Improved apparatus including hydrophilic non-ionic coating for size exclusion chromatography

Abstract

The present disclosure relates to a coating method for chromatography surfaces. Embodiments of the present disclosure feature a two-step gas-liquid phase organosilane deposition method for producing hydrophilic non-ionic surfaces in chromatography systems.

Description

[0001] Cross-reference to related applications

[0002] This patent application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 092,789, filed October 16, 2020, entitled “Device Including a Hydrophilic, Non-Ionic Coating for Size Exclusion Chromatography,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to a coating method for chromatographic applications that require or benefit from hydrophilic and / or nonionic surfaces. In particular, this disclosure relates to a two-step gas-liquid phase coating method for wettable surfaces (such as components with metallic surfaces) within chromatographic columns and systems. Furthermore, this disclosure relates to an improved size exclusion chromatography apparatus and method. Background Technology

[0004] Separating analytes that interact with metals often proves extremely challenging. Some of these challenges stem from the interaction between the analyte and the material used to construct the flow path through which the analyte travels. The desire for highly efficient chromatographic systems with minimal dispersion necessitates smaller flow path diameters and the ability to withstand increasingly higher pressures at increasingly faster flow rates. Therefore, the materials chosen for chromatographic flow paths are often inherently metallic. This is despite the fact that certain properties of biomolecules are known to have unfavorable interactions with metal surfaces, known as chromatographic secondary interactions.

[0005] Pure metals and metal alloys (along with their corresponding oxide layers) have terminal metal atoms that exhibit Lewis acid properties. More simply, these metal atoms show a tendency to accept donor electrons. This tendency is even more pronounced for any surface metal ions carrying a positive charge. Analytes with sufficient Lewis base properties (any substance that can provide non-bonding electrons) can potentially adsorb onto these sites, forming problematic non-covalent complexes. Analytes with sufficient Lewis base properties to interact in this manner are defined as metal-interacting analytes.

[0006] For example, the phosphate groups of certain biomolecules are excellent polydentate ligands capable of high-affinity metal chelation. This interaction leads to the binding of phosphorylated substances to flow path metals, thereby reducing the amount of such substances detected. This is a particularly troublesome effect because phosphorylated substances are often the most important analytes in the assay.

[0007] Carboxylic acid ester groups, commonly found in biomolecular analytes, also possess the ability to chelate metals, albeit with lower affinity than phosphate ester groups. However, the presence of a large number of carboxylic acid ester groups in some biomolecular analytes provides an opportunity for accumulated, multidentate-based adsorption losses. These complexities exist not only in peptides and proteins but also in glycans. For example, N-glycans can sometimes include one or more phosphate ester groups and one or more sialic acid residues containing carboxylic acid esters. Additionally, smaller biomolecules (such as nucleotides and sugars, like sugar phosphate esters) can exhibit behavior similar to the aforementioned N-glycan molecules. Furthermore, chromatographic secondary interactions can be particularly problematic for biomolecules, especially larger structures, due to their ability to form microenvironments (by their size and structural sequence) that can adversely interact with the surfaces of separation components and flow paths. In such cases, biomolecules can exhibit structural regions with chemical properties that amplify secondary interactions with the materials of the flow path. This amplified secondary interaction, combined with accumulated metal chelation effects, reduces the overall effective separation of biomolecules.

[0008] An alternative to using metal flow paths is to use flow paths made of polymeric materials such as polyetheretherketone (PEEK). PEEK tubing, like most polymeric materials, is formed by extrusion. This manufacturing method, utilizing polymeric resins, results in highly variable inner diameters. Consequently, PEEK column hardware produces unfavorable variations in retention times, as can be observed when switching between columns. Typically, this variation can be up to three times higher than in metal-constructed columns. Furthermore, the techniques used to manufacture polymer-based frits are not sufficiently optimized to provide suitable robust components for commercial HPLC columns. For example, commercially available PEEK frits often exhibit unacceptably low permeability.

[0009] Therefore, it is desirable in the art to provide methods and materials for reducing the chelation and secondary interactions between biomolecular analytes and the metal surfaces they are exposed to during chromatographic separation. Summary of the Invention

[0010] This invention generally relates to an apparatus and method for improved size exclusion chromatography (SEC) or anion exchange separation. In particular, the apparatus and method utilize columns that provide improved performance and reduced secondary interactions. Generally, the apparatus and method are characterized by a metal flow path in which at least a portion of its wetting surface comprises a hydrophilic nonionic coating containing polyethylene glycol silane. In some embodiments, the apparatus and method are characterized by a stationary phase comprising porous particles having a surface, wherein at least a portion thereof is modified with hydroxyl-terminated polyethylene glycol. In some embodiments, the apparatus and method are characterized by a stationary phase comprising an anion exchange resin. The method of this technology is characterized by using the apparatus for SEC or anion exchange separation of analytes to provide improved separation results.

[0011] This disclosure also relates to a method for modifying a fluid flow path disposed within the interior of a fluid system. The method typically involves contacting the fluid flow path with an organosilane in a gas phase to form a primer coating, followed by contacting the fluid flow path with a liquid-phase organosilane to provide a hydrophilic and / or nonionic surface.

[0012] According to this disclosure, it has now been discovered that coating the metallic flow path of an instrument and chromatographic apparatus with certain hydrophobic and hydrophilic organosilane reagents in a two-step gas / solution phase method provides a uniform primer layer from the gas phase coating, thereby allowing subsequent solution-based coatings to more uniformly and completely coat the flow path. The disclosed methods are applicable to stainless steel or other metal flow path components and offer manufacturing advantages over alternative non-metallic components or non-metallic lining components. That is, one or more of the methods described herein are advantageous because they allow for the manufacture of high-pressure components from stainless steel or other metals or high-pressure materials. These components made of high-pressure materials can then be customized because the internal flow path can be modified with a coating to address the hydrophobicity of the flow path.

[0013] Therefore, in one aspect, the present technology relates to a method for generating a hydrophilic nonionic surface in a chromatographic system. The method includes: depositing a primer layer on an inner surface of a chromatographic component using vapor deposition; and forming the hydrophilic nonionic surface by depositing a solution-based polyethylene glycol (PEG) silane on the primer layer.

[0014] In another aspect, this technology relates to a chromatographic apparatus for separating analytes from a sample. The apparatus includes an injector having an injection needle configured to inject a sample into a mobile phase; a sample reservoir in fluid communication with the injector; and a chromatographic column located downstream of and in fluid communication with the injector, the column having a fluid connector and a fluid conduit connecting the injector and the column. Each of the fluid conduit, injector, sample reservoir, and column has an inner surface that forms a fluid flow path having a wetting surface, at least a portion of which has a hydrophilic nonionic surface as described herein. In some embodiments, the stationary phase comprises porous particles having a surface, wherein at least a portion of the surface is modified with hydroxyl-terminated polyethylene glycol. In some embodiments, the stationary phase comprises a porous anion exchange resin.

[0015] In another aspect, this technology relates to an improved size exclusion chromatography (SEC) method comprising combining a metallic flow path component with a hydrophilic nonionic surface, as described herein, with a stationary phase material based on hydroxyl-terminated PEG-modified silica or hydroxyl-terminated PEG-modified inorganic-organic hybrid particles. Combining a metallic flow path component with a hydrophilic nonionic surface with a hydroxyl-terminated PEG-modified stationary phase material for SEC is particularly advantageous in reducing secondary interactions between the analyte and the overall system. Specifically, the combination of the coated hardware and the hydroxyl-terminated PEG-modified stationary phase material provides advantages over conventional SEC columns. These advantages include the ability to perform SEC separations with a weakly buffered mobile phase, simplified method development (including reduced dependence on buffer, pH, and column temperature), enhanced analyte recovery, improved analyte peak shape, enhanced peak resolution, and enhanced reproducibility.

[0016] In another aspect, this technology relates to an improved anion exchange separation method comprising combining a metallic flow path component with a hydrophilic nonionic surface, as described herein, with a stationary phase material based on anion exchange materials (e.g., quaternary ammonium ion exchange resins). The use of a metallic flow path component with a hydrophilic nonionic surface in anion exchange separation is particularly advantageous in reducing secondary interactions between the analyte and the system as a whole. Specifically, the use of a metallic flow path component with a hydrophilic nonionic surface provides advantages over conventional anion exchange columns. These advantages include enhanced analyte recovery, enhanced reproducibility, and reduced sample-based passivation. Attached Figure Description

[0017] To provide an understanding of embodiments of this technology, reference is made to the accompanying drawings, which are not necessarily drawn to scale. The drawings are merely illustrative and should not be construed as limiting the technology. The disclosure described herein is illustrated in the drawings by way of example, not limitation.

[0018] Figure 1 This is a schematic diagram of a chromatographic flow system including a chromatographic column and various other components, according to an exemplary embodiment of the present technology. Fluid is carried through the chromatographic flow system, wherein the fluid flow path extends from the fluid manager to the detector.

[0019] Figure 2 A non-limiting cartoon illustration depicting a modified surface according to an embodiment of the disclosed method.

[0020] Figure 3 This is a flowchart illustrating an implementation of the disclosed method.

[0021] Figure 4 This is a bar graph depicting exemplary peak area recoveries in an SEC system without using a coated frit column, and for mAb analyte standards with two reference frits, according to an embodiment of this disclosure.

[0022] Figure 5 An exemplary detector response to mAb analyte standards in a column-free SEC system using a coated ingot, according to an embodiment of the present disclosure, is described, as well as for two reference ingots.

[0023] Figures 6A to 6T An exemplary chromatographic separation of BEH200 protein standards is described, which is performed on an inorganic-organic hybrid particle SEC column with prototype hydroxyl-terminated PEG surface-modified particles using different mobile phases (pH 6.0 to 8.0 and NaCl concentrations from 0 mM to 200 mM) and using a reference stainless steel frit.

[0024] Figures 7A to 7T An exemplary chromatographic separation of the BEH200 protein standard according to an embodiment of the present disclosure is described, which is performed on a prototype hydroxyl-terminated PEG surface-modified inorganic-organic hybrid particle SEC column using different mobile phases (pH 6.0 to 8.0 and NaCl concentration 0 mM to 200 mM) and using a reference stainless steel frit in a system using a coated frit.

[0025] Figures 8A to 8TAn exemplary chromatographic separation of trastuzumab emtansine (Kadcyla; Genentech) is described, which was performed on a prototype hydroxyl-terminated PEG surface-modified inorganic-organic hybrid particle SEC column using different mobile phases (pH 6.0 to 8.0 and NaCl concentrations 0 mM to 200 mM) in a system using a reference stainless steel frit.

[0026] Figures 9A to 9T An exemplary chromatographic separation of trastuzumab emtansine (Kadcyla; Genentech) is described, which was performed on a prototype hydroxyl-terminated PEG surface-modified inorganic-organic hybrid particle SEC column using different mobile phases (pH 6.0 to 8.0 and NaCl concentrations 0 mM to 200 mM) and a reference stainless steel frit in a system using a coated frit.

[0027] Figure 10 This is an exemplary % recovery versus injection number graph depicting the complete mAb (mouse IgG1) of the reference material and the material prepared according to the embodiments of this disclosure.

[0028] Figure 11 This is an exemplary % recovery versus contact angle graph depicting the reference material and the material prepared according to embodiments of this disclosure.

[0029] Figures 12A to 12D An exemplary chromatographic separation of EPO mRNA according to an embodiment of the present disclosure is depicted on a C2PEG-coated hardware anion exchange column, wherein a series of repeated injections are performed under increased mass loading.

[0030] Figures 12E to 12H An exemplary chromatographic separation of EPO mRNA on a reference metal hardware anion exchange column is described, wherein a series of repeated injections are performed under increased mass loading.

[0031] Figure 13A This is a graph depicting the peak areas produced by repeated injections and subsequent anion exchange separation of EPO mRNA on a C2PEG-coated hardware anion exchange column according to an embodiment of this disclosure.

[0032] Figure 13B It is a graph depicting the peak areas produced by repeated injections and subsequent anion exchange separation of EPO mRNA on a reference metal hardware anion exchange column.

[0033] Figure 14A This is a graph depicting the peak areas produced by repeated injections and subsequent anion exchange separation of Cas9 mRNA on a C2PEG-coated hardware anion exchange column according to an embodiment of the present disclosure.

[0034] Figure 14B It is a graph depicting the peak areas produced by repeated injections and subsequent anion exchange separation of Cas9 mRNA on a reference metal hardware anion exchange column. Detailed Implementation

[0035] Before describing several example implementations of this technology, it should be understood that this technology is not limited to the details of the construction or process steps set forth in the following description. This technology can have other implementations and can be practiced or carried out in various ways.

[0036] Generally, this technology relates to a method for modifying a fluid flow path disposed within the interior of a fluid system. The method typically involves contacting the fluid flow path with an organosilane in the gas phase, followed by contacting the fluid flow path with a liquid-phase organosilane. Typically, a gas-phase deposition of the organosilane reagent is performed to form an organosilane primer coating on at least a portion of the wetted surface of the fluid system (e.g., a liquid chromatography system), and subsequently, a liquid-phase deposition of a different organosilane reagent is performed to form an organosilica coating on at least a portion of the wetted surface of the fluid system, in direct contact with the organosilane primer coating. Such coatings modify the fluid path and reduce secondary interactions, such as ionic and / or hydrophobic interactions, which could otherwise occur during the chromatographic separation of certain analytes (e.g., size exclusion, ion exchange, or other types of chromatography). Therefore, the modified surface is bioinert or poorly bound (meaning that the analytes in the sample do not interact with the coating or have minimal interaction with it). Furthermore, the deposited coating is highly tunable to provide a range of desired contact angles (e.g., to make the wetting surface hydrophilic or hydrophobic), chemical properties, and characteristics to achieve the desired effects on the flow path and the analytes that ultimately pass through the flow path.

[0037] Surprisingly, according to this disclosure, vapor deposition of organosilane reagents provides a uniform coating thickness within high aspect ratio components and provides a reactive primer layer for subsequent liquid deposition of the organosilane reagents. This liquid coating of the organosilane reagent (e.g., a polyethylene glycol (PEG) derivative) subsequently produces a denser and more uniform coating than achievable without a primer layer. According to this disclosure, it has also been found that in size exclusion chromatography (SEC) separation of proteins and hydrophobic analytes, combining a fluid system component coated according to the disclosed method (i.e., a denser and more uniform hydrophilic coating) with a column containing hydroxyl-terminated PEG surface-modified stationary phase particles achieves significant improvements in SEC chromatography. These improvements include further reductions in hydrophobic interactions. Specifically, the combination of the coated hardware and the hydroxyl-terminated PEG surface-modified stationary phase material provides advantages over conventional SEC columns. These advantages include the ability to perform SEC separations with weakly buffered mobile phases, simplified method development (including reduced dependence on buffer, pH, and column temperature), enhanced analyte recoveries, improved analyte peak shapes, enhanced peak resolution, and enhanced reproducibility.

[0038] According to this disclosure, significant improvements have been found in anion exchange separation of nucleic acid analytes (e.g., mRNA) using fluid system components coated according to the disclosed method (i.e., having a dense and more uniform hydrophilic coating) compared to separation performed on uncoated fluid system components. These improvements include enhanced analyte recovery, enhanced reproducibility, and reduced sample-based passivation.

[0039] Although the two-step deposition method disclosed herein for solution-phase deposition of reference nonionic hydrophilic organosilanes (e.g., polyethylene glycol) has been described, and the use of the resulting nonionic hydrophilic surfaces with respect to SEC has been described herein, those skilled in the art will recognize that other functional groups can be used instead of nonionic hydrophilic organosilanes. For example, nonionic hydrophilic liquid-phase organosilanes can be replaced by, for example, liquid-phase organosilanes with ionic functionality. Such functional groups include, but are not limited to, sulfonic acids, carboxylic acids, amines, quaternary ammonium groups, phosphate esters, and combinations thereof. Wetting surfaces modified in this way can be used, for example, in antibacterial applications, metal chelation, and ion chromatography.

[0040] definition

[0041] The following definitions are provided for the terminology used in this disclosure. Unless the context in which a term appears requires a different meaning, this application will use the terms defined below.

[0042] The articles “a” and “a (kind)” are used herein to refer to a grammatical object of one or more articles (i.e., at least one). The term “about” as used throughout this specification is used to describe and indicate small fluctuations. For example, the term “about” can refer to less than or equal to ±5%, such as less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.2%, less than or equal to ±0.1%, or less than or equal to ±0.05%. Whether explicitly stated or not, all numerical values ​​herein are modified by the term “about.” Values ​​modified by the term “about” naturally include specific values. For example, “about 5.0” must include 5.0.

[0043] Chromatography is a separation method used to concentrate or separate one or more compounds (e.g., biomolecules) present in a mixture. The compounds (e.g., biomolecules) are typically present in a sample. This disclosure extensively uses the term "sample" to refer to any mixture that an individual might wish to analyze. The term "mixture" is used to refer to a fluid containing one or more dissolved compounds (e.g., biomolecules). The compound of interest present in the sample is referred to as an analyte.

[0044] Chromatography is a differential migration process. Compounds in a mixture pass through the column at different rates, resulting in their separation. Migration occurs via convection of a fluid phase (called the mobile phase) relative to a packed bed of particles or a porous monolithic structure (called the stationary phase). In some modes of chromatography, differential migration occurs due to the difference in affinity of the analyte for the stationary and mobile phases.

[0045] Size exclusion chromatography (SEC) is a class of chromatographic methods that separate or isolate analytes in a mixture based on hydrodynamic radii. In SEC, separation occurs due to differences in the ability of analytes to detect the volume of a porous stationary phase medium. See, for example, Modern Size-Exclusion Chromatography: Practice of Gel Permeation and Gel Filtration Chromatography by A.M. Striegel et al., 2nd ed., Wiley Press, NJ, 2009. SEC is commonly used for the separation of macromolecules or molecular complexes. For example, but not limited to, many biologically derived macromolecules (“biomolecules”) are analyzed by SEC, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, antibodies, polysaccharides, antibody-drug conjugates, and fragments and complexes of any of them. Synthetic polymers, plastics, etc., are also analyzed by SEC.

[0046] SEC is typically performed using columns with a packed bed of particles. A packed bed of particles is either a separation medium or a stationary phase through which the mobile phase flows. The column is positioned in fluid communication with a pump and an injector. The sample is loaded onto the column under pressure via the injector, and the pump forces the sample components and mobile phase through the column. Components in the sample exit the column or elute from it, with the largest molecules (largest hydrodynamic radius) exiting first and the smallest molecules exiting last.

[0047] Anion exchange chromatography, as described in the SEC, differs in that the stationary phase is anion exchange resin. In anion exchange chromatography, analytes in a mixture are separated or isolated based on net surface charge. Anion exchange chromatography uses positively charged ion exchange resins that have an affinity for molecules with a net negative surface charge. Like the SEC, anion exchange chromatography can be used for the separation of macromolecules or molecular complexes. For example, but not limited to, it can separate biomolecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), amino acids, or proteins.

[0048] In both SEC and anion exchange chromatography, the respective columns are positioned in fluid communication with a detector that detects changes in the properties of the mobile phase as it leaves the column. The detector registers these changes and records them as a graph, called a chromatogram, used to determine the presence of an analyte and, in the following embodiments, to determine its concentration. The time it takes for the analyte to leave the column (retention time) indicates the size of the molecule. The molecular weight can be estimated using a standard calibration curve. Examples of detectors used in SEC and anion exchange chromatography include, but are not limited to, refractive index detectors, UV detectors, light scattering detectors, and mass spectrometers.

[0049] The terms "polyethylene glycol" and "polyethylene oxide" are used synonymously in this document; both terms refer to polyethylene glycol having the formula -(O-CH2CH2). n -OH oligomeric or polymeric polyether compounds. Therefore, the abbreviations “polyethylene glycol” and “polyethylene oxide”, “PEG” and “PEO” are used synonymously in this document.

[0050] The term "methoxy-terminated polyethylene glycol," abbreviated as "MeO-PEO" or "MeO-PEG" in this document, refers to polyethylene glycol with the formula -(O-CH2CH2). n -OMe is an oligomeric or polymeric polyether compound. Unlike hydroxyl-terminated polyethylene glycol (HO-PEG), MeO-PEG does not have available free hydroxyl groups (OH) and has been capped with methyl groups.

[0051] As used herein, the term "modifying fluid flow path" refers to the process of modifying the surface of a material to improve its properties by altering the physical and / or chemical properties of the surface (e.g., a wettable surface). As used herein, the term "modified fluid flow path" refers to a material (e.g., a metal component of a chromatography system) that has been permitted to react with at least two different organosilane reagents to covalently bond the organosilane reagents to the surface of the material, to each other, or to both. In some embodiments, the organosilane reagents are attached to the surface of the material via siloxane bonds. For example, the surface of a metallic material contains metal oxides and / or free hydroxyl groups that can react with one or more reactive organosilane reagents (e.g., halogenated or alkoxy-substituted silanes) to generate at least MO-Si-C bonds.

[0052] As used herein, the term "organic silica" refers to any of a series of hybrid materials that combine silica with various organic compounds.

[0053] As used herein, the term "organosilanes" refers to organosilicon-based monomeric chemicals that are similar to hydrocarbons and have at least one direct bond between silicon and carbon atoms in the molecule.

[0054] The embodiments of this disclosure are now described in detail, and it should be understood that such embodiments are merely exemplary. These embodiments constitute what the inventors now consider to be the best mode for practicing this technology. Those skilled in the art will recognize that such embodiments are capable of modifications and alterations.

[0055] Device

[0056] Figure 1 This is a representative schematic diagram of a chromatographic flow system / apparatus 100 for separating analytes from a sample. The chromatographic flow system 100 includes several components, including a fluid manager system 105 (e.g., controlling flow through the mobile phase of the system); tubing 110 (which may also be replaced by or used in conjunction with micromachining fluid conduits); a fluid connector 115 (e.g., a fluid cap); a frit 120; a chromatographic column 125; an injector 135 including a needle (not shown) for inserting or injecting a sample into the mobile phase; a vial, settler, or sample reservoir 130 for holding the sample prior to injection; a detector 150; and a pressure regulator 140 for controlling the flow pressure. The inner surfaces of the components of the chromatographic system / apparatus form fluid flow paths with wetting surfaces. The fluid flow paths may have a length-to-diameter ratio of at least 20, at least 25, at least 30, at least 35, or at least 40.

[0057] Detector 150 may be a mass spectrometer. The fluid flow path may include the wetted surface of the electro-spray needle (not shown).

[0058] At least a portion of the wetting surface may be coated with an organosilica coating as described in detail below to customize its hydrophobicity and reduce or eliminate ionic interactions with, for example, certain analytes. The coating is applied via a combination of gas-phase and liquid-phase organosilane deposition. Therefore, the methods and apparatus of this technique offer the advantages of being able to use high-pressure resistant materials (e.g., stainless steel) to form the flow system, while also being able to customize the wetting surface of the fluid flow path to provide appropriate hydrophobicity, thus minimizing adverse interactions with analytes or undesirable chemical effects.

[0059] The silica coating can be provided throughout the system by tubing or fluid conduit 110 extending from the fluid manager system 105 to the detector 150. The coating can also be applied to portions of the fluid path. That is, one or more components or portions of components can be coated instead of the entire fluid path. For example, the interior portion of column 125, its melt 120, and end cap 115 can be coated, while the rest of the flow path remains unmodified. Furthermore, removable / replaceable components can be coated. For example, vials or settlers containing sample reservoirs and melt 120 can be coated.

[0060] In one aspect, the flow path of the fluid system described herein is at least partially defined by the inner surface of a tubing. In another aspect, the flow path of the fluid system described herein is at least partially defined by the inner surface of a micro-machined fluid conduit. In another aspect, the flow path of the fluid system described herein is at least partially defined by the inner surface of a column. In another aspect, the flow path of the fluid system described herein is at least partially defined by a channel through a fused mass. In another aspect, the flow path of the fluid system described herein is at least partially defined by the inner surface of a needle. In another aspect, the flow path of the fluid system described herein extends from the inner surface of the needle through the inner surface of a column. In another aspect, the flow path extends from a sample reservoir container (e.g., a settler) disposed upstream of and in fluid communication with the inner surface of the needle to a port of a connector / detector.

[0061] In some implementations, only the wetting surface of the column and components located upstream of the column are coated with the organosilica coating described herein, while the wetting surface located downstream of the column is not coated.

[0062] At least a portion of the wetted surface of the fluid flow path is coated with an organosilica coating as described herein. The organosilica coating is inert to at least one of the analytes in the sample, such as biomolecules, including but not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, polysaccharides, antibodies, antibody-drug conjugates, polysaccharides, glycophosphates, etc.

[0063] In some implementations, the first (i.e., primer) coating comprises a structure according to Formula I.

[0064]

[0065] in:

[0066] R 1 R 2 R 3 R 4 R 5 and R 6 Each can be independently either OH or OR. A ;

[0067] R A Represents the attachment point of the wetted surface along the fluid flow path, where R 1 R 2 R 3 R 4 R 5 and R 6 At least one of them is OR A ;and

[0068] Z is (C1-C) 20 alkyl group, -[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 .

[0069] In some implementations, Z is (C2-C 10 )alkyl.

[0070] In some implementations, Z is -(CH2CH2)-.

[0071] exist Figure 2 Non-restrictive, simplified exemplary implementations are provided. (See references.) Figure 2 According to embodiments of this disclosure, a C2 (ethylene) bridged siloxane coating is shown as a surface hydroxyl group bonded to a metal chromatographic component.

[0072] The contact angle of water on the organic silica coating of Formula I can vary. A contact angle (also called the wetting angle) is formed when a droplet (e.g., water) is placed on a material surface (e.g., an organic silica coating as disclosed herein) and the droplet forms a dome shape on the surface. The angle formed between the surface and a line tangent to the edge of the droplet is called the contact angle Θ, as shown in the diagram below. The contact angle can be measured using, for example, a contact angle goniometer.

[0073]

[0074] When droplets spread across a surface (e.g., a hydrophilic surface) and the dome becomes flatter, the contact angle becomes smaller. Conversely, when droplets form beads on a surface (e.g., a hydrophobic surface), the dome becomes higher and the angle becomes larger.

[0075] In some embodiments, the contact angle between the organosilica coating of Formula I and water is at least about 15°. In some embodiments, the contact angle between the organosilica coating of Formula I and water may be less than or equal to 30°. In some embodiments, the contact angle between the organosilica coating of Formula I and water may be less than or equal to 90°. In some embodiments, the contact angle between the organosilica coating of Formula I and water is between about 15° and about 105°. For example, the contact angle between the organosilica coating of Formula I and water may be about 0°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, 90°, 95°, 100°, or 105°.

[0076] The thickness of the organic silica coating of Formula I can be at least approximately For example, the thickness can be between approximately to approximately Between. The thickness of the organic silica coating of Formula I can be approximately

[0077] or The thickness of the organosilica coating of Formula I (e.g., a vapor-deposited organosilica coating) can be optically detected by the naked eye. For example, higher opacity and tinting indicate a thicker coating. Therefore, coatings with a clear visual distinction are an implementation of this technology. When the coated part is observed under full-spectrum light (such as sunlight), the color changes from yellow to purple, then to blue, then to slightly green, and finally back to yellow as the thickness increases. For example, when the thickness of the organosilica coating of Formula I is... When the coating is yellow, it reflects light with peak wavelengths between 560 nm and 590 nm. When the thickness of the organic silica coating of Formula I is... When the coating is applied, it can exhibit a purple hue and reflect light with peak wavelengths between 400 nm and 450 nm. When the thickness of the organic silica coating of Formula I is... At this time, the coating can exhibit a blue color and reflect light with peak wavelengths between 450 nm and 490 nm. See, for example, Faucheu et al., Relating Gloss Loss to Topographical Features of a PVDF Coating, published October 6, 2004; Bohlin and Erik, Surface and Porous Structure of Pigment Coatings, Interactions with Flexographic Ink and Effects of Print Quality, Dissertation, Karlstad University Studies, 2013:49.

[0078] The second organosilica coating is located on at least a portion of the wetted surface of the fluid flow path and is in direct contact with the first coating. In some embodiments, the second coating comprises a structure according to Formula II.

[0079]

[0080] in:

[0081] R 7 R 8 and R 9 Each can be independently either OH or OR. B ;

[0082] R B R represents the attachment point to the wetted surface of the fluid flow path or the attachment point to the first coating, wherein in either case, R 7 R 8 and R 9 At least one of them is OR B ;

[0083] Y is a -(C1-C6)alkyl-OR 12 -[O(C1-C3)alkyl] 1-10 O(C1-C6)alkyl, -[O(C1-C3)alkyl] 1-10OH or phenyl, wherein the (C1-C6) alkyl group is substituted with one or more halogens or one or more functional groups selected from the group consisting of OH, amino, (C1-C6)alkylamino, di(C1-C6)alkylamino, cyano, -C(O)NH2, sulfonic acid, quaternary ammonium, phosphate ester and carboxyl, and wherein the phenyl group is optionally substituted with one or more groups selected from the group consisting of (C1-C3)alkyl, OH, halogen, cyano, -C(O)NH2 and carboxyl.

[0084] R 12 It is -(C1-C3) alkyl diol, -(C1-C3) alkyl-3,4-cyclohexanediol or -(C2-C4) alkyl OH;

[0085] In some implementation schemes, R B This indicates the attachment point of the wetted surface along the fluid flow path.

[0086] In some implementation schemes, R B This indicates the attachment point with the first coating. In such embodiments, those skilled in the art will recognize that when R... 1 R 2 R 3 R 4 R 5 and R 6 When one or more of the terms represent one or more hydroxyl groups, attachment refers to attachment to hydroxyl groups associated with a coating of Formula I, such as R 1 R 2 R 3 R 4 R 5 and R 6 One or more of the ones mentioned in the text.

[0087] exist Figure 2 Non-restrictive, simplified exemplary implementations are provided. (See references.) Figure 2 According to embodiments of this disclosure, an alkylsilyl polyethylene glycol coating is bonded to the free hydroxyl groups of a C2 (ethylene)-bridged siloxane coating.

[0088] In some embodiments, Y is -[O(C1-C3)alkyl] 1-10 O(C1-C6)alkyl or -[O(C1-C3)alkyl] 1- 10 OH. In some embodiments, Y is -[O(C1-C3)alkyl] 1-10 O(C1-C6)alkyl. In some embodiments, Y is -[O(C1-C3)alkyl] 1-10 OH.

[0089] In some embodiments, the second coating, including the structure according to Formula II, comprises the following structure:

[0090]

[0091] in:

[0092] R 7 R 8 and R 9 Each as defined above;

[0093] R B As defined above;

[0094] R 13 It is H or CH3;

[0095] m is an integer from approximately 1 to approximately 10; and

[0096] n is an integer ranging from approximately 2 to approximately 50.

[0097] In some embodiments, m is 3, and n is about 8 to about 12. In some embodiments, m is 3, and n is about 6 to about 9. In some embodiments, m is 3, n is about 8 to about 12, and R 13 For H. In some implementations, m is 3, n is about 6 to about 9, and R 13 It is CH3.

[0098] The contact angle of water with water on the organic silica coating of Formula II can vary. In some embodiments, the contact angle of the organic silica coating of Formula II with water can be at least about 15°. In some embodiments, the contact angle of the organic silica coating of Formula II with water can be less than or equal to 105°. The contact angle with water can be less than or equal to about 90°. In some embodiments, the contact angle with water is between about 15° and about 105°. For example, the contact angle with water can be about 0°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, 90°, 95°, 100°, or 105°.

[0099] The combined thickness of the multilayer organic silica coatings of Formula I and Formula II can be at least approximately For example, the thickness can be between approximately to approximately Between. The combined thickness of the multilayer organic silica coatings of Formula I and Formula II can be approximately... or

[0100] Methods for generating hydrophilic nonionic surfaces

[0101] In a first aspect, a method for generating a hydrophilic nonionic surface in a chromatographic system is provided, the method comprising:

[0102] a. Depositing a primer layer on the inner surface of the chromatographic component using vapor deposition; and

[0103] b. The hydrophilic nonionic surface is formed by depositing a solution-based PEG silane on the primer layer. Figure 3 A diagram of the method is provided.

[0104] On the other hand, a method is provided for modifying a fluid flow path disposed within the interior of a fluid system, the fluid flow path having a wetting surface containing hydroxyl groups, the method comprising:

[0105] a. Bring the fluid flow path into contact with the vaporized organosilane reagent of Formula I.

[0106]

[0107] in:

[0108] R 1 R 2 R 3 R 4 R 5 and R 6 Each is independently selected from the group consisting of (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N((C1-C6)alkyl)2, OH, and halogen; and

[0109] Z is (C1-C) 20 alkyl group, -[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 ;

[0110] To form a first coating deposited on at least a portion of the wetted surface of the fluid flow path; and

[0111] b. To bring the fluid flow path on which the first coating is deposited into contact with the solution containing the organosilane reagent of formula II.

[0112]

[0113] in:

[0114] R 7 R8 and R 9 Each is independently selected from C1-C6 alkyl, (C1-C6) alkenyl, -NHR 10 -NR 10 R 11 OH, OR 10 The group consisting of halogens;

[0115] R 10 and R 11 Each is independently selected from C1-C6 alkyl groups;

[0116] Y is a -(C1-C6)alkyl-OR 12 -[O(C1-C3)alkyl] 1-10 O(C1-C6)alkyl, -[O(C1-C3)alkyl] 1-10 OH or phenyl, wherein the (C1-C6)alkyl group is substituted with one or more halogens or one or more functional groups selected from the group consisting of OH, amino, (C1-C6)alkylamino, di(C1-C6)alkylamino, cyano, -C(O)NH2, sulfonic acid and carboxyl, and wherein the phenyl group is optionally substituted with one or more groups selected from the group consisting of (C1-C3)alkyl, OH, halogen, cyano, -C(O)NH2 and carboxyl;

[0117] R 12 It is -(C1-C3)alkyl ethylene oxide, -(C1-C3)alkyl-3,4-epoxycyclohexyl or -(C1-C4)alkyl OH;

[0118] A second coating is formed on at least a portion of the wetted surface in the fluid flow path and is in direct contact with the first coating.

[0119] In some implementations, Z is (C2-C 10 Alkyl group. In some embodiments, Z is -(CH2CH2)-.

[0120] In some implementation schemes, R 1 R 2 R 3 R 4 R 5 and R 6 Each is independently selected from the group consisting of (C1-C2)alkoxy, -N((C1-C2)alkyl)2, and halogens. In some embodiments, R 1 R 2 R 3 R 4 R 5 and R 6 Each is either methoxy or chlorine.

[0121] In some embodiments, the reagent of Formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

[0122] In some implementation schemes, R 7 R 8 and R 9 Each is independently selected from the group consisting of -N[(C1-C6)alkyl]2, (C1-C6)alkoxy, and halogen. In some embodiments, R 7 R 8 and R 9 Each is either ethoxy or dimethylamino.

[0123] In some embodiments, Y is -[O(C1-C3)alkyl] 1-10 O(C1-C6)alkyl or -[O(C1-C3)alkyl] 1- 10 OH. In some embodiments, Y is -[O(C1-C3)alkyl] 1-10 O(C1-C6) alkyl. In some embodiments, Y is -[O(C1-C3) alkyl]. 1-10 OH.

[0124] In some embodiments, the organosilane reagent of formula II has the following structure:

[0125]

[0126] in

[0127] m is an integer from approximately 1 to approximately 10;

[0128] n is an integer from approximately 2 to approximately 50; and

[0129] R 13 R 14 and R 15 Each is OEt.

[0130] In some implementations, m is approximately 3, or is 3.

[0131] In some embodiments, n represents the average value of the mixture of different polyethylene glycol chain lengths. For example, in some embodiments, n can be 8 to 12, meaning that the average distribution of chain lengths falls between 8 and 12 polyethylene glycol units. In other embodiments, n can be a specific value, such as about 8, about 9, or about 10 to about 11 or about 12. In some embodiments, m is 3 and n is about 8 to about 12.

[0132] In some embodiments, the organosilane reagent of formula II has the following structure:

[0133]

[0134] in

[0135] m is an integer from approximately 1 to approximately 10;

[0136] n is an integer from approximately 2 to approximately 50; and

[0137] R 13 R 14 and R 15 Each is Cl, OCH3, or dimethylamino.

[0138] In some implementations, m is approximately 3, or is 3.

[0139] In some embodiments, n represents the average value of the mixture of different polyethylene glycol chain lengths. For example, in some embodiments, n can be 8 to 12, meaning that the average distribution of chain lengths falls between 8 and 12 polyethylene glycol units. In some embodiments, n can be 6 to 9. In other embodiments, n can be a specific value, such as from about 6, about 7, about 8, about 9, or about 10 to about 11 or about 12. In some embodiments, m is 3, and n is from about 6 to about 9.

[0140] In some embodiments, the organosilane reagent of formula II is 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]tris(dimethylamino)silane.

[0141] Typically, the solution includes a solvent. Suitable solvents include volatile hydrocarbons, such as aliphatic or aromatic organic solvents. In some embodiments, the solvent is toluene.

[0142] The solution may contain organosilane reagents in a range of concentrations, such as from about 0.01% to about 10% or from about 0.05% to about 1% by volume. In some embodiments, the concentration by volume is from about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1.0% to about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, the concentration of the organosilane reagent can be higher, such as 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 99% or more by volume.

[0143] Contact with an organosilane reagent of Formula I or Formula II can last for, for example, from about 1 second to about 3 days. In some embodiments, the contact time with an organosilane reagent of Formula I is from about 1 second to about 30 minutes or from about 5 seconds to about 15 minutes.

[0144] In some embodiments, the contact time with the organosilane reagent of formula II is from about 1 hour to about 96 hours, or from about 6 hours to about 48 hours, or from about 12 hours to about 24 hours.

[0145] Contact with organosilane reagents of Formula I or Formula II can occur under a variety of different temperature conditions. In some embodiments, contact with organosilane reagents of Formula I or Formula II is at a temperature of about 0°C to about 100°C or about 10°C to about 25°C. In some embodiments, contact with organosilane reagents of Formula II is at a temperature of about 15°C to about 25°C.

[0146] In some embodiments, at least a portion of the first coating is hydrolyzed. This is typically accomplished by exposing the first coating and / or the organosilane reagent of Formula I to water during the contact.

[0147] In some embodiments, at least a portion of the second coating is hydrolyzed. This is typically accomplished by exposing the fluid flow path to water after contact with the organosilane reagent of Formula II. During such hydrolysis, any residual R... 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 Or R 9 At least a portion of the groups are converted into OH groups or crosslinked with each other through siloxane bonds.

[0148] In some embodiments, any excess organosilane reagent is removed after contact between the fluid flow path and the solution containing the organosilane reagent. Typically, such removal is accomplished by washing, rinsing, impregnation, rinsing, or otherwise exposing the fluid flow path at least once to at least one solvent. In some embodiments, the solvent is toluene. In some embodiments, the solvent is an alcohol. In some embodiments, the alcohol is isopropanol. In some embodiments, the method includes washing, rinsing, impregnation, rinsing, or otherwise exposing the fluid flow path sequentially to two different solvents, such as toluene and isopropanol.

[0149] In some embodiments, the fluid flow path is dried. Any suitable method can be used, such as exposure to a vacuum, heating, or both. In some embodiments, the method further includes drying the fluid flow path after contact with a solution containing an organosilane reagent, which includes exposing the fluid flow path to a high temperature for a period of time.

[0150] In some implementations, the first coating is annealed. Any suitable method can be used, such as exposure to heat for a period of time.

[0151] In some embodiments, stainless steel flow path components (including, but not limited to, tubing, micromachining fluid conduits, column sintering blocks, column inlet tubing, and injection needles) are coated via the disclosed methods. In one aspect, these coated components are annealed to alter their chemical or physical properties.

[0152] In some embodiments, flow path components made of materials other than stainless steel or other metals (e.g., polymers, glass, etc.) are coated via the disclosed method. Specifically, the coating is applied to the frit used within the system or to sample vials that can be connected to a syringe needle.

[0153] Chromatographic apparatus

[0154] In another aspect, the present technology relates to a chromatographic apparatus for separating analytes from a sample. The apparatus includes an injector having an injection needle configured to inject a sample into a mobile phase; a sample reservoir in fluid communication with the injector; and a chromatographic column located downstream of and in fluid communication with the injector, the column having a fluid connector and a fluid conduit connecting the injector and the column. Each of the fluid conduit, injector, sample reservoir, and column has an inner surface that forms a fluid flow path having a wetting surface, at least a portion of which has a hydrophilic nonionic surface as described herein.

[0155] Methods for size exclusion chromatography

[0156] This paper discloses an improved size exclusion chromatography (SEC) method. The method involves combining a metallic flow path component with a hydrophilic nonionic surface, as described herein, with a stationary phase material consisting of hydroxyl-terminated PEG-modified silica or hydroxyl-terminated PEG-modified inorganic-organic hybrid particles. Combining a metallic flow path component with a hydroxyl-terminated PEG-modified stationary phase material for SEC is particularly advantageous in reducing secondary interactions between the analyte and the overall system. The method typically involves contacting a sample containing at least one analyte with a column chromatography apparatus as described herein; allowing a mobile phase to flow through the stationary phase material for a period of time; and eluting the at least one analyte from the stationary phase in the mobile phase.

[0157] stationary phase materials

[0158] Size exclusion chromatography (SEC) is performed on a stationary phase material having a size-based affinity for the analyte. Metal flow path components with hydrophilic nonionic surfaces and modified size exclusion chromatography (SEC) methods utilizing metal flow path components as described herein are compatible with typical stationary phase materials suitable for SEC separations (e.g., methoxy-terminated polyethylene glycol and glycol-bonded / coated bonded phases). As mentioned above, metal flow path components with hydrophilic nonionic surfaces as disclosed herein are particularly advantageous when combined with stationary phase materials having reduced secondary (i.e., ionic and hydrophobic) interactions. An example of such stationary phase materials is porous particles comprising a surface, wherein at least a substantial portion of the surface is modified with hydroxyl-terminated polyethylene glycol (PEG). The modified porous particles can be silica or inorganic-organic hybrid particles. Non-limiting examples of such hydroxyl-terminated PEG-modified porous particles are those in which the hydroxyl-terminated polyethylene glycol has the following formula:

[0159]

[0160] in:

[0161] m is an integer from approximately 1 to approximately 10;

[0162] n is an integer from approximately 2 to approximately 50; and

[0163] The wavy line indicates the attachment point to the surface of the porous particles.

[0164] Methods for performing anion exchange chromatography

[0165] This paper discloses an improved anion exchange chromatography method. The method involves combining a metallic flow path component with a hydrophilic nonionic surface, as described herein, with a stationary phase material based on an ion exchange resin. The method typically involves contacting a sample containing at least one analyte with a column chromatography apparatus as described herein; allowing a mobile phase to flow through the stationary phase material for a period of time; and eluting the at least one analyte from the stationary phase in the mobile phase.

[0166] stationary phase materials

[0167] Anion exchange separation is performed on a stationary phase material, which is a positively charged ion exchange resin with an affinity for molecules having a net negative surface charge. Metal flow path components with hydrophilic nonionic surfaces and improved anion exchange separation methods utilizing such metal flow path components as described herein are compatible with typical anion exchange resins suitable for anion exchange chromatography, such as quaternary ammonium ion exchange resins. As stated above, the metal flow path components with hydrophilic nonionic surfaces of this disclosure are advantageous in terms of enhanced analyte recovery and improved reproducibility compared to anion exchange separation performed on systems in which the metal flow path components are not surface-modified.

[0168] Analytes

[0169] The improved methods for performing SEC or anion exchange separations disclosed herein include samples containing at least one analyte. It is noteworthy that the utility of the currently disclosed methods is not limited to biopharmaceutical or protein analytes. In some embodiments, the at least one analyte includes small molecule drugs, natural products, or polymers. In some embodiments, the at least one analyte includes one or more biomolecules. In some embodiments, the biomolecule is a nucleic acid (e.g., RNA, DNA, oligonucleotide), a protein (e.g., a fusion protein), a peptide, an antibody (e.g., a monoclonal antibody (mAb), an antibody-drug conjugate (ADC), a polysaccharide, a virus, a virus-like particle, a viral vector (e.g., a gene therapy viral vector, an adeno-associated virus vector), a biosimilar, or any combination thereof. In some embodiments, the at least one analyte comprises a nucleic acid, polysaccharide, peptide, polypeptide, protein, growth factor, carbohydrate, fatty acid, lipid, polysaccharide, ion (e.g., a metal ion), or any combination thereof. In some embodiments, the at least one analyte comprises an antibody. In some embodiments, the at least one analyte comprises a monoclonal antibody (mAb). In some embodiments, the at least one analyte comprises a high molecular weight substance or aggregate form of an antibody. In some embodiments, the at least one analyte is an antibody-drug conjugate. In some embodiments, the at least one analyte comprises a nucleic acid, which is RNA, such as mRNA. In some embodiments, the at least one analyte is a polysaccharide, peptide, herbicide, or pesticide. In some embodiments, the polysaccharide is a phosphopyran. In some embodiments, the peptide is a phosphopeptide. In some embodiments, the herbicide is glyphosate.

[0170] mobile phase

[0171] The methods disclosed herein for performing SEC and anion exchange chromatography involve passing a mobile phase through a stationary phase for a period of time. The mobile phase typically includes water, a buffer solution, and optionally one or more salts. In some specific embodiments, the mobile phase and optional sample are provided using a high-performance liquid chromatography (HPLC) system.

[0172] Buffer solutions are used to control the ionic strength and pH of the mobile phase. Many different substances can be used as buffer solutions depending on the nature of the analyte. Non-limiting examples of suitable buffer solutions include phosphates, tris(hydroxymethyl)aminomethane, and acetates. In some embodiments, the buffer solution contains phosphates. In some embodiments, the buffer solution contains acetates. In some embodiments, the buffer solution is ammonium acetate. In some embodiments, the buffer solution is an alkali metal phosphate. In some embodiments, the buffer solution is sodium phosphate or potassium phosphate. In some embodiments, the buffer solution is sodium dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof. The concentration of the buffer solution can be varied according to the desired pH and the ionic strength of the mobile phase.

[0173] In some embodiments, the mobile phase contains a salt. As used herein, the term "salt" refers to an ionic compound containing an alkali metal or alkaline earth metal and a halogen (e.g., fluoride, chloride, bromide, iodide). Undesirable interactions can be mitigated by utilizing salts to reduce secondary ionic interactions. However, increasing the salt concentration can induce aggregation and thus lead to a reduction in native monomers, and adding high concentrations of salt can exacerbate hydrophobic interactions and complicate mobile phase optimization. Suitable salts, when present, include, but are not limited to, sodium chloride and potassium chloride. Suitable salt concentrations in the mobile phase can range from about 10 mM to about 200 mM.

[0174] As described above, implementations of methods, apparatus, and processes have been discussed primarily in conjunction with SEC for the separation and reduction or elimination of hydrophobic and ionic secondary interactions. Those skilled in the art will recognize that other functional groups may be utilized in alternative implementations, and other separation methods utilizing such functional groups are envisioned herein, such as those for hydrophobic interactions and other reversed-phase chromatography.

[0175] Unless otherwise stated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise claimed, the use of any and all instances or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the materials and methods and does not constitute a limitation on the scope. The language in the specification should not be construed as indicating that any unclaimed element is necessary for the practice of the disclosed materials and methods.

[0176] Those skilled in the art will readily recognize that suitable modifications and alterations can be made to the compositions, methods, and applications described herein without departing from the scope of any embodiment or aspect thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of the claimed embodiments. All the various embodiments, aspects, and options disclosed herein can be combined in all variations. The scope of the compositions, formulations, methods, and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples, and preferred embodiments described herein.

[0177] Although the technology described herein has been illustrated with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the technology. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and apparatus of this technology without departing from the spirit and scope thereof. Therefore, this technology is intended to include modifications and variations within the scope of the appended claims and their equivalents.

[0178] Throughout this specification, the terms "an embodiment," "certain embodiments," "one or more embodiments," or "an embodiment" refer to a particular feature, structure, or characteristic described in connection with an embodiment that is included in at least one embodiment of the present invention. Therefore, the appearance of phrases such as "in one or more embodiments," "in certain embodiments," "in one embodiment," or "in an embodiment" throughout this specification does not necessarily refer to the same embodiment of the present invention. Furthermore, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. Any scope referenced herein is included.

[0179] The present invention will be described more fully with reference to the following embodiments. Before describing several exemplary embodiments of the present invention, it should be understood that the present invention is not limited to the details of the construction or process steps set forth in the following description. The present invention can have other embodiments and can be practiced or carried out in various ways. The following embodiments are set forth to illustrate certain aspects of the present invention and should not be construed as limiting thereto.

[0180] Example

[0181] The present invention can be further illustrated by the following non-limiting examples describing the chromatographic apparatus and methods.

[0182] Material

[0183] Unless otherwise stated, all reagents shall be used as is. Those skilled in the art will recognize that equivalents of the following supplies and suppliers exist, and therefore, any supplier listed below should not be construed as restrictive.

[0184] method

[0185] Example 1.C2-C 10 Vapor deposition coating (primer)

[0186] Metal components are subjected to vapor deposition of an organosilane reagent to form a primer layer. Prior to coating, all metal components are passivated using nitric acid passivation. The passivated components and silicon wafer are then introduced into the vapor deposition chamber and a vacuum is established. The first step is a plasma cleaning step. Next is a first vapor deposition cycle. Each vapor deposition cycle includes organosilane vapor deposition, followed by the introduction of water vapor for organosilane hydrolysis. The organosilane vapor is delivered at a first pressure, followed by water vapor delivery at a second pressure. After delivery, the organosilane and water are reacted with the substrate. This cycle is repeated to produce the desired number of layers and coating thickness. In this embodiment, bis(trichlorosilyl)ethanesilane is used to establish approximately... The coating is an adhesive or primer layer. A post-coating annealing step can be used to further crosslink and increase the hydrophobicity of the coating. Typically, an annealing cycle involves subjecting the coating to high temperatures under vacuum.

[0187] A silicon wafer was used as a sample block to measure the coating thickness and contact angle. To measure the thickness, a Stokes ellipsometry model (LSE) from Gaertner Scientific Corporation was used. The film thickness was determined by analyzing the polarization changes of the light and comparing them with the model.

[0188] Example 2. PEG liquid phase deposition coating on vapor phase coating (C2PEG)

[0189] The coated metal part prepared according to Example 1 was treated with a solution of polyethylene glycol (PEG) organosilane reagent. In this case, a C2-coated 4.6 mm, 0.2 μm porous titanium frit was stirred together with a dilute toluene solution of organosilane reagent, such as 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]tris(dimethylamino)silane; Gelest SIM6492.77). React the reagent solution with the melt for approximately three days. Discard the solution and wash (impregnate) the melt three times in toluene and three times in isopropanol. Dry the melt under vacuum at 70°C and then cool.

[0190] Example 3. Standalone PEG liquid phase deposition coating (reference)

[0191] Ten uncoated 4.6 mm, 0.2 μm porous titanium frits were added to a bottle containing a dilute organosilane solution, such as 2-[methoxy(polyvinyloxy)] 6-9 [Propyl]tris(dimethylamino)silane; Gelest SIM6492.77 in toluene. React the reagent solution with the melt for approximately three days. Discard the solution and wash (impregnate) the melt three times in toluene and three times in isopropanol. Dry the melt under vacuum at 70°C and then cool.

[0192] Example 4. Evaluation of secondary interactions - SEC condition, column-free

[0193] The porous titanium frits prepared according to Examples 1, 2, and 3 were respectively loaded into column-free HPLC systems ( The H-Class Bio system (purchased from Waters Corporation, Milford, MA) was tested using SEC-compliant mobile phases and protein standards at the H-Class Bio filter assembly parameters. The parameters are as follows:

[0194] • Sample: Intact mAb quality control standard, 0.667 mg / mL

[0195] • Sample diluents: 25mM sodium phosphate (pH 6.8), 75mM sodium chloride

[0196] • Mobile phase: 25 mM sodium phosphate (pH 6.8), 75 mM sodium chloride

[0197] Needle washing: 90 / 10 methanol aqueous solution

[0198] • Injection volume: 0.5 μL (0.33 μg)

[0199] • UV wavelength: 280nm

[0200] • Flow rate: 0.15 mL / min

[0201] 30℃

[0202] exist Figure 4 The results provided indicate that, under these measurement conditions, the two-step coating method according to Example 2 maximizes protein recovery. Specifically, the combination of vapor-deposited primer and liquid PEG coating produces the lowest protein adsorption. Unbound by theory, it is believed that vapor deposition produces a more reactive surface that readily accepts liquid PEG silanes via hydrolysis / condensation reactions, thereby reducing non-specific protein binding. In the absence of a primer layer (Example 3), PEG silanes fail to bind to the native metal oxide layer, resulting in a largely uncoated metal surface bound to proteins via secondary (ionic) interactions. A primer layer alone (Example 1) also produces a surface bound to proteins via non-specific binding sites, leading to... Figure 5 The representative chromatograms show poor protein recovery. Conversely, the frit prepared according to Example 2 (vapor phase primer and solution phase PEG) maximizes protein recovery, as indicated by the large analyte peaks. Figure 5 ).

[0203] Example 5. Evaluation of secondary interactions - SEC

[0204] The performance of reference (untreated) standard stainless steel hardware and hardware coated according to Example 2 was evaluated in SEC separation using prototype SEC columns (hydroxyl-terminated polyethylene glycol surface-modified hybrid particles). The stationary phase particles were prepared as follows.

[0205] In sol-gel synthesis, the procedure reported in Wyndham et al., Analytical Chemistry 2003, 75, 6781-6788 and U.S. Patent No. 6,686,035, is used to synthesize SiO2 (O2) by co-condensation of 1,2-bis(triethoxysilyl)ethane (BTEE) with tetraethyl orthosilicate (TEOS). 1.5 SiCH2CH2SiO1.5 ) 0.25 Organic / inorganic hybrid particles were obtained, and each reference is incorporated herein by reference in its entirety. The obtained inorganic-organic hybrid ethylene bridged particles have an average particle size of 1.7 μm and The average pore diameter. The surface area is 171 m². 2 / g, pore volume is 1.26cm³ 3 / g. The inorganic-organic hybrid ethylene bridged particles are then bonded to form hydroxyl-terminated PEG-bonded stationary phase particles. The inorganic-organic hybrid ethylene bridged particles are dispersed in toluene (10 mL / g). Residual water is removed from the material by azeotropic distillation (110 °C, 1 to 2 h). The reaction temperature is lowered to below 40 °C and concentrated hydrochloric acid (200 μL / g particles) is added, followed by the addition of [hydroxyl (polyvinyloxy)]. 8-12 [Propyl]triethoxysilane (8 μmol / m 2 The reaction was stirred for 5 minutes, and the temperature was increased to 110°C and maintained for 20 hours. The reaction was then cooled to room temperature, and the particles were separated by filtration. The particles were subsequently washed in the following order: 5× toluene, 1× acetone, 4× acetone / water (1:1 v / v), and 2× acetone. After the bonding reaction, residual ethoxysilyl groups were hydrolyzed with ammonium acetate. The particles were dispersed in a mixture of acetone (8.2 mL / g particles) and 0.12 M ammonium acetate solution (1.8 mL / g particles), and the mixture was stirred at 59°C for 2 hours. The reaction was then cooled to <40°C, and the particles were separated by filtration. The separated particles were then washed three times with acetone / water (1:1 v / v) and twice with acetone. The separated surface-modified particles were vacuum-dried at 70°C for 16 hours. The surface coverage of the hydroxyl-terminated PEG was 1.73 μmol / m³. 2 Hydroxyl-terminated PEG-bonded stationary phase particles were loaded into a 4.6 × 150 mm column.

[0206] Separation was performed using a commercially available high-performance liquid chromatography (HPLC) system. The H-ClassBio system (available from Waters Corporation, Milford, MA) is used. The mobile phase consists primarily of 40 mM sodium phosphate at pH 6–8 and 0 mM to 200 mM sodium chloride. Analytes are either the BEH200 protein standard mixture (thyroglobulin, IgG, BSA, myoglobin, and uracil) or trastuzumab emtansine (Kadcyla).

[0207] The SEC results using the reference hardware and the hardware coated according to Example 2, a mixture of BEH200 protein standards, were respectively... Figures 6A to 6T and Figures 7A to 7TProvided in [the document / source]. Specifically, for the reference hardware, Figures 6A to 6E The chromatograms were obtained using a NaCl-free mobile phase at pH values ​​ranging from 6.0 to 8.0. Figures 6F to 6J The chromatograms were obtained using a mobile phase containing 50 mM NaCl at pH values ​​ranging from 6.0 to 8.0. Figures 6K to 6O The chromatograms were obtained using a mobile phase containing 100 mM NaCl at pH values ​​ranging from 6.0 to 8.0; and Figures 6P to 6T The chromatograms were obtained using a mobile phase containing 200 mM NaCl at pH values ​​ranging from 6.0 to 8.0. For coated hardware, Figures 7A to 7E The chromatograms were obtained using a NaCl-free mobile phase at pH values ​​ranging from 6.0 to 8.0. Figures 7F to 7J The chromatograms were obtained using a mobile phase containing 50 mM NaCl at pH values ​​ranging from 6.0 to 8.0. Figures 7K to 7O The chromatograms were obtained using a mobile phase containing 100 mM NaCl at pH values ​​ranging from 6.0 to 8.0; and Figures 7P to 7T The chromatograms were obtained using a mobile phase containing 200 mM NaCl at pH values ​​ranging from 6.0 to 8.0. The chromatograms show that the coated hardware significantly improved peak shape and area under less-than-ideal mobile phase conditions, and that the coated hardware was less dependent on salt concentration or pH. Furthermore, when SEC separation was performed on a column of hydroxyl-terminated polyethylene glycol-bound SEC particles with the coated hardware combination according to Example 2, low buffer concentrations (e.g., 40 mM) were sufficient for the quality chromatography developed using virtually no methods for typical proteins.

[0208] The SEC results of Kadcyla analytes using the reference hardware and the hardware coated according to Example 2 were respectively in Figures 8A to 8T and Figures 9A to 9T Provided in [the document / source]. Specifically, for the reference hardware, Figures 8A to 8E The chromatograms were obtained using a NaCl-free mobile phase at pH values ​​ranging from 6.0 to 8.0. Figures 8F to 8J The chromatograms were obtained using a mobile phase containing 50 mM NaCl at pH values ​​ranging from 6.0 to 8.0. Figures 8K to 8O The chromatograms were obtained using a mobile phase containing 100 mM NaCl at pH values ​​ranging from 6.0 to 8.0; and Figures 8P to 8T The chromatograms were obtained using a mobile phase containing 200 mM NaCl at pH values ​​ranging from 6.0 to 8.0. For coated hardware, Figures 9A to 9E The chromatograms were obtained using a NaCl-free mobile phase at pH values ​​ranging from 6.0 to 8.0. Figures 9F to 9J The chromatograms were obtained using a mobile phase containing 50 mM NaCl at pH values ​​ranging from 6.0 to 8.0. Figures 9K to 9OThe chromatograms were obtained using a mobile phase containing 100 mM NaCl at pH values ​​ranging from 6.0 to 8.0; and Figures 9P to 9T The chromatogram was obtained using a mobile phase containing 200 mM NaCl at pH values ​​ranging from 6.0 to 8.0.

[0209] The chromatograms show that the coated hardware significantly improves the chromatography of the hydrophobic antibody-drug conjugate Kadcyla. Furthermore, the coated hardware allows for greater freedom in terms of mobile phase composition. Specifically, Kadcyla peaks were observed with the coated hardware without peak generation when using the uncoated reference hardware. The weakly buffered flow was sufficient for this challenging analyte when SEC separation was performed on a hybrid particle column with prototype hydroxyl-terminated polyethylene glycol surface modification in combination with the coated hardware.

[0210] In particular, Figures 7A to 7T and Figures 9A to 9T The results shown demonstrate the significant advantages associated with the combined use of coated hardware and prototype hydroxyl-terminated polyethylene glycol surface-modified hybrid particle columns.

[0211] Example 6. Evaluation of protein adsorption on various porous materials

[0212] Protein adsorption can limit the accuracy and precision of chromatographic separations. Therefore, minimal protein adsorption is desirable for more accurate and precise SEC analysis. Proteins tend to adhere to various materials through ionic and hydrophobic interactions, particularly in aqueous mobile phases used in SEC. To gain an understanding of the hydrophobicity of various materials and to determine whether a correlation exists between protein adsorption and hydrophobicity, experiments were conducted to evaluate protein recoveries from several LC filter assemblies. Specifically, the following 4.6 mm filter assemblies were evaluated:

[0213] ·0.2μm titanium (reference)

[0214] • 0.2μm titanium with C2 coating (reference)

[0215] • 0.2 μm titanium with C2-PEG coating (of the present invention; prepared by coating titanium frit according to Examples 1 and 2)

[0216] • 0.2μm titanium with C2-C10 coating (reference)

[0217] • 0.2μm 316 stainless steel (reference)

[0218] ·0.5μm PEEK (polyetheretherketone; reference)

[0219] Injection of complete mAb quality control standards (mouse IgG1; Waters PN 186006552) using a custom filter holder on an HPLC system. The H-Class Bio system (available from Waters Corporation, Milford, MA) was used. Test parameters are provided in Table 1.

[0220] Table 1. Instrument Test Parameters

[0221] mobile phase 25mM sodium phosphate, pH 6.8; 75mM sodium chloride. Flow rate 0.15 mL / min gradient isometry Sealed washing 90 / 10 water / methanol Collection time 1 minute Sample temperature 4℃ Filter assembly temperature 30℃ Injection volume 1.7μL Sample diluent 25mM sodium phosphate, pH 6.8; 75mM sodium chloride. wavelength 280nm Sampling rate 20 points / second

[0222] One filter assembly of each filter assembly type was placed in the filter holder, and 10 injections of the complete mAb quality check standard were performed. For each injection, the standard recovery was calculated using the following formula by dividing the peak area of ​​each injection by the average peak area of ​​the system:

[0223]

[0224] The system peak area was determined by removing the frit holder and replacing it with a low-volume live joint. The average area from 10 injections was used as the peak area obtained from the system. Recovery values ​​for the complete mAb were plotted for each injection and each filter assembly type. Figure 10 ).like Figure 10 As shown, all materials except the C2-PEG coating exhibited increased recovery of intact mAbs after multiple injections. Without being bound by theory, this increase in recovery is believed to be attributable to the gradual loading of the filter assembly with intact mAbs, which limits the effect of further intact mAb adsorption. In some cases, the recovery stabilized below 100%, indicating a dynamic balance between protein (intact mAb) adsorption and desorption. The C2-PEG coating of this invention allows for maximum recovery from the first to the tenth injection, indicating minimal adsorption.

[0225] Example 7. Contact Angle Measurement

[0226] The contact angles (measures of hydrophobicity) of the various reference materials and coated materials used in the filter assembly of Example 6 were determined. Flat planar sample blocks were obtained for each material to be evaluated. For the reference uncoated 316 stainless steel, an anionic detergent solution (e.g., 1% from Alconox Inc., White Plains, NY, USA) was used. A 2"×2" metal sheet sample (#8 Finish, 0.06" thick) was cleaned with solution, rinsed with deionized water, dried with nitrogen, and the contact angle was measured immediately. For the reference uncoated Grade 2 titanium, a 2"×2" metal sheet sample (matte finish, 0.063" thick) was washed, rinsed, and dried as described for the 316 stainless steel sample block, and the contact angle was measured immediately. To obtain accurate readings for the coated surfaces, silicon wafers were coated according to the procedures of Examples 1 and 2, and the contact angle was measured immediately after deposition.

[0227] For each sample block, the contact angle was measured using a Rame-Hart goniometer or a profilometer. In each case, a drop of reverse osmosis purified water (approximately 3 μL) was dispensed onto the surface of the sample block, and the resulting contact angle was measured. The contact angles for each sample block are provided in Table 2. The average intact mAb recovery obtained in Example 6 above is also provided in Table 2. Figure 11 As shown, the relationship between the contact angle and the average recovery value of ten injections is plotted.

[0228] Table 2. Contact Angle and Average Recovery Rate

[0229]

[0230]

[0231] *Contact angle obtained from Wang et al., “PEEK surface modification by fast ambient-temperature sulfonation for bone implant applications,” Journal of the Royal Society Interface, published online on March 6, 2019.

[0232] refer to Figure 11As shown in Table 2, the data indicate that, generally speaking, there is no correlation between contact angle and protein adsorption. It is generally accepted that hydrophilic surfaces are preferred for limiting the adsorption of hydrophobic proteins, but clearly more factors are involved, as surprisingly, the most hydrophilic surfaces (i.e., titanium) have the worst average recoveries. In other words, the non-fouling properties of C2-PEG coatings cannot be fully explained based solely on contact angle. For example, surface charge, surface roughness, and coating microstructure / morphology are some of the factors that cannot be detected by contact angle measurements and can further contribute to the non-fouling properties of C2-PEG coatings. In conclusion, the absence of protein (intact mAb) adsorption on C2-PEG coated surfaces illustrates the performance gains achievable in chromatographic separations through the use of hardware (e.g., filter frits) with such surface coatings.

[0233] Example 8. Evaluation of protein adsorption on various porous materials

[0234] Messenger RNA (mRNA) is an important biomolecule. mRNA is a DNA transcript used by ribosomes to express new proteins. mRNA also represents a new class of advanced therapeutic medical products. For example, mRNA is now used to vaccinate patients against SARS-CoV-2 (the beta-coronavirus responsible for COVID-19 infection). Therefore, there is a need to establish sensitive and precise methods to ensure that such mRNA therapeutics are well-characterized before clinical trials and that they can be reproducibly prepared after approval for commercialization.

[0235] Anion exchange separation is a promising method for measuring the heterogeneity of large multi-anion molecules, such as mRNA. However, challenges arise when separating mRNA using conventional metal-hard LC columns. In particular, for metal-hard columns, repeated injections of a given mass load tend to show RSD values ​​greater than 10% of the peak area. This lack of reproducibility in repeatable analyses is problematic.

[0236] Therefore, the performance of the coated metal hardware column prepared according to the embodiments of this disclosure was evaluated in representative anion exchange separation of mRNA samples. Specifically, salt gradient separation of two different mRNA samples (20 μg / mL EPO mRNA or Cas9 mRNA, available from TriLink XYZ) was performed on a strong anion exchange column with a mass loading ranging from 2 ng to 20 ng. The separation was performed using a conventional metal hardware column or a C2-PEG coated metal hardware column prepared according to this disclosure on an HPLC system (…). The separation was performed using the H-Class Bio Plus (Premier) system (purchased from Waters Corporation, Milford, MA). In each case, the mobile phase was a non-porous quaternary ammonium strong anion exchanger (Protein-Pak) with a particle size of 5 micrometers.TM Hi Res IEX; column size 100 mm × 4.6 mm; available from Waters Corporation, Milford, MA. The eluent consisted of mobile phase A (25 mM tris(hydroxymethyl)aminomethane (TRIS); pH 7.5 (1.51 g TRIS base / 0.5 L water)) and mobile phase B (2 M NaCl in mobile phase A (29.9 g NaCl / 0.5 L "A")), at a flow rate of 0.2 mL / min. Elution was performed using a gradient of 30% to 70% mobile phase B for 8 min. Injection volumes were 0.1 μL, 0.2 μL, 0.5 μL, and 1 μL (5 × replicates and repeated injections). The column temperature was 60 °C. Detection was performed by UV absorption monitored at 280 nm.

[0237] The results of separating EPO mRNA using a series of repeated injections under increased mass loading are shown below. Figures 12A to 12H In the separation performed using the C2-PEG-coated hardware anion exchange column of this disclosure. Figures 12A to 12D Provided in [the text]. Separation performed using a reference metal hardware anion exchange column in [the text]. Figures 12E to 12H Provided in [the document / reference]. The RSD values ​​for 5 replicate injections at each given mass load are reported. Figure 13A and Figure 13B The peak areas generated by repeated injections of EPO mRNA and subsequent anion exchange separation on the C2-PEG coated hardware column and the reference metal hardware column are provided respectively. Figure 14A and Figure 14B The peak areas generated by repeated injections of Cas9 mRNA and subsequent anion exchange separation are provided for the C2-PEG coated hardware column and the reference metal hardware column, respectively.

[0238] Overall, the results obtained demonstrate that higher peak areas and therefore higher recoveries are achieved at each given mass load when using C2-PEG-coated hardware. Peak area increases ranging from 15% to 200% were obtained by using C2-PEG-coated hardware surfaces compared to the reference metal hardware column. Along with improvements in recovery and consistency, it is noted that C2-PEG-coated hardware requires less sample-based passivation.

Claims

1. A chromatographic apparatus for size exclusion chromatography, the chromatographic apparatus comprising: A chromatographic column, the column comprising a compartment containing a stationary phase and an inner wall defining a wetting surface. At least a portion of the wetted surface comprises a hydrophilic nonionic coating containing a polyethylene glycol silane having the following structure: in: R 7 R 8 and R 9 Each can be independently either OH or OR. B ; R B Indicates the attachment point to the wetted surface, where R 7 R 8 and R 9 At least one of them is OR B ; R 13 It is H or CH3; m is an integer from 1 to 10; and n is an integer from 2 to 50; and The stationary phase comprises porous particles having a surface, wherein at least some portions of the surface are modified with polyethylene glycol silanes having hydroxyl-terminated ends with the following formula. ； in m is an integer from 1 to 10; n is an integer from 2 to 50; and The wavy line indicates the attachment point to the surface of the porous particles.

2. A method for modifying a fluid flow path within the interior of a fluid system disposed in a chromatographic apparatus, the chromatographic apparatus comprising a chromatographic column, the chromatographic column comprising a compartment containing a stationary phase and an inner wall defining a wetting surface, wherein the stationary phase comprises porous particles having a surface, wherein at least a portion of the surface is modified with a polyethylene glycol silane having hydroxyl-terminated ends having the following formula. ； in m is an integer from 1 to 10; n is an integer from 2 to 50; and Wherein the wavy line indicates the attachment point with the surface of the porous particles, the fluid flow path has a wetting surface containing hydroxyl groups, and the method includes: a. Contact the fluid flow path with the vaporized organosilane reagent of Formula I. (I)； in: R 1 R 2 R 3 R 4 R 5 and R 6 Each is independently selected from the group consisting of (C1-C6)alkoxy, -NH(C1-C6)alkyl, -N((C1-C6)alkyl)2, OH, and halogen; and Z is (C1-C) 20 alkyl group, -[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 ; To form a first coating deposited on at least a portion of the wetted surface along the fluid flow path; and b. Contact the fluid flow path on which the first coating is deposited with a solution containing an organosilane reagent to deposit a second coating in direct contact with the first coating, the second coating comprising the following structure: in: R 7 R 8 and R 9 Each can be independently either OH or OR. B ; R B Indicates the attachment point to the wetted surface or the attachment point to the first coating, wherein in either case, R 7 R 8 and R 9 At least one of them is OR B ; R 13 For H; m is an integer from 1 to 10; and n is an integer from 2 to 50.

3. The method according to claim 2, wherein Z is (C2-C 10 )alkyl.

4. The method according to claim 2 or 3, wherein Z is -(CH2CH2)-.

5. The method according to claim 2 or 3, wherein R 1 R 2 R 3 R 4 R 5 and R 6 Each is independently selected from the group consisting of (C1-C2)alkoxy, -N((C1-C2)alkyl)2 and halogen.

6. The method according to claim 2 or 3, wherein R 1 R 2 R 3 R 4 R 5 and R 6 Each is either methoxy or chlorine.

7. The method according to claim 2 or 3, wherein the reagent of formula I is bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane.

8. The method according to claim 2, wherein in the structure of the second coating, m is 3 and n is an integer from 8 to 12.

9. The method according to claim 2 or 3, wherein the solution further comprises an aliphatic or aromatic organic solvent.

10. The method according to claim 2 or 3, wherein the solution comprises 0.01% to 10% by volume of the organosilane reagent.

11. The method according to claim 2 or 3, wherein the solution comprises 0.05% to 1% by volume of the organosilane reagent.

12. The method according to claim 9, wherein the solvent is toluene.

13. The method according to claim 2 or 3, wherein the contact time with the organosilane reagent is from 1 hour to 96 hours.

14. The method according to claim 2 or 3, wherein the contact time with the organosilane reagent is 6 hours to 48 hours.

15. The method according to claim 2 or 3, wherein the contact time with the organosilane reagent is 12 hours to 24 hours.

16. The method according to claim 2 or 3, wherein the contact with the organosilane reagent is carried out at a temperature of 0°C to 100°C.

17. The method according to claim 2 or 3, wherein the contact with the organosilane reagent is carried out at a temperature of 10°C to 25°C.

18. The method of claim 2 or 3, further comprising removing any excess organosilane reagent after the fluid flow path comes into contact with the solution comprising the organosilane reagent, the removal comprising rinsing the fluid flow path at least once with at least one solvent.

19. The method according to any one of claims 2 or 3, further comprising drying the fluid flow path after contact with the solution comprising the organosilane reagent, wherein drying comprises exposing the fluid flow path to a high temperature for a period of time.

20. The method of claim 2 or 3, further comprising hydrolyzing at least a portion of the first coating, the second coating, or both, wherein the hydrolysis comprises bringing the fluid flow path into contact with water.

21. The method according to claim 2 or 3, further comprising annealing the first coating.

22. The method according to claim 2 or 3, wherein the first coating has a thickness of 100 Å to 1000 Å.

23. The method according to claim 2 or 3, wherein the first coating has a thickness of 400 Å to 800 Å.

24. The method of claim 2 or 3, wherein the first coating and the second coating together have a total thickness of 800 Å to 2000 Å.

25. The method of claim 2 or 3, wherein the first coating and the second coating together have a total thickness of 1000 Å to 1600 Å.

26. The method of claim 2 or 3, wherein the fluid flow path is defined at least in part by the inner surface of the pipe.

27. The method of claim 2 or 3, wherein the fluid flow path is defined at least in part by the inner surface of the microfabricated fluid conduit.

28. The method of claim 2 or 3, wherein the fluid flow path is defined at least in part by a channel through the molten metal.

29. The method of claim 2 or 3, wherein the fluid flow path is defined at least in part by the inner surface of the injection needle.

30. The method of claim 2 or 3, wherein the fluid flow path extends from a sample reservoir container disposed upstream of and in fluid communication with the inner surface of the injection needle to a port of the connector / detector.

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