Coatings with immobilized affinity ligands and enzymes and their use in liquid chromatography assays

Abstract

The present invention provides a sample preparation device for enriching components of a sample, which comprises a surface in fluid communication with the sample, an alkylsilyl coating disposed on the surface, and an affinity ligand or enzyme covalently bonded to the alkylsilyl coating.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 866,847, filed on June 26, 2019, entitled “Coatings With Immobilized Affinity Ligands and Enzymes and Use Thereof in Liquid Chromatography Assays,” the entire contents of which are hereby incorporated by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on June 24, 2020 is named W-4064-US02_(102994_1013_4)_SL.txt and is 102 kilobytes in size. Technical Field

[0005] The present disclosure relates to sample preparation devices and methods for enriching components of a sample. More specifically, the present technology relates to sample preparation devices having an alkylsilyl coating and an affinity ligand or enzyme covalently bonded to the alkylsilyl coating, and methods for enriching components (e.g., analytes) of a sample using the sample preparation devices described herein. Background Art

[0006] Liquid chromatography (LC) systems are used to perform chemical separations. A typical liquid chromatography system consists of the following main components: a pump, a syringe, a column, and a detector. The pump forces a mobile phase, such as a solution, through a fluid path including a syringe, a column, and a detector. The syringe allows the sample to be introduced into the fluid flow above the column. The column contains a medium-filled bed. The medium is usually porous and relatively inert. The compounds in the sample will show a characteristic affinity to the medium. That is, some compounds show high affinity, while some compounds show low affinity. Therefore, as the compounds are carried through the medium, the compounds are separated into bands that elute or detach from the column at different times. These bands are detected by the detector.

[0007] When biological samples are separated from liquid chromatography (LC) and detected by mass spectrometry (MS), it is sometimes necessary to enhance selectivity and improve detection limits before introducing the sample into the LC-MS system. For example, highly hydrophobic plastic plates, commonly referred to as passive adsorption immunoassay plates, can be used. This type of plate adsorbs affinity ligands through non-covalent interactions. Once activated with a ligand such as a protein, it can be used for sample processing or immunoadsorption sandwich assays such as ELISA. Similarly, streptavidin immunoassay plates that can be used with biotinylated secondary ligands can be used. These plates include immobilized streptavidin. Without being bound by theory, it is believed that streptavidin is immobilized by activation and free radical-based polymerization of polystyrene, polystyrene divinylbenzene, polycarbonate or polypropylene. In both cases, these types of plates have significant disadvantages, namely that they need to form multiple intermolecular complexes to approach the target analyte, and many applications require the use of blocking agents such as polyethylene glycol sorbitan monolaurate. In addition, due to insufficient surface area, ligand coverage and binding capacity, they often do not show an ideal dynamic range.

[0008] Glygen Corporation has commercialized the so-called NuTip device (commercially available from GlySci a Glygen Corporation company, Columbia, MD) which is manufactured with heat and pressure to have a wall surface embedded with resin. Protein A and streptavidin affinity tips can be utilized. See U.S. Patent No. 6,416,716 as an example.

[0009] In addition, we have developed Biocoat TM Laboratory ware (commercially available from Corning Incorporation, Corning, NY) to facilitate the culture of adherent cell colonies. An example is sterile plastic modified to have a poly-lysine surface. In other cases, the sterile plastic is coated with collagen or fibronectin.

[0010] Kellie and colleagues published an immunocapture method that represents a hybrid ligand binding assay / LC-MS approach. As published in Bioanalysis (2016) 8 (20), 2103-2114, entitled A Whole-Molecule Immunocapture LC-MS Approach for the in vivo Quantitation of Biotherapeutics, an adsorption immunoassay plate called MaxiSorp NUNC polystyrene plate (commercially available from Thermo Fisher Scientific, Waltham, MA) was used to adsorb anti-idiotypic antibodies and then pull down and quantify biotherapeutic mAbs by LC-MS. This plate is a product of Thermo Fisher Scientific, which has a range of immunoassay plates, including passive adsorption, covalent linker activated supports, and immobilized streptavidin, glutathione, and nickel chelate plates. Summary of the invention

[0011] What is needed are methods and devices with lower interference, improved robustness, resiliency to harsh washing conditions, finely tuned binding capacity, and reduced non-specific binding that can be used to prepare samples to be assayed by LC-MS. Adsorption-based immunocapture and enzymatic sample preparation devices are compromised by contamination, which can be exhibited by desorbing ligands and blocking agents such as polyethylene glycol sorbitan monolaurate. The methods and devices of the present technology provide materials that are easily tuned to have minimal undesirable secondary interactions with analytes of interest.

[0012] One benefit of the present technology is that the methods and devices are very robust due to the chemical connection (covalent bond) between the affinity ligand or enzyme and the underlying alkylsilyl coating. This allows for regeneration and reuse of the device as well as preventing interference or infiltration of the biological ligand into the purified analyte. Additionally, due to the silane nature of the bond, the device is highly tunable. The microenvironment can be adjusted to ensure stability of the affinity ligand or enzyme.

[0013] The present technology relates to a coating device for LC-MS sample preparation based on an alkylsilyl composition and a covalently immobilized biological ligand. It provides a ready-to-use device for anti-human Fc domain capture, where non-covalent interactions are not relied upon except for interactions corresponding to the ligand:target human Fc domain complex. In addition, a secondary resin layer can be used to provide increased surface area and binding capacity.

[0014] In one aspect, the technology features a sample preparation device for enriching components of a sample. The sample preparation device includes a surface in fluid communication with the sample, an alkylsilyl coating disposed on the surface, and an affinity ligand or enzyme covalently bonded to the alkylsilyl coating. The sample preparation device may include one or more of the embodiments described herein.

[0015] In some embodiments, the affinity ligand is bonded to the alkylsilyl coating. More than one affinity ligand can be bonded to the alkylsilyl coating. In other embodiments, the enzyme is bonded to the alkylsilyl coating. More than one enzyme can be bonded to the alkylsilyl coating.

[0016] In some embodiments, the alkylsilyl coating has at least Thickness.

[0017] The surface can be the inside surface of a well plate, the inside surface of a vial, or the inside surface of a pipette.

[0018] In some embodiments, the sample preparation device is used for chromatography.The sample preparation device can be used for mass spectrometry.

[0019] In some embodiments, the alkylsilyl coating is an inert coating, for example, the alkylsilyl coating does not interact with the analyte in the sample. The alkylsilyl coating may have Formula I:

[0020]

[0021] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Each independently selected from (C1-C6) alkoxy, -NH(C1-C6) alkyl, -N((C1-C6) alkyl)2, OH, OR A and halogen. A Indicates the point of attachment to the surface. R 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least one of them is OR A . X is (C1-C 20 )alkyl, -O[(CH2)2O] 1-20 -、-(C1-C 10 )[NH(CO)NH(C1-C 10 )] 1-20 -or-(C1-C 10)[Alkylphenyl(C1-C 10 )alkyl] 1-20 -.

[0022] In some embodiments, X is (C2-C 10 In some embodiments, X is ethyl. 1 , R 2 , R 3 , R 4 , R 5 and R 6 Each may be methoxy or chlorine. In some embodiments, the alkylsilyl coating of Formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

[0023] The sample preparation device may further include a second alkylsilyl coating in direct contact with the alkylsilyl coating of Formula I. The second alkylsilyl coating may have Formula II:

[0024]

[0025] R 7 , R 8 and R 9 R is independently selected from -NH(C1-C6)alkyl, -N[(C1-C6)alkyl]2, (C1-C6)alkoxy, (C1-C6)alkyl, (C1-C6)alkenyl, OH and halogen. 10 Selected from (C1-C6) alkyl, -OR B 、-[O(C1-C3)alkyl] 1-10 O(C1-C6)alkyl and phenyl, wherein the (C1-C6)alkyl is optionally substituted by one or more halogens, and wherein the phenyl is optionally substituted by one or more groups selected from (C1-C3)alkyl, hydroxy, fluorine, chlorine, bromine, cyano, -C(O)NH2 and carboxyl. B R is -(C1-C3)alkylethylene oxide, -(C1-C3)alkyl-3,4-epoxycyclohexyl or -(C1-C4)alkylOH. 10 The cut bond represents R 10 Optional additional covalent bonds to the carbon of the bridging silyl group to form an olefin, provided that y is not 0. y is an integer from 0 to 20. The second alkylsilyl coating may modify the alkylsilyl coating of Formula I.

[0026] In some embodiments, y is an integer from 2 to 9. In some embodiments, y is 9, R 10 is methyl, and R 7 , R 8 and R 9 Each is ethoxy or chlorine.

[0027] In some embodiments, the alkylsilyl coating of formula II is (3-glycidoxypropyl)trimethoxysilane, n-decyltrichlorosilane, trimethylchlorosilane, trimethyldimethylaminosilane, or methoxy-polyethyleneoxy (1-10) silane. In some embodiments, the alkylsilyl coating of formula II is (3-glycidoxypropyl)trimethoxysilane after hydrolysis. The alkylsilyl coating of formula I can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II can be (3-glycidoxypropyl)trimethoxysilane. In some embodiments, the alkylsilyl coating of formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II is (3-glycidoxypropyl)trimethoxysilane after hydrolysis. The alkylsilyl coating of formula I can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II can be n-decyltrichlorosilane. In some embodiments, the alkylsilyl coating of formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II is trimethylchlorosilane or trimethyldimethylaminosilane. The alkylsilyl coating of formula I can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II can be methoxy-polyethyleneoxy(3)silane.

[0028] In some embodiments, the sample preparation device further comprises an alkylsilyl coating in direct contact with the alkylsilyl coating of Formula I, the alkylsilyl coating having Formula III,

[0029]

[0030] R 11 , R 12 , R 13 , R 14 , R 15 and R 16 Each is independently selected from (C1-C6) alkoxy, -NH(C1-C6) alkyl, -N((C1-C6) alkyl)2, OH and halogen. Z is (C1-C6) alkoxy 20 )alkyl, -O[(CH2)2O] 1-20 -、-(C1-C 10 )[NH(CO)NH(C1-C 10 )] 1-20 -or-(C1-C 10 )[Alkylphenyl(C1-C 10 )alkyl] 1-20The alkylsilyl coating of formula III can modify the alkylsilyl coating of formula I.

[0031] In some embodiments, the alkylsilyl coating of formula III is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. The alkylsilyl coating of formula I can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula III can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

[0032] In some embodiments, the alkylsilyl coatings of I and III have about Total thickness.

[0033] In some embodiments, the alkylsilyl coating is an active coating, e.g., the alkylsilyl coating reacts with an analyte in the sample. The alkylsilyl coating is selected to interact with at least one analyte in the sample by (1) repulsive forces, (2) secondary interactions, or (3) a retention mechanism different from interaction with a stationary phase material.

[0034] In some embodiments, the analyte and the active coating are negatively charged. The analyte and the active coating may be positively charged. In some embodiments, the secondary interaction is an ion exchange partition.

[0035] In some embodiments, the alkylsilyl coating has Formula IV:

[0036]

[0037] Each X is independently selected from (C1-C6) alkoxy, -NH(C1-C6) alkyl, -N((C1-C6) alkyl)2, OH, OR A , R B , R C , R D and halogen. A represents an attachment point to a surface of a sample preparation device or a frit, and at least one X is OR A . R B Absent, or represents a hydrophobic modifier. C represents a charge modifier, and at least one X is R C . R D is any of the following: absent, a chelating agent or a crown ether. Y is selected from (C1-C 20 )alkyl, -O[(CH2)2O] 1-20 -、-(C1-C 10 )[NH(CO)NH(C1-C 10 )] 1-20-or-(C1-C 10 )[Alkylphenyl(C1-C 10 )alkyl] 1-20 -The bridging part.

[0038] In some embodiments, the charge modifier (R C )for

[0039]

[0040] Z is independently selected from (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N((C1-C6)alkyl)2, OH, halogen, or the point of attachment to Formula IV. At least one Z is the point of attachment to Formula IV.

[0041] In some embodiments, the charge modifier (R C )for

[0042]

[0043] Z is independently selected from (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N((C1-C6)alkyl)2, OH, halogen, or the point of attachment to Formula I. At least one Z is the point of attachment to Formula IV.

[0044] In some embodiments, the charge modifier (R C ) is a quaternary amine.

[0045] In some embodiments, a hydrophobic modifier (R B ). Hydrophobic modifier (R B ) may have a net neutral charge. In some embodiments, the hydrophobic modifier (R B )for

[0046]

[0047] Z is independently selected from (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N((C1-C6)alkyl)2, OH, halogen, or the point of attachment to Formula IV. At least one Z is the point of attachment to Formula IV.

[0048] In some embodiments, the hydrophobic modifier (R B )for

[0049]

[0050] Z is independently selected from (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N((C1-C6)alkyl)2, OH, halogen, or the point of attachment to Formula IV. At least one Z is the point of attachment to Formula IV.

[0051] In some embodiments, there is R D . R D In some embodiments, R D is a chelating agent, and the chelating agent is etidronic acid. D It may be a crown ether, and the crown ether is selected from the group consisting of 18-crown-6, 12-crown-4, 15-crown-5, dibenzo-18-crown-6 and diaza-18-crown-6.

[0052] The affinity ligand can be an antibody, a single domain antibody, an aptamer, an affibody, streptavidin, protein A, protein G or protein L or a combination thereof.

[0053] In some embodiments, the sample preparation device further comprises a linker (or spacer molecule) to covalently bond an affinity ligand or enzyme to the alkylsilyl coating. The linker can be maleimide PEG silane, NHS PEG maleimide, or PEG dialdehyde, wherein the PEG repeat is between 3 and 300. In some embodiments, the linker is glutaraldehyde or acryloxysilane.

[0054] In some embodiments, the covalent bond between the affinity ligand or enzyme and the alkylsilyl coating is formed by reductive amination.

[0055] The affinity ligand may have a concentration of about 1 nmol / m 2 to about 1000nmol / m 2 In some embodiments, the affinity ligand may have a surface coverage of about 2 nmol / m 2 to about 500 nmol / m 2 surface coverage.

[0056] In some embodiments, the enzyme is selected from a protease, a glycosidase, a ligase, a transferase, an oxidoreductase, an isomerase, a hydrolase, or a combination thereof. The enzyme may be a protease, and the protease may be selected from IdeS, IdeZ, or a combination thereof. In some embodiments, the enzyme is a β-glucuronidase. The enzyme may have a concentration of about 1 nmol / m 2 to about 1000nmol / m 2 In some embodiments, the enzyme may have a surface coverage of about 2 nmol / m 2 to about 500 nmol / m 2 surface coverage.

[0057] In some embodiments, the affinity ligand has the sequence depicted in SEQ ID NO: 1. The affinity ligand may be at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence depicted in SEQ ID NO:1.

[0058] In some embodiments, the affinity ligand has the sequence depicted by SEQ ID NO: 2. The affinity ligand may be at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence depicted by SEQ ID NO:2.

[0059] In some embodiments, the enzyme has the sequence depicted by SEQ ID NO: 3. The enzyme may be at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence depicted by SEQ ID NO:3.

[0060] In some embodiments, the enzyme has the sequence depicted by SEQ ID NO: 4. The enzyme may be at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the sequence depicted by SEQ ID NO:4.

[0061] In another aspect, the technology features a sample preparation device for enriching components of a sample. The sample preparation device includes a surface in fluid communication with the sample; an alkylsilyl coating disposed on the surface; a resin made of silica, organosilica, or a polymer, the resin covalently bonded to the alkylsilyl coating; and an affinity ligand or enzyme covalently bonded to the resin. The sample preparation device may include one or more of the embodiments described herein.

[0062] In some embodiments, the resin is covalently bonded to the alkylsilyl coating via the silane.

[0063] The sample preparation device can be used for chromatography, mass spectrometry, or a combination thereof.

[0064] On the other hand, the present technology is characterized by a method for analyzing a biological fluid. The method includes providing a sample preparation device. The sample preparation device includes a surface in fluid communication with the biological fluid, an alkylsilyl coating disposed on the surface, and an affinity ligand covalently bonded to the alkylsilyl coating. The method also includes pumping the biological fluid containing the analyte onto the surface of the sample preparation device to form an affinity complex so that the analyte is adsorbed. The adsorbed analyte is washed with a neutral isotonic buffer. The adsorbed analyte is eluted from the surface of the sample preparation device. The method also includes analyzing the eluted sample using liquid chromatography, mass spectrometry, ELISA, capillary electrophoresis, or a combination thereof. The method may include one or more embodiments of the embodiments described herein.

[0065] In some embodiments, the sample preparation device is a 96-well microelution plate, a conical bottom vial, or a pipette tip.

[0066] The affinity ligand may be capable of capturing a human immunoglobulin Fc domain. In some embodiments, the affinity ligand is a nucleic acid aptamer, an immunoglobulin, a single domain VHH camelid antibody, or a combination thereof.

[0067] In another aspect, the present technology features a method for separating a biological fluid. The method includes providing a glass frit. The glass frit includes a surface in fluid communication with the biological fluid, an alkylsilyl coating disposed on the surface, and an affinity ligand covalently bonded to the alkylsilyl coating. The glass frit is positioned at the entrance of a chromatographic column. The method also includes loading a biological fluid containing an analyte onto the surface of the glass frit, capturing the analyte to form an affinity complex, such that the analyte is adsorbed on the surface of the glass frit while the remainder of the biological fluid passes through the chromatographic column, eluting the adsorbed analyte from the surface of the glass frit, such that the analyte passes through the chromatographic column, and detecting the analyte with a detector. The method may include one or more of the embodiments described herein.

[0068] In some embodiments, the chromatography column is a size exclusion chromatography column.The detector may be a mass spectrometer.

[0069] In some embodiments, the biological fluid comprises a humanized mAb-based therapeutic agent.

[0070] The affinity ligand can be an anti-human Fc aptamer, an anti-human Fc immunoglobulin antibody, an anti-human Fc single domain antibody ligand, or a combination thereof.

[0071] In another aspect, the present technology features a method for quantifying opioid glucuronides. The method includes providing a sample preparation device. The sample preparation device includes a surface in fluid communication with a sample containing opioid glucuronides, an alkylsilyl coating disposed on the surface, and a β-glucuronidase covalently bonded to the alkylsilyl coating. The method also includes pumping a sample containing opioid glucuronides onto the surface of the sample preparation device so that the opioid glucuronides are hydrolyzed. Recover the hydrolyzed opioid glucuronides from the surface of the sample preparation device. Inject the recovered hydrolyzed opioid glucuronides onto a liquid chromatography column. The method may include one or more of the embodiments described herein.

[0072] On the other hand, the present technology is characterized in that a method for preparing a sample containing a protein for peptide mapping is provided. The method includes providing a sample preparation device. The sample preparation device includes a surface connected to a sample fluid containing the protein, an alkylsilyl coating disposed on the surface, and a protease covalently bonded to the alkylsilyl coating, wherein the protease is trypsin, Lys-C, Arg-C, Lys-N, Glu-C, Asp-N, pepsin, or a combination thereof. The method also includes aspirating the sample containing the protein onto the surface of the sample preparation device so that the protein is digested into peptide fragments. The peptide fragments are recovered from the sample preparation device and the peptide fragments are injected onto a liquid chromatography column. The method may include one or more embodiments of the embodiments described herein.

[0073] In some embodiments, the protease is trypsin, Lys-C, Asp-N, or a combination thereof.

[0074] The alkylsilyl coating can be bis(trichlorosilyl)ethane, and the protease can be trypsin, wherein the trypsin is covalently bonded to the bis(trichlorosilyl)ethane via a maleimide PEG silane linker. In some embodiments, the maleimide PEG silane linker is:

[0075]

[0076] Where n is between 3 and 300.

[0077] In some embodiments, the alkylsilyl coating is a bis(trichlorosilyl)ethane modified with triethoxysilylbutyraldehyde, and the protease is trypsin, wherein the trypsin is covalently bonded to the bis(trichlorosilyl)ethane modified with triethoxysilylbutyraldehyde by reductive amination. The alkylsilyl coating can be a bis(trichlorosilyl)ethane modified with 3-aminopropyltrimethoxysilane, and the protease can be trypsin, wherein the trypsin is covalently bonded to the bis(trichlorosilyl)ethane modified with 3-aminopropyltrimethoxysilane by reductive amination with a PEG dialdehyde. In some embodiments, the PEG dialdehyde is:

[0078]

[0079] Where n is between 3 and 300.

[0080] In some embodiments, the alkylsilyl coating is bis(trichlorosilyl)ethane modified with triethoxysilylbutyraldehyde and acrylate PEG amine, and the protease is trypsin, wherein the trypsin is covalently bonded to the modified bis(trichlorosilyl)ethane by reductive amination and Michael addition. The acrylate PEG amine can be:

[0081]

[0082] Where n is between 3 and 300.

[0083] In some embodiments, the alkylsilyl coating is bis(trichlorosilyl)ethane modified with acryloxypropyltrimethoxysilane, and the protease is trypsin, wherein the trypsin is covalently bonded to the acryloxypropyltrimethoxysilane modified bis(trichlorosilyl)ethane via Michael addition.

[0084] On the other hand, the present technology is characterized in that a method for analyzing a sample containing a monoclonal antibody (mAb) is provided. The method includes providing a sample preparation device. The sample preparation device includes a surface connected to a sample fluid containing the mAb, an alkylsilyl coating disposed on the surface, and an enzyme covalently bonded to the alkylsilyl coating, wherein the enzyme is IdeS, IdeZ, IgdE, Gingipain K, or a combination thereof. The method also includes aspirating the sample containing the mAb onto the surface of the sample preparation device so that the mAb is fragmented into subunits. The subunits of the mAb are recovered from the sample preparation device, and the subunits of the mAb are injected onto a liquid chromatography column. The method may include one or more embodiments of the embodiments described herein.

[0085] On the other hand, the present technology is characterized by a method for analyzing a sample containing a glycoprotein. The method includes providing a sample preparation device. The sample preparation device includes a surface in fluid communication with a sample containing a glycoprotein, an alkylsilyl coating disposed on the surface, and a PNGase F enzyme covalently bonded to the alkylsilyl coating. The method also includes aspirating the sample containing the glycoprotein onto the surface of the sample preparation device so that the glycans of the glycoprotein are cleaved. The deglycosylated glycoprotein is recovered from the sample preparation device and the deglycosylated glycoprotein is injected onto a liquid chromatography column. The method may include one or more of the embodiments described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 is a schematic diagram of a sample preparation device for enriching components of a sample according to an illustrative embodiment of the present technology.

[0087] Figure 2 is a schematic diagram of a microvolume well for enriching components of a sample according to an illustrative embodiment of the present technology.

[0088] Figure 3 is a schematic diagram of a microvolume well that has been derivatized to have a coated surface with an immobilized bioligand and a secondary resin layer in accordance with an illustrative embodiment of the present technology.

[0089] Figure 4 is a diagram showing an exemplary embodiment of an active coating according to an illustrative embodiment of the present technology.

[0090] Figure 5A is a table summarizing several examples of coatings and immobilized bioligands according to illustrative embodiments of the present technology.

[0091] Figure 5B yes Figure 5A , showing additional columns associated with Formula II and Formula III according to illustrative embodiments of the present technology.

[0092] Figure 5C yes Figure 5A and Figure 5B Continuation of the table showing additional columns related to linkage and bioligand according to an illustrative embodiment of the present technology. DETAILED DESCRIPTION

[0093] The present technology includes devices, compositions, and methods of using devices and LC flow path components (e.g., glass frits) that are modified with organosilica (alkylsilyl) functional groups and derivatized to support immobilized affinity ligands and / or enzymes. This technology is used to create surfaces that are beneficial for various types of offline and online liquid chromatography-mass spectrometry (LC-MS) sample preparation, ranging from quantification of monoclonal antibodies (mAbs) in biological fluids to the use of β-glucuronidase devices to assist in the quantification of opioid glucuronides. In an exemplary embodiment, the surface is coated with an alkylsilyl coating by vapor deposition and subsequently treated using a liquid phase reaction to immobilize an affinity ligand with capture selectivity for the Fc domain of a human immunoglobulin.

[0094] Device

[0095] Figure 11 is a schematic diagram of a sample preparation device 100 for enriching components of a sample. The device 100 includes a substrate 105 having a surface 110. The surface is in communication with a sample fluid. The substrate 105 can be, for example, a well plate, a vial, a pipette, or any other substrate used for sample preparation. The surface 110 can be an inner surface (e.g., a surface in communication with a sample fluid) of a well plate (also known as a microelution plate), an inner surface of a vial (e.g., a conical bottom vial), or an inner surface of a pipette (e.g., a pipette tip). The surface 110 can be any surface in communication with or in contact with a sample fluid, such as a glass frit at the entrance of a chromatographic column. An alkylsilyl (organic silica) coating 115 is disposed on the surface 110. The alkylsilyl coating 115 can be disposed on the surface 110 in a variety of ways, for example, the alkylsilyl coating 115 can be vapor deposited on the surface 110. A biological ligand 120, such as an affinity ligand and / or an enzyme, can be covalently bonded 125 to the alkylsilyl coating 115. The sample preparation device 100 may be used for chromatography and / or mass spectrometry.

[0096] Figure 2 2 is a schematic diagram of a microvolume well 200 for enriching components of a sample. The microvolume well 200 can be part of a well plate, such as a 96-well plate. As discussed above, the microvolume well 200 has a surface 205 (e.g., an inner surface) that is in fluid communication with the sample. An alkylsilyl coating 210 is disposed on the surface 205. In some embodiments, there is an optional surface modification coating 215 deposited on the alkylsilyl coating 210. The optional surface modification coating 215 can be, for example, a second alkylsilyl coating, a charge modifier, a hydrophobic modifier, a chelating agent, and / or a crown ether. A biological ligand 220, such as an affinity ligand and / or an enzyme, can be covalently bonded to the alkylsilyl coating 210 or the optional surface modification coating 215. The microvolume well 200 can be used for chromatography and / or mass spectrometry.

[0097] Figure 3 300 which has been derivatized to have a coated surface containing an immobilized bioligand and a secondary resin layer. The microvolume well 300 can be part of a well plate, such as a 96-well plate. As discussed above, the microvolume well 300 has a surface 305 (e.g., an inner surface) that is in fluid communication with the sample. An alkylsilyl coating 310 is disposed on the surface 305. In some embodiments, there is an optional surface modification coating (not shown). A resin 315 made of silica, organosilica, or a polymer is covalently bonded to the alkylsilyl coating 310 (or an optional surface modification coating). The resin 315 can be covalently bonded to the alkylsilyl coating 310 via a silane. A bioligand 320, such as an affinity ligand and / or an enzyme, is covalently bonded to the resin 315. The microvolume well 300 can be used for chromatography and / or mass spectrometry.

[0098] The resin 315 may be derived from spherical or non-spherical irregular particles made of silica, organosilica, or polymers. The resin 315 may form a layer on the alkylsilyl coating 310. The resin 315 increases the surface area where the bioligand 320 may bond and thus increases the sample preparation device ( Figure 3 The capacity of the microvolume pores in the resin (300). Non-porous resins can be used for applications that are sensitive to diffusion effects. Porous resins can be used in applications where high surface area is more critical because the pores of the resin increase the total surface area.

[0099] Alkyl silyl coating

[0100] Organosilica (alkylsilyl) is a versatile composition for coating both metal and polymer flow path components, which can range from sample preparation devices, syringe needles, and pre-column heaters to post-column tubing, column housings, frits, detector flow cells, and electrospray needles. Organosilica (alkylsilyl) coatings can be applied via gas or solution phase reactions and with a number of different silanizing agents. For example, alkylsilyl coatings can be applied via vapor deposition.

[0101] In one embodiment, it is advantageous to first apply the bis-silyl or tri-silyl bridging silane reagent via vapor deposition to achieve high build, A thick base layer (e.g., Formula I as described herein) can be formed using previously described techniques (e.g., those described in U.S. Patent Application No. 16 / 133,089, filed on September 17, 2018 and published on March 21, 2019 as U.S. Patent Publication No. 2019-0086371, the entire contents of which are incorporated herein by reference). An exemplary silane that has been demonstrated to be useful in forming the base layer is bis(trichlorosilyl)ethane. Optionally, the base layer can be treated with a charge-modified or hydrophobic / hydrophilic modified silanizing agent (including, but not limited to, the charge-modifying agents (R C ) and hydrophobic modifier (R B) reagents). These surface modifiers may be composed of strong acids / bases or weak acids / bases with dissociation constants selected within the desired pKa range, and may include, but are not limited to, trimethoxysilylpropylmethylphosphonate and N,N-(diethylaminopropyl)trimethoxysilane. Surface modifiers with fixed charges, such as quaternary amines, may also be used. Alternatively, silanes containing primary and secondary amines may be used, which also provide nucleophilic substances for subsequent fixation reactions. In addition, aldehyde-containing silanes, such as triethoxysilylbutyraldehyde, may be used to provide sites for reductive amination attachment or acryloxypropyltrimethoxysilane for Michael addition. These surface modifiers may be incorporated in situ during the vapor deposition process or by secondary liquid phase reactions. In some cases, these liquid phase reactions may need to include multiple steps in order to establish the desired thickness and / or coverage of the modified silane. The surface to be modified with a charge modifier may have a charge modifier of about 0.01 μmol / m 2 About 10 μmol / m 2 , about 0.02 μmol / m 2 About 1 μmol / m 2 In another embodiment, the bridging silane layer may alternatively be modified with a net neutral charge hydrophobic modifier (R B ) derivatized. In other embodiments, they are not derivatized. Included within these reagent options are zwitterionic modifiers such as carboxybetaine and sulfobetaine reagents. Zwitterionic modifiers can provide a macroscopic net neutral surface, but can also impart a microscopic Coulombic effect that can resist binding or repel a wide range of analytes, including those with different charge characteristics. As with charged surface modifiers, hydrophobic modifiers can range from 0.01 μmol / m 2 To 10 μmol / m 2 Surface coverage deposition.

[0102] The term "about" means that the value following it is not an exact value, but the center point of the + / -5% range of the value. If the value is a relative value given as a percentage, the term "about" also means that the value following it is not an exact value, but the center point of the + / -5% range of the value, whereby the upper limit of the range cannot exceed a value of 100%.

[0103] Exemplary alkylsilyl coatings that can be used include bioinert coatings such as those described in U.S. Patent Application No. 16 / 133,089, filed on September 17, 2018 and published on March 21, 2019 as U.S. Patent Publication No. 2019-0086371, the entire contents of which are incorporated herein by reference. In addition, active coatings (coatings that interact with the analyte or mobile phase) can also be used.

[0104] Inert coating

[0105] In some aspects, alkylsilyl coatings are used to modify sample preparation devices or flow paths of chromatographic systems to address interactions with, for example, analytes. That is, alkylsilyl coatings can be biologically inert low-binding coatings that minimize surface reactions with metal-interacting analytes and allow analytes to pass along the flow path without blocking, attaching to the surface, or changing analyte properties. Reducing / eliminating these interactions is advantageous because it allows accurate quantification and analysis of samples containing metal-interacting analytes, such as biomolecules, proteins, polysaccharides, peptides, oligonucleotides, pesticides, bisphosphonates, anionic metabolites, and zwitterions such as amino acids and neurotransmitters. Alkylsilyl coatings may have Formula I:

[0106]

[0107] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Each independently selected from (C1-C6) alkoxy, -NH(C1-C6) alkyl, -N((C1-C6) alkyl)2, OH, OR A and halogen. A Indicates the inner surface of the sample preparation device (e.g. Figure 1 The attachment point is the inner surface 110 of the glass material or the surface of the glass material. 1 , R 2 , R 3 , R 4 , R 5 and R 6 At least one of them is OR A . X is (C1-C 20 )alkyl, -O[(CH2)2O] 1-20 -、-(C1-C 10 )[NH(CO)NH(C1-C 10 )] 1-20 -or-(C1-C 10 )[Alkylphenyl(C1-C 10 )alkyl] 1-20 -.

[0108] In some embodiments, X is (C2-C 10 ) alkyl. X may be ethyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 and R6 Each is methoxy or chlorine. The alkylsilyl coating of formula I may be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

[0109] In some embodiments, the assembly further includes a second alkylsilyl coating in direct contact with the alkylsilyl coating of Formula I. The second alkylsilyl coating has Formula II:

[0110]

[0111] R 7 , R 8 and R 9 R is independently selected from -NH(C1-C6)alkyl, -N[(C1-C6)alkyl]2, (C1-C6)alkoxy, (C1-C6)alkyl, (C1-C6)alkenyl, OH and halogen. 10 Selected from (C1-C6) alkyl, -OR B 、-[O(C1-C3)alkyl] 1-10 O(C1-C6)alkyl and phenyl. (C1-C6)alkyl is optionally substituted by one or more halogens. Phenyl is optionally substituted by one or more groups selected from (C1-C3)alkyl, hydroxy, fluorine, chlorine, bromine, cyano, -C(O)NH2 and carboxyl. R B R is -(C1-C3)alkylethylene oxide, -(C1-C3)alkyl-3,4-epoxycyclohexyl or -(C1-C4)alkylOH. 10 The cut bond represents R 10 An optional additional covalent bond to the carbon of the bridging silyl group to form an olefin, provided that y is not 0. y is an integer from 0 to 20.

[0112] In some embodiments, y is an integer from 2 to 9. In some embodiments, y is 9, R 10 is methyl, and R 7 , R 8 and R 9 Each is methoxy, ethoxy or chlorine. The alkylsilyl coating of formula II can be (3-glycidyloxypropyl)trimethoxysilane, n-decyltrichlorosilane, trimethylchlorosilane, trimethyldimethylaminosilane or methoxy-polyethyleneoxy (1-10) silane. In some embodiments, the alkylsilyl coating of formula II is (3-glycidyloxypropyl)trimethoxysilane after hydrolysis.

[0113] The alkylsilyl coating of formula I may be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II may be (3-glycidyloxypropyl)trimethoxysilane. In some embodiments, the alkylsilyl coating of formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II is (3-glycidyloxypropyl)trimethoxysilane after hydrolysis. The alkylsilyl coating of formula I may be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II may be n-decyltrichlorosilane. In some embodiments, the alkylsilyl coating of formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II is trimethylchlorosilane or trimethyldimethylaminosilane. The alkylsilyl coating of formula I may be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II may be methoxy-polyethyleneoxy(3)silane.

[0114] In some embodiments, the assembly further comprises an alkylsilyl coating having Formula III in direct contact with the alkylsilyl coating of Formula I. The alkylsilyl coating of Formula III is:

[0115]

[0116] R 11 , R 12 , R 13 , R 14 , R 15 and R 16 Each is independently selected from (C1-C6) alkoxy, -NH(C1-C6) alkyl, -N((C1-C6) alkyl)2, OH and halogen. Z is (C1-C6) alkoxy 20 )alkyl, -O[(CH2)2O] 1-20 -、-(C1-C 10 )[NH(CO)NH(C1-C 10 )] 1-20 -or-(C1-C 10 )[Alkylphenyl(C1-C 10 )alkyl] 1-20 -.

[0117] In some embodiments, the alkylsilyl coating of formula III is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. The alkylsilyl coating of formula I can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula III can be bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

[0118] In the present technology, vapor deposited alkylsilyl coatings on the surface of a sample preparation device or glass frit modify the surface and reduce secondary interactions. As a result, they are biologically inert or low binding (meaning that the analyte of the sample does not interact with the alkylsilyl coating). In addition, the deposited coatings are highly tunable to provide a range of desired contact angles (e.g., making the wetting surface hydrophilic or hydrophobic), chemistries, and properties to achieve the desired effect on the flow path and ultimately the sample passing through the flow path.

[0119] The thickness of the alkylsilyl coating may be at least about For example, the thickness may be about to about The thickness of the multi-layer alkylsilyl coating of Formula I may be about

[0120]

[0121] Exemplary coatings with their corresponding approximate thicknesses and contact angles are provided in Table 1.

[0122] Table 1

[0123]

[0124]

[0125] Active coating

[0126] In addition to inert coatings, it is also beneficial to modify flow path components including sample preparation devices and glass frits with organosilica (alkylsilyl) functional groups that exhibit chemical properties that have an impact on chromatographic separations. These so-called "active" coatings can introduce Coulombic repulsion effects with analytes to prevent them from interacting with the substrate materials (e.g., metals, plastics, etc.) of the flow path in a manner that adversely affects the chromatographic separation. In addition, "active" coatings can be produced that add enhanced selectivity to the separation through subtle secondary interactions. In addition, "active" coatings that provide completely orthogonal retention mechanisms can be prepared, which are conducive to adsorption, distribution and desorption of analytes in processes different from those achieved with the stationary phase of the column. Active coatings can also interact with the mobile phase to produce desired effects on chromatographic separations, such as to enhance selectivity.

[0127] At least a portion of the surface of the sample preparation device or frit is coated with an alkylsilyl coating having Formula IV:

[0128]

[0129] Each X of Formula IV is independently selected from (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N((C1-C6)alkyl)2, OH, OR A , R B , R C , R D In some embodiments, X of Formula IV is independently selected from OR A , R B , R C and R D . (C1-C6) alkoxy, -NH(C1-C6) alkyl, -N((C1-C6) alkyl)2, OH and halogen are reactive / hydrolyzable groups. In some embodiments, these groups provide residual reactive groups, where amino>halogen>alkoxy when it comes to silane reactivity. In some embodiments, the specific coating used (e.g., the specific choice of X in Formula IV) depends on the type of chromatographic separation being performed. The goal is to form a chemically stable (good shelf life) coating on various materials present in the chromatographic flow path. However, hydrophilic coatings do not interact in reversed phase liquid chromatography, while hydrophilic coatings may show some retention in hydrophilic interaction chromatography. Although one coating may be sufficient for a variety of different chemical separations, different coatings may also be used for different liquid chromatography modes.

[0130] R A represents an attachment point to the surface of the sample preparation device, and at least one X is OR A . R B Absent, or represents a hydrophobic modifier.C represents a charge modifier, and at least one X is R C . R D is any of the following: absent, a chelating agent or a crown ether. Y is selected from (C1-C 20 )alkyl, -O[(CH2)2O] 1-20 -、-(C1-C 10 )[NH(CO)NH(C1-C 10 )] 1-20 -or-(C1-C 10 )[Alkylphenyl(C1-C 10 )alkyl] 1-20 The choice of Y can be determined by both chemical and physical considerations.

[0131] Thus, the alkylsilyl coating of formula IV has at least one A X, an attachment point to the surface of the sample preparation device and at least one R C (which represents a charge modifier) ​​X. Hydrophobic modifier R B and chelating agent or crown ether R D May not be present. In some embodiments, the alkylsilyl coating of formula IV has a charge modifier. In some embodiments, the alkylsilyl coating of formula IV has a charge modifier and a hydrophobic modifier. In some embodiments, the alkylsilyl coating of formula IV has a charge modifier and a chelating agent or a crown ether. In some embodiments, the alkylsilyl coating of formula IV has a charge modifier, a hydrophobic modifier, and a chelating agent or a crown ether.

[0132] In other embodiments, the alkylsilyl coating of Formula IV has at least one A X, an attachment point to the surface of the sample preparation device and at least one R D (which represents a chelating agent or a crown ether). In some embodiments, the alkylsilyl coating of Formula IV has a chelating agent or a crown ether and a hydrophobic modifier.

[0133] When used in the context of a chemical formula, a hyphen ("-") indicates a point of attachment. For example, when Y is -[(C1-C 10 )alkylphenyl(C1-C 10 )alkyl] 1-20 -, this means that Y is (C1-C 10 ) alkyl is attached to one SiX3 and is linked to another (C1-C 10 ) alkyl is connected to another SiX3. This applies to the remaining variables.

[0134] In one embodiment, Y in Formula IV is (C1-C15 )alkyl, (C1-C 12 )alkyl or (C1-C 10 ) alkyl. In some embodiments, Y in Formula IV is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, nonyl or decyl. On the other hand, Y in Formula IV is ethyl or decyl.

[0135] In some embodiments, X of formula IV can be another Y bridging moiety. In this way, the alkylsilyl coating can have more than two Si atoms bridged by two separate Y bridging moieties. When multiple Y bridging moieties are in the alkylsilyl coating, the Y bridging moieties can be the same Y moiety or different Y moieties. Multiple bridging moieties can cause the alkylsilyl coating to be in a linear or branched state. In addition, the alkylsilyl coating with multiple Y bridging moieties can be highly cross-linked using extensive bridging. The first bridged alkylsilyl composition can be highly cross-linked, with certain surface heterogeneity and a mixture of bridging moieties and silanols. The surface can then be modified with one or more of the modifiers described herein (e.g., charge modifiers, hydrophobic modifiers, chelating agents and / or crown ethers, or surfactants).

[0136] In some embodiments, the active alkylsilyl coating is at least partially composed of Formula IV as described herein. Thus, the alkylsilyl coating of Formula IV is at least a portion of the coating. The coating may have additional Y-bridging moieties utilizing a variety of modifiers as described herein.

[0137] In some embodiments, a single modifier is used, such as a single charge modifier. In other embodiments, multiple modifiers are used, such as multiple charge modifiers or a combination of a charge modifier and a hydrophobic modifier.

[0138] Charge modifier (R C )

[0139] Charge-modified salting agents can be applied to a suitable substrate. These charged surface modifiers are composed of strong acids / bases or weak acids / bases with dissociation constants selected within the desired pKa range, and may include but are not limited to trimethoxysilylpropylmethylphosphonate and N,N-(diethylaminopropyl)trimethoxysilane. Surface modifiers with fixed charges, such as quaternary amines, can also be used. For example, for the separation of negatively charged nucleic acids or phosphopeptides, the surface charge can be a negative charge. In such cases, the analyte is repelled from the surface and is not lost due to nonspecific adsorption.

[0140] Charge modifier (R C) may have the same charge as at least one analyte in the sample. When the charge modifier has the same charge as at least one analyte in the sample, the charge modifier introduces a Coulomb repulsion effect (i.e., such as charge repulsion) to the sample with the same charge. For example, both the analyte and the active coating may be negatively charged. Both the analyte and the active coating may be positively charged. This repulsion effect protects the analyte from interacting with the substrate material of the flow path. The selection of a suitable charge modifier may also depend on practical limitations, such as whether a suitable silane reagent has a source, or if it needs to be synthesized, it may be evaporated or prepared to be soluble in a suitable solvent system for reaction. In addition, the stability of the resulting coating should be considered. For example, a strong base that is also a nucleophilic substance may be problematic because it may autocatalyze its hydrolytic degradation.

[0141] In addition to introducing Coulomb repulsion effects to samples with the same charge, charge modifiers can also facilitate multidimensional chromatography by strongly retaining a class of analytes via a retention mechanism orthogonal to the retention mechanism of the implemented liquid chromatography column. For example, analytes adsorbed to a flow path component (e.g., a glass frit at the entrance of a chromatographic column) having an alkylsilyl coating containing a charge modifier are eluted to a downstream chromatographic column with a dispersive mobile phase change gradient or a set of dispersive mobile phase changes, and thus eluted with a secondary gradient on the packed bed of the chromatographic column. As another example, the analytes are adsorbed to a sample preparation device having an alkylsilyl coating containing a charge modifier.

[0142] The charge modifier may be, for example, any one or more of the following charge modifiers: quaternary amines,

[0143]

[0144]

[0145] Z is independently selected from (C1-C6) alkoxy, -NH(C1-C6) alkyl, -N((C1-C6) alkyl)2, OH, halogen or an attachment point to Formula IV. At least one Z is an attachment point to Formula IV. Thus, the charge modifier is covalently bonded to the alkylsilyl coating of Formula IV via the silyl ether moiety. The corresponding charge modifier can be used to obtain the charge modifier of the final coating product, for example, the charge modifier can be trimethoxysilylpropylmethylphosphonate or N,N-(diethylaminopropyl)trimethoxysilane or 2-(4-pyridylethyl)triethoxysilane.

[0146] In some embodiments, a single charge modifier is applied to the vapor deposited alkylsilyl (Formula IV) coating. In other embodiments, multiple charge modifiers are applied to the vapor deposited alkylsilyl (Formula IV) coating. The multiple charge modifiers can be the same charge modifier applied at multiple X positions of Formula IV. Alternatively, the multiple charge modifiers can be different charge modifiers applied at different X positions of Formula IV.

[0147] In some embodiments, the vapor deposited alkylsilyl coating may be intentionally doped with a charged surface modifier (R C ), such as the charged surface modifiers of U.S. Patent No. 10,159,911, entitled “High Purity Chromatographic Materials Comprising an Ionizable Modifier,” published on December 25, 2018, the entire contents of which are hereby incorporated by reference.

[0148] Charge modifier (R C ) may have a value between about 0.01 μmol / m 2 About 10 μmol / m 2 In some embodiments, the charge modifier (R C ) has a value between about 0.02 μmol / m 2 About 1 μmol / m 2 In some embodiments, the charge modifier (R C ) has a value between about 0.1 μmol / m 2 About 1 μmol / m 2 Surface coverage between.

[0149] can be Figure 4 The coating #3 obtained has a charge modifier (R C Examples of active coatings of alkylsilyl coatings of Formula IV of ) are shown below. Although the final alkylsilyl coating shown below has a usefulness for other charge modifiers (R C ), but these points can also be (C1-C6) alkoxy, -NH(C1-C6) alkyl, -N((C1-C6) alkyl)2, OH or halogen, as described herein. In addition, although the final alkylsilyl coating shown below has the advantages of being suitable for other charge modifiers (R C ) points, but these points can also be hydrophobic modifiers (R B ) and / or chelating agents or crown ethers. The charge modifiers (R C ) may also be other attachment points to the surface of the sample preparation device, i.e., RA .

[0150]

[0151] Hydrophobic modifier (R B )

[0152] In some embodiments, a charge modifier (R C ) of alkylsilyl coating and hydrophobic modifier (R B ) combination. The hydrophobic modifier may have a net charge neutrality. Included within the reagent selection of hydrophobic modifiers are zwitterionic modifiers, such as carboxybetaine and sulfobetaine reagents. Zwitterionic modifiers provide a macroscopic net neutral surface, but also impart a microscopic Coulomb effect that can repel a wide range of analytes, including those with different charge characteristics.

[0153] Hydrophobic modifier (R B ) may have a value between about 0.01 μmol / m 2 About 10 μmol / m 2 In some embodiments, the hydrophobic modifier (R B ) has a value between about 0.03 μmol / m 2 to about 0.9 μmol / m 2 In some embodiments, the hydrophobic modifier (R B ) has a value between about 0.5 μmol / m 2 About 3 μmol / m 2 Surface coverage between.

[0154] Hydrophobic modifier (R B ) is attached to Formula IV via a silyl ether moiety and has a composition selected from one or more of the following:

[0155]

[0156]

[0157] Z is independently selected from (C1-C6) alkoxy, -NH(C1-C6) alkyl, -N((C1-C6) alkyl)2, OH, halogen or an attachment point to Formula IV. At least one Z is an attachment point to Formula IV. Thus, the hydrophobic modifier is covalently bonded to the alkylsilyl coating of Formula IV via the silyl ether moiety. The charge modifier of the final coating product can be obtained using the corresponding hydrophobic modifying agent, for example, the charge modifying agent can be (3-glycidyloxypropyl) trimethoxysilane, hydrolyzed (3-glycidyloxypropyl) trimethoxysilane, n-decyltrichlorosilane or N-(3-trimethoxysilylpropyl) glucamide.

[0158] Several coatings corresponding to exemplary embodiments of alkylsilyl coatings of the present technology are shown in Figure 4 These coatings include only charge modifiers (R C ) coatings, such as coatings #1 and #3, and having a charge modifier (R C ) and hydrophobic modifier (R B ) coatings of both, such as coatings #2, #4 and #5.

[0159] The combination of a hydrophobic modifier and a charge modifier can be used to increase weak but well-controlled secondary interactions. Molecules that would otherwise co-elute due to having nearly identical hydrophobicity can be removed by, for example, coating with Figure 4 Frit of coating #4 shown, wherein when mobile phase based on gradient between water and acetonitrile is applied together with acid modifiers of different acidity, hydrophobicity and ion pairing strength, molecules can experience weak ion exchange partitioning. Mobile phase modified by formic acid is particularly useful in this regard, because it does not weaken electrostatic effects. This may be particularly useful when separating, for example, peptides, measuring citric acid cycle metabolites and polysaccharides. Combinations of hydrophobic modifiers and charge modifiers can also be used in hydrophilic interaction chromatography.

[0160] can be Figure 4 The coating #4 obtained has a charge modifier (R C ) and hydrophobic modifier (R B Examples of active coatings of alkylsilyl coatings of Formula IV with ) are shown below. Although the resulting coatings shown below have various charge modifiers (R C ), but any or all of these points may be hydrophobic modifiers (R B ) or (C1-C6) alkoxy, -NH(C1-C6) alkyl, -N((C1-C6) alkyl)2, OH, or halogen or chelating agent or crown ether, as described herein. In some embodiments, the charge modifier (R C ) and hydrophobic modifier (R B) does not need to be directly connected, for example, the charge modifier (R C ) and hydrophobic modifier (R B ) may be connected via one or more bridging silicon moieties having a chemical composition of Y (as defined herein).

[0161]

[0162] Chelating agents and crown ethers (R D )

[0163] In some embodiments, a charge modifier (R C ) with an alkylsilyl coating and an ion-scavenging ligand (e.g., a chelating agent or a crown ether) (i.e., R D The chelating agent may be, for example, ethylenediaminetetraacetic acid or etidronic acid. The crown ether may be, for example, 18-crown-6, 12-crown-4, 15-crown-5, dibenzo-18-crown-6 or diaza-18-crown-6.

[0164] Chelating agents and crown ethers can be covalently attached to the silanized / alkylsilyl coatings either by direct covalent attachment or by using a secondary linker moiety. Chelating agents and crown ethers can scavenge ions such as Ca that may be present in the mobile phase, flow path, and system. 2+ , Fe 3+ and / or K + , these ions lead to undesirable gas phase ion adducts and cause fouling and deactivation of the mass spectrometer. For example, chelating agents or crown ethers can pull metal ions out of the mobile phase to improve the MS spectrum. Typically, even MS-grade mobile phases will contain trace amounts of metal ions, such as sodium and potassium, so the quality of the mass spectrum is usually affected. Potassium at a concentration of 50 ppb can produce potassium adduct ions with a relative intensity of 2% to 10%. Active coatings such as these can multivalently chelate these metal ions, thereby ensuring that they are not present in the chromatographic effluent during ionization. Subsequently, even when a mobile phase with questionable purity is used, a mass spectrum can be obtained with an ion adduct signal with a relative intensity of less than 2%. The use of chelating agents and crown ethers may be useful when the present technology is used in combination with a glass frit at the inlet of a chromatographic column. Although not limited to theory, it is believed that a chelating agent-modified alkylsilane coating can be combined with a fixed ligand to consume certain ions from the sample. This can be used to eliminate certain interferences with downstream detection or to promote a specific type of buffer exchange.

[0165] In some embodiments, the chelating agent and crown ether can be combined directly with the alkylsilyl coating of Formula IV in the absence of a charge modifier.

[0166] In some embodiments, chelators and crown ethers may also be used in combination with the hydrophobic modifiers described herein. In some embodiments, chelators and crown ethers may be used in combination with both the charge modifiers and the hydrophobic modifiers described herein.

[0167] Thus, when a chelating agent and / or a crown ether is used as an active coating, the surface of a sample preparation device or a glass frit at the inlet of a chromatography column can be coated. At least a portion of the sample preparation device or glass frit is coated with an alkylsilyl coating having Formula IV:

[0168]

[0169] wherein each X is independently selected from (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N((C1-C6)alkyl)2, OH, OR A , R B , R C , R D and halogen. A represents an attachment point to the inner surface of the sample preparation device or frit, and at least one X is OR A . R B Absent, or represents a hydrophobic modifier. C Absent, or represents a charge modifier. D represents a chelating agent or a crown ether, and at least one X is R D Y is selected from (C1-C 20 )alkyl, -O[(CH2)2O] 1-20 -、-(C1-C 10 )[NH(CO)NH(C1-C 10 )] 1-20 -or-(C1-C 10 )[Alkylphenyl(C1-C 10 )alkyl] 1-20 -The bridging part.

[0170] Antibacterial modifier

[0171] At least a portion of the surface of the glass frit at the inlet of the sample preparation device or chromatography column is coated with an alkylsilyl coating having Formula IV:

[0172]

[0173] wherein each X is independently selected from (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N((C1-C6)alkyl)2, OH, OR A , R B , R C , R D , RE and halogen. A represents an attachment point to a surface of a sample preparation device or a frit, and at least one X is OR A . R B Absent, or represents a hydrophobic modifier. C Absent, or represents a charge modifier. D Absent, or represents a chelating agent or crown ether. E is an antibacterial moiety, and at least one X is R E Y is selected from (C1-C 20 )alkyl, -O[(CH2)2O] 1-20 -、-(C1-C 10 )[NH(CO)NH(C1-C 10 )] 1-20 -or-(C1-C 10 )[Alkylphenyl(C1-C 10 )alkyl] 1-20 -The bridging part.

[0174] In some embodiments, the antibacterial moiety is a zwitterion. Zwitterionic coatings can be prepared by silylation with bis(trichlorosilyl)ethane, followed by derivatization with N,N-(diethylaminopropyl)trimethoxysilane, and subsequent reaction with chloro- or bromo-alkylcarboxylic acids. In some embodiments, the antibacterial moiety is a quaternary amine.

[0175] The antibacterial moiety can be used alone or in combination with a charge modifier, a hydrophobicity modifier and / or a chelating agent or a crown ether.

[0176] Surfactants

[0177] In some embodiments, surfactants can be used as active coatings to solubilize proteins, including hydrophobic membrane proteins, and provide a denaturing environment that is favorable for enzymatic reactions, such as proteolysis and glycan release.

[0178] At least a portion of the surface of the sample preparation device or frit is coated with an alkylsilyl coating having Formula IV:

[0179]

[0180] wherein each X is independently selected from (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N((C1-C6)alkyl)2, OH, OR A , R B , R C , R D , R E , R F and halogen. Arepresents an attachment point to a surface of a sample preparation device or a frit, and at least one X is OR A . R B Absent, or represents a hydrophobic modifier. C Absent, or represents a charge modifier. D Absent, or represents a chelating agent or crown ether. E Not present, or indicates an antibacterial part. F represents a surfactant, and at least one X is R F Y is selected from (C1-C 20 )alkyl, -O[(CH2)2O] 1-20 -、-(C1-C 10 )[NH(CO)NH(C1-C 10 )] 1-20 -or-(C1-C 10 )[Alkylphenyl(C1-C 10 )alkyl] 1-20 -The bridging part.

[0181] In some embodiments, the surfactant (R F ) is sodium dodecyl sulfate, 3-10 (decyldimethylammonium) propane sulfonate zwittergent, sodium deoxycholate, hexadecyltrimethylammonium bromide, triton, polysorbate, or a combination thereof. In some embodiments, the surface is modified to have such a coverage of an alkylsilyl coating, wherein one region is modified to carry an immobilized bioligand, while another exhibits an immobilized surfactant that promotes denaturation of a protein analyte. In practice, this surface can then be used to induce denaturation of a protein analyte while also inducing the action of a bioligand such as a protease thereon, the protease including but not limited to trypsin.

[0182] Biological ligand

[0183] The alkylsilyl coatings described herein can be modified with biological ligands, such as affinity ligands or enzymes, to produce off-line and on-line LC sample preparation devices with significant utility. The surface coverage of the biological ligand (i.e., affinity ligand or enzyme) of the device containing the alkylsilyl coating and immobilized biological ligand ranges from about 1 nmol / m 2 to about 1000nmol / m 2 , more specifically about 2 nmol / m 2 to about 500 nmol / m 2Devices with alkylsilyl coatings and immobilized bioligands can be used to perform sample preparation for subsequent LC-based assays, including enrichment of the analyte or conversion of it into components that can be more easily or more selectively detected. Analyses facilitated using this technology can also include capillary electrophoresis and mass spectrometry.

[0184] Affinity ligand

[0185] The alkylsilyl coatings described above can be modified with biological ligands, such as affinity ligands, to create off-line and on-line LC sample preparation devices with significant utility.

[0186] The alkylsilyl surface deposited as described above is further modified using the present technology to carry a fixed affinity ligand that can facilitate the analyte sample preparation step to facilitate LC-MS determination. Affinity chromatography is used in countless determinations to capture analytes, improve selectivity and improve detection limits. Affinity capture is the starting point for analyzing biological preparations produced by cell culture and analyzing biological therapeutic agents circulating in patient biological fluids. In various options, the coating is composed of covalently linked antibodies, single domain antibodies, aptamers, affibodies, streptavidin, protein A, protein G, protein L or a combination thereof. These ligands can be directly connected to the silanization / alkylsilyl coating, which may be carried out via reductive amination, NHS-activated electrophilic substitution, carbodiimide dehydration or Michael addition reactions. In some embodiments, a spacer molecule (or linker) is used to connect the affinity ligand to the coating. For example, a protein ligand containing free thiol can be connected to the coating by silanization and a secondary reaction with maleimide PEG silane, wherein the range of PEG repeats can be from 3 to 300 (see below). Alternatively, aminosilane modified coatings can be combined with NHS activated maleimide PEG to achieve the same results. Likewise, aminosilane modified surfaces can be combined with dialdehyde reagents such as glutaraldehyde or PEG dialdehyde and reductive amination to provide immobilization that bridges the amine surface to the amine functional groups of the bioligand. Furthermore, the coating can be modified with acryloxysilanes and can then be used for Michael addition type immobilization of the bioligand.

[0187] Examples of spacer molecules (or linkers) are shown below, wherein n is from 3 to 300.

[0188]

[0189]

[0190] A selection of exemplary biological ligands is provided below. One example shows a single amino acid residue sequence of a camelid single domain antibody that can be used to selectively capture a human immunoglobulin Fc domain. For this ligand, a C-terminal cysteine ​​residue is present to facilitate directional conjugation. For a second example, the sequence of a nucleic acid aptamer is provided. This aptamer also shows affinity for the human immunoglobulin Fc domain. It is shown as an amino-terminal 5' construct through which site-specific conjugation can be implemented via reductive amination. In yet another example, the sequence of porcine trypsin that can be used for protein digestion is provided. Figure 5A-5C Several examples of coatings and immobilized bioligands that can be prepared using the above-mentioned components are summarized.

[0191] Sequence (amino acids) of a Camelidae VHH single domain antibody for capturing the Fc domain of a human immunoglobulin:

[0192] QVQLQESGGGLVQPGGSLRSCAASGGTSIRIGSINALAWYRQALGNQRELVAAVTEGGSTNYADFVKGRFTISRDQAQNMMYLQMNSLKPEDTAVYYCNADKVLYSRGGYYSVANDLWGQGTQVTVSSQAPKVDAKFDC(SEQID NO:1)

[0193] Sequence of nucleic acid aptamer for capturing human immunoglobulin Fc domain (nucleic acid):

[0194] 5'amino-GGrArGrGfUrGCfUCCGAAArGrGAAfCfUCC (SEQ ID NO: 2) wherein G represents 2'deoxyguanosine, rA represents 2'hydroxyadenine, rG represents 2'hydroxyguanosine, fU represents 2'-fluoro-2'-deoxyuridine, C represents 2'deoxycytidine, A represents 2'deoxyadenine, and 5'amino represents a 5' modification consisting of a primary amino group.

[0195] In some embodiments, the affinity ligand has the sequence depicted in SEQ ID NO: 1. The affinity ligand may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence depicted in SEQ ID NO: 1.

[0196] In some embodiments, the affinity ligand has the sequence depicted by SEQ ID NO: 2. The affinity ligand may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence depicted by SEQ ID NO: 2.

[0197] Affinity ligand method

[0198] The present technology also includes a method for analyzing a biological fluid using an affinity ligand fixed on an alkylsilyl-coated substrate (e.g., a sample preparation device). The method includes providing a sample preparation device, for example, any sample preparation device having an affinity ligand fixed on an alkylsilyl-coated surface of the sample preparation device. The method also includes pumping a biological fluid containing an analyte onto the surface of the sample preparation device to form an affinity complex so that the analyte is adsorbed. The adsorbed analyte is washed with a neutral isotonic buffer. The adsorbed analyte is eluted from the surface of the sample preparation device. The eluted sample is then analyzed using liquid chromatography, mass spectrometry, ELISA, capillary electrophoresis, or a combination thereof.

[0199] The affinity ligand used in the method can be any affinity ligand described herein. The affinity ligand can be capable of capturing the human immunoglobulin Fc domain. For example, a single acid residue sequence of a camelid single domain antibody can be used to selectively capture the human immunoglobulin Fc domain. Nucleic acid aptamers also show affinity for the human immunoglobulin Fc domain.

[0200] In another embodiment, an affinity ligand fixed on an alkylsilyl coated substrate (e.g., glass frit) is used to provide a method for separating biological fluids. The method includes providing a glass frit at the entrance of a chromatographic column. The glass frit has an affinity ligand fixed on the alkylsilyl coated surface of the glass frit, for example, any coating with a fixed affinity ligand as described herein. The biological fluid containing the analyte is loaded onto the surface of the glass frit. The analyte is captured to form an affinity complex so that the analyte is adsorbed on the surface of the glass frit, and the remainder of the biological fluid passes through the chromatographic column. The adsorbed analyte is eluted from the surface of the glass frit so that the analyte passes through the chromatographic column. The analyte is then detected by a detector.

[0201] The chromatographic column can be a size exclusion chromatography (SEC) column. The detector can be a mass spectrometer. The biofluid can be a therapeutic agent based on a humanized mAb. The affinity ligand can be an anti-human Fc aptamer, an anti-human Fc immunoglobulin antibody, an anti-human Fc single domain antibody ligand, or a combination thereof.

[0202] When separating biological samples, the alkylsilyl coating and the affinity ligand can each provide a desired effect for the overall separation. For example, when separating a sample containing a biological fluid, an affinity ligand can be used to form an affinity complex with an analyte in the sample, while the alkylsilyl coating can ensure that other analytes in the sample are not adsorbed to the surface of the sample preparation device (e.g., due to interaction with the metal substrate) and therefore are not eluted with the analyte that forms an affinity complex with the affinity ligand. In this way, the inert alkylsilyl coating together with the affinity ligand beneficially reduces the adsorption of the analyte on the substrate, while the affinity ligand selectively adsorbs a specific analyte, which can then be eluted and detected.

[0203] Enzymes

[0204] Alkylsilyl coatings as described above can be modified with bioligands such as enzymes that are helpful for sample analysis. These enzymes can be selected from proteases, glycosidases, ligases, transferases, oxidoreductases, isomerases, hydrolases, or combinations thereof. Proteases of great significance for peptide mapping, IdeS and IdeZ for antibody fragmentation, glycosidases, carboxypeptidases, and nucleases that can be used for O- and N- polysaccharide profiling can all be used to provide different embodiments of the present invention. In some cases, a mixture of enzymes can be fixed to achieve a single pot reaction and more than one chemical conversion. Alternatively, the present technology can include a β-glucuronidase for facilitating the analysis of opioid glucuronides. These can be made into a part of an off-line sample preparation device, or a sample preparation machine or a dedicated integrated LC-MS analyzer can be created. In some aspects, the surfaces coated with these fixed enzymes can be prepared and used with an LC system to make a protein digestion device or analyzer that can be used for peptide mapping of protein therapeutics.

[0205] The enzyme can be, for example, trypsin, Lys-C, Arg-C, Lys-N, Glu-C, Asp-N, pepsin, Ides, IdeZ, IgdE, Gingipain K, PNGase F, or a combination thereof.

[0206] Provided below are examples of sequences for β-glucuronidases that can be used to hydrolyze opioid metabolites. Figure 5A-5C Several examples of coatings and immobilized bioligands that can be prepared using the above-mentioned components are summarized.

[0207] Sequence (amino acids) of porcine trypsin as a protease for protein digestion:

[0208] IVGGYTCAANSIPYQVSLNSGSHFCGGSLINSQWVVSAAHCYKSRIQVRLGEHNIDVLEG

[0209] NEQFINAAKIITHPNFNGNTLDNDIMLIKLSSPATLNSRVATVSLPRSCAAAGTECLISG

[0210] WGNTKSSGSSYPSLLQCLKAPVLSDSSCKSSYPGQITGNMICVGFLEGGKDSCQGDSGGP

[0211] VVCNGQLQGIVSWGYGCAQKNKPGVYTKVCNYVNWIQQTIAAN(SEQ ID NO:3)

[0212] Sequence (amino acids) of β - glucuronidase for hydrolyzing opioid metabolites:

[0213] GLQGGMLYPQESPSRECKELDGLWSFRADFSDNRRRGFEEQWYRRPLWESGPTVDMPVPSSFNDISQDWRLRHFVGWVWYEREVILPERWTQDLRTRVVLRIGSAHSYAIVWVNGVDTLEHEGGYLPFEADISNLVQVGPLPSRLRITIAINNTLTPTTLPPGTIQYLTDTSKYPKGYFVQNTYFDFFNYAGLQRSVLLYTTPTTYIDDITVTTSVEQDSGLVNYQISVKGSNLFKLEVRLLDAENKVVANGTGTQGQLKVPGVSLWWPYLMHERPAYLYSLEVQLTAQTSLGPVSDFYTLPVGIRTVAVTKSQFLINGKPFYFHGVNKHEDADIRGKGFDWPLLVKDFNLLRWLGANAFRTSHYPYAEEVMQMCDRYGIVVIDECPGVGLALPQFFNNVSLHHHMQVMEEVVRRDKNHPAVVMWSVANEPASHLESAGYYLKMVIAHTKSLDPSRPVTFVSNSNYAADKGAPYVDVICLNSYYSWYHDYGHLELIQLQLATQFENWYKKYQKPIIQSEYGAETIAGFHQDPPLMFTEEYQKSLLEQYHLGLDQKRRKYVVGELIWNFADFMTEQSPTRVLGNKKGIFTRQRQPKSAAFLLRERYWKIANETRYPHSVAKSQCLENSLFT(SEQ ID NO:4)

[0214] In some embodiments, the enzyme has the sequence depicted by SEQ ID NO: 3. The enzyme may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence depicted by SEQ ID NO: 3.

[0215] In some embodiments, the enzyme has the sequence depicted by SEQ ID NO: 4. The enzyme may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence depicted by SEQ ID NO: 4.

[0216] Enzyme method

[0217] The present technology also includes a method for quantifying opioid glucuronides using a sample preparation device having a surface having an alkylsilyl coating (as described herein) containing immobilized β-glucuronidase. A sample containing opioid glucuronides is drawn onto the surface of the sample preparation device such that the opioid glucuronides are hydrolyzed. The hydrolyzed opioid glucuronides are recovered from the surface of the sample preparation device. The recovered hydrolyzed opioid glucuronides are injected onto a liquid chromatography column.

[0218] The present technology also includes a method for preparing a sample containing a protein for peptide mapping using a sample preparation device, the sample preparation device having a surface having an alkylsilyl coating (as described herein) containing an immobilized protease. The protease can be trypsin, Lys-C, Arg-C, Lys-N, Glu-C, Asp-N, pepsin, or a combination thereof. The sample containing the protein can be drawn onto the surface of the sample preparation device so that the protein is digested into peptide fragments. The peptide fragments are recovered from the sample preparation device and injected onto a liquid chromatography column. In some embodiments, the protease is trypsin, Lys-C, Asp-N, or a combination thereof.

[0219] In some embodiments, the alkylsilyl coating is bis(trichlorosilyl)ethane and the protease is trypsin, wherein the trypsin is linked to the alkylsilyl coating via a maleimide PEG silane linker (e.g., Figure 5A-5C #5) covalently bonded to bis(trichlorosilyl)ethane. The maleimide PEG silane linker can be:

[0220]

[0221] Where n is between 3 and 300.

[0222] The alkylsilyl coating may be bis(trichlorosilyl)ethane modified with triethoxysilylbutyraldehyde, and the protease may be trypsin, wherein trypsin is synthesized by reductive amination (e.g., Figure 5A-5C #6) covalently bonded to triethoxysilylbutyraldehyde-modified bis(trichlorosilyl)ethane.

[0223] In some embodiments, the alkylsilyl coating is bis(trichlorosilyl)ethane modified with 3-aminopropyltrimethoxysilane, and the protease is trypsin, wherein the trypsin is prepared by reductive amination with PEG dialdehyde (e.g., Figure 5A-5C #7) covalently bonded to 3-aminopropyltrimethoxysilane-modified bis(trichlorosilyl)ethane. The PEG dialdehyde can be:

[0224]

[0225] Where n is between 3 and 300.

[0226] In some embodiments, the alkylsilyl coating is bis(trichlorosilyl)ethane modified with triethoxysilylbutyraldehyde and acrylate PEG amine, and the protease is trypsin, wherein trypsin is synthesized by reductive amination and Michael addition (e.g., Figure 5A-5C #8) covalently bonded to modified bis(trichlorosilyl)ethane. The acrylate PEG amine can be:

[0227]

[0228] Where n is between 3 and 300.

[0229] In some embodiments, the alkylsilyl coating is bis(trichlorosilyl)ethane modified with acryloxypropyltrimethoxysilane, and the protease is trypsin, wherein the trypsin is catalyzed by Michael addition (e.g., Figure 5A-5C #9) covalently bonded to acryloxypropyltrimethoxysilane-modified bis(trichlorosilyl)ethane.

[0230] The present technology also includes a method for analyzing a sample containing a monoclonal antibody (mAb) using a sample preparation device having a surface having an alkylsilyl coating (as described herein) containing an immobilized enzyme, the immobilized enzyme being covalently bonded to the alkylsilyl coating. The enzyme is IdeS, IdeZ, IgdE, Gingipain K, or a combination thereof. A sample containing the mAb is drawn onto the surface of the sample preparation device so that the mAb is fragmented into subunits. The subunits of the mAb are recovered from the sample preparation device and the subunits of the mAb are injected onto a liquid chromatography column.

[0231] The present technology also includes a method for analyzing a sample containing a glycoprotein using a sample preparation device having a surface having an alkylsilyl coating (as described herein) containing an immobilized PNGase F enzyme. The sample containing the glycoprotein is drawn onto the surface of the sample preparation device so that the glycans of the glycoprotein are cleaved. The deglycosylated glycoprotein is recovered from the sample preparation device and the deglycosylated glycoprotein is injected onto a liquid chromatography column.

[0232] When separating biological samples, the alkylsilyl coating and the enzyme can each provide a desired effect for the overall separation. For example, when separating a sample containing a biological fluid, the enzyme can be used, for example, to hydrolyze or digest the analyte in the sample, and the alkylsilyl coating can ensure that the resulting digestion products are not adsorbed to the surface of the sample preparation device (e.g., due to interaction with the metal substrate) and are therefore recovered in high yield. In this way, the inert alkylsilyl coating together with the enzyme beneficially reduces the adsorption of the analyte on the substrate, while the enzyme selectively interacts with the specific analyte, which can then be recovered and detected.

[0233] Example

[0234] Example 1– Sample preparation using microelution plates with affinity surfaces

[0235] In one embodiment of the present technology, a 96-well microelution plate, a conical bottom vial or a pipette tip is coated with bis(trichlorosilyl)ethane and secondary modified to carry an affinity ligand for capturing a human immunoglobulin Fc domain, including but not limited to a nucleic acid aptamer (SEQ ID NO: 2), an immunoglobulin or a single domain VHH camelid antibody (SEQ ID NO: 1). The biological fluid is drawn onto the surface and equilibrated so that an affinity complex is formed. The adsorbed analyte is washed with a neutral isotonic buffer and then eluted with an eluent. The resulting analyte is then injected onto a liquid chromatography column and separated by the liquid chromatography column.

[0236] Example 2– Online using fixed affinity surfaces MS

[0237] In yet another embodiment, the present technology can be used as a component of an LC flow path. A 0.2 μm porosity stainless steel frit can be modified with a bis(trichlorosilyl)ethane coating and an immobilization reaction with an anti-human Fc aptamer (SEQ ID NO: 2). The frit can be used as an inlet to a size exclusion (SEC) column including but not limited to BEH SEC 1.7 μm column (available from Waters Technologies Corporation, Milford, MA). On this device, biological fluid samples containing therapeutic agents based on humanized mAbs can be loaded. The inlet glass frit and its coating will therefore selectively capture the therapeutic agent while the sample matrix will pass through the column device. Subsequently, the adsorbed mAb-based therapeutic agent will be desorbed by injection of the eluent. The SEC mobile phase composition will be selected to be composed of volatile components. The desorbed therapeutic agent will be eluted with minimal peak volume and passed through the SEC column to be able to be detected by mass spectrometry. The non-volatile eluent from the injection will be diverted to waste. Optionally, this fixed affinity ligand device can be used without secondary chromatography techniques. In some embodiments, it can be constructed with anti-human Fc immunoglobulin antibodies or anti-human Fc single domain antibody ligands. In these latter cases, the elution of the captured analyte can be promoted by a volatile eluent at a low pH.

[0238] Sequence Listing

[0239]

Claims

1. A sample preparation device for enriching components of a sample, the sample preparation device comprising: a surface, the surface being in fluid communication with the sample; an alkylsilyl coating disposed on the surface, wherein the alkylsilyl coating comprises a vapor deposition product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane; and An affinity ligand covalently bonded to the alkylsilyl coating.

2. A sample preparation device for enriching components of a sample, the sample preparation device comprising: a surface, the surface being in fluid communication with the sample; an alkylsilyl coating disposed on the surface, wherein the alkylsilyl coating comprises a vapor deposition product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane; and An enzyme covalently bonded to the alkylsilyl coating.

3. The sample preparation device according to any one of claims 1 or 2, wherein the alkylsilyl coating has at least Thickness.

4. The sample preparation device according to any one of claims 1 or 2, wherein the surface is an inner surface of a well plate.

5. The sample preparation device of any one of claims 1 or 2, wherein the surface is an interior surface of a vial.

6. The sample preparation device of any one of claims 1 or 2, wherein the surface is an inner surface of a pipette.

7. The sample preparation device according to any one of claims 1 or 2, wherein the sample preparation device is used for chromatography analysis.

8. The sample preparation device according to any one of claims 1 or 2, wherein the sample preparation device is used for mass spectrometry analysis.

9. The sample preparation device of claim 1 or 2, further comprising a second alkylsilyl coating in direct contact with the alkylsilyl coating of formula I, the second alkylsilyl coating having formula II: in R 7 , R 8 and R 9 Each is independently selected from -NH(C1-C6)alkyl, -N[(C1-C6)alkyl]2, (C1-C6)alkoxy, (C1-C6)alkyl, (C1-C6)alkenyl, OH and halogen; R 10 Selected from (C1-C6) alkyl, -OR B 、-[O(C1-C3)alkyl] 1-10 O(C1-C6)alkyl and phenyl, wherein the (C1-C6)alkyl is optionally substituted with one or more halogens, and wherein the phenyl is optionally substituted with one or more groups selected from (C1-C3)alkyl, hydroxyl, fluorine, chlorine, bromine, cyano, -C(O)NH2 and carboxyl; R B is -(C1-C3)alkylethylene oxide, -(C1-C3)alkyl-3,4-epoxycyclohexyl or -(C1-C4)alkylOH; R 10 The cut bond represents R 10 an optional additional covalent bond to the carbon bridging the silyl group to form an olefin, provided that y is not 0; and y is an integer from 0 to 20. 10 . The sample preparation device of claim 9 , wherein y is an integer from 2 to 9.

11. The sample preparation device according to claim 9, wherein y is 9, R 10 is methyl, and R 7 , R 8 and R 9 Each is ethoxy or chlorine.

12. The sample preparation device of claim 9, wherein the alkylsilyl coating of formula II is (3-glycidyloxypropyl)trimethoxysilane, n-decyltrichlorosilane, trimethylchlorosilane, trimethyldimethylaminosilane or methoxy-polyethyleneoxy (1-10) silane.

13. The sample preparation device of claim 9, wherein the alkylsilyl coating of Formula II is (3-glycidyloxypropyl)trimethoxysilane after hydrolysis.

14. The sample preparation device of claim 9, wherein the alkylsilyl coating of formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II is (3-glycidoxypropyl)trimethoxysilane.

15. The sample preparation device of claim 9, wherein the alkylsilyl coating of formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II is (3-glycidyloxypropyl)trimethoxysilane after hydrolysis.

16. The sample preparation device of claim 9, wherein the alkylsilyl coating of formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II is n-decyltrichlorosilane.

17. The sample preparation device of claim 9, wherein the alkylsilyl coating of formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II is trimethylchlorosilane or trimethyldimethylaminosilane.

18. The sample preparation device of claim 9, wherein the alkylsilyl coating of formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula II is methoxy-polyethyleneoxy(3)silane.

19. The sample preparation device of claim 1, further comprising an alkylsilyl coating in direct contact with the alkylsilyl coating of formula I, the alkylsilyl coating having formula III, in R 11 , R 12 , R 13 , R 14 , R 15 and R 16 are each independently selected from (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N((C1-C6)alkyl)2, OH and halogen; and Z is (C1-C 20 )alkyl, -O[(CH2)2O] 1-20 -、-(C1-C 10 )[NH(CO)NH(C1-C 10 )] 1-20 -or-(C1-C 10 )[Alkylphenyl(C1-C 10 )alkyl] 1-20 -.

20. The sample preparation device of claim 19, wherein the alkylsilyl coating of Formula III is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

21. The sample preparation device of claim 19, wherein the alkylsilyl coating of formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane, and the alkylsilyl coating of formula III is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

22. The sample preparation device of claim 19, wherein the alkylsilyl coatings of I and III have to Total thickness.

23. A sample preparation device according to any one of claims 1 or 2, wherein the alkylsilyl coating is selected to interact with at least one analyte in the sample through (1) repulsive forces, (2) secondary interactions, or (3) a retention mechanism that is different from interaction with a stationary phase material.

24. The sample preparation device of claim 23, wherein the analyte and the alkylsilyl coating are negatively charged.

25. The sample preparation device of claim 23, wherein the analyte and the alkylsilyl coating are positively charged.

26. The sample preparation device of claim 23, wherein the secondary interaction is ion exchange partitioning.

27. The sample preparation device of claim 1, wherein the affinity ligand is an antibody, a single domain antibody, an aptamer, an affibody, streptavidin, protein A, protein G, or protein L, or a combination thereof.

28. The sample preparation device of claim 1, further comprising a linker to covalently bond the affinity ligand to the alkylsilyl coating.

29. The sample preparation device of claim 28, wherein the linker is maleimide PEG silane, NHSPEG maleimide, or PEG dialdehyde, wherein the PEG repeats are between 3 and 300.

30. The sample preparation device of claim 28, wherein the linker is glutaraldehyde or acryloxysilane.

31. The sample preparation device of claim 1, wherein the covalent bond between the affinity ligand and the alkylsilyl coating is formed by reductive amination.

32. The sample preparation device of claim 1, wherein the affinity ligand has a concentration of 1 nmol / m 2 To 1000nmol / m 2 surface coverage.

33. The sample preparation device of claim 1, wherein the affinity ligand has a 2 nmol / m 2 To 500nmol / m 2 surface coverage.

34. The sample preparation device of claim 2, wherein the enzyme is selected from a protease, a glycosidase, a ligase, a transferase, an oxidoreductase, an isomerase, a hydrolase, or a combination thereof.

35. The sample preparation device of claim 34, wherein the enzyme is a protease, and the protease is selected from IdeS, IdeZ, or a combination thereof.

36. The sample preparation device of claim 2, wherein the enzyme is β-glucuronidase.

37. The sample preparation device of claim 2, wherein the enzyme has a concentration of 1 nmol / m 2 To 1000nmol / m 2 surface coverage.

38. The sample preparation device of claim 2, wherein the enzyme has a concentration of 2 nmol / m 2 To 500nmol / m 2 surface coverage.

39. A method of analyzing a biological fluid, comprising: A sample preparation device is provided, the sample preparation device comprising: a surface in fluid communication with the biological fluid; an alkylsilyl coating disposed on the surface, wherein the alkylsilyl coating comprises a vapor deposition product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane; and an affinity ligand covalently bonded to the alkylsilyl coating; drawing the biological fluid containing the analyte onto the surface of the sample preparation device to form an affinity complex such that the analyte is adsorbed; washing the adsorbed analyte with a neutral isotonic buffer; eluting the adsorbed analyte from the surface of the sample preparation device; and The eluted samples are analyzed using liquid chromatography, mass spectrometry, ELISA, capillary electrophoresis, or a combination thereof.

40. The method of claim 39, wherein the sample preparation device is a 96-well microelution plate, a conical bottom vial, or a pipette tip.

41. The method of claim 39, wherein the affinity ligand is capable of capturing a human immunoglobulin Fc domain.

42. The method of claim 41, wherein the affinity ligand is a nucleic acid aptamer, an immunoglobulin, a single domain VHH camelid antibody, or a combination thereof.

43. A method for separating a biological fluid, comprising: A glass frit is provided, the glass frit comprising: a surface in fluid communication with the biological fluid; an alkylsilyl coating disposed on the surface, wherein the alkylsilyl coating comprises a vapor deposition product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane; and an affinity ligand covalently bonded to the alkylsilyl coating; wherein the glass frit is positioned at the inlet of the chromatography column; loading the biological fluid comprising an analyte onto the surface of the frit; capturing the analyte to form an affinity complex such that the analyte is adsorbed on the surface of the frit while the remainder of the biological fluid passes through the chromatography column; eluting the adsorbed analyte from the surface of the frit such that the analyte passes through the chromatographic column; The analyte is detected with a detector.

44. The method of claim 43, wherein the chromatography column is a size exclusion chromatography column.

45. The method of claim 43, wherein the detector is a mass spectrometer.

46. ​​The method of claim 43, wherein the biological fluid comprises a humanized mAb-based therapeutic.

47. The method of claim 43, wherein the affinity ligand is an anti-human Fc aptamer, an anti-human Fc immunoglobulin antibody, an anti-human Fc single domain antibody ligand, or a combination thereof.

48. A method for quantifying opioid glucuronides, the method comprising: A sample preparation device is provided, the sample preparation device comprising: a surface in fluid communication with a sample comprising the opioid glucuronide; an alkylsilyl coating disposed on the surface, wherein the alkylsilyl coating comprises a vapor deposition product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane; and a β-glucuronidase covalently bonded to the alkylsilyl coating; drawing the sample comprising the opioid glucuronide onto the surface of the sample preparation device such that the opioid glucuronide is hydrolyzed; recovering the hydrolyzed opioid glucuronide from the surface of the sample preparation device; and The recovered hydrolyzed opioid glucuronide is injected onto a liquid chromatography column.

49. A method of preparing a sample containing protein for peptide mapping, the method comprising: A sample preparation device is provided, the sample preparation device comprising: a surface in fluid communication with the sample comprising the protein; an alkylsilyl coating disposed on the surface, wherein the alkylsilyl coating comprises a vapor deposition product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane; and a protease covalently bonded to the alkylsilyl coating, wherein the protease is trypsin, Lys-C, Arg-C, Lys-N, Glu-C, Asp-N, pepsin, or a combination thereof; aspirating the sample comprising the protein onto the surface of the sample preparation device such that the protein is digested into peptide fragments; recovering the peptide fragments from the sample preparation device; and The peptide fragments were injected onto a liquid chromatography column.

50. The method of claim 49, wherein the protease is trypsin, LysC, Asp N, or a combination thereof.

51. The method of claim 49, wherein the alkylsilyl coating is bis(trichlorosilyl)ethane and the protease is trypsin, wherein the trypsin is covalently bonded to the bis(trichlorosilyl)ethane via a maleimide PEG silane linker.

52. The method of claim 51, wherein the maleimide PEG silane linker is: Where n is between 3 and 300.

53. The method of claim 49, wherein the alkylsilyl coating is bis(trichlorosilyl)ethane modified with triethoxysilylbutyraldehyde, and the protease is trypsin, wherein the trypsin is covalently bonded to the triethoxysilylbutyraldehyde modified bis(trichlorosilyl)ethane by reductive amination.

54. The method of claim 49, wherein the alkylsilyl coating is bis(trichlorosilyl)ethane modified with 3-aminopropyltrimethoxysilane, and the protease is trypsin, wherein the trypsin is covalently bonded to the bis(trichlorosilyl)ethane modified with 3-aminopropyltrimethoxysilane by reductive amination with PEG dialdehyde.

55. The method of claim 54, wherein the PEG dialdehyde is: Where n is between 3 and 300.

56. The method of claim 49, wherein the alkylsilyl coating is bis(trichlorosilyl)ethane modified with triethoxysilylbutyraldehyde and acrylate PEG amine, and the protease is trypsin, wherein the trypsin is covalently bonded to the modified bis(trichlorosilyl)ethane by reductive amination and Michael addition.

57. The method of claim 56, wherein the acrylate PEG amine is: Where n is between 3 and 300.

58. The method of claim 49, wherein the alkylsilyl coating is bis(trichlorosilyl)ethane modified with acryloxypropyltrimethoxysilane, and the protease is trypsin, wherein the trypsin is covalently bonded to the acryloxypropyltrimethoxysilane modified bis(trichlorosilyl)ethane by Michael addition.

59. A method of analyzing a sample containing a monoclonal antibody, the method comprising: A sample preparation device is provided, the sample preparation device comprising: a surface in fluid communication with the sample comprising the monoclonal antibody; an alkylsilyl coating disposed on the surface, wherein the alkylsilyl coating comprises a vapor deposition product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane; and an enzyme covalently bonded to the alkylsilyl coating, wherein the enzyme is IdeS, IdeZ, IgdE, GingipainK, or a combination thereof; aspirating the sample comprising the monoclonal antibody onto the surface of the sample preparation device such that the monoclonal antibody is fragmented into subunits; recovering the subunit of the monoclonal antibody from the sample preparation device; and The subunits of the monoclonal antibodies are injected onto a liquid chromatography column.

60. A method of analyzing a sample containing a glycoprotein, the method comprising: A sample preparation device is provided, the sample preparation device comprising: a surface in fluid communication with the sample comprising the glycoprotein; an alkylsilyl coating disposed on the surface, wherein the alkylsilyl coating comprises a vapor deposition product of bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane; and a PNGase F enzyme covalently bonded to the alkylsilyl coating; aspirating the sample comprising the glycoprotein onto the surface of the sample preparation device such that glycans of the glycoprotein are cleaved; recovering the deglycosylated glycoprotein from the sample preparation device; and The deglycosylated glycoprotein is injected onto a liquid chromatography column.

61. The sample preparation device of claim 1, wherein the affinity ligand has a sequence depicted by SEQ ID NO:

1.

62. The sample preparation device of claim 1, wherein the affinity ligand is at least 80% identical to the sequence depicted by SEQ ID NO:

1.

63. The sample preparation device of claim 1, wherein the affinity ligand is at least 90% identical to the sequence depicted by SEQ ID NO:

1.

64. The sample preparation device of claim 1, wherein the affinity ligand is at least 95% identical to the sequence depicted by SEQ ID NO:

1.

65. The sample preparation device of claim 1, wherein the affinity ligand is at least 99% identical to the sequence depicted by SEQ ID NO:

1.

66. The sample preparation device of claim 1, wherein the affinity ligand has a sequence depicted by SEQ ID NO:

2.

67. The sample preparation device of claim 1, wherein the affinity ligand is at least 80% identical to the sequence depicted by SEQ ID NO:

2.

68. The sample preparation device of claim 1, wherein the affinity ligand is at least 90% identical to the sequence depicted by SEQ ID NO:

2.

69. The sample preparation device of claim 1, wherein the affinity ligand is at least 95% identical to the sequence depicted by SEQ ID NO:

2.

70. The sample preparation device of claim 1, wherein the affinity ligand is at least 99% identical to the sequence depicted by SEQ ID NO:

2.

71. The sample preparation device of claim 2, wherein the enzyme has a sequence depicted by SEQ ID NO:

3.

72. The sample preparation device of claim 2, wherein the enzyme is at least 80% identical to the sequence depicted by SEQ ID NO:

3.

73. The sample preparation device of claim 2, wherein the enzyme is at least 90% identical to the sequence depicted by SEQ ID NO:

3.

74. The sample preparation device of claim 2, wherein the enzyme is at least 95% identical to the sequence depicted by SEQ ID NO:

3.

75. The sample preparation device of claim 2, wherein the enzyme is at least 99% identical to the sequence depicted by SEQ ID NO:

3.

76. The sample preparation device of claim 2, wherein the enzyme has a sequence depicted by SEQ ID NO:

4.

77. The sample preparation device of claim 2, wherein the enzyme is at least 80% identical to the sequence depicted by SEQ ID NO:

4.

78. The sample preparation device of claim 2, wherein the enzyme is at least 90% identical to the sequence depicted by SEQ ID NO:

4.

79. The sample preparation device of claim 2, wherein the enzyme is at least 95% identical to the sequence depicted by SEQ ID NO:

4.

80. The sample preparation device of claim 2, wherein the enzyme is at least 99% identical to the sequence depicted by SEQ ID NO:4.

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