Patent Application
20250041760
The present disclosure generally relates to a column comprising particles having an average particle size ranging from about 1 μm to about 5 μm; the particles have an average pore size ranging from about 450 Å to about 3000 Å; and the particles have an average pore volume ranging from about 0.1 cm3/g to about 5 cm3/g. The column can be a size exclusion chromatography column. The column can be used in a method to separate monomers from viral analytes.
Size Exclusion Chromatography (SEC) column is a powerful analytical tool that separates molecules based on their hydrodynamic size, and it is commonly used in the purification and characterization of biomolecules. However, there is no one-size-fits-all SEC column for the separation of various biological analytes. The resolution will depend on several factors, including the size range of analytes being analyzed and the pore structure of the packing materials. The inert surface chemistry is also important to prevent non-specific interactions with the analytes.
What is needed is a size exclusion chromatography column that can deliver high throughput and high resolution for viral analyte, such as adeno-associated viruses (AAVs), aggregate analysis.
Features of the present disclosure are illustrated by way of example and not limited in the following figure(s), in which like numerals indicate like elements, in which:
In an aspect, there is disclosed a column comprising particles having an average particle size ranging from about 1 μm to about 5 μm; the particles have an average pore size ranging from about 450 Å to about 3000 Å; and the particles have an average pore volume ranging from about 0.1 cm3/g to about 5 cm3/g.
In another aspect, there is disclosed a method of using the disclosed column, 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 particles have an average pore size ranging from about 450 Å to about 3000 Å; and the particles have an average pore volume ranging from about 0.1 cm3/g to about 5 cm3/g; and adjusting a flow rate.
Additional features and advantages of various embodiments will be set forth, in part, in the description that follows, and will, in part, be apparent from the description, or can be learned by the practice of various embodiments. The objectives and other advantages of various embodiments will be realized and attained by means of the elements and combinations particularly pointed out in the description herein.
For simplicity and illustrative purposes, the present disclosure is described by referring to examples thereof. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. It will be readily apparent however, that the present disclosure may be practiced without limitation to these specific details. In other instances, some methods and structures have not been described in detail so as not to unnecessarily obscure the present disclosure.
Additionally, the elements depicted in the accompanying figures may include additional components and some of the components described in those figures may be removed and/or modified without departing from scopes of the present disclosure. Further, the elements depicted in the figures may not be drawn to scale and thus, the elements may have sizes and/or configurations that differ from those shown in the figures.
In its broad and varied embodiments, disclosed herein is a column, such as a size exclusion chromatography column, that can deliver high throughput and high resolution for viral analytes, such as adeno-associated viruses (AAVs), aggregate analysis. An end user can obtain information about the quality and purity of viral analytes more efficiently.
The present disclosure describes a column, comprising particles, such as a plurality of particles, the 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 Å to about 3000 Å; and the particles have an average pore volume ranging from about 1.0 to about 1.4. The column, such as a size exclusion chromatography column, can provide a resolution of 2.4 between AAV monomers and dimers within a short period of time, for example, about 5 minutes. The particles can be a plurality of particles. For ease of understanding, the use of the “particles” will refer to a plurality of particles, and descriptions of the particles will refer to an average of the plurality of particles. For example, a particle size would be an average particle size for the plurality of particles that can be present in the column.
The particles for use in the column can have any desired shape, which will generally depend on the targeted application. For chromatographic applications, suitable shapes include without limitation spheres, rings, polyhedra, saddles, platelets, fibers, hollow tubes, rods and cylinders, and mixtures of any two or more such shapes. In one aspect, the particles can be substantially spherical. Spherical cores can be easily packed and are thus desirable for certain applications, such as chromatography.
The composition of the particles is not critical. Suitable materials include without limitation glasses, sands, metals, metal oxides, metalloids, ceramics, and combinations thereof. In one aspect, the particles can comprise a metal oxide, such as a refractory metal oxide. In a further aspect, the particles can be a porous metal oxide particle. Exemplary metal oxides include without limitation silica, alumina, titania, zirconia, ferric oxide, antimony oxide, zinc oxide, and tin oxide. In another aspect, the particles can comprise silica, alumina, titania, zirconia, or a combination thereof. In a further aspect, the particles can comprise silica. In one aspect, the metal oxide particle can include surface hydroxyl groups can be modified with a surface modifier.
The particles have an average particle size from about 0.1 μm to about 100 μm, including without limitation particles having an average particle size from about 0.5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, and 90 μm. In specific aspects, a particle (e.g. silica) can have an average particle size of from about 1 μm to about 5 μm, including without limitation 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. Particle size can be determined using methods known in the art, for example through the use of a Coulter Counter, which can also count particles and thus provide particle size distributions.
The particle size distribution of the particles can vary depending on the composition of the particle and the method in which the particle was made and/or processed. In one aspect, the particles can have a particle size distribution of 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 a further aspect, the particles have a particle size distribution of from about 0.5% to about 10% of the average particle size, including without limitation particle size distributions of from about 0.5% to about 8%, 0.5% to about 6%, and from about 0.5% to about 5% of the median particle size.
The average pore size of the particles can affect the separation of viral analytes. Viral analytes are typically large biomolecules, and their size varies depending on the type of virus. Therefore, selecting an appropriate average pore size for the particles used in the column is needed for effective separation of the viral analytes. In particular, if the average pore size is too small, for example, less than about 475 Å, the viral analytes may not be able to enter the pores present on the particle, leading to poor separation of monomer and dimers/aggregates. On the other hand, if the average pore size is too large, for example, greater than about 3000 Å the separation may not be optimal as all analytes (e.g., monomer and dimers/aggregates) could diffuse through the column at the same rate.
The particles can have substantially ordered pores with an average pore size ranging from about 450 Å to about 3000 Å, including for example about 475, 500, 600, 700, 800, 1000, 1200, 1250, or 300 Å average pore sizes. In an aspect, the particles can have substantially ordered pores with an average pore size from about ranging from about 475 Å to about 2000 Å, including for example, about 500 Å to about 1250 Å. The particles can have an average pore size of about 500 Å. The particles can have an average pore size of about 1000 Å.
The pore volume of the particle plays a role in the separation resolution of viral analytes. Bigger pore volumes can result in wider separation windows, which can help in achieving better resolution of various-sized viral analytes. Pore volume can be important when dealing with multiple aggregates of viral analytes or when separating particles with similar sizes. The particles can have an average pore volume of from about 0.1 cm3/g to about 5 cm3/g, such as from about 0.1 cm3/g to about 3 cm3/g, and as a further example from about, 0.75 cm3/g to about 2 cm3/g, and as a further example, from about 1 cm3/g to about 1.5 cm3/g. In an aspect, the particles can have an average pore volume of from about 1.1 cm3/g to about 1.4 cm3/g.
The particle average pore size and average pore volume can vary depending on the viral analyte. In an aspect, the particles have an average pore size ranging from about 450 Å to about 3000 Å; and the particles have an average pore volume ranging from about 0.1 cm3/g to about 5 cm3/g. In another aspect, the particles have an average pore size ranging from about 475 Å to about 2000 Å; and an average pore volume ranging from about 0.1 cm3/g to about 3 cm3/g. In a further aspect, wherein the particles have an average pore size ranging from about 500 Å to about 1250 Å; and an average pore volume ranging from about 1 cm3/g to about 1.5 cm3/g. It should be appreciated that any range or specific point for the average pore size disclosed herein can be combined with any range or specific point for the average pore volume disclosed herein. However, some combinations can have enable better resolution of the monomers and aggregates of the viral analytes, as discussed further herein.
When separating viral vectors using chromatography columns, high throughput can play a role when dealing with numerous samples or when time-sensitive results are needed. High throughput can enable an end user to analyze more samples within a given timeframe, which can boost overall productivity. It can reduce time and cost involved in sample analysis and purification thereby making the process of using the chromatography column more efficient and cost-effective. High throughput chromatography columns can minimize a risk of sample degradation or contamination during prolonged analysis or purification processes, which can improve the accuracy and reproducibility of the results.
There is also disclosed herein a method of using the disclosed column, 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 particles have an average pore size ranging from about 450 Å to about 3000 Å; and the particles have an average pore volume ranging from about 0.1 cm3/g to about 5 cm3/g; and adjusting a flow rate so that monomers separate.
The viral analyte can have an average diameter 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 viral analyte can have an average diameter from about 55 nm to about 1000 nm, for example, from about 60 nm to about 100 nm.
Experiments were performed using three different pore sizes of monodispersed 3 μm SEC silica particles. The three different pore sizes were 130 Å, 300 Å, and 500 Å. Each of these particles were packed into 4.6×300 mm columns for SEC-FLD (fluorescence detector) evaluation. Additional experiments were performed using particles with a pore size of 500 Å, but with two different pore volumes, e.g., 0.75 and 1.10. The testing condition included a column compartment temperature of 25° C., 50 mM sodium phosphate pH 7.2 and 400 mM NaCl as the mobile phase, flow rate of 0.35 ml/min, and FLD: Ex 280 nm; Em 348 nm. The test samples were viral analytes, such as adeno-associated viruses (AAVs), ranging in size from approximately 20 to 25 nm in diameter for their monomers. In some aspect, the viral analytes ranged in size from 50 to 75 nm, such as 60 nm, in diameter for their monomers.
As seen in
Example 1B—AAV monomers and dimers were also able to diffuse in and out of columns with pore sizes larger than 500 Å, including 750 Å, and 1000 Å, as shown in
Example 1C—Viral analytes were run through an inventive column (Row A) and comparative columns (Rows B, C, and D). Row A utilized column with particles having an average pore size of about 500 Å. Row B is a column with particles having a particle size of 2.5 μm, and a pore size of 450 Å. Row C is a column with particles having a particle size of 5 μm, and a pore size of 500 Å. Row D is a column with particles having a particle size of 5 μm, and a pore size of 1000 Å. The resolution between monomer and dimer viral analytes is shown in
Example 1D—An inventive column with particles having a pore size of 1000 Å was used to separate a viral analyte having a diameter of about 60 nm. As shown in
A viral analyte was injected into a column comprising particles having an average pore size of about 500 Å. 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 relation of total peak area with respect to flow rate was determined where 1/F and total peak area shows good linearity. It suggested that when the viral analyte passed through the column, the viral analyte did not disintegrate or the high flow rate did not cause aggregation or breaking down of aggregates of the viral analyte.
COMPARATIVE—The 25 nm viral analyte was injected into a column made by the prior art comprising particles having an average pore size of about 750 Å (comparative. 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 is shown in
According to Table 3 the prior art column did not achieve a resolution higher than 2.0 at any flow rates studied. The particle structure in the column was not able to separate aggregates from monomers at a flow rate of 0.5 ml/min and above.
From the foregoing description, those skilled in the art can appreciate that the present teachings can be implemented in a variety of forms. Therefore, while these teachings have been described in connection with particular embodiments and examples thereof, the true scope of the present teachings should not be so limited. Various changes and modifications can be made without departing from the scope of the teachings herein.
This scope disclosure is to be broadly construed. It is intended that this disclosure disclose equivalents, means, systems and methods to achieve the devices, activities and mechanical actions disclosed herein. For each device, article, method, mean, mechanical element or mechanism disclosed, it is intended that this disclosure also encompass in its disclosure and teaches equivalents, means, systems and methods for practicing the many aspects, mechanisms and devices disclosed herein. Additionally, this disclosure regards a column and its many aspects, features and elements. Such a column can be dynamic in its use and operation, this disclosure is intended to encompass the equivalents, means, systems and methods of the use of the column and/or particles and its many aspects consistent with the description and spirit of the operations and functions disclosed herein. The claims of this application are likewise to be broadly construed. The description of the inventions herein in their many embodiments is merely exemplary in nature and, thus, variations that do not depart from the gist of the invention are intended to be within the scope of the invention. Such variations are not to be regarded as a departure from the spirit and scope of the invention.
