Characterization of gene therapy vectors

Abstract

Disclosed is a method of differentiating empty capsids from full capsids or loaded and unloaded non-viral gene therapy vectors in a viral preparation. The method comprises the steps of: a) providing a preparation of viral particles or gene therapy vectors; b) subjecting the preparation to interferometric scattering mass cytometry (ISCAMS) in an interferometric scattering microscope to generate mass distribution data of the viral particles; c) determining from the mass distribution data the level of empty capsids and capsids containing genomes or the level of loaded and unloaded vectors in the viral particles.

Description

Technical Field

[0001] This invention relates to the characterization of gene therapy vectors and viral particles, and more specifically to a method for determining the levels of empty and intact capsids in formulations of viral particles. Background Technology

[0002] Recombinant viruses have shown great promise and practicality as mediators for delivering therapeutic nucleic acids for gene therapy applications. Many different recombinant viruses are used in these gene therapy applications based on a number of factors, including the size of the nucleic acid to be delivered, the target cells or tissues for delivery, the need for short-term or long-term expression of the therapeutic nucleic acid, and the integration of the therapeutic nucleic acid into the recipient genome. Examples of viruses used in gene therapy applications include adeno-associated virus (AAV), adenovirus, lentivirus, and herpes simplex virus (HSV). Additionally, non-viral vectors can also be used for nucleic acid delivery in gene therapy. Examples of such carriers include liposomes and other lipid complexes (see, for example, US2009305409, US6749863, US6071533 and US6303378, all of which are incorporated herein by reference), polymer vesicles and polyplexes (see, for example, US20110206751, US2017000743 and US2011151013, all of which are incorporated herein by reference), as well as inorganic nanoparticles and organic nanoparticles.

[0003] Polymerase chain reaction (PCR) is a commonly used technique for determining the genomic content of adeno-associated virus (AAV), and analytical ultracentrifugation (AUC) (see, for example, US2018180525, incorporated herein by reference in its entirety) is typically used to estimate the levels of empty capsids (containing only a protein capsid and no DNA) and intact capsids (containing both protein and DNA). Additionally, spectrophotometry (e.g., WO2019212922, incorporated herein by reference in its entirety) has been used to determine the levels of empty and intact capsids. However, these techniques require multiple steps and involve complex procedures that are time-consuming and have relatively low throughput. Therefore, more efficient and better quantitative techniques are desired for sample screening, in-process sample analysis for process control and monitoring, and for determining final product concentrations and monitoring stability.

[0004] Accordingly, there is a need for simple and rapid methods to characterize viral agents regarding empty and intact capsids. Similarly, there is a need for simple and rapid methods to characterize non-viral vectors regarding whether they are loaded with the expected nucleic acid. Summary of the Invention

[0005] One aspect of the present invention is to provide a method for distinguishing between empty capsids and intact capsids in a viral preparation. This is achieved by a method comprising the following steps:

[0006] a) Preparations that provide virus particles;

[0007] b) The formulation was subjected to interferometric scattering mass spectrometry (ISCAMS) in an interferometric scattering microscope to generate mass distribution data of viral particles;

[0008] c) Determine the levels of empty capsids and genome-containing capsids in viral particles using mass distribution data.

[0009] One advantage is that the method is rapid (within minutes), simple, and highly accurate. A further advantage is that it can measure particle size from samples containing complex mixtures of varying particle sizes; smaller particles are not "hidden" by the presence of larger particles. A further advantage is that it requires a minimal sample volume (<~20 µl). These advantages make this method a particularly useful technique for in-line process analysis testing (PAT).

[0010] A second aspect of the invention is to provide a method for distinguishing between non-viral gene therapy vectors loaded with nucleic acids and vectors without nucleic acids. This is achieved by a method comprising the following steps:

[0011] a) Providing formulations with non-viral gene therapy vectors;

[0012] b) The formulation is subjected to interferometric scattering mass spectrometry (ISCAMS) in an interference scattering microscope to generate mass distribution data of the carrier;

[0013] c) Determine the levels of vectors loaded with nucleic acids and vectors without nucleic acids using mass distribution data. Attached Figure Description

[0014] Figure 1 The mass distribution of empty capsid samples of AAV serotype 5 is shown.

[0015] Figure 2 The mass distribution of intact capsid samples of AAV serotype 5 is shown.

[0016] Figure 3 The mass distribution of a 1:1 mixture of empty and intact capsids of AAV serotype 5 is shown.

[0017] Figure 4 The mass distribution of a 2:1 mixture of empty and intact capsids of AAV serotype 5 is shown.

[0018] Figure 5 shows the mass distribution of intact AAV serotype 5 capsids in the following:

[0019] a) PBS buffer + 130 mM NaCl, 0.001% (v / v) Pluronic F-188

[0020] b) PBS buffer + 130 mM NaCl, 0.001% (v / v) Pluronic F-188 (repeat)

[0021] c) Salt-free 20 mM TRIS buffer, pH 8.5

[0022] d) Salt-free 20 mM TRIS buffer, pH 8.5 (repeat).

[0023] Figure 6 The mass distribution of intact AAV serotype 5 capsids in a formulation diluted 1000x is shown.

[0024] definition

[0025] As used herein, the terms “comprises,” “comprising,” “containing,” “having,” etc., may have the meanings given to them under U.S. patent law and may mean “includes,” “including,” etc.; “consisting essentially of” or “consists essentially” also have the meanings given to them under U.S. patent law, and the term is open-ended, allowing for more content than described, as long as the basic or novel characteristics of the described content are not altered by the presence of more content than described, but excluding prior art embodiments. Detailed Implementation

[0026] In one aspect, the present invention discloses a method for characterizing a formulation of virus particles, comprising the following steps:

[0027] a) A formulation providing viral particles. The formulation may be, for example, a formulation of viral particles intended for use as a viral vector in cell / gene therapy, but they may also be intended for use as, for example, a vaccine antigen. In particular, they may be selected from adeno-associated virus (AAV), adenovirus (AV), herpes simplex virus (HSV), retrovirus, lentivirus, and alphavirus, with AAV being preferred. The viral particles may suitably be recombinant, having a modified genome in the capsid, and the formulation may also include an empty capsid. The formulation may contain at least 5 x 102 8 One virus particle / ml of formulation, for example at least 1x10 91 virus particle / ml preparation, 5x10 8 -1x10 13 Or 1x10 9 - 1x10 12 The formulation contains one virus particle per ml. The formulation may suitably contain a buffer, such as PBS or Tris buffer, having a pH of 3-9 (e.g., 6-8.5 or 7-8.5), and may further contain a salt, such as NaCl. Further, the formulation may contain a dispersant, such as poloxamer (ethylene oxide-propylene oxide block copolymer). Examples of poloxamer dispersants may be poloxamer 188 (catalog number 9003-11-6), such as Pluronic F-188 (BASF) or Pluronic F-68. The formulation may typically be a cell culture fluid (e.g., cell lysate), partially purified cell culture fluid / cell lysate, or a purified suspension of virus particles derived from cell culture fluid / cell lysate.

[0028] b) In an interference scattering microscope, the formulation is subjected to interference scattering mass spectrometry (ISCAMS) to generate mass distribution data of viral particles. In this step, the intensity of scattered light from individual viral particles on the surface can be measured, and the mass distribution data is calculated from the distribution of scattered light intensity. An interference scattering microscope can be constructed as disclosed in US20190004299 (incorporated herein by reference in its entirety), and it can be commercially available, in particular, from Refeyn Ltd., Oxford, UK, as the Refeyn OneMP microscope. In this instrument, a sample of the formulation is placed on a slide, which may be functionalized to bind or exclude certain biomolecules, and the slide is positioned above a lens of the microscope. The instrument uses a laser focused on the surface of the slide and observes the point diffusion function of molecules / particles as they approach the surface. The device can distinguish particles as they bind to and detach from the surface, and as they “wobble” across the surface. To clearly distinguish these particles, the software separates consecutive frames of a movie to provide “scaled imaging.” Microscopy detects the interference between reflected and scattered light from individual molecules / particles. The intensity, or contrast, of this interference point is then correlated with the density of the molecules. The density of proteins remains constant over a fairly large range, as does the density of DNA, and it is proportional to the particle mass. Therefore, with the aid of a calibration reference standard, the contrast of the spots can be used to calculate the particle mass. The surface holding the virus particles can suitably be a glass surface, such as a slide or coverslip. It can be an unmodified glass surface, or it can be chemically modified, for example, with functional silanes. Chemical modification can also involve derivatization with ligands capable of binding to virus particles, such as llamas antibody ligands (like those used in AVB Sepharose High Performance, GE Healthcare) that have been shown to bind to serotypes 1, 2, 3, 4, and 5 of AAV. Regarding the construction of the microscope, it typically includes:

[0029] A sample holder used to hold the sample in place;

[0030] Arranged as a lighting source to provide illumination;

[0031] Detector;

[0032] An optical system arranged to direct illumination light to a sample location, and further arranged to collect reflected output light, the output light comprising both light scattered from the sample location and illumination light reflected from the sample location, and further arranged to direct the output light to a detector; and

[0033] A spatial filter is placed to filter the output light, the spatial filter being arranged to allow the output light to pass through, but with a greater intensity reduction within a predetermined numerical aperture than at a larger numerical aperture. The sample holder may suitably include a surface (as discussed above) and viral particles interacting with that surface.

[0034] c) The levels of empty capsids and genome-containing capsids (intact capsids) within viral particles are determined using mass distribution data. Empty and intact capsids appear as distinct peaks in the mass distribution, and their relative numbers can be obtained by integrating the peaks or adding counts to each peak when presented as a histogram. Viral particles are much larger than virtually all particles within typical cell lysates, allowing for clear differentiation of viral particle peaks from other cellular protein / DNA contaminants.

[0035] In some embodiments, this method is performed for online testing in the process of manufacturing viral vectors. It can be suitably used in process analysis techniques (PAT), for example, where at least one process parameter is adjusted when a predetermined empty capsid / intact capsid ratio is reached. In this case, the sample may be complex, i.e., containing many other biomolecules. In particular, the method can be applied in conjunction with a chromatography step, for example, where the method is performed on the eluent or flow-through from the chromatography step. The process parameter can then be, for example, the amount of viral vector formulation loaded onto the chromatography column or apparatus. If the empty capsid / intact capsid ratio is too high in a chromatography cycle, the loading amount can be reduced in subsequent cycles. To increase selectivity for viral vectors (e.g., AAV vectors), using a surface derived from a ligand that selectively binds to the vector can be advantageous. Using a diluted sample to reduce the total biomolecule concentration can also be advantageous.

[0036] In a second aspect, the present invention discloses a method for distinguishing between non-viral gene therapy vectors loaded with nucleic acids and vectors not loaded with nucleic acids, comprising the following steps:

[0037] a) Formulations providing nonviral gene therapy vectors. The vectors may be, for example, liposomes or other lipid complexes, polymer vesicles and polymeric complexes, as well as inorganic and organic nanoparticles. The vectors may suitably have a diameter less than 1000 nm (e.g., less than 500 nm, less than 200 nm, or less than 100 nm). They may, for example, have a volume-weighted average diameter of 10–1000 nm (e.g., 10–500 nm, 10–200 nm, 10–100 nm, 20–500 nm, 20–500 nm, or 20–100 nm).

[0038] b) In the interference scattering microscope as discussed above, the formulation is subjected to interference scattering mass spectrometry (ISCAMS) to generate mass distribution data of the carrier. As discussed above, the intensity of scattered light from each carrier present on the surface can be measured, and the mass distribution data can be calculated from the intensity distribution of the scattered light. The carrier can be present on a glass surface, such as a glass slide or coverslip. This glass surface can be chemically modified, specifically with ligands capable of binding the carrier, or it can remain unmodified. When the carrier has a diameter of 20 nm or more (e.g., 50 nm or more or 100 nm or more), the interference scattering microscope can be equipped with a high-wavelength laser, such as a laser emitting light above 600 nm (e.g., 700 nm or more, 600–1000 nm, 600–800 nm, or 700–800 nm).

[0039] c) Determine the levels of vectors loaded with nucleic acids and vectors without nucleic acids using mass distribution data. Example

[0040] Experiments were conducted using a Refeyn OneMP microscope, with the samples placed on a non-functionalized slide above the microscope lens. ISCAMS analysis was performed according to the microscope's instructions, and the data were plotted as a histogram of particle counts versus particle mass. For each identified peak, the mean mass, standard deviation, and total peak count were calculated.

[0041] We tested purified AAV-5 samples using unfunctionalized glass slides. In our setup, the absolute count of AAV titers could not be determined, which would require surface functionalization so that AAV particles are counted only once during the sampling time. This is possible using existing or novel affinity ligands for AAV, but this was not explicitly tested here.

[0042] The detection method enables mass spectrometry to measure the mass of the AAV capsid and thus distinguish between empty and intact AAV viral particles. This is achieved without labeling, and because AAV is much larger than almost all particles in typical cell lysates, it can be clearly distinguished from other cellular protein / DNA contaminants. We found that AAV particles generally do not bind to the glass surface, so they can be counted multiple times. This is advantageous because long film collection times can be used to ensure a large number of particle counts. However, while this method provides a proportional comparison of empty particles to intact particles, it does not provide an absolute count of AAV titers. To achieve this, it would be necessary for AAV particles to bind to the imaging surface and remain bound, which is certainly possible if the surface is functionalized with affinity ligands or similar ligands to enable it to capture AAV.

[0043] Example 1

[0044] Two purification formulations of AAV serotype 5 virus particles were used: one with only an empty capsid and the other with only an intact capsid. In both cases, the virus particles were suspended in PBS buffer (pH 7.4) containing 130 mM NaCl and 0.001% (v / v) Pluronic F-188. Virus particles were purchased from Vigene Biosciences (Maryland, USA). AAV was purified and separated into empty and intact particles by density ultracentrifugation, and then percolated into buffer (PBS containing Pluronic F-188 + 130 mM NaCl). The initial concentration of intact AAV-5 particles was 2.95 x 10⁻⁶. 13 Capsule volume / ml, while empty capsule volume is 1.61 x 10⁻⁶ ml. 13 Capsule / ml. Then, in PBS + 130mM NaCl buffer, dilute both the empty and intact capsules to ~1x10⁻¹. 12 Capsule / ml.

[0045] like Figure 1 and 2 As shown, the empty capsid forms a peak at 4,000 kDa, while the complete capsid forms a peak at 5,000 kDa. The peak parameters are shown in Table 1.

[0046] Table 1. Data for empty and complete AAV-5 mantles

[0047] sample Average mass, kDa Standard deviation, kDa count AAV-5 empty shell 4024 309 938 AAV-5 complete caption 5064 428 804

[0048] Example 2

[0049] Mix the same AAV-5 formulation as in Example 1 to give i) a 1:1 empty capsid / intact capsid ratio and ii) a 2:1 ratio. Figure 3 and Figure 4 The results shown indicate that, assuming a normal distribution, the two peaks can be distinguished using curve fitting techniques. The distinguished peak data are shown in Table 2.

[0050] Table 2. Data on mixtures of empty AAV-5 capsids and intact AAV-5 capsids

[0051]

[0052] Example 3

[0053] Two AAV-5 formulations were used: one in PBS, 130 mM NaCl, 0.001% Pluronic F-188 buffer, and the other in Tris buffer (pH 8.5) without added salt. Two replicates were analyzed for each formulation. The results are shown in Figures 5a)–d) and Table 3, confirming that the mass distribution data were reproducible and that no differences were observed between the two buffers.

[0054] Table 3. Data on intact AAV-5 capsids in different buffer solutions

[0055] sample Average mass, kDa Standard deviation, kDa count PBS, 130 mM NaCl, Pluronic 5077 422 929 5064 428 804 Tris pH 8.5 5021 463 3559 5003 454 1294

[0056] Example 4

[0057] The same AAV-5 formulation as in Example 1 was diluted 1000 times with buffer (i.e., to ~1x10⁻⁶). 9 The final concentration of the capsid (capsid / mL) was measured and analyzed under a microscope. Figure 6 As shown in Table 4, although the sampling time is longer, the number of counts is smaller. However, the peak mean and width data remain consistent.

[0058] Table 4. Data on the complete AAV-5 capsid diluted 1000-fold in PBS buffer.

[0059] Average mass, kDa Standard deviation, kDa count 5067 596 191

[0060] This written specification uses examples to disclose the invention, including the best mode, and also enables any person skilled in the art to practice the invention, including preparing and using any apparatus or system and performing any incorporated method. The patentable scope of the invention is defined by the claims, but may include other examples that would occur to a person skilled in the art. Such other examples are contemplated within the scope of the claims if they have structural elements that are not different from the wording of the claims, or if they include equivalent structural elements that are not substantially different from the wording of the claims. All patents and patent applications mentioned herein are incorporated herein by reference in their entirety as if individually incorporated.

Claims

1. A method for characterizing a formulation of virus particles, comprising the following steps: a) Preparations that provide virus particles; b) The formulation is subjected to interference scattering mass spectrometry in an interference scattering microscope to generate mass distribution data of the virus particles; c) Determine the levels of empty capsids and genome-containing capsids in the viral particles from the mass distribution data, wherein the viral particles are adeno-associated virus particles.

2. The method of claim 1, wherein in step b), the intensity of scattered light from each virus particle present on the surface is measured, and the mass distribution data is calculated from the scattered light intensity distribution.

3. The method of claim 1, wherein in step b), the virus particles are present on the glass surface.

4. The method of claim 3, wherein the glass surface is the surface of a glass slide or a cover glass.

5. The method of claim 3, wherein the glass surface is chemically modified.

6. The method of any one of claims 3-5, wherein the glass surface is derivatized with a ligand capable of binding the virus particles.

7. The method of any one of claims 1-5, wherein the formulation comprises at least 5 x 10 8 viral particles per ml of formulation.

8. The method of any one of claims 1-5, wherein the formulation comprises a buffer solution.

9. The method of claim 8, wherein the buffer is PBS or Tris buffer.

10. The method of any one of claims 1-5, wherein the formulation comprises a salt.

11. The method of claim 10, wherein the formulation comprises NaCl.

12. The method of any one of claims 1-5, wherein the formulation comprises a dispersant.

13. The method of claim 12, wherein the dispersant is poloxamer.

14. The method of claim 13, wherein the dispersant is poloxamer 188.

15. The method of any one of claims 1-5, wherein the virus particle is a viral vector particle.

16. The method of any one of claims 1-5, wherein the interference scattering microscope comprises: A sample holder used to hold the sample in place; Arranged as a lighting source to provide illumination; Detector; An optical system is arranged to direct illumination light to the sample location and further arranged to collect reflected output light, the output light comprising both light scattered from the sample location and illumination light reflected from the sample location, and further arranged to direct the output light to the detector. and A spatial filter is placed to filter the output light, the spatial filter being arranged to allow the output light to pass through, but having a greater intensity reduction within a predetermined numerical aperture than at a larger numerical aperture.

17. The method of claim 16, wherein the sample holder includes a surface and virus particles are present on the surface.

18. The method of claim 17, wherein the surface is a glass surface.

19. The method of claim 18, wherein the glass surface is the surface of a glass slide or a cover glass.

20. The method of any one of claims 17-19, wherein the surface is the surface of a microfluidic channel or a porous plate.

21. The method of any one of claims 1-5, wherein the interference scattering microscope is a Refeyn OneMP microscope.

22. The method of any one of claims 1-5, performed as an online test in a process for manufacturing or using a viral vector.

23. The method of claim 22, wherein at least one process parameter is adjusted when a predetermined ratio of empty shell to complete shell is reached.

24. The method of claim 22, wherein the method is performed on the eluent or flow product from the chromatography step.

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