Rapid multiplexed analysis of released glycans by two-stage microfluidic capture and parallel fluorescent labeling prior to LC analysis

Abstract

The present technology provides a description of a system and method for purifying glycans. The system includes a polypeptide capture device fluidly coupled to a glycan capture and labeling device. The polypeptide device includes an affinity capture agent that can capture glycoproteins. The glycan capture and labeling device includes a glycan affinity capture agent (e.g., a lectin protein) that can capture specific glycans released from the glycoproteins in the polypeptide capture device. The captured glycans are then labeled with a dye. The labeled glycans are removed from the lectin and into an analysis device.

Description

Cross-references to related applications

[0001] This application claims priority and benefit to U.S. Provisional Application No. 63 / 622,702, filed January 19, 2024, entitled “Rapid Multiplexed Analysis of Released Glycans by Dual-Stage Microfluidic Capture and Parallel Fluorescent Tagging Prior to LC Analysis,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure relates to the use of affinity chromatography for the analysis of polysaccharides. Background Technology

[0003] Protein glycosylation plays a crucial role in determining the in vivo bioactivity, stability, solubility, and metabolic fate of biotherapeutic agents. Glycosylation is a post-translational modification, and unlike transcription, it is a non-template-driven enzymatic modification process—its structural state is highly dependent on the production method and conditions.

[0004] Many novel recombinant glycoprotein therapeutics—such as glycoprotein hormones, cytokines, coagulation factors, and monoclonal antibodies—are in clinical use. The glycan profiles of these products are often an important consideration in the development of biotherapeutic agents. Glycan profiles are also unique because they have been shown to fluctuate with variations in cell culture medium composition, temperature, pH, culture time, clonal line, and other variables during glycoprotein synthesis. For novel therapies, the presence of immunogenic glycoforms (such as high-mannose or fucoidan-free glycosylation) can derail drug candidates in the development pipeline. Contract organizations and biosimilar projects must additionally achieve robust and similar glycoform distributions to match regulatory submissions. However, glycan characterization can be challenging. Therefore, improved and efficient methods for glycan characterization are becoming increasingly important. Summary of the Invention

[0005] Problems associated with glycan characterization are mitigated by using pre-fractionation and labeling of glycans prior to chromatographic analysis. In one embodiment, a method for capturing and labeling glycans released from a protein includes passing the protein through a peptide capture device containing an affinity trapping agent, wherein at least a portion of the protein passing through the peptide capture device is captured by the affinity trapping agent. The captured protein is treated with a glycanase, thereby removing one or more glycans from the captured protein. The released glycan is then introduced into a glycan capture and labeling device. The glycan capture and labeling device contains one or more glycan affinity trapping agents (e.g., lectin proteins) that have an affinity for specific structural features of the glycan. The captured glycan is cleaned and labeled with a dye (e.g., a fluorescent dye).

[0006] In a preferred embodiment, the glycan capture and labeling device includes multiple wells, each containing a different type of glycan affinity trapping agent. For example, each well may contain a different class of lectin proteins. When glycans released from proteins pass through the wells, the glycan affinity trapping agents in the wells selectively bind to one or more types of glycans. In this way, glycans can be pre-fractionated prior to chromatographic analysis.

[0007] In some embodiments, the glycan trapping and labeling device includes one or more reagent reservoirs coupled to one or more wells. Each reagent reservoir contains a different dye. During use, the dye is released from each reagent reservoir into a single well among the one or more wells. In this way, each well receives a unique dye that will label only the glycan trapped in the well. When multiple wells are present, a reagent reservoir is fluidly coupled to each well, wherein each reagent reservoir contains a different dye. Some embodiments are characterized by two wells, each well having a different glycan affinity trapping agent coupled to an inner surface. Some embodiments are characterized by four or more wells (e.g., six wells), wherein each well is associated with a unique glycan affinity trapping agent.

[0008] Using different dyes for each well of a glycan capture and labeling device can provide enhanced detection of different glycans. For example, the fluorescent dyes used in the wells can have different fluorescence, which allows for the monitoring of different classes of glycans using different wavelengths. Additionally, dyes coupled to glycans can alter the chromatographic behavior of the glycans (e.g., retention time), depending on the analytical method used to study the glycan. Changes in retention time can facilitate the separation of glycans that typically have similar elution rates. Attached Figure Description

[0009] The present technology will be more fully understood through the following detailed description taken in conjunction with the accompanying drawings, in which:

[0010] Figure 1A Typical release glycan patterns of NIST monoclonal antibodies were depicted;

[0011] Figure 1B Typical release glycan patterns of antibodies with double N-linked glycosylation sites were depicted.

[0012] Figure 2 A system for separating and labeling glycans is described.

[0013] Figure 3 A top view depicting the glycan markers and the trapping device is shown.

[0014] Figure 4 A projected view of the glycan capture and labeling device is shown.

[0015] Figure 5 An exploded view of the glycan capture and labeling device is shown.

[0016] Figure 6 The chromatograms shown depict the enrichment of the high-mannose glycoform after lectin affinity binding and elution with retention times from 0 to 40 minutes. Figure 6 It also provides magnified views of portions of the chromatogram between 11 and 30 minutes.

[0017] Figure 7 The chromatograms shown depict the unbound “penetrating” material generated during the binding step with retention times of 0 to 40 minutes during enrichment of the high mannose glycotype. Figure 7 It also provides magnified views of portions of the chromatogram between 11 and 30 minutes.

[0018] Figure 8A This demonstrates the separation and measurement via HILIC-FLR. Figure 6 The chromatogram of the residual dye peaks in the eluted material.

[0019] Figure 8B This demonstrates the separation and measurement via HILIC-FLR. Figure 7 The chromatogram of residual dye peaks penetrating the material.

[0020] Figure 8C It shows Figure 8A and Figure 8B The quantification of dye peaks provided in the text.

[0021] Figure 9 A graph illustrating the results of the binding kinetics experiment is provided. This graph plots the percentage of bound material versus binding time (in minutes) and demonstrates that binding is completed within approximately 5 minutes. The glycans bound to the mannose lectin, were washed, and then eluted. The substances were quantified using subsequent HILIC-FLR separation. Detailed Implementation

[0022] Glycoproteins consist of one or more oligosaccharide chains covalently attached to the amino acid side chains of a basic protein. The oligosaccharide chains attached to the basic protein are called "glycans".

[0023] Typically, glycoproteins are produced during the synthesis of glycoprotein therapeutics. The glycan profile (defined herein as the number and type of glycans attached to the underlying protein) can be unique to glycoprotein products. However, the glycan profile can vary based on cell culture medium composition, temperature, pH, culture time, cloning line, and other variables during glycoprotein synthesis.

[0024] Glycans are composed of multiple basic carbohydrate units. The presence or absence of certain carbohydrates in glycans can produce significant differences in the pharmacological properties of glycoproteins. Table 1 shows the effects of various oligosaccharides present in glycans on the pharmacological properties of glycoproteins.

[0025]

[0026] Typical procedures for glycan characterization involve purifying the target glycoprotein, enzymatically releasing the glycan, buffer exchange, adding a fluorescent mass tag, and finally analyzing the sample using liquid chromatography combined with fluorescence and mass detection. While this assay is extremely accurate and powerful, it requires extensive sample preparation and chromatographic runs of up to one hour or longer. Faster chromatography has been achieved using mass detection alone; however, this approach trades quantitative accuracy for analysis time and has robustness issues.

[0027] The standard approach to managing the demand for glycan data in large experimental designs is to measure only the first 3-5 candidates from each round of parallel bioreactor experiments, or to limit data collection to a sampling period of a few days. Such methods not only risk selecting suboptimal candidates in terms of product quality, but also limit the use of time-parameterized parameters for feed, temperature, pH, and dissolved oxygen.

[0028] Eliminating the need for a mass spectrometer significantly reduces the complexity of the instruments required for glycan determination, thus removing a barrier to its widespread use. A typical glycan release spectrum is shown in... Figure 1A (for NIST monoclonal antibodies) and Figure 1B (For antibodies with double N-linked glycosylation sites). These chromatograms show that each antibody contains 5-10 high-abundance glycoforms and dozens of much lower-abundance glycoforms. In the case of antibodies with disaccharylation sites ( Figure 1B The number of low-abundance sugars is much higher, and many of these substances co-elute from each other. In the absence of quality verification capabilities, the confidence level for identifying these substances solely based on retention time is extremely low.

[0029] In one embodiment, the chromatographic complexity of separating released glycans is reduced by using pre-fractionation with lectins. Lectins are a class of naturally occurring proteins that exhibit specificity for different glycoforms and structures. Pre-fractionation of released glycans is achieved by coupling the released glycans to a structured array via a device that couples the released glycans to one or more of these lectin proteins. In this way, the released glycan sample can be pre-fractionated before optical detection alone.

[0030] The preparation of released glycan samples comprises five stages: enzymatic cleavage of glycans from proteins / peptides; separation of protein / peptide components from glycan components; labeling of the newly released glucamine with NHS carbamate groups linked to mass and fluorescence tags; separation of unlabeled material from labeled glycans; and analysis by LC-FLR-MS. The sample preparation process also includes the initial separation of target proteins / peptides from background host cell protein material; this is typically achieved through affinity separation. This paper describes an apparatus that utilizes combined affinity purification and enzymatic release of glycans, along with combined glycan fractionation based on glycan affinity groups, to produce samples suitable for analysis by LC.

[0031] A system for separating and labeling polysaccharides is shown in Figure 2 The system includes a peptide capture device and a glycan capture and labeling device. For example... Figure 2 As shown, the peptide capture device is fluidly connected to the glycan capture and labeling device. Specifically, the outlet of the peptide capture device is fluidly connected to the inlet of the glycan capture and labeling device. One or more tubes or valves may form a fluid passage between the peptide capture device and the glycan capture and labeling device.

[0032] The sample preparation process involves the initial isolation of the target peptide (e.g., protein) from the background host cell material using affinity separation. Figure 2 In the depicted system, the peptide capture device comprises an affinity trapping agent. An affinity trapping agent is an agent that selectively binds to a protein / peptide of interest. In a preferred embodiment, the affinity trapping agent binds to a broad class of proteins / peptides. Specifically, the affinity trapping agent binds to many different peptides. For example, protein A, protein G, protein A / G, and protein L are proteins that bind to most species and IgG antibody subclasses. In an alternative embodiment, an antigen against the captured antibody can be used as the affinity trapping agent. In another embodiment, affinity for scaffold systems (such as monoclonal antibodies) can be developed via an evolutionary display approach. Other types of affinity trapping agents used in peptide capture devices include platform affinity agents that can be optimized for proteins / peptides of a given target or target class, such as DARPin, affinity bodies, repeatbodies, fynomers, anticalins, His tags, and aptamers.

[0033] In one embodiment, an affinity trapping agent is immobilized to the inner surface of a peptide trapping device. The peptide trapping device includes an inlet, an inner chamber, and an outlet. The affinity trapping agent is immobilized to the surface of the inner chamber. The affinity trapping agent can be immobilized to the surface through physical interaction, covalent binding, or bioaffinity. In a preferred embodiment, the affinity agent is covalently coupled to the surface of the inner chamber. Covalent coupling of a protein-based affinity agent to a surface can be achieved through the direct reaction of one or more amino acid side chains of the protein-based affinity agent with the surface. Amino acids having hydroxyl side groups (e.g., serine) or amine-terminal side groups (e.g., lysine) are used to covalently link the protein-based affinity agent to the surface. Alternatively, a linker group can be coupled to the surface of the inner chamber. The linker group can be a bifunctional group capable of forming covalent bonds with both the surface and the affinity trapping agent.

[0034] In another embodiment, the chamber of the peptide capture device may encapsulate resin particles, wherein one or more affinity trapping agents are bound to the resin. For example, the resin may be formed from cross-linked agarose beads, wherein an affinity trapping agent (e.g., protein A) is covalently bound to the resin. The affinity trapping agent may be bound to the resin using a reductive amination coupling strategy. The particles may be dispersed within the inner chamber of the peptide capture device. The inlet and outlet of the chamber may include one or more sieve plates for retaining the resin particles within the chamber. In an alternative embodiment, nanoparticles may be dispersed on the inner surface of the peptide capture device. The nanoparticles are selected from materials (metals, polymers, silica / glass, etc.) that capture peptides of interest when a sample passes through the peptide capture device.

[0035] During use, the sample is introduced into the peptide capture device through the inlet and into the inner chamber. The sample can be unclarified or clarified biological material. The sample can be a blood sample or a sample taken from a bioreactor. The sample is introduced into and passes through the chamber, where it comes into contact with an affinity trap. As the sample passes through the chamber, antibodies and other glycosylated peptides present in the sample are captured by the affinity trap. In some embodiments, the flow rate of the sample through the chamber can be adjusted to allow sufficient time for proteins / peptides or antibodies in the sample to bind to the affinity trap. In other embodiments, a ridged structure can be used to facilitate mixing and binding of the material.

[0036] One or more valves may be used to introduce fluid into a peptide capture device. For example, a multi-port valve may be coupled to the inlet of the peptide capture device. A multi-port valve may include multiple inlet ports and an outlet port. The outlet port of the valve may be coupled to the inlet of the peptide capture device to allow selective introduction of fluid into the peptide capture device. The multi-port valve may also include multiple inlet ports. In one embodiment, the inlet port of the multi-port valve may be coupled to one or more of the following: equilibration buffer; enzyme solution (e.g., glycanase solution); elution buffer; sample introduction (e.g., glycosylated proteins / peptides in a biological sample); and wash solution.

[0037] The first step in capturing glycans released from glycoproteins / glycopeptides is introducing the sample into the peptide capture device. A solvent or sample is chosen to promote the binding of the glycoprotein / glycopeptide under investigation to the affinity trapping agent. For example... Figure 2 As shown, the sample passes through the first valve and enters the peptide capture device. A pump connected to the valve provides pressure, forcing the sample through the valve into the peptide capture device. In an alternative method, after the sample is introduced into the peptide capture device, centrifugal force can be applied to the peptide capture device to provide positive pressure to the sample, pushing it through the affinity trapping agent.

[0038] The sample can be dissolved or mixed in a solvent that promotes the binding of the glycoprotein / glycopeptide of interest. Some parameters that promote the binding of the glycoprotein / glycopeptide to the affinity trap include salt concentration and buffer pH. In some embodiments, a salt concentration of <50 mM is preferred, more preferably <5 mM. Preferably, the pH of the sample is maintained below 8. In the absence of a structure designed to promote mixing of fluid components, the flow rate of the sample through the peptide trap is determined in part by the release kinetics of the affinity trap and the trapped protein / peptide. To ensure the capture of the desired protein / peptide in the sample, the sample flow rate is reduced to allow a residence time of less than 50% of the kinetic release half-life. For example, for most proteins / peptides, the kinetic release time (half-life) of protein A is about 50 minutes. To ensure that the protein / peptide is adequately captured by protein A, the residence time of the sample in contact with the affinity trap should be set to about 25 minutes or less.

[0039] Depending on the configuration of the affinity trap and the peptide trapping device, flow rates from 0.1 μL / s to 1.0 mL / s can be used. In the absence of any specific structures in the peptide trapping device that provide mixing and increase the effective surface area of ​​the inner surface, the flow rate will be relatively slow to ensure that the protein of interest has sufficient time to diffuse to the affinity trap (e.g., 0.1 μL / s to 10 μL / s). Using structures that promote mixing and / or flow pathways through the peptide trapping device allows for even faster flow rates (e.g., 0.1 mL / s to 1.0 mL / s).

[0040] Second valve ( Figure 2 Valve 2) can be used to control the location where fluid flows out of the outlet of the peptide capture device. In an embodiment, valve 2 has at least one input and two outputs. Valve 2 can be a three-way valve or a multi-port valve, depending on the required outputs and inputs of the specific system. Figure 2In the depicted embodiment, the outlet of the peptide capture device is coupled to valve 2. Valve 2 may have two outlet positions. The first outlet position of valve 2 fluidly transfers fluid from the peptide capture device to a waste container. During use, fluid typically transfers to the waste container as sample or wash fluid flows through the peptide capture device. The second outlet position of valve 2 leads to the input of the glycan capture and labeling device. Valve 2 is positioned in the second outlet position when fluid transfer from the peptide capture device to the glycan capture and labeling device occurs.

[0041] After the protein binds to the affinity trap, one or more optional washes can be performed to help remove host cell proteins and other interfering substances. In one embodiment, a series of washes utilizing different pH buffers are used to remove unwanted material from the peptide capture device. For example, a “high pH” buffer may be used for washing, wherein the pH of the buffer solution is greater than 7, greater than 8, greater than 9, or greater than 10. In some embodiments, a series of high pH buffer solutions may be used to wash the peptide capture device, wherein the pH increases or decreases with each consecutive wash between pH 7 and pH 10 or higher.

[0042] Another possible wash uses a detergent to remove unwanted material from the peptide capture device. Exemplary detergents include, but are not limited to, 1M urea + isopropanol, Tween 80, and polysorbate. The use of a detergent can be the sole washing step or can be combined with a high-pH washing step.

[0043] After the desired protein / peptide binds to the affinity trap, the glycan is cleaved from the glycosylated protein / peptide. In one embodiment, the process of cleaving the glycan from the bound protein / peptide is achieved by passing a solution containing a glycanase through a peptide trapping device. The glycanase is an enzyme that cleaves the glycan from the glycosylated protein / peptide. In one embodiment, the glycanase is an N-glycanase. The N-glycanase cleaves the N-linked glycan from the glycosylated protein / peptide. The N-glycanase cleaves the glycan from the glycosylated protein / peptide by hydrolyzing the amide bond between the innermost glucose unit and the amino acid side chain bonded to the glycan. Exemplary glycanases include, but are not limited to, PNGase F, PNGase A, endoglycosidase H, and endoglycosidase F.

[0044] The cleavage of glycans from bound proteins / peptides can be achieved using a flow-through process. In this process, a glycanase solution is passed through a peptide capture device. The glycanase releases the glycan from the bound peptide / peptide, and the released glycan is carried out of the peptide capture device by the flowing fluid. Using a flow-through enzymatic digestion strategy allows for easy removal of the glycan without the need to remove the protein / peptide. When using N-hydroxysuccinimide (NHS) carbamate to conjugate a label to the glycan, the labeling step requires an amine-free buffer. However, elution buffers used to remove proteins / peptides from affinity columns typically utilize amine-containing buffer salts. Typical processes that rely on eluting proteins / peptides from affinity columns before glycan removal and analysis face the problem of removing or replacing the buffer after the protein / peptide has been eluted. Therefore, the claimed flow-through process offers the benefit of cleanly separating the glycan from the underlying protein / peptide in an amine-free buffer, thereby allowing for easy labeling of the glycan in subsequent processing steps. A second benefit of the claimed process is the elimination of the acid neutralization step typically required after low-pH elution of proteins from the peptide capture step.

[0045] Once cut, the glycans flow into the glycan capturing and labeling device. Figure 2 In the embodiment shown, valve 2 is set to a second position that leads to the input of the glycan capture and labeling device.

[0046] After removing the glycans from the peptide capture device, the device is prepared to capture samples by removing the bound proteins / peptides. Deglycosylated proteins / peptides can be removed by elution with a suitable elution solution containing an eluent. In one embodiment, the elution solution is an acidic solution containing an eluent. The acidic elution solution has a pH less than 7, less than 5, or less than 3. Typical elution solutions used to wash away deglycosylated proteins / peptides have a pH of 2.0 to 3.2. The concentration of the eluent is from about 50 mM to about 150 mM. In one embodiment, the eluent is citric acid. Other eluents include, but are not limited to, aqueous solutions containing phosphoric acid, hydrochloric acid, or sulfuric acid.

[0047] Figure 3 A top view of a glycan labeling and trapping device is depicted. The device includes one or more pores disposed within a reaction chamber; each pore contains one or more glycan affinity trapping agents coupled to the inner surface of the pore. In a preferred embodiment, the one or more glycan affinity agents are lectins. Figure 4 A projected view of the glycan capture and labeling device 100 is shown. Figure 5An exploded view of a glycan capture and labeling device 100 is shown. The glycan capture and labeling device 100 includes a body 110 having an inlet 120, a reaction chamber 130, a reagent reservoir 140, and an outlet 150. The reaction chamber 130 includes a plurality of orifices 135. Each of the plurality of orifices 135 is fluidly coupled to one of the plurality of reagent reservoirs 140, as depicted in the figures. A reservoir conduit 125 fluidly connects the reagent reservoir to the orifice 135. The reaction chamber 130 is an open chamber that allows fluid from the inlet 120 to enter the orifice 135 disposed within the reaction chamber. Fluid in the reaction chamber 130 will flow out of the reaction chamber through the outlet 150. The fluid reaction chamber and orifices may be made of a material capable of being directly or indirectly bonded to an affinity agent via a connector. Suitable materials include, but are not limited to, cyclic olefin polymers (COPs), cyclic olefin copolymers (COCs), and epoxy resins. In other embodiments, the body of the glycan capturing and labeling device is made of silica / glass or metal (e.g., steel or titanium), and a layer of reactive material is applied to the body to allow coupling of the glycan affinity capture agent. For example, a layer of COP, COC, or epoxy resin may be applied to the body to allow coupling of the glycan capture agent.

[0048] Figure 5 One embodiment of the construction of a glycan capturing and labeling device 100 is shown. In this embodiment, the device consists of multiple layers connected to each other. Figure 5 In the illustrated embodiment, the device includes a base layer 200, a porous layer 210, a reaction chamber layer 220, and a reservoir layer 230. These layers are bonded together with an adhesive that is resistant to the fluids (primarily water) used during testing. The adhesive forms a seal that retains the fluid within the structure formed by these layers. These layers can be formed by etching the depicted pathways and openings into the material. Alternatively, one or more of these layers can be formed using chemical etching, laser ablation and injection molding, 3D printing, or additive manufacturing processes. In some embodiments, these layers are made individually and then bonded together. In some embodiments, the layers of the device are constructed sequentially, stacked one layer at a time. Sealing of these layers can be performed thermally, using an adhesive, or through diffusion bonding of the layers.

[0049] The basement 200 includes a basement inlet passage 202 and a reservoir conduit 204 formed in the body.

[0050] The pore layer 210 includes a pore inlet passage 212. The pore layer also includes a plurality of openings 214 that define pores 135 in the glycan trapping and labeling device. When the pore layer is attached to the base layer, the sidewalls of the openings define the boundaries of the pores, while the base layer 200 defines the bottom of the pores. The pore layer also includes a reagent reservoir base 216 and a reservoir upper conduit 218. When the pore layer is attached to the base layer, the reservoir lower conduit 208 and the reservoir upper conduit 218 combine to form a reservoir conduit 125. Figure 4 (As depicted in the text).

[0051] The reaction chamber layer 220 includes a reaction chamber inlet passage 222, a reaction chamber outlet passage 224, and a reservoir compartment 226. The reaction chamber layer also includes a reaction chamber opening 228 that defines a sidewall of the reaction chamber of the glycan capture and labeling device. When these layers are attached to each other, the base inlet passage 202, the orifice inlet passage 212, and the reaction chamber inlet passage 222 combine to form inlet 120. Figure 4 (As depicted in the image). The reaction chamber outlet passage 224 defines the outlet 150 of the glycan capture and labeling device. When these layers are attached to each other, the reaction chamber opening 228 defines the outer wall of the reaction chamber.

[0052] Reservoir layer 230 includes a plurality of reservoirs 236. When these layers are attached to each other, the reservoirs 236 are aligned with reservoir compartments 226 and reservoir base 216. Reservoirs 236 contain different dyes for marking the glycans trapped in the pores. In an embodiment, reservoirs 236 may be removable, allowing the dyes to be replaced or changed. Reservoirs 236 may be sealed to contain the dyes. In one embodiment, when pressure is applied to the reservoir, an opening may be formed in the reservoir, allowing dyes from the reservoir to enter the reservoir compartments and the base. Pressure from opening the reservoir pushes the dyes into the pores, where the dyes react with the glycans.

[0053] Each pore 135 includes a glycan affinity trap. In embodiments, each pore 135 includes a different glycan affinity trap that is specific to different classes of glycans. Exemplary glycan affinity traps include lectin proteins and scaffolds engineered to have specific binding behavior to certain glycan subclasses. The glycan-specific scaffold may be formed from DARPin, affinity molecules, repeatbodies, fynomers, anticalins, and aptamers.

[0054] In this implementation, lectin proteins can be used as glycan affinity traps. Lectin proteins exhibit specific selectivity for glycans. Each different type of lectin protein binds to a specific class of glycans based on their structure. Lectin proteins bind to specific classes of glycans in a mixture of glycans. Furthermore, lectin proteins are multivalent, resulting in high affinity binding to glycans. By selecting specific lectin proteins, multiple glycans can be separated in pores within a glycan trapping and labeling device, with each pore containing a specific class of glycans.

[0055] Lectin proteins that can be used in glycan capture and labeling devices can be selectively targeted at specific glycans of interest. Glycan-binding lectin proteins can be classified according to the specific carbohydrate structure types exhibited by the lectin.

[0056] Mannose-binding lectins include, but are not limited to, Amaranthus variegatedus lectin (AMA), Concanavalin A (ConA), Snowdrop lectin (GNA, GNL), Amaryllis hybrid lectin (HHA, HHL), Moriga M lectin (MNA-M), Narcissus lectin (NPA), Elderberry lectin II (SNA-II), and Utricular lectin (UDA), as well as Lentil hemagglutinin (LcH).

[0057] Complex N-glycan-bound lectins include, but are not limited to, button mushroom lectin (ABA, ABL), colchicine lectin (CA), broom lectin (CAA), tulip lectin (TL), datura lectin (DSA), common bean-E lectin (PHA-E), common bean-L lectin (PHA-L) and black locust lectin (RPA).

[0058] O-glycan-bound lectins include, but are not limited to, amaranth lectin (ACA, ACL), peanut lectin (PNA), jackfruit lectin (AIA, Jacalin), fragile coral lectin (CF), cassia lectin (MPA, MPL), snail lectin (HPA), and spotted snail lectin (HAA). Figure 5 ).

[0059] Fucose-binding lectins include, but are not limited to, Alanium cirrhosa lectin (AAL), Aspergillus oryzae lectin (AOL), LAA, LcH, LcA, LTL, PSA, PTL / PTA I, PTL / PTA II, TJA-II, and UEA-I.

[0060] Sialic acid-binding lectins include, but are not limited to, cholera toxin B (CTB), sophora japonica lectin-I (MAL-I, MAM, MAL), sophora japonica lectin-II (MAL-II, MAH), scabra polypore lectin (PSL), trichosanthes kirilowii lectin-I (TJA-I), and elderberry lectin-I (SNA-I).

[0061] Terminal GlcNAc and chitin-binding lectins include, but are not limited to, hyacinth bean lectin-II (GS-II), pokeweed lectin (PWA), vitex lectin-II (UEA-II), and wheat germ lectin (WGA).

[0062] Terminal galactose and LacNAc-binding lectins include, but are not limited to, Bauhinia purpurea lectin (BPA, BPL), Erythrina crista-gallicu lectin (ECA, ECL), Hyacinthus spp. lectin-I (GS-I), Tomato lectin (LEA), Lycoperdon spp. lectin (MOA), Pseudomonas aeruginosa-IL lectin (PA-IL), Ricinus spp. lectin (RCA-I, RCA120), Sophora japonica lectin (SJA), and Potato lectin (STA, STL).

[0063] Terminal GalNAc-binding lectins include, but are not limited to, broom lectin (CSA), clover lectin (DBA), soybean lectin (SBA), vitex lectin (VVL, VVA) and wisteria lectin (WFA, WFL).

[0064] In one embodiment, the glycan trapping device includes a plurality of pores. Each of the plurality of pores includes a different glycan affinity trapping agent. In an embodiment, each pore may include a different lectin protein selected from one of the lectin types described above. For example, one pore may include mannose-binding lectin, and another pore may include fucose-binding lectin. Additional pores may include other types of lectin proteins. In a preferred embodiment, each pore contains a lectin protein from the different classes of lectin proteins described above. In another embodiment, an affinity agent derived from a monoclonal antibody or other scaffold design may be developed to be specific to a particular glycan and may be used as a glycan affinity agent.

[0065] Glycan affinity traps are coupled to the inner surface of pores via a coupling agent. Alternatively, glycan affinity traps can be directly coupled to the inner surface of pores via the binding of their functional groups. For lectin proteins, streptavidin can be used to bind the lectin protein to the surface. Streptavidin can bind to the inner surface of pores. Biotinylated lectin proteins bind to streptavidin, thus essentially immobilizing the lectin protein to the surface. Biotinylation-streptavidin coupling of lectins to the surface allows for the use of a universal immobilization scheme. Any lectin protein can be biotinylated so that when the biotinylated lectin protein is introduced into the pore, it binds to streptavidin. In this way, a single immobilization strategy can be used for a variety of different lectins.

[0066] Glycan capture is achieved by directly introducing a glycan solution generated in a peptide capture device into the reaction chamber of a glycan capture and labeling device. As the solution enters and passes through the reaction chamber, the glycans corresponding to specific glycan affinity traps disposed in the pores are captured through affinity binding to the glycan affinity traps. For example... Figure 3 The depicted glycan capture and labeling device includes an inlet and an outlet. The inlet is fluidly coupled to the outlet of the peptide capture device, such as... Figure 2As shown. The outlet is fluidly connected to an analytical system (e.g., a liquid chromatography system). A valve (not shown) may be present between the outlet and the analytical system to allow the fluid flowing from the glycan capture and labeling device to be sent to a waste container or directly to the analytical device, depending on the processing steps performed.

[0067] After the polysaccharide-containing solution passes through the reaction chamber, one or more washing steps are performed to remove any unbound polysaccharides. The washing steps are carried out using an amine-free buffer (e.g., sodium bicarbonate) to remove unbound polysaccharides. After the washing steps are completed, air is passed through the reaction chamber to remove all remaining liquid.

[0068] After the glycans are captured, cleaned, and dried, they can be released and transferred to the analytical device. Alternatively, the glycans can be labeled with a suitable label before release and transfer to the analytical device, which will improve the ability to distinguish different glycans in the analytical device.

[0069] In one embodiment, the glycan capture and labeling device includes one or more reagent reservoirs containing dyes that can bind to glycans, such as... Figure 3 As shown. Each reagent reservoir is fluidly coupled to a single orifice. After the glycan is captured, each reagent reservoir is activated such that the dye in each reservoir is transferred to the orifice fluidly coupled to the reservoir. In one embodiment, the fluid can be transferred using a pump coupled to the reagent reservoir. The pump can be activated to transfer fluid from the reagent reservoir to the orifice. Alternatively, the reagent reservoir can be a pressurized reservoir. Applying pressure to the pressurized reservoir forces the dye into the orifice fluidly coupled to the reservoir.

[0070] In one embodiment, the reagent reservoir relies on the hydrophilic interaction between the dye-containing liquid and the device's cap to contain the dye within each orifice. To suppress leakage and spread between orifices, the captured polysaccharide is air-dried before being treated with the dye in the reservoir, as discussed above.

[0071] The glycans in the wells are incubated with the dye for a sufficient time to allow the dye to bind to the glycans in the wells. Labeling glycans with dyes is beneficial for their detection because it improves both detection sensitivity and selectivity by altering the chromatographic behavior of the glycans. Strategies and methods for labeling glycans are described in U.S. Patent Nos. 8,124,792 and 11,352,325, and U.S. Patent Application Publications Nos. 2018 / 0094293, 2019 / 0331669, and 2020 / 0332028, all of which are incorporated herein by reference.

[0072] The dye can selectively attach to specific carbohydrate groups of the glycan. In one embodiment, the dye is coupled to one or more terminal carbohydrate groups of the glycan. The dye can be selectively coupled to the terminal carbohydrates of the glycan via an enzyme-mediated reaction. For example, the enzymatic labeling of the glycan can be tailored to label only specific terminal carbohydrates. In one embodiment, the dye can specifically target fucose, sialic acid, high-mannose, and terminal GlcNAc carbohydrates. In some embodiments, the glycans bound to the glycan affinity trap will have different terminal carbohydrates. Therefore, the specificity of the dye can increase the additional pre-fractionation of the glycan by labeling only some, but not all, of the glycans trapped in the pores.

[0073] After the glycan is labeled, the labeled glycan is washed with buffer to remove excess and unreacted dye from the wells and reaction chamber. Following washing, the glycan is removed from the glycan affinity trap and enters the analytical device (e.g., a liquid chromatography system). The glycan is removed by applying a carbohydrate solution to the reaction chamber and wells. The carbohydrates in the solution replace the glycan bound to the glycan affinity trap. The released labeled glycan is transferred out of the reaction chamber through the outlet of the glycan trap and label device. The outlet of the glycan trap and label device is coupled to the analytical device, allowing the glycan to be directly transferred to the analytical device as it is removed from the wells.

[0074] In one embodiment, the dye is a fluorescent compound. Using a different fluorescent label for each well of the glycan provides enhanced detection of different glycan substances. For example, the fluorescent label used in each well may have different fluorescence, which allows for the monitoring of different classes of glycans using different wavelengths. In this embodiment, the glycan can be monitored on multiple fluorescence channels corresponding to the fluorescent labels used to label the glycan. The number of channels monitored will correspond to the number of pre-classified and differentially labeled glycan subtypes.

[0075] Furthermore, dyes coupled to glycans can alter the chromatographic behavior of glycans (e.g., retention time), depending on the analytical method. Changes in retention time can facilitate the separation of glycans that typically have similar elution rates.

[0076] In one example, rhodamine labeling is used to label high-mannose glycans, and fluorescein labeling is used to label N-linked glycans containing a core fucose. The two subclasses (rhodamine- and fluorescein-labeled glycans) undergo differential chromatographic retention and differential excitation-emission transitions during HILIC separation, providing two parallel dimensions of separation. The differential retention as a function of glycan subclasses reduces the local complexity of the simplified glycan profile; combined with the multi-channel nature of the detection, this scheme accelerates the gradient and reduces the required assay time.

[0077] Example

[0078] Polystyrene beads with lectin attachment

[0079] Carboxylated polystyrene beads were coupled to (5S)-N-(5-amino-1-carboxypentyl)iminodiacetic acid (NTA-NH2) using standard EDAC chemistry. The NTA-coupled beads were washed and incubated in 100 mM NiCl2 for 1 hour to occupy the hyponitrotriacetic acid moiety. His-labeled lectin was added to the particles in a 5-molar excess. The lectin-attached beads were washed three times with PBS, followed by incubation for 1 hour in a mixture containing 2 mM calcium acetate, 2 mM magnesium acetate, and 2 mM manganese acetate. The lectin-attached beads were then washed four times with PBS to remove excess salt ions.

[0080] lectin specificity of high-mannose polystyrene beads attached with lectin

[0081] The specificity of lectin for high-mannoglycans was tested using lectin-attached beads. A heterogeneous mixture of high-mannoglycans was prepared from stock proteins via glycolysis using PNGase F followed by labeling with Rapifluor-MS tags. The resulting slurry contained labeled glycans, residual glycans, unreacted tags, deglycosylated proteins, and a mixture of deglycosylated and variable-labeled proteins. The slurry was incubated with 50 μL aliquots of lectin-attached polystyrene beads for 20 min. The liquid containing unbound components was removed, and the beads were washed four times with PBS. The glycans were eluted from the lectin-attached beads using 50 μL of 1M α-methylmannose incubated with the beads for 30 min. The supernatant was analyzed by HILIC-FLR. Figure 6 The chromatograms are shown in the figure. The liquid containing unbound material after the initial slurry reacts with the polystyrene beads attached to the lectin was also analyzed by HILIC-FLR and is shown in the figure. Figure 7 middle.

[0082] Figure 6 The enrichment of high-mannose glycoforms after lectin affinity binding and elution is shown. Figure 7 Unbound material from the lectin affinity binding step of this treatment is shown. (Comparison) Figure 6 and Figure 7 It can be seen that the sample obtained by eluting the bound polystyrene from the polystyrene beads attached to lectin provides a sample with a significantly reduced amount of undesirable compounds.

[0083] Figures 8A to 8C The residual dye peaks separated and measured by HILIC-FLR are depicted, and the magnitude of excess dye removal is illustrated. Figure 8A and Figure 8B The regions where dyes and proteins eluted in the chromatogram are highlighted (0 to 11 minutes). Compare the elution fractions ( Figure 8A) and unbound fractions ( Figure 8B The signal of the dye substance in that region. Figure 8C It shows Figure 8A and Figure 8B Quantification of the dye peaks in the middle of the spectrum demonstrated that unreacted dye and labeled proteins were reduced by more than 200-fold.

[0084] Using the same preparation method described above, the binding kinetics were studied by incubating sugar- and lectin-attached beads for variable times. Figure 9 The kinetic curves shown demonstrate the rapid binding of sugar to the protein, even with a fluorescent tag attached. This kinetic binding experiment shows that binding is completed within approximately 5 minutes.

[0085] In view of this specification, further modifications and alternative embodiments of various aspects of the present technology will be apparent to those skilled in the art. Therefore, this specification should be construed as illustrative only and for the purpose of teaching those skilled in the art the general manner of implementing the present technology. It should be understood that the forms of the present technology shown and described herein should be considered as examples of embodiments. Elements and materials may be substituted for those shown and described herein, parts and processes may be reversed, and certain features of the present technology may be utilized independently, all of which will be apparent to those skilled in the art upon obtaining the benefits of this specification of the present technology. Various changes may be made to the elements described herein without departing from the spirit and scope of the present technology as set forth in the following claims.

Claims

1. A method for capturing glycans released from proteins or peptides, the method comprising: The protein or polypeptide is passed through a polypeptide capture device containing an affinity trapping agent, wherein at least a portion of the protein or polypeptide passed through the polypeptide capture device is captured by the affinity trapping agent; The captured protein or polypeptide is treated with a polysaccharide enzyme to remove one or more polysaccharides from the captured protein or polypeptide. as well as The released glycan is introduced into a glycan capturing and labeling device, wherein the glycan capturing and labeling device comprises one or more glycan affinity trapping agents, wherein the one or more glycan affinity trapping agents have an affinity for specific structural features of the glycan.

2. The method of claim 1, wherein the affinity trapping agent comprises one or more proteins selected from the group consisting of: protein A, protein G, protein A / F, and protein L.

3. The method of claim 1, wherein the affinity trapping agent comprises an antibody or nanoparticle bound to the protein or polypeptide.

4. The method according to any one of claims 1 to 3, wherein the glycanase is PNGase F, PNGase A, endoglycosidase H, or endoglycosidase F.

5. The method according to any one of claims 1 to 4, wherein the polypeptide capturing device is fluidly coupled to the glycan capturing and labeling device.

6. The method according to any one of claims 1 to 5, wherein the glycan removed from the captured protein is fluidly transferred from the polypeptide capturing device to the glycan capturing and labeling device.

7. The method according to any one of claims 1 to 6, wherein the glycan affinity trapping agent is a lectin protein.

8. The method of claim 7, wherein the lectin protein is selected from the group consisting of: mannose-binding lectin, complex N-glycan-binding lectin, O-glycan-binding lectin, fucose-binding lectin, sialic acid-binding lectin, terminal GlcNAc and chitin-binding lectin, terminal galactose and LacNAc-binding lectin, and terminal GalNAc-binding lectin.

9. The method of claim 7, wherein the lectin protein comprises one or more lectin proteins selected from the group consisting of: mannose-binding lectin, complex N-glycan-binding lectin, O-glycan-binding lectin, fucose-binding lectin, sialic acid-binding lectin, terminal GlcNAc-binding lectin, chitin-binding lectin, terminal galactose-binding lectin, LacNAc-binding lectin, and terminal GalNAc-binding lectin.

10. The method according to any one of claims 1 to 9, the method further comprising removing the protein or polypeptide from the polypeptide capture device.

11. The method according to any one of claims 1 to 10, the method further comprising incubating each glycan bound to the glycan affinity trap with a dye bound to the glycan.

12. The method of claim 11, wherein a different dye is applied to the glycan of each glycan affinity trapping agent that is bound to a different glycan affinity trapping agent.

13. The method according to claim 11 or 12, further comprising eluting the polysaccharide from the polysaccharide capture and labeling device.

14. A system for separating and labeling polysaccharides, the system comprising: A peptide capture device comprising an affinity capture agent; and A glycan trapping and labeling device, the glycan trapping and labeling device comprising one or more pores, each pore containing a glycan affinity trapping agent coupled to the inner surface of the pore; The polypeptide capturing device is fluidly coupled to the glycan capturing and labeling device such that the outlet of the polypeptide capturing device is coupled to the inlet of the glycan capturing and labeling device.

15. The system of claim 14, further comprising a valve positioned between the polypeptide capture device and the glycan capture and labeling device.

16. The system of claim 15, wherein the valve comprises at least one input port and at least two output ports, wherein the input port is coupled to the outlet of the polypeptide capture device, and one of the output ports is coupled to the input of the glycan capture and labeling device, and one of the output ports is coupled to a waste collection container.

17. The system according to any one of claims 14 to 16, wherein the glycan capturing and labeling device comprises one or more orifices, wherein the glycan affinity capturing agent is coupled to the surface of the orifices.

18. The system of claim 17, wherein the glycan capture and labeling device comprises one or more reagent reservoirs containing a dye capable of binding to the glycan, wherein each reagent reservoir is fluidly coupled to a single pore.

19. The system according to any one of claims 14 to 18, wherein the glycan capturing and labeling device comprises a variety of glycan affinity capture agents.

20. The system of claim 19, wherein the glycan capturing and labeling device comprises a plurality of pores, wherein each of the plurality of pores contains a different glycan affinity capturing agent.

21. The system according to any one of claims 14 to 20, wherein the glycan affinity trapping agent is a lectin protein.

22. The system of claim 21, wherein the lectin protein is selected from the group consisting of: mannose-binding lectin, complex N-glycan-binding lectin, O-glycan-binding lectin, fucose-binding lectin, sialic acid-binding lectin, terminal GlcNAc and chitin-binding lectin, terminal galactose and LacNAc-binding lectin, and terminal GalNAc-binding lectin.

23. The system of claim 21, wherein the glycan affinity trap is selected from the group consisting of: DARPin, affinity, repeatbody, fynomer, anticalin, and aptamer.

24. The system according to any one of claims 20 to 23, wherein the glycan capture and labeling device comprises a plurality of reagent reservoirs, each reagent reservoir containing a different dye capable of binding to the glycan, wherein each reagent reservoir is fluidly coupled to a single pore.

25. The system according to any one of claims 14 to 24, wherein the glycan capture and labeling device comprises an inlet conduit coupled to a reaction chamber and an outlet conduit coupled to the reaction chamber, wherein the reaction chamber comprises a plurality of pores, each pore containing a different class of lectin proteins; wherein during use, fluid reaches the reaction chamber through the inlet conduit, the fluid is collected in the pores in the reaction chamber, and wherein during use, the fluid flows out of the reaction chamber through the outlet conduit.

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