This disclosure provides a novel label-free N-glycan analysis method to detect and quantify N-glycans and N-linked glycosylation profiles without using a label, such as a fluorescent label. This method allows for reduced sample preparation and chromatographic separation times, and can be used for product batch release.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application No. 62/938,803 filed Nov. 21, 2019, which is herein incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

[0002] The content of the electronically submitted sequence listing in ASCII text file (Name: 3338_190PC01_SL_ST25.txt; Size: 14,351 bytes; and Date of Creation: Nov. 20, 2019), filed with the application, is incorporated herein by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

[0003] Glycosylation often plays a significant role in the biological function(s) of glycoconjugates (e.g., glycoproteins). For example, a glycoprotein's glycosylation pattern can affect its ability to fold correctly, its stability (e.g., resistance to proteolytic and/or other degradation), catalytic activity, pharmacodynamic and/or pharmacokinetic properties, and/or the ability of that glycoprotein to properly interact with other molecules. Alternatively or additionally, a glycoprotein's glycosylation pattern can affect transport and targeting of the glycoprotein, e.g., determining whether the glycoprotein remains intracellular (including, e.g., the correct targeting of the glycoprotein to the proper subcellular compartment or compartments), whether the glycoprotein will be membrane-bound and/or whether the glycoprotein will be secreted from the cell.

[0004] Monoclonal antibodies and other proteins are complex glycoproteins that are developed for treatment of various indications such as cancer and autoimmune diseases. Specifically, monoclonal antibodies are commonly glycosylated at a conserved asparagine residues of the heavy chain. Glycans are important in governing the function and efficacy of the monoclonal antibody therapeutics, and as such, are generally required to be part of the critical quality attribute panel for release testing for use in humans. Depending on the terminal sugar residues, “N-linked glycans” or “N-glycans” are classified into complex, high-mannose, and hybrid N-glycans.

[0005] Traditionally, N-linked glycans are released from the glycoprotein after denaturation and enzymatic digestion with PNGase F, followed by fluorescent labeling (such as with 2-aminobenzamide) of the liberated glycans. The labeled glycans are then separated using hydrophilic liquid chromatography (HILIC) using fluorescence detection to generate a chromatographic profile of the glycans present in the antibody samples. Despite decades of use, this process remains cumbersome, using toxic reagents and extended sample preparation time. Therefore, there are needs to efficiently quantifying a glycosylation profile of a protein.

SUMMARY OF THE DISCLOSURE

[0006] The present disclosure is directed to a method of quantifying a glycosylation profile of a recombinant protein, comprising analyzing the one or more N-linked glycans without any label, wherein the N-linked glycans are released from the recombinant protein by enzymatic digestion prior to the analysis. The present disclosure is also directed to a method of quantifying a glycosylation profile of a recombinant protein, comprising analyzing the one or more N-linked glycans without any fluorophore, wherein the N-linked glycans are released from the recombinant protein by enzymatic digestion prior to the analysis. In some aspects, the recombinant protein is 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% pure. In some aspects, the analysis comprises separation of one or more N-linked glycans during a chromatography comprising a column. In some aspects, the column is a mixed mode column. In some aspects, the separation is done using one or more mobile phases. In some aspects, the column is a mixed mode porous graphite carbon (PGC) column. In some aspects, the enzyme comprises peptide N-glycosidase F (PNGaseF). In some aspects, the enzyme is incubated with the recombinant protein, wherein the one or more N-linked glycans are released from the recombinant protein prior to the separation. In some aspects, the enzyme is diluted in buffer. In some aspects, the one or more N-linked glycans that are separated are measured by a mass spectrometer, ELSD, NQAD, or refractive index detector. In some aspects, the one or more N-linked glycans that are separated are measured by a charged aerosol detector (CAD).

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

[0008] In some aspects, the temperature is between about 50° C. and about 70° C., about 50 ° C. and about 60° C., about 60° C. and about 70° C., about 55° C. and about 65° C., about 55° C. and about 60° C., about 60° C. and about 65° C., about 65° C. and about 70° C., about 50° C. and about 55° C. In some aspects, 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 aspects, the separation is based on a gradient. In some aspects, the gradient is from about 95% to about 5%. In some aspects, 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%, from about 90% to about 75%, from about 87% to about 50%, from about 87% to about 55%, from about 87% to about 60%, from about 87% to about 65%, from about 87% to about 70%, from about 87% to about 75%, from about 85% to about 50%, from about 85% to about 55%, from about 85% to about 60%, from about 85% to about 65%, from about 85% to about 70%, or from about 85% to about 75%. In some aspects, the gradient is from about 87% to about 75%.

[0010] The present disclosure is also directed to a method of analyzing the glycan profile of the protein of interest. In some aspects, 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-deoxynononic acid (Kdn), Fucose (Fuc), Glucuronic Acid (GlcA), Iduronic acid (IdoA), Galacturonic acid (GalA), Mannuronic acid (ManA), or any combination thereof. In some aspects, the one or more N-glycans comprise one or more bi-antennary glycans. In some aspects, the bi-antennary glycans are selected from a group consisting of G0F, G0, G1F, G1, G2F, G2, S1G2F, S1G2, S2G2F, S2G2 and any combination thereof. In some aspects, the glycosylation profile comprises one or more asialylated glycans, mono-sialylated glycans, di-sialylated glycans, and/or tri-sialylated and tetra-sialylated glycans. In some aspects, the recombinant protein is an antibody. In some aspects, the antibody is an isotype selected from IgM, IgA, IgE, IgD, and IgG. In some aspects, the antibody is isotype IgG. In some aspects, the IgG antibody is selected from IgG1, IgG2, IgG3, and IgG4. In some aspects, 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 aspects, the antibody has a single N-linked glycosylation site. In some aspects, the single N-linked glycosylation site is Asparagine 297 (N297).

[0011] In some aspects, 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 aspects, the recombinant protein is a fusion protein. In some aspects, the fusion protein is fused to a heterologous moiety. In some aspects, the heterologous moiety is a half-life extending moiety. In some aspects, the half-life extending moiety comprises albumin, 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, albumin-binding small molecules, Fc, or a combination thereof In some aspects, the half-life extending moiety is an Fc. In some aspects, the method is a batch release.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 shows a sample glycan chromatogram produced from a separation analysis as shown in Example 1 (mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in acetonitrile).

[0013] FIG. 2 shows a sample glycan chromatogram produced from a separation analysis as shown in Example 2(mobile phase A: 0.1% triethylamine (TEA) in water; mobile phase B: 0.1% triethylamine (TEA) in acetonitrile).