Tagged Exoglycosidase Enzymes and Immobilized Glycan Sequencing Methods

Abstract

The present invention relates to a set of exoglycosidase enzymes that can be used and / or are suitable for N-glycan sequencing, each of which has a different exoglycosidase activity specific for cleaving different terminal carbohydrates, each of which comprises a peptide tag, preferably a HIS tag, which can be used for potential immobilization to ensure the best accessibility to the active site of the enzyme for automation purposes and targeted special workflows. The present invention provides rapid enzyme digestion performance in both aqueous phase and immobilized forms. The immobilized enzymes allow for long term storage and ready to use premixes. The immobilization method of the present invention opens up the possibility of automation and of meeting special experimental needs where enzyme immobilization is important.

Description

[Technical field]

[0001] The present inventors have designed a pure, soluble and functional exoglycosidase enzyme that can be produced in a high yield and cost-effective manner using a bacterial expression system.

[0002] The present invention relates to a set of exoglycosidase enzymes that can be used and / or are suitable for N-glycan sequencing, each of which has a different exoglycosidase activity specific for cleaving different terminal carbohydrates, each of which comprises a peptide tag, preferably a HIS tag, which can be used for potential immobilization to ensure the best accessibility to the active site of the enzyme for automation purposes and targeted special workflows.

[0003] The present invention provides rapid enzyme digestion performance in both aqueous phase and immobilized forms. The immobilized enzymes allow for long term storage and ready to use premixes.

[0004] The immobilization method of the present invention opens up the possibility of automation and of meeting special experimental needs where enzyme immobilization is important. [Background technology]

[0005] Protein glycosylation is one of the most common post-translational modifications in eukaryotic cells, and carbohydrate structures are known to contain a large amount of biological information. They play important roles in cell-cell interactions, signaling pathways, and are involved in disease progression [Gabius, HJ The sugar code: Why glycans are so important, Biosystems 164 (2018) 102-111.]. N- and O-glycosylation are important for proper folding, stability, and functionality of proteins [Vliegenthart, JF The complexity of glycoprotein-derived glycans, Proc Jpn Acad Ser B Phys Biol Sci 93(2) (2017) 64-86.]. Such glycan moieties are composed of monosaccharide units, and the monomer sequence, linkage type, and position imply complexity and diversity. Thus, structural analysis of glycans is a challenging task, but crucial in understanding their biological functions.

[0006] Biologics have surpassed small molecules thanks to their superior specificity and efficacy. In most cases, biotherapeutics are glycosylated monoclonal antibodies or fusion proteins. The carbohydrate moiety of biologics has an important role in the biological activity, solubility and immunogenicity of the therapeutic. Therefore, it is essential to characterize the glycan profile of glycoproteins.

[0007] Exoglycosidase digestion is a common method for characterizing glycans. Archer Hartmann et al. [Archer Hartmann et al. Microscale exoglycosidase processing and lectin capture of glycans with phospholipid assisted capillary electrophoresis separations, Anal Chem 83(7) (2011) 2740-7.] teaches a method for in-capillary cleavage of terminal glycan residues with exoglycosidases.

[0008] Yamagami, M. et al. also described an in-capillary method combining online exoglycosidase digestion with the plug-plug kinetic mode of capillary electrophoresis (CE) for the analysis of glycoprotein-derived oligosaccharides. [Yamagami, M. et al. Plug-plug kinetic capillary electrophoresis for in-capillary exoglycosidase digestion as a profiling tool for the analysis of glycoprotein glycans, J Chromatogr A 1496 (2017) 157-162.]

[0009] Song T et al. [Song T. et al. In-depth method for the characterization of glycosylation in manufactured recombinant monoclonal antibody drugs, Anal Chem 86(12) (2014) 5661-6.] described a liquid chromatography-mass spectrometry (LC-MS) method for preparing an N-glycan library based on eight commercially available rMab drugs. Complete glycan structures were obtained by exoglycosidase sequencing.

[0010] Oligosaccharide sequencing by exoglycosidase digestion is one of the most commonly used techniques to determine the structures of complex glycans [Guttman, M. et al. Comparative glycoprofiling of HIV gp120 immunogens by capillary electrophoresis and MALDI mass spectrometry, Electrophoresis 36(11-12) (2015) 1305-13.; Varadi, C. Analysis of cetuximab N-Glycosylation using multiple fractionation methods and capillary electrophoresis mass spectrometry, J Pharm Biomed Anal 180 (2020) 113035.].

[0011] Enzymatic digestion provides precise sequence and linkage information of oligosaccharide chains. Exoglycosidase enzymes have monosaccharide unit and bond orientation (α vs. β) specificity and can therefore reveal not only the sequence of a glycan but also its anomeric configuration [Gattu S. et al. Microscale Measurements of Michaelis-Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage, Anal Chem 89(1) (2017) 929-936., Lu, CL et al. Capillary Electrophoresis Separations of Glycans, Chem Rev 118(17) (2018) 7867-7885][11,21]. Glycan sequencing requires multiple separations of the reaction mixture, and CE is frequently used due to its low sample volume requirements and short separation times. Structural information can be derived from the peak shifts of successive exoglycosidase treatments.

[0012] One of the limitations of enzymes for industrial applications is that they lose their activity rapidly, which can significantly increase the cost of analysis. Enzyme immobilization can avoid the efficiency loss [Sheldon RA, S. van Pelt, Enzyme immobilization in biocatalysis: why, what and how, Chem. Soc. Rev. 42(15) (2013) 6223-6235.]. Furthermore, immobilization allows repeated use and improves resistance to denaturation. Immobilization may increase enzyme activity, but this is not clear. Numerous immobilization techniques have been reported for various applications [Krenkova J., F. Foret, Immobilized microfluidic enzymatic reactors, Electrophoresis 25(21-22) (2004) 3550-63.].

[0013] developed an on-line immobilized enzyme reactor technique coupled with HPLC-MS / MS techniques for the simultaneous characterization of glycan and peptide moieties in pronase-generated glycopeptides. [Temporini, C. et al. Pronase-Immobilized Enzyme Reactor: an Approach for Automation in Glycoprotein Analysis by LC / LC-ESI / MSn, Analytical Chemistry 79(1) (2007) 355-363.] Pronase enzyme was immobilized on an epoxy-silica monolithic material. The immobilization of pronase reduced the reaction time from 48 hours to 40 minutes.

[0014] Krogh, TN et al. [Protein Analysis Using Enzymes Immobilized to Paramagnetic Beads Analytical Biochemistry 274, (1999) 153-162] use chemical immobilization of trypsin and exo- and endoglycosidases to paramagnetic beads to demonstrate a protein analysis method that combines protein chemistry and enzymes. The immobilization is said to have resulted in high sensitivity of glycopeptides, faster glycosidase digestion, and reduced sample contamination. MALDI-MS is used as the analytical method.

[0015] Immobilized enzyme pipettes have been developed as efficient tools for microvolume chromatography analysis as well as enzyme microreactors fit in modern laboratory environments. In glycoscience research, immobilization of enzymes such as PNGase F in pipette tips has been previously applied to rapidly analyze glycoproteins [Chen, J. et al. Solid phase extraction of N-linked glycopeptides using hydrazide tip, Anal Chem 85(22) (2013) 10670-4., Yamamoto, S. et al. A fast and convenient solid phase preparation method for releasing N-glycans from glycoproteins using trypsin- and peptide-N-glycosidase F (PNGase F)-impregnated polyacrylamide gels fabricated in a pipette tip, Journal of Pharmaceutical and Biomedical Analysis 179 (2020) 112995.].

[0016] Automation and speed of analysis, including the development of high throughput techniques, are continuing challenges in the field of glycan sequencing.

[0017] A recent study described carbohydrate sequencing based on rapid and automated exoglycosidase digestion of human immunoglobulin G (IgG) and Enbrel (etanercept) fusion protein N-glycans, in which the sample storage compartment of the CE instrument was used for reaction temperature control and the separation capillary was used as the enzyme delivery device. [Szigeti, M and Guttman, A Automated N-Glycosylation Sequencing Of Biopharmaceuticals By Capillary Electrophoresis, Sci Rep 7(1) (2017) 11663.; Guttman A, Szigeti M: Fast Glycan Sequencing Using a Fully Automated Carbohydrate Sequencer, Sciex, https: / / sciex.com / products / consumables / fast-glycan-analysis-and-labeling-for-the-pa-800-plus 2017-09-14] However, no fixation was applied and the method consisted of a series of separate digestion and sequencing steps.

[0018] Although glycan sequencing methodologies have undergone significant development over the past few years, there remains a need in the art to provide a rapid and robust sequencing methodology based on immobilized exoglycanases.

[0019] The present inventors provide a glycan sequencing system based in particular on the use of a series of engineered, tagged exoglycanases immobilized to a solid matrix support via a peptide tag, and a system for N-glycan sequencing. Summary of the Invention

[0020] 1. The present invention relates to a set of exoglycosidase enzymes that can be used and / or are suitable for N-glycan sequencing, each of which has a different exoglycosidase activity specific for cleaving a different terminal carbohydrate from the non-reducing end of a glycan, each of which comprises a peptide tag for immobilizing the exoglycosidase on a matrix support, wherein the peptide tag is present at a position in the exoglycosidase sequence that does not affect the exoglycosidase activity.

[0021] Alternatively expressed, a set of exoglycosidase enzymes is a plurality (ie, more than one) of exoglycosidase enzymes, or a series or system of exoglycosidase enzymes.

[0022] The peptide tag is preferably a methyl-chelating peptide tag, very preferably a His tag.

[0023] In a highly preferred embodiment, the set of exoglycosidase enzymes comprises at least two enzymes selected from the following enzyme group: neuraminidase, galactosidase and hexosaminidase.

[0024] Preferably, the set of exoglycosidases comprises: a neuraminidase, preferably α-neuraminidase, having the activity of cleaving terminal sialic acids from glycans and having a peptide tag at its C-terminus; β-galactosidase with a peptide tag at its N-terminus; A hexosaminidase with a peptide tag at its C-terminus. At least The levels of hexosaminidase are increased relative to neuraminidase and β-galactosidase so as to have sufficient hexosaminidase activity to achieve complete cleavage of terminal carbohydrates in a given period defined for each exoglycosidase in the set.

[0025] In a preferred embodiment, the exoglycosidases (and preferably each of them) are immobilized on a matrix support.

[0026] In a preferred embodiment, the set of exoglycosidases comprises two or more subsets of exoglycosidases, each subset differing from each other subset by at least one exoglycosidase, and a subset may comprise a single exoglycosidase; Within the subset, each exoglycosidase has a different exoglycosidase activity specific for cleaving a different terminal carbohydrate from the non-reducing end of the glycan; Preferably, in the subset, each exoglycosidase is immobilized on the same matrix support.

[0027] In a preferred embodiment, each of the exoglycosidases is immobilized on a matrix support, and preferably in a subset, each exoglycosidase is immobilized on the same matrix support.

[0028] 2. Preferably, the invention relates to a set of exoglycosidases according to paragraph 1, The levels of exoglycosidases in the set refer to a set of exoglycosidases that are adjusted such that each exoglycosidase has sufficient exoglycosidase activity to achieve complete cleavage of the terminal carbohydrate within a given temperature range and time period defined for each exoglycosidase in the set.

[0029] Preferably, the predetermined period is up to 1.0 hour, preferably up to 40 minutes, preferably up to 30 minutes, and preferably the temperature range is 37-60° C. Preferably, the temperature range is 37-60° C. and the time range is 5.0 minutes to 30 minutes.

[0030] Highly preferably, the predetermined period is up to 30 minutes and the temperature range is 37-60°C.

[0031] Preferably, the levels of the exoglycosidases are regulated, i.e., their levels are adjusted relative to one another to demonstrate equivalent activity or activity such that each is capable of effecting complete digestion within a certain period or range of time.

[0032] 3. In a preferred embodiment, the present invention relates to a set of exoglycosidases according to any one of paragraphs 1 to 2, The set of exoglycosidases relates to a set of exoglycosidases that includes exoglycosidases that have the activity of cleaving terminal carbohydrates from N-glycans.

[0033] Preferably, the set of exoglycosidases comprises at least a neuraminidase, preferably an alpha-neuraminidase, having activity to cleave terminal sialic acids from glycans, and at least one further exoglycosidase, and preferably the peptide tag is provided at the C-terminus of the neuraminidase.

[0034] 4. In a preferred embodiment, the present invention relates to a set of exoglycosidases according to paragraphs 1, 2, 3, preferably 3, wherein at least one further exoglycosidase comprises at least β-galactosidase, and preferably the peptide tag is provided at the N-terminus of the β-galactosidase.

[0035] In a preferred embodiment, the at least one additional exoglycosidase is selected from the group consisting of β-galactosidase and hexosaminidase.

[0036] In a preferred set of exoglycosidases, the peptide tag is a His tag and the matrix support is a transition metal-containing matrix support, the transition metal being more preferably Cu. 2+ , Ni 2+ , Zn 2+ and Co 2+ is selected from.

[0037] 5. In a preferred embodiment, the present invention relates to a set of exoglycosidases according to any one of paragraphs 3 to 4, the set of exoglycosidases, wherein the at least one further exoglycosidase comprises at least a hexosaminidase; Preferably, the peptide tag is provided at the C-terminus of the hexosaminidase; the set of exoglycosidases, wherein the at least one further exoglycosidase comprises at least a hexosaminidase; Preferably, the peptide tag is provided at the C-terminus of the hexosaminidase, which relates to a set of exoglycosidases.

[0038] 6. In a preferred embodiment, the invention relates to a set of exoglycosidases according to paragraph 5, wherein the levels of hexosaminidase are increased relative to neuraminidase and β-galactosidase so as to have sufficient hexosaminidase activity to achieve complete cleavage of terminal carbohydrates during a predefined time period defined for each exoglycosidase in the set, and the levels of the enzymes are therefore adjusted and therefore regulated to complete the cleavage reaction in essentially the same predefined time period.

[0039] 7. In a preferred embodiment, the present invention relates to a set of exoglycosidases according to any one of paragraphs 1 to 6, wherein the peptide tags are metal-chelating peptide tags and the matrix support is a metal-containing matrix support.

[0040] In certain embodiments, the peptide tag is selected from the group consisting of a metal chelating peptide tag, an epitope peptide tag, a substrate peptide tag, a ligand peptide tag, and a modified peptide tag.

[0041] Preferably, the matrix support comprises a binding moiety for binding of a peptide tag, and in certain embodiments the matrix support comprises a metal ion, an antibody, or any binding molecule having an epitope binding site, a substrate binding molecule, a receptor that binds to a ligand, a binding molecule that binds to a modified peptide tag.

[0042] Optionally, the modified peptide tag is modified to allow for covalent attachment to a matrix support.

[0043] 8. In a preferred embodiment, the present invention relates to a set of exoglycosidases according to any of paragraphs 1 to 7, wherein the exoglycosidases are immobilized on a matrix support, and preferably the exoglycosidases mixed together are dialysed together (co-dialysis) to reduce negative salt effects.

[0044] 9. In a preferred embodiment, the present invention relates to a set of exoglycosidases according to any one of paragraphs 1 to 8, wherein each of the exoglycosidases is immobilized on a matrix support, in particular by the means described in paragraph 7.

[0045] 10. In a preferred embodiment, the invention relates to a set of exoglycosidases according to any one of paragraphs 1 to 9, wherein two or more exoglycosidases are immobilized on the same matrix support.

[0046] 11. In a preferred embodiment, the present invention relates to a set of exoglycosidases according to any one of paragraphs 1 to 10, comprising two or more subsets of exoglycosidases, each subset differing from each other subset by at least one exoglycosidase, and a subset may comprise a single exoglycosidase; Within a subset, each exoglycosidase relates to a set of exoglycosidases having a different exoglycosidase activity specific for cleaving a different terminal carbohydrate from the non-reducing end of the glycan.

[0047] 12. In a preferred embodiment, the invention relates to a set of exoglycosidases according to paragraph 11, wherein within a subset each exoglycosidase is immobilised on the same matrix support.

[0048] 13. In a further aspect, the present invention relates to the use of a set of exoglycosidases according to any one of paragraphs 1 to 12 for N-glycan sequencing. Preferably, glycan cleavage reactions with multiple subsets of exoglycosidases are performed simultaneously and each reaction mixture is analyzed to obtain glycan sequence information. Preferably, the exoglycosidase is as defined in any of paragraphs 1 to 12, in particular 1, preferably a preferred option therein, or in any of paragraphs 6 to 12, in particular or any of paragraphs 7 to 12 or 8 to 12.

[0049] 14. In a preferred embodiment, the present invention relates to the use of a set of exoglycosidases according to paragraph 13 for analyzing the result (reaction mixture) of a glycan cleavage reaction (digestion) to obtain glycan sequence information. Preferably, the result (obtained reaction mixture, i.e. the cleaved glycan products) is analyzed by a separation method selected from the group consisting of HPLC, UPLC and capillary electrophoresis, preferably with fluorescence detection.

[0050] Preferably, the analysis is carried out by capillary electrophoresis, preferably with fluorescence detection.

[0051] 15. In a preferred embodiment, the present invention relates to the use of a set of exoglycosidases as defined in any of paragraphs 13-14, in which glycan cleavage reactions (for N-glycan sequencing) using multiple subsets of exoglycosidases as defined in any of paragraphs 11-12 are carried out simultaneously (i.e. in parallel) and each of the reaction mixtures is analysed to obtain glycan sequence information.

[0052] In a preferred embodiment of this analysis, the separation results (e.g., chromatograms or electropherograms) are run to obtain the sequence. When multiple cleavage reactions are run, the results of these reactions are compared and assembled to obtain the sequence information.

[0053] In an exemplary embodiment, sequential cleavage reactions are performed and the sugar moieties are identified in each reaction mixture.

[0054] In a preferred embodiment, the sequencing is carried out using a set of exoglycosidases and a subset thereof, Each subset differs from each other subset by at least one exoglycosidase, and a subset may contain a single exoglycosidase; Within the subset, each exoglycosidase has a different exoglycosidase activity specific for cleaving a different terminal carbohydrate from the non-reducing end of the glycan; Each reaction mixture was analyzed to obtain glycan sequence information; Preferably, in the subset, each exoglycosidase is immobilized on the same matrix support.

[0055] In another exemplary embodiment, the results of cleavage reactions with various enzyme mixtures are analyzed and the sugar compositions are assessed (measured or identified), thereby determining the glycan sequence when these compositional data are compared and assembled.

[0056] See Table 1 and associated discussion for examples for assessing sugar composition.

[0057] 16. In a further aspect, the present invention relates to a method for preparing a set of exoglycosidases according to any of paragraphs 1 to 12, comprising: A nucleic acid sequence encoding an exoglycosidase is provided, a nucleic acid sequence encoding a peptide tag is inserted (e.g., by molecular cloning) into a nucleic acid sequence encoding an exoglycosidase such that, upon expression, the peptide tag is present in a position in the exoglycosidase sequence that does not affect exoglycosidase activity; Each exoglycosidase is expressed in an expression system, preferably a bacterial expression system; Each exoglycosidase is isolated (highly preferably using an engineered His peptide tag on a metal chelate column), Preferably, the isolation comprises binding the exoglycosidase to a chromatography matrix via a peptide tag; The method relates to methods in which the levels of exoglycosidases are adjusted such that each exoglycosidase has sufficient exoglycosidase activity to achieve complete cleavage of the terminal carbohydrate in a given time period defined for each exoglycosidase in the set at a given temperature range.

[0058] Preferably, the predetermined period is up to 1.0 hour, preferably up to 40 minutes, preferably up to 30 minutes, and preferably the temperature range is 37-60° C. Preferably, the temperature range is 37-60° C. and the time range is 5.0 minutes to 30 minutes.

[0059] Preferably, the levels of the exoglycosidases are regulated, i.e., their levels are adjusted relative to one another to demonstrate equivalent activity or activity such that each is capable of effecting complete digestion within a certain period or range of time.

[0060] Preferably, the matrix support comprises a binding moiety for binding of a peptide tag, and in certain embodiments the matrix support comprises a metal ion, an antibody, or any binding molecule having an epitope binding site, a substrate binding molecule, a receptor that binds to a ligand, a binding molecule that binds to a modified peptide tag.

[0061] Optionally, the modified peptide tag is modified to allow for covalent attachment to a matrix support.

[0062] 17. In a preferred embodiment, the present invention relates to a method for preparing a set of exoglycosidases as defined in paragraph 16, wherein at least a portion of the exoglycosidases are mixed together to form a single cleavage reaction mixture, preferably a subset of the exoglycosidases are mixed together as defined in any of paragraphs 11-12.

[0063] 18. In a preferred embodiment, the present invention relates to a method for preparing a set of exoglycosidases according to paragraph 17, wherein the exoglycosidases mixed together are dialyzed together (co-dialysis) to reduce negative salt effects on signal intensity during capillary electrophoresis analysis.

[0064] In a preferred embodiment, co-dialysis is highly preferred for CE and / or HPLC separation analysis.

[0065] 19. In a preferred embodiment, the present invention relates to a method for preparing a set of exoglycosidases according to any of paragraphs 16 to 18, wherein the exoglycosidases are immobilized on a matrix support via a peptide tag. Preferably, the peptide tag is a tag according to claim 16.

[0066] 20. In a preferred embodiment, the present invention relates to a method for preparing a set of exoglycosidases according to paragraph 19, comprising: Two or more subsets of exoglycosidases are mixed together and co-dialyzed, Each subset differs from the others in at least one exoglycosidase, and the subsets may comprise a single exoglycosidase; Within the subset, each exoglycosidase has a different exoglycosidase activity specific for cleaving a different terminal carbohydrate from the non-reducing end of the glycan; The method relates to a method in which, within a subset, each exoglycosidase is immobilized on the same matrix support.

[0067] Preferably, the peptide tag is a tag according to claim 16.

[0068] In a highly preferred embodiment, the peptide tag is a metal chelate tag, in particular a His tag.

[0069] 21. A kit for preparing an immobilized set of exoglycosidases as defined in any of paragraphs 11 to 12, comprising: A set of soluble exoglycosidases as defined in paragraphs 1 to 10, wherein the exoglycosidases are present in soluble form in a buffer solution as a stock exoglycosidase solution or a set of stock exoglycosidase solutions; a matrix support functionalized to bind to or be bound by a peptide tag engineered into the exoglycosidase; Optionally, buffers and reagents for carrying out the immobilization, buffers for washing the matrix support before or after immobilization, and a storage buffer, preferably containing a stabilizing agent, preferably glycerin. Includes.

[0070] Abbreviation APTS: 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt; AmAc: ammonium acetate; PNGase F: peptide-N-glycosidase F; CE: capillary electrophoresis; MD: maltodextrin; NANase: neuraminidase-TEV-6HIS enzyme; GALase: 6HIS-TEV-β-galactosidase enzyme; HEXase: hexosaminidase-TEV-6HIS enzyme; hIgG1: human immunoglobulin G1.

[0071] definition An "exoglycosidase enzyme" (or exoglycosidase for short) is a glycoside hydrolase (EC 3.2.1) enzyme that breaks (or cleaves) a glycosidic bond at a terminal residue.

[0072] Exoglycosidases include, inter alia, enzymes with the following specificities: Neuraminidase, particularly α-neuraminidase; galactosidase, particularly β-galactosidase; glucosaminidase, particularly N-acetylglucosaminidase, more particularly βN-acetylglucosaminidase; mannosidase, particularly α- and / or β-mannosidase; fucosidase, particularly α-fucosidase; and the like.

[0073] Without limitation, exoglycosidases have been described in the art [Kobata A. (2013). Exo- and endoglycosidases revisited. Proceedings of the Japan Academy. Series B, Physical and biological sciences, 89(3), 97-117.].

[0074] "N-glycan sequencing" is a process in which exoglycosidase enzymes (exoglycosidases) remove terminal carbohydrates from the non-reducing end of a glycan, but do not cleave internal bonds between carbohydrates, thereby allowing the removed glycan residues to be identified, preferably by bond and sugar, by using positionally specific exoglycosidases.

[0075] A "peptide tag" as defined herein is a peptide whose coding sequence has been inserted or added to a protein, here the coding sequence of an exoglycosidase enzyme, such that the expressed protein also contains a peptide tag, which peptide tag can bind to a binding moiety carried by a matrix (matrix support) to form, in technical terms, a resin or matrix support with binding moieties. Preferably, the "peptide tag" is used for immobilization to a matrix support. Preferably, the "peptide tag" is used to isolate or purify a protein, and the matrix support is a chromatography matrix. The matrix support comprises an inert support material and a binding moiety that can be bound or attached to the peptide tag to form a resin. The support material and the binding moiety may be linked by a linker (moiety). In the case of a metal chelating tag, such as a His tag, the resin is a metal-containing resin.

[0076] Peptide tags suitable for immobilization and preferably purification include inter alia epitope tags bound by antibodies or other epitope-binding molecules such as a FLAG tag, an HA tag, a Myc tag, a NE tag, or tags of ligand-binding protein pairs such as a calmodulin tag, peptides bound by calmodulin or an SBP tag or a Strep tag, peptides that bind to streptavidin or modified versions thereof, or modified peptide tags, e.g. peptide tags useful for covalent binding, or metal chelating tags such as a His tag, preferably comprising or consisting of 5-10 His amino acids, preferably a 6His tag having 6His amino acids.

[0077] A "set" of enzymes as used herein is a group or collection of enzymes that are considered as an entity in themselves. Thus, in a preferred embodiment, the members of the set can work together and / or are useful to achieve the result that each member of the set is utilized for. A set is composed of similar enzymes (a set of enzymes with common properties). However, a set is a kit, and the components within the kit may differ from each other in basic properties (e.g., the kit may include enzymes, buffers, some devices, resins, etc.).

[0078] A "subset" is a set that is a part of a set, i.e., it can be the same as the set, or (in the narrower sense) a smaller portion of the set that contains fewer members than the set itself (the whole).

[0079] The meaning of "level" of an exoglycanase enzyme in a set or subset of enzymes can refer to the amount or concentration of individual exoglycanases with different substrate (carbohydrate bond) specificities to adjust the activity and ideally reach complete cleavage of the terminal sugar moiety in the digestion reaction of each exoglycosidase.

[0080] A "group" (or "moiety") is used herein as a portion of a molecule or complex that can in principle be derivatized by the retransfer of another or other moiety, such as a hydrogen atom (the moiety is usually a negatively charged ion) or a metal ion (the moiety may be a chelator) or an organic part of a complex (the moiety may be a metal ion).

[0081] A "chelate" contains at least two "coordinative" or "dative" bonds, i.e., two-center, two-electron bonds, where both electrons come from the same atom, and typically the non-bonding pair is coordinated to a metal ion. Such bonds are indicated in the formula by wavy lines.

[0082] The singular forms "a," "an," and "the," or at least "one," include plural references unless the context clearly dictates otherwise.

[0083] The terms "comprises" or "comprising" or "including" are to be construed herein to have a non-exhaustive meaning, allowing for the addition or inclusion of additional features or method steps or components to those including the recited features or method steps or components. "Comprising" may be replaced with "including" where required by the implementation of a given language variant, and may be limited to "consisting essentially of" where other elements or components are not essential to the practice of the invention. [Brief description of the drawings]

[0084] [Figure 1] Genetic design of NANase (left), GALase (middle) and HEXase (right). For protein expression, the genes were integrated into an engineered pET23b plasmid between NdeI and XhoI sites to obtain pET23b-NdeI-neuraminidase-TEV-6HIS-XhoI ("NANase") and NdeI-hexosaminidase-SacI-TEV-6HIS-XhoI constructs ("HEXase"). Meanwhile, for expression of β-galactosidase, fusion of an N-terminal 6HIS-TEV tag to its functional part using an engineered pET17b-based plasmid [Materials and Methods] was applied ("GALase"). [Diagram 2] N-glycan sequencing of a hIgG1 glycoprotein sample where enzymes were applied to the sample individually (panel A) and premixed and co-dialysed (panel B) and digested overnight at 37° C. The efficiency of the resulting co-dialysed enzyme mixture (in 50% glycerol after 10-fold dilution) was compared to the mixtures used individually and the enzymes without glycerol. Separation conditions: BFS capillary with total length of 50 cm (effective length of 40 cm), NCHO resolving gel buffer, capillary and sample temperatures of 20° C., and pressure injection for 5.0 seconds using 5.0 psi. [Diagram 3]Figure 1. Exoglycosidase digestion of glycans of hIgG1 sample with immobilized enzyme mixture. Enzymes were immobilized on PhyNexus microcolumns. Separation conditions were the same as in Figure 2. By increasing the concentration of HEXase to 0.6 μM (4-fold), a complete reaction was obtained in 30 min with immobilized enzymes. [Figure 4] N-glycan sequencing of Daratumumab mAb glycan (Panel A), human serum glycan (Panel B) and Synagis mAb glycan (Panel C) with premixed and codialysed enzymes in aqueous phase and in immobilized form using a 30 min temperature gradient method. Separation conditions were the same as in Figure 2. In all cases, complete exoglycosidase digestion was obtained in the given time frame. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] In accordance with the present invention, a set of exoglycosidases for use in glycan sequencing is provided, the set of exoglycosidases comprising two or more exoglycosidases, Each of these exoglycosidases has a different exoglycosidase activity specific for cleaving a different terminal carbohydrate from the non-reducing end of the glycan, Each of these exoglycosidases contains a peptide tag for immobilizing the exoglycosidase on a matrix support; The peptide tag is located in a position in the exoglycosidase sequence that does not affect exoglycosidase activity.

[0086] In a particular embodiment, we demonstrate the production and application of peptide-tagged, highly preferably His-tagged or 6HIS-tagged exoglycosidase enzymes, such as neuraminidase, β-galactosidase and hexosaminidase, for rapid N-glycan sequencing of glycoproteins. The enzymes are capable of high performance digestion in aqueous phase and in immobilized form for e.g. automated solutions.

[0087] Whether it be β-glucosidase [Zhou Y et al: Synchronized purification and immobilization of His-tagged beta-glucosidase via Fe3O4 / PMG core / shell magnetic nanoparticles. Sei Rep 7, 41741 (2017)], β-galactosidase-1 [R&D Systems: recombinant human β-galactosidase-1, His-tagged (cat.&6464-GH)] or hexosaminidase [R&D Systems: recombinant human hexosaminidase A / HEXA, His-tagged (cat.#6237-GH)], His-tagging is generally known in the art.

[0088] In a further preferred embodiment, the enzymes may be premixed to form a subset of exoglycosidases, which upon digestion may result in a reaction mixture resulting from the activity of multiple exoglycosidases.

[0089] In a highly preferred embodiment, all applied enzyme mixtures were co-dialysed together to reduce negative salt effects on signal intensity during capillary electrophoresis analysis. In one embodiment, the co-dialysis was performed to exactly the same concentrations.

[0090] In a further preferred embodiment, the mixture as a subset is used in simultaneous reactions of glycan digestion (terminal carbohydrate cleavage).

[0091] Since immobilization affects the catalytic activity of the enzyme, it is particularly important to select the appropriate immobilization technique to achieve the most efficient enzymatic reaction. In the present invention, peptide tags are used for immobilization of exoglycosidases. Peptide tags can be engineered on any part of the molecule and are therefore universally applicable in any exoglycosidase. This is important in the current set of exoglycosidases to be treated similarly, i.e. in the same way. Each recombinant exoglycosidase must be designed to some extent to introduce the peptide tag at a site that does not interfere with the enzyme activity.

[0092] In a preferred embodiment, a tag is used that can bind to a metal-containing resin that comprises a matrix (or a matrix support and a metal ion immobilized thereon), where immobilization specifically involves the use of a chelating ligand.

[0093] The metal ions immobilized on the metal-containing resin are, for example, Cu. 2+ ,Ni 2+ ,Zn 2+ and Co 2+ A transition metal selected from the group consisting of, preferably Ni 2+ For example, the HIS tag has a high affinity for these metal ions and binds strongly to these resins.

[0094] It is an advantage of peptide tags that they are also useful for protein purification, so that the preparation method of the exoglycosidase set of the present invention is simple and straightforward, since the same tag can be applied for isolation (or purification) and immobilization.

[0095] For example, during the isolation of a protein of interest from a cell lysate, for example a bacterial lysate, most other proteins in the lysate do not bind to the resin or only weakly bind to it, for example in the case of His tag.In one example, low concentrations of imidazole are added to both the binding buffer and the washing buffer to disrupt the weak binding of other proteins and to elute any weakly binding proteins.Then, the tagged protein, for example the His tagged protein, can be eluted with a higher concentration of the agent that destroys tag-matrix binding, for example in the case of His tag imidazole.

[0096] The same methods can be used to regenerate matrix supports. Tagged proteins are immobilized on the matrix supports to form resins that can be used for multiple cleavage reactions and are stable. However, they can be regenerated by removing and re-adding a fresh preparation of tagged exoglycanase, thereby regenerating the resin.

[0097] The matrix support can be made from any suitable matrix, such as, but not limited to, agarose, for example, 4% agarose or 6% highly cross-linked agarose.

[0098] With regard to exemplary additional matrices, such supports include, but are not limited to, inorganic materials such as metal oxides, minerals, carbon materials, organic materials such as cellulose, biopolymers, chitosan, agarose, and synthetic polymers without imprints.

[0099] In certain embodiments, wi, such as nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA).

[0100] The combination of these three variables (matrix, ligand, and ion) provides the resin assembly.

[0101] Other tags are also applicable, for example artificial tags that are subsequently chemically modified. Such tags are, for example, aldehyde tags. For example, formylglycine generating enzyme (FGE) can convert cysteine ​​from the conversed 6 amino acid sequence CXPXR to formylglycine with an aldehyde group (also called "aldehyde tag"). [Wang F, Li R, Jian H, Huang Z, Wang Y, Guo Z, Gao R. Design and Construction of an Effective Expression System with Aldehyde Tag for Site-Specific Enzyme Immobilization. Catalysts. 2020; 10(4):410].

[0102] Other types of tags useful for immobilization and attachment are well known and are defined or listed in the . Exoglycosidase digestion of glycans, typically with overnight incubation at 37°C, is listed. In this invention, a fast and robust exoglycosidase sequential method by CE was developed. A preferred embodiment of this method was achieved by fluorescence detection of aminopyrene trisulfonate (APTS)-labeled N-glycans. Exoglycosidase enzymatic digestion was rapidly performed by adjusting the pH of the buffer, applying a temperature gradient, and immobilizing the enzyme.

[0103] In certain embodiments, exoglycanase enzymes in an enzyme set or subset are used together by adjusting the levels (amounts or concentrations) of individual exoglycanases with different substrate (carbohydrate bond) specificities to tailor activity and ideally reach complete cleavage of the terminal sugar moiety in the digestion reaction of each exoglycosidase.

[0104] It is also important to engineer a peptide tag into a position on the exoglycosidase that does not interfere with (impair) or reduce exoglycosidase activity and also allows for binding to a matrix support. This can be done for any exoglycosidase, where the structure of the exoglycosidase is known to a sufficient degree to determine the catalytic site and the part of the molecule that is sufficiently exposed to be suitable for binding to a matrix via the tag. If the 3D structure is not known from measurements, homology models or even sequence analysis to assign function and structural information to parts of the molecule may be suitable for this purpose.

[0105] A useful part of this engineering method is to remove portions of the exoglycosidase that are not necessary for activity, i.e., to "simplify" the molecule. As examples, the following enzymes are used herein:

[0106] β-galactosidase - We used a bacterial (here, Streptococcus pneumoniae) enzyme to achieve high-level production in bacterial expression systems, e.g., E. coli. The enzyme is a cell surface protein and is useful for extracellular use. From the 17 subunits, the first five (subunits 1-5) are responsible for catalytic activity, but only these five subunits have been cloned. Based on the 3D, i.e., X-ray crystal structure (PDB: 4cu6) of the part (137-985 aminosabac) with β-galactosidase activity, it was found that the peptide tag (e.g., 6His) added to the N-terminus was suitable for immobilization and the active center was sufficiently available for the exoglycanase reaction. The structure, origin and function of the complete enzyme (2233 amino acids) have been described by Singh AK et al. [Singh AK et al. Unravelling the multiple functions of the architecturally intricate Streptococcus pneumoniae beta-galactosidase, BgaA PLoS Pathog. 10:e1004364-e1004364(2014)].

[0107] Design of neuraminidase and hexosaminidase - the sequences of these enzymes are also published. In this approach, export signals and peptides that anchor the proteins to the cell wall were omitted from the constructs.

[0108] The polypeptide chain and structure of (bacterial) neuraminidase used herein as a starting point for the design are disclosed by Christensen S and Egebjerg J [Christensen S and Egebjerg J. Cloning, expression and characterization of a sialidase gene from Arthrobacter ureafaciens. Biotechnol Appl Biochem. 2005 Jun;41(Pt 3):225-31. doi: 10.1042 / BA20040144. PMID: 15461582.].

[0109] For the 3D structure, a homology model is available in the SWISS-MODEL repository (Q5W7Q2(Q5W7Q2_FLASK) Flavobacterium sp(strain K172), https: / / swissmodel.expasy.org / repository / uniprot / Q5W7Q2?template=2bf6.1.A&range=69-422).

[0110] Based on the homology model 3D structure, the catalytic domain of the enzyme can be found at the N-terminus of the enzyme, although the C-terminal domain also plays a role in activity.

[0111] [Iwamori M et al. Involvement of the C-terminal tail of Arthrobacter ureafaciens sialidase isoenzyme M in cleavage of the internal sialic acid of ganglioside GM1. J Biochem. 2005 Sep;138(3):327-34. doi: 10.1093 / jb / mvi126. PMID: 16169883.]. The 6His tag (and TEV-protease cleavage site) was engineered to be located near the C-terminus of the molecule so that the active center of the enzyme would be available to the substrate after immobilization. Surprisingly, the construct yielded an active protein.

[0112] In the case of hexosaminidase-hexosaminidase, the crystal structure of the catalytic domain of the enzyme from Streptococcus pneumoniae was available, but not for the entire molecule. Structural information is available [Pluvinage B, et al. Conformational analysis of StrH, the surface-attached exo-β-DN-acetylglucosaminidase from Streptococcus pneumoniae. J Mol Biol. 2013 Jan 23;425(2):334-49. doi: 10.1016 / j.jmb.2012.11.005. Epub 2012 Nov 12. PMID: 23154168.]. In the enzyme redesigned by the inventors as a spacer, the G5 domain was maintained to provide better accessibility of the catalytic domain to the substrate after immobilization. A 6His tag and a TEV enzyme cleavage site were engineered into the C-terminal part of the molecule.

[0113] While the specific and particular design of an exoglycosidase may not be obvious a priori based on the art, one of skill in the art will understand that some solution can be achieved for any exoglycosidase, provided that a suitable site exists in the enzyme for the peptide tag to be sufficiently exposed and located sufficiently far from the catalytic domain.

[0114] Results and Discussion Gene design and construction In the present invention, the exoglycosidase gene is engineered to contain a peptide tag for immobilization.

[0115] The genes of neuraminidase (EC 3.2.1.18), hexosaminidase (EC 3.2.1.52) and β-galactosidase (EC 3.2.1.23) exoglycosidases were designed to allow the expressed proteins to be immobilized by their HIS-tags along the above principles. Taking into account the localization of the active centers, a 6HIS-tag followed by a TEV protease cleavage site was fused to the C-terminus of the enzymatically functional parts of the neuraminidase and hexosaminidase proteins. For protein expression, the genes were integrated into an engineered pET23b plasmid between the NdeI and XhoI sites, resulting in the pET23b-NdeI-neuraminidase-TEV-6HIS-XhoI and NdeI-hexosaminidase-SacI-TEV-6HIS-XhoI constructs, respectively (Figure 1). The expressed proteins are referred to herein as "NANase" and "HEXase." In the case of β-galactosidase, similar structural considerations led to the use of a pET17b-based plasmid engineered for the expression of "GALase" [PNGaseF cikk hiv], fusing an N-terminal 6HIS-TEV tag to its functional portion.

[0116] Protein expression in E. coli BL21 strain The six cysteines of NANase form three disulfide bridges upon proper folding by oxidation, which is not supported in the reducing environment of the bacterial cytosol. Engineered E. coli cells such as SHuffle T7 Express can support proper disulfide bridge formation, resulting in a soluble and catalytically active enzyme. Our attempts to express NANase in the correct form were very successful, resulting in approximately 40 mg of pure protein per liter of culture. HEXase contains only a single Cys. SHuffle T7 Express can assist in the correct folding of the protein by preventing the formation of intermolecular SS bonds, resulting in a soluble and catalytically active enzyme. For the properly folded form of HEXase, 35 mg of pure protein per liter of culture was achieved. Since SHuffle usually produces a significantly smaller amount of cell mass, induced cultures were harvested at a higher centrifugal force (10,000 g). Because GALase does not contain cysteine, expression of the enzyme was attempted in four different BL21(DE3) strains, and BL21(DE3)Codon+RIL was found to be the most efficient.

[0117] The recombinant NANase, HEXase and GALase were purified using a HiTrap Chelating Ni-affinity column after sample preparation steps, and elution was performed with a buffered imidazole solution.

[0118] The purity of the proteins in the eluted fractions was assessed by SDS-PAGE and the protein concentrations were calculated.

[0119] Long-term storage and overnight reference isolation The optimal concentration of each enzyme for complete APTS-labeled glycan digestion was determined by preliminary experiments in aqueous phase, one by one, without buffering the denatured protein samples. It is important to note that in the case of exoglycosidase enzymatic digestion for N-glycan sequencing, only complete digestion is acceptable for intact peak identification. The optimal concentration was 0.15 μM for each enzyme, which was premixed and codialyzed in a buffer of 20 mM Tris-HCl, 50 mM NaCl, pH=7.5, containing the same amount of salt with 20-fold enzyme concentration. The mixture was then diluted 1:1 with glycerol and stored at −20°C when used. This approach resulted in 1) long-term storage of enzyme ready for use after dilution, 2) negligible salt effect during injection for CE analysis, and 3) high performance enzymatic reaction even in the presence of glycerol in the storage mixture, since a 10-fold dilution of the mixture before use was sufficient to reduce its inhibitory effect to a negligible level.

[0120] Three different compositions of solutions were prepared: A) NANase only (0.3 mg / mL) - Mixture 1; B) NANase (0.3 mg / mL) and GALase (0.3 mg / mL) - Mixture 2; and C) NANase (0.3 mg / mL) and GALase (0.3 mg / mL) and HEXase (0.42 mg / ml) - Mixture 3. The concentration of HEXase was slightly higher than the other two enzymes.

[0121] The efficiency of the resulting co-dialysed enzyme mixture (in 50% glycerol after 10-fold dilution) was compared to the mixture used individually and the enzymes without glycerol using the exact same enzyme concentrations overnight at 37° C. (FIG. 2). Panel A shows N-glycan sequencing of an exemplary hIgG1 glycoprotein sample using an overnight digestion at 37° C. where the enzymes were applied individually to the sample, and panel B shows an experiment where pre-mixed and co-dialysed enzymes were applied.

[0122] The results shown in Figure 2 and Table 1 indicate that there is no significant difference in area % between the standard and co-dialysis (stored in 1:1 glycerol) methods. Therefore, the established storage and premixing method can be used for further use and testing.

[0123] [Table 1]

[0124] High-throughput aqueous phase and immobilized exoglycosidase digestion All enzymatic reactions using the three 6HIS-tagged enzyme mixtures were carried out using a temperature gradient method.

[0125] Additionally, samples were buffered at pH=4.5 with 10 mM ammonium acetate to maximize the overall reaction rate of the enzymes working in parallel.6 Due to the properties of the HIS-tagged enzyme, the temperature gradient step was further optimized and reduced to 30 min, with 1) 37°C for 5.0 min, 2) heating to 50°C for 3.0 min, 3) 50°C for 12 min, 4) heating to 60°C for 3.0 min, and 5) heating at 60°C for 7.0 min, obtaining complete N-glycan sequencing in an excellent time frame.

[0126] The experiment was then repeated with the same parameters using immobilized enzyme on Ni-IMAC resin. In the examples, PhyNexus microcolumns were used for this purpose. A bed volume of 40 μL with a pipette tip capacity of 1 ml was sufficient for the analysis. In this method, a bidirectional flow can be applied, which improves the reaction efficiency.

[0127] The pipetting operations and methods therewith can be automated using controller software.

[0128] While the immobilized enzymes used individually resulted in complete digestion in 30 min using the temperature gradient method, the HEXase enzyme showed incomplete digestion when it was immobilized and mixed with the GALase and NANase enzymes. We hypothesize that the three enzymes compete for available Ni ions, with HEXase lagging behind. By increasing the concentration of HEXase to 0.6 μM (4-fold), a complete reaction was obtained in 30 min with the immobilized enzyme (Figure 3).

[0129] Thus, the inventors found that when applying a mixture of exoglycanases for sequencing purposes, the concentrations or ratios of the various types of exoglycanases can be adjusted to reach complete digestion within the limited time frame available for analysis.

[0130] This immobilization method opens up the possibility of automation and of meeting special experimental needs where enzyme immobilization is important.

[0131] Test methods using mAb and biological samples Other glycoprotein samples from different origins, such as daratumumab mAb glycan (panel A), human serum glycan (panel B) and Synagis mAb glycan (panel C), were tested with the premixed and codialysed enzymes in aqueous phase and in immobilized form for 30 min using the temperature gradient method. Separation conditions were the same as in Figure 2. In all cases, complete exoglycosidase digestion was obtained in the given time frame (Figure 4).

[0132] Glycan sequencing by separation methods As a separation method, the reaction mixture was analyzed using capillary gel electrophoresis (CE) with laser-induced fluorescence (LIF) as detection for glycan sequencing.

[0133] CE-LIF is a well-known technique and its modifications are within the skill of the person skilled in the art. Modifications of such methods are described, for example, in reviews. [Lu, CL et al. Capillary Electrophoresis Separations of Glycans, Chem Rev 118(17) (2018) 7867-7885;]

[0134] Those skilled in the art will appreciate that this method is highly preferred due to its speed, reliability and small sample volume. Currently, several analytical methods are applied to identify the structure and linkage composition of glycans in biological samples, notably liquid chromatography (HPLC) or capillary electrophoresis (CE) separation w / o mass spectrometry (MS) analysis [Qin, W. et al. Alteration of Serum IgG Galactosylation as a Potential Biomarker for Diagnosis of Neuroblastoma, J Cancer 9(5) (2018) 906-913.; Ashline, DJ et al. Carbohydrate structural isomers analyzed by sequential mass spectrometry, Anal Chem 79(10) (2007) 3830-42.; Reinhold, VN et al. Structural characterization of carbohydrate sequence, linkage, and branching in a quadrupole ion trap mass spectrometer: neutral oligosaccharides and N-linked glycans, Anal Chem 70(14) (1998) 3053-9.] followed by glucose unit (GU) calculations [Mittermayr, S. and Guttman, A. Influence of molecular configuration and conformation on the electromigration of oligosaccharides in narrow bore capillaries, Electrophoresis 33(6) (2012) 1000-7.; Jarvas, G. et al.Structural identification of N-linked carbohydrates using the GUcal application: A tutorial, J Proteomics 171 (2018) 107-115. and Jarvas, G. et al. Triple-Internal Standard Based Glycan Structural Assignment Method for Capillary Electrophoresis Analysis of Carbohydrates, Analytical Chemistry 88(23) (2016) 11364-11367.] can be selected. .

[0135] example material and method Chemicals and Reagents Water (HPLC grade), acetonitrile, dithiothreitol (DTT), ammonium acetate, acetic acid, sodium dodecyl sulfate (SDS), sodium cyanoborohydride (1 M in THF), ethylenediaminetetraacetic acid (EDTA), glycine, tetramethylethylenediamine (TEMED), ammonium persulfate (APS) and Nonidet P-40 (NP-40) were obtained from Sigma Aldrich (St. Louis, MO, USA). Aminopyrene-1,3,6-trisulfonic acid (APTS), N-linked carbohydrate separation buffer (NCHO) and M1 magnetic beads from the Fast Glycan kit were obtained from Sciex (Brea, CA, USA). hIgG1 glycoprotein was obtained from Molecular Innovations (Peary, MI, USA). Daratumumab (Darazalex) and palivizumab (Synagis) glycoproteins and human serum were kindly provided by the University of Debrecen (Debrecen, Hungary). PNGase F enzyme was produced in-house but is also available from ThermoFisher Scientific or Gibco. Tris-HCL and NaCl were obtained from VWR (Radnor, PA, USA). NaH2PO4 was obtained from Spektrum 3D (Debrecen, Hungary). Glycerol, Coomassie Brilliant Blue R250 and imidazole were manufactured by Merck (Darmstadt, Germany). 40% (37.5:1) acrylamide and bisacrylamide solutions were from Bio-Rad Laboratories (Hercules, CA, USA) and boric acid was from Scharlab (Debrecen, Hungary). Agarose was obtained from Nippon Genetics Europe (Duren, Germany).

[0136] Exoglycosidase enzyme production Microorganisms, vectors, media and enzymes Expression vectors pET23b and pET17b were purchased from Novagen (Madison, WI, USA). Cloning E. coli TOP10 strain was from Invitrogen (Carlsbad, CA, USA). Restriction endonucleases AgeI HF, SacI HF, NdeI and XhoI, and expression host SHuffle® T7 Express Competent E. coli were purchased from New England Biolabs (Ipswich, MA, USA). BL21-CodonPlus(DE3)-RIL E. coli cells were obtained from Agilent Technologies (Santa Clara, CA, USA). T4 DNA ligase was from Thermo Scientific (Waltham, MA, USA). Cells were cultivated in LB broth medium and LB agar (Scharlau, Barcelona, ​​Spain). Ampicillin (Amp) and chloramphenicol (Chl) were obtained from Sigma Aldrich (St. Louis, MO, USA). Amp stock solutions of 100 g / L and Chl stock solutions of 30 g / L were prepared and used at 1,000-fold dilution in culture medium. Coding DNA sequences for the functional parts of neuraminidase (or sialidase) and hexosaminidase were synthesized by Biomatik (Cambridge, ON, Canada) and provided in pUC57 plasmids. Coding sequences for the functional parts of β-galactosidase were synthesized by Twist Bioscience (San Francisco, CA, USA) and provided in pTwist plasmids. DNA sequencing was performed by Macrogen Europe (Amsterdam, The Netherlands).

[0137] Construction of different exoglycosidase expression plasmids The genes for neuraminidase (EC 3.2.1.18), hexosaminidase (EC 3.2.1.52) and β-galactosidase (EC 3.2.1.23) exoglycosidases were designed to allow the expressed proteins to be immobilized by their HIS-tags. Taking into account the localization of the active center, a 6HIS-tag followed by a TEV protease cleavage site was fused to the C-terminus of the enzymatically functional parts of the neuraminidase and hexosaminidase proteins.

[0138] The coding sequence of the 39-990 polypeptide segment of neuraminidase (or sialidase, the native protein without its release signal peptide) from the sia-AU gene from Paenarthrobacter ureafaciens (UniProt ID: Q5W7Q2) and the coding sequence of the 34-1,280 polypeptide segment of β-N-acetylhexosaminidase from the strH gene from Streptococcus pneumoniae serotype 4 (the native protein without its release signal peptide, the LPXTG recognition signal of the sortase and the C-terminal peptidoglycan) (UniProt ID: P49610) were codon-optimized for E. coli and extended at their 3' ends with SacI nuclease and TEV protease cleavage site coding sequences. The synthesized genes were provided in pUC57 between its EcoRV sites. These plasmids and the pET23b expression vector were digested with NdeI and XhoI restriction enzymes, purified from agarose gel, and the C-terminal 6HIS tag coding sequence was fused to the designed gene and ligated by T4 ligase. TOP10 E. coli cells were transformed with the ligation mixture and spread on LB agar plates containing 100 μg / mL ampicillin. Plasmid DNA from selected colonies was purified by Monarch Plasmid Miniprep Kit (New England Biolabs) and analyzed by restriction digestion and sequencing.

[0139] The coding sequence of the 137-985 polypeptide segment of β-galactosidase (catalytic domain of the native enzyme) from the bgaA gene from Streptococcus pneumoniae serotype 4 (UniProt ID: Q8DQP4) was first codon-optimized for E. coli. The synthesized gene was provided in a pTwist plasmid and digested by AgeI HF and SacI HF restriction enzymes. The pET17b plasmid was modified in our laboratory by the insertion of the coding sequence of an N-terminal 6-HIStidine tag, a TEV protease cleavage site, a folding enhancer glycine-serine-HIStidine (GSH) tripeptide and an AgeI restriction nuclease cleavage site by stepwise mutagenesis after the start codon, and the N-terminal polypeptide MHHHHHHENLYFQGSHTG was fused to the catalytic domain of β-galactosidase after the AgeI cleavage site.

[0140] Expression of exoglycosidase proteins The pET23b plasmid encoding neuraminidase-TEV-6HIS (NANase) and hexosaminidase-TEV-6HIS (HEXase) was used to transform SHuffle T7 Competent Escherichia coli (E. coli) cells applying the standard protocol recommended by the manufacturer. Protein expression was performed as follows: 5.0 mL of LB / Amp medium was inoculated and grown overnight at 30 °C and 250 rpm. 0.5–0.75 L of LB / Amp was supplemented with 1.0% (v / v) of the overnight culture and incubated at OD 600The cells were grown at 30°C until the OD reached 0.6-0.8. The cell culture was induced with 0.4 mM IPTG and further incubated at 16°C for 16 h. The cells were harvested by centrifugation at 10,000g and 6.0°C for 30 min in a Heraus Biofuge primo R centrifuge device, followed by freezing at -20°C and then -80°C. The pET17b plasmid encoding 6HIS-TEV-GSH-galactosidase (GALase) was used to transform BL21-CodonPlus(DE3)-RIL Escherichia coli (E. coli) competent bacteria. The cells were grown overnight in 5.0 ml of LB / Amp,Chl medium at 37°C and 250 rpm. 0.25 L of LB / Amp,Chl was inoculated with 0.5% (v / v) of the overnight culture and grown at 37°C and 250 rpm. The OD of the culture was 600 When the β-actin concentration reached 0.6–0.8, it was induced with 0.5 mM IPTG and further incubated for 16 h at 30 °C. Cells were harvested by centrifugation at 4,370 g and 6.0 °C for 30 min in a Heraus Multifuge 3S-R centrifuge and frozen at −20 °C and then −80 °C.

[0141] Purification of recombinant exoglycosidases Cell pellets from 0.5 L SHuffle / Neuraminidase-TEV-6HIS and 0.75 L SHuffle / HEXase producing cell cultures were thawed and suspended in 10 mL and 15 mL ice-cold buffer "A" (20 mM NaH2PO4, 500 mM NaCl, 25 mM imidazole, pH=7.5) supplemented with Complete protease inhibitors, respectively, and incubated on ice for 1 h. After addition of 2.0% (w / v) glass beads (0.5 mm diameter, Sigma-Aldrich), cells were disrupted by 10 sonications of 30 or 45 s each at 10 W power (Cole-Parmer, Vernon Hills, IL, USA). The remaining cell debris was sedimented by ultracentrifugation at 111,000 g for 30 min at 10°C in a Beckman Coulter Optima™ Max-XP ultracentrifuge using an MLA-80 fixed angle rotor. The supernatant was filtered through a 0.45 μm diameter syringe filter and applied directly to a pre-equilibrated 5 mL HiTrap Chelating Ni-affinity column (GE Healthcare, Chicago, IL, USA). NANase eluted at 50% B (buffer B: 20 ​​mM NaH2PO4, 500 mM NaCl, 500 mM imidazole, pH = 7.5) buffer, which corresponds to approximately 260 mM imidazole, while HEXase eluted in a single peak at 25% B (approximately 140 mM imidazole).

[0142] A cell pellet from a 0.25 L cell culture producing BL21-CodonPlus(DE3)-RIL / GALase was thawed, suspended in 10 mL buffer "A" supplemented with Complete protease inhibitors, and incubated on ice for 1 h. 2% (w / v) glass beads were added and cells were disrupted by 10 rounds of sonication for 30-45 s at 10 W power. The remaining cell debris was sedimented by ultracentrifugation at 111,000 g in a Beckman Coulter Optima™ Max-XP ultracentrifuge for 30 min at 10 °C. The supernatant was filtered through a 0.45 μm diameter syringe filter and applied directly to a pre-equilibrated 5.0 mL HiTrap Chelating Ni-affinity column (GE Healthcare, Chicago, IL, USA). The main fraction of pure protein was eluted with 125 mM buffered imidazole, equivalent to 20% B buffer.

[0143] The purity of the proteins in the eluted fractions was checked by 8–10% SDS PAGE, and the protein concentrations were calculated from the absorbance measured at 280 nm with a UV-Vis spectrophotometer (Jasco V-630, Tokyo, Japan) using the following parameters: molecular weight MW = 102.14 kDa and molar extinction coefficient ε = 114,515 M for NANase; -1 cm -1 , and for HEXase, MW = 139.65 kDa and ε = 154,940 M -1 cm -1 , MW = 98.37 kDa, ε = 175,560 M for GALase -1 cm -1 .

[0144] N-glycan sample preparation N-glycan sample preparation was performed using 10 μL of a 10 mg / mL glycoprotein solution (hIgG1, daratumumab, Synagis) or applying 10 μL of human serum at a 50-fold dilution based on Reider et al. [B. Reider, M. Szigeti, A. Guttman, Evaporative fluorophore labeling of carbohydrates via reductive amination, Talanta 185 (2018) 365-369.]. Briefly, glycoprotein samples were denatured for 10 min at 80 °C using 2.0 μL of denaturation mixture (12.5 mM DTT, 0.6% SDS and 0.06% NP40).

[0145] After the incubation step, the samples were digested with 1.0 μL of PNGase F (0.1 mg / mL) in 20 μL of 20 mM AmAc for 2.0 hours at 37° C. After the digestion step, 20 μL of labeling solution was added (containing 6 mM APTS, 100 mM sodium cyanoborohydride and 24% acetic acid in 1 M THF) and labeled overnight at 37° C. and uncapped. Excess dye was then removed using alternating wash cycles of 20 μL of 10x concentrated M1 beads (Sciex) and 185 μL of acetonitrile for a total of four wash cycles. The APTS-labeled samples were then eluted with 100 μL of HPLC grade water.

[0146] Enzyme premixing and codialysis First, enzyme solutions were prepared in mixtures containing 20-fold higher concentrations of enzyme than required for the deglycosylation reaction (based on preliminary experiments targeting optimal enzyme concentrations for a complete enzymatic reaction at 37°C for 1.0 h). Three different compositions of solutions were prepared: A) NANase only (0.3 mg / mL) - Mixture 1; B) NANase (0.3 mg / mL) and GALase (0.3 mg / mL) - Mixture 2; and C) NANase (0.3 mg / mL) and GALase (0.3 mg / mL) and HEXase (0.42 mg / ml) - Mixture 3. The exoglycosidase solutions were dialyzed together overnight at 4.0°C in a buffer of 20 mM Tris-HCl, 50 mM NaCl, pH=7.5. After dialysis, glycerol was added to the protein solution at a final concentration of 50% to facilitate long-term storage (up to 2 years) at -20°C.

[0147] Enzyme immobilization and exoglycosidase digestion For all N-glycan sequencing using immobilized 6HIS-tagged exoglycosidase enzymes, 1 mL size Ni-IMAC pipette tips from PhyNexus (San Jose, CA, USA) were used with a bed volume of 40 μL. Pipetting was performed automatically using over 1000 pipette heads with controller software (Capture-Purify-Enrich, version 2.2.3, PhyNexus). Tips (three tips for each of the three enzyme mixtures) were washed with 1.0 mL of 20 mM ammonium acetate solution (pH = 7.0) for 2.0 min using aspiration and dispensing speeds of 1.4 mL / min and 2.8 mL / min, respectively (total volume 700 μL) before use. Then, 10 μL of solution from each enzyme mixture was added to 90 μL of HPLC grade water (10-fold dilution) and immobilized on the pre-cleaned tip for 20 min using an aspiration rate of 0.6 mL / min and a dispense rate of 1.2 mL / min (300 μL pipetting volume to ensure extensive contact of the enzyme with the resin). Finally, the pipette tip was washed again with 1.0 mL of fresh ammonium acetate using the same conditions as for tip washing. In case of longer storage required, the prepared tips were stored at 2-8 °C for up to 1 month (test period) using 50% glycerol in HPLC grade water. During the preparation of the tips, 100 μL of sample from the N-glycan sample preparation was divided into 20 μL portions and diluted to 100 μL with 10 mM ammonium acetate (pH = 4.5). The three prepared tips were then used using the same conditions as for immobilization for digestion, applying a temperature gradient method for 30 min. Aqueous phase reference digestion experiments were performed using the same parameters, where the same amount of enzyme was added to the samples in a 5.0 μL volume.

[0148] Glycan sequencing by capillary gel electrophoresis All capillary electrophoresis separations were performed on a PA800 Plus capillary electrophoresis system equipped with a laser-induced fluorescence (LIF) detection system with 488 nm excitation and 520 nm emission from Sciex. For all separations, NCHO gel was used as the separation medium, and a bare fused silica capillary with an inner diameter of 50 μm and a total length of 50 cm (effective length of 40 cm) was used. Sample injection was performed by applying a pressure of 5.0 psi for 5.0 seconds for all measurements. The temperature of both the capillary and the sample storage chamber was 20 °C. The applied electric field strength during separation was 600 V / cm (30 kV) with reverse polarity (anode at the inlet end of the capillary).

[0149] Industrial Applications The comprehensive analysis of N-linked carbohydrates of glycoproteins has attracted great interest in recent years in both the biopharmaceutical and biomedical fields. Glycan sequencing has nowadays gained great importance and is applied in several areas of biological and medical research as well as diagnostics.

[0150] Carbohydrate sequencing is a well-established method, but is still mostly performed by laborious manual processes. The present invention is useful for streamlining this process and its adaptation into an automated carbohydrate sequencing method using appropriate exoglycosidase enzymes in conjunction with some of the features of advanced analytical methods, especially the utilization of capillary electrophoresis (CE) equipment. Furthermore, the immobilization of exoglycosidase enzymes, especially in a subset, has the advantage of speeding up and simplifying the process, making the product more easily usable in the laboratory.

[0151] Carbohydrate sequencing finds applications in, among other things, research, diagnostics including personalized diagnostics [Hennig, R. et al. Towards personalized diagnostics via longitudinal study of the human plasma N-glycome, Biochim Biophys Acta 1860(8) (2016) 1728-38.], and biological drug production for analysis and monitoring, etc. References JPEG2024538794000003.jpg241160 JPEG2024538794000004.jpg216160

Claims

1. A set of exoglycosidase enzymes (exoglycosidases) for use in N-glycan sequencing, each of the exoglycosidases having a different exoglycosidase activity specific for cleaving a different terminal carbohydrate from the non-reducing end of a glycan, each of the exoglycosidases comprising a peptide tag for immobilizing the exoglycosidase on a matrix support, the peptide tag being located at a position in the exoglycosidase sequence that does not affect exoglycosidase activity; the set of exoglycosidases a neuraminidase, preferably α-neuraminidase, having the activity of cleaving terminal sialic acids from glycans and having the peptide tag at its C-terminus; β-galactosidase with the peptide tag attached to its N-terminus; a hexosaminidase having the peptide tag attached to the C-terminus of the hexosaminidase; At least A set of exoglycosidases, wherein the level of hexosaminidase is increased relative to neuraminidase and β-galactosidase, such that each exoglycosidase in the set has sufficient hexosaminidase activity to achieve complete cleavage of terminal carbohydrates within a defined period of time.

2. the predetermined period of time is at most 1.0 hour, preferably at most 40 minutes, preferably at most 30 minutes, and preferably at a temperature range of 37-60°C; The set of exoglycosidases according to claim 1.

3. The predetermined period is up to 30 minutes and the temperature range is 37 to 60°C. The set of exoglycosidases according to claim 1.

4. The set of exoglycosidases according to claim 1 , wherein the peptide tags are metal-chelating peptide tags and the matrix support is a metal-containing matrix support.

5. The peptide tag is a His tag, the matrix support is a transition metal-containing matrix support, and the transition metal is more preferably Cu. ２＋ , Ni ２＋ , Zn ２＋ and Co ２＋ The set of exoglycosidases according to claim 4, selected from:

6. The set of exoglycosidases according to any one of claims 1 to 5, wherein each of the exoglycosidases is immobilized on the matrix support.

7. The method comprises the steps of: (a) preparing a medicament for use in a method for treating a medicament ... Within the subset, each exoglycosidase has a different exoglycosidase activity specific for cleaving a different terminal carbohydrate from the non-reducing end of the glycan; The set of exoglycosidases according to any one of claims 1 to 5, preferably wherein in the subset each exoglycosidase is immobilised on the same matrix support.

8. each of said exoglycosidases is immobilized on said matrix support; 8. The set of exoglycosidases of claim 7, wherein in the subset, each exoglycosidase is immobilized on the same matrix support.

9. 9. The set of exoglycosidases of claim 8, wherein the exoglycosidases mixed together are dialyzed together (co-dialyzed) to reduce negative salt effects.

10. Glycan cleavage reactions using multiple subsets of exoglycosidases as defined in claim 7 are performed simultaneously, and each of the reaction mixtures is analyzed to obtain glycan sequence information. Use of the set of exoglycosidases according to claim 7 for glycan sequencing.

11. The use according to claim 10, wherein the result of the glycan cleavage reaction is analyzed to obtain glycan sequence information by a separation method selected from the group consisting of HPLC, UPLC and capillary electrophoresis, preferably with fluorescence detection, preferably by capillary electrophoresis with fluorescence detection.

12. The use according to claim 11, wherein the exoglycosidases mixed together are dialyzed together (co-dialyzed) to reduce negative salt effects in the capillary electrophoresis analysis.

13. A method for preparing the set of exoglycosidases according to any one of claims 1 to 5, comprising: a nucleic acid sequence encoding the exoglycosidase is provided, the exoglycosidase comprising at least one neuraminidase, preferably α-neuraminidase, β-galactosidase and hexosaminidase, having the activity of cleaving terminal sialic acid from glycans; a nucleic acid sequence encoding the peptide tag is inserted into the nucleic acid sequence encoding the exoglycosidase, such that the peptide tag, upon expression, is present in a position in the exoglycosidase sequence that does not affect exoglycosidase activity; the peptide tag is provided at the C-terminus of the neuraminidase; the peptide tag is provided at the N-terminus of the β-galactosidase; the peptide tag is provided at the C-terminus of the hexosaminidase; each exoglycosidase is expressed in an expression system, preferably a bacterial expression system; Each exoglycosidase is isolated, Preferably, said isolating comprises binding said exoglycosidase to a chromatography matrix via said peptide tag; the levels of the exoglycosidases are adjusted so that each exoglycosidase has sufficient exoglycosidase activity to achieve complete cleavage of the terminal carbohydrate within a predetermined time period defined for each exoglycosidase in the set at a predetermined temperature range; and the levels of hexosaminidase are increased relative to neuraminidase and β-galactosidase so that each exoglycosidase has sufficient hexosaminidase activity to achieve complete cleavage of the terminal carbohydrate within a predetermined time period defined for each exoglycosidase in the set; Preferably, said predetermined period of time is at most 1.0 hour, preferably at most 40 minutes, preferably at most 30 minutes; preferably, said temperature range is 37-60°C; A method wherein at least some of said exoglycosidases are mixed together to form a single cleavage reaction mixture.

14. 14. A method for preparing a set of exoglycosidases according to claim 13, wherein a subset of exoglycosidases are mixed together as defined in claim 7, A method wherein the exoglycosidases mixed together are dialyzed together (co-dialyzed) to reduce negative salt effects on signal intensity during capillary electrophoresis analysis.

15. 15. A method for preparing a set of exoglycosidases according to claim 13 or 14, wherein the exoglycosidases are immobilized on a matrix support via the peptide tags, Preferably, Two or more subsets of exoglycosidases are mixed together and co-dialyzed; each subset differs from the others in at least one exoglycosidase, and the subsets may comprise a single exoglycosidase; Within the subset, each exoglycosidase has a different exoglycosidase activity specific for cleaving a different terminal carbohydrate from the non-reducing end of the glycan; A method wherein within a subset, each exoglycosidase is immobilized on the same matrix support.

16. A kit for preparing an immobilized set of exoglycosidases as defined in any one of claims 1 to 5, said kit comprising: a set of exoglycosidases as defined in claims 1 to 5, wherein the exoglycosidases are present in soluble form in a buffer solution as a stock exoglycosidase solution or a set of stock exoglycosidase solutions; a matrix support functionalized to bind to or be capable of being bound by a peptide tag engineered into said exoglycosidase; Optionally, buffers and reagents for carrying out immobilization, buffers for washing said matrix support before or after immobilization, and said storage buffer, preferably containing a stabilizing agent, preferably glycerin. Includes a kit.

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