Column comprising particles

Abstract

Disclosed is a column comprising particles having an average particle size ranging from about 1 [mu] m to about 5 [mu] m; the particles have an average pore size ranging from about 450 to about 3000; and the particles have an average pore volume ranging from about 0.1 cm < 3 > / g to about 5 cm < 3 > / g. The column may be a size exclusion chromatography column. The column can be used in a method for separating a monomer from a viral analyte, and the method is also disclosed.

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Application No. 18 / 362,691, filed July 31, 2023, the entire disclosure of which is incorporated by reference herein in its entirety. TECHNICAL FIELD

[0002] The present disclosure relates generally to a column comprising particles having an average particle size ranging from about 1 pm to about 5 pm; an average pore size of the particles ranging from about 450 A to about 3000 A; and an average pore volume of the particles ranging from about 0.1 cm 3 / g to about 5 cm 3 / g. The column can be a size exclusion chromatography column. The column can be used in a method of separating monomers from a viral analyte. BACKGROUND

[0003] Size exclusion chromatography (SEC) columns are a powerful analytical tool that separates molecules based on their hydrodynamic size and are commonly used for purification and characterization of biomolecules. However, there is no one size fits all SEC column for separating a variety of biological analytes. Resolution will depend on several factors including the size range of the analytes being analyzed and the pore structure of the packing material. Inert surface chemistry is also important for preventing non-specific interactions with the analytes.

[0004] What is needed is a size exclusion chromatography column that can achieve high throughput and high resolution for viral analyte (such as adeno-associated virus (AAV)) aggregate analysis. BRIEF DESCRIPTION OF DRAWINGS

[0005] The features of the present disclosure are illustrated by way of example and not limited to one or more of the following figures in which like numbers refer to like elements, wherein:

[0006] Figure 1A is a chromatogram illustrating the effect of a comparative pore size and the inventive pore size on separation of monomer and dimer from a viral analyte;

[0007] Figure 1B is a chromatogram illustrating the effect of the inventive pore size on separation of monomer and dimer from a viral analyte;

[0008] Figure 1C is a calibration curve using a viral analyte from Figure 1B and particles having a pore size of 3000 A;

[0009] Figure 1Dis a chromatogram of a column of particles having different pore sizes showing separation of monomer and dimer from a viral analyte;

[0010] Figure 1E is a chromatogram of a column of particles having an average pore size of about 1000 A showing separation of monomer and dimer from a 60 nm viral analyte;

[0011] Figure 1F is a chromatogram of a column of particles having different average pore sizes showing separation of monomer and dimer from a 60 nm viral analyte;

[0012] Figure 2 is a calibration curve and related chromatogram illustrating the effect of particle pore volume on separation of monomer and dimer from a viral analyte;

[0013] Figure 3 is a chromatogram illustrating separation of a 25 nm viral analyte using a column comprising particles having an average particle size of 500 A and different flow rates;

[0014] Figure 4 is a chromatogram illustrating separation of a 60 nm viral analyte using a column comprising particles having an average particle size of 500 A and different flow rates; and

[0015] Figure 5 is a chromatogram illustrating separation of a 25 nm viral analyte using a column comprising particles having an average particle size of 750 A and different flow rates.

[0016] Figure 6 is a schematic according to an embodiment of the present disclosure illustrating a cross-sectional view of a column comprising particles.

[0017] Figure 7 is a schematic according to an embodiment of the present disclosure illustrating a longitudinal view of a column, including a cross-sectional view showing a profile of the particles. SUMMARY

[0018] In one aspect, a column is disclosed, the column comprising particles having an average particle size ranging from about 1 μm to about 5 μm; the particles having an average pore size ranging from about 450 A to about 3000 A; and the particles having an average pore volume ranging from about 0.1 cm 3 / g to about 5 cm 3 / g.

[0019] In another aspect, a method of using the disclosed column is disclosed, the method comprising injecting a viral analyte into a column comprising particles having an average particle size ranging from about 1 pm to about 5 pm; the particles having an average pore size ranging from about 450 A to about 3000 A; and the particles having an average pore volume ranging from about 0.1 cm 3 / g to about 5 cm 3 / g; and adjusting the flow rate.

[0020] Additional features and advantages of the various embodiments will be set forth in part in the description that follows, and in part will be apparent from the description, or can be learned by practice of various embodiments. The objectives and other advantages of the various embodiments will be realized and attained by means of the elements and combinations particularly pointed out in the description that follows. DETAILED DESCRIPTION

[0021] For purposes of simplicity and illustration, the present disclosure is described by reference to examples of the present disclosure. In the following description numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. It will be apparent, however, to one skilled in the art that the present disclosure can be practiced without limitation to these specific details. In other instances, some methods and structures have not been described in detail in order not to unnecessarily obscure aspects of the present disclosure.

[0022] Additionally, the elements depicted in the figures can include additional components and some of the components described in these figures can be removed and / or modified and replaced by other components without departing from the scope of the present disclosure. Furthermore, the elements depicted in the figures can not be drawn to scale and, as such, the dimensions and / or configurations of the elements can be different than those shown in the figures.

[0023] Among its broad and varied embodiments, disclosed herein is a column, such as a size exclusion chromatography column, that can provide high throughput and high resolution for aggregate analysis of viral analytes, such as adeno-associated virus (AAV). End users can more efficiently obtain information about the quality and purity of viral analytes.

[0024] The present disclosure describes a column 100 (100) Figure 6 and Figure 7The column comprises particles 102, such as a plurality of particles, wherein the average particle size ranges from about 1 μm to about 5 μm; the average pore size ranges from about 450 Å to about 3000 Å; and the average pore volume ranges from about 1.0 to about 1.4. The column, such as a size exclusion column, can provide a resolution of 2.4 between AAV monomers and dimers over a short time period (e.g., about 5 minutes). The particles may be a plurality of particles. For ease of understanding, the use of "particles" will refer to a plurality of particles, and the description of particles will refer to the average of the plurality of particles. For example, particle size will be the average particle size of the plurality of particles that may be present in the column.

[0025] The particles used in the column can have any desired shape, which will generally depend on the target application. For chromatographic applications, suitable shapes include, but are not limited to, spheres, rings, polyhedra, saddles, platelets, fibers, hollow tubes, rods, and cylinders, as well as any mixture of two or more such shapes. In one respect, the particles can be substantially spherical. Spherical cores can be easily packed and are therefore desirable for certain applications such as chromatography.

[0026] The composition of the particles is not critical. Suitable materials include, but are not limited to, glass, sand, metals, metal oxides, metalloids, ceramics, and combinations thereof. In one aspect, the particles may include metal oxides, such as refractory metal oxides. In another aspect, the particles may be porous metal oxide particles. Exemplary metal oxides include, but are not limited to, silica, alumina, titanium dioxide, zirconium oxide, iron oxide, antimony oxide, zinc oxide, and tin oxide. In yet another aspect, the particles may include silica, alumina, titanium dioxide, zirconium oxide, or combinations thereof. In yet another aspect, the particles may include silica. In one aspect, the metal oxide particles may include surface hydroxyl groups, which may be modified with a surface modifier.

[0027] The average particle size is from about 0.1 μm to about 100 μm, including but not limited to particles with average particle sizes of about 0.5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, and 90 μm. Specifically, the average particle size (e.g., silica) can be from about 1 μm to about 5 μm, including but not limited to about 1, 1.2, 1.5, 1.8, 1.9, 2, 2.2, 2.5, 2.7, 3, 3.2, 3.3, 3.5, 3.6, 3.7, 3.8, 4, 4.2, 4.4, 4.6, 4.8, and 5 μm. The particle size can be determined using methods known in the art, such as by using a Coulter counter, which can also count the particles and thus provide a particle size distribution.

[0028] The particle size distribution of the particles can vary depending on the composition of the particles and the method of preparation and / or processing of the particles. In one aspect, the particle size distribution of the particles can be less than about 20% of the average particle size, including, for example, less than about 15%, less than about 10%, or less than about 5% of the average particle size. In further aspects, the particle size distribution of the particles is about 0.5% to about 10% of the average particle size, including, but not limited to, about 0.5% to about 8%, 0.5% to about 6%, and about 0.5% to about 5% of the median particle size.

[0029] The average pore size of the particles can affect the separation of the viral analytes. Viral analytes are typically large biomolecules, and their size varies depending on the type of virus. Thus, to effectively separate the viral analytes, it is desirable to select an appropriate average pore size for the particles used in the column. In particular, if the average pore size is too small, e.g., less than about 475 A, the viral analytes can not be able to enter the pores present on the particles, resulting in poor separation of monomers and dimers / aggregates. On the other hand, if the average pore size is too large, e.g., greater than about 3000 A, the separation can not be optimal because all of the analytes, e.g., monomers and dimers / aggregates, can diffuse through the column at the same rate.

[0030] The particles can have substantially ordered pores with an average pore size ranging from about 450 A to about 3000 A, including, for example, an average pore size of about 475, 500, 600, 700, 800, 1000, 1200, 1250, or 3000 A. In one aspect, the particles can have substantially ordered pores with an average pore size ranging from about 475 A to about 2000 A, including, for example, about 500 A to about 1250 A. The average pore size of the particles is about 500 A. The average pore size of the particles is about 1000 A.

[0031] The pore volume of the particles plays a role in the resolution scheme of the separation of the viral analytes. A larger pore volume can result in a wider separation window, which can help achieve better resolution of viral analytes of various sizes. The pore volume can be important when dealing with multiple viral analyte aggregates or when separating particles of similar sizes. The average pore volume of the particles can be about 0.1 cm 3 / g to about 5 cm 3 / g, such as about 0.1 cm 3 / g to about 3 cm 3 / g, and as a further example, about 0.75 cm 3 / g to about 2 cm 3 / g, and as a further example, about 1 cm 3 / g to about 1.5 cm 3 / g. In one aspect, the average pore volume of the particles can be about 1.1 cm3 from about 0.1 cm3 / g to about 1.4 cm3 / g 3 / g.

[0032] The average pore size and average pore volume of the particles can vary depending on the viral analyte. In one aspect, the average pore size of the particles ranges from about 450 A to about 3000 A; and the average pore volume of the particles ranges from about 0.1 cm3 / g to about 5 cm3 / g. 3 from about 0.1 cm3 / g to about 1.4 cm3 / g 3 / g. In another aspect, the average pore size of the particles ranges from about 475 A to about 2000 A; and the average pore volume ranges from about 0.1 cm3 / g to about 3 cm3 / g. 3 from about 0.1 cm3 / g to about 1.4 cm3 / g 3 / g. In a further aspect, the average pore size of the particles ranges from about 500 A to about 1250 A; and the average pore volume ranges from about 1 cm3 / g to about 1.5 cm3 / g. 3 from about 0.1 cm3 / g to about 1.4 cm3 / g 3 / g. It should be understood that any range or specific point of average pore size disclosed herein can be combined with any range or specific point of average pore volume disclosed herein. However, some combinations enable better resolution of monomers and aggregates of viral analytes, as discussed further herein.

[0033] High throughput can be useful when processing large numbers of samples or when time-sensitive results are needed when using a chromatography column to separate viral vectors. High throughput can enable an end user to analyze more samples within a given timeframe, which can enhance overall productivity. It can reduce the time and cost involved in sample analysis and purification, making the process of using a chromatography column more efficient and cost-effective. High throughput chromatography columns can minimize the risk of sample degradation or contamination during extended analysis or purification processes, which can improve the accuracy and reproducibility of results.

[0034] Also disclosed herein is a method of using the disclosed column, the method comprising injecting a viral analyte into a column comprising particles having an average particle size ranging from about 1 pm to about 5 pm; the particles having an average pore size ranging from about 450 A to about 3000 A; and the particles having an average pore volume ranging from about 0.1 cm3 / g to about 5 cm3 / g; and adjusting the flow rate such that monomers are separated. 3 from about 0.1 cm3 / g to about 1.4 cm3 / g 3 / g; and adjusting the flow rate such that monomers are separated.

[0035] The average diameter of the viral analyte can be from about 10 nm to about 55 nm, and for example, from about 15 nm to about 40 nm, and as another example, from about 20 nm to about 30 nm. In another aspect, the average diameter of the viral analyte can be from about 55 nm to about 1000 nm, for example, from about 60 nm to about 100 nm.

[0036] Examples

[0037] Experiments were performed using monodisperse 3 pm SEC silica particles of three different pore sizes. The three different pore sizes were 130 A, 300 A, and 500 A. Each of these particles was packed into a 4.6 x 300 mm column for SEC-FLD (fluorescence detector) evaluation. Additional experiments were performed using particles with a pore size of 500 A but with two different pore volumes (e.g., 0.75 and 1.10). Test conditions included a column compartment temperature of 25 °C, 50 mM sodium phosphate pH 7.2 and 400 mM NaCl as the mobile phase, a flow rate of 0.35 ml / min, FLD: Ex 280 nm; Em 348 nm. The test sample was a viral analyte, such as an adeno-associated virus (AAV), for which the monomer has a size range of about 20 to 25 nm in diameter. In some aspects, the viral analyte has a size range of 50 to 75 nm, such as for which the monomer has a diameter of 60 nm.

[0038] Example 1A - Pore Size Effect

[0039] As shown in Figure 1A , AAV monomer and dimer were unable to diffuse through the SEC column, which included particles with an average particle size of about 2.7 pm and an average pore size of 130 A or 300 A, resulting in their being rejected and co-eluting. However, when entering a particle pore size of 500 A, the AAV monomer and dimer were able to diffuse in and out, resulting in a clear separation.

[0040] Example IB - AAV monomer and dimer were also able to diffuse in and out of columns with a pore size greater than 500 A (including 750 A and 1000 A), as shown in Figure 1B . The size of the viral analyte varied from 11 nm to 165 nm, but viral analytes with an internal diameter of about 23 nm to about 50 nm were separated, as shown in Figure 1B , and the retention time ranged from about 3 min to about 6 min, as shown in Figure 1C .

[0041] Example 1C - Viral analytes through the inventive column (row A) and comparative columns (rows B, C, and D). Row A used a column with particles having an average pore size of about 500 A. Row B was a column with particles having a particle size of 2.5 pm and a pore size of 450 A. Row C was a column with particles having a particle size of 5 pm and a pore size of 500 A. Row D was a column with particles having a particle size of 5 pm and a pore size of 1000 A. The resolution between monomer and dimer viral analytes is shown in Figure 1D .

[0042] Example 1D - Separation of a viral analyte having a diameter of about 60 nm using a column of the present application with particles having a pore size of 1000 A. As shown in Figure 1E monomers, dimers, and higher order aggregates are well resolved. The same viral analyte was separated by columns having particles of various average pore sizes as shown in Figure 1F Row A is a column of particles having an average pore size of about 750 A, Row B is a column of particles having an average pore size of about 1000 A, Row C is a column of particles having an average pore size of about 1000 A, Row D is a column of particles having an average pore size of about 1250 A, and Row E is a column of particles having an average pore size of about 1500 A. Rows B and C establish that the same column can be used to produce nearly identical results, establishing reproducibility. The monomers and dimers of Row E are nearly co-eluted, likely because the average pore size is too large relative to the size of the diameter of the viral analyte.

[0043] Example 2 - Pore Volume Effect

[0044] Figure 2 It is shown that increasing the pore volume from 0.75 to 1.10 results in a wider separation window. This enables better baseline separation and resolution between AAV dimers and higher order aggregates. In particular, aggregate analytes having an estimated diameter size of about 30-60 nm diffuse through a column having particles with an average pore volume of 0.75 and tend to co-elute (as shown in the middle graph of Figure 2 ). Analytes having the same diameter size that diffuse through a column having particles with an average pore volume of 1.10 exhibit a clear separation between higher order aggregates, dimers, and monomers in the analyte (as shown in the bottom graph of Figure 2 ). The higher pore volume (1.10) results in a flatter linear curve, resulting in a wider separation window and thus better separation of aggregates, dimers, and monomers of the viral analyte.

[0045] Example 3 - Flow Rate / High Throughput Test

[0046] The viral analyte was injected into a column comprising particles having an average pore size of about 500 A. The flow rate of the column was varied (0.1 ml / min, 0.2 ml / min, 0.35 ml / min, 0.5 ml / min, and 0.7 ml / min). The total peak area was determined as a function of flow rate, where 1 / F and total peak area showed good linearity. This indicates that the viral analyte does not break down as it passes through the column, or that high flow rates do not cause aggregation or fragmentation of the viral analyte aggregates.

[0047] Figure 3is a chromatogram showing elution of a viral analyte with a diameter of 25 nm in a particle column with an average pore size of about 500 A. The data shown in Table 1 establishes that the biological analyte remains stable even at higher flow rates. A resolution of 2.40 between AAV monomer and dimer is achieved in 5 minutes. Table 1.

[0048] Figure 4 is a chromatogram showing elution of a viral analyte with a diameter of 60 nm in a particle column with an average pore size of about 500 A. The data shown in Table 2 establishes that the monomer and dimer / aggregates remain stable even at higher flow rates. Table 2.

[0049] Comparison - 25 nm viral analyte was injected into a column made from prior art that included particles with an average pore size of about 750 A (Comparison). The flow rate of the column was varied (0.1 ml / min, 0.2 ml / min, 0.35 ml / min, 0.5 ml / min, and 0.7 ml / min). The chromatogram shows that at Figure 5 .

[0050] According to Table 3, the prior art column did not achieve a resolution higher than 2.0 at any of the flow rates studied. The particle structure in the column was not able to separate the aggregates from the monomer at flow rates of 0.5 ml / min and higher. Table 3.

[0051] From the foregoing description, one skilled in the art can understand that the present teachings can be implemented in various forms. Therefore, although the present teachings have been described in connection with particular embodiments and examples thereof, it should be understood that the true scope of the present teachings is not limited to such. Various changes and modifications can be made without departing from the scope of the teachings herein.

[0052] The scope of the present disclosure should be construed broadly. The present disclosure is intended to encompass equivalents, modifications, and other alternatives falling within the scope of the appended claims. For each of the devices, articles, methods, means, mechanisms, and elements disclosed herein, additional equivalent means, mechanisms, articles, and elements falling within the scope of the present disclosure are intended to be embraced. Furthermore, the present disclosure is intended to encompass many aspects, features, and elements of the column and its many aspects, features, and elements. Such columns can be dynamic in their use and operation, and the present disclosure is intended to encompass equivalents, means, systems, and methods of use of the column and / or particles, as well as many aspects thereof consistent with the description and spirit of the operations and functions disclosed herein. The claims of the present application are likewise intended to be construed broadly. The description of the application in this document is merely exemplary in nature and, thus, variations that do not depart from the essence of the application are intended to be within the scope of the application. Such variations are not to be regarded as a departure from the spirit and scope of the application.

Claims

1. A column comprising: particles having an average particle size ranging from about 1 μm to about 5 μm; the particles having an average pore size ranging from about 450 A to about 3000 A; and The average pore volume of the particles ranges from about 0.1 cm 3 / g to about 5 cm 3 / g.

2. The column of claim 1, wherein the particles have an average pore diameter ranging from about 475 A to about 2000 A; and an average pore volume ranging from about 0.1 cm 3 / g to about 3 cm 3 / g.

3. The column of claim 1, wherein the particles have an average pore diameter ranging from about 500 A to about 1250 A; and an average pore volume ranging from about 1 cm 3 / g to about 1.5 cm 3 / g.

4. The column of claim 1, wherein the particles have an average pore diameter of about 500 A; and an average pore volume of about 1.1 cm3 / g. 3 / g.

5. The column of claim 1, wherein the particles have an average pore diameter of about 1000 A; and an average pore volume of about 1.1 cm3 / g. 3 / g.

6. The column of claim 1, wherein the particles are metal oxide particles.

7. The column of claim 1, wherein the particles are silica.

8. The column of claim 1, wherein the particles have an average particle size ranging from 2.5 μm to 3.5 μm.

9. A method of using a size exclusion chromatography column, the method comprising: injecting a viral analyte into a column comprising particles having an average particle size ranging from about 1 μm to about 5 μm; The average pore diameter of the particles ranges from about 450 A to about 3000 A; and the average pore volume of the particles ranges from about 0.1 cm 3 / g to about 5 cm 3 / g; and adjusting a flow rate.

10. The method of claim 9, wherein the viral analyte has an average diameter ranging from about 10 nm to about 55 nm.

11. The method of claim 9, wherein the viral analyte has an average diameter ranging from about 60 nm to about 100 nm.

12. The method of claim 9, wherein the flow rate is greater than about 0.1 ml / min.

13. The method of claim 9, wherein the flow rate is greater than about 0.35 ml / min.

14. The method of claim 9, wherein monomers from the viral analyte are separated from higher order aggregates of the viral analyte.

15. The method of claim 9, wherein the viral analyte does not dissociate at a flow rate greater than about 0.35 ml / min.

16. The method of claim 9, wherein the particles have an average pore size ranging from about 475 A to about 2000 A; and an average pore volume ranging from about 0.1 cm 3 / g to about 3 cm 3 / g.

17. The method of claim 9, wherein the particles have an average pore size ranging from about 500 A to about 1250 A; and an average pore volume ranging from about 1 cm 3 / g to about 1.5 cm 3 / g.

18. The method of claim 9, wherein the particles have an average pore diameter of about 500 A; and an average pore volume of about 1.1 cm3 / g. 3 / g.

19. The method of claim 9, wherein the particles have an average pore diameter of about 1000 A; and an average pore volume of about 1.1 cm3 / g. 3 / g.

20. The method of claim 9, wherein the column is a size exclusion chromatography column.

Images