Label-free n-glycan quantification method

Abstract

To provide a method for efficiently quantifying a glycosylation profile of a protein.SOLUTION: A method of quantifying the glycosylation profile of a recombinant protein, comprising analyzing one or more N-linked glycans without labeling, wherein the N-linked glycans are released from the recombinant protein by an enzyme prior to analysis.EFFECT: It allows for shorter sample preparation and chromatographic separation times and can be used for batch release of products.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 938,803, filed November 21, 2019, the entire contents of which are incorporated herein by reference.

[0002] References to sequence listings submitted electronically via EFS-WEB The contents of the Sequence Listing, an electronically submitted ASCII text file (filename: 3338_190PC01_SL_ST25.txt; size: 14,351 bytes; and creation date: November 20, 2019), are filed herewith and are incorporated herein by reference in their entirety. [Background technology]

[0003] Glycosylation often plays an important role in the biological function of glycoconjugates (e.g., glycoproteins). For example, the glycosylation pattern of a glycoprotein can affect its ability to fold correctly, its stability (e.g., resistance to proteolysis and / or other degradation), catalytic activity, pharmacokinetic and / or pharmacodynamic properties, and / or the ability of the glycoprotein to properly interact with other molecules. Alternatively or additionally, the glycosylation pattern of a glycoprotein can affect the trafficking and targeting of the glycoprotein, determining, for example, whether the glycoprotein remains intracellularly (e.g., including the correct targeting of the glycoprotein to the appropriate intracellular compartment(s)), whether the glycoprotein is membrane-bound, and / or whether the glycoprotein is secreted from the cell.

[0004] Monoclonal antibodies and other proteins are complex glycoproteins developed for the treatment of various indications, including cancer and autoimmune diseases. Specifically, monoclonal antibodies are typically glycosylated at conserved asparagine residues in the heavy chain. Glycans are important in governing the function and efficacy of monoclonal antibody therapeutics and are therefore typically required to be part of a panel of critical quality attributes in release testing for human use. "N-linked glycans," or "N-glycans," are classified according to the terminal sugar residue into complex, high-mannose, and hybrid N-glycans. Summary of the Invention [Problem to be solved by the invention]

[0005] Traditionally, N-linked glycans are released from glycoproteins after denaturation and enzymatic digestion with PNGase F, followed by fluorescent labeling of the released glycans (e.g., with 2-aminobenzamide). The labeled glycans are then separated by hydrophilic liquid chromatography (HILIC) with a fluorescence detector to generate a chromatographic profile of the glycans present in the antibody sample. Despite being used for decades, this process remains cumbersome, requiring toxic reagents and lengthy sample preparation times. Therefore, there is a need for an efficient method to quantify the glycosylation profile of proteins. [Means for solving the problem]

[0006] The present disclosure is directed to a method for quantifying the glycosylation profile of a recombinant protein, comprising analyzing one or more N-linked glycans without labeling, wherein the N-linked glycans are released from the recombinant protein by enzymatic digestion prior to analysis. The present disclosure is also directed to a method for quantifying the glycosylation profile of a recombinant protein, comprising analyzing one or more N-linked glycans without a fluorophore, wherein the N-linked glycans are released from the recombinant protein by enzymatic digestion prior to analysis. In some embodiments, the recombinant protein has a purity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%. In some embodiments, the analysis comprises separating one or more N-linked glycans during chromatography, including a column. In some embodiments, the column is a mixed-mode column. In some embodiments, the separation is performed using one or more mobile phases. In some embodiments, the column is a mixed-mode porous graphitic carbon (PGC) column. In some embodiments, the enzyme comprises peptide N-glycosidase F (PNGase F). In some embodiments, the enzyme is incubated with the recombinant protein, and one or more N-linked glycans are released from the recombinant protein before separation. In some embodiments, the enzyme is diluted in a buffer. In some embodiments, the separated one or more N-linked glycans are measured by a mass spectrometer, an ELSD, an NQAD, or a refractive index detector. In some embodiments, the separated one or more N-linked glycans are measured by a charged particle detector (CAD).

[0007] In some embodiments, the chromatography comprises a first mobile phase and a second mobile phase, and the first mobile phase and the second mobile phase are different. In some embodiments, the first mobile phase comprises water. In some embodiments, the second mobile phase comprises acetonitrile. In some embodiments, the first mobile phase comprises formic acid (FA), trifluoroacetic acid (TFA), triethylamine (TEA), or any combination thereof. In some embodiments, the first mobile phase comprises 0.1% FA. In some embodiments, the first mobile phase comprises 0.1% TEA. In some embodiments, the second mobile phase comprises formic acid (FA), trifluoroacetic acid (TFA), triethylamine (TEA), or any combination thereof. In some embodiments, the second mobile phase comprises 0.1% FA. In some embodiments, the second mobile phase comprises 0.1% TEA. In some embodiments, the separation is performed at a temperature below 70°C.

[0008] In some embodiments, the temperature is between about 50° C. and about 70° C., between about 50° C. and about 60° C., between about 60° C. and about 70° C., between about 55° C. and about 65° C., between about 55° C. and about 60° C., between about 60° C. and about 65° C., between about 65° C. and about 70° C., between about 50° C. and about 55° C. In some embodiments, the temperature is about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., or about 69° C.

[0009] In some embodiments, the separation is based on a gradient. In some embodiments, the gradient is from about 95% to about 5%. In some embodiments, the gradient is from about 95% to about 50%, from about 95% to about 55%, from about 95% to about 60%, from about 95% to about 65%, from about 95% to about 70%, from about 95% to about 75%, from about 95% to about 80%, from about 95% to about 85%, from about 90% to about 50%, from about 90% to about 55%, from about 90% to about 60%, from about 90% to about 65%, from about 90% to about 70%, about 90% to about 75%, about 87% to about 50%, about 87% to about 55%, about 87% to about 60%, about 87% to about 65%, about 87% to about 70%, about 87% to about 75%, about 85% to about 50%, about 85% to about 55%, about 85% to about 60%, about 85% to about 65%, about 85% to about 70%, or about 85% to about 75%. In some embodiments, the gradient is about 87% to about 75%.

[0010] The present disclosure also relates to a method for analyzing the glycan profile of a protein of interest. In some embodiments, one or more N-glycans are galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (Glc), N-acetylglucosamine (GlcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxynonanoic acid (Kdn), fucose (Fuc), glucuronic acid (GlcA), iduronic acid (IdoA), galacturonic acid (GalA), mannuronic acid (ManA), or any combination thereof. In some embodiments, the one or more N-glycans comprise one or more biantennary glycans. In some embodiments, the biantennary glycans are selected from the group consisting of GOF, GO, GIF, GI, G2F, G2, S1G2F, S1G2, S2G2F, S2G2, and any combination thereof. In some embodiments, the glycosylation profile comprises one or more asialylated glycans, monosialylated glycans, disialylated glycans, and / or trisialylated and tetrasialylated glycans. In some embodiments, the recombinant protein is an antibody. In some embodiments, the antibody is of an isotype selected from IgM, IgA, IgE, IgD, and IgG. In some embodiments, the antibody is of the IgG isotype. In some embodiments, the IgG antibody is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the antibody is an antibody against GITR, an antibody against CXCR4, an antibody against CD73, an antibody against TIGIT, an antibody against OX40, an antibody against LAG3, an antibody against CSF1R, and / or an antibody against IL8. In some embodiments, the antibody has a single N-linked glycosylation site. In some embodiments, the single N-linked glycosylation site is asparagine 297 (N297).

[0011] In some embodiments, the recombinant protein comprises an enzyme, a hormone, a cytokine, a cell surface receptor, a protease, a cytokine receptor, or any combination thereof. In some embodiments, the recombinant protein is a fusion protein. In some embodiments, the fusion protein is fused to a heterologous moiety. In some embodiments, the heterologous moiety is a half-life extending moiety. In some embodiments, the half-life extending moiety comprises albumin, an albumin-binding polypeptide, a fatty acid, PAS, the beta subunit of the C-terminal peptide (CTP) of human chorionic gonadotropin, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN, an albumin-binding small molecule, Fc, or a combination thereof. In some embodiments, the half-life extending moiety is Fc. In some embodiments, the method is batch release. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows a sample glycan chromatogram generated from the separation analysis shown in Example 1 (Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile). [Figure 2] FIG. 2 shows a sample glycan chromatogram generated from the separation analysis shown in Example 2 [Mobile phase A: 0.1% triethylamine (TEA) in water; Mobile phase B: 0.1% triethylamine (TEA) in acetonitrile]. DETAILED DESCRIPTION OF THE INVENTION

[0013] This disclosure introduces a novel label-free method for detecting and quantifying N-glycans without the use of fluorescent or UV labels. By using a reversed-phase porous graphitic carbon column and charged particle detection to generate glycan profiles, sample preparation and chromatographic separation time are significantly reduced, resulting in significant cost savings. The label-free method yielded quantitative results similar to those of the fluorescently labeled method, confirming the method's suitability for product release.

[0014] I. Definition The term "and / or" as used herein is to be taken as a specific disclosure of each of two particular features or components, with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0015] Wherever embodiments are described herein with the term "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many terms used in this disclosure.

[0017] Units, prefixes, and symbols are expressed in their International System of Units (SI) recognized form. Numerical ranges are inclusive of the numbers defining the range. The headings used herein do not limit the various aspects of this disclosure, which are provided by reference to the specification as a whole. Accordingly, the terms defined below are fully defined by reference to the specification in its entirety.

[0018] The use of the alternative (e.g., "or") should be understood to mean one, both, or any combination thereof of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "one or more" of any cited or listed components.

[0019] The terms "about" or "comprising essentially of" refer to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "essentially comprising" can mean within 1 or more than 1 standard deviation according to the practice in the art. Alternatively, "about" or "essentially comprising" can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5-fold. When a particular value or composition is provided in this application and claims, unless otherwise stated, the meaning of "about" or "essentially comprising" should be assumed to be within an acceptable error range of that particular value or composition.

[0020] As described herein, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the recited range and fractions thereof, where appropriate (such as integer tenths and hundredths), unless otherwise indicated.

[0021] As used herein, the term "protein of interest" is used to include any protein (either natural or recombinant) present in a mixture for which purification is desired. Such proteins of interest include, but are not limited to, enzymes, hormones, growth factors, cytokines, immunoglobulins (e.g., antibodies), and / or any fusion proteins.

[0022] As used herein, a "biomarker" is almost any detectable compound, such as a protein, peptide, proteoglycan, glycoprotein, lipoprotein, carbohydrate, lipid, nucleic acid (e.g., DNA, such as cDNA or amplified DNA, or RNA, such as mRNA), organic or inorganic chemical, natural or synthetic polymer, small molecule (e.g., metabolite), or a discriminating molecule or discriminating fragment of any of the foregoing present in or derived from a biological sample, or any other characteristic or indicia thereof that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmaceutical response to a therapeutic intervention.

[0023] As used herein, the term "analyte" is used to include any molecule or protein (either natural or recombinant) present in a mixture for which analysis or quantitation is desired. Such analytes include, but are not limited to, small molecules, enzymes, hormones, growth factors, cytokines, immunoglobulins (e.g., antibodies), and / or any fusion proteins.

[0024] The terms "purification," "separation," or "isolation," as used interchangeably herein, refer to increasing the degree of purity of a molecule from a composition or sample that contains the molecule and one or more impurities. Typically, the degree of purity of a molecule is increased by removing (fully or partially) at least one impurity from the composition.

[0025] As used herein, the term "mass spectrometry" refers to a highly sensitive technique used to detect, identify, and quantify molecules based on their mass-to-charge ratio (m / z). As ions pass along the central axis of four parallel, equidistant poles or rods, such as a quadrupole, an electric field is used to separate ions according to their mass-to-charge ratio (m / z), the ratio of mass to integral charge (z). Two voltages are applied to each rod: one fixed direct current and one circulating alternating current with superimposed radio frequency. The magnitude of the applied electric field can be adjusted to ensure that only ions with a specific m / z ratio pass through the quadrupole and are detected. Ions with other m / z values ​​are deflected into trajectories that either collide with the quadrupole rods and are discharged, or are ejected from the mass analyzer field and removed via vacuum. Because only ions of a specific m / z are stable in the quadrupole at any one time, quadrupoles are sometimes called exclusive detectors. Ions with stable trajectories are sometimes referred to as having collisionless, resonant, or stable trajectories.

[0026] Typically, in a triple quadrupole mass spectrometer experiment, the first quadrupole (Q1) is configured to pass only ions of a specific m / z (precursor ions) of the expected species in the sample. The second quadrupole (i.e., Q2 or collision cell) is used to fragment the ions that pass through Q1. The third quadrupole (Q3) is configured to pass only ions of a specific m / z (fragment ions) corresponding to the expected fragmentation products of the expected species to the detector. In some embodiments, the sample is ionized in the mass spectrometer to generate one or more protonated or deprotonated molecular ions. In some embodiments, the one or more protonated or deprotonated molecules have a single, double, triple, or higher charge. In some embodiments, the mass spectrometer is a triple quadrupole mass spectrometer. In some embodiments, the resolution used for Q1 and Q3 is unit resolution. In other embodiments, the resolution used for Q1 and Q3 is different. In other embodiments, the resolution used in Q1 is higher than the unit resolution of Q3.

[0027] As used herein, the term "charged aerosol detection" or "CAD" refers to an analytical technique based on nebulizing an eluate with a nitrogen (or air) carrier gas to form droplets, which are then dried to remove the mobile phase and produce analyte particles. A primary stream of analyte particles is contacted with a positively charged secondary stream by passing it through a high-voltage platinum corona wire. The charge is diffusively transferred to the opposing analyte particle stream, which then travels to a collector where it is measured with a sensitive electrometer, producing a signal proportional to the amount of analyte present.

[0028] As used herein, the term "fluorophore" refers to a fluorescent chemical compound that can re-emit light upon photoexcitation. Fluorophores typically contain several attached aromatic groups or planar or cyclic molecules with several π-bonds. Two commonly used fluorophores are 2-AB (2-aminobenzamide) and 2-AA (anthranilic acid or 2-aminobenzoic acid). Other fluorophores include PA (2-aminopyridine), AMAC (2-aminoacridone), ANDS (7-amino-1,3-naphthalenedisulfonic acid), ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid), APTS (9-aminopyrene-1,4,6-trisulfonic acid), and 3-(acetylamino)-6-aminoacridine.

[0029] As used herein, a "glycan profile" is understood to be any defined set of quantitative values ​​for glycans that can be used for comparison with a reference value or profile obtained from another sample or group of samples. For example, the glycan profile of a sample from a protein sample may differ significantly from the glycan profile of a sample from another source. By comparing the profile with a reference or standard profile, the glycan profile may be useful for predicting or forecasting the pharmacokinetic (PD) or pharmacokinetic (PK) therapeutic effect of a protein. Reference and sample glycan profiles can be generated using any analytical device capable of detecting glycans, such as a charged particle detector (CAD).

[0030] The term "chromatography" refers to any technique for separating a protein of interest (e.g., an antibody) from other molecules (e.g., contaminants) present in a mixture. Typically, the protein of interest is separated from other molecules (e.g., contaminants) as a result of differences in the rate at which individual molecules of the mixture migrate through a stationary medium under the influence of a mobile phase, or as a result of binding and elution processes. The terms "matrix" or "chromatography matrix" are used interchangeably herein and refer to any type of adsorbent, resin, or solid phase that separates the protein of interest (e.g., an Fc region-containing protein such as an immunoglobulin) from other molecules present in the mixture during the separation process. Non-limiting examples include particulate resins, monolithic or fibrous resins, and membranes that can be placed in columns or cartridges. Examples of materials for forming the matrix include polysaccharides (such as agarose and cellulose) and other mechanically stable matrices such as silica (e.g., controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles, and derivatives of any of the above. Typical matrix types suitable for the methods of the present disclosure include cation exchange resins, affinity resins, anion exchange resins, and mixed-mode resins. A "ligand" is a functional group attached to a chromatography matrix and determines the binding properties of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed-mode groups (combinations of the above). Preferred ligands that can be used herein include, but are not limited to, strong cation exchange groups such as sulfopropyl and sulfonic acid; strong anion exchange groups such as trimethylammonium chloride; weak cation exchange groups such as carboxylic acid; weak anion exchange groups such as N5N-diethylamino or DEAE; hydrophobic interaction groups such as phenyl, butyl, propyl, and hexyl; and affinity groups such as Protein A, Protein G, and Protein L. To make this disclosure more readily understandable, certain terms will first be defined.As used in this application, unless stated otherwise herein, each of the following terms shall have the meaning defined below. Additional definitions are defined throughout this application.

[0031] The term "affinity chromatography" refers to a protein separation technique in which a protein of interest (e.g., a protein of interest or an antibody having an Fc region) specifically binds to a ligand specific for the protein of interest. Such ligands are commonly referred to as biospecific ligands. In some embodiments, the biospecific ligand (e.g., Protein A or a functional variant thereof) is covalently attached to a chromatography matrix material, making it accessible to the protein of interest in solution when the solution contacts the chromatography matrix. The protein of interest generally retains its specific binding affinity for the biospecific ligand during the chromatography step, while other solutes and / or proteins in the mixture do not significantly or specifically bind to the ligand. Binding of the protein of interest to the immobilized ligand allows contaminating proteins or protein impurities to pass through the chromatography matrix while the protein of interest remains specifically bound to the immobilized ligand on the solid phase material. The specifically bound protein of interest is then removed from the immobilized ligand in its active form under appropriate conditions (e.g., low pH, high pH, ​​high salt, competing ligand, etc.) and passed through the chromatography column with an elution buffer, free of the contaminating proteins or protein impurities that previously passed through the column. Any component can be used as a ligand for purifying its respective specific binding protein, for example, an antibody. However, in various methods according to the present disclosure, Protein A is used as a ligand for a target protein having an Fc region. The conditions for eluting a target protein (e.g., a protein having an Fc region) from a biospecific ligand (e.g., Protein A) can be easily determined by those skilled in the art. In some embodiments, Protein G or Protein L or functional variants thereof can be used as a biospecific ligand.In some embodiments, a biospecific ligand such as Protein A is used in the pH range of 5-9 to bind to proteins with Fc regions, and the biospecific ligand / target protein complex is washed or re-equilibrated and then eluted with a buffer having a pH above or below 4 that contains at least one salt.

[0032] As used herein, the term "buffer" refers to a substance whose presence in a solution increases the amount of acid or alkali that must be added to produce a unit change in pH. Buffer solutions resist changes in pH through the action of their acid-base complex components. Buffer solutions used with biological reagents are generally capable of maintaining a constant concentration of hydrogen ions so that the pH of the solution is within the physiological range. Traditional buffer components include, but are not limited to, organic salts, inorganic salts, acids, and bases.

[0033] The term "conductivity," as used herein, refers to the ability of an aqueous solution to pass an electric current between two electrodes. In an aqueous solution, electric current flows by ion transport. Therefore, as the amount of ions present in an aqueous solution increases, the solution will have a higher conductivity. The unit of measurement for conductivity is millisiemens per centimeter (mS / cm) and can be measured using a conductivity meter.

[0034] As used herein, the term "mobile phase" refers to the liquid or gas that flows through a chromatography system, causing the substances being separated to move at different velocities over the stationary phase. Mobile phases can be polar or non-polar. Polar mobile phases are commonly used in combination with non-polar stationary phases, and these chromatographic separations are known as reversed-phase chromatography. Conversely, non-polar mobile phases are often used in combination with polar stationary phases, and are commonly known as normal-phase chromatography.

[0035] As used herein, the term "stationary phase" refers to the solid or liquid phase of a chromatography system onto which the substances to be separated are selectively adsorbed. Silica is commonly used as the stationary phase.

[0036] As used herein, the term "chromatography column" or "column" in relation to chromatography refers to a container, often in the form of a cylinder or hollow post, that is filled with a chromatography matrix or resin, a material that provides the physical and / or chemical properties used in purification.

[0037] The terms "ion exchange" and "ion exchange chromatography" refer to a chromatography process in which an ionizable solute of interest (e.g., a protein of interest in a mixture) interacts with oppositely charged ligands bound (e.g., by covalent attachment) to a solid-phase ion exchange material under appropriate conditions of pH and conductivity, and the solute of interest interacts nonspecifically with more or less charged compounds than solute impurities or contaminants in the mixture. Contaminant solutes in the mixture can be washed from the column of ion exchange material or bind to or are excluded from the resin faster or slower than the solute of interest. "Ion exchange chromatography" specifically includes cation exchange (CEX), anion exchange (AEX), and mixed-mode chromatography.

[0038] A "cation exchange resin" or "cation exchange membrane" refers to a negatively charged solid phase having free anions for exchange with cations in an aqueous solution passing over or through the solid phase. Any negatively charged ligand attached to the solid phase suitable for forming a cation exchange resin can be used, including, for example, carboxylates, sulfonates, and the like, as described below. Commercially available cation exchange resins include, for example, sulfonate-based groups (e.g., MonoS, MiniS, Source 15S and 30S from GE Healthcare, SP SEPHAROSE® Fast Flow, SP SEPHAROSE® High Performance, Capto S, Capto SP ImpRes from Tosoh, TOYOPEARL® SP-650S and SP-650M from Tosoh, MACRO-PREP® High S from BioRad, Ceramic HyperD S, TRISACRYL® M and LS SP, and Spherodex LS SP from Pall Technologies); sulfoethyl-based groups (e.g., FRACTOGEL® SE from EMD, POROS® S-10 and S-20 from Applied Biosystems); sulfopropyl-based groups (e.g., TSK Gel SP 5PW and SP-5PW-HR from Tosoh, Life Technologies, Inc., and others). POROS® HS-20, HS50, and POROS® XS from Technologies; sulfoisobutyl-based groups (e.g., FRACTOGEL® EMD SO3 from EMD); -sulfoxyethyl-based groups (e.g., SE52, SE53, and Express-Ion S from Whatman); carboxymethyl-based groups (e.g., CM SEPHAROSE® Fast Flow from GE Healthcare, Hydrocell CM from Biochrom Labs Inc., MACRO-PREP® CM from BioRad, Ceramic HyperD CM, TRISACRYL® M CM, TRISACRYL® LS CM from Pall Technologies, Matrx CELLUFINE® C500 and C200 from Millipore, CM52, CM32, CM23, and Express-Ion C from Whatman, TOYOPEARL® CM-650S, CM-650M, and CM-650C from Tosoh); sulfonic acid-based and carboxylic acid-based groups (e.g., BAKERBOND® Carboxy-Sulfon from JT Baker); carboxylic acid-based groups (e.g., WP from JT Baker) CBX, DOWEX® MAC-3 from Dow Liquid Separations, AMBERLITE® Weak Cation Exchangers, DOWEX® Weak Cation Exchanger, and DIAION® Weak Cation Exchangers from Sigma-Aldrich, and FRACTOGEL® EMDCOO from EMD; sulfonic acid-based groups (e.g., Hydrocell SP from Biochrom Labs Inc., DOWEX® Fine Mesh Strong Acid Cation Resin from Dow Liquid Separations, JTExamples of suitable cation exchange resins include, but are not limited to, Baker's UNOsphere S, WP Sulfonic, Sartorius' SARTOBIND® S membrane, Sigma-Aldrich's AMBERLITE® Strong Cation Exchangers, DOWEX® Strong Cation, and DIAION® Strong Cation Exchanger; or those with orthophosphate groups (e.g., Whatman's P11). Other cation exchange resins include carboxymethylcellulose, BAKERBOND ABX™, Ceramic HyperD Z, Matrex Cellufine C500, and Matrex Cellufine C200.

[0039] An "anion exchange resin" or "anion exchange membrane" refers to a positively charged solid phase, and therefore has one or more positively charged ligands attached thereto. Any positively charged ligand attached to a solid phase suitable for forming an anion exchange resin can be used, including, for example, a quaternary amino group. Commercially available anion exchange resins include DEAE cellulose, POROS® PI 20, PI 50, HQ 10, HQ 20, HQ 50, and D 50 from Applied Biosystems, SARTOBIND® Q, MonoQ, MiniQ, Source 15Q and 30Q from Sartorius, Q, DEAE, and ANX SEPHAROSE® Fast Flow, Q SEPHAROSE® High Performance, QAE SEPHADEX®, and FAST Q SEPHAROSE® (GE Healthcare), WP PEI, WP DEAM, and WP QUAT from JT Baker, Hydrocell DEAE and Hydrocell QA from Biochrom Labs Inc., UNOsphere Q, MACRO-PREP® DEAE, and MACRO-PREP® High Q from Biorad, and Ceramic HyperD Q and Ceramic HyperD from Pall Technologies. DEAE, TRISACRYL® M and LS DEAE, Spherodex LS DEAE, QMA SPHEROSIL® LS, QMA SPHEROSIL® M and MUSTANG® Q, Dow Liquid Separations' DOWEX® Fine Mesh Strong Base Type I and Type II Anion Resins and DOWEX® MONOSPHER E 77 weak base anion, Millipore's INTERCEPT® Q membrane, Matrex CELLUFINE® A200, A500, Q500, and Q800, EMD's FRACTOGEL® EMD TMAE, FRACTOGEL® EMDExamples include DEAE and FRACTOGEL® EMD DMAE, Sigma-Aldrich's AMBERLITE® weak and strong anion exchangers type I and II, DOWEX® weak and strong anion exchangers type I and II, DIAION® weak and strong anion exchangers type I and II, DUOLITE®, Tosoh's TSK gel Q and DEAE 5PW and 5PW-HR, TOYOPEARL® SuperQ-650S, 650M and 650C, QAE-550C and 650S, DEAE-650M and 650C, Whatman's QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D or Express-Ion Q, and SARTOBIND® Q (Sartorius Corporation, New York, USA). Other anion exchange resins include POROS XQ, SARTOBIND® Q, Q SEPHAROSE™ XL, Q SEPHAROSE™ big beads, DEAE Sephadex A-25, DEAE Sephadex A-50, QAE Sephadex A-25, QAE Sephadex A-50, Q SEPHAROSE™ high performance, Q SEPHAROSE™ XL, Resource Q, Capto Q, Capto DEAE, Toyopearl GigaCap Q, Fractogel EMD TMAE HiCap, Nuvia Q, or PORGS PI.

[0040] "Porous graphitic carbon (PGC) columns" or "porous graphitic carbon (PGC) reversed-phase columns" exhibit unique properties as stationary phases, providing retention of highly polar analytes and separation of structurally related substances. At the microscale, PGC surfaces consist of flat sheets of hexagonally arranged carbon atoms, similar to very large polynuclear aromatic molecules. This surface is crystalline and highly reproducible, lacking micropores or chemically bonded phases. By utilizing this column's properties as a stationary phase for HPLC, a wide range of separations that would normally be considered problematic can be resolved. These columns offer unique retention and separation of highly polar compounds, and the surface is stereoselective, enabling the separation of geometric isomers and other closely related compounds. PGC also provides retention of native glycan species. Separations in PGC are facilitated by a combination of reversed-phase behavior, based on the hydrophobicity of the analyte, and the polar retaining effect of graphite, based on the high polarizability of graphitic carbon materials. PGC is sensitive to structural as well as linkage isomers and is capable of resolving isomeric glycans. An example of a porous graphitic carbon column is HYPERCARB™ (THERMO SCIENTIFIC™).

[0041] As used herein, "N-linked glycan" refers to a protein modification in which a glycan is attached to a glycoconjugate via a nitrogen bond. The acceptor for the glycan is a selected asparagine residue on a polypeptide chain that has entered the periplasm or the lumen of the ER, respectively. Oligosaccharyltransferase, a central enzyme in the N-glycosylation pathway, catalyzes the formation of the N-glycosidic bond of an oligosaccharide to the side chain amide of an asparagine residue, specified by the consensus sequence NXS / T, where X can be any amino acid residue. All eukaryotic N-glycans share a common core sequence, Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr, and are classified into three types: (1) oligomannose type, in which only Man residues extend the core; (2) complex type, in which an "antenna" initiated by GlcNAc extends the core; and (3) hybrid type, in which Man extends the Manα1-6 arm of the core and one or two GlcNAcs extend the Manα1-3 arm.

[0042] As used herein, "N-linked glycosylation" refers to the attachment of an oligosaccharide to a nitrogen atom, usually the N4 of an asparagine residue. N-glycosylation can occur on secreted or membrane-bound proteins, primarily in eukaryotes and archaea.

[0043] As used herein, the term "contaminants" is used to cover any undesired components or compounds in a mixture. In cell cultures, cell lysates, or clarified bulk products (e.g., clarified cell culture supernatants), contaminants include, for example, host cell nucleic acids (e.g., DNA) and host cell proteins present in the cell culture medium. Host cell contaminant proteins include, but are not limited to, proteins naturally or recombinantly produced by host cells, as well as proteins related to or derived from the protein of interest (e.g., proteolytic fragments) and other process-related contaminants. In certain embodiments, contaminant precipitates are separated from the cell culture using other means, such as centrifugation, sterile filtration, depth filtration, or tangential flow filtration.

[0044] The term "antibody" refers, in some embodiments, to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In some antibodies, e.g., naturally occurring IgG antibodies, the heavy chain constant region is composed of a hinge and three domains, CH1, CH2, and CH3. In some antibodies, e.g., naturally occurring IgG antibodies, each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The heavy chain may or may not have a C-terminal lysine. The term "antibody" can include bispecific or multispecific antibodies.

[0045] As used herein, "IgG antibodies," e.g., human IgG1, IgG2, IgG3, and IgG4 antibodies, in some embodiments, have the structure of naturally occurring IgG antibodies, i.e., have the same number of heavy and light chains and disulfide bonds as naturally occurring IgG antibodies of the same subclass. For example, an IgG1, IgG2, IgG3, or IgG4 antibody can consist of two heavy chains (HC) and two light chains (LC), with the two HCs and LCs each linked by the same number and positions of disulfide bridges as occur in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies (unless the antibody is mutated to modify the disulfide bridges).

[0046] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. The IgG isotype is divided into subclasses in specific species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. Immunoglobulins, such as IgG1, exist in several allotypes, which differ from each other by at most a few amino acids. "Antibodies" include, by way of example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, and completely synthetic antibodies.

[0047] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment (a papain fragment) or a similar monovalent fragment consisting of the VL, VH, LC, and CH1 domains; (ii) a F(ab')2 fragment (a pepsin fragment) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a combination of two or more isolated CDRs, which can be optionally linked by a synthetic linker. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows the VL and VH regions to pair and be produced as a single protein chain to form a monovalent molecule (known as a single-chain Fv (scFv)). See Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0048] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom; (b) antibodies isolated from host cells, e.g., transfectomas, that have been transformed to express the antibody; (c) antibodies isolated from recombinant, combinatorial human antibody libraries; and (d) antibodies prepared, expressed, created or isolated by other means, including splicing of human immunoglobulin gene sequences into other DNA sequences.

[0049] As used herein, "isotype" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies) encoded by heavy chain constant region genes.

[0050] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.

[0051] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. As used herein, the term "protein" is intended to encompass molecules composed of one or more polypeptides, which may, in some cases, be associated by bonds other than amide bonds. Alternatively, a protein may be a single polypeptide chain. In this latter example, the single polypeptide chain may, in some cases, comprise two or more polypeptide subunits fused to form the protein. The terms "polypeptide" and "protein" also refer to products of post-expression modifications, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide or protein may be derived from a natural biological source or produced by recombinant technology, but need not necessarily be translated from a designated nucleic acid sequence. It may be produced by any method, including chemical synthesis.

[0052] As used herein, the term "abatacept" refers to a genetically engineered fusion protein consisting of the functional binding domain of human cytotoxic T-lymphocyte antigen-4 (CTLA-4) and the Fc domain of a human monoclonal immunoglobulin of the IgG1 class. Abatacept consists of two homologous glycosylated polypeptide chains of approximately 46 kDa, covalently linked via a single disulfide bond.

[0053] As used herein, the term "belatacept" refers to a mutant CTLA4-Fc polypeptide that contains two mutations relative to abatacept: the amino acid at position 29 is mutated to tyrosine and the amino acid at position 104 is mutated to glutamic acid. In some embodiments, for example, the beta polypeptide contains at least one CTLA-4A29YL104E - The amino acid sequence of the extracellular domain of Fc is included. A non-limiting example of belatacept includes SEQ ID NO: 4. For example, belatacept is further described in U.S. Provisional Application No. 2009-0252749(A1), published October 8, 2009, the entire contents of which are incorporated herein by reference.

[0054] As used herein, the term "polynucleotide" or "nucleotide" is intended to encompass a single nucleic acid as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), complementary DNA (cDNA), or plasmid DNA (pDNA). In certain embodiments, a polynucleotide contains conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds such as those found in peptide nucleic acids (PNAs)).

[0055] The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA, cDNA, or RNA fragments, present in a polynucleotide. When applied to a nucleic acid or polynucleotide, the term "isolated" refers to a nucleic acid molecule, DNA, or RNA, that has been removed from its original environment; for example, a recombinant polynucleotide encoding an antigen-binding protein contained in a vector is considered isolated for purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. Additionally, polynucleotides or nucleic acids can include regulatory elements such as promoters, enhancers, ribosomal binding sites, or transcription termination signals.

[0056] Various aspects of the disclosure are described in further detail in the following subsections.

[0057] II. Glycan Release and Separation Methods The present disclosure is directed to methods for analyzing glycans, e.g., N-linked glycans, in proteins. All eukaryotic N-glycans share a common core sequence, Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-X-Ser / Thr, and are classified into three types: (1) oligomannose-type, in which only Man residues extend the core; (2) complex-type, in which a GlcNAc-initiated "antenna" extends the core; and (3) hybrid-type, in which Man extends the Manα1-6 arm of the core and one or two GlcNAc extend the Manα1-3 arm. After initial processing in the endoplasmic reticulum, glycoproteins are transported to the Golgi apparatus, where further processing occurs. The trimmed N-linked oligosaccharide chains can be modified by the addition of several mannose residues to generate "high-mannose oligosaccharides." Alternatively or additionally, one or more monosaccharide units of N-acetylglucosamine can be added to the core mannose subunit to form "complex oligosaccharides." Galactose can be added to the N-acetylglucosamine subunit, and sialic acid subunits can be added to the galactose subunit, resulting in chains that terminate in sialic acid, galactose, or N-acetylglucosamine residues. Fucose residues can be added to the N-acetylglucosamine residue of the core oligosaccharide. Each of these additions is catalyzed by a specific glycosyltransferase.

[0058] Hybrid glycans contain characteristics of both high-mannose and complex glycans. For example, one branch of a hybrid glycan may contain primarily or exclusively mannose residues, while another branch may contain N-acetylglucosamine, sialic acid, galactose, and / or fucose sugars. N-linked glycans are involved in various cellular processes. For example, N-linked glycans contribute to the proper folding of proteins in eukaryotic cells. Chaperone proteins in the endoplasmic reticulum (endoplasmic reticulum) bind to the three glucose residues present on the core of N-linked glycans. Chaperone proteins typically assist in the folding of proteins to which the glycans are attached. If folding is successful, the three glucose residues are removed, allowing the N-linked glycan to proceed to further processing. If the protein is not properly folded, the three glucose residues are reattached, allowing the protein to reassociate with the chaperone. This cycle is repeated multiple times until the protein achieves the proper conformation. If a protein fails to fold properly multiple times, it is usually exported from the endoplasmic reticulum and degraded by cytoplasmic proteases. Alternatively or additionally, N-linked glycans can contribute to protein folding through steric effects. For example, cysteine ​​residues in peptides can be temporarily inhibited from forming disulfide bonds with other cysteine ​​residues depending on the size of nearby glycans. Thus, the presence of N-linked glycans allows cells to control which cysteine ​​residues form disulfide bonds. The initial oligosaccharide chain is usually trimmed by specific glycosidase enzymes in the endoplasmic reticulum to yield a short, branched core oligosaccharide consisting of two N-acetylglucosamine and three mannose residues.

[0059] N-linked glycans may be involved in cell-cell interactions. For example, tumor cells frequently produce abnormal N-glycan structures, which can be recognized by the CD337 receptor on natural killer cells as a sign that the cells in question are cancerous. N-linked glycans may be involved in targeting degradative lysosomal enzymes to lysosomes. In particular, modification of N-linked glycans with mannose-6-phosphate residues can serve as a signal that the protein to which the glycan is attached should be targeted to lysosomes. Thus, the present disclosure recognizes the importance of determining the glycosylation pattern of N-linked glycans (e.g., N-linked glycans conjugated to glycoproteins). The methods described herein can be used to analyze the properties (e.g., composition and / or structure) of any N-linked glycan.

[0060] The first step in glycan analysis of glycoconjugates such as glycoproteins is the release of sugars from the molecule to which they are attached. N-linked glycans on glycoproteins can be released by amidases such as peptide-N-glycosidase F (PNGase F).

[0061] Glycans are commonly derivatized with labels such as fluorophores to enhance their analytical potential. Traditionally, mass spectrometry or fluorescence detection techniques have been used to analyze released glycans labeled with fluorescent compounds such as 2-AB. Mass spectrometry ("MS" or "mass-spec") is an analytical technique used to measure mass-to-charge ratio ions. This is achieved by ionizing a sample, separating ions of different masses, and recording their relative abundances by measuring the intensity of the ion flux. A typical mass spectrometer consists of three components: an ion source, a mass analyzer, and a detector system. The ion source is the part of the mass spectrometer that ionizes the target substance (analyte). The ions are then transported by magnetic or electric fields to the mass analyzer, where they are separated according to their mass-to-charge ratio (m / z). Many mass spectrometers use two or more mass analyzers for tandem mass spectrometry (MS / MS). The detector records the charge or current generated when ions pass through or impact a surface. A mass spectrum is the result of measuring the signal generated by a detector when m / z ions are scanned by a mass analyzer. However, in some embodiments, the present disclosure is directed to a method for analyzing glycan profiles without using labels or a mass spectrometer. Charged particle detection (CAD) is essentially a three-step process. First, the detector converts analyte molecules eluting from the column into dry particles. The number of particles increases proportionally to the amount of analyte. Second, a positively charged gas stream impinges on the analyte particles. Charge is then transferred to the particles, with larger particles carrying a greater charge. Finally, the particles are transported to a collector, where the charge is measured using a sensitive electrometer.

[0062] The disclosed methods are useful for releasing and separating N-linked glycans without derivatization. The present disclosure is directed to a method for quantifying the glycosylation profile of a recombinant protein, comprising analyzing one or more N-linked glycans without labeling, wherein the N-linked glycans are enzymatically released from the recombinant protein prior to analysis. In some embodiments, the methods described herein can provide one or more of glycoconjugate digestion and / or glycan release, fluorescent labeling, and glycan purification for subsequent analysis. Methods for analyzing compounds from biological sources often include a derivatization step to introduce a fluorophore that facilitates detection after chromatographic separation. In one embodiment, a label is used to tag the released glycans prior to analysis. In various embodiments of the reaction mixture, the N-glycans are attached to a label via the reducing end of the N-glycan. One type of label is a fluorophore, which is a molecule that absorbs light at one wavelength and emits at another wavelength. In some embodiments, the label is a fluorophore.

[0063] The disclosed methods are useful for releasing N-glycans attached to proteins at asparagine residues. In some embodiments, the enzyme comprises peptide N-glycosidase F (PNGase F). PNGase F is an enzyme generally derived from the bacterium Elizabethkingia miricola. It cleaves entire glycans from glycoproteins and requires that glycosylated asparagine moieties be substituted with polypeptide chains at their amino (R1) and carboxyl (R2) termini. The sequence of the PNGase F protein can be found at UniProt ID: P21163. Natural variants of the PNGase F enzyme are known in the art. For example, natural variants of PNGase F can contain one or more amino acid substitutions selected from D100N, E158Q, E246Q, or a combination thereof. The disclosed methods can also be achieved using functional fragments of the PNGase F enzyme to release N-glycans from asparagine residues.

[0064] The methods of the present disclosure also contemplate the use of other enzymes to release N-glycans from amino acids. In some embodiments, the enzyme can be endoglycosidase F1, endoglycosidase F2, endoglycosidase F3, endoglycosidase H, or a functional fragment thereof. In some embodiments, the enzyme is endoglycosidase F1. In some embodiments, the enzyme is endoglycosidase F2. In some embodiments, the enzyme is endoglycosidase F3. In some embodiments, the enzyme is endoglycosidase H. In other embodiments, two or more enzymes can be used to cleave N-glycans.

[0065] The disclosed methods are useful for further separating N-glycans from proteins and from each other. In some embodiments, the analysis involves separating one or more N-linked glycans during chromatography, including a column. In some embodiments, the column is a hydrophilic interaction liquid chromatography (HILIC)-UPLC column. In some embodiments, the column is a reversed-phase (RP)-UPLC column. Other examples of typical matrix types suitable for the columns of the disclosed methods are cation exchange resins, affinity resins, anion exchange resins, or mixed-mode resins. Other binding ligands can be attached to the chromatography resin to modify the binding properties of the column. Examples include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed-mode groups (combinations of the above). The disclosed methods can also be used to separate N-glycans using electrophoresis. Electrophoresis has been used to separate and analyze mixtures. Electrophoresis involves the migration and separation of molecules in an electric field based on differences in mobility. Various types of electrophoresis are known, including free zone electrophoresis, gel electrophoresis, isoelectric focusing, and capillary electrophoresis. Capillary electrophoresis (CE) is directed to the separation of free and bound labels. CE generally involves introducing a sample into a capillary tube. Sample components each have their own electrophoretic mobility, with faster-moving samples moving faster through the capillary tube than slower-moving samples. Thus, sample components are resolved into distinct zones within the capillary tube as they migrate through the capillary tube. In some embodiments, the electrophoresis is capillary electrophoresis. In some embodiments, the electrophoresis is gel electrophoresis.

[0066] The disclosed methods are useful for analyzing N-glycans after separation using chromatography involving a column. In some embodiments, the methods include using mixed-mode chromatography. Mixed-mode chromatography (MMC), or multimodal chromatography, refers to a chromatographic method that utilizes multiple modes of interaction between a stationary phase and an analyte to achieve its separation, which is distinct from traditional single-mode chromatography, which separates components based on one key attribute. In some embodiments, the column is a mixed-mode column. In some embodiments, the separation is performed using one or more mobile phases. In some embodiments, the column is a mixed-mode porous graphitic carbon (PGC) column. In some embodiments, the disclosure provides a method for measuring the glycosylation profile of a recombinant protein, comprising (i) releasing one or more N-linked glycans using an enzyme, e.g., PNGase F, and (ii) separating the N-linked glycans in the mobile phase of the mixed-mode chromatography. In some aspects, the present disclosure provides methods for quantifying the glycosylation profile of a recombinant protein, comprising: (i) releasing one or more N-linked glycans with an enzyme, e.g., PNGase F, (ii) separating the N-linked glycans in a mobile phase of mixed-mode chromatography, and (iii) measuring the separated N-linked glycans with a detector. In some aspects, the present disclosure provides methods for characterizing N-linked glycans of a recombinant protein, comprising: (i) releasing one or more N-linked glycans with an enzyme, e.g., PNGase F, (ii) separating the N-linked glycans by electrophoresis, and (iii) measuring the separated N-linked glycans with a detector.

[0067] In some aspects, the present disclosure provides methods for measuring the glycosylation profile of a recombinant protein, comprising: (i) releasing one or more N-linked glycans with an enzyme, e.g., PNGase F, and (ii) separating the N-linked glycans by electrophoresis. In some aspects, the present disclosure provides methods for quantifying the glycosylation profile of a recombinant protein, comprising: (i) releasing one or more N-linked glycans with an enzyme, e.g., PNGase F, (ii) separating the N-linked glycans by electrophoresis, and (iii) measuring the separated N-linked glycans with a detector. In some aspects, the present disclosure provides methods for characterizing N-linked glycans of a recombinant protein, comprising: (i) releasing one or more N-linked glycans with an enzyme, e.g., PNGase F, (ii) separating the N-linked glycans by electrophoresis, and (iii) measuring the separated N-linked glycans with a detector.

[0068] In some embodiments, the enzyme is incubated with the recombinant protein and one or more N-linked glycans are released from the recombinant protein prior to isolation. In some embodiments, the enzyme is diluted with a buffer. In some embodiments, the buffer is selected from the group consisting of phosphate, citrate, formate, acetate, and tris(hydroxymethyl)-aminomethane ("Tris"). In some embodiments, the buffer is phosphate. In some embodiments, the buffer is citrate. In some embodiments, the buffer is formate. In some embodiments, the buffer is acetate. In some embodiments, the buffer is Tris.

[0069] The disclosed methods are useful for analyzing N-linked glycans via mass spectrometry. One common mass spectrometry approach is the use of three mass analyzers in tandem, known as mass spectrometry / mass spectrometry (MS / MS). For example, in a triple quadrupole mass spectrometer experiment, the first quadrupole (Q1) is configured to pass only ions of a specific m / z (precursor ions) of the expected species in the sample. The second quadrupole (i.e., Q2 or collision cell) is used to fragment the ions that pass through Q1. The third quadrupole (Q3) is configured to pass only ions of a specific m / z (fragment ions) corresponding to the expected fragmentation products of the expected species through to the detector. One example of a mass spectrometer that can be used in the current process is an NQAD, which is a device used to monitor the mass / charge ratio of components passing through the detector as described above. In some embodiments, the separated N-linked glycans are measured by a mass spectrometer. In some embodiments, the separated N-linked glycans are characterized by a mass spectrometer. In some embodiments, the separated N-linked glycan or glycans are detected by mass spectrometry.

[0070] The disclosed method is also useful for analyzing N-linked glycans via an evaporative light scattering detector, or ELSD. An evaporative light scattering detector (ELSD) is a detector used in conjunction with high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography (UHPLC). An ELSD analyzes the solvent after elution from HPLC. The solvent eluted from HPLC is mixed with an inert carrier gas and passed through a nebulizer, breaking the liquid into tiny aerosolized droplets. The spray is then heated to evaporate the mobile phase, and the remaining target substance is exposed to light and its scattered light is detected. ELSD detectors are useful for identifying samples that cannot be identified via other common methods, such as ultraviolet (UV) detection, because the components do not absorb ultraviolet light. In some embodiments, one or more separated N-linked glycans are measured using an evaporative light scattering detector. In some embodiments, one or more separated N-linked glycans are characterized using an evaporative light scattering detector. In some embodiments, one or more separated N-linked glycans are detected using an evaporative light scattering detector.

[0071] The disclosed methods are also useful for analyzing N-linked glycans using a refractive index detector. A refractive index detector is a device used in conjunction with high-performance liquid chromatography (HPLC) or ultra-high-performance liquid chromatography (UHPLC). The device operates on a detection principle that involves measuring the change in refractive index of the eluate passing through a column, and can detect the difference in refractive index between the sample and the mobile phase. The refractive index detector has a flow cell with two sections, one for the sample and one for the reference solvent. The detector measures the refractive index of both components, essentially subtracting the background signal of the mobile phase from the sample signal. In some embodiments, one or more separated N-linked glycans are measured by the refractive index detector. In some embodiments, one or more separated N-linked glycans are characterized by the refractive index detector. In some embodiments, one or more separated N-linked glycans are detected by the refractive index detector.

[0072] The disclosed methods are also useful for analyzing N-linked glycans using a charged particle detector. Charged particle detectors refer to an analytical technique based on nebulizing an eluate with a nitrogen (or air) carrier gas to form droplets, which are then dried to remove the mobile phase and produce analyte particles. A primary stream of analyte particles contacts a positively charged secondary stream by passing through a high-voltage platinum corona wire. The charge is diffusively transferred to the opposing analyte particle stream, which then travels to a collector and is measured with a highly sensitive electrometer, generating a signal proportional to the amount of analyte present. In some embodiments, one or more separated N-linked glycans are measured by a charged particle detector (CAD). In other embodiments, one or more separated N-linked glycans are measured by a mass spectrometer, an ELSD, an NQAD, or a refractive index detector.

[0073] The disclosed method involves the separation of released glycans via chromatography, e.g., liquid chromatography (LC). LC is a well-established analytical technique for separating components of a fluid mixture for subsequent analysis and / or identification. LC involves packing a column, microfluidic chip-based channel, or tube with a stationary phase material, typically a finely divided solid or gel, such as small particles with a diameter of a few microns. The small particle size provides a large surface area, allowing the stationary phase to be modified with a variety of chemicals. A liquid eluent is pumped through a liquid chromatography column ("LC column") at a desired flow rate based on the column's dimensions and particle size. This liquid eluent is sometimes referred to as the mobile phase. The sample to be analyzed is introduced (e.g., injected) in small volumes into the mobile phase stream prior to the LC column. The migration rates of analytes in the sample are influenced by specific chemical and / or physical interactions with the stationary phase as they travel the length of the column. The time at which a particular analyte elutes or elutes from the end of the column, known as the retention time or elution time, allows for reasonable identification of the characteristics of a given analyte.

[0074] In some embodiments, the chromatography for separating N-linked glycans for the present methods comprises one or more mobile phases. In some embodiments, the chromatography comprises at least two mobile phases, at least three mobile phases, at least four mobile phases, or at least five mobile phases. In other embodiments, the chromatography comprises a first mobile phase and a second mobile phase. In other embodiments, the chromatography comprises a first mobile phase, a second mobile phase, and a third mobile phase. In other embodiments, the chromatography comprises a first mobile phase, a second mobile phase, a third mobile phase, or a fourth mobile phase. In other embodiments, the chromatography comprises a first mobile phase, a second mobile phase, a third mobile phase, a fourth mobile phase, or a fifth mobile phase. In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, and / or the fifth mobile phase are different.

[0075] In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase is selected from the group consisting of hexane, methanol, water, acetonitrile, ethyl acetate, benzene, chloroform, benzene, ether, or a mixture thereof. In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase is water. In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase is acetonitrile. In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase is ethyl acetate. In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase is benzene. In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase is chloroform. In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase is benzene. In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase is ether.

[0076] In some embodiments, the first mobile phase comprises water. In some embodiments, the first mobile phase comprises water and the second mobile phase comprises acetonitrile. In some embodiments, the first mobile phase is water and further comprises formic acid (FA), and the second mobile phase is acetonitrile. In some embodiments, the first mobile phase is water and the second mobile phase is acetonitrile and further comprises formic acid (FA). In some embodiments, the first mobile phase is water and further comprises formic acid (FA), and the second mobile phase is acetonitrile and further comprises formic acid (FA). In some embodiments, the first mobile phase is water and further comprises about 0.01% to about 1% formic acid (FA), and the second mobile phase is acetonitrile. In some embodiments, the first mobile phase is water and further comprises about 0.01% to about 1% formic acid (FA), and the second mobile phase is acetonitrile. In some embodiments, the first mobile phase is water and further comprises about 0.01% to about 1% formic acid (FA), and the second mobile phase is acetonitrile and further comprises about 0.01% to about 1% formic acid (FA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.01% formic acid (FA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.02% formic acid (FA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.03% formic acid (FA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.04% formic acid (FA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.05% formic acid (FA). In some embodiments, the first and second mobile phases comprise about 0.06% formic acid (FA). In some embodiments, the first and second mobile phases comprise about 0.07% (0.07) formic acid (FA). In some embodiments, the first and second mobile phases comprise about 0.08% formic acid (FA). In some embodiments, the first and second mobile phases comprise about 0.09% formic acid (FA). In some embodiments, the first and second mobile phases comprise about 0.1% formic acid (FA).

[0077] In some embodiments, the first mobile phase comprises water. In some embodiments, the first mobile phase comprises water, and the second mobile phase comprises acetonitrile. In some embodiments, the first mobile phase is water and further comprises trifluoroacetic acid (TFA), and the second mobile phase is acetonitrile. In some embodiments, the first mobile phase is water, and the second mobile phase is acetonitrile and further comprises trifluoroacetic acid (TFA). In some embodiments, the first mobile phase is water and further comprises trifluoroacetic acid (TFA), and the second mobile phase is acetonitrile and further comprises trifluoroacetic acid (TFA). In some embodiments, the first mobile phase is water and further comprises about 0.01% to about 1% trifluoroacetic acid (TFA), and the second mobile phase is acetonitrile. In some embodiments, the first mobile phase is water, and further comprises about 0.01% to about 1% trifluoroacetic acid (TFA), and the second mobile phase is acetonitrile. In some embodiments, the first mobile phase is water and further comprises about 0.01% to about 1% trifluoroacetic acid (TFA), and the second mobile phase is acetonitrile and further comprises about 0.01% to about 1% trifluoroacetic acid (TFA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.01% trifluoroacetic acid (TFA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.02% trifluoroacetic acid (TFA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.03% trifluoroacetic acid (TFA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.04% trifluoroacetic acid (TFA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.05% trifluoroacetic acid (TFA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.06% trifluoroacetic acid (TFA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.07% (0.07) trifluoroacetic acid (TFA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.08% trifluoroacetic acid (TFA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.09% trifluoroacetic acid (TFA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.1% trifluoroacetic acid (TFA).

[0078] In some embodiments, the first mobile phase comprises water. In some embodiments, the first mobile phase comprises water, and the second mobile phase comprises acetonitrile. In some embodiments, the first mobile phase is water and further comprises triethylamine (TEA), and the second mobile phase is acetonitrile. In some embodiments, the first mobile phase is water, and the second mobile phase is acetonitrile and further comprises triethylamine (TEA). In some embodiments, the first mobile phase is water and further comprises triethylamine (TEA), and the second mobile phase is acetonitrile and further comprises triethylamine (TEA). In some embodiments, the first mobile phase is water and further comprises about 0.01% to about 1% triethylamine (TEA), and the second mobile phase is acetonitrile. In some embodiments, the first mobile phase is water, and the second mobile phase is acetonitrile and further comprises about 0.01% to about 1% triethylamine (TEA). In some embodiments, the first mobile phase is water and further comprises about 0.01% to about 1% triethylamine (TEA), and the second mobile phase is acetonitrile and further comprises about 0.01% to about 1% triethylamine (TEA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.01% triethylamine (TEA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.02% triethylamine (TEA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.03% triethylamine (TEA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.04% triethylamine (TEA). In some embodiments, the first mobile phase and the second mobile phase comprise about 0.05% triethylamine (TEA). In some embodiments, the first and second mobile phases comprise about 0.06% triethylamine (TEA). In some embodiments, the first and second mobile phases comprise about 0.07% triethylamine (TEA). In some embodiments, the first and second mobile phases comprise about 0.08% triethylamine (TEA). In some embodiments, the first and second mobile phases comprise about 0.09% triethylamine (TEA). In some embodiments, the first and second mobile phases comprise about 0.1% triethylamine (TEA).

[0079] In some embodiments, the first mobile phase is water and further comprises triethylamine (TEA), and the second mobile phase is acetonitrile. In some embodiments, the first mobile phase is water and further comprises triethylamine (TEA), and the second mobile phase is acetonitrile and further comprises triethylamine (TEA). In some embodiments, the first mobile phase is water and further comprises about 0.05% to about 1.5% triethylamine (TEA), and the second mobile phase is acetonitrile, and the glycans S1G2, S2G2, and S2G2F, in any combination, have increased affinity for the chromatography column during separation compared to 0.1% formic acid in mobile phase A and / or mobile phase B. In some embodiments, the first mobile phase is water and further comprises triethylamine (TEA), and the second mobile phase is acetonitrile and further comprises about 0.05% to about 1.5% triethylamine (TEA), and the glycans S1G2, S2G2, S2G2F, in any combination, have increased affinity for the chromatographic column during separation compared to 0.1% formic acid in mobile phase A and / or mobile phase B. In some embodiments, the first mobile phase is water and further comprises about 0.05% to about 1.5% triethylamine (TEA), and the second mobile phase is acetonitrile and further comprises about 0.05% to about 1.5% triethylamine (TEA), and the glycans S1G2, S2G2, S2G2F, in any combination, have increased affinity for the chromatographic column during separation compared to 0.1% formic acid in mobile phase A and / or mobile phase B. In some embodiments, the first mobile phase comprises about 0.1% trimethylamine (TEA) in water, and the second mobile phase comprises about 0.1% triethylamine (TEA) in acetonitrile. In some embodiments, the first mobile phase comprises about 0.05% triethylamine (TEA). In some embodiments, the first mobile phase comprises about 0.06% triethylamine (TEA). In some embodiments, the first mobile phase comprises about 0.07% triethylamine (TEA). In some embodiments, the first mobile phase comprises about 0.08% triethylamine (TEA). In some embodiments, the first mobile phase comprises about 0.09% triethylamine (TEA). In some embodiments, the first mobile phase comprises about 0.1% triethylamine (TEA).In some embodiments, the first mobile phase comprises about 0.11% triethylamine (TEA). In some embodiments, the first mobile phase comprises about 0.12% triethylamine (TEA). In some embodiments, the first mobile phase comprises about 0.13% triethylamine (TEA). In some embodiments, the first mobile phase comprises about 0.14% triethylamine (TEA). In some embodiments, the first mobile phase comprises about 0.15% triethylamine (TEA). In some embodiments, the second mobile phase comprises about 0.05% triethylamine (TEA). In some embodiments, the second mobile phase comprises about 0.06% triethylamine (TEA). In some embodiments, the second mobile phase comprises about 0.07% triethylamine (TEA). In some embodiments, the second mobile phase comprises about 0.08% triethylamine (TEA). In some embodiments, the second mobile phase comprises about 0.09% triethylamine (TEA). In some embodiments, the second mobile phase comprises about 0.10% triethylamine (TEA). In some embodiments, the second mobile phase comprises about 0.11% triethylamine (TEA). In some embodiments, the second mobile phase comprises about 0.12% triethylamine (TEA). In some embodiments, the second mobile phase comprises about 0.13% triethylamine (TEA). In some embodiments, the second mobile phase comprises about 0.14% triethylamine (TEA). In some embodiments, the second mobile phase comprises about 0.15% triethylamine (TEA).

[0080] In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase comprises formic acid (FA), trifluoroacetic acid (TFA), triethylamine (TEA), or any combination thereof. In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase comprises about 0.01% to about 2% formic acid (FA), trifluoroacetic acid (TFA), triethylamine (TEA), or any combination thereof.

[0081] In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase comprises about 0.01% to about 1% or about 1% to about 2% formic acid (FA), e.g., 0.1% FA. In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase comprises about 0.01% to about 1% or about 1% to about 2% trifluoroacetic acid (TFA), e.g., 0.1% TFA. In some embodiments, the first mobile phase, the second mobile phase, the third mobile phase, the fourth mobile phase, or the fifth mobile phase comprises about 0.01% to about 1% or about 1% to about 2% triethylamine (TEA), e.g., 0.1% TEA, and the glycans S1G2, S2G2, and / or S2G2F have increased affinity for the chromatographic column during separation compared to 0.1% formic acid in mobile phase A and / or mobile phase B.

[0082] In some embodiments, the methods of the present disclosure use one or more agents for reductive amination to improve peak symmetry and reduce peak tailing during chromatographic separation. In some embodiments, the one or more agents include 2-picoline borane (pic-BH) and / or sodium cyanoborohydride (NaBHCN).

[0083] The method of the present disclosure is also useful for separating N-glycans using chromatography. Chromatographic separation can be improved by adjusting the temperature of the chromatography column during separation. In some embodiments, the separation is performed at a temperature below 70°C. In some embodiments, the separation is performed at a temperature below 60°C. In some embodiments, the separation is performed at a temperature below 50°C. In some embodiments, the separation is performed at a temperature below 40°C.

[0084] In some embodiments, the temperature is between about 50°C and about 70°C, between about 50°C and about 60°C, between about 60°C and about 70°C, between about 55°C and about 65°C, between about 55°C and about 60°C, between about 60°C and about 65°C, between about 65°C and about 70°C, between about 50°C and about 55°C, between about 50°C and about 60°C, between about 50°C and about 59°C, between about 51°C and about 59°C, between about 51°C and about 58°C, between about 52°C and about 58°C, between about 52°C and about 57°C, between about 53°C and about 57°C, between about 53°C and about 56°C, and between about 54°C and about 56°C.

[0085] In some embodiments, the temperature is between about 50°C and about 70°C. In some embodiments, the temperature is between about 50°C and about 60°C. In some embodiments, the temperature is between about 60°C and about 70°C. In some embodiments, the temperature is between about 55°C and about 65°C. In some embodiments, the temperature is between about 55°C and about 60°C. In some embodiments, the temperature is between about 60°C and about 65°C. In some embodiments, the temperature is between about 65°C and about 70°C. In some embodiments, the temperature is between about 50°C and about 55°C. In some embodiments, the temperature is between about 50°C and about 60°C. In some embodiments, the temperature is between about 50°C and about 59°C. In some embodiments, the temperature is between about 51°C and about 59°C. In some embodiments, the temperature is between about 51°C and about 58°C. In some embodiments, the temperature is between about 52°C and about 58°C. In some embodiments, the temperature is between about 52°C and about 57°C. In some embodiments, the temperature is between about 53° C. and about 57° C. In some embodiments, the temperature is between about 53° C. and about 56° C. In some embodiments, the temperature is between about 54° C. and about 56° C.

[0086] In some embodiments, the temperature is between about 45° C. and about 55° C., between about 45° C. and about 54° C., between about 46° C. and about 54° C., between about 46° C. and about 53° C., between about 47° C. and about 53° C., between about 47° C. and about 52° C., between about 48° C. and about 52° C., between about 49° C. and about 52° C., or between about 49° C. and about 51° C. In some embodiments, the temperature is between about 55° C. and about 65° C., between about 55° C. and about 64° C., between about 56° C. and about 64° C., between about 56° C. and about 63° C., between about 57° C. and about 63° C., between about 57° C. and about 62° C., between about 58° C. and about 62° C., between about 59° C. and about 62° C., or between about 59° C. and about 61° C.

[0087] In some embodiments, the temperature is about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, or about 69°C.

[0088] In some embodiments, the temperature is between about 45°C and about 55°C. In some embodiments, the temperature is between about 45°C and about 54°C. In some embodiments, the temperature is between about 46°C and about 54°C. In some embodiments, the temperature is between about 46°C and about 53°C. In some embodiments, the temperature is between about 47°C and about 53°C. In some embodiments, the temperature is between about 47°C and about 52°C. In some embodiments, the temperature is between about 48°C and about 52°C. In some embodiments, the temperature is between about 49°C and about 52°C. In some embodiments, the temperature is between about 49°C and about 51°C. In some embodiments, the temperature is between about 55°C and about 65°C. In some embodiments, the temperature is between about 55°C and about 64°C. In some embodiments, the temperature is between about 56°C and about 64°C. In some embodiments, the temperature is between about 56°C and about 63°C. In some embodiments, the temperature is between about 57°C and about 63°C. In some embodiments, the temperature is between about 57° C. and about 62° C. In some embodiments, the temperature is between about 58° C. and about 62° C. In some embodiments, the temperature is between about 59° C. and about 62° C. In some embodiments, the temperature is between about 59° C. and about 61° C.

[0089] The disclosed methods are also useful for separating N-linked glycans using chromatography. Gradients in reversed-phase HPLC typically involve online (dynamic) mixing of solvents (mobile phases), achieving a steady increase in one solvent over the course of the separation, which helps to change the elution strength of the eluate over time. In some embodiments, the separation is based on a gradient. In some embodiments, the separation is based on a gradient of one or more mobile phases. In some embodiments, the gradient is from a first mobile phase to a second mobile phase. In some embodiments, the gradient is from a first mobile phase to a second mobile phase to a third mobile phase. In some embodiments, the gradient is from a first mobile phase to a second mobile phase to a third mobile phase to a fourth mobile phase. In some embodiments, the gradient is from a first mobile phase to a second mobile phase to a third mobile phase to a fifth mobile phase. In some embodiments, the gradient is from about 95% to about 5%. In some embodiments, the gradient is from about 95% to about 50%, from about 95% to about 55%, from about 95% to about 60%, from about 95% to about 65%, from about 95% to about 70%, from about 95% to about 75%, from about 95% to about 80%, from about 95% to about 85%, from about 90% to about 50%, from about 90% to about 55%, from about 90% to about 60%, from about 90% to about 65%, from about 90% to about 70%, about 90% to about 75%, about 87% to about 50%, about 87% to about 55%, about 87% to about 60%, about 87% to about 65%, about 87% to about 70%, about 87% to about 75%, about 85% to about 50%, about 85% to about 55%, about 85% to about 60%, about 85% to about 65%, about 85% to about 70%, or about 85% to about 75%. In some embodiments, the gradient is about 87% to about 75%.

[0090] In some embodiments, the gradient is from about 95% to about 50%. In some embodiments, the gradient is from about 95% to about 51%. In some embodiments, the gradient is from about 95% to about 52%. In some embodiments, the gradient is from about 95% to about 53%. In some embodiments, the gradient is from about 95% to about 54%. In some embodiments, the gradient is from about 95% to about 55%. In some embodiments, the gradient is from about 95% to about 56%. In some embodiments, the gradient is from about 95% to about 57%. In some embodiments, the gradient is from about 95% to about 58%. In some embodiments, the gradient is from about 95% to about 59%. In some embodiments, the gradient is from about 95% to about 60%. In some embodiments, the gradient is from about 90% to about 74%. In some embodiments, the gradient is from about 90% to about 75%. In some embodiments, the gradient is from about 90% to about 76%. In some embodiments, the gradient is from about 90% to about 77%. In some embodiments, the gradient is from about 90% to about 78%. In some embodiments, the gradient is from about 90% to about 79%. In some embodiments, the gradient is from about 90% to about 80%. In some embodiments, the gradient is from about 90% to about 81%. In some embodiments, the gradient is from about 90% to about 82%. In some embodiments, the gradient is from about 90% to about 83%. In some embodiments, the gradient is from about 90% to about 84%. In some embodiments, the gradient is from about 90% to about 85%. In some embodiments, the gradient is from about 87% to about 50%. In some embodiments, the gradient is from about 87% to about 51%. In some embodiments, the gradient is from about 87% to about 52%. In some embodiments, the gradient is from about 87% to about 53%. In some embodiments, the gradient is from about 87% to about 54%. In some embodiments, the gradient is from about 87% to about 55%. In some embodiments, the gradient is from about 87% to about 56%. In some embodiments, the gradient is from about 87% to about 57%. In some embodiments, the gradient is from about 87% to about 58%. In some embodiments, the gradient is from about 87% to about 59%. In some embodiments, the gradient is from about 87% to about 60%. In some embodiments, the gradient is from about 87% to about 61%. In some embodiments, the gradient is from about 87% to about 62%. In some embodiments, the gradient is from about 87% to about 63%. In some embodiments, the gradient is from about 87% to about 64%. In some embodiments, the gradient is from about 87% to about 65%. In some embodiments, the gradient is from about 87% to about 66%.In some embodiments, the gradient is from about 87% to about 67%. In some embodiments, the gradient is from about 87% to about 68%. In some embodiments, the gradient is from about 87% to about 69%. In some embodiments, the gradient is from about 87% to about 70%. In some embodiments, the gradient is from about 87% to about 71%. In some embodiments, the gradient is from about 87% to about 72%. In some embodiments, the gradient is from about 87% to about 73%. In some embodiments, the gradient is from about 87% to about 74%. In some embodiments, the gradient is from about 87% to about 75%.

[0091] III. Methods of Glycan Analysis The methods of the present disclosure are useful for analyzing released and separated N-linked glycans, as described above. N-linked glycosylation is the attachment of oligosaccharides, sometimes called glycans, to nitrogen atoms (the amide nitrogens of asparagine (Asn) residues in proteins) in a process called N-glycosylation. The present disclosure is also directed to methods for analyzing the N-linked glycan profile of a protein of interest. In some embodiments, one or more N-linked glycans are galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (Glc), N-acetylglucosamine (GlcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), fucose (Fuc), glucuronic acid (GlcA), iduronic acid (IdoA), galacturonic acid (GalA), mannuronic acid (ManA), or any combination thereof. In some embodiments, the N-glycan is Gal. In some embodiments, the N-glycan is GalNac. In some embodiments, the N-glycan is GalN. In some embodiments, the N-glycan is Glc. In some embodiments, the N-glycan is GlcNAc. In some embodiments, the N-glycan is GlcN. In some embodiments, the N-glycan is Man. In some embodiments, the N-glycan is ManNAc. In some embodiments, the N-glycan is ManN. In some embodiments, the N-glycan is Xyl. In some embodiments, the N-glycan is Fuc. In some embodiments, the N-glycan is GlcA. In some embodiments, the N-glycan is IdoA. In some embodiments, the N-glycan is GalA. In some embodiments, the N-glycan is ManA. In some embodiments, one or more N-glycans comprise one or more biantennary glycans. In some embodiments, the biantennary glycan is selected from the group consisting of GOF, GO ... In some embodiments, the biantennary glycan is G1F or G1.In some embodiments, the biantennary glycan is G2F or G2. In some embodiments, the biantennary glycan is S1G2F or S1G2. In some embodiments, the biantennary glycan is S2G2F or S2G2.

[0092] In some embodiments, one or more N-linked glycans are high mannose glycans. High mannose glycans contain unsubstituted terminal mannose sugars. These glycans typically contain 5 to 9 mannose residues attached to a chitobiose (GlcNAc2) core. In some embodiments, one or more high mannose N-linked glycans are mannose-5. In some embodiments, one or more high mannose N-linked glycans are mannose-6. In some embodiments, one or more high mannose N-linked glycans are mannose-7. In some embodiments, one or more high mannose N-linked glycans are mannose-8. In some embodiments, one or more high mannose N-linked glycans are mannose-9.

[0093] High-mannose N-linked glycans can also be phosphorylated. Mannose-6-phosphate (M6P) is an important signal for the transport of lysosomal enzymes to lysosomes. Many lysosomal enzymes are glycoproteins with high-mannose and complex glycans. For example, FABRAZYME™ is an example of a lysosomal protein, α-galactosidase A. The ability of FABRAZYME™ to be taken up by cells and subsequently transported to lysosomes is due to the presence of mannose 6-phosphate (M6P) on its N-linked glycan; FABRAZYME™ binds to the mannose 6-phosphate / IGF-II receptor, which is present on the cell surface of most cell types, and is delivered to lysosomes. In some embodiments, one or more mannose residues of the high-mannose N-linked glycan are phosphorylated.

[0094] The disclosed methods are also useful for separating sialylated glycans. Sialylation is the process by which a sialic acid group is introduced as a terminal monosaccharide onto molecules such as oligosaccharides and carbohydrates. Two common mammalian sialic acids are N-acetylneuraminic acid (Neu5Ac or NANA) and N-glycolylneuraminic acid (Neu5Gc or NGNA). In some embodiments, the sialic acid is Neu5Ac. In some embodiments, the sialic acid is Neu5Gc. In some embodiments, the sialic acid is 2-keto-3-deoxynonanoic acid (Kdn). In some embodiments, the glycosylation profile includes one or more nonsialylated glycans, monosialylated glycans, disialylated glycans, and / or trisialylated and tetrasialylated glycans. In some embodiments, the glycosylation profile includes one or more nonsialylated glycans. In some embodiments, the glycosylation profile comprises one or more monosialylated glycans. In some embodiments, the glycosylation profile comprises one or more disialylated glycans. In some embodiments, the glycosylation profile comprises one or more trisialylated glycans. In some embodiments, the glycosylation profile comprises one or more tetrasialylated glycans.

[0095] The disclosed methods are useful for characterizing the glycosylation profile of a protein. In some embodiments, the protein is a recombinant protein. In some embodiments, the recombinant protein has a purity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

[0096] In some embodiments, the recombinant protein is a fusion protein. In some embodiments, the fusion protein is an anti-myostatin fusion protein. In some embodiments, the fusion protein is talditercept alfa. In other embodiments, the fusion protein is an Fc fusion protein. In some embodiments, the fusion protein is an Fc fusion protein homodimer. In some embodiments, the fusion protein comprises a CTLA-4 extracellular fusion protein. In some embodiments, the CTLA-4Fc fusion protein is abatacept. In other embodiments, the CTLA-4Fc fusion protein is belatacept. CTLA4-Ig molecules, their uses, and methods are also described in U.S. Patent Nos. 5,434,131, 5,851,795, 5,885,796, 5,885,579, and 7,094,874, the entire contents of which are incorporated herein by reference. In some embodiments, the CTLA-4-Fc fusion protein comprises at least one N-linked glycan, at least two N-linked glycans, or at least three N-linked glycans, e.g., T5, T7, and T15. As used herein, "T5," "T7," and "T15" refer to specific glycosylation sites present on the abatacept or belatacept molecule. These tags correspond to asparagine 76, asparagine 108, and asparagine 207, respectively, which correspond to residues in SEQ ID NO:3 and SEQ ID NO:5 (bold). SEQ ID NO: 1 [CTLA4 extracellular domain sequence] MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD Abatacept (SEQ ID NO: 3) [amino acids 27-383 of SEQ ID NO: 2] MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Belatacept (SEQ ID NO: 5) [amino acids 27-383 of SEQ ID NO: 4] MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0097] In some embodiments, the recombinant protein is an antibody. In some embodiments, the antibody is an isotype selected from IgM, IgA, IgE, IgD, and IgG. In some embodiments, the antibody is an isotype IgM. In some embodiments, the antibody is an isotype IgA. In some embodiments, the antibody is an isotype IgE. In some embodiments, the antibody is an isotype IgD. In some embodiments, the antibody is an isotype IgG. In some embodiments, the IgG antibody is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the antibody is bispecific. In some embodiments, the antibody is multispecific.

[0098] The disclosed methods are useful for analyzing antibodies useful for therapeutic purposes. In some embodiments, the antibody is an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody, an anti-CSF1R antibody, or an anti-IL8 antibody. In some embodiments, the antibody has a single N-linked glycosylation site. In some embodiments, the single N-linked glycosylation site is asparagine 297 (N297). In some embodiments, the antibody has at least one N-linked glycosylation site, at least two N-linked glycosylation sites, at least three N-linked glycosylation sites, at least four N-linked glycosylation sites, or at least five N-linked glycosylation sites.

[0099] The disclosed methods are also useful for analyzing N-linked glycans of other recombinant proteins. In some embodiments, the recombinant protein comprises an enzyme, a hormone, a cytokine, a cell surface receptor, a protease, a cytokine receptor, or any combination thereof. In some embodiments, the recombinant protein is a fusion protein. In some embodiments, the fusion protein is fused to a heterologous moiety. In some embodiments, the heterologous moiety is a half-life extending moiety. In some embodiments, the half-life extending moiety comprises albumin, an albumin-binding polypeptide, a fatty acid, PAS, the β subunit of the C-terminal peptide (CTP) of human chorionic gonadotropin, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN, an albumin-binding small molecule, Fc, or a combination thereof. In some embodiments, the half-life extending moiety is Fc.

[0100] In some aspects, the methods of the invention are useful for analyzing N-linked glycans of fusion proteins, such as anti-PD-L1 / TGFβR2 bispecific fusion proteins. See Jochems et al., Oncotarget. 2017 Sep 26; 8(43): 75217-75231.

[0101] In some embodiments, the methods of the present invention are useful for analyzing BsAb-HAS (human serum albumin) fusion proteins. Various BsAbs include, but are not limited to, BsAb fragments such as scFv-HSA-scFv, scDiabody-HSA, and tandem scFv-HSA.

[0102] In some embodiments, the methods of the present invention are useful for analyzing BsAb-toxin fusion proteins. BsAb-toxin fusion proteins are an extended version of immunotoxins, in which an antibody moiety is fused with a protein toxin that acts as a cytotoxic moiety. Among them, tandem scFv-toxins are the most frequently reported type and are known as dual-specific ligand-directed toxins (BLTs). Typically, catalytic toxins such as Pseudomonas exotoxin (PE) and diphtheria toxin (DT) are selected as the cytotoxic moiety.

[0103] The method of the present disclosure is also useful for biomanufacturing analysis and batch release analysis. Batch release testing is generally required by Good Manufacturing Practice (GMP) and is a necessary requirement for ensuring high quality pharmaceuticals and biopharmaceuticals before release for sale, supply, or export. In some embodiments, the method is a batch release assay.

[0104] The present disclosure is further illustrated by the following examples, which should in no way be construed as further limiting. The contents of all references cited throughout this application are expressly incorporated herein by reference. [Example]

[0105] [Example 1] Reagents, Columns and Systems Recombinant PNGase F and Rapid PNGase F were purchased from New England Biolabs, Inc. (Ipswich, MA). Glycan standards were purchased from Prozyme (Hayward, CA). Mobile phase water and acetonitrile were purchased from JT Baker via Fisher Scientific (Hampton, NH). All mobile phase additives (e.g., formic acid, trifluoroacetic acid, triethylamine) were purchased from Sigma-Aldrich (St. Louis, MO). AdvanceBio N-Glycan Cleanup Cartridges were purchased from Agilent Technologies (Santa Clara, CA).

[0106] Various columns were screened during development, including HILIC, reversed-phase, and porous graphite carbon separations. For HILIC separations, XBridge Glycan BEH Amide™ 130 Å, 2.5 μm (2.1 × 150 mm) and Acquity UPLC Glycan BEH Amide™ 130 Å, 1.7 μm (2.1 × 150 mm) columns were purchased from Waters Corporation (Milford, MA). Additionally, a PolyGLYCOPLEX™ A 3 μm (2.1 × 100 mm) column was purchased from PolyLC Inc. (Columbia, MD), and a TSKGel Amide-80 2 μm (2.0 × 150 mm) column was purchased from Tosoh Biosciences LLC (Tokyo, Japan). For reversed-phase evaluation, an XBridge BEH C18 2.5 μm (3.0 × 150 mm) column, an Acquity UPLC Peptide CSH C18 130 Å, 1.7 μm (2.1 × 100 mm) column, and a CORTECS UPLC C18+™, 1.6 μm (2.1 × 100 mm) column were all purchased from Waters Corporation. A HYPERCARB™ 3 μm (2.1 × 100 mm) column was purchased from Thermo Scientific™ (Waltham, MA). All chromatographic separations were performed on an Alliance 2695 HPLC™ system or an Acquity H-Class UPLC™ system interfaced with a Thermo Scientific™ CORONA™ VEO™ charged aerosol detector system for glycan detection.

[0107] Optimal release of oligosaccharides Oligosaccharides were released from mAb samples by diluting 1 mg of mAb with 10 μL Rapid PNGase F buffer and 20 mM Tris buffer, pH 7.5, in a final volume of 180 μL. The sample was then briefly vortexed, centrifuged, and the supernatant collected. Rapid PNGase F was diluted 1:10 with HPLC water, and 20 μL of this solution was added to each reaction vial, vortexed, and centrifuged. The Rapid PNGase F solution was incubated at 50°C for 60 minutes and then cooled to room temperature. To remove protein and buffer components, oligosaccharides were extracted using Agilent AdvanceBio N-Glycan Deglycosylation Cleanup Cartridges™. The cartridges were first equilibrated twice with 500 μL water and then twice with 500 μL of 85% acetonitrile, and the vacuum was set to -0.05 bar. The sample was then diluted 1:5 with acetonitrile (to 80% ACN) and loaded onto the pre-equilibrated cartridge. The solution was drawn through the cartridge, and the cartridge was washed twice with 600 μL of a 1:9:90 formic acid:water:acetonitrile solution. The purified oligosaccharides were then collected using a new collection vial. The oligosaccharides were eluted from the cartridge by washing twice with 50 μL of water. The flow-through fraction was lyophilized using a vacuum concentrator, and the sample was reconstituted with 25 μL of 5% acetonitrile containing 0.1% formic acid in water and transferred to a vial suitable for LC analysis.

[0108] Optimal separation of unlabeled oligosaccharides The purified oligosaccharides were separated using a porous graphite carbon column (HYPERCARB™ 2.1 x 100 mm, 3 μm, THERMO SCIENTIFIC™) connected to a Waters Acquity UPLC™ system. Mobile phases A and B consisted of 0.1% formic acid and 100% acetonitrile, respectively. The column temperature was set at 60°C, and the flow rate was maintained at 0.3 mL / min. Samples were injected at 95% A (5 μL volume), and a gradient from 87% A to 78% A was applied over 10 min, resulting in a total run time of 30 min, including equilibration. The charged aerosol detector settings were: evaporation temperature 50°C, power function 1, data acquisition rate 25 Hz, and filter constant 10 s.

[0109] Assessment of the degree of deglycosylation To assess the extent of deglycosylation, a time course study was performed on three different monoclonal antibodies of different IgG subclasses: mAbA, mAbB, and mAbC. Samples were prepared using an optimized procedure, with time points taken at t = 0, 5, 60, 240, and 1440 min. For the non-rapid PNGase F treatment, samples were prepared identically to their rapid counterparts, except that the non-rapid PNGase F reaction was performed at 37°C for 1440 min. These samples were then evaluated using reduced CE-SDS and an optimized chromatographic method to assess the extent of deglycosylation, confirming that 60 min was sufficient for deglycosylation of these molecules.

[0110] Chromatography optimization Historically, glycan separations have been performed using hydrophilic interaction liquid chromatography (HILIC) because glycans are polar compounds that bind to amides present on the resin surface under high organic concentrations and can be eluted with increasing aqueous concentrations (Veillon et al., 2017). Typically, these separations have been performed using purified glycans labeled with fluorescent tags such as 2-AA or 2-AB, which affect binding and separation. To understand the interaction between unlabeled glycans and the column, two HILIC HPLC columns (XBridge Glycan BEH Amide and PolyGLYCOPLEX A) were evaluated as a starting point. Mobile phase A was 100% acetonitrile, and mobile phase B was 50 mM ammonium formate, pH 4.4, providing a typical mobile phase composition for HILIC separations. Both columns were evaluated using Prozyme's unlabeled glycan standard library. Four major glycans contained in the standard (G0F, G1F, G2F, and Man5) were observed on both columns, but the low resolution and weak signal limited the development potential of these columns. On both columns, a shoulder was observed upon injection of the individual standards, which had not previously been observed using fluorescently labeled standards.

[0111] In the next step, a UPLC-based HILIC column was evaluated to determine whether it could improve resolution and peak shape. UPLC HILIC columns (Acquity Glycan BEH Amide column and TSKgel Amide column) were screened under the same conditions as the HPLC experiments. Superior separations were obtained with the UPLC system and BEH column due to their higher resolution. Interestingly, a shoulder was still observed for each individual glycan standard, which was unexpected at the start of the study. The interaction of polar glycan analytes with the HILIC stationary phase was further analyzed. When using HILIC for analysis, the released glycan is typically tagged with a fluorescent label at the free reducing end. However, in this case, without the tag, this reducing end is exposed, allowing for anomeric equilibrium (ref). Because these two species of the same glycan interact differently with the stationary phase depending on whether they are in the closed or open conformation, we can conclude that the shoulder represents the anomeric form of the same glycan. After evaluating the available literature, we determined that adding triethylamine to the mobile phase could increase the pH of the solution and shift the equilibrium to one side (ref). However, when TEA was used in the HILIC mobile phase, the glycans did not bind to the column because the pH adjustment was outside the functional range of the HILIC column. This led us to evaluate reversed-phase as an alternative, as the glycans become nonpolar in the presence of TEA.

[0112] Four C18 columns were evaluated without success (Peptide CSH, Acquity BEH, CORTECS C18+, and Kinetex C18). This was attributed to the fact that glycans cannot bind as effectively to the C18 stationary phase as proteins or peptides, and these columns are marketed for protein-based applications. A new column from Thermo Scientific, the porous graphitic carbon (PGC) Hypercarb™ column, was evaluated. Unlike typical reversed-phase columns, this column's stationary phase consists of carbon atoms arranged in a flat hexagon, which can promote interactions between planar compounds (such as glycans). PGC is superior for isomeric separation of glycans compared to any other stationary phase (Song et al., 2015). Initial screening, following the manufacturer's mobile phase recommendations, yielded promising results, as a glycan standard library showed good resolution and signal intensity for all peaks when screened with a very broad gradient (95% to 5%) of mobile phase A (0.1% TFA in water) and mobile phase B (acetonitrile). Narrowing the gradient resulted in better separation and no shoulder peaks were observed. The absence of shoulders in these glycan peaks is likely due to a different mode of interaction, where the free reducing ends of the sugars do not interact significantly with the stationary phase as they do in HILIC separations.

[0113] Next, we optimized the mobile phase and separation conditions. Specifically, we evaluated the ion-pairing reagents in the mobile phase. Initially, trifluoroacetic acid (TFA) and triethylamine (TEA) were evaluated as ion-pairing reagents in the mobile phase. Separations were performed using the same gradient with mobile phase A supplemented with 0.1% TFA or 0.1% TEA. Higher resolution and better peak shape were observed with 0.1% TFA compared to 0.1% TEA. Furthermore, the chromatograms with TFA added showed additional glycan peaks (which were not observed in the mobile phase or assay blank injections). Since adding 0.1% TFA to both mobile phases did not significantly improve separation compared to adding it to mobile phase A, we changed the mobile phase composition to 0.1% TFA in water for mobile phase A and 100% acetonitrile for mobile phase B. Column temperatures of 60 and 70 °C were evaluated, and it was determined that 70 °C provided better separation under these conditions. Finally, the gradient was optimized by comparing 95% to 75% A or 95% to 87%, then to 75% A. The second gradient evaluated provided better resolution between minor peaks and was therefore designated the optimal gradient.

[0114] Reoptimizing the separation for MS compatibility Because the glycans were unlabeled, a method suitable for mass spectrometry was developed. Five parameters were then optimized: mobile phase composition (MPA and MPB), column temperature, separation time, and sample preparation and injection volume. Because TFA is known to suppress mass spectral signals, it was desirable to evaluate formic acid as an ion-pairing reagent instead of TFA. This provided an opportunity to redevelop the assay to further improve separation performance and throughput. A preliminary comparison of mobile phase A (0.1% TFA or 0.1% FA) yielded comparable results, so further improvements in separation efficiency were pursued. Next, the addition of 0.1% FA to mobile phase B (acetonitrile) was evaluated. A more stable baseline was observed, as judged by less baseline drift, and the addition of formic acid resulted in better peak separation in both mobile phases. Based on these results, it was concluded that the addition of 0.1% formic acid to both mobile phase A (water) and mobile phase B (acetonitrile) was desirable. The next parameter optimized was column temperature, with separations performed at 50, 60, 70, and 80 °C. The results showed that temperatures of 70°C and 80°C resulted in significantly poorer resolution than temperatures of 50°C and 60°C. At higher temperatures, many peaks tended to overlap rather than be resolved. After examining peaks near the baseline, 60°C was determined to provide the highest peak resolution across the entire chromatogram and was selected as the optimal column temperature. The next parameter evaluated was the gradient run time from 87% to 78% mobile phase A, selecting 10, 20, and 30 minutes. The results strongly indicated that a 10-minute gradient time provided significantly better separation than longer gradients. A 10-minute gradient time resolved more peaks than longer run times (Figure 1).

[0115] The final parameter we optimized was the deglycosylation conditions. The manufacturer recommends deglycosylating 100 μg of protein with 1 μL of Rapid PNGase F at 50°C for 5 minutes. Initial evaluation indicated that this was insufficient to obtain a signal using CAD. After preliminary optimization, 1 mg of protein was digested with 20 μL of a 1:10 diluted Rapid PNGase F for 60 minutes. To assess the extent of deglycosylation, we set up a 60-minute reaction and a 24-hour non-rapid PNGase F control. These reactions were then analyzed by CE-SDS (reduced with a non-deglycosylated sample control) and the optimized glycan method. The CE-SDS data indicated that after 60 minutes, >98% of the protein was deglycosylated, as judged by the disappearance of the main non-reduced peak, the disappearance of the reduced heavy chain peak, and the formation of the main non-glycosylated heavy chain peak in each electropherogram. These findings were supported by the glycan separation, where signal intensity reached a plateau after 60 minutes. Because deglycosylation was complete after 60 minutes in both CE-SDS and chromatography, 60 minutes was chosen to maximize deglycosylation.

[0116] [Example 2] Oligosaccharides were released from mAb samples and analyzed according to Example 1, with the following changes to the mobile phase conditions: Mobile phase A: 0.1% triethylamine (TEA) in water; and Mobile phase B: 0.1% triethylamine (TEA) in acetonitrile (Figure 2). Other experiments were repeated as described in Example 1. A sample glycan chromatogram generated from the separation analysis is shown in Figure 2.

[0117] Throughout this application, various publications are referenced by author name and date in parentheses, or by patent or patent publication number. The disclosures of these publications in their entireties are incorporated herein by reference into this application in order to more fully describe the state of the art known to those skilled in the art as of the date of the present disclosure as described and claimed herein. However, the citation of a reference herein should not be construed as an admission that the reference is prior art to the present disclosure.

Claims

1. A method for quantifying the glycosylation profile of a recombinant protein, comprising label-free analysis of one or more N-linked glycans, wherein the N-linked glycans are enzymatically released from the recombinant protein prior to analysis.

2. A method for quantifying the glycosylation profile of a recombinant protein, comprising analyzing one or more N-linked glycans without a fluorophore, wherein the N-linked glycans are enzymatically released from the recombinant protein prior to analysis.

3. 3. The method of claim 1 or 2, wherein the recombinant protein has a purity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

4. 4. The method of any one of claims 1 to 3, wherein the analysis comprises separating one or more N-linked glycans during chromatography comprising a column.

5. The method of claim 4, wherein the column is a mixed-mode column.

6. 6. The method of claim 4 or 5, wherein the separation is carried out using one or more mobile phases.

7. 7. The method of any one of claims 4 to 6, wherein the column is a mixed-mode porous graphitic carbon (PGC) column.

8. 8. The method of any one of claims 1 to 7, wherein the enzyme comprises peptide N-glycosidase F (PNGase F).

9. 9. The method of claim 8, wherein an enzyme is incubated with the recombinant protein and one or more N-linked glycans are liberated from the recombinant protein prior to isolation.

10. 10. The method of claim 9, wherein the enzyme is diluted in a buffer.

11. 11. The method of any one of claims 4 to 10, wherein the separated N-linked glycan or glycans are measured by a mass spectrometer, an ELSD, an NQAD, or a refractive index detector.

12. 12. The method of claim 11, wherein the separated N-linked glycan or glycans are measured by a charged particle detector (CAD).

13. 13. The method of any one of claims 4 to 12, wherein the chromatography comprises a first mobile phase and a second mobile phase, the first mobile phase and the second mobile phase being different.

14. 14. The method of claim 13, wherein the first mobile phase comprises water.

15. 15. The method of claim 13 or 14, wherein the second mobile phase comprises acetonitrile.

16. 16. The method of any one of claims 13 to 15, wherein the first mobile phase comprises formic acid (FA), trifluoroacetic acid (TFA), triethylamine (TEA), or any combination thereof.

17. 17. The method of claim 16, wherein the first mobile phase comprises 0.1% FA.

18. 18. The method of any one of claims 13 to 17, wherein the second mobile phase comprises formic acid (FA), trifluoroacetic acid (TFA), triethylamine (TEA), or any combination thereof.

19. 20. The method of claim 18, wherein the second mobile phase comprises 0.1% FA.

20. 20. The process of any one of claims 4 to 19, wherein the separation is carried out at a temperature below 70°C.

21. 21. The method of claim 20, wherein the temperature is between about 50°C and about 70°C, between about 50°C and about 60°C, between about 60°C and about 70°C, between about 55°C and about 65°C, between about 55°C and about 60°C, between about 60°C and about 65°C, between about 65°C and about 70°C, between about 50°C and about 55°C.

22. 21. The method of claim 20, wherein the temperature is about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, or about 69°C.

23. 23. The method of any one of claims 4 to 22, wherein the separation is on a gradient.

24. 24. The method of claim 23, wherein the gradient is from about 95% to about 5%.

25. The gradient may be from about 95% to about 50%, from about 95% to about 55%, from about 95% to about 60%, from about 95% to about 65%, from about 95% to about 70%, from about 95% to about 75%, from about 95% to about 80%, from about 95% to about 85%, from about 90% to about 50%, from about 90% to about 55%, from about 90% to about 60%, from about 90% to about 65%, from about 90% to about 70%, from about 90% to about 25. The method of claim 24, wherein the solubility is about 75%, about 87% to about 50%, about 87% to about 55%, about 87% to about 60%, about 87% to about 65%, about 87% to about 70%, about 87% to about 75%, about 85% to about 50%, about 85% to about 55%, about 85% to about 60%, about 85% to about 65%, about 85% to about 70%, or about 85% to about 75%.

26. 26. The method of claim 25, wherein the gradient is from about 87% to about 75%.

27. 27. The method of any one of claims 1 to 26, wherein the one or more N-glycans are galactose (Gal), N-acetylgalactosamine (GalNAc), galactosamine (GalN), glucose (Glc), N-acetylglucosamine (GlcNAc), glucosamine (GlcN), mannose (Man), N-acetylmannosamine (ManNAc), mannosamine (ManN), xylose (Xyl), N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxynonanoic acid (Kdn), fucose (Fuc), glucuronic acid (GlcA), iduronic acid (IdoA), galacturonic acid (GalA), mannuronic acid (ManA), or any combination thereof.

28. 28. The method of any one of claims 1 to 27, wherein the one or more N-glycans comprise one or more biantennary glycans.

29. 29. The method of claim 28, wherein the biantennary glycan is selected from the group consisting of G0F, G0, G1F, G1, G2F, G2, S1G2F, S1G2, S2G2F, S2G2 and any combination thereof.

30. 30. The method of any one of claims 1 to 29, wherein the glycosylation profile comprises one or more non-sialylated glycans, monosialylated glycans, disialylated glycans, and / or trisialylated and tetrasialylated glycans.

31. 31. The method of any one of claims 1 to 30, wherein the recombinant protein is an antibody.

32. 32. The method of claim 31, wherein the antibody is of an isotype selected from IgM, IgA, IgE, IgD, and IgG.

33. 33. The method of claim 32, wherein the antibody is of the isotype IgG.

34. 34. The method of claim 33, wherein the IgG antibody is selected from IgG1, IgG2, IgG3, and IgG4.

35. 35. The method of any one of claims 31 to 34, wherein the antibody is an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody, an anti-CSF1R antibody, or an anti-IL8 antibody.

36. 36. The method of any one of claims 31 to 35, wherein the antibody has a single N-linked glycosylation site.

37. 37. The method of claim 36, wherein the single N-linked glycosylation site is asparagine 297 (N297).

38. 38. The method of any one of claims 1 to 37, wherein the recombinant protein comprises an enzyme, a hormone, a cytokine, a cell surface receptor, a protease, a cytokine receptor, or any combination thereof.

39. 39. The method of any one of claims 1 to 38, wherein the recombinant protein is a fusion protein.

40. 40. The method of claim 39, wherein the fusion protein is fused to a heterologous moiety.

41. 41. The method of claim 40, wherein the heterologous moiety is a half-life extending moiety.

42. 42. The method of claim 41, wherein the half-life extending moiety comprises albumin, albumin-binding polypeptide, fatty acid, PAS, the beta subunit of the C-terminal peptide of human chorionic gonadotropin (CTP), polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN, an albumin-binding small molecule, Fc, or a combination thereof.

43. 43. The method of claim 42, wherein the half-life extending moiety is Fc.

44. 44. The method of any one of claims 1 to 43, which is a batch release assay.

Images