EP-3541835-B1 - RAPID AND EFFICIENT DE-GLYCOSYLATION OF GLYCOPROTEINS

Inventors

• THAKUR, Anushikha
• ROHIL, Sheija
• PATIL, Shrivardhan
• SUDHAKARAN, Meenakshi
• ADHIKARY, LAXMI

Dates

Publication Date

20260513

Application Date

20171117

Claims (9)

1. A method of assessing molecular similarity by comparing the tertiary structure of monoclonal antibodies, biosimilars of monoclonal antibodies, monoclonal antibody fragments, or antibody fusion proteins with multiple glycan sites, via partial de-glycosylation, the method comprising: (a) combining a native monoclonal antibody, biosimilar of a monoclonal antibody, monoclonal antibody fragment, or antibody fusion protein with endoglycosidase Peptide-N-Glycosidase F (PNGaseF) to partially cleave N-linked glycans in an amount from 1 unit to 10 units per 1 mg of glycoprotein; (b) incubating components of step (a) under native conditions at a temperature of about 37 °C for a pre-selected limited period in the range from 45 mins to 8 hrs to partially cleave N-linked glycans in order to obtain sub-populations of partially de-glycosylated protein, thereby generating a fingerprint of partially de-glycosylated protein sub-populations at a particular time point; and (c) segregating the partially de-glycosylated protein sub-populations using reduced capillary electrophoresis SDS, thereby allowing a comparison of the tertiary structure of the monoclonal antibody, biosimilar of a monoclonal antibody, monoclonal antibody fragment or antibody fusion protein.
2. The method of claim 1, comprising comparing lots of a monoclonal antibody, biosimilar of a monoclonal antibody, monoclonal antibody fragment or antibody fusion protein.
3. A method of complete de-glycosylation of a monoclonal antibody, monoclonal antibody fragment, biosimilar of a monoclonal antibody, or antibody fusion protein with multiple glycan sites, the method comprising: (a) combining a monoclonal antibody, monoclonal antibody fragment, biosimilar of a monoclonal antibody or antibody fusion protein with sodium dodecyl sulfate (SDS) and a reducing agent, wherein the reducing agent is in a sufficient amount to denature the monoclonal antibody, monoclonal antibody fragment, biosimilar of a monoclonal antibody, or antibody fusion protein, and wherein SDS is in a concentration of 0.1% to 0.8%; (b) incubating components of step (a) at a temperature from 90 °C to 100 °C for 2 minutes to 5 minutes to provide for a denatured monoclonal antibody, monoclonal antibody fragment, biosimilar of a monoclonal antibody, or antibody fusion protein; (c) cooling the denatured monoclonal antibody, monoclonal antibody fragment, biosimilar of a monoclonal antibody, or antibody fusion protein; (d) combining the denatured monoclonal antibody, monoclonal antibody fragment, biosimilar of a monoclonal antibody, or antibody fusion protein with Triton-X 100 ® in an amount to counter the inhibitory effects of SDS, wherein Triton-X 100 ® is in a concentration of 0.6% to 1.2%; (e) introducing endoglycosidase PNGaseF to cleave N-linked glycans in an amount from 0.66 unit to 10 units per 1 mg of denatured monoclonal antibody, monoclonal antibody fragment, biosimilar of a monoclonal antibody, or antibody fusion protein; (f) incubating the components of step (e) at 37 °C for 1 to 15 minutes to provide for a completely de-glycosylated protein; (g) separating the released glycans from the completely de-glycosylated protein; and (h) separating the released glycans by liquid chromatography.
4. The method of claim 3, wherein the method further comprises adding an exoglycosidase in an amount of 0.1 units per 1 mg of denatured glycoprotein after step (e) or after step (g), and incubating components at 37 °C for 30 minutes to provide a de-sialylated protein.
5. The method of claim 3, wherein the reducing agent is in an amount to break disulphide bonds and selected from the group consisting of β-mercaptoethanol, dithiothreitol, or tris (2-carboxyethyl) phosphine.
6. The method of claim 5, wherein β-mercaptoethanol is in an amount of 100 mM to 150 mM.
7. The method of claim 4, wherein the exoglycosidase is Sialidase which has the ability to cleave terminal sialic acid from both N- and O-linked glycans of glycoproteins.
8. The method of claim 3, wherein the enzymatic activity is completed in 1 to 15 minutes.
9. The method of claim 8, wherein the enzymatic activity is completed in 30 minutes.

Description

FIELD OF THE INVENTION The present invention relates to rapid and reliable analytical methods, which are required by the industry to establish molecular similarity. It presents a method for quick and efficient de-glycosylation of glycoproteins. It also relates to a method of assessing molecular similarity by comparing tertiary structure of glycoproteins utilizing partial de-glycosylation as a tool. BACKGROUND OF THE INVENTION Glycosylation plays a critical role in protein folding, trafficking, and stability as well as cellular events such as receptor binding, cell signalling, immune recognition, inflammation and pathogenicity. Proteins of eukaryotic origin are often glycosylated as a result of post translational modification. Changes in specific glycan levels are often used as biomarkers in several diseases including diabetes, cancer, and infectious diseases. Since glycosylation is complex and heterogeneous, mapping the glycome can be an extremely challenging task and is generally done by liquid chromatography LC profiling of released glycan. Out of the two viz. N-linked and O-linked; the N-linked glycans are detached from glycoproteins by enzymatic cleavage with PNGaseF. The enzyme O-Glycosidase is commonly used for cleaving core 1 O-glycans, however pre-treatment with the Neuraminidase enzyme is required to remove terminal sialic acids from O-Glycan. The secondary and tertiary structures of a protein blocks access of the enzyme to the carbohydrates unless the protein is first denatured. Known protocols for denaturing involve the use of detergents or reducing agents, with an overnight incubation at 37°C. The de-glycosylated protein can be useful for intact/reduced mass analysis in case of large and complex monoclonal antibodies which suffers due to inherent heterogeneity and insufficient ionization due to glycans. The released glycans can be labelled at their free-reducing terminus with a fluorescent dye for N-glycan profiling by methods such as high performance liquid chromatography (HPLC), capillary electrophoresis (CE), or mass spectrometry (MS). The de-glycosylation of proteins by PNGaseF depends upon factors like: surface accessibility of glycans and steric hindrance by bulky and highly branched glycans. These factors are indirectly dependent on protein global conformation and glycan site occupancy, respectively. All these factors will determine the rate of de-glycosylation of a particular site on protein, which can be monitored by CE-SDS (capillary electrophoresis sodium dodecyl sulphate) utilizing difference in the molecular weight of de-glycosylated species. Thus, a partial de-glycosylation profile containing information on rate of de-glycosylation of different sites on the glycoprotein can serve as a fingerprint of its tertiary/quaternary conformation. The glycans released after protein de-glycosylation is useful both for quality control and often for determining whether a protein will have a desired therapeutic efficacy or other effect. For a chromatographic mapping protocol, complete de-glycosylation of both proteins and peptides is often desirable. De-glycosylation may reduce smearing during protein separation by SDS-PAGE or may allow easier ionization and spectral interpretation during mass spectrometric analysis. This may be particularly useful when looking at intact molecular weights of proteins that may be skewed due to heterogeneity from abundance of post translation modifications. In the case of therapeutic antibodies, de-glycosylation is often necessary in characterizing modifications such as the presence of C-terminal lysine, or for labelled or drug-conjugated monoclonal antibodies, to monitor the number of small molecules coupled to the immunoglobulin. In bio-pharma industries, criteria for approval include quality, efficacy and safety. Thus, assessing the molecular similarity of a candidate biosimilar to the innovator product is a critical task during development of a biosimilar product. For this purpose, rapid and reliable analytical methods are required by the industry to establish molecular similarity required by regulators. Kaschak, T. et al. (Analytical Biochemistry 2011, 417, 2, 256-263) disclose the characterization of a non-enzymatically glycated IgGl using reducing capillary electrophoresis-SDS and mass spectrometry. Liu, J., et al. (BioDrugs 2016, 30, 321-338) describe a method of assessing analytical similarity between a biosimilars and the monoclonal antibody adalimumab, which includes deglycosylation. International patent application WO 2008/128220 A1 discloses strategies for analyzing protein-linked glycans, particularly glycans on cell surface glycoproteins, which include treatment with sialidase. Szabo, Z. et al. (Analytical Chemistry 2010, 82, 2588-2593) relates to releasing N-linked glycans from glycoproteins, employing the endoglycosidase PNGase F. OBJECT OF THE INVENTION The object of present invention is to compare tertiary structure of glycoproteins utilizing par