
  • the calibration function is obtained by determining the same NMR parameter for two calibration samples, the first comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 10:90 to 30:70, in one preferred embodiment at about 25:75, and the second comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 70:30 to 90:10, in one preferred embodiment at about 75:25, and fitting to a linear function to provide a calibration function.
  • the calibration function is obtained by determining the same NMR parameter for two calibration samples, the first comprising only DNA-containing AAV particles or only empty AAV particles and the second being selected from the group of calibration samples consisting of samples comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 10:90 to 30:70, in one preferred embodiment at about 25:75, samples comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 40:60 to 60:40, in one preferred embodiment at about 50:50, and samples comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 70:30 to 90:10, in one preferred embodiment at about 75:25, and fitting to a linear function to provide a calibration function.
  • the calibration function is obtained by determining the same NMR parameter for at least three calibration samples, the first comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 0:100 to 30:70, in one preferred embodiment at about 0:100 or 25:75, the second comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 40:60 to 60:40, in one preferred embodiment at about 50:50, and the third comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 70:30 to 100:0, in one preferred embodiment at about 75:25 or 100:0, and fitting to a linear function to provide a calibration function.
  • the calibration function is obtained by determining the same NMR parameter for at least three calibration samples, the first comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 0:100 to 30:70, in one preferred embodiment at about 0:100 or 25:75, the second comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 40:60 to 60:40, in one preferred embodiment at about 50:50, and the third comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 70:30 to 100:0, in one preferred embodiment at about 75:25 or 100:0, and fitting the first and the second calibration sample to a first linear calibration function for the ratio from 0:100 up to and including the ratio 50:50 and fitting the second and the third calibration samples to a second linear calibration function for the ratio from and excluding 50:50 to 100:0.
  • the method is for determining the ratio/fraction/concentration of loaded viral particles in a sample, wherein the sample comprises between 75% and 100% of DNA-containing viral particles, in one preferred embodiment between 50% and 100% of DNA-containing AAV particles, and the calibration function is a linear function.
  • the calibration function is obtained by determining the same NMR parameter for two calibration samples, the first comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 40:60 to 60:40, in one preferred embodiment at about 50:50, and the second comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 70:30 to 90:10, in one preferred embodiment at about 75:25, and fitting to a linear function to provide a calibration function.
  • the calibration function is a second order polynomial function.
  • the method is for determining the ratio/fraction/concentration of loaded viral particles in a sample, wherein the sample comprises up to 100% of DNA-containing viral particles, in one preferred embodiment up to 100% of DNA-containing AAV particles and the calibration function is a second order polynomial function.
  • the calibration function is obtained by determining the same NMR parameter for at least three calibration samples, the first comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 0:100 to 30:70, in one preferred embodiment at about 0:100 or 25:75, the second comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 40:60 to 60:40, in one preferred embodiment at about 50:50, and the third comprising DNA-containing AAV particles and empty AAV particles at a ratio in the range of 70:30 to 100:00, in one preferred embodiment at about 75:25 or 100:0, and fitting to a linear function to provide a calibration function.
  • the calibration function is then used to obtain the respective ratio/fraction/concentration based on the determined NMR parameter for an unknown sample.
  • the total concentration of the viral particles in the aqueous solution is at least 1*10 12 viral particles per mL (vp/mL). In one embodiment, the total concentration of the viral particles in the aqueous solution is at least 5*10 12 vp/mL. In one preferred embodiment, the total concentration of the viral particles in the aqueous solution is at least 1*10 13 vp/mL. In one embodiment, the total concentration of the viral particles in the aqueous solution is at least 2*10 13 vg/mL.
  • the aqueous solution has a volume of at least 900 ⁇ L.
  • the method according to the current invention is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to producing an aqueous solution comprising (a mixture of) DNA-containing and empty AAV particles, and/or the steps indicated elsewhere herein. Moreover, one or more of said steps may be performed by automated equipment.
  • the present invention also relates to a use of the method according to the present invention, for a purpose selected from the group consisting of producing an AAV particle, purifying an AAV particle, removing impurities from a preparation of an AAV particle, and release of recombinant AAV particle preparations for therapeutic application, especially real-time release.
  • one aspect of the current invention is the use of the transverse nuclear magnetic spin relaxation time T 2 for the determination of the loading status of AAV particles.
  • the loading status is the ratio of DNA-containing AAV particles to empty AAV particles.
  • the loading status is the fraction of DNA-containing AAV particles in a sample.
  • the loading status is the concentration of DNA-containing AAV particles in a sample.
  • one preferred aspect of the current invention is a method for determining the concentration of DNA-containing AAV particles in a sample
  • one preferred aspect of the current invention is a method for determining the fraction of DNA-containing AAV particles in a sample
  • one preferred aspect of the current invention is a method for determining the ratio of DNA-containing AAV particles to empty AAV particles in a sample
  • FIG. 1 Dependency of T 2 on the DNA-loading of AAV particles of the serotype 2 (AAV2).
  • FIG. 2 Dependency of T 2 on the DNA-loading of AAV particles of the serotype 6 (AAV6).
  • FIG. 3 Dependency of T 2 on the DNA-loading of AAV particles of the serotype 8 (AAV8)
  • DNA-containing AAV particles as well as empty AAV particles of different serotypes were purchased from Virovek, Hayward, Calif., United States of America, at a concentration of 2*10 13 vg/mL.
  • concentration of vg/mL equals the concentration of vp/mL (virus particles per mL) as one genome is packaged in one particle.
  • the DNA of the DNA-containing AAV particles comprised an expression cassette for green fluorescent protein with a CMV promoter (CMV-GFP):
  • Transverse relaxation rates (R 2 ) or times (T 2 ) were recorded with a Bruker mini spec mq20 spectrometer (20 MHz; Bruker BioSpin GmbH, Rheinstetten, Germany).
  • the spectrometer was equipped with a 0.47 T magnet and a H2O-10-25AVGX4 probe. At least 4 acquisitions were measured for each sample with sample volumes of 900 ⁇ l at 20° C. To determine T 2 or R 2 , signal decay was followed for at least 5 sec.
  • the samples were generated by mixing full and empty AAV2, AAV6 and AAV8, respectively, having the same concentration (2*10 13 vg/ml) dissolved in the same aqueous solution comprising 1 ⁇ PBS buffer containing 0.001% (w/v) Pluronic F-68 (for AAV8), and containing 0.001% (w/v) Pluronic F-68 as well as 100 mM sodium citrate (for AAV2 and AAV6) at different ratios. Samples were measured without further longtime storage, e.g. ⁇ 0° C.
  • DNA-containing AAV particles and empty AAV particles were mixed in aqueous solution (AAV2 and AAV6: 1 ⁇ PBS buffer containing 0.001% (w/v) Pluronic F-68, 100 mM sodium citrate; AAV8 without sodium citrate) to result in different full/empty ratios spanning the range from 0% to 100% DNA-containing AAV particle.
  • AAV2 and AAV6 1 ⁇ PBS buffer containing 0.001% (w/v) Pluronic F-68, 100 mM sodium citrate; AAV8 without sodium citrate
  • T 2 transverse relaxation times
  • AAV linear fitting serotype function R 2 value 2 1.9088*x + 1543.7 0.9208 6 1.5298*x + 1615.1 0.9905 (1.3646*x + 1615.1) (0.8984) 8 ⁇ 1.8916*x + 1717.4 0.9026
  • AAV 2 nd order polynomial fitting serotype function R 2 value 2 0.0184*x 2 + 0.0642*x + 1566.7 0.996 6 0.0047*x 2 + 1.0479*x + 1620.9 0.9987 (0.0089*x 2 + 0.4749*x + 1626.2) (0.9318) 8 ⁇ 0.0166*x 2 ⁇ 0.2333*x + 1696.7 0.9633

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