Silica-based particles with hydroxyl-terminated peg bonding and methoxy-terminated peg surface modification

Abstract

The present disclosure relates to stationary phase materials for performing size exclusion chromatography. Embodiments of the present disclosure feature a hydroxyl-terminated polyethylene glycol surface-modified silica particle stationary phase material, which is optionally also methoxyl-terminated polyethylene glycol surface-modified.

Description

[0001] Cross-reference to related applications

[0002] This patent application claims priority and benefit to U.S. Provisional Application No. 63 / 079,301, filed September 16, 2020, entitled “Sorbent Used to Improve Chromatographic Separations in Size Exclusion Chromatography Via Reduced Secondary Interactions,” which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to stationary phase materials for size exclusion chromatography. In particular, this disclosure relates to stationary phase materials comprising porous silica particles having hydroxyl-terminated polyethylene glycol ligands on the particle surface. In some embodiments, this disclosure also relates to stationary phase materials comprising porous silica particles having hydroxyl-terminated ligands on the particle surface and methoxyl-terminated polyethylene glycol ligands on the particle surface. Background Technology

[0004] Size exclusion chromatography (SEC) is a commonly used separation technique that uses differences in hydrodynamic radii to separate dissolved analytes on a stationary phase. Theoretically, perfect SEC is based solely on hydrodynamic radius separation; however, secondary interactions, such as ionic and hydrophobic interactions, can cause undesirable effects, including peak broadening, tailing, and loss of resolution and separation efficiency. For the separation of biopharmaceutical materials, such as monoclonal antibodies, antibody-drug conjugates, or fusion proteins, these secondary interactions pose significant analytical challenges. Traditional methods for reducing these secondary interactions include adding salts, such as sodium chloride or potassium chloride, or including organic co-solvents, such as methanol, ethanol, isopropanol, or acetonitrile. However, there is no universal solution for all target analytes, and each desired separation requires optimization of the mobile phase composition. Mobile phase optimization is often lengthy, time-consuming, and lacks ease of use for novice users.

[0005] Introducing polar surface modifications onto the stationary phase used in SEC minimizes nonspecific secondary interactions, providing high efficiency and the ability to remain unaffected by variations in method parameters. Commonly used surface modifications are based on the bonding and coating of glycols and polyethylene glycols. While these surface modifications can reduce secondary interactions caused by size exclusion separation, they have their own drawbacks. The main disadvantages of glycol-bonded / coated surfaces are the persistent presence of hydrophobic secondary interactions and the associated requirements for mobile phase development.

[0006] Surface modification with polyethylene glycol (PEG) (also known as polyethylene oxide (PEO)) is another common strategy for reducing nonspecific interactions, and such modified materials have been incorporated into many different products and platforms. This prevalence is partly due to performance and partly due to the ease of manipulation of the strategy. PEG is suitable for both two-dimensional and three-dimensional applications, providing a coating of porous particles used in materials such as SEC. PEG produces a hydrated layer that repels nonspecific interactions, including ionic and hydrophobic interactions. Compared to glycol-bonded surfaces, PEG provides more effective resistance to nonspecific secondary interactions. On the other hand, when used alone, methoxy-terminated PEG bonding (the most common surface modification) can lead to poor peak shape and low efficiency in SEC separation. Summary of the Invention

[0007] This disclosure generally relates to stationary phase materials for size exclusion chromatography. Typically, the stationary phase materials have been surface-modified with polyethylene glycol (PEG) and are hydroxyl-terminated. Surprisingly, according to this disclosure, hydroxyl-terminated PEG surface-modified particles have been found to provide reduced analyte secondary interactions and decreased dependence on buffer, pH, and column temperature for SEC separation. Furthermore, according to this disclosure, it has been surprisingly found that the presence of methoxy-terminated PEG groups does not lead to poor peak shapes and, in fact, significantly increases the stability of the stationary phase material to alkaline conditions, compared to stationary phase materials modified only with methoxy-terminated PEG groups, or stationary phase materials modified with hydroxyl-terminated PEG bonds and further modified with methoxy-terminated PEG groups. In particular, the combination of hydroxyl-terminated PEG and methoxy-terminated PEG groups on the particle surface provides reduced analyte secondary interactions, decreased dependence on buffer, pH, and column temperature for SEC separation, and increased overall chemical stability of the stationary phase material, all without sacrificing peak shape or efficiency. When the stationary phase material can withstand high pH conditions (e.g., pH above about 8), the combination of hydroxyl and methoxy-terminated PEG groups on the silica surface can be particularly useful.

[0008] In one aspect, a stationary phase material is provided comprising porous particles having a surface, at least a substantial portion of which is modified with hydroxyl-terminated polyethylene glycol (PEG), wherein the porous particles comprise silica. Furthermore, the stationary phase may be additionally modified with methoxyl-terminated PEG. These stationary phase materials can be used in size exclusion chromatography.

[0009] Therefore, in another aspect, a stationary phase material is provided comprising porous silica particles having a surface, at least a substantial portion of which is modified with hydroxyl-terminated polyethylene glycol (PEG), and at least a portion of which is modified with methoxyl-terminated polyethylene glycol. These stationary phase materials can be used in size exclusion chromatography.

[0010] An implementation of one or more of the above aspects may include one or more of the following features.

[0011] In some embodiments, the porous silica particles have a diameter with an average size distribution of about 1 μm to about 50 μm. In some embodiments, the porous silica particles have a diameter with an average size distribution of about 1 μm to about 20 μm. In some embodiments, the porous silica particles have a diameter with an average size distribution of about 1.5 μm to about 5 μm.

[0012] In some implementations, the porous silica particles have approximately to approximately about to approximately or about to approximately The average aperture.

[0013] In some embodiments, hydroxyl-terminated polyethylene glycol has the following formula:

[0014]

[0015] in:

[0016] m is an integer from approximately 1 to approximately 10;

[0017] n is an integer from approximately 2 to approximately 50; and

[0018] The wavy line indicates the attachment point to the surface of the porous silica particles.

[0019] In some embodiments, m is 2 or 3. In some embodiments, n is about 5 to about 15 or about 8 to about 12. In some embodiments, m is 3 and n is about 8 to about 12. In some embodiments, hydroxyl-terminated polyethylene glycol is at about 0.5 μmol / m 2 Approximately 15 μmol / m 2 The density exists on the surface of porous silica particles. In some embodiments, hydroxyl-terminated polyethylene glycol is present at approximately 0.5 μmol / m³. 2 To approximately 5.0 μmol / m 2 or approximately 1.0 μmol / m 2 To approximately 2.0 μmol / m 2The density exists on the surface of porous silica particles.

[0020] In some embodiments, the surface modified with methoxy-terminated polyethylene glycol is a result of treating the stationary phase material with a methoxy-terminated polyethylene glycol reagent having the following formula:

[0021]

[0022] in:

[0023] At least one of R1, R2 and R3 is OMe, OEt, Cl or N(CH3)2;

[0024] m is an integer from approximately 1 to approximately 10; and

[0025] n is an integer from approximately 2 to approximately 20.

[0026] In some implementations, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some implementations, m is 2 or 3. In some implementations, m is 3 (i.e., propyl).

[0027] In some implementations, n is about 5 to about 15. In some implementations, n is about 6 to about 12, such as about 6 to about 9.

[0028] In some implementations, the methoxy-terminated PEG reagent is 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]trichlorosilane or 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]tris(dimethylamino)silane.

[0029] In some embodiments, at least a portion of the surface of the porous silica particles modified with hydroxyl-terminated polyethylene glycol and methoxyl-terminated polyethylene glycol reagents comprises a structure represented by one of the following formulas:

[0030] WO 2022 / 061039A1

[0031]

[0032] In some embodiments, at least a portion of the surface of the porous silica particles modified with hydroxyl-terminated polyethylene glycol and methoxyl-terminated polyethylene glycol reagents comprises a structure represented by the following formula:

[0033]

[0034] In another aspect, a column is provided that comprises a stationary phase material as described herein, the column having an interior for receiving the stationary phase material.

[0035] In some embodiments, compared to a reference column having a stationary phase comprising porous silica particles with a surface modified with polyethylene glycol without hydroxyl end caps, this column provides one or more of the following:

[0036] In SEC separation performed on the column, the secondary ionic interactions between the analyte and the stationary phase material are reduced;

[0037] During SEC separation performed on the column, the hydrophobic secondary interactions between the analyte and the stationary phase material are reduced;

[0038] The dependence on mobile phase pH is reduced in SEC separation performed on the column;

[0039] The dependence on column temperature is reduced in SEC separation performed on the column;

[0040] The reduction is determined by the improvement of peak shape calculated based on the USP peak tail, asymmetry at 4.4, peak width at 50%, or a combination thereof.

[0041] In some embodiments, compared to a reference column having a stationary phase comprising porous silica particles with a polyethylene glycol-modified surface without hydroxyl end caps, the column provides: enhanced peak resolution of SEC separations performed on the column; enhanced reproducibility of SEC separations performed on the column; or both.

[0042] In some embodiments, the column comprises a stationary phase material comprising porous silica particles having a surface, at least a substantial portion of which is modified with hydroxyl-terminated polyethylene glycol (PEG), and wherein at least a portion of which is modified with methoxyl-terminated PEG, and provides enhanced stability to alkaline conditions compared to a column having a stationary phase comprising porous silica particles having only a hydroxyl-terminated PEG-modified surface.

[0043] In some embodiments, alkaline conditions are a pH above about 8. In some embodiments, alkaline conditions are a pH between about 8 and about 9.

[0044] In another aspect, a method is provided for preparing a stationary phase material for size exclusion chromatography, the stationary phase having a surface modified with polyethylene glycol and at least a substantial portion thereof by hydroxyl-terminated poly(ethylene glycol), the method comprising:

[0045] A stationary phase material comprising porous particles containing silica is provided;

[0046] Contact the stationary phase material with a polyethylene glycol reagent having hydroxyl-terminated ends in the following formula:

[0047]

[0048] in:

[0049] At least one of R1, R2 and R3 is OMe, OEt, Cl or N(CH3)2;

[0050] m is an integer from approximately 1 to approximately 10; and

[0051] n is an integer from approximately 2 to approximately 50.

[0052] To form a reaction mixture; and

[0053] The reaction mixture was treated with an aqueous hydrolysate to modify at least a substantial portion of the surface of the porous silica particles.

[0054] In some embodiments, R1, R2, and R3 are each OMe, OEt, Cl, or N(CH3)2. In some embodiments, R1, R2, and R3 are each OMe or each OEt, and the contact further includes adding a catalyst to the reaction mixture.

[0055] In some implementations, the catalyst is hydrochloric acid (HCl).

[0056] In some implementations, hydroxyl-terminated polyethylene glycol is used at approximately 0.5 μmol / m 2 Approximately 15 μmol / m 2 The density exists on the surface of porous silica particles. In some embodiments, hydroxyl-terminated polyethylene glycol is present at approximately 0.5 μmol / m³. 2 To approximately 5.0 μmol / m 2 or approximately 1.0 μmol / m 2 To approximately 2.0 μmol / m 2 The density exists on the surface of porous silica particles.

[0057] In some embodiments, the method further includes contacting a hydroxyl-terminated polyethylene glycol-modified stationary phase material with a non-hydroxyl-terminated polyethylene glycol reagent. In some embodiments, the non-hydroxyl-terminated polyethylene glycol reagent is a methoxyl-terminated polyethylene glycol silane reagent. In some embodiments, the methoxyl-terminated polyethylene glycol silane reagent has the following formula:

[0058]

[0059] in:

[0060] At least one of R1, R2 and R3 is OMe, OEt, Cl or N(CH3)2;

[0061] m is an integer from approximately 1 to approximately 10; and

[0062] n is an integer from approximately 2 to approximately 20.

[0063] In some implementations, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some implementations, m is 2 or 3. In some implementations, m is 3 (i.e., propyl).

[0064] In some implementations, n is about 5 to about 15. In some implementations, n is about 6 to about 12, such as about 6 to about 9.

[0065] In some implementations, the methoxy-terminated PEG silane is 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]trichlorosilane or 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]tris(dimethylamino)silane.

[0066] In another aspect, the present technology relates to a method for forming a stationary phase material. The method includes: providing a stationary phase material comprising porous particles containing silica; contacting the stationary phase material with a hydroxyl-terminated polyethylene glycol reagent; treating the reaction mixture with a hydrolyzed aqueous solution to surface-modify the porous silica particles; and further treating the stationary phase material with a methoxyl-terminated polyethylene glycol reagent.

[0067] Another aspect of this technology relates to a stationary phase material for size exclusion chromatography, the stationary phase material being produced by the methods described above (e.g., contacting with a hydroxyl-terminated polyethylene glycol reagent; treatment with a hydrolyzed aqueous solution; and further treatment with a methoxyl-terminated polyethylene glycol reagent). In some embodiments, the stationary phase particles are produced using the methods described above, wherein contacting with the hydroxyl-terminated polyethylene glycol reagent and treatment with a hydrolyzed aqueous solution are repeated / performed for a period of time sufficient to establish one or more coatings (i.e., a hydroxyl-terminated PEG coating) prior to treatment with a methoxyl-terminated polyethylene glycol reagent (e.g., bonding).

[0068] The above aspects, features, and implementation schemes are further described in conjunction with the accompanying figures and examples provided below. Attached Figure Description

[0069] To provide an understanding of embodiments of this technology, reference is made to the accompanying drawings, which are not necessarily drawn to scale. The drawings are merely illustrative and should not be construed as limiting the technology. The disclosure described herein is illustrated in the drawings by way of example, not limitation.

[0070] Figure 1A Describing the modification of PEG surfaces by NISTmAb with proto-hydroxyl-terminated surfaces An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0071] Figure 1B Depicting NISTmAb in reference BTEE / TEOS coated hydroxyl-terminated PEG bonds An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0072] Figure 1C Depicting NISTmAb in reference An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0073] Figure 1D Describing the modification of trastuzumab emtansine (Kadcyla; Genentech) on PEG surface with proto-hydroxyl-terminated PEG. An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and acetonitrile at varying concentrations.

[0074] Figure 1E Describing trastuzumab emtansine (Kadcyla; Genentech) in a BTEE / TEOS-coated / hydroxyl-capped PEG bond. An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and acetonitrile at varying concentrations.

[0075] Figure 1F Describing trastuzumab emtansine (Kadcyla; Genentech) in reference An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and acetonitrile at varying concentrations.

[0076] Figure 2A Describing the modification of PEG surfaces by NISTmAb with proto-hydroxyl-terminated surfaces An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0077] Figure 2B Depicting the modification of NISTmAb on reference methoxy-terminated PEG surfaces An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0078] Figure 2C Depicting NISTmAb in reference An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0079] Figure 2D Describing the modification of trastuzumab emtansine (Kadcyla; Genentech) on PEG surface with proto-hydroxyl-terminated PEG. An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and acetonitrile at varying concentrations.

[0080] Figure 2E Describing the modification of trastuzumab emtansine (Kadcyla; Genentech) on a reference methoxyl-terminated PEG surface. An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and acetonitrile at varying concentrations.

[0081] Figure 2F Describing trastuzumab emtansine (Kadcyla; Genentech) in reference An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and acetonitrile at varying concentrations.

[0082] Figure 3A Depicting the modification of NISTmAb on the surface of prototype hydroxyl-terminated polyethylene glycol (PEG) An exemplary chromatographic separation was performed on a silica particle SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0083] Figure 3B Describing the surface modification of trastuzumab emtansine (Kadcyla; Genentech) on hydroxyl-terminated polyethylene glycol (PEG) surfaces. An exemplary chromatographic separation was performed on a silica particle SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and acetonitrile at varying concentrations.

[0084] Figure 3C Depicting the effect of NISTmAb on the modification of prototype hydroxyl-terminated polyethylene glycol (PEG) surfaces after high pH application. An exemplary chromatographic separation was performed on a silica particle SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0085] Figure 3D Depicting the modification of trastuzumab emtansine (Kadcyla; Genentech) on the surface of hydroxyl-terminated polyethylene glycol (PEG) after high pH application. An exemplary chromatographic separation was performed on a silica particle SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and acetonitrile at varying concentrations.

[0086] Figure 4A Depicting NISTmAb in reference BTEE / TEOS coated hydroxyl-terminated PEG bonds An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0087] Figure 4B Describing trastuzumab emtansine (Kadcyla; Genentech) in a BTEE / TEOS-coated / hydroxyl-capped PEG bond. An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and acetonitrile at varying concentrations.

[0088] Figure 4C Depicting NISTmAb in reference BTEE / TEOS coated hydroxyl-terminated PEG bonds after high pH application An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0089] Figure 4D Describing the effect of trastuzumab emtansine (Kadcyla; Genentech) on PEG-terminated / hydroxyl-capped BTEE / TEOS coated substrates after high pH application. An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and acetonitrile at varying concentrations.

[0090] Figure 5A Describing the NISTmAb in PEG-bonded / methoxy-terminated structures with protohydroxyl end-capped PEG. An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0091] Figure 5B Describing the PEG-bonded / methoxylated modification of trastuzumab emtansine (Kadcyla; Genentech) with proto-hydroxyl-terminated PEG and PEG-bonded / methoxyl-terminated PEG. An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0092] Figure 5C Depicting the effects of NISTmAb on PEG-bonded / methoxy-terminated PEG after high pH application An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0093] Figure 5D Describing the modification of trastuzumab emtansine (Kadcyla; Genentech) with PEG-bonded / methoxy-terminated PEG after high pH application, specifically the modification of PEG with PEG-bonded / methoxy-terminated PEG at the protohydroxyl end. An exemplary chromatographic separation was performed on a silica SEC column, with the mobile phase comprising an aqueous sodium phosphate buffer and sodium chloride of varying concentrations.

[0094] Figure 6 This depicts an exemplary chromatographic separation of adeno-associated virus on a prototype SEC column, which is filled with columns having an average pore size of [missing information]. The 3 μm particles were modified at least partially by OH-terminated polyethylene oxide (PEO) bonding, and the mobile phase contained 10 mM sodium phosphate buffer, 3 mM potassium chloride and 137 mM sodium chloride. Detailed Implementation

[0095] Before describing several example implementations of this technology, it should be understood that this technology is not limited to the details of the construction or process steps set forth in the following description. This technology can have other implementations and can be practiced or carried out in various ways.

[0096] definition

[0097] The following definitions are provided for the terminology used in this disclosure. Unless the context of the text in which a term appears requires a different meaning, this application will use the terms defined below.

[0098] The articles “a” and “a (kind)” are used herein to refer to a grammatical object of one or more articles (i.e., at least one). The term “about” as used throughout this specification is used to describe and indicate small fluctuations. For example, the term “about” can refer to less than or equal to ±5%, such as less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.2%, less than or equal to ±0.1%, or less than or equal to ±0.05%. Whether explicitly stated or not, all numerical values ​​herein are modified by the term “about.” Values ​​modified by the term “about” naturally include specific values. For example, “about 5.0” must include 5.0.

[0099] Chromatography is a separation method used to concentrate or separate one or more compounds (e.g., biomolecules) present in a mixture. The compounds (e.g., biomolecules) are typically present in a sample. This disclosure extensively uses the term "sample" to refer to any mixture that an individual might wish to analyze. The term "mixture" is used to refer to a fluid containing one or more dissolved compounds (e.g., biomolecules). The compound of interest present in the sample is referred to as an analyte.

[0100] Chromatography is a differential migration process. Compounds in a mixture pass through the column at different rates, resulting in their separation. Migration occurs via convection of a fluid phase (called the mobile phase) relative to a packed bed of particles or a porous monolithic structure (called the stationary phase). In some modes of chromatography, differential migration occurs due to the difference in affinity of the analyte for the stationary and mobile phases.

[0101] Size exclusion chromatography (SEC) is a class of chromatographic methods that separate or isolate analytes in a mixture based on hydrodynamic radii. In SEC, separation occurs due to differences in the ability of analytes to detect the volume of a porous stationary phase medium. See, for example, Modern Size-Exclusion Chromatography: Practice of Gel Permeation and Gel Filtration Chromatography by A.M. Striegel et al., 2nd ed., Wiley Press, NJ, 2009. SEC is commonly used for the separation of macromolecules or molecular complexes. For example, but not limited to, many biologically derived macromolecules (“biomolecules”) are analyzed by SEC, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, polysaccharides, antibody-drug conjugates, and fragments and complexes of any of them. Synthetic polymers, plastics, etc., are also analyzed by SEC.

[0102] SEC is typically performed using columns with a packed bed of particles. A packed bed of particles is either a separation medium or a stationary phase through which the mobile phase flows. The column is positioned in fluid communication with a pump and an injector. The sample is loaded onto the column under pressure via the injector, and the pump propels the sample components and mobile phase through the column. Components in the sample exit the column or elute from it, with the largest molecules (largest hydrodynamic radius) exiting first and the smallest molecules exiting last.

[0103] The column is positioned in fluid communication with the detector, which detects changes in the properties of the mobile phase as it leaves the column. The detector registers these changes and records them as a graph, called a chromatogram, used to determine the presence of an analyte and, in the following embodiments, to determine its concentration. The time it takes for the analyte to leave the column (retention time) indicates the size of the molecule. The molecular weight can be estimated using a standard calibration curve. Examples of detectors used in SECs include, but are not limited to, refractive index detectors, UV detectors, light scattering detectors, and mass spectrometers.

[0104] "Hybrid" (including "inorganic-organic hybrid materials") refers to inorganic structures in which organic functional groups are integrated with the internal or "skeleton" inorganic structure and the surface of the hybrid material. The inorganic portion of the hybrid material can be, for example, alumina, silicon dioxide, titanium, cerium, or zirconium or oxides thereof, or ceramic materials. Exemplary hybrid materials are shown in U.S. Patents 4,017,528, 6,528,167, 6,686,035, and 7,175,913, each of which is incorporated herein by reference in its entirety. A non-limiting example of an inorganic-organic hybrid material is an empirically formulated SiO2(O 1.5 SiCH2CH2SiO 1.5 ) 0.25 Ethylene-bridged hybrid materials.

[0105] The terms "polyethylene glycol" and "polyethylene oxide" are used synonymously in this document; both terms refer to polyethylene glycol having the formula -(O-CH2CH2). n -OH oligomeric or polymeric polyether compounds. Therefore, the abbreviations “polyethylene glycol” and “polyethylene oxide”, “PEG” and “PEO” are used synonymously in this document.

[0106] The term "methoxy-terminated polyethylene glycol," abbreviated as "MeO-PEO" or "MeO-PEG" in this document, refers to polyethylene glycol with the formula -(O-CH2CH2). n -OMe is an oligomeric or polymeric polyether compound. Unlike hydroxyl-terminated polyethylene glycol (HO-PEG), MeO-PEG does not have available free hydroxyl groups (OH) and has been capped with methyl groups.

[0107] As used herein, the term "surface modification" refers to the process of modifying a material surface to improve its properties by altering its physical and / or chemical properties. As used herein, the term "surface modified" refers to a material (e.g., porous stationary phase particles or core material) that has been reacted with surface-modifying groups ("surface modifiers") to covalently, non-covalently, adsorb, or otherwise attach the surface modifiers to the surface of a core material or a stationary phase material. In some embodiments, the surface-modifying groups are attached to the surface of the material via siloxane bonds. For example, the surface of a silica or hybrid silica material contains silanol groups that can react with one or more reactive organosilane reagents (e.g., halogenated or alkoxy-substituted silanes) to create Si-O-Si-C bonds. Surface modification can be a bonded surface or a coated surface.

[0108] The term "bonded surface" refers to a material (e.g., porous stationary phase particles or core materials) that has a silane molecule monolayer primarily covalently attached due to the bonding reaction between surface-modifying groups and available hydroxyl groups on the material surface.

[0109] The term "coated surface" refers to a material (e.g., porous stationary phase particles or core material) that has multiple layers of surface-modifying groups due to the formation of oligomers and polymers of surface-modifying groups and horizontal and vertical polymerization reactions on the material surface.

[0110] The phrase “at least a substantial portion” used in this document to describe the degree of modification (i.e., bonding or coating) means that the surface density of the modifier (e.g., hydroxyl-terminated polyethylene glycol) on the surface of the stationary phase particles is at least about 0.5 micromoles (0.5 μmol / m²) per square meter of particle surface area. 2 The surface density of the modified material can be determined by calculating the difference in carbon percentage between the particles before and after surface modification, as measured by elemental analysis. The surface density, as reported herein, is determined based on this calculation.

[0111] Unless otherwise indicated or contradicted by the context, the term “surface” for stationary phase particles as used herein is intended to refer to the outermost extent of the particle surface.

[0112] The embodiments of this disclosure are now described in detail, and it should be understood that such embodiments are merely exemplary. These embodiments constitute what the inventors now consider to be the best mode for practicing this technology. Those skilled in the art will recognize that such embodiments are capable of modifications and alterations.

[0113] stationary phase materials

[0114] Size exclusion chromatography (SEC) is performed on a stationary phase material that has a size-based affinity for the analyte. Ideally, SEC separations would occur solely based on the size of the analyte molecules; however, non-specific secondary interactions between the analyte and the stationary phase reduce separation efficiency and quality. The most common secondary interactions are ionic and hydrophobic interactions, both of which lead to poor chromatographic performance, including peak broadening, peak tailing, and losses in resolution and separation efficiency.

[0115] At least two types of ion interactions can occur. When the protein analyte and the stationary phase carry the same charge, ion repulsion occurs due to electrostatic repulsion (reducing protein elution time). When the protein and the stationary phase carry opposite charges, ion exchange occurs (increasing elution time). To improve the ionic properties of the stationary phase surface, it is common practice to derivatize the stationary phase material with hydrophobic silanes (e.g., silica or inorganic-organic hybrid particles). Increased particle hydrophobicity reduces ion interactions but can introduce additional hydrophobic interactions.

[0116] Compared to unmodified proteins, antibody-drug conjugates (ADCs) often exhibit increased hydrophobicity due to their payload conjugation. This payload can interact with the hydrophobic regions of the modified particles, leading to poor separation quality. Surface modifications (e.g., glycol bonding, polyethylene glycol (PEG)-based bonding) can improve these interactions to varying degrees. While these surface modifications can reduce secondary interactions in size exclusion separation, they have their own drawbacks. The main disadvantages of glycol-bonded / coated surfaces are the persistent presence of hydrophobic secondary interactions and the associated need for complex mobile phase development.

[0117] Current PEG-based modifications, such as methoxy-terminated polyethylene glycol bonding (MeO-PEG), have resulted in more hydrophilic, protein-resistant, non-toxic, and biocompatible surfaces. PEG-based modifications reduce non-specific interactions and have been incorporated into many different products and platforms. This ubiquity is partly due to its chemical capabilities and partly due to its ease of manipulation. PEG is suitable for both two-dimensional and three-dimensional applications, providing a coating for materials such as porous particles used in SEC. PEG possesses the chemical and structural properties common to most protein-resistant materials and molecules: hydrophilicity, electroneutrality, and hydrogen bond acceptor / donor properties. PEG is known to produce a very stable hydration layer due to the almost matching hydrogen bond network of water between adjacent ether oxygen atoms. See, for example, Daley et al., The Journal of Physical Chemistry B 2017, 121(46), 10574-10582. This hydration layer serves to repel non-specific interactions, including ionic and hydrophobic interactions. In addition to surface hydration, the flexibility of PEG chains is also considered to play an important role in protein resistance. Optimal protein resistance can be achieved when the surface hydration and steric repulsion of flexible chains work together (Chen et al., Polymer 2010, 51(23), 5283-5293). Molecular simulations and numerous experimental studies have confirmed that grafting density, chain length, steric hindrance, and chain conformation play important roles in resistance performance. See, for example, Szleifer, I. Biophysical Journal 1997, 72, 595-612; Oelmeier et al., BMC Biophysics 2012, 5, 14-26; Bernhard et al., Physical Chemistry Chemical Physics 2017, 19, 28182-28188; and Sanchez-Cano, C. et al., International Journal of Molecular Sciences 2020, 21, 1007). Although there are different ideas about the effect of chain length on protein resistance, the reported results and models demonstrate the importance of high grafting density. Generally, PEG-based modifications offer more effective resistance to nonspecific secondary interactions compared to glycol-bonded surfaces. However, the most common PEG-based bonding phase, methoxy-terminated PEG, can lead to poor peak shape and low efficiency in SEC when used as the sole surface modifier.

[0118] Surprisingly, according to this disclosure, it has been found that modification of porous silica particles with short-chain, hydroxyl-terminated polyethylene glycol (HO-PEG) bonding allows for a significant reduction in secondary interactions, improved peak shapes, and increased versatility of the mobile phase, without negatively impacting the chromatographic performance of potential analytes in a wide array.

[0119] Without being bound by theory, it is believed that for low molecular weight PEG chains, the conformation of the PEG chain on the surface depends on the chain length and the end-capping groups of the chain, and that the difference in chain conformation of the hydroxyl-capped PEG chains of the present invention, relative to methoxy-capped PEG chains, results in differences in mass transfer and chromatographic performance.

[0120] This disclosure provides a stationary phase material for size exclusion chromatography. The stationary phase material comprises particles with a hydroxyl-terminated polyethylene glycol (PEG) modified surface. While the hydroxyl-terminated PEG modified surface offers certain advantages, these advantages may not be realized under all operating conditions. For example, although in some embodiments the hydroxyl-terminated PEG modified porous silica particles of this disclosure exhibit improved peak shape relative to reference methoxy-terminated PEG modified porous silica particles, the hydroxyl-terminated PEG modified porous silica particles lack stability under alkaline conditions, making them potentially unsuitable for use with higher pH mobile phases.

[0121] However, further modification of the silica particles according to this disclosure has been found to have surprising advantages. For example, hydroxyl-terminated polyethylene glycol-modified silica particles can be further modified with methoxyl-terminated PEG. Such modification can provide certain additional advantages in different operating environments and applications, such as enhanced stability under alkaline conditions.

[0122] The properties of the stationary phase material and its surface modification are further described below.

[0123] Particles

[0124] This disclosure provides a stationary phase material for size exclusion chromatography. Such a material may consist of one or more particles (i.e., "base material"), such as one or more spherical particles. The particles are typically spherical, but may also be of any shape suitable for chromatographic applications.

[0125] The particles have a particle size or particle size distribution. Particle size can be measured, for example, using a Beckman Coulter Multisizer 3 instrument as follows: The particles are uniformly suspended in a 5% lithium chloride methanol solution. For each sample, a count of more than 70,000 particles can be run in volume mode using a 30 μm well. Using the Coulter principle, the volume of the particle is converted to its diameter, where the particle diameter is the equivalent sphere diameter, which is the diameter of a sphere with the same volume as the particle. Particle size can also be determined by optical microscopy.

[0126] The particles disclosed herein typically have an average diameter of about 1 μm to about 50 μm, such as a size distribution of about 1 μm, about 2 μm, about 5 μm, about 10 μm, or about 20 μm to about 30 μm, about 40 μm, or about 50 μm. In some embodiments, the particles have a diameter with an average size distribution of about 1 μm to about 20 μm. In some embodiments, the particles have a diameter with an average size distribution of about 1.7 μm to about 5 μm. In some embodiments, the particles have a size distribution with an average diameter of about 3 μm.

[0127] In some embodiments, the particles comprise silica. The particles may be porous or non-porous. In some embodiments, the silica particles are porous and may be fully porous or surface porous. Porous materials have pore size or pore size distribution. The average pore size (pore diameter) may vary depending on the intended analyte. As described in U.S. Patent No. 5,861,110, the pore diameter may be calculated from 4V / s BET, pore volume, or pore surface area.

[0128] Pore ​​diameters are typically chosen to allow molecules to diffuse freely within the analyte and mobile phase, enabling them to interact with the stationary phase. In some embodiments, porous silica particles have a pore size of approximately [missing information]. to approximately or about to approximately The average pore size. For example, the average pore size could be approximately... about about about about about or about to approximately about about about about about about about about or about In some implementations, the average aperture is approximately to approximately In some implementations, the average aperture is approximately In some implementations, the average aperture is approximately In some implementations, the average aperture is approximately In some implementations, the average aperture is approximately In some implementations, the average aperture is approximately In some implementations, the average aperture is approximately to approximately or about to approximately In some implementations, the average aperture is approximately In some implementations, the average aperture is approximately

[0129] The porous silica particles have a surface, and at least a substantial portion of this surface is modified with hydroxyl-terminated polyethylene glycol (PEG). The coverage density of the hydroxyl-terminated PEG on the surface of the modified porous silica particles can vary. For example, in some embodiments, the hydroxyl-terminated PEG modifier is at approximately 0.5 μmol / m³. 2 Approximately 15 μmol / m 2 The density exists on the surface of porous silica particles. In some embodiments, the hydroxyl-terminated polyethylene glycol modifier is present at approximately 0.5 μmol / m³. 2 Approximately 5 μmol / m 2 or about 1 μmol / m 2 To approximately 2.0 μmol / m 2 The density exists on the surface of porous silica particles.

[0130] In some embodiments, hydroxyl-terminated polyethylene glycol has the following formula:

[0131]

[0132] in:

[0133] m is an integer from approximately 1 to approximately 10;

[0134] n is an integer from approximately 2 to approximately 50; and

[0135] The wavy line indicates the attachment point to the surface of the porous silica particles.

[0136] Not wanting to be bound by theory, it is believed that the apparent chain conformation of polyethylene glycol units depends at least in part on the chain length.

[0137] In some implementations, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some implementations, m is 2 or 3. In some implementations, m is 3 (i.e., propyl).

[0138] In some embodiments, n is about 2, about 5, about 10, about 15, or about 20 to about 25, about 30, about 35, about 40, about 45, or about 50. In some embodiments, n is about 5 to about 15. In some embodiments, n is about 8 to about 12. In a particular embodiment, m is 3, and n is about 8 to about 12. In some embodiments, n is an average value representing a mixture of different polyethylene glycol chain lengths. For example, in some embodiments, n can be 8 to 12, meaning that the average distribution of chain lengths falls between 8 and 12 polyethylene glycol units. Such embodiments reflect the average chain length distribution in commercially available trialkoxysilyl alkyl polyethylene glycols, for example, those that can be used as surface-modifying agents in embodiments of this disclosure. In other embodiments, n can be a specific value, such as about 8, about 9, or about 10 to about 11 or about 12.

[0139] In some embodiments, the hydroxyl-terminated polyethylene glycol is bifunctional, forming a bridged (“bridged”) polyethylene glycol when attached to the surface of porous particles. In some embodiments, the bridged polyethylene glycol comprises a polyethylene glycol unit and also comprises two alkyl moieties, each alkyl moieties having exposed hydroxyl groups. In such embodiments, the exposed hydroxyl groups are the hydroxyl-terminated of the hydroxyl-terminated polyethylene glycol. In some embodiments, the bridged polyethylene glycol has the following formula:

[0140]

[0141] The wavy line indicates the attachment point to the surface of the porous silica particles; and m and n are each as defined above. In such embodiments, the modifier is bis-(silylalkyl-2-hydroxyalkoxy)polyethylene glycol. In some embodiments, m is 3, and n is 5 to 8.

[0142] In some embodiments, hydroxyl-terminated polyethylene glycol is directly attached to the hydroxyl groups on the initial surface (i.e., the natural or synthetic surface) of the porous silica particles. The initial surface refers to the porous particles that have not undergone any coating or bonding treatment and are in their natural state at the time of preparation. In such embodiments, this surface can be described as a bonded surface after reaction with the hydroxyl-terminated polyethylene glycol reagent. A non-limiting description of the hydroxyl-terminated polyethylene glycol bonded particles (1) is illustrated below:

[0143]

[0144] In other embodiments, the natural or synthetic surface is modified with a coating layer before or simultaneously with the attachment of the hydroxyl-terminated polyethylene glycol. In such embodiments, the hydroxyl-terminated polyethylene glycol is attached to the natural surface of the porous silica particles via a complex network of siloxane bonds.

[0145] In some embodiments, the hydroxyl-terminated polyethylene glycol reagent is partially polymerized by hydrolytic condensation with itself or with TEOS prior to reaction with the hydroxyl groups on the initial surface of the porous particles. In such embodiments, the resulting surface-modified particles can be described as hydroxyl-terminated polyethylene glycol coated surfaces. A non-limiting description of hydroxyl-terminated polyethylene glycol coated particles (2) is illustrated below:

[0146]

[0147] The following is a non-limiting description of hydroxyl-terminated polyethylene glycol / TEOS coated particles (3):

[0148]

[0149] In some embodiments, the initial surface of the porous particles is coated with a silane reagent to form a secondary surface of oligomeric and / or polymeric siloxane multilayers on the particles. Such oligomeric and / or polymeric siloxane multilayers include those produced by the reaction of the particle surface with, for example, 1,2-bis(triethoxysilane)ethane (BTEE), tetraethyl orthosilicate (TEOS), or a partially hydrolyzed condensation product of BTEE and TEOS. A hydroxyl-terminated polyethylene glycol reagent is then bonded to the coated surface. A non-limiting depiction of hydroxyl-terminated polyethylene glycol-bonded and BTEE / TEOS-coated particles (4) is illustrated below:

[0150]

[0151] In some embodiments, the hydroxyl-terminated polyethylene glycol-modified porous silica particles also include a surface coating derived from the reaction of the porous particle surface with BTEE, TEOS, or a partially hydrolyzed condensation product of BTEE and TEOS.

[0152] In some embodiments, the porous silica particles comprise a surface coating derived from the reaction of the porous particle surface with a partially hydrolyzed condensation product of a hydroxyl-terminated polyethylene glycol reagent, a partially hydrolyzed condensation product of a hydroxyl-terminated polyethylene glycol reagent and TEOS, or a combination thereof.

[0153] In some embodiments, the porous silica particles comprise or further comprise a surface coating derived from the reaction of the porous particle surface with a partially hydrolyzed condensation product of a polyethylene glycol silane reagent, a partially hydrolyzed condensation product of a polyethylene glycol silane reagent and TEOS, or a combination thereof. Suitable polyethylene glycol-based reagents include, but are not limited to, bridged polyethylene glycol-based reagents as discussed above and polyethylene glycol-based reagents having masked or protected hydroxyl groups. In some embodiments, the hydroxyl groups may be terminal or may be otherwise attached to the main chain of the reagent (e.g., hydroxyl groups exposed on the carbon chain). In some embodiments where the hydroxyl groups are masked or protected, the masking or protecting groups may be removed prior to chromatography with particles having a surface modified with such reagents (i.e., providing exposed or terminal hydroxyl groups). Those skilled in the art will recognize such protecting groups and understand how to maintain or remove such protecting groups using standard chemical conditions known to those skilled in the art. Table 1 provides a non-limiting list of suitable polyethylene glycol-based silane reagents that may be used as a supplement to or alternative to the hydroxyl-terminated polyethylene glycol reagents described above.

[0154] Table 1. Exemplary Additional or Alternative Polyethylene Glycol Silane Reagents

[0155]

[0156]

[0157] In some embodiments, the porous silica particles with hydroxyl-terminated polyethylene glycol-modified surfaces further comprise surface bonding or coating derived from the reaction of hydroxyl-terminated polyethylene glycol-modified porous silica particles with a non-hydroxyl-terminated polyethylene glycol reagent. In some embodiments, the reagent is selected from Table 1. In some embodiments, the reagent is 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]trichlorosilane or 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]tris(dimethylamino)silane.

[0158] In some embodiments, the porous silica particles with hydroxyl-terminated polyethylene glycol (PEG) modified surfaces are bonded with hydroxyl-terminated PEG. In some embodiments, the porous silica particles with hydroxyl-terminated PEG modified surfaces are coated with hydroxyl-terminated PEG. In some embodiments, the porous silica particles with hydroxyl-terminated PEG modified surfaces are coated with hydroxyl-terminated PEG / TEOS. In some embodiments, the porous silica particles with hydroxyl-terminated PEG modified surfaces are hydroxyl-terminated PEG bonded to a BTEE / TEOS coating.

[0159] In any of these embodiments, the modified porous particles may further comprise a methoxy-terminated polyethylene glycol surface modification (e.g., bonding). Non-limiting sketches illustrating possible configurations of such bonding and coating arrangements are provided below with structures 4, 5, 6, and 7. As those skilled in the art will recognize, such structures would have a very complex network of silicon-oxygen bonds that cannot be adequately represented structurally. Therefore, structures 4, 5, 6, and 7 are provided only to illustrate the general concept of the coating and bonding combinations disclosed herein. Structure 4 represents a porous particle surface coated with BTEE / TEOS and bonded with hydroxyl-terminated polyethylene glycol as described above. Structure 5 represents a porous particle surface coated with both methoxy-terminated and hydroxyl-terminated polyethylene glycol. Structure 6 represents a porous particle surface coated with both methoxy-terminated and hydroxyl-terminated polyethylene glycol / TEOS. Structure 7 represents a porous particle surface that is already hydroxyl-terminated polyethylene glycol bonded and further modified with methoxyl-terminated polyethylene glycol.

[0160]

[0161] In certain embodiments, the porous silica particles are hydroxyl-terminated polyethylene glycol (PEG) bonded, hydroxyl-terminated PEG coated, or hydroxyl-terminated PEG bonded and BTEE / TEOS coated, and further surface-modified with a methoxyl-terminated PEG reagent. Thus, in some embodiments, the porous silica particles are hydroxyl-terminated PEG bonded, hydroxyl-terminated PEG coated, or hydroxyl-terminated PEG bonded and BTEE / TEOS coated, and further methoxyl-terminated PEG modified.

[0162] In some embodiments, the methoxy-terminated PEG modifying agent is a methoxy-terminated polyethylene glycol silane agent. In some embodiments, the methoxy-terminated polyethylene glycol silane agent has the following formula:

[0163]

[0164] in:

[0165] At least one of R1, R2 and R3 is OMe, OEt, Cl or N(CH3)2;

[0166] m is an integer from approximately 1 to approximately 10; and

[0167] n is an integer from approximately 2 to approximately 20.

[0168] In some implementations, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some implementations, m is 2 or 3. In some implementations, m is 3 (i.e., propyl).

[0169] In some implementations, n is about 5 to about 15. In some implementations, n is about 6 to about 12, such as about 6 to about 9.

[0170] In some implementations, the methoxy-terminated PEG modifier is 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]trichlorosilane or 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]tris(dimethylamino)silane. Without wishing to be bound by any particular theory, it is believed that further modification with methoxy-terminated polyethylene glycol reagents of hydroxyl-terminated polyethylene glycol-bonded, hydroxyl-terminated polyethylene glycol-coated, or hydroxyl-terminated polyethylene glycol-bonded and BTEE / TEOS-coated silica particles enhances the stability of porous silica particles to alkaline conditions (e.g., above 7, such as about 8, about 9, or even about 10 pH values).

[0171] As demonstrated herein, porous silica particles with hydroxyl-terminated polyethylene glycol bonds alone exhibit excellent peak shapes (significantly better than reference methoxy-terminated PEG-bonded silica); however, after exposure to alkaline conditions (pH 8.5 at 40°C), the peak shapes significantly degraded in subsequent lower pH separations (Example 11). Figure 3C and Figure 3D contrast Figure 3A and Figure 3B Similarly, silica particles with BTEE / TEOS coating and individual hydroxyl-terminated polyethylene glycol bonds (i.e., silica particles with inorganic-organic hybrid coatings such as ethylene-bridged hybrid coatings and hydroxyl-terminated polyethylene glycol bonds) exhibited excellent peak shapes (again, much better than reference methoxy-terminated PEG-bonded silica), but after exposure to alkaline conditions (pH 8.5), the peak shapes degraded in subsequent lower pH separations (Example 12). Figure 4C and Figure 4D contrast Figure 4A and Figure 4B The peak shape is not as damaged as that of silica particles with individual hydroxyl-terminated polyethylene glycol bonds. Figure 4C and Figure 4D contrast Figure 3C and Figure 3D However, the damaged peak shape indicates the sensitivity of the stationary phase material to degradation under certain conditions.

[0172] In contrast, silica particles with hydroxyl-terminated polyethylene glycol bonds and further modified with methoxyl-terminated polyethylene glycol exhibited superior peak shape and also demonstrated excellent high pH stability. Specifically, refer to Example 13. Figures 5A to 5DThe results shown demonstrate that the column of Example 8 (PEG modified with hydroxyl and methoxy end caps) exhibits outstanding high pH stability (e.g., no significant difference in chromatographic results of NISTmAb and Kadcyla analytes before and after high pH application). Figure 5A and Figure 5B contrast Figure 5C and Figure 5D ).

[0173] The ratio of hydroxyl-terminated polyethylene glycol groups to methoxyl-terminated polyethylene glycol groups on the porous silica surface can vary. For example, in some embodiments, the molar ratio is about 2:1 or about 1:1.

[0174] Methods for preparing stationary phase materials for SEC

[0175] This disclosure provides a simple and universal method for bonding hydroxyl-terminated polyethylene glycol (PEG) layers to a silica surface, wherein the PEG grafting density is remarkably high. The method typically involves allowing porous silica particles to react with a silylalkyl PEG reagent having one or more leaving groups on a silane. The distance between some adjacent ortho-hydroxyl groups on the porous particle surface allows for bifunctional reactions between the ortho-hydroxyl groups and bifunctional or trifunctional reagents. When adjacent hydroxyl groups on the surface are not properly spaced for bifunctional reactions, only monofunctional reactions occur.

[0176] The reaction is typically carried out according to standard methods, for example, by reacting porous silica particles with a suitable reagent in an organic solvent under reflux conditions. Organic solvents such as toluene are commonly used for this reaction.

[0177] In some embodiments, the method includes: providing a stationary phase material comprising porous particles containing silica (or silica particles already coated with an inorganic / organic hybrid material such as BTEE / TEOS partially hydrolyzed condensate); contacting the stationary phase material with a hydroxyl-terminated polyethylene glycol reagent; treating the reaction mixture with a hydrolyzed aqueous solution to surface-modify the porous silica particles; and further treating the stationary phase material with a methoxyl-terminated polyethylene glycol reagent. The stationary phase material produced by this method (contacting with a hydroxyl-terminated polyethylene glycol reagent; treating with a hydrolyzed aqueous solution; and further treating with a methoxyl-terminated polyethylene glycol reagent) is within the scope of this technology. In some embodiments, the stationary phase particles are produced using the above method, wherein contacting with the hydroxyl-terminated polyethylene glycol reagent and treating with a hydrolyzed aqueous solution are repeated / performed for a period of time sufficient to establish one or more coatings (i.e., hydroxyl-terminated coatings) prior to treatment with the methoxyl-terminated polyethylene glycol reagent (e.g., bonding).

[0178] While the above methods include contacting hydroxyl-terminated polyethylene glycol (PEG) reagents prior to treatment with methoxy-terminated PEG reagents, other production methods are also within the scope of this disclosure. For example, in some embodiments, porous silica particles are first contacted with methoxy-terminated PEG reagents, followed by a reaction with hydroxyl-terminated PEG reagents. In other embodiments, the porous silica particles may be contacted simultaneously or substantially simultaneously with both hydroxyl-terminated and methoxy-terminated PEG reagents.

[0179] In some embodiments, the method includes providing a stationary phase material comprising porous particles containing silica; and contacting the stationary phase material with a polyethylene glycol reagent having hydroxyl-terminated caps having the following formula:

[0180]

[0181] in:

[0182] At least one of R1, R2 and R3 is OMe, OEt, Cl or N(CH3)2;

[0183] m is an integer from approximately 1 to approximately 10; and

[0184] n is an integer from approximately 2 to approximately 50.

[0185] To form a reaction mixture; and

[0186] The reaction mixture was treated with a hydrolyzed aqueous solution to modify the surface of the porous silica particles.

[0187] In some embodiments, R1, R2, and R3 are each OMe, OEt, Cl, or N(CH3)2. In some embodiments, R1, R2, and R3 are each OMe or each OEt.

[0188] In some implementations, m is approximately 3, or is 3.

[0189] In some embodiments, n represents the average value of a mixture of different polyethylene glycol chain lengths. For example, in some embodiments, n can be 8 to 12, meaning that the average distribution of chain lengths falls between 8 and 12 polyethylene glycol units. In other embodiments, n can be a specific value, such as about 8, about 9, or about 10 to about 11 or about 12.

[0190] In some embodiments, R1, R2, and R3 are each OMe, m is 3, and n is 3. In some embodiments, R1, R2, and R3 are each OMe, m is 3, and n is 8 to 12. In some embodiments, R1, R2, and R3 are each OMe, m is 3, and n is 4.

[0191] In some embodiments, the method includes providing a stationary phase material comprising porous particles containing silica; and contacting the stationary phase material with a polyethylene glycol reagent having bifunctional hydroxyl-terminated caps having the following formula:

[0192]

[0193] R1, R2, and R3, m and n are each as defined above. In some implementations, m is 3, n is 5 to 8, and R1, R2, and R3 are each OMe or each OEt.

[0194] The amount of hydroxyl-terminated polyethylene glycol reagent used can vary depending on the desired surface coverage. In some embodiments, this amount is based on the surface area of ​​the particles. In some embodiments, the amount is about 1 μmol / m². 2 Approximately 40 μmol / m 2 .

[0195] In some embodiments, a catalyst is added to the reaction mixture to promote the reaction between hydroxyl groups on the surface of the porous particles and hydroxyl-terminated polyethylene glycol. It is believed that, without being bound by theory, in the case of less reactive hydroxyl-terminated polyethylene glycol reagents (e.g., trimethoxy or triethoxysilylpropyl polyethylene glycol), the presence of a suitable catalyst is a decisive factor in the extent of the reaction, and therefore a decisive factor in the coverage density of the hydroxyl-terminated polyethylene glycol modifier on the surface of the porous silica particles. In some embodiments, the catalyst is an organic base such as pyridine or imidazole. In some embodiments, the catalyst is an acid. Particularly suitable catalysts are inorganic acids, such as hydrochloric acid (HCl). The amount of catalyst added can vary depending on the particles, the modifier, and the desired surface coverage. In some embodiments, the amount of catalyst is based on the weight of the porous silica particles. In some embodiments, the amount of catalyst is from about 50 μL / g particles to about 1000 μL / g particles. In some embodiments, the amount of catalyst is about 200 μL / g particles.

[0196] After the reaction with the hydroxyl-terminated polyethylene glycol reagent is complete, the product mixture is typically treated with an aqueous hydrolysis solution to hydrolyze any remaining excess reagent and any residual alkoxysilyl functional groups on the surface-modified particles. In some embodiments, the hydrolysis solution is an aqueous ammonium acetate solution. In some embodiments, the hydrolysis solution is an aqueous ammonium bicarbonate solution. The resulting product is then typically washed with water and acetone and dried under reduced pressure at 70°C for 16 hours.

[0197] In some embodiments, a hydroxyl-terminated polyethylene glycol (PEG) reagent is reacted with hydroxyl groups on the initial surface of porous particles. The initial surface refers to the porous particles that have not undergone any coating or bonding treatment and are in their natural state at the time of preparation. In such embodiments, this surface can be described as a bonded surface after reaction with the hydroxyl-terminated PEG reagent. A non-limiting description of hydroxyl-terminated PEG bonded particles (1) is illustrated below:

[0198]

[0199] In some embodiments, the method includes partially polymerizing a hydroxyl-terminated polyethylene glycol (PEG) reagent with itself via hydrolytic condensation prior to reaction with hydroxyl groups on the initial surface of the porous particles. Typically, the incomplete (~50%) hydrolytic condensation product can be obtained by reacting the hydroxyl-terminated PEG reagent in ethanol (3.1 mol ethanol / mol silane) and 0.1 M HCl (13.5 g / mol silane). In some embodiments, the hydroxyl-terminated PEG reagent is [hydroxyl (polyvinyloxy)]. 8-12 [Propyl]triethoxysilane.

[0200] The solution was heated at 70°C under an inert atmosphere for 18 hours. The reaction medium temperature was then increased to 90°C for atmospheric distillation to strip the ethanol. The temperature was then increased to 100°C under an inert atmosphere and held for 1 hour. Finally, the reaction medium was cooled to room temperature to obtain the incomplete condensation reaction product. The hydroxyl-terminated polyethylene glycol reagent was then reacted with the hydroxyl groups on the coated surface.

[0201] In some embodiments, the method includes partially polymerizing a hydroxyl-terminated polyethylene glycol reagent with TEOS via hydrolytic condensation prior to reaction with hydroxyl groups on the initial surface of the porous particles. Typically, the incomplete (~50%) hydrolytic condensation product can be obtained by reacting the hydroxyl-terminated polyethylene glycol reagent with tetraethoxysilane (TEOS) (1:1 mol / mol) in ethanol (3.1 mol ethanol / mol silane) and 0.1 HCl (13.5 g / mol silane). In some embodiments, the hydroxyl-terminated polyethylene glycol reagent is [hydroxyl (polyvinyloxy)]. 8-12 [Propyl]triethoxysilane.

[0202] The solution was heated at 70°C under an inert atmosphere for 18 hours. The reaction medium temperature was then increased to 90°C for atmospheric distillation to strip the ethanol. The temperature was then increased to 100°C under an inert atmosphere and held for 1 hour. Finally, the reaction medium was cooled to room temperature to obtain the incomplete condensation reaction product. The hydroxyl-terminated polyethylene glycol reagent was then reacted with the hydroxyl groups on the coated surface.

[0203] In each of the above embodiments describing the partially hydrolyzed condensation product, the partially hydrolyzed condensation product is then reacted with porous particles, which may be silica particles or silica particles already coated with, for example, the BTEE / TEOS partially hydrolyzed condensation product described herein. For example, the BTEE / TEOS partially hydrolyzed condensation product provides an inorganic / organic hybrid coating on the silica particles. U.S. Utility Application Serial No. 16 / 082,823, published March 28, 2019 as US 2019 / 0091657A1, describes the coating of porous silica particles with an inorganic / organic hybrid material, and that application is incorporated herein by reference in its entirety. Typically, the particles are dispersed in a solvent and any residual water is removed by azeotropic distillation. A suitable, non-limiting example of a solvent is toluene. Typically, the reaction mixture is cooled to, for example, below about 40°C, and an appropriate amount of the partially hydrolyzed condensation product is added at a rate of about 1 g / g of particles. A catalyst is then added. In a specific embodiment, the catalyst is an aqueous solution of ammonium hydroxide (NH4OH) at a concentration of approximately 0.05 g base / g particles. After the reaction is complete, the particles are separated by filtration, washed, and dispersed in a 70 / 30 (v / v) water / ethanol mixture (10 mL / g). Ammonium hydroxide (1 g NH4OH / g particles) is added, and the mixture is stirred at 50°C for 2 hours. The reaction is then cooled to below approximately 40°C, and the particles are separated by filtration. The separated particles are washed and vacuum dried at 70°C for 16 hours. The above process can be repeated as needed.

[0204] The modified particles were then exposed to high temperatures (100-140°C) and high pH (8-9.8) according to the hydrothermal treatment method described in Jiang's U.S. Patents Nos. 6,686,035, 7,223,473, and 7,919,177 and Wyndham's International Patent Application Publication No. WO2008 / 103423. The modified particles were then dispersed in a 1.0 M HCl solution (8.4 mL / g particles) and the mixture was stirred at 100°C for 20 hours. The reaction was then cooled to <40°C and the particles were separated by filtration. The separated particles were washed with water until the pH of the filtrate was above 5, and then washed with methanol. Finally, the separated particles were vacuum dried at 70°C for 16 hours.

[0205] In some embodiments, the method includes first coating the initial surface of the porous particles with a silane reagent (i.e., reacting the initial surface of the porous particles) to form an oligomeric and / or polymeric siloxane multilayer on the particle surface. Suitable silane reagents include 1,2-bis(triethoxysilane)ethane (BTEE), tetraethyl orthosilicate (TEOS), and partially hydrolyzed condensation products of BTEE and TEOS. In some embodiments, the silane reagent is BTEE, TEOS, partially hydrolyzed condensation products of BTEE and TEOS, partially hydrolyzed condensation products of hydroxyl-terminated polyethylene glycol reagents, partially hydrolyzed condensation products of hydroxyl-terminated polyethylene glycol reagents and TEOS, or combinations thereof.

[0206] Typically, the incomplete (~68%) hydrolysis-condensation product can be obtained by reacting a hydroxyl-terminated polyethylene glycol reagent with tetraethoxysilane (TEOS) (1:4 mol / mol) in ethanol (3.1 mol ethanol / mol silane) and 0.1 M HCl (19.7 g / mol silane). The solution was heated at 70 °C under an inert atmosphere for 18 hours. The temperature of the reaction medium was then increased to 90 °C for atmospheric distillation to strip the ethanol. The temperature was then increased to 100 °C under an inert atmosphere and held for 1 hour. Finally, the reaction medium was cooled to room temperature to obtain the incomplete condensation product.

[0207] In some embodiments, the method further includes reacting the hydroxyl-terminated polyethylene glycol-modified surface with an additional polyethylene glycol silane reagent. A non-limiting list of suitable example reagents is provided in Table 1. Typically, reacting the hydroxyl-terminated polyethylene glycol-modified surface with an additional polyethylene glycol silane reagent involves dispersing porous silica particles in a solvent and removing any residual water by azeotropic distillation. A suitable non-limiting example of a solvent is toluene. Typically, the reaction mixture is cooled to, for example, below about 40°C, and a catalyst is added. In a particular embodiment, the catalyst is hydrochloric acid. In some embodiments, HCl is added as a dilute solution such as 1M or 0.1M. In some embodiments, the amount of acid added is about 200 μL / g of particles.

[0208] After adding the catalyst, at approximately 1 μmol / m 2 Approximately 40 μmol / m 2An appropriate amount of silane reagent is added. In some embodiments, the additional polyethylene glycol silane is selected from Table 1. In some embodiments, the reagent is a methoxy-terminated polyethylene glycol silane reagent. The reaction is typically stirred for about 5 minutes and the temperature is increased for a period of time (e.g., 110°C for 20 hours). After cooling, the particles are separated by filtration and washed to remove residual solvent and reagent. The particles are then dispersed in a hydrolysis mixture. In a specific embodiment, the mixture comprises an aqueous solution of acetone and ammonium acetate. The mixture is typically heated for a period of time to complete the reaction, after which the particles are separated by filtration, washed, and dried.

[0209] In a particular implementation, porous particles are particles with a pore size of approximately to approximately The silica particles are hydroxyl-terminated polyethylene glycol (PEG) bonded, hydroxyl-terminated PEG coated, or hydroxyl-terminated PEG bonded and BTEE / TEOS coated, and further surface-modified with a methoxyl-terminated PEG reagent. Therefore, in some embodiments, the porous silica particles are hydroxyl-terminated PEG bonded, hydroxyl-terminated PEG coated, or hydroxyl-terminated PEG bonded and BTEE / TEOS coated, and further methoxyl-terminated PEG modified.

[0210] In some embodiments, the methoxy-terminated PEG modifying agent is a methoxy-terminated polyethylene glycol silane agent. In some embodiments, the methoxy-terminated polyethylene glycol silane has the following formula:

[0211]

[0212] in:

[0213] At least one of R1, R2 and R3 is OMe, OEt, Cl or N(CH3)2;

[0214] m is an integer from approximately 1 to approximately 10; and

[0215] n is an integer from approximately 2 to approximately 20.

[0216] In some implementations, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some implementations, m is 2 or 3. In some implementations, m is 3 (i.e., propyl).

[0217] In some implementations, n is about 5 to about 15. In some implementations, n is about 6 to about 12, such as about 6 to about 9.

[0218] In some implementations, the methoxy-terminated PEG modifier is 2-[methoxy(polyvinyloxy)] 6-9[Propyl]trichlorosilane or 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]tris(dimethylamino)silane.

[0219] The stationary phase material produced by any of the above methods (e.g., contact with a hydroxyl-terminated polyethylene glycol reagent; treatment with a hydrolyzed aqueous solution; and further treatment with a methoxyl-terminated polyethylene glycol reagent) is within the scope of this technology.

[0220] column

[0221] For use in SEC, the stationary phase is typically fixed within a housing with walls defining a chamber, such as a column having an interior for receiving the stationary phase material. This column will have a length and a diameter.

[0222] In some embodiments, the length of the column is about 300 mm or about 150 mm. In some embodiments, the length of the column is less than about 300 mm, less than about 150 mm, less than about 100 mm, or less than about 50 mm. In some embodiments, the length of the column is about 50 mm, about 30 mm, about 20 mm, or about 10 mm.

[0223] In some embodiments, the column has a bore size of approximately 4.6 mm inner diameter (id). In some embodiments, the column has a bore size greater than 4.6 mm inner diameter. In some embodiments, the column has a bore size of approximately 7.8 mm inner diameter. In some embodiments, the column has a bore size greater than 7.8 mm inner diameter. In some embodiments, the column has a bore size greater than approximately 4 mm inner diameter, greater than approximately 5 mm inner diameter, greater than approximately 6 mm inner diameter, or greater than approximately 7 mm inner diameter.

[0224] Methods for size exclusion chromatography

[0225] This article discloses a method for performing size exclusion chromatography (SEC). The method typically involves contacting a sample containing at least one analyte with a stationary phase stationary within a column, allowing a mobile phase to flow through the stationary phase for a period of time, and eluting the at least one analyte from the stationary phase in the mobile phase.

[0226] Typically, SEC (Separation of Protein Analytes) utilizes a mobile phase containing buffers and salts, and may include mild dissociation agents, surfactants, or organic solvents. The mobile phase composition serves to maintain the analyte in its native form, prevent or reduce aggregation, and produce mass separation and peak shape. Undesirable (e.g., hydrophobic) interactions that lead to poor chromatography are often mitigated through mobile phase optimization, particularly by utilizing various salts or organic co-solvents at multiple concentrations to attempt to reduce ionic and hydrophobic secondary interactions. However, such optimization is not always direct, and increasing salt concentration or adding organic co-solvents can induce aggregation or denaturation, leading to a reduction in native monomers. Furthermore, adding high concentrations of salt can exacerbate hydrophobic interactions. For example, a mobile phase with sufficient ionic strength to ensure analyte stability and solubility can unintentionally induce secondary interactions, resulting in poor peak shape and recovery. The problem of hydrophobic interactions is most readily demonstrated when separating analytes with hydrophobic moieties, such as antibody-drug conjugates (ADCs).

[0227] In one aspect, a method is provided for size exclusion chromatography of a sample containing at least one analyte, the method comprising:

[0228] a. Contact the sample with a column chromatography apparatus comprising a column having an interior for receiving a stationary phase and a stationary phase fixed within the interior of the column, wherein the fixed stationary phase comprises porous silica particles having a diameter with an average size distribution between about 1 μm and about 20 μm; to approximately The average pore size; and the porous silica particles have a surface concentration of approximately 0.5 μmol / m². 2 To approximately 5.0 μmol / m 2 Surface modification of hydroxyl-terminated polyethylene oxide;

[0229] b. Allow the mobile phase to flow through a stationary phase for a period of time, the mobile phase comprising water; a buffer solution; and optionally, an amino acid or a derivative thereof; and

[0230] c. Elute the at least one analyte from the stationary phase in the mobile phase.

[0231] Each component of the disclosed method is further described below.

[0232] Analytes

[0233] The methods for size exclusion chromatography disclosed herein include samples containing at least one analyte. It is noteworthy that the utility of the currently disclosed methods is not limited to biopharmaceutical or protein analytes. In some embodiments, the at least one analyte includes small molecule drugs, natural products, or polymers. In some embodiments, the at least one analyte includes one or more biomolecules. In some embodiments, the biomolecule is a nucleic acid (e.g., RNA, DNA, oligonucleotide), a protein (e.g., fusion protein), a peptide, an antibody (e.g., monoclonal antibody (mAb)), an antibody-drug conjugate (ADC), a polysaccharide, a virus, virus-like particles, a viral vector (e.g., gene therapy viral vector, adeno-associated virus vector), a biosimilar, or any combination thereof. In some embodiments, the at least one analyte includes nucleic acids, polysaccharides, peptides, polypeptides, proteins, or any combination thereof. In some embodiments, the at least one analyte includes adeno-associated virus, adenovirus, mRNA, DNA, plasmid, exosome, extracellular vesicle, nucleic acid encapsulated in lipid nanoparticles, or a combination thereof.

[0234] In some embodiments, the at least one analyte comprises an antibody. In some embodiments, the at least one analyte comprises a monoclonal antibody (mAb). In some embodiments, the at least one analyte comprises a high molecular weight substance or aggregate form of an antibody. In some embodiments, the at least one analyte is an antibody-drug conjugate.

[0235] mobile phase

[0236] Methods for performing SEC typically involve flowing a mobile phase through a stationary phase for a period of time. In some specific embodiments, the mobile phase and optional sample are provided using a high-performance liquid chromatography (HPLC) system.

[0237] Mobile phase compositions are used to retain analytes in their native form, prevent or reduce aggregation, and produce mass separation and peak shape. Undesirable (e.g., hydrophobic) interactions that lead to poor chromatography are often mitigated through mobile phase optimization, particularly by utilizing various salts or organic co-solvents at multiple concentrations to attempt to reduce ionic and hydrophobic secondary interactions. Surprisingly, according to this disclosure, it has been found that when using novel hydroxyl-terminated polyethylene glycol surface-modified stationary phase materials as disclosed herein, secondary interactions are reduced, simplifying the optimization of mobile phase composition and providing separation that is less sensitive to changes in mobile phase composition, pH, temperature, etc.

[0238] buffer solution

[0239] In some embodiments, the mobile phase comprises a buffer. The buffer is used to control the ionic strength and pH of the mobile phase. Many different substances can be used as buffers depending on the nature of the analyte. Non-limiting examples of suitable buffers include phosphates, tris(hydroxymethyl)aminomethane, and acetates. In some embodiments, the buffer comprises a phosphate. In some embodiments, the buffer comprises an acetate. In some embodiments, the buffer is ammonium acetate. In some embodiments, the buffer is an alkali metal phosphate. In some embodiments, the buffer is sodium phosphate or potassium phosphate. In some embodiments, the buffer is sodium dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof.

[0240] The concentration of the buffer solution can be varied depending on the desired pH and the ionic strength of the mobile phase. In some embodiments, the buffer solution is present at a concentration of about 10 mM to about 100 mM, such as about 10 mM, about 20 mM, about 40 mM, or about 50 mM to about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0241] The pH of the mobile phase can vary. In some embodiments, the pH of the mobile phase is from about 5.0 to about 8.0. In some embodiments, the pH of the mobile phase is from about 6.0 to about 7.5. In some embodiments, the pH is from about 6.0 or about 6.5 to about 7.0 or about 7.5. In some embodiments, the pH is about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5.

[0242] Salt

[0243] In some embodiments, the mobile phase contains a salt. As used herein, the term "salt" refers to an ionic compound containing an alkali metal or alkaline earth metal and a halogen (e.g., fluoride, chloride, bromide, iodide). Undesirable interactions can be mitigated by utilizing salts to reduce secondary ionic interactions. However, increasing the salt concentration can induce aggregation and thus lead to a reduction in native monomers, and adding high concentrations of salt can exacerbate hydrophobic interactions and complicate mobile phase optimization. Suitable salts, when present, include, but are not limited to, sodium chloride and potassium chloride. Suitable salt concentrations in the mobile phase range from about 10 mM to about 200 mM. In other embodiments, the mobile phase is salt-free.

[0244] Cosolvent

[0245] In some embodiments, the mobile phase contains an organic co-solvent. Organic co-solvents such as methanol, ethanol, isopropanol, or acetonitrile are common additives in SEC mobile phases. However, such co-solvents can cause protein denaturation of the protein analyte. In some embodiments, the mobile phase does not contain an organic co-solvent. In some embodiments, the mobile phase does not contain an organic co-solvent and does not contain salts. In other embodiments, the mobile phase contains a co-solvent. In the case of PEO-modified stationary phase surfaces as described herein, conformational changes in polymer chains can occur depending on the method conditions, which can lead to an increase in the hydrophobic characteristics of these surfaces. In some embodiments, for example, in the separation of antibody-drug conjugates, such increased hydrophobic characteristics can lead to poor peak shapes and reduced resolution. Therefore, in some embodiments, the mobile phase contains an organic co-solvent in an amount of up to about 15 vol% of the mobile phase. In some embodiments, the co-solvent is acetonitrile. In some embodiments, the co-solvent is isopropanol. In some embodiments, isopropanol is present in an amount of about 5 vol% to about 15 vol%.

[0246] condition

[0247] Flow rate

[0248] SEC separation using stationary phase materials as disclosed herein can be performed by flowing the mobile phase through the stationary phase at a variety of different flow rates, which can be determined by those skilled in the art based on scale, stationary phase particle size, difficulty of separation, etc. In some embodiments, the mobile phase is flowed through the stationary phase at a flow rate of about 0.2 mL / min to about 3 mL / min. In some embodiments, the flow rate is about 1 mL / min. In some embodiments, the flow rate is about 2 mL / min. In some embodiments, the flow rate is about 3 mL / min. In some embodiments, the flow rate is less than 1 mL / min, such as about 0.05 mL / min, about 0.1 mL / min, about 0.2 mL / min, about 0.3 mL / min, about 0.4 mL / min, or about 0.5 mL / min to about 0.6 mL / min, about 0.7 mL / min, about 0.8 mL / min, about 0.9 mL / min, or about 1 mL / min. In some implementations, the flow rate is about 0.25 mL / min, about 0.3 mL / min, or about 0.35 mL / min.

[0249] temperature

[0250] The temperature at which the chromatography is performed (i.e., the column temperature) can be varied. In some embodiments, the column temperature is from about 20°C to about 50°C, such as about 30°C, about 35°C, about 40°C, about 45°C, or about 50°C. In some embodiments, the methods disclosed herein are insensitive to changes in column temperature, meaning that retention times, peak shapes and heights, and analyte stability are maintained within a certain temperature range (e.g., from about 20°C to about 50°C). In other embodiments, the column temperature is less than about 45°C, less than about 35°C, or less than about 25°C, such as from about 15°C to about 25°C, or about 20°C.

[0251] time

[0252] The time required for SEC separation will vary depending on many factors, but will typically be less than approximately 60 minutes, less than approximately 50 minutes, less than approximately 40 minutes, less than approximately 30 minutes, less than approximately 20 minutes, less than approximately 10 minutes, less than approximately 5 minutes, less than approximately 4 minutes, less than approximately 3 minutes, less than approximately 2 minutes, or less than approximately 1 minute. Specifically, this time will be determined by the elution time of the analyte of interest. In some implementations, the retention time is repeatable between runs and relatively unaffected by variations in temperature, pH, buffer concentration, etc.

[0253] Detection

[0254] For methods of detecting the presence of analytes in the mobile phase leaving the stationary phase material, there are many suitable options. In some embodiments, the detector is a refractive index detector, a UV detector, a light scattering detector, a mass spectrometer, or a combination thereof. In a specific embodiment, the detector is a UV detector. Many detectors are available; however, the specific detector is a Waters detector. Adjustable UV detector (Waters Corporation, Milford, Mass., USA).

[0255] Reduction of secondary interactions

[0256] Compared to methoxy-terminated PEG surfaces, the hydroxyl-terminated PEG surfaces of this disclosure offer significant chromatographic performance advantages in terms of reduced secondary interactions (i.e., surface resistance to nonspecific adsorption of SEC analytes). Improvements in chromatographic performance can be characterized by improvements in one or more of the following: peak shape, peak area, peak tailing, analyte recovery, or reduced run-to-run variability. Such improvements can be quantified by calculating factors such as USP tailing and asymmetry at 4.4, as well as the half-width at half-maximum (FWHM) of the analyte peaks.

[0257] Providing such improvements does not require complex mobile phase optimization, which for typical bioanalyses involves adjusting pH, modulating ionic strength via salt addition, adding ionizing agents to reduce aggregation and stabilize protein conformation, and adding organic co-solvents. The need for individual optimization for each analyte increases the technical difficulty of separation. Reducing unwanted secondary interactions thus increases the value of the separation method. Furthermore, reduced mobile phase changes enable the use of a wider range of detectors. Lower salt loadings, especially non-volatile salts such as potassium chloride or sodium chloride, make mass spectrometer connections easier. Moreover, lower salt loadings reduce the likelihood of particulates, thus allowing orthogonal assistance to keep the system in optimal operating condition. Reducing the use of organic co-solvents for protein analytes lowers the chance of irreversible denaturation. A wider pH range allows for greater harmony between analyte solubility and native structure requirements and the mobile phase. This ultimately leads to broader applicability.

[0258] In some embodiments, compared to a reference column having a stationary phase comprising porous particles with a polyethylene glycol-modified surface without hydroxyl-terminated ends, a column comprising a stationary phase material as disclosed herein provides one or more of the following:

[0259] In SEC separation performed on the column, the secondary ionic interactions between the analyte and the stationary phase material are reduced;

[0260] During SEC separation performed on the column, the hydrophobic secondary interactions between the analyte and the stationary phase material are reduced;

[0261] The dependence on mobile phase pH is reduced in SEC separation performed on the column;

[0262] The dependence on column temperature is reduced in SEC separation performed on the column;

[0263] The reduction is determined by the improvement of peak shape calculated based on the USP peak tail, asymmetry at 4.4, peak width at 50%, or a combination thereof.

[0264] In some embodiments, compared to a reference column having a stationary phase comprising porous particles with a polyethylene glycol-modified surface without hydroxyl end caps, the column provides: enhanced peak resolution of SEC separation performed on the column, increased reproducibility of SEC separation, or both.

[0265] Unless otherwise stated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise claimed, the use of any and all instances or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the materials and methods and does not constitute a limitation on the scope. The language in the specification should not be construed as indicating that any unclaimed element is necessary for the practice of the disclosed materials and methods.

[0266] Those skilled in the art will readily recognize that suitable modifications and alterations can be made to the compositions, methods, and applications described herein without departing from the scope of any embodiment or aspect thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of the claimed embodiments. All the various embodiments, aspects, and options disclosed herein can be combined in all variations. The scope of the compositions, formulations, methods, and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples, and preferred embodiments described herein.

[0267] Although the technology described herein has been illustrated with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the technology. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and apparatus of this technology without departing from the spirit and scope thereof. Therefore, this technology is intended to include modifications and variations within the scope of the appended claims and their equivalents.

[0268] Throughout this specification, the terms "an embodiment," "certain embodiments," "one or more embodiments," or "an embodiment" refer to a particular feature, structure, or characteristic described in connection with an embodiment that is included in at least one embodiment of the present invention. Therefore, the appearance of phrases such as "in one or more embodiments," "in certain embodiments," "in one embodiment," or "in an embodiment" throughout this specification does not necessarily refer to the same embodiment of the present invention. Furthermore, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. Any scope referenced herein is included.

[0269] The present invention will be described more fully with reference to the following embodiments. Before describing several exemplary embodiments of the present invention, it should be understood that the present invention is not limited to the details of the construction or process steps set forth in the following description. The present invention can have other embodiments and can be practiced or carried out in various ways. The following embodiments are set forth to illustrate certain aspects of the present invention and should not be construed as limiting thereto.

[0270] Example

[0271] The present invention can be further illustrated by the following non-limiting examples describing the chromatographic apparatus and methods.

[0272] Material

[0273] Unless otherwise stated, all reagents shall be used as is. Those skilled in the art will recognize that equivalents of the following supplies and suppliers exist, and therefore the suppliers listed below should not be construed as restrictive.

[0274] The NISTmAb reference standard RM8671 (referred to as "NISTmAb" in this document) is obtained from the National Institute of Standards and Technology (NIST; Gaithersburg, MD). NISTmAb material is recombinant humanized IgG1κ expressed in mouse suspension cultures. This material is a ~150 kDa homodimer of two identical light chains and two identical heavy chains linked by chain bonds and intrachain disulfide bonds. One vial of RM 8671 contains 800 μL of 10 mg / mL IgG1κ monoclonal antibody in 12.5 mmol / L L-histidine, 12.5 mmol / L L-histidine HCl (pH 6.0).

[0275] Trastuzumab emtansine (Kadcyla, 2 mg / mL) was prepared from Genentech and diluted to concentrations of 2 mg / mL–5 mg / mL. Kadcyla was chosen due to the difficulty in mobile phase optimization caused by hydrophobic secondary interactions commonly encountered in SEC separation.

[0276] Silica particles ( and Purchased from Daiso Fine Chem USA, INC (Daisogel; 3848 W Carson Street, Suite 105, Torrance, CA 90503) and used as is or treated with a dilute acid solution (1M HCl, 20 hours, 100°C) before use.

[0277] method

[0278] The surface area (SA), pore volume (PV), and pore diameter (PD) of the materials presented in this paper were measured using a multi-point nitrogen adsorption method (Micromeritics ASAP 2400; Micromeritics Instruments Inc., Norcross, Ga.). SA was calculated using the Brunauer–Emmett–Teller (BET) method, PV is a single-point value determined for P / Pd from -0.98 to 0.99, and PD was calculated from the desorption section of the isotherm using the Barrett, Joyner, and Halenda (BJH) method. For values ​​higher than... The average PD value was obtained, and the pore diameter and pore volume were measured by mercury porosity method (Micromeritics AutoPore IV. Micromeritics, Norcross, Ga.). The skeletal density was measured using a Micromeritics AccuPyc1330 helium hydrometer (V2.04N, Norcross, Ga.).

[0279] Particle size was measured using a Beckman Coulter Multisizer 3 analyzer (Miami, Fla.; 30 μm well, 70,000 counts). Particle size (dp) was measured as the 50% cumulative diameter of the volumetric particle size distribution. The width of the distribution was measured as the 90% cumulative volumetric diameter divided by the 10% cumulative volumetric diameter (expressed as a 90 / 10 ratio).

[0280] Surface coverage was determined by the difference in % carbon content of particles before and after surface modification, measured by elemental analysis. The percentage of carbon (%C) and nitrogen (%N) was measured by combustion analysis using a LECO TruMac carbon-nitrogen / sulfur analyzer (Leco Corporation, Michigan, US).

[0281] The following embodiments describe the preparation of the stationary phase material. The SEC performance of the stationary phase materials in Examples 1, 2, 4, 7, and 8 was evaluated and compared with several reference columns (Reference Examples 5 and 6, and several commercial reference columns). Specifically, the ion and hydrophobic secondary interactions of the stationary phase material for each column were evaluated.

[0282] When the protein analyte and the stationary phase carry the same charge, ion repulsion occurs due to electrostatic repulsion (reduced protein elution time). When the protein and the stationary phase carry opposite charges, ion exchange occurs (increased elution time). To evaluate the effect of such secondary ion interactions, NIST mAb was injected into mobile phases with increasing salt concentrations (100 mM disodium hydrogen phosphate, pH 6.8, containing 0 mM, 50 mM, 100 mM, and 200 mM NaCl), and the percentage change in USP tailing was calculated.

[0283] Hydrophobic secondary interactions between proteins and hydrophobic sites on the stationary phase can lead to increased protein retention and poor peak shape. To evaluate the effect of such hydrophobic secondary interactions, Kadcyla antibody-drug conjugates (ADCs) were injected into mobile phases with increasing acetonitrile percentages (100 mM disodium hydrogen phosphate, 200 mM NaCl, pH 6.8, containing 0%, 5%, 10%, and 15% ACN), and the percentage change in USP tailing was calculated.

[0284] All separations were performed using a commercially available high-performance liquid chromatography (HPLC) system. Class H biological system (available from Waters Corporation, Milford, MA). Separation was performed at 30°C and a flow rate of 0.35 mL / min unless otherwise instructed, and detected by UV absorption at 280 nm.

[0285] Stationary phase preparation

[0286] A series of prototype (Examples 1 to 4 and Examples 7 to 8) and reference (Examples 5 and 6) stationary phase materials with different base particle materials and pore diameters were prepared. The base particles, modifications, and surface coverage are summarized in Table 2 below.

[0287] Example 1. Hydroxyl-terminated PEG, silica, average pore diameter is

[0288] With an average particle size of 3 μm and an average pore diameter of A stationary phase consisting of hydroxyl-terminated polyethylene glycol (PEG)-bonded silica particles was prepared. The surface area was 28 m². 2 / g, and the pore volume is 0.82cm³. 3 / g.

[0289] Silica particles were bonded to form hydroxyl-terminated PEG-bonded stationary phase particles. The silica particles were dispersed in toluene (10 mL / g). Residual water was removed from the material by azeotropic distillation (110 °C, 1-2 h). The reaction temperature was lowered to below 40 °C and concentrated hydrochloric acid (200 μL / g particles) was added, followed by the addition of [hydroxyl (polyvinyloxy)]. 8-12 [Propyl]triethoxysilane (30 μmol / m 2 The reaction was stirred for 5 minutes, and the temperature was increased to 110°C and maintained for 20 hours. The reaction was then cooled to room temperature, and the particles were separated by filtration. The particles were then washed in the following order: 5× toluene, 1× acetone, 4× acetone / water (1:1 v / v), and 2× acetone.

[0290] Following the bonding reaction, the residual ethoxysilyl group was hydrolyzed using ammonium acetate or ammonium bicarbonate. The particles were dispersed in a mixture of acetone (8.2 mL / g particles) and 0.12 M ammonium acetate solution (1.8 mL / g particles), and the mixture was stirred at 59 °C for 2 hours. The reaction was then cooled to <40 °C, and the particles were separated by filtration. The separated particles were subsequently washed three times with acetone / water (1:1 v / v) and twice with acetone. The separated surface-modified particles were vacuum-dried at 70 °C for 16 hours. The surface coverage of the hydroxyl-terminated PEG was 1.46 μmol / m³. 2 Hydroxyl-terminated PEG-bonded stationary phase particles were loaded into a 4.6 × 150 mm column.

[0291] Example 2. Hydroxyl-terminated PEG, silica, average pore diameter is

[0292] With an average particle size of 3 μm and an average pore diameter of The preparation of silica particles into a stationary phase comprising hydroxyl-terminated polyethylene glycol (PEG)-bonded silica particles.

[0293] Silica particles were dispersed in toluene (10 mL / g). Residual water was removed from the material by azeotropic distillation (110 °C, 1-2 h). The reaction temperature was lowered to below 40 °C and concentrated hydrochloric acid (200 μL / g particles) was added, followed by the addition of [hydroxyl (polyvinyloxy)]. 8-12 [Propyl]triethoxysilane (40 μmol / m 2 The reaction was stirred for 5 minutes, and the temperature was increased to 110°C and maintained for 20 hours. The reaction was then cooled to room temperature, and the particles were separated by filtration. The particles were then washed in the following order: 5× toluene, 1× acetone, 4× acetone / water (1:1 v / v), and 2× acetone.

[0294] Following the bonding reaction, the residual ethoxysilyl group was hydrolyzed using ammonium acetate or ammonium bicarbonate. The particles were dispersed in a mixture of acetone (8.2 mL / g particles) and 0.12 M ammonium acetate solution (1.8 mL / g particles), and the mixture was stirred at 59 °C for 2 hours. The reaction was then cooled to <40 °C, and the particles were separated by filtration. The separated particles were subsequently washed three times with acetone / water (1:1 v / v) and twice with acetone. The separated surface-modified particles were vacuum-dried at 70 °C for 16 hours. The surface coverage of the hydroxyl-terminated PEG was 1.67 μmol / m³. 2 Hydroxyl-terminated PEG-bonded stationary phase particles were loaded into a 4.6 × 150 mm column.

[0295] Example 3: BTEE / TEOS coated silica with an average pore diameter of

[0296] Silane reagent (3A) was prepared by incomplete (~68%) hydrolytic condensation of 1,2-bis(triethoxysilane)ethane (BTEE) and tetraethyl orthosilicate (TEOS). Ethanol (3.1 mol ethanol / mol silane reagent), TEOS (molar ratio to BTEE 1:4), and 0.1 M HCl (19.7 g / mol silane reagent) were added to BTEE. The solution was heated at 70 °C under an inert atmosphere for 18 hours. The reaction temperature was then increased to 90 °C, and the ethanol was removed by atmospheric distillation. The temperature was then increased to 100 °C under an inert atmosphere and held for 1 hour. The reaction mixture was cooled to room temperature to obtain the condensation product 3A.

[0297] With an average particle size of 3 μm and an average pore diameter of The silica particles were completely dispersed in toluene (21 mL / g particles). The surface area was 28 m². 2 / g, and the pore volume is 0.82cm³. 3 / g. Residual water was removed from the material by azeotropic distillation (110°C, 1 h). The reaction temperature was maintained at 40°C while silane reagent 3A (1.0 g / g particles) was added and stirred for 10 min. Catalytic NH4OH aqueous solution (0.05 g / g particles) was added. The reaction was stirred at 40°C for another 10 min, then increased to 60°C and held for 2 h. The reaction was then cooled to room temperature and the particles were separated by filtration. The particles were then washed twice with ethanol (10 mL / g) and dispersed in 70 / 30 (v / v) water / ethanol (10 mL / g). Ammonium hydroxide solution (1 g NH4OH / g particles) was added and the mixture was stirred at 50°C for 2 h. The reaction was then cooled to <40°C and the particles were separated by filtration. The separated particles were washed in the following order (10 mL / g): 2× methanol / water (1:1 v / v) and 2× methanol. The separated surface-modified particles were vacuum dried at 70°C for 16 h. Repeat the process as needed to obtain the desired concentration of surface modifier.

[0298] To ensure the uniformity of the hybrid coating, after the hydrothermal treatment process, the modified particles were exposed to high temperatures (100°C–140°C) and high pH (8–9.8) according to the procedures reported in Jiang (US Patents 6,686,035; 7,223,473; and 7,919,177) and Wyndham (International Patent Application Publication WO2008 / 103423).

[0299] The modified particles were then dispersed in a 1.0 M HCl solution (8.4 mL / g particles), and the mixture was stirred at 100 °C for 20 hours. The reaction was then cooled to below 40 °C, and the particles were separated by filtration. The separated particles were washed with water until the pH of the filtrate was above 5, and then washed three times with methanol. The separated surface-modified particles were vacuum-dried at 70 °C for 16 hours.

[0300] Example 4. Coated and bonded silica with an average pore diameter of

[0301] The porous coated silica particles prepared according to Example 3 were completely dispersed in toluene (20 mL / g). Residual water was removed from the material by azeotropic stripping (110 °C, 3 h). The reaction temperature was cooled to below 40 °C and concentrated hydrochloric acid (200 μL / g particles) was added, followed by the addition of [hydroxyl (polyvinyloxy)]. 8-12 [Propyl]triethoxysilane (30 μmol / m 2The reaction was stirred for 5 minutes, and the temperature was increased to 110°C and maintained for 20 hours. The reaction was then cooled to room temperature, and the particles were separated by filtration. The particles were subsequently washed in the following order: 5× toluene, 1× acetone, 4× acetone / water (1:1 v / v), and 2× acetone. After the bonding reaction, the residual ethoxysilyl group was hydrolyzed with ammonium acetate or ammonium bicarbonate. The particles were dispersed in an acetone solution (8.2 mL / g particles) and a 0.12 M ammonium acetate solution (1.8 mL / g particles), and the mixture was stirred at 59°C for 2 hours. The reaction was then cooled to below 40°C, and the particles were separated by filtration. The separated particles were then washed three times with acetone / water (1:1 v / v) and twice with acetone, and then vacuum dried at 70°C for 16 hours. The surface coverage of the modified particles was 1.41 μmol / m². 2 Hydroxyl-terminated PEG-bonded stationary phase particles were loaded into a 4.6 × 150 mm column.

[0302] Example 5. PEG-modified silica with reference to methoxyl-terminated silica, with an average pore diameter of [missing information].

[0303] With an average particle size of 3 μm and an average pore diameter of A reference stationary phase for preparing silica particles containing methoxy-terminated polyethylene glycol (PEG) bonds was used. The surface area was 14 m². 2 / g, and the pore volume is 0.69cm³. 3 / g.

[0304] Residual water was removed from the particles by azeotropic stripping (110°C, 3 hours). The reaction temperature was cooled to below 40°C and 2-[methoxy(polyvinyloxy)] was added. 6-9 [Propyl]tris(dimethylamino)silane (40 μmol / m 2 The reaction was stirred for 5 minutes, and the temperature was increased to 110°C and maintained for 20 hours. The reaction was then cooled to room temperature, and the particles were separated by filtration. The particles were subsequently washed in the following order: 7× toluene, 1× acetone, 6× acetone / water (1:1 v / v), and 2× acetone. The particles were then dispersed in an acetone solution (8.2 mL / g particles) and a 0.12 M ammonium acetate solution (1.8 mL / g particles), and the mixture was stirred at 59°C for 2 hours. The reaction was then cooled to below 40°C, and the particles were separated by filtration. The separated particles were then washed three times with acetone / water (1:1 v / v) and twice with acetone, and then vacuum dried at 70°C for 16 hours. The surface coverage of the modified particles was 2.35 μmol / m³. 2 Methoxy-terminated PEG-bonded stationary phase particles were loaded into a 4.6 × 150 mm column.

[0305] Example 6. Referring to BTEE / TEOS coated silica, the average pore diameter is

[0306] Silane reagent (6A) was prepared by incomplete (~68%) hydrolytic condensation of 1,2-bis(triethoxysilane)ethane (BTEE) and tetraethyl orthosilicate (TEOS). Ethanol (3.1 mol ethanol / mol silane reagent), TEOS (molar ratio to BTEE 1:4), and 0.1 M HCl (19.7 g / mol silane reagent) were added to BTEE. The solution was heated at 70 °C under an inert atmosphere for 18 hours. The reaction temperature was then increased to 90 °C, and the ethanol was removed by atmospheric distillation. The temperature was then increased to 100 °C under an inert atmosphere and held for 1 hour. The reaction mixture was cooled to room temperature to obtain the condensation product 6A.

[0307] With an average particle size of 3 μm and an average pore diameter of The silica particles were completely dispersed in toluene (21 mL / g particles). The surface area was 14 m². 2 / g, and the pore volume is 0.69cm³. 3 / g. Residual water was removed from the material by azeotropic distillation (110°C, 1 h). The reaction temperature was maintained at 40°C while silane reagent 6A (1.0 g / g particles) was added and stirred for 10 min. Catalytic NH4OH aqueous solution (0.05 g / g particles) was added. The reaction was stirred at 40°C for another 10 min, then increased to 60°C and held for 2 h. The reaction was then cooled to room temperature and the particles were separated by filtration. The particles were then washed twice with ethanol (10 mL / g) and dispersed in 70 / 30 (v / v) water / ethanol (10 mL / g). Ammonium hydroxide solution (1 g NH4OH / g particles) was added and the mixture was stirred at 50°C for 2 h. The reaction was then cooled to <40°C and the particles were separated by filtration. The separated particles were washed in the following order (10 mL / g): 2× methanol / water (1:1 v / v) and 2× methanol. The separated surface-modified particles were vacuum dried at 70°C for 16 h. Repeat the process as needed to obtain the desired concentration of surface modifier.

[0308] To ensure the uniformity of the hybrid coating, after the hydrothermal treatment process, the modified particles were exposed to high temperatures (100°C–140°C) and high pH (8–9.8) according to the procedures reported in U.S. Patents 6,686,035, 7,223,473, and 7,919,177 to Jiang and in International Patent Application Publication WO2008 / 103423 to Wyndham.

[0309] The modified particles were then dispersed in a 1.0 M HCl solution (8.4 mL / g particles), and the mixture was stirred at 100 °C for 20 hours. The reaction was then cooled to below 40 °C, and the particles were separated by filtration. The separated particles were washed with water until the pH of the filtrate was above 5, and then washed three times with methanol. The separated surface-modified particles were vacuum-dried at 70 °C for 16 hours. The stationary phase particles were loaded into a 4.6 × 150 mm column.

[0310] Example 7. Coated and bonded silica with an average pore diameter of

[0311] The porous coated particles prepared according to Example 6 were completely dispersed in toluene (10 mL / g). Residual water was removed from the material by azeotropic distillation (110 °C, 1 h–2 h). The reaction temperature was lowered to below 40 °C and concentrated hydrochloric acid (200 μL / g particles) was added, followed by the addition of [hydroxyl (polyvinyloxy)]. 8-12 [Propyl]triethoxysilane (40 μmol / m 2 The reaction was stirred for 5 minutes, and the temperature was increased to 110°C and maintained for 20 hours. The reaction was then cooled to room temperature, and the particles were separated by filtration. The particles were then washed in the following order: 5× toluene, 1× acetone, 4× acetone / water (1:1 v / v), and 2× acetone.

[0312] Following the bonding reaction, the residual ethoxysilyl group was hydrolyzed using ammonium bicarbonate or ammonium acetate. The particles were dispersed in a mixture of acetone (8.2 mL / g particles) and 0.12 M ammonium acetate solution (1.8 mL / g particles), and the mixture was stirred at 59 °C for 2 hours. The reaction was then cooled to <40 °C, and the particles were separated by filtration. The separated particles were subsequently washed three times with acetone / water (1:1 v / v) and twice with acetone. The separated surface-modified particles were vacuum-dried at 70 °C for 16 hours. The surface coverage of the hydroxyl-terminated PEG was 1.67 μmol / m³. 2 The hydroxyl-capped PEG-bonded stationary phase particles were loaded into a 4.6 × 150 mm column.

[0313] Example 8. Hydroxyl-terminated PEG-bonded and MeO-terminated PEG-modified silica, average pore diameter for

[0314] The porous bonded particles prepared according to Example 2 were completely dispersed in toluene (20 mL / g). Residual water was removed from the material by azeotropic stripping (110 °C, 3 h). The reaction temperature was cooled to below 40 °C and 2-[methoxy(polyvinyloxy)] was added. 6-9 [Propyl]tris(dimethylamino)silane (40 μmol / m 2The reaction was stirred for 5 minutes, and the temperature was increased to 110°C and maintained for 20 hours. The reaction was then cooled to room temperature, and the particles were separated by filtration. The particles were subsequently washed in the following order: 7× toluene, 1× acetone, 6× acetone / water (1:1 v / v), and 2× acetone. The particles were then dispersed in an acetone solution (8.2 mL / g particles) and a 0.12 M ammonium acetate solution (1.8 mL / g particles), and the mixture was stirred at 59°C for 2 hours. The reaction was then cooled to below 40°C, and the particles were separated by filtration. The separated particles were then washed three times with acetone / water (1:1 v / v) and twice with acetone, and then vacuum dried at 70°C for 16 hours. The surface coverage of the modified particles was 0.83 μmol / m². 2 Hydroxyl-terminated PEG-bonded, methoxy-terminated PEG-modified stationary phase particles were loaded into a 4.6 × 150 mm column.

[0315] Table 2. Properties of stationary phase materials .

[0316]

[0317]

[0318] result

[0319] Example 9. NIST mAb and Kadcyla analytes in SEC columns and reference columns of Examples 1 and 4 two Separation on silica columns

[0320] The chromatographic performance of the prototype SEC columns of Examples 1 and 4 for NISTmAb and Kadcyla was evaluated and compared with that of the commercial reference Sepax silica SRT SEC1000 column (Sepax Technologies, Inc., Newark, Delaware). The mobile phase composition consisted of 100 mM disodium hydrogen phosphate buffer, 200 mM NaCl, 15% acetonitrile aqueous solution, and water. The mobile phase composition was adjusted to provide 0 mM, 50 mM, 100 mM, and 200 mM NaCl concentrations at pH 6.8 for ion interaction assays (NISTmAb) and 0%, 5%, 10%, or 15% acetonitrile concentrations for hydrophobic interaction assays (Kadcyla). NISTmAb and Kadcyla analytes (1 μL) were injected into the mobile phase.

[0321] The results of these experiments ( Figures 1A-1FThis demonstrates that hydroxyl-terminated PEG surface modification is also compatible with wider diameter silica materials. Specifically, when compared to commercially available Sepax reference columns, the columns of Examples 1 and 4 showed significant improvements in SEC for NISTmAb and Kadcyla (respectively...). Figure 1A / Figure 1D and Figure 1B / Figure 1E contrast Figure 1C / Figure 1F The peak shape results are provided in Table 3, which show that the prototype column provides a narrower Kadcyla peak with significantly reduced secondary interactions compared to a commercially available reference column with the same pore diameter but lacking hydroxyl end capping and modified PEG.

[0322] Table 3. Peak shape results of Examples 1 and 4 and the reference column. .

[0323]

[0324] USP tailing % change between 0 mM and 200 mM NaCl

[0325] *USP tailing % variation between 0% and 15% acetonitrile

[0326] Example 10. NIST mAb and Kadcyla analytes on an SEC column as described in Example 2, with reference methoxy-terminated PEG-modified... The silica pillar (Example 5) and reference Separation on silica columns

[0327] The chromatographic performance of the prototype SEC column from Example 2 for NISTmAb and Kadcyla was evaluated and compared with that of a reference methoxy-terminated PEG-modified silica column (Example 5) and a commercial reference Sepax silica SRT SEC2000 column (Sepax Technologies, Inc., Newark, Delaware). The mobile phase consisted of 100 mM disodium hydrogen phosphate buffer, 200 mM NaCl, 15% acetonitrile aqueous solution, and water. The mobile phase was adjusted to provide 0 mM, 50 mM, 100 mM, and 200 mM NaCl concentrations at pH 6.8 for ion interaction assays (NISTmAb) and 0%, 5%, 10%, or 15% acetonitrile concentrations for hydrophobic interaction assays (Kadcyla). NISTmAb and Kadcyla analytes (1 μL) were injected into the mobile phase.

[0328] The results of these experiments ( Figures 2A-2FThis demonstrates that hydroxyl-terminated PEG surface modification is also compatible with silica materials with wider pore diameters. Specifically, when compared to the reference column of Example 5 and the commercially available Sepax reference column, the column of Example 2 showed significant improvements in SEC of NISTmAb and Kadcyla (respectively...). Figure 2A and Figure 2D contrast Figure 2B / 2E and Figure 2C / 2F). Peak shape results are provided in Table 4, which shows that the prototype column provides a narrower Kadcyla peak with significantly reduced secondary interactions compared to a reference column with the same pore diameter but lacking hydroxyl end capping and modified with PEG.

[0329] Table 4. Peak shape results of Example 2 and the reference column .

[0330]

[0331] USP tailing % change between 0 mM and 200 mM NaCl

[0332] *USP tailing % variation between 0% and 15% acetonitrile

[0333] Example 11. NIST mAb and Kadcyla analytes on an SEC column from Example 2 before and after high pH application. Separation

[0334] The high pH stability of the prototype SEC column of Example 2 was tested by evaluating the chromatographic performance of the column against NISTmAb and Kadcyla before and after high pH application. The mobile phase composition consisted of 100 mM disodium hydrogen phosphate buffer, 200 mM NaCl, 15% aqueous acetonitrile solution, and water. The mobile phase composition was adjusted to provide 0 mM, 50 mM, 100 mM, and 200 mM NaCl concentrations at pH 6.8 for ion interaction assays (NISTmAb) and 0%, 5%, 10%, or 15% acetonitrile concentrations for hydrophobic interaction assays (Kadcyla). NISTmAb and Kadcyla analytes (1 μL) were injected into the mobile phase.

[0335] High pH applications involved injecting a combined sample of thyroglobulin and uracil 500 times at 40°C using a mobile phase of 100 mM ammonium acetate at pH 8.5. At the end of the high pH application, the column was adjusted at 30°C using 10% acetonitrile / 90% 25 mM sodium phosphate at pH 7.0 + 100 mM potassium chloride.

[0336] The results of these experiments ( Figures 3A-3D This indicates that the column of Example 2 failed at the end of the high pH application. Specifically, during the high pH application ( Figure 3C and Figure 3DAfter that, chromatographic results for NISTmAb and Kadcyla could not be obtained. Figure 3A and Figure 3B ).

[0337] Example 12. NIST mAb and Kadcyla analytes on the SEC column of Example 7 before and after high pH application. Separation

[0338] The high pH stability of the prototype SEC column of Example 7 was tested by evaluating the chromatographic performance of the column against NISTmAb and Kadcyla before and after high pH applications. The mobile phase composition consisted of 100 mM disodium hydrogen phosphate buffer, 200 mM NaCl, 15% aqueous acetonitrile solution, and water. The mobile phase composition was adjusted to provide 0 mM, 50 mM, 100 mM, and 200 mM NaCl concentrations at pH 6.8 for ion interaction assays (NISTmAb) and 0%, 5%, 10%, or 15% acetonitrile concentrations for hydrophobic interaction assays (Kadcyla). NISTmAb and Kadcyla analytes (1 μL) were injected into the mobile phase.

[0339] High pH applications involved injecting a combined sample of thyroglobulin and uracil 500 times at 40°C using a mobile phase of 100 mM ammonium acetate at pH 8.5. At the end of the high pH application, the column was adjusted at 30°C using 10% acetonitrile / 90% 25 mM sodium phosphate at pH 7.0 + 100 mM potassium chloride.

[0340] The results of these experiments ( Figures 4A-4D This demonstrates that the column of Example 7 exhibits a significant improvement in pH stability compared to the column of Example 2. Specifically, compared to the column of Example 2 ( Figure 3C and Figure 3D Compared to Example 7, the column in Example 7 showed better performance after high pH application. Figure 4C and Figure 4D Peak shape results before and after high pH application are provided in Table 5.

[0341] Table 5. Peak shape results of Example 7 before and after high pH application. .

[0342]

[0343] USP tailing % change between 0 mM and 200 mM NaCl

[0344] *USP tailing % variation between 0% and 15% acetonitrile

[0345] Example 13. NIST mAb and Kadcyla analytes on the SEC column of Example 8 before and after high pH application. Separation

[0346] The high pH stability of the prototype SEC column of Example 8 was tested by evaluating the chromatographic performance of the column against NISTmAb and Kadcyla before and after high pH application. The mobile phase composition consisted of 100 mM disodium hydrogen phosphate buffer, 200 mM NaCl, 15% aqueous acetonitrile solution, and water. The mobile phase composition was adjusted to provide 0 mM, 50 mM, 100 mM, and 200 mM NaCl concentrations at pH 6.8 for ion interaction assays (NISTmAb) and 0%, 5%, 10%, or 15% acetonitrile concentrations for hydrophobic interaction assays (Kadcyla). NISTmAb and Kadcyla analytes (1 μL) were injected into the mobile phase.

[0347] High pH applications involved injecting a combined sample of thyroglobulin and uracil 500 times at 40°C using a mobile phase of 100 mM ammonium acetate at pH 8.5. At the end of the high pH application, the column was adjusted at 30°C using 10% acetonitrile / 90% 25 mM sodium phosphate at pH 7.0 + 100 mM potassium chloride.

[0348] The results of these experiments ( Figures 5A-5D This demonstrates that the column of Example 8 exhibits significantly higher pH stability compared to the columns of Examples 2 and 7. Specifically, there was no significant difference in the chromatographic results of NISTmAb and Kadcyla before and after high pH application. Figure 5A and Figure 5B contrast Figure 5C and Figure 5D Peak shape results before and after high pH application are provided in Table 6.

[0349] Table 6. Peak shape results of Example 8 before and after high pH application .

[0350]

[0351] USP tailing % change between 0 mM and 200 mM NaCl

[0352] *USP tailing % variation between 0% and 15% acetonitrile

[0353] Summary of Results

[0354] In summary, the PEG column with prototype hydroxyl-terminated ends provides a reduction in ionic and hydrophobic secondary interaction performance. Regarding hydrophobic secondary interactions, the improved peak shape of Kadcyla on the prototype column... to The pore diameter is consistent across the range. For each prototype variant (HO-terminated PEG-bonded and HO-terminated PEG-coated, with and without TEOS, and HO-terminated PEG-bonded hybrid BTEE / TEOS-coated), Kadcyla's SEC performance is improved relative to commercial columns and reference columns (methoxy-terminated PEG-bonded). For wide-pore-based (e.g., ) Columns containing silica particles were surprisingly found to have significantly higher pH stability compared to columns containing silica particles without further methoxy-terminated PEG surface modification.

[0355] Example 14

[0356] Size-based separation is becoming increasingly important in the emerging fields of cell and gene therapy. High-resolution, high-throughput separation is required to confirm the efficacy and safety of candidate therapeutics and vaccines. These advanced therapeutics, as defined by the FDA and EMA, almost exclusively correspond to those with a diameter greater than [missing information]. And sometimes as high as Large molecular complexes. Measuring the heterogeneity of these substances using analytical ultracentrifugation (AUC) is standard practice. However, the long turnaround time for generating AUC data has arguably hampered the development of novel forms, including AAV and lentiviral vector gene therapies, adenovirus vector vaccines, and lipid nanoparticle mRNA. Therefore, size exclusion chromatography (SEC)-based assays are needed, which can more rapidly generate the size heterogeneity of these substances without compromising measurement accuracy and fidelity. To achieve this, highly efficient SEC columns with highly inert surfaces are required.

[0357] Adenovirus has a diameter of approximately SEC separation requires the use of wide-pore particles and columns. Although adeno-associated viruses (AAVs) are smaller than adenoviruses (approximately one-third their size), AAV separation also requires special consideration in method development. Due to their smaller size, AAVs can be separated using smaller pore sizes. However, highly detailed aggregate information is often sought, and therefore, it is necessary to resolve aggregates with diameters close to... Methods for dimer and trimer AAV. Therefore, having ≥ Porous particles may be advantageous for the SEC separation of AAVs because they are less likely to filter out large subvisible aggregates, thus avoiding bias and providing a more accurate size distribution of the sample. Furthermore, AAVs are often prepared from expression systems that include another larger virus such as adenovirus or baculovirus. Therefore, separation with wide-pore SEC particles is desirable for detecting these impurities.

[0358] To determine the suitability of columns containing the particles disclosed herein, AAV separation was performed. Specifically, CMV-GFP adeno-associated virus (AAV) serotype 2 (1x10⁻¹⁰) was separated. 13 Samples (5 μL) of 10 mM genomic copies / mL were separated into two sizes using the column described in Example 2 on a Waters H-Class Bio system at a flow rate of 0.25 mL / min using a mobile phase containing 10 mM sodium phosphate pH 7.4 buffer, 3 mM potassium chloride, and 137 mM sodium chloride (phosphate-buffered saline). The column temperature was 30 °C. Detection was performed by UV absorbance at 280 nm and a scan rate of 10 Hz using ACQUITY TUV.

[0359] Figure 6 The chromatograms illustrating the separation are provided. A magnified view shows the resolution of substances in the high molecular weight elution window. Resolution of multiple substances began with symmetrical peaks just 6 minutes prior to elution, continuing until the AAV monomer eluted at approximately 7.5 minutes. Overall, the PEG-bonded 3 μm... The use of pore-sized particles is well-suited to the use of simple mobile phases containing only phosphate-buffered brine.

Claims

1. A chromatographic stationary phase material comprising porous silica particles having a surface, at least a substantial portion of said surface being modified with hydroxyl-terminated polyethylene glycol, wherein said hydroxyl-terminated polyethylene glycol is at a concentration of 0.5 µmol / m 2 Up to 15µmol / m 2 The density exists on the surface, and the surface is further modified with methoxy-terminated polyethylene glycol.

2. The chromatographic stationary phase material according to claim 1, wherein the porous silica particles have a diameter with an average size distribution of 1µm to 50µm.

3. The chromatographic stationary phase material according to claim 1, wherein the porous silica particles have a diameter with an average size distribution of 1µm to 20µm.

4. The chromatographic stationary phase material according to claim 1, wherein the porous silica particles have a diameter with an average size distribution of 1.5 µm to 5 µm.

5. The chromatographic stationary phase material according to claim 1, wherein the porous silica particles have an average pore size of 40 Å to 3000 Å.

6. The chromatographic stationary phase material according to claim 1, wherein the porous silica particles have an average pore size of 1000 Å to 3000 Å.

7. The chromatographic stationary phase material according to claim 1, wherein the porous silica particles have an average pore size of 1000 Å to 2000 Å.

8. The chromatographic stationary phase material according to claim 1, wherein the hydroxyl-terminated polyethylene glycol-modified surface has the following formula: ； in: m is an integer from 1 to 10; n is an integer from 2 to 50; and The wavy line indicates the attachment point to the surface of the porous silica particles.

9. The chromatographic stationary phase material according to claim 8, wherein m is 2 or 3.

10. The chromatographic stationary phase material according to claim 8, wherein n is 5 to 15.

11. The chromatographic stationary phase material according to claim 10, wherein n is 8 to 12.

12. The chromatographic stationary phase material according to claim 8, wherein m is 3 and n is 8 to 12.

13. The chromatographic stationary phase material according to claim 1, wherein the hydroxyl-terminated polyethylene glycol is at a concentration of 0.5 µmol / m 2 Up to 5µmol / m 2 The density exists on the surface of the porous silica particles.

14. The chromatographic stationary phase material according to claim 1, wherein the hydroxyl-terminated polyethylene glycol is at a concentration of 1.0 µmol / m 2 Up to 2.0 µmol / m 2 The density exists on the surface of the porous silica particles.

15. The chromatographic stationary phase material according to claim 1, wherein the portion of the surface modified with the methoxy-terminated polyethylene glycol is the result of treating the stationary phase material with a methoxy-terminated polyethylene glycol reagent having the following formula: ； in: At least one of R1, R2 and R3 is OMe, OEt, Cl or N(CH3)2; m is an integer from 1 to 10; and n is an integer from 2 to 20.

16. The chromatographic stationary phase material of claim 15, wherein at least a portion of the surface of the porous silica particles modified with the hydroxyl-terminated polyethylene glycol and the methoxyl-terminated polyethylene glycol reagent comprises a structure represented by one of the following formulas: (5) (6) or (7)。 17. The chromatographic stationary phase material of claim 16, wherein at least a portion of the surface of the porous silica particles modified with the hydroxyl-terminated polyethylene glycol and the methoxyl-terminated polyethylene glycol reagent comprises a structure represented by the following formula: (7)。 18. A column comprising the chromatographic stationary phase material according to any one of claims 1 to 17, the column having an interior for receiving the chromatographic stationary phase material.

19. The column according to claim 18, wherein, Compared to a reference column having a stationary phase comprising porous silica particles with a surface modified with polyethylene glycol without hydroxyl end caps, the column provides one or more of the following: In size exclusion chromatography separation performed on the column, the secondary ionic interactions between the analyte and the stationary phase material are reduced; In size exclusion chromatography separation performed on the column, the hydrophobic secondary interactions between the analyte and the stationary phase material are reduced; In size exclusion chromatography separation performed on the column, the dependence on the mobile phase pH is reduced; In size exclusion chromatography separation performed on the column, the dependence on column temperature is reduced; The reduction is determined by the improvement of the peak shape calculated based on the USP peak tail, the peak width at 50%, or a combination thereof.

20. The column according to claim 18, wherein, Compared to a reference column having a stationary phase comprising porous silica particles with a surface modified with polyethylene glycol without hydroxyl end caps, the column provides one or more of the following: Enhanced peak resolution is achieved in size exclusion chromatography separation performed on the column; Enhanced reproducibility of size exclusion chromatography separations performed on the column.

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