Compositions of chromatography adsorbents and methods for analyzing antibody drug conjugates

Abstract

The present disclosure provides chromatographic compositions and methods for separation of biomolecules, in particular stationary phases and chromatographic columns for improved separation and resolution of DAR species in antibody drug conjugates (ADCs). In one example, a method comprises: (a) exposing a sample to a chromatography stationary phase of a reverse phase chromatography column, and (b) applying an eluent to the chromatography column to elute a DAR species of an antibody-drug conjugate in its native state, where the chromatography stationary phase comprises: a solid support; and a polymer coating on a surface of the solid support; wherein the polymer coating comprises a copolymer comprising a first hydrophilic monomer and a second hydrophilic monomer, where the first hydrophilic monomer and the second hydrophilic monomer have different hydrophilicity, where the copolymer has a tuned hydrophilicity determined by a molar ratio of the first hydrophilic monomer to the second hydrophilic monomer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 374,382, filed on September 2, 2022, entitled “Compositions of Chromatographic Adsorbents and Methods for Analyzing Antibody Drug Conjugates” and U.S. Provisional Application No. 63 / 505,876, filed on June 2, 2023, entitled “Compositions of Chromatographic Adsorbents and Methods for Analyzing Antibody Drug Conjugates,” the contents of which are hereby incorporated by reference in their entirety. Background Art

[0003] Antibody-drug conjugates (ADCs) are highly potent biopharmaceutical drugs constructed by attaching small molecule therapeutic agents to antibodies with permanent or unstable linkers. Antibodies target specific antigens found only on target cells. Once the ADC binds to the cell, it triggers the internalization of the antibody along with the drug. This delivers the drug to the diseased cells with very high specificity, maximizing its efficacy and minimizing systemic exposure and associated side effects.

[0004] The application of ADC is one of the research and development hotspots in the field of medicine and targeted immunotherapy. There are currently more than 100 ADCs in different stages of clinical development, and there are hundreds of ongoing clinical trials. Therefore, the demand for innovative solutions for ADC purification and analysis is increasing. Chromatographic columns have been used for the analysis of ADC. However, current chromatographic columns cannot provide sufficient resolution of ADC component species. Improved chromatographic separation columns and methods for analyzing ADC DAR species are very useful for improving product quality, efficacy and safety. Summary of the invention

[0005] In one aspect, the present disclosure relates to chromatography compositions and methods for separation of biomolecules, in particular stationary phases and chromatography columns for improved separation and resolution of ADCs using mass spectrometry compatible conditions.

[0006] Selected Definition

[0007] As used herein, "weight percent," "wt %," "percent by weight," "% by weight," and variations thereof refer to the concentration of a substance as the weight of the substance divided by the total weight of the composition and multiplied by 100.

[0008] As used herein, "mole percent," "mol %," "percent by mole," and variations thereof refer to the relative amount of a substance as the moles of that substance divided by the total moles of the composition and multiplied by 100.

[0009] As used herein, "volume percent," "vol %," "percent by volume," and variations thereof refer to the relative amount of a substance as the volume of the substance divided by the total volume of the composition and multiplied by 100.

[0010] As used herein, "g" stands for gram; "L" stands for liter; "mg" stands for "milligram (10-3 gram)"; "µg" is equal to one microgram (10-6 gram). "mL" or "cc" stands for milliliter (10-3 liter). One "µL" is equal to one microliter (10-6 liter). The unit "mg / 100g", "mg / 100mL" or "mg / L" is the unit of concentration or amount of a component in a composition. One "mg / L" is equal to one ppm (parts per million). "Da" refers to Dalton, which is the unit of molecular weight; one Da is equal to one g / mol. The temperature unit used herein is degrees Celsius (°C).

[0011] The term “about” is used in conjunction with a numerical value to include normal variations in measurements as would be expected by those skilled in the art and is understood to have the same meaning as “approximately” and to cover typical margins of error, such as ±15%, ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the stated value.

[0012] The term "about" also encompasses amounts that vary due to different equilibrium conditions of compositions resulting from a particular starting composition.Whether or not modified by the term "about," the claims include equivalents.

[0013] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" includes having two or more compounds that are the same or different from each other. It should also be noted that the term "or" is generally used in its sense that includes "and / or" unless the context clearly dictates otherwise. As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in an alternative ("or").

[0014] For the sake of brevity and simplicity, any range of values ​​shown in this specification contemplates all values ​​within that range and will be interpreted as support for claims reciting any sub-ranges with the following endpoints, which are real values ​​within the specified range in question. By way of hypothetical illustrative example, the disclosure of a range of 1 to 5 in this specification should be considered support for a claim to any of the following ranges: 1-5; 1-4; 1-3; 1-2; 2-5; 2-4; 2-3; 3-5; 3-4; and 4-5.

[0015] As used herein, reference to column "Prototype 1" or prototype column "V1" refers to the same tested prototype, and these names are used interchangeably. Similarly, "Prototype 2" or column "V2" also refers to the same prototype column and are used interchangeably throughout the disclosure.

[0016] The term "substantially" is used herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also used herein to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0017] The term "substantially free of" may refer to any component that the composition of the present disclosure lacks or almost lacks. When referring to "substantially free of", it means that the component is not intentionally added to the composition of the present disclosure. The use of the term "substantially free of" a component allows the composition of the present disclosure to include trace amounts of the component because trace amounts of the component are present in another component. However, it should be recognized that when a composition is referred to as "substantially free of" a component, only trace amounts or the minimum amount of the component will be allowed. In addition, if a composition is referred to as "substantially free of" a component, if the component is present in trace amounts or the minimum amount, it should be understood that it will not affect the effectiveness of the composition. It should be understood that if a component is not explicitly included in this article or it is not stated in this article that it may include, then the composition of the present disclosure may be substantially free of the component. Similarly, the explicit inclusion of a component allows it to be explicitly excluded, thereby allowing the composition to be substantially free of the explicitly stated component.

[0018] As used herein, the terms "comprise", "comprises" and "comprising" specify the presence of stated features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0019] As used herein, the transition phrase “consisting essentially of” means that the scope of a claim should be interpreted to include the specified materials or steps recited in the claim and those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0020] Therefore, the term “consisting essentially of”, when used in the claims of the present invention, is not intended to be interpreted as being equivalent to “comprising”.

[0021] As used herein, the terms "increase," "increasing," "increased," "enhance," "enhanced," "enhancing," and "enhancement" (and grammatical variations thereof) describe an increase of at least about 1%, 5%, 10%, 15%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more compared to a control.

[0022] As used herein, the terms "reduce," "reduced," "reducing," "reduction," "diminish," and "decrease" (and grammatical variations thereof) describe, for example, a decrease of at least about 1%, 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% compared to a control. In certain embodiments, reduction can result in no or substantially no (i.e., an insignificant amount, e.g., less than about 10% or even 5% or even 1%) detectable activity or amount.

[0023] The terms "preferred" and "preferably" refer to embodiments that may provide certain benefits in certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not mean that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the present disclosure.

[0024] As used herein, an "antibody drug conjugate" or "ADC" is a biologically active drug attached to a monoclonal antibody (mAb) via a chemical linker or linker unit with a labile bond.

[0025] "Antibody" is used in the broadest sense herein, and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments. Antibodies can be murine, human, humanized, chimeric or derived from other species. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. (Janeway et al. (2001) "Immunobiology", 5th edition, Garland Publishing, New York). Target antigens generally have numerous binding sites, also referred to as epitopes, recognized by CDRs on a variety of antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen can have more than one corresponding antibody. Antibodies also refer to full-length immunoglobulin molecules, or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules containing antigen binding sites that specifically bind to an antigen or a portion thereof of a target of interest, such targets including but not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can belong to any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. The immunoglobulins can be derived from any species. However, in one aspect, the immunoglobulins are of human, mouse, or rabbit origin.

[0026] The term "Linker unit" refers to the direct or indirect linkage of an antibody to a drug.

[0027] Attachment of the linker to the mAb can be achieved in a variety of ways, such as through surface lysines, reductive coupling to oxidized carbohydrates, and via cysteine ​​residues released by reduction of interchain disulfide bonds. Various ADC linkage systems are known in the art, including hydrazone-based linkages, disulfide-based linkages, and peptide-based linkages.

[0028] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique that identifies a compound by its mass. MS refers to a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z." MS techniques generally include (1) ionizing a compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating the mass-to-charge ratio. The compound may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector.

[0029] Typically, one or more molecules of interest are ionized and the ions are then introduced into a mass spectrometer where they follow a path in space that depends on mass ("m") and charge ("z") due to a combination of magnetic and electric fields.

[0030] As used herein, the term "ionization" or "ionizing" refers to the process of generating analyte ions having a net charge equal to one or more electron units. Negative ions are ions having a net negative charge of one or more electron units, while positive ions are ions having a net positive charge due to the lack of one or more electron units.

[0031] As used herein, the term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into its components due to the differential distribution of the chemical entities as they flow around or through a stationary liquid or solid phase.

[0032] As used herein, the term "liquid chromatography" or "LC" means the process of selective delay of one or more components of a fluid solution as the fluid uniformly permeates through a column of finely separated substances or through a capillary passage. The delay is due to the distribution of the components of the mixture between one or more stationary phases and a bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase. Examples of "liquid chromatography" include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), turbulent flow liquid chromatography (TFLC) (sometimes referred to as high turbulence liquid chromatography (HTLC) or high throughput liquid chromatography).

[0033] As used herein, the term "high performance liquid chromatography" or "HPLC" (sometimes also referred to as "high pressure liquid chromatography") refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase (usually a densely packed column). As used herein, the term "ultra-high performance liquid chromatography" or "UHPLC" (sometimes referred to as "ultra-high pressure liquid chromatography") refers to HPLC that occurs at much higher pressures than traditional HPLC techniques.

[0034] The term "LC / MS" refers to a liquid chromatograph (LC) interfaced with a mass spectrometer.

[0035] As used herein, reference to a "(meth)acrylate" compound (where the "meth" is bracketed) is meant to include both acrylate and methacrylate compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Shown is an example of a traditional method chromatographic separation of DAR in its native form, HIC-UV, with a gradient from high to low salt concentration.

[0037] Figure 2A -B shows an example from the literature of a method to make the effluent from a HIC separation compatible with MS by splitting the post-column flow and greatly diluting it.

[0038] Figure 3AShown are chemical structures of exemplary first hydrophilic monomers or exemplary hydrophilic functional groups of the first hydrophilic monomers described herein according to various embodiments of the present disclosure.

[0039] Figure 3B Shown are chemical structures of exemplary second hydrophilic monomers or exemplary less hydrophilic functional groups of the second hydrophilic monomers described herein according to various embodiments of the present disclosure.

[0040] Figure 4A Shown are chemical structures of exemplary initiators or initiating groups / sites covalently attached to the surface of a solid support for ATRP polymerization according to various embodiments of the present disclosure.

[0041] Figure 4B Shown are chemical structures of exemplary RAFT chain transfer agents having initiating groups / sites covalently attached to a solid support surface for RAFT polymerization according to various embodiments of the present disclosure.

[0042] Figure 4C Shown are chemical structures of exemplary NMP agents having initiating groups / sites covalently attached to a solid support surface for NMP polymerization according to various embodiments of the present disclosure.

[0043] Figure 5 Exemplary ADCs according to various embodiments of the present disclosure and abbreviations of their constituents are shown.

[0044] Figure 6 Shown are chromatograms obtained from analyzing a sample of a model ADC (brentuximab vedotin) using a control chromatography column V1 (having silica particles coated with 100% MMA homopolymer) according to Example 2, wherein the top chromatogram is a chromatogram using MS detection, the middle chromatogram is a chromatogram using UV detection at a wavelength of 280 nm, and the bottom chromatogram is a chromatogram using UV detection at a wavelength of 215 nm.

[0045] Figures 7A-7E Shows Figure 6 Deconvoluted mass spectra of the peaks marked in. 7A shows the mass spectrum of D0; Figure 7B The mass spectrum of D1 is shown; Figure 7C The mass spectrum of D2 is shown; Fig.7D The mass spectrum of D3 is shown; Fig. 7E The mass spectrum of D4 is shown.

[0046] Figures 8A-8GShown are chromatograms obtained from analyzing a proprietary ADC according to Example 2 using a prototype chromatography column V2 (having silica particles coated with a 1:1.3 molar ratio of HEMA:MMA), wherein the top graph is a chromatogram using MS detection, the middle graph is a chromatogram using UV detection at a wavelength of 280 nm, and the bottom graph is a chromatogram using UV detection at a wavelength of 215 nm.

[0047] Figures 9A-9F Results obtained from the analysis of ADC mimetics according to Example 2 using the Prototype 2 (or V2) chromatography column (surface chemistry HEMA:MMA, 1:1.3 molar ratio) are shown. Fig.9A : HRMS (top) and UV (bottom) chromatograms at 280 nm. Figures 9B-9F are the deconvoluted spectra of various DAR species.

[0048] Figures 10A-10F Results obtained from the analysis of ADC mimetics using Prototype 3 chromatography columns according to Example 2 (with surface chemistry HEMA:MMA, 1:2 molar ratio) are shown. Fig. 10A : HRMS (top) and UV (bottom) chromatograms at 280 nm. Figures 10B-10F are the deconvoluted spectra of various DAR species.

[0049] Figures 11A-11C Results from the analysis of the commercial ADC Polivy using three different prototype columns, each with a different polymer composition, are shown. TM LC-HRMS XIC (XIC = extracted ion current) chromatogram of (polituzumab vedotin-piiq).

[0050] Figures 12A-12I The commercial ADC Polivy was analyzed on the prototype 2 column. TM Detailed LC-HRMS results of (polituzumab vedotin-piiq). DETAILED DESCRIPTION

[0051] The present disclosure relates generally to chromatography compositions and methods for separation of biomolecules, and in particular to stationary phases and chromatography columns for improved separation and resolution of ADCs.

[0052] The average DAR of ADCs and the relative amounts of each ADC species (e.g., D0-D8) are important quality parameters in the design, manufacture, and therapeutic use of ADCs. Traditional liquid chromatography (LC) methods have been used to separate ADCs and to characterize DAR and drug loading distribution. For example, reverse phase liquid chromatography coupled to mass spectrometry (RPLC-MS) has been used to determine the average drug-to-antibody ratio (DAR) by separating and reducing the denatured subunits of ADCs (Fekete, 2016), but this method loses information about the drug loading distribution. Hydrophobic interaction chromatography (HIC) based on non-denaturing separation has been used to analyze the drug distribution of ADCs (Wiggins, 2015). However, HIC-based methods use a gradient of decreasing salt concentration for elution ( Figure 1 ), and the high initial concentration and low volatility of the salt prevent its direct coupling to mass spectrometry for peak identification.

[0053] Based on various designs of post-column makeup and splitting platforms, another HIC-MS method using makeup and splitting strategy was developed (Y. Yan, 2020) to reduce the salt concentration by six times and reduce the flow rate to 1-5 μL / min for NSI-MS detection. Figure 2A One design of the post-column dilution setup is shown, while Figure 2B Results of ADC separation are shown.

[0054] Recently, a non-denaturing version of reversed-phase liquid chromatography (RPLC) or native RPLC has been developed to separate intact ADCs and determine their molecular weights by online mass spectrometry (Chen, 2019). Chen reported a native RPLC method that used a chromatographic stationary phase containing poly(alkyl methacrylate) homopolymer-coated silica particles to determine the molecular weight and DAR of a model ADC.

[0055] In the present disclosure, it is found that a polymer coating with tuned hydrophilicity covalently attached to the surface of a solid support for a chromatographic stationary phase can significantly improve the separation, resolution and quantification of intact ADCs and positional isomers with different drug to antibody ratios (DARs) in their native state under MS-compatible and native reversed phase conditions. Copolymerization of a first hydrophilic monomer (e.g., 2-hydroxyethyl methacrylate (HEMA)) and a second hydrophilic monomer (e.g., methyl methacrylate (MMA)) at various molar ratios on a solid support (e.g., nonporous silica support particles) advantageously allows tuning of the hydrophilicity of the stationary phase.

[0056] Retention of native antibodies (DAR=0 or D0) outside the column void and baseline resolution of ADCs with various DARs can be achieved using a complementary phase comprising a copolymer-coated solid support, with lower test pressures, faster analysis and / or less organic modifiers. By incorporating hydrophilic functional groups into the copolymer and the hydrophilicity of the tuned polymer coating, the stationary phase of the present application allows elution of high DAR species (e.g., DAR=8 or D8) under native reverse phase conditions, which is different from stationary phases coated with poly(alkyl methacrylate) without a first hydrophilic monomer and / or without tuned hydrophilicity. In addition, the traditional separation method for determining the DAR value of an ADC is hydrophobic interaction chromatography or HIC with UV detection. However, the HIC method uses high concentrations of salt, which makes it incompatible with online mass spectrometry. By HIC, such as Figure 1 Fractions are collected as indicated by the vertical lines in , and those fractions are desalted prior to off-line MS analysis. In contrast, the chromatography and related methods of the present invention advantageously avoid the use of high salt concentrations, which allows direct coupling with MS for on-line operation, thereby greatly simplifying analysis and improving data quality.

[0057] Chromatographic stationary phase

[0058] In some aspects, the present disclosure provides a chromatography stationary phase comprising: a solid support; and a polymer coating on the surface of the solid support. The polymer coating comprises a copolymer, and the copolymer comprises a first hydrophilic monomer (monomeric unit) and a second hydrophilic monomer (monomeric unit), wherein the copolymer has a tuned hydrophilicity determined by a molar ratio of the first hydrophilic monomer to the second hydrophilic monomer. The first hydrophilic monomer and the second hydrophilic monomer are different monomers and have different hydrophilicities with respect to each other. The second hydrophilic monomer has a lower hydrophilicity than the first hydrophilic monomer.

[0059] The solid support may be selected from a plurality of support particles, a capillary column, a multicapillary column, a glass column, a stainless steel column, a monolith, or any combination thereof.

[0060] In some embodiments, the solid support of the present invention includes a plurality of support particles. The support particles can be any support particles that can be used with one of the chromatography techniques and can form or grow a polymer coating thereon. The support particles can be organic or inorganic or composite particles, including but not limited to: silica particles, polymer beads, glass beads, aluminum oxide particles, titanium dioxide particles, zirconium oxide particles, magnetite particles, iron oxide particles or a combination thereof, such as silica coated with zirconium oxide, or iron oxide-silicon dioxide composite particles (e.g., silica particles coated on the surface of iron oxide particles). In certain embodiments, the support particles include silicon dioxide (silicon dioxide particles) or are substantially composed of silicon dioxide (silicon dioxide particles). In some embodiments, supports other than particles can be used, including integral silicon dioxide and 3D printing columns.

[0061] The support particles used herein may have a certain hardness or rigidity, such as hard spherical silica particles. In some embodiments, the particles may be porous or non-porous. In other embodiments, the particles are non-porous or substantially non-porous. Hard particles are particles that are not compressed under the pressure used in chromatography, for example, under a pressure of about 1,000 to about 25,000 psi. The size of the support particles may vary, depending on the size of the column to be filled. In certain embodiments, the particle size is at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 5 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm. In certain embodiments, the support particles have an average particle size of about 0.5 μm to about 50 μm, or about 1 μm to about 40 μm, or about 5 μm to about 30 μm, or about 10 μm to about 20 μm. The support particles may be spherical or irregular in shape.

[0062] The solid support may have a surface comprising a plurality of functional groups covalently attached thereto. The functional groups may include at least one of hydroxyl, amino, carboxylic, sulfonic, phosphoric, or any combination thereof. For example, the support particles may be nonporous silica particles having a plurality of silanol groups (Si-OH) exposed on the particle surface.

[0063] The surface of the solid support can be at least partially functionalized with a polymer coating (or copolymer) by covalent bonding. Covalent bonding can include at least one joint or spacer with 1 atom to about 100 atoms, or 2 atoms to about 50 atoms, or 3 atoms to about 20 atoms. The covalent joint can have at least two ends, respectively connected to the polymer coating at one end, and connected to at least one of the functional groups on the solid support surface at the other end. The non-limiting examples of covalent bonding include: ether (-O-), amine (-N (R)-), carbonyl (-C (=O)-), siloxane (-Si-O-Si-), carboxylate (-C (=O) O-), alkyl (-(CH2) n-), ethylene glycol (-(CH2-O)-), carboxylic acid amide (-C (=O) N (R)-), aliphatic bonding, aromatic bonding, or any combination thereof. In some embodiments, the polymer coating includes a plurality of copolymers, wherein each copolymer is covalently attached to the surface of the solid support via at least one of a covalent joint or spacer.

[0064] The copolymer of the polymer coating of the present invention is a copolymerization product of at least two monomers, wherein at least one monomer is a first hydrophilic monomer and at least one monomer is a second hydrophilic monomer.

[0065] The copolymers may be linear copolymers, branched copolymers, star polymers, random copolymers, statistical copolymers, block copolymers, bottle brush polymers, or any polymer architecture common in the art.

[0066] In some embodiments, copolymer is the free radical copolymerization product of at least one first hydrophilic monomer and at least one second hydrophilic monomer. The first hydrophilic monomer comprises at least one polymerizable group covalently coupled to at least one hydrophilic functional group. The second hydrophilic monomer comprises at least one polymerizable group covalently coupled to at least one less hydrophilic functional group. In some embodiments, the polymerizable group of the monomer is an ethylenically (-C (R) = C (R) -, R = H, methyl or alkyl) unsaturated group, including but not limited to vinyl groups, acrylate groups, methacrylate groups, acrylamide groups, methacrylamide groups, styrene groups or derivatives thereof and analogs. Non-limiting examples of hydrophilic functional groups include polyethylene oxide, hydroxyl, dihydroxy, polyhydroxy, carboxyl (carboxylic), sulfonic acid (sulfonic), phosphoric acid (phosphoric), amine, acyl, amine or any combination thereof. Non-limiting examples of less hydrophilic functional groups include: linear or branched or cyclic (n>3) C1 to C18 saturated or unsaturated alkyl groups, aryl groups, aromatic groups, fluorocarbon groups, or any combination thereof. In some embodiments, the copolymer of the present invention is a substantially linear polymer comprising a main chain having hydrophilic functional groups and less hydrophilic functional groups attached to the main chain on side chains.

[0067] In a specific embodiment, the first hydrophilic monomer is selected from 2-hydroxyethyl (meth)acrylate, 3-hydroxypropyl (meth)acrylate, 2,3-dihydroxypropyl (meth)acrylate, vinyl acetate, vinyl alcohol, sodium (meth)acrylate, tetrahydrofurfuryl (meth)acrylate, furfuryl (meth)acrylate, glycidyl (meth)acrylate, 2-(diethylamino)ethyl (meth)acrylate, 2-aminoethyl (meth)acrylate hydrochloride, 2-ethoxyethyl (meth)acrylate, potassium salt of 3-sulfopropyl (meth)acrylate, glycosyloxyethyl (meth)acrylate, or a derivative thereof, or any combination thereof.

[0068] In a specific embodiment, the second hydrophilic monomer is selected from alkyl (meth)acrylates (alkyl = Me, Et, n-Pr, i-Pr, n-Bu, s-Bu, t-Bu, etc.), hexyl (meth)acrylate, cyclohexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, isodecyl (meth)acrylate, isobornyl (meth)acrylate, phenyl (meth)acrylate, 2-naphthyl (meth)acrylate, 9-anthrylmethyl (meth)acrylate, 2,2,3,4,4,4-hexafluorobutyl (meth)acrylate, octadecyl (meth)acrylate, styrene, or a derivative thereof, or any combination thereof.

[0069] Figure 3A Chemical structures of non-limiting examples of the first hydrophilic monomer or the hydrophilic functional group of the first hydrophilic monomer are provided in . Figure 3B Chemical structures of non-limiting examples of the second hydrophilic monomer or the less hydrophilic functional group of the second hydrophilic monomer are provided in .

[0070] In some embodiments, the copolymers of the present invention have a molar ratio of the first hydrophilic monomer (monomer unit) to the second hydrophilic monomer (monomer unit) of 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to about 25:1, 1:10 to about 10:1, about 1:5 to about 5:1, or about 1:2 to about 2:1, about 1:1.5 to about 1.5:1, about 1:1.2 to about 1.2:1, about 1.1:1 to about 1:1.1, or about 1:1. In some embodiments, the second hydrophilic monomer unit has a mole percentage of at least about 1 mol%, at least about 5 mol%, at least about 10 mol%, at least about 20 mol%, at least about 30 mol%, at least about 40 mol%, at least about 50 mol%, at least about 60 mol%, at least about 70 mol%, at least about 80 mol%, at least about 90 mol%, at least about 95 mol%, at least about 99 mol%, or at least about 99.9 mol%, based on the total moles of the monomer units of the copolymer. In certain embodiments, the first hydrophilic monomer unit has a mole percentage of at most about 99 mol%, at most about 95 mol%, at most about 90 mol%, at most about 80 mol%, at most about 70 mol%, at most about 60 mol%, at most about 50 mol%, at most about 40 mol%, at most about 30 mol%, at most about 20 mol%, at most about 10 mol%, at most about 5 mol%, at most about 1 mol%, or at most about 0.1 mol%, based on the total moles of the monomer units of the copolymer.

[0071] In some embodiments, the copolymers of the present invention have a molar ratio of hydrophilic functional groups (derived from the first hydrophilic monomer) to less hydrophilic functional groups (derived from the second hydrophilic monomer) of 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to about 25:1, 1:10 to about 10:1, about 1:5 to about 5:1, or about 1:2 to about 2:1, about 1:1.5 to about 1.5:1, about 1:1.2 to about 1.2:1, about 1.1:1 to about 1:1.1, or about 1:1.

[0072] By selecting the type of hydrophilic monomer / second hydrophilic monomer and controlling / adjusting its molar ratio, the copolymer of the present application advantageously provides a tuned hydrophilicity of the polymer coating on the surface of the solid support. As described above, in the case of improved separation and resolution of ADC, tuned hydrophilicity may be critical. Depending on the properties and chemical / physical properties of the ADC or its constituent antibodies and drugs, the hydrophilicity of the copolymer (or polymer coating) can be tuned / adjusted in response to the surface properties of the ADC to reduce or minimize the adverse interactions between the ADC and the solid support and to maintain the structural integrity of the ADC. Therefore, the polymer coating with tuned hydrophilicity provides an effective solution for separating and resolving natural ADCs without structural damage, dissociation, and antibody denaturation.

[0073] In some embodiments, the solid support is coated on its surface with a copolymer having a first hydrophilic monomer and a second hydrophilic monomer at different molar ratios. In some examples, at least a first portion of the surface is coated with a copolymer having a first molar ratio of the first hydrophilic monomer to the second hydrophilic monomer, and at least a second portion of the surface is coated with a copolymer having a second molar ratio of the first hydrophilic monomer to the second hydrophilic monomer, wherein the first molar ratio and the second molar ratio are not the same.

[0074] In certain embodiments, the solid support comprises a plurality of support particles having a polymer coating covalently attached to the surface of each support particle. The polymer coating may have copolymers having different molar ratios of a first hydrophilic monomer to a second hydrophilic monomer. In some instances, at least a first portion of the support particles is coated with a copolymer having a first molar ratio of a first hydrophilic monomer to a second hydrophilic monomer, and at least a second portion of the support particles is coated with a copolymer having a second molar ratio of a first hydrophilic monomer to a second hydrophilic monomer, wherein the first molar ratio and the second molar ratio are not the same. For example, the first molar ratio is about 1:2, and the second molar ratio is about 2:1. In some embodiments, the molar ratio of the hydrophilic monomer / second hydrophilic monomer on the surface of the support particles varies between support particles, ranging from 1:100 to about 100:1, from about 1:50 to about 50:1, from about 1:25 to about 25:1, from 1:10 to about 10:1, from about 1:5 to about 5:1, or from about 1:2 to about 2:1, from about 1:1.5 to about 1.5:1, from about 1:1.2 to about 1.2:1, from about 1.1:1 to about 1:1.1. In some embodiments, the molar ratio of the first hydrophilic functional group / the second hydrophilic functional group derived from the polymer coating varies between support particles, ranging from 1:100 to about 100:1, from about 1:50 to about 50:1, from about 1:25 to about 25:1, from 1:10 to about 10:1, from about 1:5 to about 5:1, or from about 1:2 to about 2:1, from about 1:1.5 to about 1.5:1, from about 1:1.2 to about 1.2:1, from about 1.1:1 to about 1:1.1.

[0075] In certain embodiments, the polymer coating of the present invention comprises a copolymer of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), wherein the molar ratio of HEMA to MMA ranges from 1:100 to about 100:1, from about 1:50 to about 50:1, from about 1:25 to about 25:1, from 1:10 to about 10:1, from about 1:5 to about 5:1, or from about 1:2 to about 2:1, from about 1:1.5 to about 1.5:1, from about 1:1.2 to about 1.2:1, from about 1.1:1 to about 1:1.1.

[0076] A polymer coating comprising a copolymer having tuned hydrophilicity can be generated using any method known to those skilled in the art and attached to the surface of a solid support. In some embodiments, the polymer coating can be formed or grown on a solid support. The solid support can have surface chemistry and / or initiating groups / sites that enable the attachment of polymer chains via a "grafting from" or "grafting onto" strategy known in the art.

[0077] In some embodiments, the support particles can have initiating groups / sites that can generate initiating free radicals, initiate polymerization, and propagate polymer chains to form copolymers of the present invention. In some embodiments, the initiating groups / sites are introduced to the surface of the solid support by covalently attaching a free radical initiator with initiating groups / sites to at least one functional group on the solid surface, such as a hydroxyl group (-OH) or a silanol group (-Si-OH). For example, the initiator can contain a chlorosilane group or an alkoxysilane group that allows covalent bonding to the silica surface, or a functional group that can be bonded to a silane or silanol on the solid support surface. Initiators or initiating groups / sites can be used to initiate the formation of a polymer coating on the support particles.

[0078] In some embodiments, the copolymer is grafted from the surface of the solid support via conventional (uncontrolled) free radical copolymerization. In certain embodiments, the solid support has polymerizable groups covalently attached to its surface, and the copolymer is generated by copolymerization of a first hydrophilic monomer and a second hydrophilic monomer initiated from the polymerizable groups on the surface of the solid support. For example, the polymerizable group can be an olefinic (-C=C) unsaturated group that generates an initiating free radical (-CC) under polymerization conditions, and the initiating free radical initiates and propagates polymer chain growth to generate a copolymer.

[0079] In some embodiments, the copolymer is grafted from the surface of the solid support via living / controlled radical copolymerization (L / CRP) of a first hydrophilic monomer and a second hydrophilic monomer. Non-limiting examples of L / CRP include atom transfer radical polymerization (ATRP) or activator regenerated ATRP, nitroxide-mediated radical polymerization (NMP), or reversible addition-fragmentation chain transfer (RAFT). Surface-initiated L / CRP methods are generally known in the art and can be found in a publication entitled "Functional Interfaces Constructed by Controlled / Living Radical Polymerization for Analytical Chemistry" (Wang, 2016), which is hereby incorporated by reference in its entirety.

[0080] In some embodiments, copolymer is grafted from the surface of solid support via ATRP.ATRP used in the present disclosure also covers any enhanced or modified ATRP method, including but not limited to initiator ATRP (ICAR-ATRP) for continuous activator regeneration, activator (ARGET) ATRP regenerated by electron transfer, activator (AGET) ATRP generated by electron transfer, supplementary activator and reductant ATRP (SARA-ATRP), electrochemically mediated atom transfer radical polymerization (eATRP) and light-induced ATRP.The method of surface-initiated ATRP is generally known in the art, and can be found in the publication entitled "Surface-Initiated Radical Polymerization on Porous Silica" (Huang, 1997), and its relevant parts are hereby incorporated by reference.ATRP initiator can be any compound capable of attaching or binding to a solid support and initiating the formation of a polymer coating on a solid support. Generally known initiators are provided in the publication entitled "Functional polymers by atom transfer radical polymerization," (Coessens, 2001), which is hereby incorporated by reference in its entirety. The ATRP initiator may be selected from, but is not limited to: (chloromethyl) phenethyl) trimethoxysilane, (3-trimethoxysilyl) propyl 2-bromo 2-methylpropionate, [11-(2-bromo-2-methyl) propionyloxy] undecyl trichlorosilane, cyanomethyl [3 (trimethoxysilyi) propyl] trithiocarbonate (which is a RAFT agent), trimethoxyaminopropylsilane, glycidoxypropyltrimethoxysilane (these are not ATRP initiators) or a combination thereof. As described above, the ATRP initiator can be covalently attached to the surface of the solid support, and the ATRP initiating group / site initiates polymer chain growth to generate the desired copolymer. The ATRP initiator can become part or all of the linker / spacer connecting the copolymer and the solid support. ATRP polymerization can utilize transition metal complexes known in the art as catalysts, wherein alkyl halides are used as initiators (R-X).Non-limiting examples of ATRP catalysts include transition metal complexes such as those of Cu, Fe, Ru, Ni, and Os.

[0081] In some embodiments, copolymers are grafted from the surface of a solid support via RAFT. Similar to ATRP, one or more RAFT chain transfer agents can be covalently attached to the surface of a solid support. RAFT chain transfer agents can be activated to generate RAFT radicals, which mediate and propagate copolymerization to produce desired copolymers on the surface of a solid support. RAFT chain transfer agents used herein include but are not limited to dithioesters, dithiocarbamates, trithiocarbonates, xanthates, or any derivatives thereof. In specific embodiments, the RAFT chain transfer agent is cyanomethyl [3-(trimethoxysilylpropyl] trithiocarbonate.

[0082] In some embodiments, the copolymer is grafted from the surface of the solid support via NMP. Similar to ATRP, one or more NMP agents containing nitroxide groups (such as alkoxyamines) can be covalently attached to the surface of the solid support. The NMP agent can be activated to generate nitroxide radicals (R2N-Oꞏ), which mediate and propagate copolymerization to produce the desired copolymer on the surface of the solid support. The NMP agent used herein includes, but is not limited to, N-tert-butyl-N-(1-diethylphosphono-2,2-dimethylpropyl)nitroxide (SG1) and / or its alkoxy derivatives, 2,2,5-trimethyl-4-phenyl-3-azahexane-3-nitroxide (TIPNO), and alkoxyamines based on 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO). Figure 2A Non-limiting examples of ATRP initiators, RAFT chain transfer agents, and NMP agents, which are attached to the surface of a solid support, are provided in -2C.

[0083] The amount of initiating groups / sites on the surface of the solid support can vary, depending on the type of initiator, the solid support particle, the polymer coating and other factors. The amount of initiating groups / sites can be about at least about 0.05, at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6. At least about 0.7, at least about 0.8, at least about 0.9, at least about 1, at least about 1.1, at least about 1.2, at least about 1,3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 2.0, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3, at least about 3.1, at least about 3.2, at least about 3.3, at least about 3.4, or about at least about 3.5 μm / m. These values ​​can define a range, such as about 0.1 to about 3 μm / m.

[0084] In some embodiments, the polymer coating can be internally crosslinked via the interchain crosslinking reaction of the copolymer by chain end coupling, main chain coupling, side chain coupling or a combination thereof. The crosslinking reaction can be mediated or controlled by a thermal process or a photoprocess or spontaneously during or after copolymerization, with or without a catalyst or initiator. The crosslinked polymer coating can have improved hardness, stability or mechanical durability. In some embodiments, the polymer coating is not crosslinked or further processed after copolymerization.

[0085] The polymer coating of the present application can have the thickness of about 10 nm to about 500 nm, about 50 nm to about 400 nm or about 100 nm to about 300 nm.The thickness of polymer coating can be changed by adjusting the concentration of initiating group / site and / or copolymerization conditions (such as temperature, time, monomer concentration, etc.).In some embodiments, the thickness of polymer is at least about 0.1, at least about 0.5, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140 or at least about 150 nm. These values ​​can be used to define ranges, such as from about 3 nm to about 20 nm, or from about 20 nm to about 25 nm. The density of the polymer coating can be at least about 0.005, at least about 0.01, at least about 0.05, at least about 0.1, at least about 0.5, at least about 1, at least about 2, at least about 3, or at least about 4 μmol / m2. These values ​​can be used to define ranges, such as from about 0.01 μmol / m2 to about 1 μmol / m2.

[0086] In a particular embodiment, the solid support comprises a plurality of nonporous silica particles. At least one of the silica particles is coated with a polymer coating comprising a copolymer of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), wherein

[0087] The molar ratio of HEMA to MMA ranges from 1:100 to about 100:1, from about 1:50 to about 50:1, from about 1:25 to about 25:1, from 1:10 to about 10:1, from about 1:5 to about 5:1, or from about 1:2 to about 2:1, from about 1:1.5 to about 1.5:1, from about 1:1.2 to about 1.2:1, from about 1.1:1 to about 1:1.1. The silica particles prior to copolymerization have at least one ATRP initiating group / site covalently attached to their surface, and the copolymer is prepared by initiating copolymerization of HEMA and MMA and propagating the copolymer chain from the ATRP initiating site / group.

[0088] In some embodiments, the present disclosure provides a chromatography column comprising a stationary phase as described herein. Methods for preparing / packing a chromatography column are described below. In some embodiments, the chromatography column has a length of about 1 to about 50 cm and an inner diameter of about 25 μm to about 10 cm.

[0089] It should be noted that although the stationary phases of the present invention are found to be particularly effective for the resolution of native ADCs, they can also be adapted for other chromatographic applications, including but not limited to size exclusion chromatography, ion exchange chromatography, affinity chromatography, hydrophilic interaction chromatography, hydrophobic chromatography, perfusion chromatography, and reversed phase liquid chromatography for the separation of small molecules, peptides, sugars and polysaccharides, glycans, monoclonal antibodies, proteins, and other macromolecular compounds and combinations thereof. The columns are suitable for high pressure liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), fast protein liquid chromatography (FPLC), solid phase extraction (SPE), liquid chromatography coupled to mass spectrometry (LC-MS), and ultrafiltration, and can also be used to separate compounds by electrophoresis, isoelectric focusing, and capillary electrochromatography.

[0090] The choice of separation material depends on the intended use of the column and the size of the sample. The particles of the separation material may range in size from about 100 nm to about 10 μm, or from about 300 nm to about 2 μm, or from about 300 nm to about 900 nm.

[0091] Method for preparing the stationary phase

[0092] In some aspects, the present disclosure provides a method for preparing a chromatography column described herein. In one example, the method includes: (1) providing a solid support for a chromatography column; and (2) forming a polymer coating on the surface of the solid support, wherein the polymer coating comprises a copolymer comprising a first hydrophilic monomer and a second hydrophilic monomer, wherein the copolymer has a tuned hydrophilicity determined by a molar ratio of the first hydrophilic monomer to the second hydrophilic monomer. In some embodiments, forming the polymer coating comprises a copolymer covalently attached to the surface of the solid support via a linker / spacer.

[0093] In some embodiments, forming a polymer coating includes using an initiator or initiating group / site covalently attached to the surface of the support particle, via a free radical copolymerization of a hydrophilic monomer and a second hydrophilic monomer, from the surface grafting / growth copolymer of the solid support. In some embodiments, the method further includes covalently attaching at least one initiator or a molecule with at least one initiating group / site to the surface of the solid support before copolymerization. Free radical copolymerization can be any conventional polymerization method or living / controlled polymerization (L / CRP) method described above. As described above, the initiating group / site attached to the surface of the solid support can be derived from a polymerizable group (such as a C=C bond), an ATRP initiator, a RAFT chain transfer agent, an NMP agent, or any combination thereof.

[0094] In some embodiments, the solid support comprises a plurality of support particles, and the method comprises: packing the chromatography column with the support particles; and forming a polymer coating on the surface of at least one support particle. In some embodiments, forming the polymer coating further comprises grafting / growing copolymers from the surface of the support particles. The copolymers may be grafted onto the support particles before, after, or before and after the chromatography column is packed. Conventional column packing methods may be used in combination with the present disclosure to prepare the chromatography columns of the present application. A common technique for forming packed chromatography columns is to produce a slurry of support particles within the column, and then use a high solvent flow through the column to push the support particles toward the outlet of the column. In order to produce these columns using slurry technology, the chromatography column is open at the top during the packing process, and the end is attached to one end of the slurry compartment. The chromatography medium slurry fills the slurry compartment and the column tube. The outlet end of the column has a frit that retains the chromatography medium but allows the liquid to leave the column. The inlet end of the slurry chamber is attached to a high pressure, high volume pump which forces the liquid through the chamber.

[0095] The high flow rate of liquid forces the particles to migrate toward the outlet of the column, and the chromatography medium is slowly compressed into a uniform and dense bed. In some embodiments, the method for preparing a chromatography column comprises: (1) providing a plurality of non-porous silica particles for a chromatography column, wherein the non-porous silica particles have at least one initiating group / site covalently attached to the surface thereof; (2) initiating copolymerization of a first hydrophilic monomer and a second hydrophilic monomer from the initiating group / site to generate a polymer coating comprising a copolymer of the first hydrophilic monomer and the second hydrophilic monomer on the surface of the support particle; (3) placing the copolymer-coated non-porous silica particles on the support particle; and (4) placing the copolymer-coated non-porous silica particles on the support particle. The copolymer is introduced into a chromatography column and packed into the chromatography column, wherein the copolymer has a tuned hydrophilicity determined by the molar ratio of the first hydrophilic monomer to the second hydrophilic monomer, wherein the molar ratio is 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to about 25:1, 1:10 to about 10:1, about 1:5 to about 5:1, or about 1:2 to about 2:1, about 1:1.5 to about 1.5:1, about 1:1.2 to about 1.2:1, about 1.1:1 to about 1:1.1. In some embodiments, the first hydrophilic monomer is HEMA and the second hydrophilic monomer is MMA. In some embodiments, the copolymer is generated by surface-initiated ATRP.

[0096] Alternatively, the polymer coating can be formed or grown on the packed support particles using an "in situ" method known in the art. Typically, the chromatography column is packed with support particles to form packed support particles within the column; and the polymer coating is then formed on the packed support particles by directly grafting the polymer from the packed support particles. General examples of "in situ" methods can be found in US Patent Publication No. 20190257801, the relevant parts of which are incorporated herein.

[0097] In certain embodiments, the method for preparing a chromatography column comprises:

[0098] (1) providing a plurality of non-porous silica particles for a chromatography column, wherein the non-porous silica particles have at least one initiating group / site covalently attached to a surface thereof; (2) placing the coated non-porous silica particles into a chromatography column and packing the chromatography column, and (3) initiating copolymerization of a first hydrophilic monomer and a second hydrophilic monomer from the initiating group / site to generate a polymer coating comprising a copolymer of the first hydrophilic monomer and the second hydrophilic monomer on the surface of the packed support particles; wherein the copolymer has a tuned hydrophilicity determined by a molar ratio of the first hydrophilic monomer to the second hydrophilic monomer, wherein the molar ratio is 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to about 25:1, 1:10 to about 10:1, about 1:5 to about 5:1, or about 1:2 to about 2:1, about 1:1.5 to about 1.5:1, about 1:1.2 to about 1.2:1, about 1.1:1 to about 1:1.1. In some embodiments, the first hydrophilic monomer is HEMA and the second hydrophilic monomer is MMA. The second hydrophilic monomer is considered to be less hydrophilic than the first hydrophilic monomer. In some embodiments, the copolymer is generated by surface-initiated ATRP.

[0099] Monomer or prepolymer solution containing hydrophilic monomer / second hydrophilic monomer, catalyst, part, initiator, cosolvent etc. can be introduced into the support particles in the column, such as by introducing monomer or prepolymer solution into the packed column.Polymer coating can be affected by the reaction conditions and reaction time that allow polymerization.Polymer can grow approximately linearly with reaction time.For example, monomer solution with about 3 mol / L concentration can experience polymerization reaction for about 30 to about 60 minutes to generate polymer coating.It should be noted that those skilled in the art can reach the desired molar ratio (partly depending on the reactivity ratio (reactivity ratio), copolymerization parameters and reaction conditions) of the first hydrophilic monomer unit / second hydrophilic monomer unit of copolymer by controlling the molar ratio of the corresponding first hydrophilic monomer / second hydrophilic monomer in the monomer solution.For example, using the copolymerization of the surface-initiated monomer solution of HEMA:MMA containing the molar ratio of about 1:1, the following copolymer can be generated, which has about the same molar ratio of HEMA unit:MMA unit in the copolymer.

[0100] In some embodiments, the polymer coating can be monitored by measuring the back pressure (backpressure) over time during the "in-situ" copolymerization step. The polymer coating can be grown by making the prepolymer solution flow through the post at a low rate. After the solution is initially pumped into the post, the flow rate can be reduced, for example, reduced by about 50%. Along with the formation of the polymer coating, the pressure of the speed of reduction can be gradually raised. The reaction can be allowed to proceed until the time or pressure reaches the desired value, such as up to 1 hour or about 20,000 psi. The reaction time can be about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0, 8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5 or about 5 hours. These values ​​can be used to define a range, such as about 0.5 to about 2 hours. The reaction pressure can be about 1000, about 2000, about 3000, about 4000, about 5000, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 11,000, about 12,000, about 13,000, about 14,000, about 15,000 psi or about 20,000 psi. These values ​​can be used to define ranges, such as about 1000 to about 12000 psi.

[0101] In some embodiments, copolymerization reaction can be allowed to carry out under constant pressure (such as defined above), and the reaction can be stopped under the desired flow rate (flow rate). The desired flow rate can be about 0.1, about 0.5, about 1, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, about 300, about 350, about 400, about 450 or about 500 μ L / min. These values ​​can be used to define a scope, such as about 50 to about 300 μ L / min.

[0102] In some embodiments, the polymer coating can be formed without flowing a solution (e.g., a prepolymer solution or a reagent solution) through the column. The solution can be introduced into the column (initially flowing through, etc.) and then the polymer coating can be allowed to form without flowing or with constant flow or continuous flow through the column. As described above, the column can still be kept under pressure and allowed to react.

[0103] Compared with the column disclosed in the art, the chromatography column of the present disclosure can improve resolution. Resolution is measured by the ratio of peak distance to peak base. Compared with the column disclosed in the art (such as the column without the composition of the present disclosure), the chromatography column of the present disclosure can improve resolution by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or improve by about 100%. These values ​​can be used to define a range, such as about 10% to about 30%. Compared with the column disclosed in the art (such as the column without the composition of the present disclosure), the chromatography column of the present disclosure can improve resolution by about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times or about 10 times. These values ​​can be used to define a range, such as about 2 times to about 4 times.

[0104] Compared with posts disclosed in the art, chromatography column of the present disclosure can have improved inter-column reproducibility.Compared with posts disclosed in the art (such as posts without compositions of the present disclosure), chromatography column of the present disclosure can improve reproducibility by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or improve by about 100%.These values ​​can be used to define a range, such as about 20% to about 40%.Compared with posts disclosed in the art (such as posts without compositions of the present disclosure), chromatography column of the present disclosure can improve reproducibility by about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times or about 10 times.These values ​​can be used to define a range, such as about 2 times to about 4 times.

[0105] The chromatography columns of the present disclosure can be operated at flow rates up to or greater than about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 250, about 300, about 350, about 400, about 450, and up to about 500 μL / min. These values ​​can be used to define ranges, such as about 160 to about 200 μL / min.

[0106] Compared with posts disclosed in the art, chromatography columns of the present disclosure can improve column stability. Column stability can be measured by the onset of column failure (i.e., bed collapse) under high flow rates, which can cause accelerated aging due to shearing. Compared with posts disclosed in the art (such as posts without compositions of the present disclosure), chromatography columns of the present disclosure can improve column stability by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or improve by about 100%. These values ​​can be used to define a range, such as about 20% to about 50%. Compared with posts disclosed in the art (such as posts without compositions of the present disclosure), chromatography columns of the present disclosure can improve column stability by about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times or about 10 times. These values ​​can be used to define a range, such as about 2 times to about 4 times.

[0107] Methods used to analyze ADC

[0108] The chromatography column according to the present disclosure is particularly suitable for native reverse phase chromatography of antibody-drug conjugates (ADC). In some aspects, the present disclosure provides a method for analyzing ADC using the chromatography stationary phase described herein. In one example, the method for analyzing ADC in a sample includes: (a) exposing the sample to the chromatography stationary phase of a reverse phase chromatography column, and (b) applying an eluent to the chromatography column to elute the native antibody-drug conjugate. Wherein the chromatography stationary phase comprises: a solid support and a polymer coating on the surface of the solid support; wherein the polymer coating comprises a copolymer of at least one first hydrophilic monomer and at least one second hydrophilic monomer, wherein the copolymer has a tuned hydrophilicity determined by a molar ratio of the first hydrophilic monomer to the second hydrophilic monomer. In certain embodiments, the polymer coating comprises a copolymer of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), and wherein the molar ratio of HEMA to MMA is 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to about 25:1, 1:10 to about 10:1, about 1:5 to about 5:1, or about 1:2 to about 2:1, about 1:1.5 to about 1.5:1, about 1:1.2 to about 1.2:1, about 1.1:1 to about 1:1.1.

[0109] In some embodiments, the method further comprises at least one of the following: separation and resolution of eluted native ADCs having the same DAR or different DARs (0 to 20), separation and resolution of positional isomers of ADCs having the same DAR; detection and quantification of eluted native ADCs, unconjugated antibodies, fragments and dissociated ADCs and unconjugated drugs using detection methods such as UV spectroscopy or mass spectrometry; establishment of a mass-to-charge (m / z) ratio or molecular weight and DAR of at least one of the eluted native ADCs at different DAR ratios; determination of the relative amount or concentration of various native ADCs in the sample; determination of the average DAR of the sample, identification / quantification of unconjugated antibodies and / or unconjugated drugs in the sample. In some embodiments, the sample is not treated to denature or dissociate the ADC and / or its antibody, and the ADC remains intact after elution.

[0110] In some embodiments, mass spectrometry is used to establish the mass-to-charge ratio of each ADC, native antibody, and unconjugated drug in the sample. This is particularly useful in monitoring the formation and stability of ADCs and / or quality control of ADC production. In certain embodiments, spectroscopy is used, such as ultraviolet (UV) spectroscopy and / or visible spectroscopy.

[0111] In some embodiments, the eluent (mobile phase) used in the method of the present application has a salt concentration of about 1 mM to about 100 mM, or about 20 mM to about 80 mM, or about 30 mM to about 60 mM. In certain embodiments, the salt concentration is no more than about 100 mM, or no more than about 80 mM, or no more than about 60 mM. In some embodiments, the salt includes ammonium acetate. The low salt concentration of the eluent advantageously improves MS compatibility without the need to desalt the analyte solution prior to MS analysis.

[0112] In some embodiments, the eluent further comprises an organic solvent such as isopropanol, wherein the organic solvent has a volume of no more than about 80 vol%, no more than about 60 vol%, no more than about 50 vol%, no more than about 40 vol%, no more than about 30 vol%, no more than about 20 vol%, no more than about 10 vol%, no more than about 1 vol%, or about 0% based on the total volume of the eluent. These values ​​can be used to define ranges, such as from about 0 to about 80 vol%. In some embodiments, the eluent comprises a gradient of about 0 vol% to about 50 vol% of an organic solvent.

[0113] Various mass spectrometry systems capable of high mass accuracy, high sensitivity and high resolution are known in the art and can be used in the method of the present invention. The mass analyzer of such mass spectrometers includes, but is not limited to, quadrupole (Q), time of flight (TOF), ion trap (orbital trap), magnetic sector or FT-ICR, or a combination thereof.

[0114] The ion source of a mass spectrometer should mainly produce sample molecular ions or pseudomolecular ions and some characterizable fragment ions. Examples of such ion sources include atmospheric pressure ionization sources, such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). ESI and MALDI are the two most commonly used methods for protein ionization for mass spectrometry. ESI and APC1 are the most commonly used ion source technologies for LC / MS (Lee, M. "LC / MS Applications in Drug Development" (2002) J. Wiley & Sons, New York).

[0115] Commercially available mass spectrometers can sample and record the entire mass spectrum simultaneously and at a frequency that allows sufficient spectra to be obtained for multiple components in a mixture to ensure that the mass spectral signal intensities or peak areas are quantitatively representative. This will also ensure that the elution times observed for all masses are not modified or distorted by the mass analyzer, and this will help ensure that quantitative measurements are not compromised by the need to measure the abundance of transient signals.

[0116] Figure 5 Examples of ADCs with different DARs are shown. L0 refers to an antibody light chain with no drug loading. L1 refers to an antibody light chain with one drug / linker attached. H0, H1, H2, and H3 refer to antibody heavy chains with 0, 1, 2, and 3 drug / linker attachments, respectively. D0 refers to an antibody with no drug attached; D2, D4, D6, and D8 refer to ADCs with 2, 4, 6, and 8 drug molecules attached, respectively.

[0117] Analyzing ADCs using the prototype chromatography column and method can generate chromatograms, such as total ion chromatograms (TIC) or extracted ion chromatograms (EIC), which show the chromatographic separation and resolution of intact ADCs with different DARs (i.e., D0, D1, D2, D3, D4, D5, D6, D7, D8, etc.). In certain embodiments, ADCs with high DARs (e.g., DARs of 8 or more) can be separated from other ADCs, easily observed, and reliably identified from the chromatograms and accompanying mass spectra. From the chromatograms, the concentration of each ADC in the sample and the relative ratios of ADCs with different DARs can be determined and quantified, for example, by using commercial analytical software.

[0118] In certain embodiments, the prototype chromatography column provides improved separation and resolution of ADCs with high DARs compared to chromatography columns disclosed in the art (such as those chromatography columns without the compositions of the present disclosure). In some embodiments, the prototype chromatography column provides improved retention of native antibodies (DO) outside the column void compared to chromatography columns disclosed in the art (such as chromatography columns without the compositions of the present disclosure).

[0119] like Figure 5 As shown, some ADCs (e.g., D2, D4, and D6) can each have various positional isomers. These ADC positional isomers associated with the same DAR can have different surface properties and interaction levels. When contacted with the stationary phase of the present invention, improved separation and resolution of these positional isomers can be achieved.

[0120] Example

[0121] Certain embodiments of the present disclosure are further described with reference to the following experiments and examples. These experiments, examples and samples are intended only to illustrate the present disclosure, but are not intended to limit or restrict the scope of the present disclosure in any way, and should not be interpreted as providing conditions, parameters, reagents or starting materials that must be used specifically to practice the technology of the present disclosure.

[0122] Example 1

[0123] The sample chromatography column was prepared according to the following general procedure. Installation of ATRP initiator on the surface of the support particles: Nonporous silica (average particle size of about 3.1 μm, about 150 g) was added to a 1 L round-bottom flask and dried in a convection oven at about 80°C for about 3 h. Toluene (about 500 g) was added to the same flask and stirred with an overhead stirrer for 5 min. The ATRP initiator 3-trimethoxysilylpropyl-2-bromo-2-methylpropionate (about 10 mL) was diluted with toluene (about 10 mL) and added to the same flask. The reaction was stirred at room temperature for about 5 min, then heated to reflux for about 12 h and stirred overnight. The reaction mixture was filtered under vacuum, washed with toluene (about 100 mL), and vacuum filtration was continued until the silica particles were dry. The silica particles were removed from the filter paper and re-slurried in toluene (about 500 g) in a 1 L round-bottom flask. Trimethylsilyl chloride (about 11 mL) was added to the flask, and the reaction was heated to reflux for about 6 h and stirred overnight. The reaction mixture was filtered, washed with toluene (about 1.5 L), and vacuum filtration was continued until the silica particles were dry. The surface functionalized silica particles with ATRP initiating groups (Si-Br) were further dried in a vacuum oven at about 60 ° C for about 2 days.

[0124] Preparation of polymer-coated silica particles: Three solutions, solution A, solution B, and solution C, were prepared separately. Solution A was prepared by mixing water (about 250 mL) and isopropanol (about 250 mL), and then degassed with N2 at about 4 mL / min for about 2 h. Solution B (blue solution) was prepared by dissolving cupric chloride (about 360 mg) in about 18 mL of degassed solution A and adding tris[2-(dimethylamino)ethyl]amine (Me6TREN, about 800 μL) to the same solution. Solution C was prepared by dissolving sodium ascorbate (about 180 mg) in about 18 mL of degassed solution A.

[0125] Surface functionalized silica particles (Si-Br, about 30 g) and solution A (about 300 mL) were added to a 500 mL round-bottom flask. 2-Hydroxyethyl methacrylate (HEMA, about 12.5 g) and methyl methacrylate (MMA, about 12.5 g) were added to the same flask. It should be noted that the ratio of HEMA to MMA can be changed to tune the hydrophilicity of the resulting copolymer, depending on the requirements of HPLC separation. The mixture was degassed with N2 bubbling for about 1 hour. Solution B and solution C were added to a round-bottom flask at the same time, and the reaction mixture was stirred at room temperature under N2 atmosphere for about 10 min. The reaction mixture was then filtered immediately, washed with solution A (about 30 mL), then washed twice with water (about 500 mL), and the crude product was continued to be vacuum filtered overnight to dryness. The next day, the crude product was washed with 0.25 wt% EDTA solution (about 100 mL), twice with water (about 500 mL), solution A (about 100 mL), methanol (about 100 mL), and vacuum filtration was continued for 1 h to produce copolymer-coated nonporous silica particles. The final product was further dried in a convection oven at about 80°C for about 1 h.

[0126] Preparation of chromatography column: The copolymer-coated silica particles were slurry-packed into a stainless steel column (50 x 2.1 mm) by a downward flow method. The slurry of the copolymer-coated silica particles was prepared by dispersing about 0.3 g of the copolymer-coated silica particles in about 15 ml of acetonitrile, stirring the mixture for a few minutes, and optionally sonicating it for a few minutes. Acetonitrile (about 50 mL) was used as the packing solvent, and the packing pressure was about 10,000 psi to about 20,000 psi.

[0127] Three sample chromatography columns were prepared. A control (reference) column V1 (or prototype 1) coated with a homopolymer of 100% MMA (without the first hydrophilic monomer) was prepared. Prototype column V2 (or prototype 2) had silica particles coated with a copolymer of a 1:1.3 HEMA:MMA molar ratio (this is equivalent to 50 / 50 wt% HEMA and MMA, as shown in Example 1). Prototype column V3 (or prototype 3) had silica particles coated with a copolymer of a 1:2.0 HEMA:MMA molar ratio (this is equivalent to 40 wt% / 60 wt% HEMA and MMA, respectively).

[0128] Example 2

[0129] Agilent 1290 Infinity II liquid chromatograph is used for chromatography measurement. Model ADC (Brentuximab-Vedotin conjugate) is used for the development of chromatographic separation. ADC sample is obtained from Seattle Genetics. ADC comprises antibody Brentuximab and drug conjugate Vedotin. Sample chromatography columns 1 and 2 and control chromatography columns prepared according to Example 1 are used as analytical columns, and their separation performance is compared. Mass spectrometer is coupled online with liquid chromatograph. Elution is performed using a gradient mobile phase based on ammonium acetate (50 mM) and isopropanol (IPA). Mobile phase A (MPA) contains 50 mM ammonium acetate aqueous solution, does not carry out pH adjustment (pH about 7), and mobile phase (MPB) contains 50 v / v% IPA and 50 v / v% 50 mM ammonium acetate aqueous solution, does not carry out pH adjustment. Gradient starts from 100 vol% MPA and is maintained for 2 minutes at 100% MPA. MPB was increased to 15 vol% MPB in 3 minutes (5 minutes total elapsed time), then increased to 50 vol% MPB in 15 minutes (20 minutes total elapsed time), maintained at 50 vol% MPB for 6 minutes (26 min total elapsed time), then returned to 100% MPA in 0.1 minutes and maintained there for 6 minutes (about 32 minutes total elapsed time). The flow rate was 0.2 mL / min and the column temperature was 30°C. Peak identities were assigned by matching the deconvoluted mass with the theoretical mass.

[0130] Figure 6 Shown are chromatograms obtained from analyzing a model ADC using control chromatography column V1 according to Example 2, wherein the top chromatogram shows MS detection, the middle chromatogram shows UV detection at a wavelength of 280 nm, and the bottom chromatogram shows UV detection at a wavelength of 215 nm. Figure 6 In Figure 1, the chromatograms represent the analysis of commercially available ADCs and illustrate the elution and resolution of DAR 0 to 4 drug conjugates (D0-D4). ADC species with high drug loading (i.e., DAR 5-8) do not elute from the control column under native conditions. In particular, Figure 6 As shown in the middle chromatogram of , chromatographic resolution of intact ADC with different DARs (e.g., D0, D1, D2, D3, and D4) and positional isomers in their native state using MS-compatible native reversed-phase conditions.

[0131] In the example of prototype column, the chromatographic elution and resolution of higher DAR species (DAR4 - DAR8) are realized by copolymerization of HEMA and MMA on non-porous silica in various ratios, which allows tuning the hydrophilicity of the stationary phase. In various examples, surface properties are controlled by using HEMA and MMA in various ratios. The retention of natural antibodies (D0 or DAR0) outside the column void and the baseline resolution of D1-D8 (or DAR1-DAR8) can be realized using complementary phases. Particle size can be optimized to allow lower test pressures, which allows faster analysis with less organic modifiers. In various examples, due to the incorporation of HEMA in the surface coating, the stationary phase allows elution of high DAR species (up to DAR8 or D8) under natural reverse phase conditions, which may be impossible for 100% MMA.

[0132] In various examples of the present disclosure, determination of the average DAR and the relative amount of each ADC species (e.g., D0-D8) may be possible. Complementary stationary phases provide new tools to reliably determine the average DAR and the relative amount of each ADC species. Complementary phases can provide appropriate retention and resolution of all DAR species under native reversed phase conditions. In addition, the eluents used in these separations are compatible with mass spectrometry for identification and characterization. Other techniques, such as reversed phase mode, can be combined with mass spectrometry, but the protein can be denatured and may no longer be in a native conformation.

[0133] Figures 7A-7E Shows Figure 6 The mass spectrum of the peaks marked in . For example, Fig. 7A The mass spectrum of D0 is shown; Figure 7B The mass spectrum of D1 is shown; Figure 7C The mass spectrum of D2 is shown; Fig.7D The mass spectrum of D3 is shown; Fig. 7E The mass spectrum of D4 is shown.

[0134] Figures 8A-8G Shown are chromatograms obtained from the analysis of a proprietary ADC according to Example 2 using a prototype column V2 (having silica particles coated with a copolymer having a HEMA:MMA molar ratio of -1:1.3), wherein the top graph is a chromatogram using MS detection, the middle graph is a chromatogram using UV detection at a wavelength of 280 nm, and the bottom graph is a chromatogram using UV detection at a wavelength of 215 nm.

[0135] Figures 9A-9F Results obtained from the analysis of ADC mimetics using the prototype chromatography column V2 according to Example 2 are shown. 9A: HRMS (top) and UV (bottom) chromatograms at 280 nm. 9B-9F Deconvoluted spectra of various DAR species

[0136] Figures 10A-10F Results obtained from the analysis of ADC mimetics using the prototype chromatography column V3 according to Example 2 are shown. 10A: HRMS (top) and UV (bottom) chromatograms at 280 nm. 10B-10F Deconvoluted spectra of various DAR species

[0137] Figures 11A-11C Results from the analysis of the commercial ADC Polivy using three different prototype columns, each with a different polymer composition, are shown. TM LC-HRMS XIC (XIC = extracted ion current) chromatogram of (polituzumab vedotin-piiq). Prototype-2 ( Fig.11A )、Prototype-4 ( Fig. 11B ) and Prototype-6 ( Fig. 11C ) columns have bonding polymer compositions of 50 / 50, 75 / 25 and 1 / 99 by weight of HEMA / MMA, respectively. This series of chromatograms shows that the polarity of the column can be adjusted by changing the polymer composition, and therefore the analyte retention is adjusted. This control of the binding phase polarity allows the retention of the column to be tuned to match the hydrophilicity of the analyte, which allows the analyte to be eluted with a relatively low organic solvent in its native intact state in the mobile phase. The calculated drug to antibody ratio (DAR) values ​​are displayed together with the XIC parameters and the separation equipment and conditions.

[0138] Figures 12A-12I The commercial ADC Polivy was analyzed on column prototype-2 (50 / 50 by weight HEMA / MMA). TM Detailed LC-HRMS results for (polituzumab vedotin-piiq). XIC chromatogram from native region (m / z 5000-8000) shows retention times of all DAR species. Raw HRMS spectra and deconvoluted neutral mass spectra of all DAR species are shown and demonstrate that all eluted species are in native intact state.

[0139] The present invention and the examples herein are further illustrated and embodied in the following clauses:

[0140] Item 1. A chromatographic stationary phase comprising:

[0141] a solid support; and

[0142] a polymer coating on the surface of a solid support;

[0143] Wherein the polymer coating comprises a copolymer comprising a first hydrophilic monomer and a second hydrophilic monomer, wherein the copolymer has a tuned hydrophilicity determined by a molar ratio of the first hydrophilic monomer and the second hydrophilic monomer.

[0144] Item 2. The chromatographic stationary phase of Item 1, wherein the first hydrophilic monomer and the second hydrophilic monomer are different.

[0145] Item 3. The chromatographic stationary phase of Item 1, wherein the second hydrophilic monomer has a lower hydrophilicity than the first hydrophilic monomer.

[0146] Item 4. The chromatographic stationary phase of Item 1, wherein the solid support comprises a multicapillary column, a monolithic column, a plurality of support particles, or any combination thereof.

[0147] Item 5. The chromatographic stationary phase of Item 1, wherein the solid support has a surface comprising a plurality of functional groups covalently attached thereto.

[0148] Item 6. The chromatographic stationary phase of Item 5, wherein the functional group is selected from at least one of hydroxyl, silanol, amino, or any combination thereof.

[0149] Item 7. The chromatographic stationary phase of Item 5, wherein the copolymer is attached to the surface of the solid support via a linker / spacer covalently linking the copolymer and at least one of the functional groups.

[0150] Item 8. The chromatographic stationary phase of Item 1, wherein the solid support comprises a plurality of inorganic particles selected from at least one of the following: silica particles, alumina particles, titania particles, zirconia particles, iron oxide products, glass beads, or a combination thereof.

[0151] Item 9. The chromatographic stationary phase of Item 1, wherein the support particles comprise a plurality of non-porous silica particles having a diameter of about 0.5 µm to about 50 µm.

[0152] Item 10. The chromatographic stationary phase of Item 1, wherein the first hydrophilic monomer comprises an ethylenic (-C=C) unsaturated group coupled to a hydrophilic functional group, wherein the hydrophilic functional group is selected from polyethylene oxide, hydroxyl, dihydroxyl, polyhydroxyl, carboxylic acid, sulfonic acid, phosphoric acid, amine, amide or any combination thereof.

[0153] Item 11. The chromatographic stationary phase of Item 1, wherein the second hydrophilic monomer comprises an ethylenically unsaturated group coupled to a linear or branched or cyclic (n>3) C1 to C18 saturated or unsaturated alkyl group, an aryl group, an aromatic group, a fluorocarbon group or any combination thereof.

[0154] Item 12. The chromatographic stationary phase of Item 1, wherein the first hydrophilic monomer is selected from 2-hydroxyethyl (meth)acrylate, 3-hydroxypropyl (meth)acrylate, 2,3-dihydroxypropyl (meth)acrylate, vinyl acetate, vinyl alcohol, sodium (meth)acrylate, tetrahydrofurfuryl (meth)acrylate, furfuryl (meth)acrylate, glycidyl (meth)acrylate, 2-(diethylamino)ethyl (meth)acrylate, 2-aminoethyl (meth)acrylate hydrochloride, 2-ethoxyethyl (meth)acrylate, potassium salt of 3-sulfopropyl (meth)acrylate, glycosyloxyethyl (meth)acrylate, or derivatives thereof, or any combination thereof.

[0155] Item 13. The chromatographic stationary phase of Item 1, wherein the second hydrophilic monomer is selected from alkyl (meth)acrylates (alkyl = Me, Et, n-Pr, i-Pr, n-Bu, s-Bu, t-Bu, etc.), hexyl (meth)acrylate, cyclohexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, isodecyl (meth)acrylate, isobornyl (meth)acrylate, phenyl (meth)acrylate, 2-naphthyl (meth)acrylate, 9-anthrylmethyl (meth)acrylate, 2,2,3,4,4,4-hexafluorobutyl (meth)acrylate, octadecyl (meth)acrylate, styrene, or a derivative thereof, or any combination thereof.

[0156] Item 14. The chromatographic stationary phase of Item 1, wherein the molar ratio of the first hydrophilic monomer to the second hydrophilic monomer ranges from 1:100 to about 100:1, from about 1:50 to about 50:1, from about 1:25 to about 25:1, from 1:10 to about 10:1, from about 1:5 to about 5:1, or from about 1:2 to about 2:1, from about 1:1.5 to about 1.5:1, from about 1:1.2 to about 1.2:1, from about 1.1:1 to about 1:1.1 or about 1:1.

[0157] Clause 15. The chromatographic stationary phase of clause 1, wherein the polymer coating comprises a copolymer of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), and wherein the molar ratio of HEMA to MMA is 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to about 25:1, 1:10 to about 10:1, about 1:5 to about 5:1, or about 1:2 to about 2:1, about 1:1.5 to about 1.5:1, about 1:1.2 to about 1.2:1, about 1.1:1 to about 1:1.1, or about 1:1.

[0158] Item 16. The chromatographic stationary phase of Item 1, wherein the polymer coating has a thickness of about 1 nm to about 500 nm.

[0159] Item 17. The chromatographic stationary phase of Item 1, wherein the polymer coating is formed by grafting / growing the copolymer from the surface of the solid support.

[0160] Item 18. The chromatographic stationary phase of Item 17, wherein the copolymer is grafted from the surface of the solid support via living / controlled radical copolymerization (L / CRP) of the first hydrophilic monomer and the second hydrophilic monomer.

[0161] Item 19. The chromatographic stationary phase of Item 18, wherein the copolymer is grafted from the surface of the solid support via atom transfer radical polymerization (ATRP) or nitroxide-mediated radical polymerization (NMP) or reversible addition-fragmentation chain transfer (RAFT).

[0162] Item 20. The chromatographic stationary phase of Item 19, wherein ATRP or AGET-ATRP is initiated by an initiator covalently attached to the surface of the solid support, and wherein the initiator comprises: (chloromethylphenethyl)trimethoxysilane, 3-trimethoxysilyl propyl 2-bromo 2-methylpropionate, [11-(2-bromo-2-ethyl)propionyloxy]undecyltrichlorosilane, or a combination thereof.

[0163] Item 21. The chromatographic stationary phase of Item 20, wherein the initiator is covalently attached to at least one functional group on the surface of the solid support, wherein the functional group is selected from hydroxyl, silanol, amino, or any combination thereof.

[0164] Item 22. A chromatography column comprising a chromatographic stationary phase according to Item 1.

[0165] Item 23. The chromatography column of Item 22, wherein the chromatography column has a length of 1 to 50 cm and an internal diameter of 25 μm to 10 cm.

[0166] Item 24. The chromatography column of Item 22, wherein the solid support comprises a plurality of support particles that are uniformly packed, and the support particles exhibit no cracks or uninterrupted contact between the inner surface of the chromatography column and the support particles.

[0167] Item 25. The chromatography column of Item 22, wherein the chromatography column is suitable for HPLC, UHPLC, FPLC, SPE, LC-MS.

[0168] Item 26. The chromatography column of Item 22, wherein the chromatography column is suitable for native hydrophobic interaction chromatography and native reverse phase chromatography of antibody-drug conjugate (ADC).

[0169] Clause 27. A method for preparing a chromatography column, the method comprising:

[0170] providing a solid support for a chromatography column; and forming a polymer coating on a surface of the solid support,

[0171] Wherein the polymer coating comprises a copolymer comprising a first hydrophilic monomer and a second hydrophilic monomer, wherein the copolymer has a tuned hydrophilicity determined by a molar ratio of the first hydrophilic monomer and the second hydrophilic monomer.

[0172] Item 28. The method of Item 27, wherein the first hydrophilic monomer and the second hydrophilic monomer are different.

[0173] Item 29. The method of Item 27, wherein the second hydrophilic monomer is less hydrophilic than the first hydrophilic monomer.

[0174] Clause 30. The method of Clause 27, wherein the solid support comprises a multicapillary column, a monolithic column, a plurality of support particles, or any combination thereof.

[0175] Clause 31. The method of Clause 27, wherein the solid support has a surface comprising a plurality of functional groups attached thereto.

[0176] Clause 32. The method of Clause 27, wherein the functional group is selected from at least one of hydroxyl, silanol, amino, or any combination thereof.

[0177] Clause 33. The method of Clause 27, wherein the solid support comprises a plurality of support particles, and wherein the method further comprises:

[0178] packing the chromatography column with support particles; and forming a polymer coating on the surface of at least one of the support particles.

[0179] Clause 34. The method of Clause 33, wherein forming the polymer coating comprises forming a copolymer covalently attached to the surface of the solid support via a linker / spacer covalently linking the copolymer to the surface.

[0180] Clause 35. The method of Clause 33, wherein the solid support comprises a plurality of inorganic particles selected from at least one of the following: silica particles, alumina particles, titania particles, zirconia particles, iron oxide products, glass beads, or combinations thereof.

[0181] Clause 36. The method of Clause 35, wherein the support particles comprise a plurality of non-porous silica particles having a diameter of about 0.5 μm to about 50 μm.

[0182] Clause 37. The method of Clause 27, wherein the first hydrophilic monomer comprises an ethylenic (-C=C) unsaturated group coupled to a hydrophilic functional group selected from polyethylene oxide, hydroxyl, dihydroxyl, polyhydroxyl, carboxylic acid, sulfonic acid, phosphoric acid, amine, amide, or any combination thereof.

[0183] Clause 38. The method of Clause 27, wherein the second hydrophilic monomer comprises an ethylenically unsaturated group coupled to a linear or branched or cyclic (n>3) C1 to C18 saturated or unsaturated alkyl group, an aryl group, an aromatic group, a fluorocarbon group, or any combination thereof.

[0184] Clause 39. The method of Clause 27, wherein the first hydrophilic monomer is selected from 2-hydroxyethyl (meth)acrylate, 3-hydroxypropyl (meth)acrylate, 2,3-dihydroxypropyl (meth)acrylate, vinyl acetate, vinyl alcohol, sodium (meth)acrylate, tetrahydrofurfuryl (meth)acrylate, furfuryl (meth)acrylate, glycidyl (meth)acrylate, 2-(diethylamino)ethyl (meth)acrylate, 2-aminoethyl (meth)acrylate hydrochloride, 2-ethoxyethyl (meth)acrylate, potassium salt of 3-sulfopropyl (meth)acrylate, glycosyloxyethyl (meth)acrylate, or a derivative thereof, or any combination thereof.

[0185] Clause 40. The method of Clause 27, wherein the second hydrophilic monomer is selected from alkyl (meth)acrylates (alkyl = Me, Et, n-Pr, i-Pr, n-Bu, s-Bu, t-Bu, etc.), hexyl (meth)acrylate, cyclohexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, isodecyl (meth)acrylate, isobornyl (meth)acrylate, phenyl (meth)acrylate, 2-naphthyl (meth)acrylate, 9-anthrylmethyl (meth)acrylate, 2,2,3,4,4,4-hexafluorobutyl (meth)acrylate, octadecyl (meth)acrylate, styrene, or a derivative thereof, or any combination thereof.

[0186] Clause 41. The method of clause 27, wherein the polymer coating comprises a copolymer of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), and wherein the molar ratio of HEMA to MMA is 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to about 25:1, 1:10 to about 10:1, about 1:5 to about 5:1, or about 1:2 to about 2:1, about 1:1.5 to about 1.5:1, about 1:1.2 to about 1.2:1, about 1.1:1 to about 1:1.1, or about 1:1.

[0187] Clause 42. The method of Clause 27, wherein the polymer coating has a thickness of about 1 nm to about 500 nm.

[0188] Clause 43. The method of Clause 27, wherein forming the polymer coating comprises grafting / growing the copolymer from the surface of the support particle via living / controlled polymerization (L / CRP) of the hydrophilic monomer and the second hydrophilic monomer using an initiator covalently attached to the surface of the support particle.

[0189] Item 44. The method of Item 43, wherein the copolymer is grafted from the surface of the support particle via atom transfer radical polymerization (ATRP) or activator regenerated ATRP, nitroxide mediated radical polymerization (NMP) or reversible addition-fragmentation chain transfer (RAFT).

[0190] Clause 45. The method of Clause 44, further comprising covalently attaching an initiator or initiating groups / sites to the surface of the solid particles prior to copolymerization, wherein the initiator is used to initiate the formation of the polymer coating on the support particles.

[0191] Item 46. The method of Item 45, wherein the initiator comprises: (chloromethylphenethyl)trimethoxysilane, 3-trimethoxysilyl propyl 2-bromo 2-methylpropionate, [11-(2-bromo-2-ethyl)propionyloxy]undecyltrichlorosilane, or a combination thereof.

[0192] Clause 47. The method of Clause 45, wherein the initiator is covalently attached to at least one functional group on the surface of the solid support, wherein the functional group is selected from hydroxyl, silanol, amino, or any combination thereof.

[0193] Item 48. The method of Item 27, wherein the chromatography column is packed after forming the polymer coating.

[0194] Clause 49. The method of Clause 27, wherein the chromatography column is packed to form packed support particles prior to forming the polymer coating, and wherein forming the polymer coating comprises grafting / growing the copolymer on the surface of at least one packed support particle.

[0195] Clause 50. The method of Clause 27, further comprising introducing a monomer or prepolymer solution to the packed support particles.

[0196] Clause 51. The method of Clause 27, wherein the polymer coating has a substantially uniform density throughout the support particle.

[0197] Item 52. A chromatography column prepared by the method of Item 27.

[0198] Clause 53. A method of preparing a chromatography column, the method comprising: providing a solid support comprising a plurality of support particles;

[0199] forming a polymer coating on the surface of at least one support particle; and filling a chromatography column with the support particles,

[0200] wherein the polymer coating comprises a copolymer of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), wherein the copolymer has a tuned hydrophilicity determined by a molar ratio of HEMA to MMA in a range of about 1:100 to about 100:1.

[0201] Clause 54. The method of clause 52, further comprising:

[0202] The initiator was covalently attached to the solid particle surface; the copolymer was grafted / grown from the surface via living / controlled radical copolymerization (L / CRP) of HEMA and MMA using the initiator.

[0203] Clause 55. A method for analyzing an antibody-drug conjugate (ADC) in a sample, the method comprising:

[0204] (a) exposing the sample to a chromatographic stationary phase of a reverse phase chromatography column, the chromatographic stationary phase comprising:

[0205] a solid support; and

[0206] a polymer coating on the surface of a solid support;

[0207] wherein the polymer coating comprises a copolymer comprising a first hydrophilic monomer and a second hydrophilic monomer, wherein the copolymer has a tuned hydrophilicity determined by a molar ratio of the first hydrophilic monomer and the second hydrophilic monomer;

[0208] (b) Applying an eluent to a chromatography column to elute the antibody-drug conjugate in its native intact form.

[0209] Clause 56. The method of Clause 55, wherein the solid support comprises a plurality of support particles having a diameter of about 0.5 μm to about 50 μm.

[0210] Clause 57. The method of Clause 55, wherein the first hydrophilic monomer comprises an ethylenic (-C=C) unsaturated group coupled to a hydrophilic functional group selected from polyethylene oxide, hydroxyl, dihydroxyl, polyhydroxyl, carboxylic acid, sulfonic acid, phosphoric acid, amine, amide, or any combination thereof.

[0211] Item 58. The method of Item 55, wherein the second hydrophilic monomer comprises an ethylenically unsaturated group coupled to a linear or branched or cyclic (n>3) C1 to C18 saturated or unsaturated alkyl group, an aryl group, an aromatic group, a fluorocarbon group, or any combination thereof.

[0212] Clause 59. The method of Clause 55, wherein the molar ratio of the first hydrophilic monomer to the second hydrophilic monomer ranges from 1:100 to about 100:1, from about 1:50 to about 50:1, from about 1:25 to about 25:1, from 1:10 to about 10:1, from about 1:5 to about 5:1, or from about 1:2 to about 2:1, from about 1:1.5 to about 1.5:1, from about 1:1.2 to about 1.2:1, from about 1.1:1 to about 1:1.1, or about 1:1.

[0213] Clause 60. The method of clause 55, wherein the polymer coating comprises a copolymer of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), and wherein the molar ratio of HEMA to MMA is 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to about 25:1, 1:10 to about 10:1, about 1:5 to about 5:1, or about 1:2 to about 2:1, about 1:1.5 to about 1.5:1, about 1:1.2 to about 1.2:1, about 1.1:1 to about 1:1.1, or about 1:1.

[0214] Clause 61. The method of Clause 55, wherein the polymer coating is formed by grafting / growing the copolymer from the surface of the solid support.

[0215] Clause 62. The method of Clause 55, wherein the copolymer is grafted from the surface via living / controlled radical copolymerization (L / CRP) of the first hydrophilic monomer and the second hydrophilic monomer.

[0216] Clause 63. The method of Clause 55, further comprising separating and resolving the eluted DAR species of the ADC in its native form.

[0217] Clause 64. The method of Clause 55, wherein the eluted DAR species of the ADC have the same or different drug to antibody ratios (DARs).

[0218] Clause 65. The method of Clause 55, further comprising detecting the eluted DAR species in the ADC in its native intact form using mass spectrometry.

[0219] Clause 66. The method of Clause 55, wherein mass spectrometry is used to establish a mass-to-charge (m / z) ratio or a molecular weight and a DAR value for at least one of the eluted ADCs across various DAR ratios.

[0220] Clause 67. The method of Clause 55, wherein the method is used to determine the relative amount of each eluted DAR species in an ADC sample.

[0221] Clause 68. The method of clause 55, wherein the method is used to separate positional isomers of DAR species in ADCs having the same DAR value.

[0222] Clause 69. The method of Clause 55, wherein the eluent has a salt concentration of no more than about 100 mM.

[0223] The above specification, examples and data provide a complete description of the manufacture and use of the composition of the disclosure.Since many embodiments of the disclosure can be made without departing from the spirit and scope of the disclosure, the disclosure resides in the claims hereinafter appended.

Claims

1. A chromatographic stationary phase comprising: a solid support; and a polymer coating on the surface of the solid support; Wherein the polymer coating comprises a copolymer comprising a first hydrophilic monomer and a second hydrophilic monomer, wherein the copolymer has a tuned hydrophilicity determined by a molar ratio of the first hydrophilic monomer and the second hydrophilic monomer.

2. The chromatographic stationary phase of claim 1, wherein the first hydrophilic monomer and the second hydrophilic monomer are different.

3. The chromatographic stationary phase of claim 1, wherein the second hydrophilic monomer has a lower hydrophilicity than the first hydrophilic monomer.

4. The chromatographic stationary phase of claim 1, wherein the solid support comprises a multicapillary column, a monolithic column, a plurality of support particles, or any combination thereof.

5. The chromatographic stationary phase of claim 4, wherein the support particles comprise a plurality of non-porous silica particles having a diameter of about 0.5 µm to about 50 µm.

6. The chromatographic stationary phase of claim 1, wherein the first hydrophilic monomer comprises an olefinic (-C=C) unsaturated group coupled to a hydrophilic functional group, wherein the hydrophilic functional group is selected from polyethylene oxide, hydroxyl, dihydroxyl, polyhydroxyl, carboxylic acid, sulfonic acid, phosphoric acid, amine, amide or any combination thereof.

7. The chromatographic stationary phase of claim 1, wherein the second hydrophilic monomer comprises an ethylenically unsaturated group coupled to a linear or branched or cyclic (n>3) C1 to C18 saturated or unsaturated alkyl group, an aryl group, an aromatic group, a fluorocarbon group or any combination thereof.

8. The chromatographic stationary phase of claim 1, wherein the first hydrophilic monomer is selected from 2-hydroxyethyl (meth)acrylate, 3-hydroxypropyl (meth)acrylate, 2,3-dihydroxypropyl (meth)acrylate, vinyl acetate, vinyl alcohol, sodium (meth)acrylate, tetrahydrofurfuryl (meth)acrylate, furfuryl (meth)acrylate, glycidyl (meth)acrylate, 2-(diethylamino)ethyl (meth)acrylate, 2-aminoethyl (meth)acrylate hydrochloride, 2-ethoxyethyl (meth)acrylate, potassium salt of 3-sulfopropyl (meth)acrylate, glycosyloxyethyl (meth)acrylate, or derivatives thereof, or any combination thereof.

9. The chromatographic stationary phase of claim 1, wherein the second hydrophilic monomer is selected from alkyl (meth)acrylates (alkyl = Me, Et, n-Pr, i-Pr, n-Bu, s-Bu, t-Bu, etc.), hexyl (meth)acrylate, cyclohexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, isodecyl (meth)acrylate, isobornyl (meth)acrylate, phenyl (meth)acrylate, 2-naphthyl (meth)acrylate, 9-anthrylmethyl (meth)acrylate, 2,2,3,4,4,4-hexafluorobutyl (meth)acrylate, octadecyl (meth)acrylate, styrene, or derivatives thereof, or any combination thereof.

10. The chromatographic stationary phase of claim 1, wherein the polymer coating comprises a copolymer of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), and wherein the molar ratio of HEMA to MMA is 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to about 25:1, 1:10 to about 10:1, about 1:5 to about 5:1, or about 1:2 to about 2:1, about 1:1.5 to about 1.5:1, about 1:1.2 to about 1.2:1, about 1.1:1 to about 1:1.1, or about 1:

1.

11. The chromatographic stationary phase of claim 1, wherein the polymer coating has a thickness of about 1 nm to about 500 nm.

12. The chromatographic stationary phase of claim 1, wherein the copolymer is grafted from the surface of the solid support via atom transfer radical polymerization (ATRP) or nitroxide-mediated radical polymerization (NMP) or reversible addition-fragmentation chain transfer (RAFT).

13. A chromatography column comprising the chromatographic stationary phase according to claim 1.

14. A method for analyzing an antibody-drug conjugate (ADC) in a sample, the method comprising: (a) exposing the sample to a chromatographic stationary phase of a reverse phase chromatography column, the chromatographic stationary phase comprising: Solid support; and a polymer coating on the surface of the solid support; wherein the polymer coating comprises a copolymer comprising a first hydrophilic monomer and a second hydrophilic monomer, wherein the copolymer has a tuned hydrophilicity determined by a molar ratio of the first hydrophilic monomer and the second hydrophilic monomer; (b) applying an eluent to the chromatography column to elute the antibody-drug conjugate in its native intact form.

15. The method of claim 14, wherein the polymer coating comprises a copolymer of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), and wherein the molar ratio of HEMA to MMA is 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to about 25:1, 1:10 to about 10:1, about 1:5 to about 5:1, or about 1:2 to about 2:1, about 1:1.5 to about 1.5:1, about 1:1.2 to about 1.2:1, about 1.1:1 to about 1:1.1, or about 1:

1.

16. The method of claim 14, further comprising isolating and resolving the eluted DAR species of the ADC in its native form.

17. The method of claim 14, wherein the method is used to separate positional isomers of DAR species in ADC having the same DAR value.

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