Pore structures for separating adeno-associated viruses from aggregates thereof

Abstract

Methods of preparing porous particulate materials for use as stationary media and related chromatographic separation apparatuses utilizing the disclosed stationary media are disclosed. The porous particulate material has a pore volume that results in improved stability and column life, and additionally has a modified surface, resulting in a surface-modified porous particulate material that improves the separation of AAV from its aggregates in a sample to be tested.

Description

[0001] Introduction

[0002] The present invention relates to a size exclusion chromatography column that uses porous particles with pore sizes as a stationary phase for separating adeno-associated virus from its aggregates. Technical Field

[0003] The present disclosure relates to chromatography columns. More specifically, the present disclosure relates to a size exclusion chromatography column that uses porous particles with pore sizes as a stationary phase for separating adeno-associated virus from its aggregates. Background Art

[0004] Size exclusion chromatography (hereinafter referred to as "SEC") is a general name given to chromatographic separation techniques that involve liquid chromatography for separating macromolecules based on their molecular size. SEC has been synonymous with other such chromatographic technique names, including gel permeation chromatography (GPC), gel filtration chromatography (GFC), and steric exclusion chromatography.

[0005] The main purpose and use of SEC technology is to provide molecular weight distribution data on biomolecules or synthetic molecules (such as monoclonal antibodies, proteins, and polymer molecules). If the sample of interest contains a mixture of multiple molecules, the sample is injected at the head of the SEC column, and the liquid mobile phase is passed through the column at a fixed flow rate, thereby establishing a pressure gradient along its length. The column includes a packing material, which is typically porous and has a controlled pore size. When the sample passes through the packing material (stationary phase), small macromolecules are able to penetrate the pores of the packing material, but larger molecules are too large to enter the pores and remain in the interstitial space. Therefore, the larger molecules flow down the length of the column faster, while the smaller molecules are able to reside within the pores of the packing material and enter through the pores of the packing material. Due to their size differences, the various molecules are separated as they move down the column and leave (elute) at different times. Thus, these molecules of different sizes are separated and presented as different chromatographic bands.

[0006] When selecting a stationary phase medium, several criteria are considered in SEC separation technology. First, the packing material should not chemically interact with the sample. Second, it must be mechanically stable and able to withstand the operating temperature and high operating pressures. In addition, it must have a sufficient pore volume and a sufficient pore size range to resolve the molecular weight distribution of the sample.

[0007] For high-performance SEC, which implies high pressure, the typical packing materials used are semi-rigid polymer gels or rigid modified silica particles. Generally, rigid silica packings have several advantages over semi-rigid gel packings; they are tolerant of a greater variety of mobile phases, they are stable at the elevated temperatures required to characterize certain polymers, and their pore sizes are more easily defined. However, silica-based packings do have certain disadvantages, such as unwanted adsorption effects, and some stability issues at high operating pressures, especially for smaller-sized particles. This is of particular concern because the silica particles used in modern packing materials need to have specific size parameters to achieve the separation of biologics from their aggregates. For example, one parameter is size, such as 1.8 μm.

[0008] As the technology in this field has evolved, so has the demand for chromatographic equipment. To speed up run times and obtain faster results, higher-pressure operation and reduced column sizes have been introduced. Currently, typical column lengths are 30 - 50 mm, diameters are 2.0 - 10.0 mm, and the particle size used for the stationary phase is generally 3 μm and below. The reduction in particle size allows for a reduction in column length, which in turn results in faster runs and overall time savings in addition to increased chromatographic resolution.

[0009] However, there are various challenges associated with the reduced particle size of the stationary phase. Since modern chromatographic equipment operates at higher pressures than before, the structural and mechanical integrity of the stationary-phase particles has become increasingly important. The particles need to have mechanical strength to handle the repeated high pressures they experience over the life of the column. The life of the column is directly affected by the stability of the packed particles, and over the course of increasing numbers of runs, the packed particles in the stationary phase may experience particle breakage.

[0010] In particular, with regard to fully porous particles, the structure and / or mechanical stability of the particles are affected by the pore volume present in the particles. As a general principle, the lower the pore volume of the particles, the stronger and more stable the particles are in a high-pressure environment. Conventional fully porous silica particles commonly used in current chromatographic applications can have a porosity characterized by a pore volume of 1.4 - 1.8 cc / g. For smaller particles in the range of 1.6 - 1.8 μm, the mechanical stability is highly affected by the particle porosity because these particles are more prone to experiencing breakage, which affects the column life and result reliability. Therefore, the particle porosity has become an important factor in determining the equipment life and result reliability. Thus, reducing the porosity of fully porous particles to make them more mechanically stable, but also including the required specific pore sizes, is of great interest in the industry.

[0011] In addition, silica is known to have a strong affinity for polar solutes, which results in less than ideal packing material properties when size exclusion chromatography is involved. The amorphous nature of silica is reflected in the random distribution of various chemical structures on the surface. Silica particles typically have silanol groups (Si-OH) on the surface. Generally, heating silica can cause the bonded silanol groups to condense and result in the formation of siloxane bonds. Free and bonded silanol groups are present on the surface, and the free silanol groups constitute the main adsorption and reaction sites on the surface of the silica particles.

[0012] When size exclusion of molecules such as proteins is involved, these potential reaction sites contribute to an effect commonly referred to as the "non-size effect", which generally includes all factors that affect the retention of proteins on an SEC column, rather than the classical partitioning of solutes between the pore volume and the interstitial volume. These non-size effects include, for example, attractive interactions such as ion exchange and hydrophobic bonding, which tend to increase the elution volume of the solute, resulting in them appearing smaller than they actually are. Another interaction that occurs is electrostatic repulsion (ion exclusion), which has the effect of excluding the volume that the solute would otherwise be able to access, making them appear larger than their size.

[0013] For the reasons described above, there is a need for a more mechanically stable SEC packing material, especially in high-pressure operations, and the packing material is modified to avoid ion exchange interactions and reverse-phase interactions, which are known to distort chromatographic results and accuracy. In addition, there is a need to provide a column with a packing material in which the pore structure for separating biologic agents such as adeno-associated virus (AAV) from their aggregates will provide additional run time and improved resolution. The description herein addresses these current deficiencies in the SEC market. Summary of the Invention

[0014] The invention described herein relates to devices and methods for performing SEC. Embodiments of the present disclosure operate at normal high-performance liquid chromatography pressures (HPLC) as well as ultra-high-performance liquid chromatography pressures (UHPLC), which range from about 1,000 psi to about 10,000 psi and greater and faster flow rates to accelerate analysis time. Embodiments described herein are characterized by a stationary phase having a well-defined pore structure and particle size to produce highly reproducible results. The invention described herein also includes a method for preparing an SEC stationary phase material having increased mechanical stability and modified surface chemistry that minimizes adsorption interactions of the sample with the stationary phase material. A chromatographic separation device using the improved stationary phase material is also disclosed.

[0015] In one embodiment of the present disclosure, a method for preparing a stationary medium (column packing material) is disclosed. In this embodiment, the stationary medium is a porous particulate material. The porous particulate material has initial properties and morphology, such as initial pore volume, initial pore size, initial pore size and initial average diameter, initial surface chemistry, etc. The method comprises the following steps:

[0016] a. reducing the pore volume of the particulate material from an initial pore volume to a final pore volume;

[0017] b. hydrating the porous particulate material; and

[0018] c. chemically modifying the porous particulate material with a hydrophilic silane compound to obtain a surface-modified porous particulate material.

[0019] In one embodiment, in the first step of the disclosed method, a raw material of porous particulates having an initial size and morphology is provided. In some embodiments, the porous particulate material comprises silica particles (SiO 2 ). For a particular size exclusion separation, depending on the molecule of interest, specific parameters are required in terms of the stationary phase particulate material. In certain embodiments, those parameters include a specific desired pore volume, which is less than the pore volume of currently available raw silica materials. Accordingly, a pore volume reduction step is carried out to reduce the pore volume of the particulate material from the initial pore volume to the final pore volume.

[0020] In one embodiment, the step of reducing the pore volume of the particulate material comprises treating the particulate material. This treatment of the surface of the particulate material results in dehydration of the particulate material. Specifically with respect to silica particles, the surface treatment step results in a dehydroxylation reaction occurring on the surface of the silica particles. The surface of the particles has silanol groups (Si-OH), which can condense after treatment.

[0021] The step of reducing the pore volume of the particulate material is crucial for providing a stationary phase material that is more structurally and mechanically stable than currently available materials and capable of withstanding high operating pressures without experiencing particle breakage. A more mechanically stable particulate material allows the separation device to operate at higher pressures and thus produce faster results. It also results in a longer column life, which allows more samples to be run before the column must be replaced.

[0022] The particulate material can consist of particles having an average diameter size in the range of about 2.8 - 3.2 μm. In one embodiment, the particle diameter is in the range of about 2.9 - 3.1 μm. In another embodiment, the particle diameter is about 3.0 μm. In related embodiments, the average particle diameter is about 2.9 μm. In another embodiment, the average particle diameter is about 3.0 μm. In even further embodiments, the average particle diameter is about 3.1 μm.

[0023] In other related embodiments, the particulate material comprises particles having a porosity characterized by an initial pore volume in the range of 0.4 cc / g to 1.1 cc / g. In other embodiments, the initial pore volume is in the range of 0.6 to 1.0 cc / g, or any value therebetween. In additional embodiments, the initial pore volume is in the range of 0.7 cc / g to 0.9 cc / g, 0.8 cc / g to 0.9 cc / g, or any value therebetween.

[0024] In some related embodiments, the particulate material comprises particles having a porosity characterized by an initial pore size in the range of 500 - 800 angstroms In other embodiments, the initial pore size is in the range of or any value therebetween. In further embodiments, the initial pore size is in the range of or any value therebetween.

[0025] When performing the first step of the disclosed method, the resulting particulate material has a reduced porosity characterized by a final pore volume in the range of 0.5 cc / g to 1.0 cc / g or any value therebetween. In some embodiments, the final pore volume can be reduced in the range of 0.6 cc / g to 0.9 cc / g, or any value therebetween.

[0026] After processing the particulate material, the final pore size has a porosity characterized by a final pore size in the range of 600 to 800 angstroms In other embodiments, the final pore size is in the range of 650 to or any value therebetween. In further embodiments, the final pore size is in the range of 630 to or any value therebetween.

[0027] An optional second step of the method disclosed herein includes the step of hydrating the particulate material that has undergone optional pore reduction by a method such as heat treatment. The step of hydrating the particulate material is performed to reintroduce functional groups onto the surface of the particulate material for further chemical bonding that occurs in subsequent chemical surface modification steps.

[0028] Once the hydration step is complete, the silica particulate material can be treated with a bonding step. The bonding step is carried out to ensure that the surface functionality of the particulate material prevents ion-exchange interactions and reversed-phase interactions during the time of interaction between the sample and the particulate material within the separation column.

[0029] In one embodiment, the bonding step uses a hydrophilic silane compound as a reagent for performing chemical modification of the silica particulate surface. The resulting hydrophilic surface has -OH groups, such that the particulates match the aqueous mobile phase in polarity, thereby reducing chemical interactions with the molecules contained within the sample. The surface modification also sterically blocks the silanol groups retained on the silica surface, thereby reducing ion-exchange interactions with the molecules of interest contained within the sample to be assayed.

[0030] The bonding step in the present disclosure is carried out in an aqueous solution. Most silane bonding known in the art is carried out in an aprotic solvent; however, the inventors of the present disclosure have found that this step can alternatively simply be carried out in an aqueous solution within a controlled pH range.

[0031] In one embodiment of the present disclosure, the step of chemically modifying the surface of the porous particulate material with a hydrophilic compound comprises:

[0032] a. Preparing a bonding aqueous solution having a controlled pH range;

[0033] b. Adding the hydrophilic silane compound to the aqueous solution;

[0034] c. Adding the porous particulate material to the mixture of step b) and causing the hydrophilic silane compound to bond to the surface of the porous particulate material and obtaining a surface-modified porous particulate adsorbent.

[0035] In other embodiments, the step of chemically modifying the surface of the porous particulate material with a hydrophilic compound comprises combining the reaction solvent and reagents in the following specific amounts:

[0036] a. Preparing 6X amount of DI water;

[0037] Mixing 1 / 3× amount of the hydrophilic silane compound with DI water

[0038] b. Adding the aqueous mixture to 1X amount of the porous silica particulates

[0039] c. Adjusting the pH of the aqueous slurry mixture to 7 with approximately 0.15×1N NaOH

[0040] d. Reacting the hydrophilic silane compound with 1X of the porous particulate material to obtain a surface-modified porous particulate material.

[0041] The value X represents the amount of the porous particulate material by weight. The following examples illustrate in more detail the process of chemical surface modification performed on the porous particulate material.

[0042] In other embodiments, a chromatographic separation device is disclosed, comprising:

[0043] - at least one columnar member having internal voids;

[0044] - at least one stationary packing material within the internal voids;

[0045] wherein according to the methods disclosed herein, the stationary packing material comprises a particulate material with a reduced pore volume and surface modification.

[0046] In certain embodiments, the stationary packing material comprises silica particles having an average pore volume of about 0.5 cc / g to 1.0 cc / g or any value therebetween. In some embodiments, the final pore volume can be reduced in the range of 0.7 cc / g to 0.9 cc / g or any value therebetween.

[0047] In some embodiments, the chromatographic separation device is an SEC device or a gel filtration chromatography device (GFC). GFC is used to separate large macromolecules such as antibodies, immunoglobulins, protein complexes, protein aggregates, peptides, and other biomolecules. In gel filtration chromatography, the compounds of interest in the sample move and filter through the stationary phase based on their molecular size. An aqueous solvent is typically used in the mobile phase to ensure that the compounds of interest maintain their biological integrity. Gel filtration columns can separate biomolecules in the range of 10 to 5000 kD. Gel filtration chromatography columns require GFC stationary packing materials with low surface activity, high efficiency, and consistent and uniform pore sizes. In some exemplary embodiments, an SEC column is capable of separating biomolecules in the range of 10 to 4000 kD. In other related embodiments, an SEC column is capable of separating biomolecules in the range of 100 to 3000 kD. In other related embodiments, an SEC column is capable of separating biomolecules in the range of 200 to 2000 kD. In other related embodiments, an SEC column is capable of separating biomolecules in the range of 300 to 1,500 kD. In other related embodiments, an SEC column is capable of separating biomolecules in the range of 1,000 to 3,000 kD.

[0048] In some embodiments described herein, the columnar member of the chromatographic separation device has a length of 100 mm, 150 mm, or 300 mm.

[0049] Symbols and nomenclature

[0050] The term "about" is used in connection with a numerical value to include the normal variations in measurements as would be expected by a person skilled in the art, and is understood to have the same meaning as "approximately" and to cover typical margins of error, such as ±15%, ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the value. The term "about" also covers amounts that differ due to different equilibrium conditions of the compositions resulting from a particular initial composition. Whether or not modified by the term "about", the claims include equivalents of the stated amounts.

[0051] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a composition containing "a compound" includes compositions having two or more compounds that are the same as or different from each other. It should also be noted that the term "or" is generally used in its inclusive sense of "and / or" unless the context clearly dictates otherwise. As used herein, "and / or" means and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0052] For the sake of brevity and clarity, any range of values set forth in this specification contemplates all values within that range and should be interpreted as supporting claims reciting any sub-range having endpoints that are real values within the specified range being discussed. As an illustrative hypothetical example, a range of 1 to 5 disclosed in this specification should be considered to support claims for any of the following ranges: 1 - 5; 1 - 4; 1 - 3; 1 - 2; 2 - 5; 2 - 4; 2 - 3; 3 - 5; 3 - 4; and 4 - 5.

[0053] The term "substantially" is used herein to denote the degree of inherent uncertainty that may be attributed to any quantitative comparison, value, measurement or other representation. The term "substantially" is also used herein to denote the degree to which a quantitative representation may vary from the stated reference without resulting in a change in the basic function of the subject matter being discussed.

[0054] As used herein, the terms "comprise", "comprises" and "comprising" specify the presence of the stated features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0055] As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim is to be interpreted to cover the specified materials or steps recited in the claim and those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. Thus, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising".

[0056] The terms "preferred" and "preferably" refer to embodiments that may provide certain benefits in certain circumstances. However, in the same or other circumstances, other embodiments may also be preferred. Moreover, the recitation of one or more preferred embodiments does not imply that other embodiments are not available and is not intended to exclude other embodiments from the scope of the present disclosure.

[0057] As used throughout this specification and the claims, a list of items joined by the terms "at least one of" or "one or more of" may represent any combination of the listed items. For example, the phrase "at least one of X, Y, or Z" may represent X; Y; Z; X and Y; X and Z; Y and Z; or X, Y, and Z.

[0058] All details of the present disclosure are set forth in the following description and shown in the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Chromatograms are depicted that show the separation of AAV2 using various embodiments of the present invention compared to alternative SEC columns.

[0060] Figure 2 Chromatograms are depicted that show the separation of AAV5 using various embodiments of the present invention compared to alternative SEC columns.

[0061] Figure 3 Chromatograms are depicted that show the separation of AAV8 using various embodiments of the present invention compared to alternative SEC columns.

[0062] Figure 4 Chromatograms are depicted that show the separation of AAV9 using various embodiments of the present invention compared to alternative SEC columns.

[0063] Figure 5A -H Chromatograms are depicted that show the separation of AAV using embodiments of the present invention. DETAILED DESCRIPTION

[0064] The present disclosure relates to a method for preparing an SEC packing material having increased mechanical stability and modified surface chemistry that minimizes the adsorption interaction of a sample with the packing material. Also disclosed is a chromatographic separation apparatus using the improved stationary phase material. The packing material is particularly advantageous in separating AAV from its aggregates.

[0065] In one embodiment of the present disclosure, a method for preparing a stationary medium (column packing material) is disclosed. In this embodiment, the stationary medium is a porous particulate material. The porous particulate material has initial characteristics and morphology, such as initial pore volume, initial pore size, initial pore size and initial average diameter, initial surface chemistry, etc. The method comprises the following steps:

[0066] a. The particulate material has an initial pore volume;

[0067] b. Optionally, hydrating the porous particulate material; and

[0068] c. Chemically modifying the surface of the porous particulate material with a hydrophilic compound to obtain a surface-modified porous particulate material.

[0069] In one embodiment, in the first step of the disclosed method, a raw material of porous particles having an initial size and morphology is provided. In some embodiments, the porous particulate material comprises silica particles (SiO 2 ). For a particular size exclusion separation, depending on the molecule of interest, specific parameters are required in terms of the stationary phase particulate material. In certain embodiments, those parameters include a specific desired pore volume that is less than the pore volume of currently available raw silica materials. Accordingly, a pore volume reduction step is carried out to reduce the pore volume of the particulate material from the initial pore volume to the final pore volume.

[0070] The present disclosure provides size exclusion separation of aggregates from a molecule selected from the group consisting of adeno-associated virus (AAV), monoclonal antibody, immunoglobulin, protein complex, protein aggregate, parvovirus, peptide, biomolecule, or a combination thereof.

[0071] In one embodiment, the step of reducing the pore volume of the particulate material comprises heat treating the particulate material. This heat treatment of the particulate material causes dehydration of the particulate material. Specifically with respect to silica particles, the heat treatment step causes a dehydroxylation reaction to occur on the surface of the silica particles. The surface of the particles contains silanol groups (Si-OH), which can be removed during thermal processing, typically at temperatures above 400 °C, through a dehydroxylation process. During this process, the bond between the OH group and the hydrogen from an adjacent silanol breaks due to a condensation reaction to form water. This produces siloxane Si-O-Si bridges. When the particles undergo this heat treatment and when the silanol groups are removed, it is believed that the resulting siloxane bridges (Si-O-Si) formed within the pores at the pore-solid interface may create a rougher pore-solid interface and reduce the internal pore size, thereby reducing the pore size and pore volume.

[0072] The step of reducing the pore volume of the particulate material is crucial for providing a stationary phase material that is structurally and mechanically more stable than currently available materials and capable of withstanding high operating pressures without experiencing particle breakage. A more mechanically stable particulate material allows the separation equipment to operate at higher pressures and thus produces faster results. It also results in a longer lifespan of the separation column, which allows more samples to be run before the column must be replaced.

[0073] In some related embodiments, the particulate material comprises particles having a porosity characterized by an initial pore size in the range of 500 - 800 angstroms In other embodiments, the initial pore size is in the range of or any value therebetween. In further embodiments, the initial pore size is in the range of or any value therebetween. After surface treatment of the particulate material, the final pore size has a porosity characterized by a final pore size in the range of 600 to 800 angstroms In other embodiments, the final pore size is in the range of 650 to or any value therebetween. In further embodiments, the final pore size is in the range of 630 to or any value therebetween.

[0074] When the method disclosed above is carried out, the resulting particulate material has a reduced porosity, characterized by a final pore volume in the range of 0.5 cc / g to 1.0 cc / g or any value therebetween. In some embodiments, the final pore volume can be reduced to be in the range of 0.6 cc / g to 0.9 cc / g or any value therebetween.

[0075] The particulate material can consist of particles having an average diameter size in the range of about 1.0 - 10 μm. In one embodiment, the particle diameter is in the range of about 1.2 - 8.0 μm. In another embodiment, the particle diameter is in the range of about 1.4 - 7.0 μm. In another embodiment, the particle diameter is in the range of about 1.6 - 6.0 μm. In another embodiment, the particle diameter is in the range of about 1.8 - 4.5 μm. In an even further embodiment, the particle diameter is in the range of about 2.5 - 3.5 μm.

[0076] In other related embodiments, the particulate material comprises particles having a porosity characterized by an initial pore volume in the range of 0.4 cc / g to 1.1 cc / g. In other embodiments, the initial pore volume is in the range of 0.6 to 1.0 cc / g, or any value therebetween. In additional embodiments, the initial pore volume is in the range of 0.7 cc / g to 0.9 cc / g, 0.8 cc / g to 0.9 cc / g, or any value therebetween.

[0077] When performing the first step of the disclosed method, the resulting particulate material has a reduced porosity characterized by a final pore volume in the range of 0.5 cc / g to 1.0 cc / g or any value therebetween. In some embodiments, the final pore volume can be reduced in the range of 0.6 cc / g to 0.9 cc / g, or any value therebetween.

[0078] The method disclosed herein includes the step of hydrating a particulate material that has undergone a pore reduction heat treatment step. The step of hydrating the particulate material is performed in order to reintroduce functional groups to the surface of the particulate material for further chemical bonding that occurs during a subsequent bonding step. Since -OH groups are removed during the dehydroxylation reaction that occurs during the heat treatment of the particulate material, once pore reduction has occurred, it is necessary to reintroduce some -OH groups onto the surface of the silica particles so that the hydrophilic substances used in the bonding step have reaction sites to bond to the surface of the silica particles.

[0079] Accordingly, in one embodiment of the present disclosure, the particulate material now having a reduced pore volume undergoes an optional hydration step, wherein the porous particulate material is ultrasonically treated with water for a period of time and then the aqueous particulate mixture is added to a reactor and refluxed with a hydrofluoric acid (HF) solution. In the case of silica particles, the hydrofluoric acid solution breaks the strong Si - O bonds on the surface of the silica particles and this allows reaction sites to be available for a subsequent bonding step.

[0080] Thus, in another embodiment of the present disclosure, the particulate material is not a thermal process and undergoes a hydration step where the porous particulate material is ultrasonically treated with water for a period of time, and then the aqueous particulate mixture is added to a reactor and refluxed with a hydrofluoric acid (HF) solution. In the case of silica particles, the HF solution breaks the strong Si-O bonds on the surface of the silica particles, and this allows reaction sites to be available in subsequent bonding steps.

[0081] Once this optional hydration step is completed, the particulate material has post-hydration parameters, which include post-hydration pore volume (cc / g) and post-hydration pore size (angstroms), examples of which are shown in Table 1 below. Table 1 outlines various particulate material parameters, including silica particle pore volume (P.V), pore size (P.S), and internal pore surface area (S.A).

[0082] Table 1

[0083]

[0084] In embodiments where the optional hydration step has been completed, the silica particulate material can be further processed with a bonding step. The bonding step is carried out to ensure that the surface functionality of the particulate material prevents ion-exchange interactions and reversed-phase interactions during the time the sample interacts with the particulate material within the separation column.

[0085] When the optional hydration step is not carried out, the silica particulate material is also treated with a chemical surface modification step. The chemical surface modification step is carried out to ensure that the surface functionality of the particulate material prevents ion-exchange interactions and reversed-phase interactions during the time the sample interacts with the particulate material within the separation column.

[0086] In one embodiment, the chemical surface modification step uses a hydrophilic silane compound as a reagent for the chemical surface modification step of the silica particles. The hydrophilic silane has multiple -OH groups and causes the surface of the silica particles to become similar to the aqueous mobile phase, thereby reducing chemical interactions with the molecules contained in the sample. The silane chemical surface modification also ensures that the silanol groups remaining on the silica surface are sterically blocked and do not have ion-exchange interactions with the molecules of interest contained in the sample to be measured. The chemical surface modification step in this method is simply carried out in the presence of water with the aid of an acidic catalyst. Existing silane chemical surface modifications known in the art have been synthesized in toluene, however, the inventors of the present disclosure surprisingly found that this step can alternatively be simply carried out in an aqueous solution.

[0087] In one embodiment of the present disclosure, the step of chemically surface modifying the porous particulate material with a hydrophilic compound comprises:

[0088] a. Prepare an aqueous bonding solution with a controlled pH range;

[0089] b. Add the hydrophilic organosilane compound to the aqueous solution;

[0090] c. Add the porous particulate material to the mixture of step b), and bond the hydrophilic organosilane compound to the surface of the porous particulate material to obtain a surface-modified porous particulate adsorbent.

[0091] The hydrophilic compound added to the aqueous solution can be a very hydrophilic silane compound. In a particular embodiment, the hydrophilic silane compound is glycidoxypropyltrimethoxysilane (GTMPS) or diethoxy(3-glycidoxypropyl)methylsilane. When GTMPS is added to the aqueous solution, this causes hydrolysis of GTMPS, resulting in hydrolyzed GTMPS, which can more readily adsorb to the silica surface and react with the silica surface.

[0092] Then the porous particulate material (i.e., porous silica particles) is added to the aqueous mixture containing GTMPS and allowed to react at a temperature of about 100 °C for about 12 hours. During this reaction, GTMPS adsorbs onto the surface of the silica particles in a silylation reaction. The silylation of the silica surface by GTMPS significantly reduces the number of charged surface groups and silanol groups on the silica particles. GTMPS covalently binds to the silica surface, and the epoxy ring on GTMPS opens and is converted to a diol (which can also be referred to as a diol-bonded phase). The more GTMPS added to the aqueous solution, the higher the degree of silylation that can occur, and thus the thicker the bond on the silica particle surface. The thickness of the bond can be analyzed based on % carbon readings. This can be measured by an elemental analyzer. In some embodiments, the % carbon value of the bond is 0.5 - 1.5% carbon.

[0093] In one embodiment, the step of bonding the porous particulate material with the hydrophilic compound comprises combining the reaction solvent and reagents in the following specific amounts:

[0094] a. Prepare 6X amount of DI water;

[0095] Mix 1 / 3× amount of the hydrophilic organosilane compound with the DI water

[0096] b. Add the aqueous mixture to 1X amount of the porous silica particles

[0097] c. Adjust the pH of the aqueous slurry mixture to 7 with 0.15×1N NaOH

[0098] React the hydrophilic organosilane compound with 1X of the porous particulate material to obtain a surface-modified porous particulate material.

[0099] The value X herein represents the amount by weight of the porous particulate material. The following examples illustrate in more detail the bonding process carried out on the porous particulate material, such as chemical surface modification.

[0100] In some embodiments, the use of the porous particulate material includes at least two columns in series, each column containing the porous particulate material. As described herein, the first column or the second column can be interchangeable such that the first column member and the second column member only define one of the two column members, without defining the orientation or placement in the series. In the first column member, one of the porous particulate materials has particles with an average diameter size in the range of about 2.8 μm to 3.2 μm. In one embodiment, the particle diameter is in the range of about 2.9 - 3.1 μm. In another embodiment, the particle diameter is about 3.0 μm. In a related embodiment, the average particle diameter is about 2.9 μm. In another embodiment, the average particle diameter is about 3.0 μm. In an even further embodiment, the average particle diameter is about 3.1 μm.

[0101] In other related embodiments, the particulate material comprises particles having a porosity characterized by an initial pore volume in the range of 0.4 cc / g to 1.1 cc / g. In other embodiments, the initial pore volume is in the range of 0.6 to 1.0 cc / g, or any value therebetween. In additional embodiments, the initial pore volume is in the range of 0.7 cc / g to 0.9 cc / g, 0.8 cc / g to 0.9 cc / g, or any value therebetween.

[0102] In some related embodiments, the particulate material comprises particles having a porosity characterized by an initial pore size in the range of 500 - 800 angstroms In other embodiments, the initial pore size is in the range of or any value therebetween. In further embodiments, the initial pore size is in the range of or any value therebetween.

[0103] When the first step of the disclosed method is carried out, the resulting particulate material has a reduced porosity, characterized by a final pore volume in the range of 0.5 cc / g to 1.0 cc / g or any value therebetween. In some embodiments, the final pore volume can be reduced in the range of 0.6 cc / g to 0.9 cc / g, or any value therebetween.

[0104] After processing the particulate material, the final pore size has a range from 600 to 800 angstroms Porosity characterized by a final pore size within the range. In other embodiments, the final pore size is in the range of 650 to and any value therebetween. In a further embodiment, the final pore size is in the range of 630 to and any value therebetween.

[0105] Another porous particulate material in the second column member comprises particles having an average diameter size in the range of about 1.0 - 3.0 μm. In one embodiment, the particle diameter is in the range of about 1.2 - 2.8 μm. In another embodiment, the particle diameter is in the range of about 1.4 - 2.6 μm. In another embodiment, the particle diameter is in the range of about 1.6 - 2.4 μm. In another embodiment, the particle diameter is in the range of about 1.8 - 2.2 μm. In an even further embodiment, the particle diameter is in the range of about 2.0 - 2.2 μm.

[0106] In some embodiments, the particulate material comprises particles having a porosity characterized by an initial pore volume in the range of 1.0 cc / g to 1.8 cc / g. In other embodiments, the initial pore volume is in the range of 1.1 - 1.7 cc / g, or any value therebetween. In a further embodiment, the initial pore volume is in the ranges of 1.2 - 1.6 cc / g, 1.3 - 1.5 cc / g, or any value therebetween.

[0107] During the production process of one of the porous particulate materials, the resulting particulate material has a reduced porosity characterized by a final pore volume in the range of 0.7 cc / g to 1.1 cc / g or any value therebetween. In some embodiments, the final pore volume can be reduced in the range of 0.8 to 1.0 cc / g, or any value therebetween. This same porous particulate material can also include a step of hydrating the particulate material that has undergone a pore reducing heat treatment step. The step of hydrating the particulate material is carried out in order to reintroduce functional groups to the surface of the particulate material for further chemical bonding during a subsequent coating step. Once the hydration step is complete, the silica particulate material can be processed with a coating step. The coating step is carried out to ensure that the surface functionality of the particulate material prevents ion exchange interactions and reverse phase interactions during the time of interaction between the sample and the particulate material within the separation column.

[0108] It will be appreciated that a system comprising at least two column members in series can allow for increased separation efficiency, where each column member comprises a porous particulate material. However, it will be appreciated that the separation efficiency of an SEC column increases with an increase in column length. However, under the current system described herein, the ability to obtain a high resolution separation efficiency can be fine-tuned by connecting multiple column members in series according to the test sample.

[0109] Example

[0110] 6X deionized water is placed in a reactor, and the reactor temperature can optionally be set to 25°C, although an increase in temperature is not necessary. In embodiments where temperature is required, once the temperature has stabilized and the reading is in the range of 25 - 30°C, 1 / 3X volume of GTMPS is added to the reactor. The resulting mixture is stirred and the temperature is monitored. Due to the exothermic reaction occurring, there will be an instantaneous temperature increase (in the range of 1.5 - 4°C). The mixture becomes opaque but becomes transparent after approximately 10 minutes. Once the mixture has clarified, 30 grams of silica particles are added. The pH of the aqueous slurry is measured and adjusted to approximately 6.75 - 7.25 using approximately 0.15X of 1N NaOH. The temperature set point of the reactor is set to 100°C, where the heating function is 50°C per hour, and the reaction is allowed to proceed for a period of approximately 12 hours. The silica particles used in this example are particles of 3.0 μm size that have previously undergone a hydration process. Once the reaction is complete, a washing step is performed on the silica particles using filter paper and a Buchner funnel. The silica particles are washed using 6X amount of methanol. The silica particles are then dried in an 80°C environment for 8 hours. Subsequently, the % carbon of the bonded silica particles is measured using an elemental analyzer, allowing the silane chemical surface modification on the particles to be quantified. It should be understood that the amount of silica used depends on the final amount of the desired porous particle material.

[0111] In another exemplary method, 6X deionized water is placed in a reactor and the reactor temperature is set to 25°C. Once the temperature stabilizes and the reading is in the range of 25 - 30°C, 1 / 3X volume of GTMPS is added to the reactor. The resulting mixture is stirred and the temperature is monitored. Due to the exothermic reaction, there will be an instantaneous temperature increase (in the range of 1.5 - 4°C). The mixture becomes opaque but becomes transparent after about 10 minutes... Once the mixture is clarified, a predetermined amount of silica particles is added, such as 30 g for small batch manufacturing or 300 g for full-scale production. The pH of the aqueous slurry is measured. The pH reading is close to a pH value between 2 - 9, preferably between 4 - 8, and more preferably adjusted to about 6.75 - 7.25 with 0.15X of 1N NaOH. The temperature set point of the reactor is set to 100°C, where the heating function is 50°C per hour, and the reaction is allowed to proceed for a period of about 12 hours. The initial pore volume of the silica particles used in this example is 0.4 cc / g to 1.1 cc / g, preferably 0.7 cc / g to 0.9 cc / g. Once the reaction is complete, a washing step is performed on the silica particles using filter paper and a Buchner funnel. The silica particles are washed with 10X volume of deionized water and then with 5X volume of methanol. Then the silica particles are dried in an 80°C environment for about 8 hours. Subsequently, the % carbon of the bonded silica particles is measured using an elemental analyzer, allowing the quantification of the silane chemical surface modification on the particles.

[0112] After completing the chemical surface modification step, the resulting particles are now silane-coated silica particles with diol surface modification, which allows for reduced ion exchange and reversed-phase interactions with the molecules in the sample to be tested. The surface modification that occurs due to the silane chemical surface modification gives the silica particles a surface functionality similar to water, thus making the surface chemistry of the packing material similar to the mobile phase chemistry. Therefore, any interaction that occurs between the sample and the packing material is now only due to the size exclusion principle and is not altered by non-size-dependent factors and interactions (such as the adsorption of molecules with reactive silanol groups on the particle surface). Since the silica particles are now diol-modified surfaces or diol-bonded phase particles, they no longer have negatively charged silanol groups on the surface, which would interfere with the retention time of the analyte of interest. The diol ligands cover the silica surface and exhibit polar functional groups that mimic water, thus making the surface of the silica particles similar to the aqueous mobile phase that carries the analyte / molecules through and past the stationary phase packing material.

[0113] The present disclosure also relates to a chromatographic separation device disclosed as part of the present invention, particularly an SEC device, wherein the packing material used contains a porous particulate material. These porous packing materials have a pore volume, which provides the separation device with increased mechanical stability and a longer lifespan, and they also have a chemical surface modification thereon, resulting in a surface-modified particulate material with reduced interaction with the analyte of interest. In some embodiments, the surface-modified porous particulate material has a ligand density of 2.5 - 4.5 umol / m 2 of chemical surface modification.

[0114] The chromatographic separation device disclosed herein is particularly useful in the analysis of monoclonal antibodies, biosimilars, and other biomolecules.

[0115] In one embodiment, a chromatographic separation device is disclosed, comprising:

[0116] - at least one columnar member having an internal void;

[0117] - at least one stationary packing material within the internal void;

[0118] wherein the stationary packing material comprises a pore volume and a surface-modified particulate material according to the method disclosed herein.

[0119] In certain embodiments, the stationary packing material comprises silica particles having an average pore volume of about 0.5 to 1.2 cc / g. In another embodiment, the silica particles have an average pore volume of about 0.6 to 1.1 cc / g. In a further embodiment, the silica particles have an average pore volume of about 0.6 cc / g to 0.9 cc / g.

[0120] In some embodiments, the chromatographic separation device is a SEC device or a gel filtration chromatography (GFC) device. GFC is used to separate large macromolecules such as antibodies, immunoglobulins, protein complexes, protein aggregates, peptides, and other biomolecules. In gel filtration chromatography, the compounds of interest in the sample move and filter through the stationary phase based on their molecular size. An aqueous solvent is typically used in the mobile phase to ensure that the compounds of interest maintain their biological integrity. Gel filtration columns can separate biomolecules within a size range. Gel filtration chromatography columns require GFC stationary packing materials with low surface activity, high efficiency, and consistent and uniform pore sizes. In some exemplary embodiments, the SEC column is capable of separating biomolecules in the range of 10 to 4000 kD. In other related embodiments, the SEC column is capable of separating biomolecules in the range of 100 to 3000 kD. In other related embodiments, the SEC column is capable of separating biomolecules in the range of 200 to 2000 kD. In other related embodiments, the SEC column is capable of separating biomolecules in the range of 300 to 1,500 kD. In other related embodiments, the SEC column is capable of separating biomolecules in the range of 1,000 to 3,000 kD.

[0121] In some embodiments, the columnar member of the chromatographic separation device has a length of about 100 mm, about 150 mm, or about 300 mm.

[0122] The columnar member has an internal void with an inner diameter of about 1 mm, about 1.5 mm, about 2.1 mm, about 4.6 mm, about 7.8 mm, or about 10 mm, which houses the porous packing material of the present disclosure. The particle size of the stationary packing material housed in the columnar member can vary between 1.6 - 3.0 μm. In some embodiments, the average particle size of the packing material is greater than 1.8 μm, and in other embodiments, a packing material with a particle size of 3.0 μm is used. In addition to the column size parameters, this will depend on the type of analyte to be tested and the size of the molecules of interest. As described above, the porous packing material within the columnar member of the separation device has a reduced pore volume, which results in increased stability of the packing material and increased lifespan of the columnar member of the chromatographic separation device disclosed herein.

[0123] In at least one exemplary embodiment, a chromatographic separation device for molecules includes: at least one columnar member having an internal void; and at least one stationary phase packing material within the internal void. In at least this exemplary embodiment, the stationary phase packing material comprises a porous particulate material, wherein the porous particulate material comprises silica particles having an average final pore size of about 600 to preferably 650 to more preferably 630 to .

[0124] In related embodiments, the chromatographic separation device comprises a porous particulate material that is packed into a column having a diameter of about 4.6 mm at a flow rate of 0.35 mL / min, and the resolution Rs of AAV monomer / aggregate is > 1.2. In other related embodiments, when the porous particulate material is packed into a column having a diameter of about 7.8 mm at a flow rate of 1.0 mL / min, the chromatographic separation device has a resolution Rs of AAV monomer / aggregate > 1.2. In another related embodiment, the chromatographic separation device comprises a porous particulate material that is packed into a column having a diameter of about 4.6 mm at a flow rate of 0.35 mL / min, wherein the chromatographic separation device has a molecular recovery rate of > 90%. In another related embodiment, the chromatographic separation device comprises a porous particulate material that is packed into a column having a diameter of about 7.8 mm at a flow rate of 0.35 mL / min, wherein the chromatographic separation device has a molecular recovery rate of > 90%.

[0125] Now refer to Figure 1 , which shows a chromatogram of AAV2 comparing the porous packing materials in chromatographic separation devices with 4.6 mm column diameters (dashed lines) and 7.8 mm column diameters (dotted lines) to other separation columns. These comparison columns are represented by (1) double dotted lines; (2) short dashes, long dashes; and (3) solid lines. Each comparison column used has a diameter of 7.8 mm. The samples were run as received, at 0.35 mL / min and 0.45 mL / min, and had varying column pressures, as shown on the Y-axis. As shown, the flow rate and pressure conditions shown in the figure remained stable. These results demonstrate a high level of stability of the packing material and a high degree of reliability of the results over hundreds of runs. When compared to the comparative SEC columns (comparison column 1, comparison column 2, and comparison column 3), the SEC devices (experimental column 1 and experimental column 2) designed and operated with the packing materials disclosed herein showed a high degree of superiority in terms of column stability and resolution when separating AAV from aggregates.

[0126] Table 2 - AAV2

[0127]

[0128] From Figure 1 the analysis is shown in Table 2, where the comparison data between two experimental columns 1 and 2 is compared to comparative examples 1, 2, and 3 that ran AAV2 as a test sample. Table 2 shows significant differences between experimental columns 1 and 2 in analyzing AAV high molecular weight samples and AAV monomers.

[0129] Now turn to Figure 2, which shows the chromatogram of AAV5, comparing the porous packing materials in chromatographic separation devices with 4.6 mm column diameter (dashed line) and 7.8 mm column diameter (dash-dotted line) being compared with other separation columns. These comparison columns are represented by (1) double dash-dotted line; (2) short dash, long dash; and (3) solid line. Each of the comparison columns used has a diameter of 7.8 mm. The samples were run as received, at 0.35 mL / min and 0.45 mL / min, and had varying column pressures, as shown on the Y-axis. As shown, the flow rate and pressure conditions shown in the figure remained stable. These results demonstrate a high level of stability of the packing material and a high degree of reliability of the results over hundreds of runs. When compared with competing SEC columns (comparison columns 1, 2, and 3), the SEC devices (experimental columns 1 and 2) designed and operated with the packing materials disclosed herein showed a high degree of superiority in terms of column stability and resolution in separating AAV from aggregates.

[0130] Table 3 - AAV5

[0131]

[0132] from Figure 2 The analysis of is shown in Table 3, where the comparison data between two experimental columns 1 and 2 is compared with comparison examples 1, 2, and 3 that ran AAV5 as a test sample. Table 3 shows a significant difference between experimental columns 1 and 2 in analyzing AAV high molecular weight samples and AAV monomers.

[0133] Now turning to Figure 3 , which shows the chromatogram of AAV8, comparing the porous packing materials in chromatographic separation devices with 4.6 mm column diameter (dashed line) and 7.8 mm column diameter (dash-dotted line) being compared with other separation columns. These comparison columns are represented by (1) double dash-dotted line; (2) short dash, long dash; and (3) solid line. Each of the comparison columns used has a diameter of 7.8 mm. The samples were run as received, at 0.35 mL / min and 0.45 mL / min, and had varying column pressures, as shown on the Y-axis. As shown, the flow rate and pressure conditions shown in the figure remained stable. These results demonstrate a high level of stability of the packing material and a high degree of reliability of the results over hundreds of runs. When compared with competing SEC columns (comparison columns 1, 2, and 3), the SEC devices (experimental columns 1 and 2) designed and operated with the packing materials disclosed herein showed a high degree of superiority in terms of column stability and resolution in separating AAV from aggregates.

[0134] Table 4 - AAV8

[0135]

[0136] from Figure 3The analysis is shown in Table 4, where the comparison data between the two experimental columns 1 and 2 are compared with Comparative Examples 1, 2, and 3 running AAV8 as a test sample. Table 4 shows significant differences between experimental columns 1 and 2 for the analysis of AAV high molecular weight samples and AAV monomers.

[0137] Now turning to Figure 4 , which shows the chromatogram of AAV9, comparing the porous packing materials in chromatographic separation devices with 4.6 mm column diameter (dashed line) and 7.8 mm column diameter (dash-dot line) being compared with other separation columns. These comparison columns are represented by (1) double dash-dot line; (2) short dash, long dash; and (3) solid line. Each of the comparison columns used has a diameter of 7.8 mm. The samples were run as received, at 0.35 mL / min and 0.45 mL / min, and had varying column pressures, as shown on the Y-axis. As shown, the flow rate and pressure conditions shown in the figure remained stable. These results demonstrate a high level of stability of the packing material and a high degree of reliability of the results over hundreds of runs. When compared to competing SEC columns (Comparative Columns 1, 2, and 3), the SEC devices (Experimental Columns 1 and 2) designed and operated with the packing materials disclosed herein showed a high degree of superiority in terms of column stability and resolution when separating AAV from aggregates.

[0138] Table 5 - AAV9

[0139]

[0140] From Figure 4 The analysis is shown in Table 5, where the comparison data between the two experimental columns 1 and 2 are compared with Comparative Examples 1, 2, and 3 running AAV9 as a test sample. Table 5 shows significant differences between experimental columns 1 and 2 for the analysis of AAV high molecular weight samples and AAV monomers.

[0141] Now referring to Figure 5A -H, where multiple serotypes were analyzed. The column can be used to view capsid differences. AAV 1, 2, 3, 5, 6, 8, 9, rh10 were run, and the RT of the monomers is shown in Table 6. The largest retention time (RT) differences were observed for AAV1 and AAV5. Although smaller differences were noted between comparisons between AAVs (e.g., AAV1 and AAV6), these serotype samples differed by only a few amino acids in their capsid structures. Additionally, RT can be used to identify empty AAVs from full AAVs. Using the porous particulate materials described herein, these small RT shifts are highly reproducible and can also be used for identification purposes, considering the specificity of the porous particulate materials used in the column.

[0142] Table 6 Monomeric peak retention times of serotypes observed on the SEC column

[0143] Serotype Retention time (min) AAV1-CMV-GFP( Figure 5A ) 5.633 AAV6-CMV-GFP( Figure 5E ) 5.651 AAV8-CMV-GFP( Figure 5F ) 5.655 AAV2-CMV-GFP( Figure 5B ) 5.708 AAVrh10-CMV-GFP( Figure 5H ) 5.722 AAV9-CMV-GFP( Figure 5G ) 5.737 AAV3-CMV-GFP( Figure 5C ) 5.827 AAV4-CMV-GFP 6.008 AAV5-CMV-GFP( Figure 5D ) 6.190

[0144] Although the present invention has been described with respect to one or more particular embodiments, it should be understood that other embodiments of the present invention can be made without departing from the scope of the present invention. Accordingly, the present disclosure is considered to be limited only by the appended claims.

[0145] Other examples and embodiments of the present disclosure are disclosed in the following recited clauses:

[0146] 1. A chromatographic separation device for molecules, comprising:

[0147] - at least one columnar member having an internal void;

[0148] - at least one stationary phase packing material within the internal void;

[0149] wherein the stationary phase packing material comprises surface-modified porous particulate material.

[0150] 2. The chromatographic separation device according to clause 1, wherein the chromatographic separation device is a size exclusion chromatography device.

[0151] 3. The chromatographic separation device according to any one of clauses 1-2, wherein the surface-modified porous particulate material comprises silica particles.

[0152] 4. The chromatographic separation device according to any one of clauses 1-3, wherein the surface-modified porous particulate material comprises a chemical surface modification having 0.5-1.5% carbon.

[0153] 5. The chromatographic separation device according to any one of clauses 1-4, wherein the surface-modified porous particulate material comprises a chemical surface modification having a diol-bonded phase.

[0154] 6. The chromatographic separation device according to any one of clauses 1-5, wherein the surface-modified porous particulate material comprises a chemical surface modification having a ligand density of 2.5-4.5 umol / m 2 of.

[0155] 7. The chromatographic separation device according to any one of clauses 1-6, wherein the porous particulate material comprises silica particles having a final pore volume of 0.50-1.00 cc / g.

[0156] 8. The chromatographic separation device according to any one of clauses 1-6, wherein the porous particulate material comprises silica particles having a final pore volume of 0.6-0.8 cc / g.

[0157] 9. The chromatographic separation device according to any one of clauses 1-8, wherein the porous particulate material comprises silica particles having an average particle size of 3.0 μm.

[0158] 10. The chromatographic separation device according to any one of clauses 1-9, wherein the porous particulate material comprises silica particles having an average initial pore size of about 500 to .

[0159] 11. The chromatographic separation device according to any one of clauses 1-9, wherein the porous particulate material comprises silica particles having an average final pore size of about 600 to .

[0160] 12. The chromatographic separation device according to any one of clauses 1-11, wherein at least one columnar member has a length of 100 mm, 150 mm or 300 mm.

[0161] 13. The chromatographic separation device according to any one of clauses 1-12, wherein the internal void of at least one columnar member has a diameter between 1 mm and 10 mm.

[0162] 14. The chromatographic separation device according to any one of clauses 1-13, wherein the molecules are selected from the group consisting of: adeno-associated virus (AAV), monoclonal antibody, immunoglobulin, protein complex, protein aggregate, parvovirus, peptide, biomolecule, or a combination thereof.

[0163] 15. A chromatographic separation device for molecules, comprising:

[0164] - at least one columnar member having an internal void;

[0165] - at least one stationary phase packing material within the internal void;

[0166] wherein the stationary phase packing material comprises a porous particulate material, and wherein the porous particulate material comprises silica particles having an average final pore size of about 600 to .

[0167] 16. The chromatographic separation device according to clause 15, wherein when the porous particulate material is filled into a column having a diameter of about 4.6 mm at a flow rate of 0.35 mL / min, the chromatographic separation device has a resolution Rs of AAV monomer / aggregate > 1.2.

[0168] 17. The chromatographic separation device according to clause 15, wherein when the porous particulate material is filled into a column with a diameter of about 7.8 mm at a flow rate of 1.0 mL / min, the chromatographic separation device has a resolution Rs of AAV monomer / aggregate > 1.2.

[0169] 18. The chromatographic separation device according to any one of clauses 15-16, wherein when the porous particulate material is filled into a column with a diameter of about 4.6 mm at a flow rate of 0.35 mL / min, the chromatographic separation device has a molecular recovery rate of > 90%.

[0170] 19. The chromatographic separation device according to any one of clauses 15 or 17, wherein when the porous particulate material is filled into a column with a diameter of about 7.8 mm at a flow rate of 1.0 mL / min, the chromatographic separation device has a molecular recovery rate of > 90%.

[0171] 20. A material for a chromatographic separation device, comprising:

[0172] At least one stationary phase packing material, wherein the stationary phase packing material comprises a surface-modified porous particulate material.

[0173] 21. The material for a chromatographic separation device according to clause 20, wherein the surface-modified porous particulate material comprises silica particles.

[0174] 22. The material for a chromatographic separation device according to any one of clauses 20-21, wherein the surface-modified porous particulate material comprises a chemical surface modification having 0.8-1.2% carbon.

[0175] 23. The material for a chromatographic separation device according to any one of clauses 20-22, wherein the surface-modified porous particulate material comprises a chemical surface modification having a diol bonding phase.

[0176] 24. The material for a chromatographic separation device according to any one of clauses 20-23, wherein the surface-modified porous particulate material comprises a chemical surface modification having a ligand density of 2.5-4.5 umol / m 2 of.

[0177] 25. The material for a chromatographic separation device according to any one of clauses 20-24, wherein the porous particulate material comprises silica particles having a final pore volume of 0.50-1.00 cc / g.

[0178] 26. The material for a chromatographic separation device according to any one of clauses 20-25, wherein the porous particulate material comprises silica particles having a final pore volume of 0.7-0.9 cc / g.

[0179] 27. The material for a chromatographic separation device according to any one of clauses 20 - 26, wherein the porous particulate material comprises silica particles having an average particle size of 3.0 μm.

[0180] 28. The material for a chromatographic separation device according to any one of clauses 20 - 27, wherein the porous particulate material comprises silica particles having an average initial pore size of about 500 to .

[0181] 29. The material for a chromatographic separation device according to any one of clauses 20 - 28, wherein the porous particulate material comprises silica particles having an average final pore size of about 600 to .

[0182] 30. The material for a chromatographic separation device according to any one of clauses 20 - 28, wherein the porous particulate material comprises silica particles having an average final pore size of about 650 to .

[0183] 31. The material for a chromatographic separation device according to any one of clauses 20 - 28, wherein the porous particulate material comprises silica particles having an average final pore size of about 630 to .

[0184] 32. The material for a chromatographic separation device according to any one of clauses 20 - 31, wherein the material is capable of separating molecules, wherein the molecules are selected from the group consisting of: adeno-associated virus (AAV), monoclonal antibody, immunoglobulin, protein complex, protein aggregate, parvovirus, peptide, biomolecule, and combinations thereof.

[0185] 33. The material for a chromatographic separation device according to any one of clauses 20 - 32, wherein the particulate material can be composed of particles having an average diameter size in the range of about 1.0 - 10 μm, or about 1.2 - 8.0 μm, or about 1.4 - 7.0 μm, or about 1.6 - 6.0 μm, or about 1.8 - 4.5 μm, or about 2.5 - 3.5 μm.

[0186] 34. The material for use in a chromatographic separation device according to any one of clauses 20 - 33, wherein when the material is packed into a separation column having a column diameter of 4.6 mm at a running rate of 0.35 mL / min, molecules having a USP half-height at or above 1.7 or 2.0 or 2.1 will be separated.

[0187] 35. A material for use in a chromatographic separation device according to any one of clauses 20 - 34, wherein when the material is packed into a separation column with a column diameter of 7.8 mm at a running rate of 1.0 mL / min, molecules with a USP half-height of 1.7 or higher or 2.0 or 2.5 will be separated out.

[0188] 36. A material for a chromatographic separation device according to any one of clauses 20 - 35, wherein the particulate material comprises particles having an average diameter size in the range of about 1.0 - 10 μm.

[0189] 37. A material for a chromatographic separation device according to any one of clauses 20 - 35, wherein the particulate material comprises particles having an average diameter size in the range of about 1.2 - 8.0 μm.

[0190] 38. A material for a chromatographic separation device according to any one of clauses 20 - 35, wherein the particulate material comprises particles having an average diameter size in the range of about 1.4 - 7.0 μm.

[0191] 39. A material for a chromatographic separation device according to any one of clauses 20 - 35, wherein the particulate material comprises particles having an average diameter size in the range of about 1.6 - 6.0 μm.

[0192] 40. A material for a chromatographic separation device according to any one of clauses 20 - 35, wherein the particulate material comprises particles having an average diameter size in the range of about 1.8 - 4.5 μm.

[0193] 41. A material for a chromatographic separation device according to any one of clauses 20 - 35, wherein the particulate material comprises particles having an average diameter size in the range of about 2.5 - 3.5 μm.

[0194] 42. A material for a chromatographic separation device according to any one of clauses 20 - 41, wherein the porous particulate packing material is for an SEC column, and the porous particulate packing material is capable of spating out biomolecules in the range of 10 to 4000 kD.

[0195] 43. A material for a chromatographic separation device according to any one of clauses 20 - 41, wherein the porous particulate packing material is for an SEC column, and the porous particulate packing material is capable of spating out biomolecules in the range of 100 to 3000 kD.

[0196] 44. A material for a chromatographic separation device according to any one of clauses 20 - 41, wherein the porous particulate packing material is for an SEC column, and the porous particulate packing material is capable of spating out biomolecules in the range of 200 to 2000 kD.

[0197] 45. A material for a chromatographic separation device according to any one of clauses 20 - 41, wherein the porous particulate packing material is for an SEC column, and the porous particulate packing material is capable of eluting biomolecules in the range of 300 to 1,500 kD.

[0198] 46. A material for a chromatographic separation device according to any one of clauses 20 - 41, wherein the porous particulate packing material is for an SEC column, and the porous particulate packing material is capable of eluting biomolecules in the range of 1,000 to 3,000 kD.

[0199] 47. A chromatographic separation system, comprising:

[0200] - At least two columnar members positioned in series, each of the first columnar member and the second columnar member having an internal void;

[0201] - At least one stationary phase packing material in each of the first internal void and the second internal void;

[0202] wherein the stationary phase packing material comprises a surface - modified porous particulate material.

[0203] 48. The chromatographic separation system according to clause 47, wherein the chromatographic separation system is a size - exclusion chromatography system.

[0204] 49. The chromatographic separation system according to any one of clauses 47 - 48, wherein the surface - modified porous particulate material comprises silica particles.

[0205] 50. The chromatographic separation system according to any one of clauses 47 - 49, wherein the surface - modified porous particulate material in the first columnar member comprises a coating having 3.0 - 7.0% carbon, and wherein the surface - modified porous particulate material in the second columnar member comprises a chemical surface modification having 0.5 - 1.5% carbon.

[0206] 51. The chromatographic separation system according to any one of clauses 47 - 50, wherein the surface - modified porous particulate material in at least one columnar member comprises a coating having a diol bonding phase.

[0207] 52. The chromatographic separation system according to any one of clauses 47 - 51, wherein the surface - modified porous particulate material in at least one columnar member comprises a coating having a thickness of 3.0 - 3.7 umol / m 2 of coating.

[0208] 53. The chromatographic separation system according to any one of clauses 47 - 52, wherein the porous particulate material in the first columnar member comprises silica particles having a final pore volume of 0.7 to 1.1 cc / g, and wherein the porous particulate material in the second columnar member comprises silica particles having a final pore volume of 0.50 - 1.00 cc / g.

[0209] 54. The chromatographic separation system according to any one of clauses 47 - 52, wherein the porous particulate material in the first columnar member comprises silica particles having a final pore volume of 0.7 to 1.1 cc / g, and wherein the porous particulate material in the second columnar member comprises silica particles having a final pore volume of 0.6 - 0.8 cc / g.

[0210] 55. The chromatographic separation system according to any one of clauses 47 - 52, wherein the porous particulate material in the second columnar member comprises silica particles having an average initial pore size of about 225 - 280 Å and an average final pore size of about 195 - 270 Å.

[0211] 56. The chromatographic separation system according to any one of clauses 47 - 55, wherein the porous particulate material in the first columnar member comprises silica particles having an average final pore size of about 600 to and wherein the porous particulate material in the second columnar member comprises silica particles having an average final pore size of about 195 - 270 Å.

[0212] 57. The chromatographic separation system according to any one of clauses 47 - 56, wherein at least one columnar member has a length of 100 mm, 150 mm or 300 mm.

[0213] 58. The chromatographic separation system according to any one of clauses 47 - 57, wherein the internal void of at least one columnar member has a diameter between 2.1 mm - 7.8 mm.

[0214] 59. The chromatographic separation system according to any one of clauses 47 - 58, which is used to separate molecules selected from monoclonal antibodies, immunoglobulins, protein complexes, oligonucleotides, oligosaccharides, protein aggregates, peptides, and / or other biomolecules or combinations thereof.

[0215] The above description, examples, and data provide a complete description of the composition, manufacture, and use of the present invention. Since many embodiments of the present invention can be made without departing from the spirit and scope of the present invention, the present invention resides in the claims appended hereto.

Claims

1. A chromatographic separation device for molecules, comprising: - at least one columnar member having an internal void; - at least one stationary phase filler material within the interior void; The stationary phase packing material comprises a surface-modified porous particle material.

2. The chromatographic separation device according to claim 1, wherein the chromatographic separation device is a size exclusion chromatography device.

3. A chromatographic separation device according to any one of claims 1-2, wherein the surface modified porous particulate material comprises silica particles.

4. A chromatographic separation device according to any one of claims 1 to 3, wherein the surface modified porous particulate material comprises a chemical surface modification having 0.5-1.5% carbon.

5. A chromatographic separation device according to any one of claims 1 to 4, wherein the surface modified porous particulate material comprises a chemical surface modification with a diol bonded phase.

6. The chromatographic separation device according to any one of claims 1 to 5, wherein the surface-modified porous particulate material comprises a ligand density of 2.5 to 4.5 umol / m 2 Chemical surface modification.

7. The chromatographic separation device of any one of claims 1 to 6, wherein the porous particulate material comprises silica particles having a final pore volume of 0.50-1.00 cc / g.

8. The chromatographic separation device of any one of claims 1-6, wherein the porous particulate material comprises silica particles having a final pore volume of 0.6-0.8 cc / g.

9. The chromatographic separation device according to any one of claims 1 to 8, wherein the porous particulate material comprises silica particles having an average particle size of 3.0 μm.

10. The chromatographic separation device according to any one of claims 1 to 9, wherein the porous particulate material comprises a particle size of about 500 to about 1000 microns. The average initial pore size of the silica particles.

11. The chromatographic separation device according to any one of claims 1 to 9, wherein the porous particulate material comprises a porous particle having a particle size of about 600 to about 100 μm. The average final pore size of the silica particles.

12. The chromatographic separation device according to any one of claims 1 to 11, wherein at least one columnar member has a length of 100 mm, 150 mm or 300 mm.

13. The chromatographic separation device according to any one of claims 1 to 12, wherein the internal void of at least one columnar member has a diameter between 1 mm and 10 mm.

14. The chromatographic separation device according to any one of claims 1-13, wherein the molecule is selected from the group comprising adeno-associated virus (AAV), monoclonal antibodies, immunoglobulins, protein complexes, protein aggregates, parvoviruses, peptides, biomolecules or combinations thereof.

15. A chromatographic separation apparatus for molecules, comprising: - at least one columnar member having an internal void; - at least one stationary phase filler material within the interior void; The stationary phase filling material comprises a porous granular material, wherein the porous granular material comprises a particle having a diameter of about 600 to about The average final pore size of the silica particles.

16. The chromatographic separation device of claim 15, wherein when the porous particulate material is filled into a column having a diameter of about 4.6 mm at a flow rate of 0.35 mL / min, the chromatographic separation device has a resolution Rs>1.2 for AAV monomers / aggregates.

17. The chromatographic separation device of claim 15, wherein when the porous particulate material is filled into a column with a diameter of about 7.8 mm at a flow rate of 1.0 mL / min, the chromatographic separation device has a resolution Rs>1.2 for AAV monomers / aggregates.

18. The chromatographic separation device according to any one of claims 15-16, wherein when the porous particulate material is filled into a column having a diameter of about 4.6 mm at a flow rate of 0.35 mL / min, the chromatographic separation device has a molecular recovery rate of >90%.

19. The chromatographic separation device according to any one of claims 15 or 17, wherein when the porous particulate material is packed into a column having a diameter of about 7.8 mm at a flow rate of 1.0 mL / min, the chromatographic separation device has a molecular recovery rate of >90%.

20. A material for a chromatographic separation device, comprising: At least one stationary phase packing material, wherein the stationary phase packing material comprises a surface modified porous particulate material.

21. The material for a chromatographic separation device of claim 20, wherein the surface modified porous particulate material comprises silica particles.

22. The material for a chromatographic separation device according to any one of claims 20-21, wherein the surface modified porous particulate material comprises a chemical surface modification with 0.5-1.5% carbon.

23. The material for a chromatographic separation device according to any one of claims 20 to 22, wherein the surface modified porous particulate material comprises a chemical surface modification with a glycol bonded phase.

24. The material for chromatographic separation device according to any one of claims 20 to 23, wherein the surface-modified porous particulate material comprises a ligand density of 2.5 to 4.5 umol / m 2 Chemical surface modification.

25. The material for a chromatographic separation device according to any one of claims 20 to 24, wherein the porous particulate material comprises silica particles having a final pore volume of 0.50 to 1.00 cc / g.

26. The material for a chromatographic separation device according to any one of claims 20 to 25, wherein the porous particulate material comprises silica particles having a final pore volume of 0.7 to 0.9 cc / g.

27. The material for a chromatographic separation device according to any one of claims 20 to 26, wherein the porous particulate material comprises silica particles having an average particle size of 3.0 μm.

28. The material for a chromatographic separation device according to any one of claims 20 to 27, wherein the porous particulate material comprises a porous particle having a diameter of about 500 to about 100 mm. The average initial pore size of the silica particles.

29. The material for a chromatographic separation device according to any one of claims 20 to 28, wherein the porous particulate material comprises a porous particle having a diameter of about 600 to about 100 mm. The average final pore size of the silica particles.

30. The material for a chromatographic separation device according to any one of claims 20 to 28, wherein the porous particulate material comprises a porous particle having a diameter of about 650 to about 100 mm. The average final pore size of the silica particles.

31. The material for a chromatographic separation device according to any one of claims 20 to 28, wherein the porous particulate material comprises a porous particle having a diameter of about 630 to about 100 mm. The average final pore size of the silica particles.

32. A material for a chromatographic separation device according to any one of claims 20-31, wherein the material is capable of separating molecules, wherein the molecules are selected from the group consisting of adeno-associated virus (AAV), monoclonal antibodies, immunoglobulins, protein complexes, protein aggregates, parvoviruses, peptides, biomolecules and combinations thereof.

33. A material for use in a chromatographic separation device according to any one of claims 20-32, wherein the particulate material can be composed of particles having an average diameter size in the range of about 1.0-10 μm, or about 1.2-8.0 μm, or about 1.4-7.0 μm, or about 1.6-6.0 μm, or about 1.8-4.5 μm, or about 2.5-3.5 μm.

34. A material for use in a chromatographic separation device according to any one of claims 20-33, wherein when the material is filled into a separation column having a column diameter of 4.6 mm at a running rate of 0.35 mL / min, molecules with a USP half height at or above 1.7 or 2.0 or 2.1 will be separated.

35. A material for use in a chromatographic separation device according to any one of claims 20-34, wherein when the material is filled into a separation column having a column diameter of 7.8 mm at a running rate of 1.0 mL / min, molecules with a USP half height at or above 1.7 or 2.0 or 2.5 will be separated.

36. A material for a chromatographic separation device according to any one of claims 20 to 35, wherein the particulate material comprises particles having an average diameter size in the range of about 1.0 to 10 μm.

37. The material for a chromatographic separation device according to any one of claims 20 to 35, wherein the particulate material comprises particles having an average diameter size in the range of about 1.2 to 8.0 μm.

38. The material for a chromatographic separation device according to any one of claims 20-35, wherein the particulate material comprises particles having an average diameter size in the range of about 1.4-7.0 μm.

39. The material for a chromatographic separation device according to any one of claims 20 to 35, wherein the particulate material comprises particles having an average diameter size in the range of about 1.6 to 6.0 μm.

40. The material for a chromatographic separation device according to any one of claims 20-35, wherein the particulate material comprises particles having an average diameter size in the range of about 1.8-4.5 μm.

41. A material for a chromatographic separation device according to any one of claims 20-35, wherein the particulate material comprises particles having an average diameter size in the range of about 2.5-3.5 μm.

42. The material for a chromatographic separation device according to any one of claims 20 to 41, wherein the porous particle packing material is used in a SEC column, and the porous particle packing material is capable of precipitating biomolecules in the range of 10 to 4000 kD.

43. The material for a chromatographic separation device according to any one of claims 20 to 41, wherein the porous particle packing material is used in a SEC column, and the porous particle packing material is capable of precipitating biomolecules in the range of 100 to 3000 kD.

44. The material for a chromatographic separation device according to any one of claims 20 to 41, wherein the porous particle packing material is used in a SEC column, and the porous particle packing material is capable of precipitating biomolecules in the range of 200 to 2000 kD.

45. The material for a chromatographic separation device according to any one of claims 20 to 41, wherein the porous particle packing material is used in a SEC column, the porous particle packing material being capable of precipitating biomolecules in the range of 300 to 1,500 kD.

46. ​​The material for a chromatographic separation device according to any one of claims 20 to 41, wherein the porous particle packing material is used in a SEC column, the porous particle packing material being capable of precipitating biomolecules in the range of 1,000 to 3,000 kD.

47. A chromatographic separation system comprising: - at least two columnar members positioned in series, each of the first columnar member and the second columnar member having an internal void; - at least one stationary phase filler material within each of the first interior void and the second interior void; The stationary phase filling material comprises a surface-modified porous particle material.

48. The chromatographic separation system of claim 47, wherein the chromatographic separation system is a size exclusion chromatography system.

49. The chromatographic separation system of any one of claims 47-48, wherein the surface modified porous particulate material comprises silica particles.

50. A chromatographic separation system according to any one of claims 47-49, wherein the surface modified porous particulate material in the first columnar member comprises a coating having 3.0-7.0% carbon, and wherein the surface modified porous particulate material in the second columnar member comprises a chemical surface modification having 0.5-1.5% carbon.

51. A chromatographic separation system according to any one of claims 47-50, wherein the surface modified porous particulate material in at least one columnar member comprises a coating having a glycol bonded phase.

52. The chromatographic separation system according to any one of claims 47 to 51, wherein the surface modified porous particulate material in at least one columnar member comprises a surface modified porous material having ... 2 The thickness of the coating.

53. A chromatographic separation system according to any one of claims 47-52, wherein the porous particulate material in the first columnar member comprises silica particles having a final pore volume of 0.7 to 1.1 cc / g, and wherein the porous particulate material in the second columnar member comprises silica particles having a final pore volume of 0.50-1.00 cc / g.

54. A chromatographic separation system according to any one of claims 47-52, wherein the porous particulate material in the first columnar member comprises silica particles having a final pore volume of 0.7 to 1.1 cc / g, and wherein the porous particulate material in the second columnar member comprises silica particles having a final pore volume of 0.6-0.8 cc / g.

55. The chromatographic separation system of any one of claims 47-52, wherein the porous particulate material in the second columnar member comprises silica particles having an average initial pore size of about 225-280 angstroms and an average final pore size of about 195-270 angstroms.

56. The chromatographic separation system of any one of claims 47-55, wherein the porous particulate material in the first columnar member comprises a porous particle having a diameter of about 600 to about 100 mm. and wherein the porous particulate material in the second columnar member comprises silica particles having an average final pore size of about 195-270 angstroms.

57. A chromatographic separation system according to any one of claims 47 to 56, wherein at least one columnar member has a length of 100 mm, 150 mm or 300 mm.

58. The chromatographic separation system of any one of claims 47-57, wherein the internal void of at least one columnar member has a diameter between 2.1 mm and 7.8 mm.

59. The chromatographic separation system according to any one of claims 47-58, which is used to separate molecules selected from monoclonal antibodies, immunoglobulins, protein complexes, oligonucleotides, oligosaccharides, protein aggregates, peptides and / or other biomolecules or combinations thereof.

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