Improved size exclusion chromatography using low concentrations of amino acids in the mobile phase.

Abstract

This disclosure relates to methods for performing size exclusion chromatography. Embodiments of this disclosure are characterized by methods for improving the separation of protein analytes in size exclusion chromatography, for example, by using a low concentration of amino acids or derivatives thereof in the mobile phase and using a stationary phase comprising porous particles with surface-modified polyethylene groups capped with hydroxyl groups.

Description

[0001] Cross-references to related applications

[0002] This patent application claims priority and benefit to U.S. Provisional Application No. 63 / 079,303, filed September 16, 2020, entitled “Size Exclusion Chromatography Utilizing Low Concentration Amino Acid Size Exclusion Chromatography Mobile Phase,” which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to methods for performing size exclusion chromatography. In particular, this disclosure relates to methods for improving the separation of protein analytes in size exclusion chromatography, for example by using low concentrations of amino acids in the mobile phase. Background Technology

[0004] Size exclusion chromatography (SEC) is a commonly used separation technique that uses differences in hydrodynamic radii to separate dissolved analytes. 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] Containing medium to high levels of salt, while potentially beneficial in reducing secondary interactions, may require a desalting step during purification or preclude the use of certain types of detectors, such as mass spectrometry (MS). The use of organic cosolvents burdens the separation of native proteins; there is a pervasive threat that proteins will irreversibly denature or present in conformations that diminish or eliminate analyte value. Summary of the Invention

[0006] This disclosure generally relates to methods for performing size exclusion chromatography (SEC). Methods for performing SEC, for example, to separate, decompose, and / or analyze biomolecules, are disclosed herein. Generally, the methods of this disclosure provide SEC separation with reduced secondary interactions, while exhibiting compatibility with a wide range of analytes and the ability to use standard detection methods. These methods utilize a mobile phase supplemented with low concentrations of amino acids (including, but not limited to, modified amino acids) to help stabilize the target analyte and improve chromatographic performance. Such methods are particularly suitable with stationary phase materials bonded to or coated with polyethylene oxide (PEO) (also known as polyethylene glycol (PEG)). Surprisingly, according to this disclosure, it has been found that adding low concentrations of certain amino acids or their derivatives to a buffered mobile phase improves peak characteristics during SEC on PEO-modified SEC particles. Advantageously, supplementing with low concentrations of certain amino acids or their derivatives does not impair the sensitivity of standard optical and mass spectrometry (MS) detection methods. Furthermore, in some embodiments, chromatographic improvement is maintained using various buffers, pH values, and column temperatures.

[0007] In one aspect, a method is provided for size exclusion chromatography of a sample containing at least one analyte, the method comprising:

[0008] 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 particles having a diameter with an average size distribution between about 1 μm and about 20 μm; to approximately The average aperture; and

[0009] Furthermore, the porous 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 glycol;

[0010] b. Passing a mobile phase through a stationary phase for a period of time, the mobile phase comprising water; a buffer solution; and an amino acid or a derivative thereof, wherein the amino acid or a derivative thereof is present in the mobile phase at a concentration of about 5 mM to about 40 mM; and

[0011] c. Elute the at least one analyte from the stationary phase in the mobile phase.

[0012] In some implementations, elution involves separating the sample into one or more analytes based on a reduced hydrodynamic radius.

[0013] In some embodiments, the amino acid or its derivative is present in the mobile phase at a concentration of about 5 mM to about 20 mM. In some embodiments, the amino acid or its derivative is present in the mobile phase at a concentration of about 10 mM. In some embodiments, the amino acid is selected from the group consisting of L-arginine, L-ornithine, and L-lysine. In some embodiments, the amino acid derivative is an alkyl ester or an N-acylated amino acid. In some embodiments, the amino acid derivative is L-arginine methyl ester.

[0014] In some embodiments, the at least one analyte comprises a nucleic acid, polysaccharide, peptide, polypeptide, or protein. In some embodiments, the at least one analyte comprises an antibody. In some embodiments, the at least one analyte is an antibody-drug conjugate. In some embodiments, the at least one analyte comprises adenovirus, adeno-associated virus (AAV), mRNA, DNA, plasmid, exosome, extracellular vesicle, nucleic acid encapsulated in lipid nanoparticles, or combinations thereof. In some embodiments, the at least one analyte comprises adenovirus. In some embodiments, the at least one analyte comprises AAV.

[0015] In some embodiments, the method further includes detecting whether the at least one analyte is present or absent in the sample. In some embodiments, the detection is performed using a refractive index detector, a UV detector, a light scattering detector, a mass spectrometer, or a combination thereof. In some embodiments, the detection is performed using a UV detector.

[0016] In some embodiments, the mobile phase is flowed through a stationary phase at a flow rate of about 0.2 mL / min to about 3 mL / min.

[0017] In some implementations, the time period is less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 10 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute.

[0018] In some embodiments, the buffer solution is present at a concentration of about 10 mM to about 100 mM. In some embodiments, the buffer solution is an alkali metal phosphate. In some embodiments, the buffer solution is sodium dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof.

[0019] In some implementations, the pH of the mobile phase is from about 6.0 to about 7.5.

[0020] In some implementations, the column temperature is from about 20°C to about 50°C.

[0021] In some implementations, the mobile phase does not include an organic cosolvent, does not include a salt, or does not include either.

[0022] In some embodiments, the porous particles comprise silica, an inorganic-organic hybrid material, or a polymer. In some embodiments, the porous particles comprise silica. In some embodiments, the porous particles comprise an inorganic-organic hybrid material. In some embodiments, the porous particles comprise SiO2(O) having the empirical formula... 1.5 SiCH2CH2SiO 1.5 ) 0.25 The porous particles are inorganic-organic hybrid particles. In some embodiments, the porous particles comprise inorganic-organic hybrid materials. The porous hybrid material particles have approximately to approximately about to approximately or about to approximately The average aperture.

[0023] In some embodiments, hydroxyl-terminated polyethylene glycol has the following formula:

[0024]

[0025] in:

[0026] m is an integer from approximately 1 to approximately 10;

[0027] n is an integer from approximately 2 to approximately 50; and

[0028] The wavy line indicates the attachment point to the surface of the porous particles.

[0029] In some implementations, m is 2 or 3.

[0030] In some implementations, n is about 5 to about 15 or about 8 to about 12.

[0031] In some implementations, m is 3, and n is about 8 to about 12.

[0032] In some embodiments, the porous particles comprise porous silica particles having a surface, at least a substantial portion of which is modified with hydroxyl-terminated polyethylene glycol. In some embodiments, the surface-modified porous silica particles have approximately to approximately or about to approximately or about to approximately The average pore size, and at least a portion of the surface is modified with methoxy-terminated polyethylene glycol. In some embodiments, the methoxy-terminated polyethylene glycol-modified portion of the surface is the result of treating the stationary phase material with a methoxy-terminated polyethylene glycol reagent having the following formula:

[0033]

[0034] in:

[0035] At least one of R1, R2 and R3 is OMe, OEt, Cl or N(CH3)2;

[0036] m is an integer from approximately 1 to approximately 10; and

[0037] n is an integer ranging from approximately 3 to approximately 20.

[0038] 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).

[0039] 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.

[0040] 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.

[0041] In some embodiments, compared to size exclusion chromatography using a mobile phase that does not contain amino acids or their derivatives, the secondary interaction between the at least one analyte and the stationary phase is reduced, and the reduction in secondary interaction is characterized by one or more improvements in peak shape, peak area, peak tailing, analyte recovery, or reduced inter-run variability.

[0042] In another aspect, a method for reducing secondary interactions in size exclusion chromatography is provided, the method comprising:

[0043] a. Provide a sample comprising at least one analyte;

[0044] b. A column chromatography apparatus configured to detect the presence or absence of at least one analyte in a sample, the 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 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 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 glycol;

[0045] c. Provide a mobile phase comprising water; a buffer solution; and an amino acid or a derivative thereof, wherein the amino acid or a derivative thereof is present in the mobile phase at a concentration of about 5 mM to about 40 mM;

[0046] d. Inject the sample onto the fixed stationary phase;

[0047] e. Allow the mobile phase to flow through a fixed stationary phase for a period of time;

[0048] f. Elute the at least one analyte from the stationary phase in the mobile phase; and

[0049] g. Detecting the presence of at least one analyte in the sample, wherein a peak in the chromatogram indicates the presence of at least one analyte in the sample, and wherein the reduction of the secondary interaction is characterized by an improvement in one or more of peak shape, peak area, peak tailing, analyte recovery, or reduced inter-run variability. Attached Figure Description

[0050] 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.

[0051] Figure 1 Describing trastuzumab emtansine (Kadcyla; Genentech) in pores with an average pore size of [missing information]. Exemplary chromatographic separation of 1.7 μm particles on a prototype SEC column, the particles having MeO-PEO(6-9EO)propyltris(dimethylamino)silane bonds on a HO-PEO(8-12EO)-TEOS coating, the mobile phase containing sodium phosphate aqueous buffer and different concentrations of L-lysine.

[0052] Figure 2 Depicting Kadcyla in filled with an average pore size of Exemplary chromatographic separation of 1.7 μm particles on a prototype SEC column, the particles having MeO-PEO(6-9EO)propyltris(dimethylamino)silane bonds on a HO-PEO(8-12EO)-TEOS coating, the mobile phase containing sodium phosphate aqueous buffer and different concentrations of L-ornithine.

[0053] Figure 3 Depicting Kadcyla in filled with an average pore size of Exemplary chromatographic separation of 1.7 μm particles on a prototype SEC column, the particles having HO-PEO(8-12EO)triethoxysilane bonds, the mobile phase containing sodium phosphate aqueous buffer and different concentrations of L-arginine.

[0054] Figure 4 Depicting Kadcyla in filled with an average pore size of Exemplary chromatographic separation of 1.7 μm particles on a prototype SEC column, the particles having MeO-PEO(6-9EO)propyltris(dimethylamino)silane bonds on a HO-PEO(8-12EO)-TEOS coating, the mobile phase containing sodium phosphate aqueous buffer and different concentrations of L-arginine methyl ester.

[0055] Figure 5A Peak tailing depicts an exemplary chromatographic separation of Kadcyla on a prototype polyethylene oxide (PEO) bonded SEC column packed with an average pore size of [missing information]. The 1.7 μm particles were used, and the mobile phase contained sodium phosphate aqueous buffer and different concentrations of L-arginine.

[0056] Figure 5B The half-peak width depicts an exemplary chromatographic separation of Kadcyla on a prototype polyethylene oxide (PEO) bonded SEC column packed with an average pore size of [missing information]. The 1.7 μm particles were used, and the mobile phase contained sodium phosphate aqueous buffer and different concentrations of L-arginine.

[0057] Figure 6A Depicting Kadcyla in commercially available SEC columns (BEH200; Waters Inc., aperture: The exemplary chromatographic separation peak tailing at 1.7 μm, with the mobile phase containing sodium phosphate aqueous buffer and different concentrations of L-arginine.

[0058] Figure 6B Depicting Kadcyla in commercially available SEC columns (BEH200; Waters Inc., aperture: The exemplary chromatographic separation at 1.7 μm shows a half-peak width, with the mobile phase containing sodium phosphate aqueous buffer and different concentrations of L-arginine.

[0059] Figure 7 Depicting Kadcyla in commercially available SEC columns (BioSuite; Waters Inc., aperture: An exemplary chromatographic separation was performed on 10 μm silica particles, with the mobile phase containing an aqueous sodium phosphate buffer and different concentrations of L-arginine.

[0060] Figure 8 Depicting Kadcyla in filled with an average pore size of Exemplary chromatographic separation of 1.7 μm particles on a prototype SEC column, the particles having HO-PEO(8-12EO) triethoxysilane bonds, the mobile phase containing 30 mM L-arginine and sodium phosphate aqueous buffer at different pH values.

[0061] Figure 9 Depicting Kadcyla in commercially available SEC columns (BEH200; Waters Inc., aperture: An exemplary chromatographic separation was performed at 1.7 μm, with the mobile phase containing 30 mM L-arginine and sodium phosphate aqueous buffer at different pH values.

[0062] Figures 10A-10O A standard (Waters, Inc.) describing a BEH200 protein mixture containing thyroglobulin, IgG, BSA, myoglobin, and uracil was prepared in pores with an average pore size of [missing information]. Exemplary chromatographic separation of 1.7 μm particles on a prototype SEC column, these particles having HO-PEO(8-12EO)triethoxysilane bonds, was performed at five different temperatures (30°C–50°C) using three different mobile phases (40 mM sodium phosphate, 40 mM sodium phosphate with 40 mM L-arginine, and 40 mM sodium phosphate with 50 mM sodium chloride). Figure 10A , Figure 10B , Figure 10C , Figure 10D and Figure 10E Results at a series of different temperatures depict the mobile phase of 40 mM sodium phosphate. Figure 10F , Figure 10G , Figure 10H , Figure 10I and Figure 10J Results at a series of different temperatures were depicted for the mobile phase of 40 mM sodium phosphate and 40 mM L-arginine. Figure 10K , Figure 10L , Figure 10M , Figure 10N and Figure 10O The results at various temperatures are depicted for the mobile phases of 40 mM sodium phosphate and 50 mM sodium chloride.

[0063] Figure 11A Depicting Kadcyla in filled with an average pore size of Exemplary chromatographic separation of 3 μm HO-PEO(8-12EO)triethoxysilane-bonded silica particles on a prototype SEC column, with the mobile phase containing sodium phosphate aqueous buffer and different concentrations of L-arginine.

[0064] Figure 11B Depicting Kadcyla in a 3μm-filled, Exemplary chromatographic separation of silica particles on a prototype SEC column, the particles having HO-PEO(8-12EO)triethoxysilane bonds on a hybrid coating of (1,2-bis(triethoxysilyl)ethane (BTEE) and tetraethyl orthosilicate (TEOS), the mobile phase containing sodium phosphate aqueous buffer and different concentrations of L-arginine.

[0065] Figure 12A Depicting Kadcyla in filled with an average pore size of Exemplary chromatographic separation of 1.7 μm particles on a prototype SEC column, the particles having MeO-PEO(6-9EO)propyltris(dimethylamino)silane bonds on a HO-PEO(8-12EO)-TEOS coating, the mobile phase containing sodium phosphate aqueous buffer and different concentrations of γ-aminobutyric acid.

[0066] Figure 12B Depicting Kadcyla in filled with an average pore size of Exemplary chromatographic separation of 1.7 μm particles on a prototype SEC column, the particles having MeO-PEO(6-9EO)propyltris(dimethylamino)silane bonds on a HO-PEO(8-12EO)-TEOS coating, the mobile phase containing sodium phosphate aqueous buffer and poly-L-histidine at different concentrations.

[0067] Figure 12C Depicting Kadcyla in filled with an average pore size of Exemplary chromatographic separation of 1.7 μm particles on a prototype SEC column, the particles having MeO-PEO(6-9EO)propyltris(dimethylamino)silane bonds on a HO-PEO(8-12EO)-TEOS coating, the mobile phase containing sodium phosphate aqueous buffer and poly-L-lysine of varying concentrations.

[0068] Figure 12D Depicting Kadcyla in filled with an average pore size of Exemplary chromatographic separation of 1.7 μm particles on a prototype SEC column, the particles having MeO-PEO(6-9EO)propyltris(dimethylamino)silane bonds on a HO-PEO(8-12EO)-TEOS coating, the mobile phase containing sodium phosphate aqueous buffer and different concentrations of α-cyclodextrin.

[0069] Figure 13 Depicted in a filling with an average pore size of In an exemplary chromatographic separation of prototype polyethylene oxide (PEO) particles bonded to an SEC column, the peak area of ​​Kadcyla was measured, with the mobile phase containing sodium phosphate aqueous buffer and different concentrations of L-lysine, 4-guanidinobutyric acid, L-arginine, γ-aminobutyric acid, L-cysteine, or creatine anhydride.

[0070] Figure 14 Depicting replication-defective human adenovirus type 5 in a cavity filled with an average pore size of Exemplary chromatographic separation of 3 μm particles on a prototype SEC column, the particles being at least partially modified by OH-terminated polyethylene glycol (PEG) bonding, the mobile phase comprising sodium phosphate aqueous buffer, sodium chloride and 30 mM L-arginine.

[0071] Figure 15 Depicting replication-defective human adenovirus type 5 in a cavity filled with an average pore size of Exemplary chromatographic separation of 3 μm particles on a prototype SEC column, the particles being at least partially modified with OH-terminated polyethylene glycol (PEG) bonds, the mobile phase comprising sodium phosphate buffer, sodium chloride, and arginine. Detailed Implementation

[0072] 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.

[0073] definition

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] "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.

[0082] 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.

[0083] 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.

[0084] 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 reactive organosilanes (e.g., halogenated or alkoxy-substituted silanes) to form Si-O-Si-C bonds. Surface modification can be a bonded surface or a coated surface.

[0085] The term "bonded surface" refers to a material (e.g., porous stationary phase particles or core materials) that has a monolayer of silane molecules with covalent attachment due to the bonding reaction between surface-modifying groups and available hydroxyl groups on the material surface.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] The embodiments of this disclosure will now be described in detail as methods for performing SEC, and it should be understood that such methods are exemplary. These methods constitute embodiments that the inventors now consider to be the best mode for practicing this technology. Those skilled in the art will recognize that such methods are capable of modifications and alterations.

[0090] Methods for size exclusion chromatography

[0091] 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.

[0092] 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).

[0093] The use of the amino acid arginine as an alternative mobile phase additive, or in combination with other mobile phase additives, to improve chromatography has been previously reported. See, for example, Yumioka et al., J Pharm Sci 2010, 99(2), 618-20; Ejima et al., Journal of Chromatography A 2005, 1094(1), 49-55; and U.S. Patent No. 7,501,495 to Ajinomoto Co. It is believed that the presence of such amino acids helps reduce aggregate formation by inhibiting protein-protein interactions. See, for example, Schneider et al., J Phys Chem B 2011, 115(22), 7447-7458. However, the effect on protein aggregation requires high concentrations of approximately 200 mM–500 mM. Such high amino acid concentrations would burden many conventional chromatographic detectors (including mass spectrometers) and would likely require additional sample purification steps using amino acid additives that are not well-suited as mobile phase modifiers.

[0094] Surprisingly, according to this disclosure, it has been found that when novel SEC particles bonded or coated with PEO are used in a stationary phase that reduces secondary interactions, the threshold for improved chromatography with amino acid supplementation in the mobile phase (e.g., better peak shape and peak area) is much lower than the concentration required for protein aggregate stability. Therefore, in one aspect, a method is provided for size exclusion chromatography of a sample containing at least one analyte, the method comprising:

[0095] 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 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 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 glycol;

[0096] b. Passing a mobile phase through a stationary phase for a period of time, the mobile phase comprising water; a buffer solution; and an amino acid or a derivative thereof, wherein the amino acid or a derivative thereof is present in the mobile phase at a concentration of about 5 mM to about 40 mM; and

[0097] c. Elute the at least one analyte from the stationary phase in the mobile phase.

[0098] Each component of the disclosed method is further described below.

[0099] Analytes

[0100] 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 adenovirus, adeno-associated virus, mRNA, DNA, plasmid, exosome, extracellular vesicle, nucleic acid encapsulated in lipid nanoparticles, or a combination thereof. In some embodiments, the at least one analyte includes adenovirus or AAV. In some embodiments, the at least one analyte includes 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.

[0101] mobile phase

[0102] The methods for performing SEC as disclosed herein involve flowing a mobile phase through a stationary phase for a period of time. The mobile phase contains an amino acid or a derivative thereof, water, and a buffer solution. In some specific embodiments, the mobile phase and optional sample are provided using a high-performance liquid chromatography (HPLC) system.

[0103] amino acids

[0104] Amino acids are molecules containing an amino group, a carboxylic acid group, and a side chain specific to each amino acid. As used herein, the term "amino acid" includes known naturally occurring protein amino acids, referred to by their common three-letter abbreviations and full names. The term "amino acid" also includes stereoisomers and modifications of naturally occurring protein amino acids, as well as non-protein amino acids, post-translational modified amino acids, enzymatically synthesized amino acids, and derived amino acids.

[0105] In some implementations, the amino acid is an alpha(α)-amino acid. α-amino acids have the general formula H₂N-C. αHR-COOH, where R is the side chain moiety and the amino group is attached to the carbon atom immediately adjacent to the carboxylate group (i.e., the α-carbon). The differences between various α-amino acids lie in the side chain moiety attached to the α-carbon. The side group can include charged groups (positive, negative, or zwitterionic), polar uncharged groups, nonpolar (e.g., alkyl) groups, hydrophobic moieties, cyclic groups, aromatic groups, or any combination thereof.

[0106] Other suitable types of amino acids include those in which the amino group is attached to a different carbon atom. For example, beta(β)-amino acids, where the carbon atom to which the amino group is attached is surrounded by a carboxylate group by a carbon atom (C). β Separate. For example, although α-alanine has the formula H2N-C α H(CH3)-COOH, but β-alanine has the general formula H2N-C β H2-C α H2-COOH (i.e., 3-aminopropionic acid). Gamma (γ)-amino acids are those in which the carbon atom to which the amino group is attached is separated from the carboxylate group by two carbon atoms. For example, γ-aminobutyric acid has the formula H2N-C. γ H2-C β H2-C α H2-COOH.

[0107] In some embodiments, the amino acid is a proteogenic amino acid. The term "proteogenic amino acid" means that the amino acid is one of 20 naturally occurring amino acids encoded and incorporated into proteins. These amino acids are often referred to as "natural" amino acids and have an L-stereochemical structure. In some embodiments, the amino acid is alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or combinations thereof. In some embodiments, the amino acid is L-arginine. In some embodiments, the amino acid is L-lysine.

[0108] In some embodiments, the amino acid is a non-natural amino acid or a non-proteinogenic amino acid. Non-limiting examples of suitable such amino acids include sarcosine, creatine, 4-guanidinobutyric acid, taurine, γ-aminobutyric acid, and ornithine. In some embodiments, the amino acid is L-ornithine.

[0109] In some implementations, the amino acid is selected from the group consisting of arginine, ornithine, lysine, and combinations thereof.

[0110] In some embodiments, the amino acid is an amino acid derivative. As used herein, the term "derivative" includes any modification or variation of the amino acid moiety, including modifications to the side chain moiety of naturally occurring amino acids. Modifications to amino acids include, but are not limited to, esterification, alkylation, acylation, halogenation, sulfonation, nitration, and carboxylation.

[0111] In some implementations, the amino acid derivative is an N-acylated amino acid.

[0112] In some embodiments, the amino acid derivative is an alkyl ester of an amino acid. In a particular embodiment, the amino acid derivative is arginine methyl ester.

[0113] The concentration of the amino acid or its derivative in the mobile phase can vary. In some embodiments, the amino acid or its derivative is present in the mobile phase at a concentration of about 5 mM to about 50 mM, such as about 5 mM, about 10 mM, about 20 mM, or about 30 mM to about 40 mM or about 50 mM. In some embodiments, the amino acid or its derivative is present in the mobile phase at a concentration of about 5 mM to about 20 mM. In some embodiments, the amino acid or its derivative is present in the mobile phase at a concentration of about 10 mM. In embodiments utilizing a combination of amino acids, such concentration refers to the total concentration, not the concentration of a single amino acid.

[0114] buffer solution

[0115] The mobile phase contains a buffer solution. The buffer solution is used to control the ionic strength and pH of the mobile phase. Many different substances can be used as buffer solutions depending on the nature of the analyte. Non-limiting examples of suitable buffer solutions include phosphates, tris(hydroxymethyl)aminomethane, and acetates. In some embodiments, the buffer solution contains phosphates. In some embodiments, the buffer solution contains acetates. In some embodiments, the buffer solution is ammonium acetate. In some embodiments, the buffer solution is an alkali metal phosphate. In some embodiments, the buffer solution is sodium phosphate or potassium phosphate. In some embodiments, the buffer solution is sodium dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof.

[0116] 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.

[0117] 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.

[0118] Salt

[0119] 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.

[0120] In other embodiments, the mobile phase is salt-free. Surprisingly, according to this disclosure, it has been found that in some embodiments, the presence of salt is detrimental to separation (e.g., reduced peak shape and tailing). The absence of salt is beneficial in reducing the complexity of mobile phase optimization.

[0121] Cosolvent

[0122] 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. When present, co-solvents such as acetonitrile are typically present in the mobile phase at less than about 15% by volume. However, such co-solvents can cause protein denaturation of protein analytes. In some embodiments, the mobile phase does not contain an organic co-solvent. Surprisingly, according to this disclosure, it has been found that in some embodiments, the presence of an organic co-solvent is detrimental to separation (e.g., peak shape and tailing reduction). Eliminating the need for organic co-solvents is beneficial in reducing the complexity of mobile phase optimization. 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, and these conformational changes 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 up to about 15% by volume of an organic co-solvent. 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 from about 5% to about 15% by volume.

[0123] condition

[0124] Flow rate

[0125] The separation methods disclosed herein can be performed by flowing the mobile phase through a 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 embodiments, the flow rate is about 0.35 mL / min.

[0126] temperature

[0127] 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 20°C, about 25°C, 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 time, peak shape and peak height, and analyte stability are maintained within a certain temperature range (e.g., from about 30°C to about 50°C). In some embodiments, higher monomer peak efficiencies can be obtained by using column temperatures below ambient temperature. Therefore, in some 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.

[0128] time

[0129] 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.

[0130] stationary phase materials

[0131] The method disclosed herein utilizes a stationary phase material. Such a material may consist of one or more particles, such as one or more spherical particles. The particles are typically spherical, but may also be of any shape suitable for chromatography.

[0132] 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.

[0133] The particles typically have a certain size distribution, wherein the average (mean) diameter is from about 1 μm to about 50 μm, such as 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 1.7 μm. In some embodiments, the particles have a size distribution with an average diameter of about 3 μm.

[0134] Particles are typically porous and can be fully porous or surface porous. Porous materials have pore size or pore size distribution. The average pore size (pore diameter) can vary depending on the analyte. As described in U.S. Patent No. 5,861,110, the pore diameter can be calculated from 4V / s BET, pore volume, or pore surface area.

[0135] The pore diameter is typically chosen to allow molecules to diffuse freely in the analyte and mobile phase, enabling them to interact with the stationary phase.

[0136] In some implementations, the porous particles have approximately 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 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

[0137] Porous particles may comprise any suitable material. Suitable materials include, but are not limited to, silica, inorganic / organic hybrid materials, and polymeric materials. In some embodiments, the porous particles comprise silica, inorganic / organic hybrid materials, or polymers. In some embodiments, the porous particles comprise silica. In some embodiments, the porous particles comprise inorganic / organic hybrid materials. In some embodiments, the porous particles comprise or have the empirical formula SiO2(O) 1.5 SiCH2CH2SiO 1.5 ) 0.25 Inorganic-organic hybrid ethylene-bridged particles. Such materials can be prepared in a sol-gel synthesis via the co-condensation of 1,2-bis(triethoxysilyl)ethane (BTEE) with tetraethyl orthosilicate (TEOS). Suitable procedures are reported in Wyndham et al., Analytical Chemistry 2003, 75, 6781-6788, and U.S. Patent No. 6,686,035, each of which is incorporated herein by reference in its entirety.

[0138] The porous 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 modified porous particle surface 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 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 particles.

[0139] In some embodiments, the porous particles have a hydroxyl-terminated polyethylene glycol (PEG) modified surface. In some embodiments, the hydroxyl-terminated PEG has the following formula:

[0140]

[0141] m is an integer from approximately 1 to approximately 10;

[0142] n is an integer from approximately 2 to approximately 50; and

[0143] The wavy line indicates the attachment point to the surface of the porous particles.

[0144] 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.

[0145] 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).

[0146] 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. Such embodiments reflect the average chain length distribution in commercially available polyethylene glycols that can be used as surface-modifying agents in the embodiments of this disclosure. In other embodiments, n may be a specific value, such as about 8, about 9, or about 10 to about 11 or about 12.

[0147] 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:

[0148]

[0149] The wavy line indicates the attachment point to the surface of the porous particles; and m and n are each as defined above. In such embodiments, the modifier is bis-(silylalkyl-2-hydroxy-alkoxy)polyoxyethylene. In some embodiments, m is 3, and n is 5 to 8.

[0150] 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 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:

[0151]

[0152] 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 particles via a complex network of siloxane bonds.

[0153] 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:

[0154]

[0155] The following is a non-limiting description of hydroxyl-terminated polyethylene glycol / TEOS coated particles (3):

[0156]

[0157] 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 reacting 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:

[0158]

[0159] In some embodiments, the hydroxyl-terminated polyethylene glycol-modified porous 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.

[0160] In some embodiments, the porous 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.

[0161] In some embodiments, the porous 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.

[0162] Table 1. Exemplary Additional or Alternative Polyethylene Glycol Silane Reagents

[0163]

[0164]

[0165] In some embodiments, the porous particles with hydroxyl-terminated polyethylene glycol (PEG) modified surfaces further comprise surface bonding or coating derived from the reaction of the hydroxyl-terminated PEG modified porous particles with a non-hydroxyl-terminated PEG 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.

[0166] In some embodiments, the porous particles with hydroxyl-terminated polyethylene glycol (PEG) modified surfaces are bonded with hydroxyl-terminated PEG. In some embodiments, the porous particles with hydroxyl-terminated PEG modified surfaces are coated with hydroxyl-terminated PEG. In some embodiments, the porous particles with hydroxyl-terminated PEG modified surfaces are coated with hydroxyl-terminated PEG / TEOS. In some embodiments, the porous particles with hydroxyl-terminated PEG modified surfaces are hydroxyl-terminated PEG bonded to a BTEE / TEOS coating.

[0167] In any of these embodiments, the modified porous particles may further comprise a methoxy-terminated polyethylene glycol surface modification (e.g., bonding). Non-limiting cartoon illustrations representing possible configurations of such bonding and coating arrangements are provided below in the form of 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. Structure 7 represents a porous particle surface modified with both hydroxyl-terminated and methoxy-terminated polyethylene glycol.

[0168]

[0169] In some embodiments, the porous particles are porous silica particles. In specific 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 methoxy-terminated PEG agent. 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 methoxy-terminated PEG modified.

[0170] 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:

[0171]

[0172] in:

[0173] At least one of R1, R2 and R3 is OMe, OEt, Cl or N(CH3)2;

[0174] m is an integer from approximately 1 to approximately 10; and

[0175] n is an integer from approximately 2 to approximately 20.

[0176] 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).

[0177] 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.

[0178] 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.

[0179] Hydroxyl-terminated polyethylene glycol surface-modified stationary phase materials can be prepared, for example, by reacting porous particles with a reagent such as trimethoxysilylpropyl polyethylene glycol and hydrolyzing any remaining alkoxy groups. The distance between some adjacent ortho-hydroxyl groups on the surface of the porous particles 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.

[0180] The reaction is typically carried out according to standard methods, for example, by reacting porous particles with a suitable reagent in an organic solvent under reflux conditions. Organic solvents such as toluene are commonly used for this reaction.

[0181] In some embodiments, the stationary phase material is prepared by 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 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.

[0182] In some embodiments, the stationary phase material is prepared by partially polymerizing a hydroxyl-terminated polyethylene glycol (PEG) reagent with TEOS via hydrolytic condensation prior to reaction with hydroxyl groups on the initial surface of porous particles. Typically, the incomplete (~50%) hydrolytic condensation product can be obtained by reacting the hydroxyl-terminated PEG reagent with tetraethoxysilane (TEOS) (1:1 mol / mol) 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.

[0183] 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 hybrid particles, silica particles, or hybrid or silica particles already coated with, for example, the BTEE / TEOS partially hydrolyzed condensation product as described herein. For example, the BTEE / TEOS partially hydrolyzed condensation product provides an inorganic / organic hybrid material coating on silica particles. U.S. Utility Application Serial No. 16 / 082,823, published March 28, 2019 as US 2019 / 0091657A1, describes coating porous silica particles with an inorganic / organic hybrid material, and is hereby incorporated by reference in its entirety.

[0184] In some embodiments, after modifying the polyethylene glycol surface to provide hydroxyl-terminated polyethylene glycol, the porous particles are reacted with an additional polyethylene glycol silane reagent. In some embodiments, this reagent is a methoxy-terminated polyethylene glycol silane reagent. In some embodiments, the reagent is 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]trichlorosilane or 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]tris(dimethylamino)silane. Typically, methods for reacting hydroxyl-terminated polyethylene glycol-modified surfaces with additional polyethylene glycol silane reagents involve dispersing porous particles (e.g., hybrid particles as described herein or silica particles) in a solvent and removing any residual water by azeotropic distillation.

[0185] In a particular implementation, porous particles are particles with a pore size of approximately to approximately or about to approximately 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. 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.

[0186] In some embodiments, the methoxy-terminated PEG modifying agent is a methoxy-terminated polyethylene glycol (PEG) agent. In some embodiments, the methoxy-terminated PEG agent has the following formula:

[0187]

[0188] in:

[0189] At least one of R1, R2 and R3 is OMe, OEt, Cl or N(CH3)2;

[0190] m is an integer from approximately 1 to approximately 10; and

[0191] n is an integer ranging from approximately 3 to approximately 20.

[0192] 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).

[0193] 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.

[0194] 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.

[0195] The ratio of hydroxyl-terminated polyethylene glycol groups to methoxyl-terminated polyethylene glycol groups on the surface of the porous particles can vary. For example, in some embodiments, the molar ratio is about 2:1 or about 1:1.

[0196] column

[0197] For use in SEC, the stationary phase is typically secured in a housing with walls defining a chamber, for example, in a column with an interior for receiving the stationary phase. Such a column will have a length and a diameter.

[0198] In some embodiments, the column length is approximately 300 mm. In some embodiments, the column length is approximately 150 mm. In some embodiments, the column length is less than approximately 300 mm, less than approximately 150 mm, less than approximately 100 mm, or less than approximately 50 mm. In some embodiments, the column length is approximately 50 mm, approximately 30 mm, approximately 20 mm, or approximately 10 mm.

[0199] 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.

[0200] Detection

[0201] In some embodiments, the method further includes detecting whether the at least one analyte is present or absent in the sample. Many suitable options exist for the detection method. In some embodiments, detection is performed using a refractive index detector, a UV detector, a light scattering detector, a mass spectrometer, or a combination thereof. In a specific embodiment, detection is performed using a UV detector. Many detectors are available; however, a specific detector is the Waters detector. Adjustable UV detector (Waters Corporation, Milford, Mass., USA).

[0202] Reduction of secondary interactions

[0203] Ideally, SEC separation will separate only by size; however, nonspecific secondary interactions with 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 loss of resolution and separation efficiency. At least two types of ionic interactions can occur. Ion repulsion occurs due to electrostatic repulsion when the protein analyte and stationary phase carry the same charge (reduced protein elution time). Ion exchange occurs when the protein and stationary phase carry opposite charges (increased elution time). To improve the ionic properties of the stationary phase surface, it is common practice to derivatize the material with hydrophobic silanes (e.g., silica). Increased particle hydrophobicity reduces ionic interactions but can introduce additional hydrophobic interactions. Antibody-drug conjugates (ADCs) often exhibit increased hydrophobicity compared to unmodified proteins due to their payload conjugation. 3 The payload can interact with the hydrophobic regions of the modified particles, leading to poor separation quality. Other surface modifications (e.g., glycol bonding, methoxy-terminated polyethylene glycol bonding) are known to improve such interactions to varying degrees. These undesirable interactions can be mitigated through mobile phase optimization, particularly by using salts or organic co-solvents to reduce ionic and hydrophobic secondary interactions, respectively; however, such optimization is not always straightforward. For example, a mobile phase with sufficient ionic strength to ensure analyte stability and solubility can inadvertently induce secondary interactions, resulting in poor peak shape and recovery.

[0204] Surprisingly, according to this disclosure, supplementing the SEC mobile phase with low concentrations of amino acids or their derivatives leads to a reduction in secondary interactions between the analyte and the stationary phase material. This reduction in secondary interactions is relative to SEC performed using a mobile phase that does not contain amino acids or their derivatives. The reduction in secondary interactions can be characterized by improvements in one or more of the following: peak shape, peak area, peak tailing, analyte recovery, or reduced inter-run variability. Such improvements can be quantified by calculating factors such as USP tailing and asymmetry at 4.4, as well as the full width at half maximum (FWHM) of the analyte peak.

[0205] In another aspect, a method for reducing secondary interactions in size exclusion chromatography is provided, the method comprising:

[0206] a. Provide a sample comprising at least one analyte;

[0207] b. A column chromatography apparatus configured to detect the presence or absence of at least one analyte in a sample, the column chromatography apparatus comprising a column having an interior for receiving a stationary phase and a stationary phase fixed within the column, wherein the fixed stationary phase comprises porous 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 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 glycol;

[0208] c. Provide a mobile phase comprising water; a buffer solution; and an amino acid or a derivative thereof, wherein the amino acid or a derivative thereof is present in the mobile phase at a concentration of about 5 mM to about 40 mM;

[0209] d. Inject the sample onto the fixed stationary phase;

[0210] e. Allow the mobile phase to flow through a fixed stationary phase for a period of time;

[0211] f. Elute the at least one analyte from the stationary phase in the mobile phase; and

[0212] g. Detecting the presence of at least one analyte in the sample, wherein a peak in the chromatogram indicates the presence of at least one analyte in the sample, and wherein the reduction of the secondary interaction is characterized by an improvement in one or more of peak shape, peak area, peak tailing, analyte recovery, or reduced inter-run variability.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] Example

[0219] The present invention can be further illustrated by the following non-limiting examples describing the chromatographic apparatus and methods.

[0220] Material

[0221] 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.

[0222] Silica particles ( to Purchased from Daiso Fine Chem USA, INC (Daisogel; 3848 W Carson Street, Suite 105, Torrance, CA 90503) and used as is or treated with dilute acid solution (1M HCl, 20 hours, 100°C) before use.

[0223] The prepared trastuzumab emtansine (Kadcyla, 2 mg / mL) was obtained from Genentech and diluted to concentrations of 2 mg / mL-5 mg / mL.

[0224] method

[0225] 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.).

[0226] 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).

[0227] 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).

[0228] A series of prototype (Examples 1-4 and Examples 6-7) and reference (Example 5) 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.

[0229] Example 1. Coated and bonded inorganic-organic hybrid ethylene bridged particles with an average pore diameter of

[0230] From [hydroxyl (polyvinyloxy)] 8-12 The incomplete (~50%) hydrolytic condensation of [propyl]triethoxysilane and tetraethyl orthosilicate (TEOS) to prepare a silane reagent (1A). [Hydroxy] 8-12 [Propyltriethoxysilane] was reacted with ethanol (3.1 mol ethanol / mol silane reagent), TEOS (molar ratio of TEOS to PEO reagent 1:1), and 0.1 M HCl (15.6 g / mol silane reagent). The solution was heated at 70 °C under an inert atmosphere for 18 hours. The reaction temperature was then increased to 90 °C and distilled off the ethanol under normal pressure. 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 give product 1A.

[0231] Inorganic-organic hybrid ethylene bridging particles ( Prepared according to the method described in US Patent 6,686,035, the particles were completely dispersed in toluene (21 mL / g particles). The surface area was 168 m². 2 / g, pore volume is 1.19cm³ 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 1A (1.0 g / g particles) was added and stirred for 10 min. Catalytic NH4OH aqueous solution (0.05 g / g - 0.1 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 hours. This process was repeated as needed to obtain the desired concentration of surface modifier.

[0232] To ensure the uniformity of the PEO 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 Patent Nos. 6,686,035; 7,223,473; and 7,919,177) and Wyndham (International Patent Application Publication No. WO2008 / 103423).

[0233] 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 particles were vacuum-dried at 70 °C for 16 hours. The surface coverage of the modified particles was 0.86 μmol / m³. 2 .

[0234] The porous particles 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)6-9propyl]tris(dimethylamino)silane (8 μmol / g) was added. 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 1.03 μmol / m². 2 Hydroxyl-terminated PEO-bonded stationary phase particles were loaded into a 4.6 × 150 mm column.

[0235] Example 2. Hydroxyl-terminated PEO-bonded, inorganic-organic hybrid ethylene bridged particles with an average pore diameter of

[0236] A stationary phase comprising hydroxyl-terminated polyethylene oxide (PEO)-bonded inorganic-organic hybrid ethylene bridging particles was prepared. In the sol-gel synthesis, the procedure reported in Wyndham et al., Analytical Chemistry 2003, 75, 6781-6788 and U.S. Patent No. 6,686,035, was used to synthesize SiO2 (O2) via the co-condensation of 1,2-bis(triethoxysilyl)ethane (BTEE) with tetraethyl orthosilicate (TEOS). 1.5 SiCH2CH2SiO 1.5 ) 0.25 Inorganic / organic hybrid particles were obtained, and each reference is incorporated herein by reference in its entirety. The obtained inorganic-organic hybrid ethylene bridged particles have an average particle size of 1.7 μm and The average pore diameter. The surface area is 171 m². 2 / g, pore volume is 1.26cm³ 3 / g.

[0237] The inorganic-organic hybrid ethylene bridged particles were then bonded to form hydroxyl-terminated PEO-bonded stationary phase particles. The inorganic-organic hybrid ethylene bridged particles were 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 (8 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 then washed in the following order: 5× toluene, 1× acetone, 4× acetone / water (1:1 v / v), and 2× acetone.

[0238] Following the bonding reaction, the residual ethoxysilyl group was hydrolyzed using 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 PEO was 1.73 μmol / m³. 2 Hydroxyl-terminated PEO-bonded stationary phase particles were loaded into a 4.6 × 150 mm column.

[0239] Example 3. Hydroxyl-terminated PEO-bonded silica with an average pore diameter of

[0240] With an average particle size of 3 μm and an average pore diameter of A stationary phase consisting of hydroxyl-terminated polyethylene oxide (PEO)-bonded silica particles was prepared. The surface area was 28 m². 2 / g, pore volume is 0.82cm³ 3 / g.

[0241] 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.

[0242] Following the bonding reaction, the residual ethoxysilyl group was hydrolyzed using 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 PEO was 1.46 μmol / m³. 2 Hydroxyl-terminated PEO-bonded stationary phase particles were loaded into a 4.6 × 150 mm column.

[0243] Example 4. Coated and bonded silica with an average pore diameter of

[0244] Silane reagent (4A) was prepared by incomplete (~68%) hydrolytic condensation of 1,2-bis(triethoxysilane)ethane (BTEE) with 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 4A.

[0245] 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, 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 adding silane reagent 4A (1.0 g / g particles) and stirring 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.

[0246] To ensure coating uniformity, 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 Patent Nos. 6,686,035; 7,223,473; and 7,919,177) and Wyndham (International Patent Application Publication No. WO2008 / 103423).

[0247] 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 then vacuum-dried at 70 °C for 16 hours.

[0248] The porous silica-coated particles 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. 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 1.03 μmol / m². 2 Hydroxyl-terminated PEO-bonded stationary phase particles were loaded into a 4.6 × 150 mm column.

[0249] Example 5. Referring to BTEE / TEOS coated silica, the average pore diameter is

[0250] Silane reagent (4A) was prepared by incomplete (~68%) hydrolytic condensation of 1,2-bis(triethoxysilane)ethane (BTEE) with 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 4A.

[0251] 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, 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 adding silane reagent 4A (1.0 g / g particles) and stirring 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.

[0252] 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. Patent Nos. 6,686,035, 7,223,473, and 7,919,177 in Jiang and in Wyndham's International Patent Application Publication No. WO2008 / 103423.

[0253] 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.

[0254] Example 6. Coated and bonded silica with an average pore diameter of

[0255] The porous coated particles prepared according to Example 5 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 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 then washed in the following order: 5× toluene, 1× acetone, 4× acetone / water (1:1 v / v), and 2× acetone.

[0256] 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.

[0257] Example 7. Hydroxyl-terminated PEO-bonded silica with an average pore diameter of

[0258] With an average particle size of 3 μm and an average pore diameter of A stationary phase consisting of hydroxyl-terminated polyethylene oxide (PEO)-bonded silica particles was prepared. The surface area was 14 m². 2 / g, pore volume is 0.69cm³ 3 / g.

[0259] 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.

[0260] 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 1.56 μmol / m³. 2 Hydroxyl-terminated PEG-bonded stationary phase particles were loaded into a 4.6 × 150 mm column.

[0261] Example 8. Hydroxyl-terminated PEO-bonded / MeO-terminated PEG-modified silica, with an average pore diameter of

[0262] The porous bonded particles prepared according to Example 7 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 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 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.

[0263] Table 2. Prototype Columns

[0264]

[0265] SEC Method

[0266] Example 9. Mobile phase supplemented with lysine

[0267] The prepared trastuzumab emtansine (Kadcyla, 2 mg / mL, Genentech) was diluted to 2 mg / mL and injected into the column of Example 1 at a 1 μL injection volume. Separation was performed using a commercially available high-performance liquid chromatography (HPLC) system. Class H biological system (available from Waters Corporation, Milford, MA) was operated at 0.35 mL / min at 30 °C. Detection was performed by UV absorbance at 280 nm. The mobile phase composition contained A) 250 mM sodium phosphate buffer, pH 6.8; B) 200 mM L-lysine; and D) water (18.2 megohms). The mobile phase composition was adjusted to provide 40 mM sodium phosphate (pH 6.8) and 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM L-lysine.

[0268] A significant improvement in peak shape was observed when compared to a mobile phase consisting only of 40 mM sodium phosphate (pH 6.8). Figure 1 ). refer to Figure 1 Stacked chromatograms show that the presence of lysine approximately doubles the analyte recovery when present at concentrations from 10 mM to 50 mM. Peak height increases, tailing decreases, and the half-peak width is significantly reduced. Furthermore, unlike the mobile phase lacking L-lysine, the peak area returns to baseline. Notably, when sodium phosphate buffer is supplemented with 10 mM L-lysine, the peak area of ​​the analyte Kadcyla significantly increases. As the concentration of L-lysine increases, the peak area actually decreases until it is indistinguishable from separation performed using only the buffer as the mobile phase.

[0269] Example 10. Mobile phase supplemented with ornithine

[0270] Separation was performed as in Example 9, but L-lysine was replaced with 200 mM L-ornithine. Stacked chromatogram ( Figure 2 The results showed that the presence of L-ornithine increased the analyte recovery by approximately three times when it was present in concentrations ranging from 10 mM to 50 mM, with the 10 mM concentration exhibiting the best performance.

[0271] Example 11. Mobile phase supplemented with arginine

[0272] Separation was performed as in Example 9, but with 200 mM L-arginine instead of L-lysine, and using the column from Example 2. Stacked chromatogram ( Figure 3 The results show that the presence of L-arginine increases the analyte recovery when present at concentrations from 10 mM to 50 mM. A stable level is reached at a concentration of approximately 30 mM.

[0273] Example 12. Mobile phase supplemented with arginine methyl ester

[0274] Separation was performed as in Example 9, but L-lysine was replaced with 200 mM L-arginine methyl ester. Stacked chromatogram ( Figure 4 The results showed that the presence of L-arginine methyl ester at concentrations ranging from 10 mM to 50 mM increased analyte recovery by approximately three times. These results were comparable to those observed with supplementation of L-lysine or L-ornithine (Examples 1 and 2); the recovered peak area was significantly higher when the mobile phase was supplemented with 10 mM L-arginine methyl ester compared to other concentrations tested.

[0275] Example 13. Mobile phase supplemented with arginine

[0276] The separation was performed as in Example 9, and then 100 mM L-arginine was added. The tailing and peak width of the peak were analyzed at 50% concentration, comparing the L-arginine concentration. Figure 5A and Figure 5B The results show that the presence of L-arginine at each concentration reduced tailing and half-peak width. Adding L-arginine provided slightly different results compared to the aforementioned examples, as supplementing sodium phosphate with increasing concentrations of L-arginine did not result in a decrease in peak area. However, the improvement was observed to saturate at approximately 30 mM L-arginine. Not wishing to be bound by theory, this effect is believed to be due to the specific chemical properties of the prototype SEC column.

[0277] Example 14. Mobile phase supplemented with arginine (BEH200 column)

[0278] Separation was performed as in Example 13, but using a commercially available SEC column (BEH200; Waters Corporation, pore size...). (1.7 μm, 4.6 mm × 150 mm). Peak tailing and width were analyzed at 50% concentration compared to L-arginine concentration. Figure 6A and Figure 6B The results shown in the paper indicate that the presence of L-arginine reduces tailing and half-peak width for each concentration. The data suggest that higher concentrations of L-arginine are required to obtain the optimal Kadcyla peak shape using a BEH column. Specifically, the effect appears to be maximized at L-arginine concentrations between 50 mM and 100 mM.

[0279] Example 15. Mobile phase supplemented with arginine (BioSuite column)

[0280] Separation was performed as in Example 14, but using a different commercially available SEC column (BioSuite; Waters Corporation, pore size: 10μm silica particles, 1.7μm, 7.5mm × 300mm. Stacked chromatogram ( Figure 7The data show that the presence of L-arginine improves peak shape with increasing concentration. The data also indicate that significantly higher concentrations of L-arginine (e.g., ≥100 mM) are required to generate high-quality Kadcyla peaks.

[0281] Example 16. Mobile phase supplemented with arginine; pH study

[0282] The prepared trastuzumab emtansine (Kadcyla, 2 mg / mL, Genentech) was diluted to 2 mg / mL and injected into the column of Example 2 in 1 μL. Separation was performed using an HClassBio1 (Waters Corporation, Milford, MA) at 30 °C and a flow rate of 0.35 mL / min. Detection was performed by UV absorbance at 280 nm. The mobile phase composition contained A) 125 mM sodium dihydrogen phosphate buffer; B) 125 mM disodium hydrogen phosphate buffer; C) 200 mM L-arginine; and D) water (18.2 megohms). The mobile phase composition was adjusted to provide 40 mM sodium phosphate (pH range 6.0 to 7.5) and 30 mM L-arginine. Stacked chromatograms ( Figure 8 The results show that a favorable peak shape was provided by supplementing the mobile phase with L-arginine within this pH range.

[0283] Example 17. Mobile phase supplemented with arginine; pH study (BEH200 column)

[0284] Separation was performed as in Example 16, but using a commercially available SEC column (BEH200; Waters Corporation, pore size...). 1.7 μm, 4.6 mm × 150 mm). Stacked chromatogram ( Figure 9 The results show that the presence of L-arginine improves the peak shape within this pH range, and peak characteristics generally improve with increasing pH. Notably, within the same pH range, the peak shape changes slightly compared to separation on a prototype polyethylene oxide (PEO)-bonded SEC column (Example 8). It is not desirable to be bound by theory, but it is believed that such alternative conventional columns may require higher concentrations of L-arginine in the mobile phase.

[0285] Example 18. Mobile phase supplemented with arginine; BEH200 protein standard.

[0286] A BEH200 protein mixture standard (Waters, Inc.) containing thyroglobulin, IgG, BSA, myoglobin, and uracil was injected into the column of Example 2 at a volume of 1 μL. Separation was performed using an HClassBio1 (Waters, Milford, MA) at a flow rate of 0.35 mL / min. Detection was performed by UV absorbance at 280 nm. The mobile phase composition contained A) 250 mM sodium phosphate pH 6.8 buffer; B) 200 mM L-arginine; C) 1 M sodium chloride; and D) water (18.2 megohms). The mobile phase composition was adjusted to provide three different mobile phases: 40 mM sodium phosphate at pH 6.8 (see [link to article]). Figures 10A-10E ); 40 mM sodium phosphate at pH 6.8, containing 40 mM L-arginine (see...) Figures 10F-10J ); and 40 mM sodium phosphate at pH 6.8, containing 50 mM sodium chloride (see Figure 10K-Figure 10O Separation was performed at column temperatures of 30℃, 35℃, 40℃, 45℃, and 50℃. Results ( Figures 10A to 10O This demonstrates the supplementation of the mobile phase with L-arginine (see [link]). Figures 10F-10J Peak height, peak shape, and elution time were stabilized within this temperature range. This was particularly evident for myoglobin.

[0287] Example 19. Mobile phase supplemented with arginine; two different column chemistry properties

[0288] The prepared trastuzumab emtansine (Kadcyla, 2 mg / mL, Genentech) was diluted to 2 mg / mL and injected into the column of Example 3 or Example 4 at a volume of 1 μL. Separation was performed using an HClassBio1 (Waters, Milford, MA) at 30 °C and a flow rate of 0.35 mL / min. Detection was performed by UV absorbance at 280 nm. The mobile phase composition contained A) 250 mM sodium phosphate pH 6.8 buffer; B) 200 mM L-arginine; and D) water (18.2 megohms). The mobile phase composition was adjusted to provide 40 mM sodium phosphate and 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM L-arginine. Stacked chromatograms ( Figure 11A and Figure 11B The results demonstrate that for the columns of Examples 3 and 4 (with and without the hybrid coating, respectively), supplementing the mobile phase with L-arginine provides favorable peak characteristics, exhibiting stable levels at concentrations of approximately 30 mM L-arginine. Both columns illustrate the observations made in the prototype PEO-bonded inorganic-organic hybrid ethylene bridging particles, namely, that concentrations of L-arginine in the mobile phase greater than 30 mM do not provide any additional benefit regarding peak shape, peak area, or any other measurable chromatographic characteristics.

[0289] Example 20. Supplementing the mobile phase with various additives (1)

[0290] The prepared trastuzumab emtansine (Kadcyla, 2 mg / mL, Genentech) was diluted to 2 mg / mL and injected into the column of Example 1 at a volume of 1 μL. Separation was performed using an HClassBio1 (Waters, Milford, MA) at 30 °C and a flow rate of 0.35 mL / min. Detection was performed by UV absorbance at 280 nm. The mobile phase composition contained A) 500 mM sodium phosphate pH 6.8 buffer; B) 200 mM poly-L-histidine or 5.01 g / 100 mL α-cyclodextrin; C) 200 mM γ-aminobutyric acid or poly-L-lysine; and D) water (18.2 megohms). The mobile phase composition was adjusted to provide 40 mM sodium phosphate and various concentrations of additives. Chromatograms of separation with γ-aminobutyric acid, poly-L-histidine, poly-L-lysine, or α-cyclodextrin (respectively...) Figure 12A , Figure 12B , Figure 12C and Figure 12D The results showed that adding these additives to the mobile phase did not produce improved peak characteristics.

[0291] Example 21. Supplementing the mobile phase with various additives (2)

[0292] The separation was performed as in Example 20, but different concentrations (10mM-50mM) of L-lysine, 4-guanidinobutyric acid, L-arginine, γ-aminobutyric acid, L-cysteine ​​or creatine anhydride were used as additives. Figure 13 The bar chart shows that the most favorable peak areas for separation were obtained with lysine or arginine, especially with low concentrations (e.g., 10 mM) of lysine.

[0293] Example 22. Adenovirus incubation using a hydroxyl-terminated PEO-bonded stationary phase and an arginine-supplemented mobile phase. Separation

[0294] 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 entirely correspond to sizes larger than [missing information - likely a specific size or diameter]. And sometimes as high as Large molecular complexes. Measuring the heterogeneity of these substances using analytical ultracentrifugation (AUC) has become standard practice. However, the long turnaround time for generating AUC data has arguably hindered the development of novel forms, including adeno-associated virus (AAV) and lentiviral vector gene therapies, adenovirus vector vaccines, and lipid nanoparticle mRNAs. Therefore, size exclusion chromatography (SEC)-based assays are needed to 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.

[0295] To determine the particles (3 μm) disclosed herein, The suitability of a column with a pore size (at least partially modified with HO-terminated PEG bonds) for this type of separation was demonstrated for adenovirus isolation. Specifically, a sample (5 μL) of replication-defective human adenovirus type 5 (packaged with CMV-GFP plasmid; 1 × 10⁻⁶) was used for this purpose. 12 Size separation was performed on a Waters H-grade biological system using the column described in Example 7 at a flow rate of 0.2 mL / min using a mobile phase containing 40 mM sodium phosphate pH 7.0 buffer, 50 mM sodium chloride, and 30 mM arginine. The column temperature was 30 °C. Detection was performed using ACQUITY FLR at a scan rate of 10 Hz via natural fluorescence, with an excitation wavelength of 260 nm and an emission wavelength of 350 nm.

[0296] exist Figure 14 An exemplary chromatogram is provided, showing the detection of monomeric substances with a retention time of approximately 5 minutes. High molecular weight substances are elucidated and observed, eluting between 3 and 5 minutes, and fine structures are visible in the chromatogram, indicating dimer and trimer breakdown as well as potential protein impurities. Two peaks eluting after the monomeric substance, commonly observed in adenovirus preparations and formulations, may be attributed to incomplete formation or partial dissociation of the capsid. In summary, the separation provides symmetrical peaks at neutral pH and relatively low ionic strength.

[0297] Example 23. Using a hydroxyl-terminated PEO-bonded stationary phase, in the absence of arginine in the mobile phase, the following was carried out. adenovirus isolation

[0298] Using a mobile phase containing arginine is generally beneficial for the separation of large molecules, but it may not always be necessary for effective separation. To investigate the dependence of the separation performed in Example 22 on the presence of arginine, the separation was performed in the absence of arginine in the mobile phase. Figure 15 The document provides exemplary chromatograms illustrating non-ideal chromatographic behavior. Specifically, high molecular weight substances exhibit abnormal peak shapes and tailing effects, making this method unsuitable for pharmaceutical product characterization.

Claims

1. A method for performing size exclusion chromatography on a sample containing at least one analyte, the method comprising: 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 particles having a surface area and having a diameter with an average size distribution between 1 µm and 20 µm; an average pore size between 40 Å and 3000 Å; and wherein the porous particles have a surface area concentration of 0.5 µmol / m². 2 Up to 5.0 µmol / m 2 Surface modification of hydroxyl-terminated polyethylene glycol; b. Allowing a mobile phase to flow through the stationary phase for a period of time, the mobile phase comprising water; a buffer solution; and an amino acid or a derivative thereof, wherein the amino acid or the derivative thereof is present in the mobile phase at a concentration of 5 mM to 40 mM; and c. Elute the at least one analyte from the stationary phase in the mobile phase.

2. The method of claim 1, wherein elution comprises separating the sample into the one or more analytes based on a reduced hydrodynamic radius of one or more analytes.

3. The method according to claim 1 or 2, wherein the amino acid or its derivative is present in the mobile phase at a concentration of 5 mM to 20 mM.

4. The method according to claim 1 or 2, wherein the amino acid or its derivative is present in the mobile phase at a concentration of 10 mM.

5. The method according to claim 1 or 2, wherein the amino acid is selected from the group consisting of L-arginine, L-ornithine and L-lysine.

6. The method according to claim 1 or 2, wherein the amino acid derivative is an alkyl ester or an N-acylated amino acid of the amino acid.

7. The method according to claim 1 or 2, wherein the amino acid derivative is L-arginine methyl ester.

8. The method according to claim 1 or 2, wherein the at least one analyte comprises a nucleic acid, a polysaccharide, a peptide, or a protein.

9. The method according to claim 1 or 2, wherein the at least one analyte comprises adenovirus, adeno-associated virus, mRNA, DNA, plasmid, exosome, extracellular vesicle, nucleic acid encapsulated by lipid nanoparticles, or a combination thereof.

10. The method according to claim 1 or 2, wherein the at least one analyte comprises an antibody.

11. The method according to claim 1 or 2, wherein the at least one analyte is an antibody-drug conjugate.

12. The method according to claim 1 or 2, wherein the at least one analyte comprises adenovirus or adeno-associated virus.

13. The method according to claim 1 or 2, further comprising detecting whether the at least one analyte is present or absent in the sample.

14. The method of claim 13, wherein the detection is performed using a refractive index detector, a UV detector, a light scattering detector, a mass spectrometer, or a combination thereof.

15. The method of claim 13, wherein the detection is performed using a UV detector.

16. The method according to claim 1 or 2, wherein the mobile phase is flowed through the stationary phase at a flow rate of 0.2 mL / min to 3 mL / min.

17. The method according to claim 1 or 2, wherein the time is less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 10 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute.

18. The method according to claim 1 or 2, wherein the buffer solution is present at a concentration of 10 mM to 100 mM.

19. The method according to claim 1 or 2, wherein the buffer solution is an alkali metal phosphate.

20. The method according to claim 1 or 2, wherein the buffer solution is sodium dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof.

21. The method according to claim 1 or 2, wherein the pH of the mobile phase is from 6.0 to 7.

5.

22. The method according to claim 1 or 2, wherein the column temperature is 20°C to 50°C.

23. The method according to claim 1 or 2, wherein the mobile phase does not include an organic co-solvent, does not include a salt, or does not include either an organic co-solvent or a salt.

24. The method according to claim 1 or 2, wherein the porous particles comprise silica, an inorganic-organic hybrid material, or a polymer.

25. The method according to claim 1 or 2, wherein the porous particles comprise silicon dioxide.

26. The method according to claim 1 or 2, wherein the porous particles comprise an inorganic-organic hybrid material.

27. The method according to claim 1 or 2, wherein the porous particles comprise inorganic-organic hybrid ethylene bridged particles having the empirical formula SiO2(O 1.5 SiCH2CH2SiO 1.5 ) 0.25 .

28. The method according to claim 1 or 2, wherein the hydroxyl-terminated polyethylene glycol 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 particles.

29. The method of claim 28, wherein m is 2 or 3.

30. The method of claim 28, wherein n is 5 to 15.

31. The method of claim 28, wherein m is 3 and n is 8 to 12.

32. The method of claim 25, wherein the porous particles have an average pore size of 1000 Å to 2000 Å, and wherein at least a portion of the surface of the porous particles is modified with methoxy-terminated polyethylene glycol.

33. The method of claim 32, wherein the portion of the surface modified with the methoxy-terminated polyethylene glycol is the result of treating the porous particles 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 3 to 20.

34. The method according to claim 1 or 2, wherein, relative to size exclusion chromatography using a mobile phase not containing the amino acid or its derivatives, the secondary interaction between the at least one analyte and the stationary phase is reduced, the reduction of the secondary interaction being characterized by one or more of improved peak shape, increased peak area, reduced peak tailing, increased analyte recovery, or reduced inter-run variability.

35. The method according to claim 1 or 2, wherein the at least one analyte comprises a polypeptide.

36. The method of claim 28, wherein n is 8 to 12.

37. A method for reducing secondary interactions in size exclusion chromatography, the method comprising: a. Provide a sample comprising at least one analyte; b. A column chromatography apparatus configured to detect the presence or absence of at least one analyte in a sample, the 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 particles having a surface and the porous particles having a diameter with an average size distribution between 1 µm and 20 µm; an average pore size of 40 Å to 2000 Å; and wherein the porous particles have a surface concentration of 0.5 µmol / m 2 Up to 5.0 µmol / m 2 Surface modification of hydroxyl-terminated polyethylene glycol; c. Providing a mobile phase comprising water; a buffer solution; and an amino acid or a derivative thereof, wherein the amino acid or a derivative thereof is present in the mobile phase at a concentration of 5 mM to 40 mM; d. Inject the sample onto the fixed stationary phase; e. Allow the mobile phase to flow through the stationary phase for a period of time; f. Elute the at least one analyte from the stationary phase in the mobile phase; and g. Detecting the presence of at least one analyte in the sample, wherein a peak in the chromatogram indicates the presence of at least one analyte in the sample, and wherein the reduction of the secondary interaction is characterized by one or more of improved peak shape, increased peak area, reduced peak tailing, increased analyte recovery, or reduced inter-run variability.

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