Fucosidase mutants and uses thereof

Abstract

The present disclosure relates to a novel mutant form of alpha-fucosidase (alpha-L-fucosidase) that exhibits enhanced alpha-(1,6) fucosidase activity. The present disclosure also relates to compositions comprising the novel mutant form of alpha-fucosidase, and methods of using the novel mutant form of alpha-fucosidase to cleave alpha-(1,6)-linked fucose in a glycoconjugate.

Description

Technical Field

[0001] This disclosure relates to a novel mutant form of α-fucosidase (α-L-fucosidase) that exhibits enhanced α-(1,6)fucosidase activity. This disclosure also relates to compositions comprising the novel mutant form of α-fucosidase, and methods for cleaving α-(1,6)-linked fucose in glycoconjugates using the novel mutant form of α-fucosidase. Background Technology

[0002] N-glycosylation is one of the most common post-translational modifications observed in mammalian proteins and plays a crucial role in regulating the intrinsic properties and biological functions of these proteins. For example, the attachment of N-glycans can significantly affect protein folding, stability, antigenicity, and immunogenicity. Furthermore, N-glycans can directly participate in a wide range of biological recognition processes, including cell adhesion, host-pathogen interactions, cancer metastasis, and immune responses. Although mammalian N-glycans share a basic oligosaccharide core structure, introducing additional modifications to the core structure (such as sialylation and fucosylation) helps to increase the level of structural diversity affecting biological functions.

[0003] The physiological activity of therapeutic antibodies is mediated by two distinct mechanisms. The first involves target antigen neutralization or apoptosis, which contributes to enhanced therapeutic efficacy. The second involves antibody effector functions activated through the formation of immune complexes, antibody-dependent cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). Genetic analysis of patient leukocyte receptor (FcγR) polymorphisms has elucidated the significance of ADCC in the clinical efficacy of therapeutic antibodies, particularly anticancer antibodies. Consequently, the bioindustry has been actively developing ADCC enhancement technologies involving the modification of N-glycans attached to the antibody constant region (Fc). It is known that the amount of α-(1,6)-linked fucose (core fucose) on the innermost GlcNAc of the N-glycan attached to the antibody Fc region affects ADCC activity. Removal of core fucose from the innermost GlcNAc of the N-glycan on the Fc region significantly enhances the ADCC activity of IgG because the binding affinity of non-fucosylated antibodies to the FcγRIIIα receptor increases.

[0004] Numerous methods have been developed to enhance FcγRIIIα binding and ADCC by reducing IgG fucosylation. Some strategies involve developing production cell lines that eliminate or reduce α-(1,6)fucosyltransferase expression. Alternative strategies for reducing fucosylation include silencing the α-(1,6)fucosyltransferase gene using RNAi. However, enzymatic defucosylation of N-glycans remains challenging in vitro because N-glycans are embedded between two Fc domains. The presence of the Fc domains restricts α-fucosidase access to fucose residues, creating steric hindrance and resulting in low efficiency of enzymatic defucosylation.

[0005] Only a few α-L-fucosidases, such as α-L-fucosidase from bovine kidney, α-L-fucosidase from humans (FucA1), α-L-fucosidase from Bacteroides fragilis (BfFuc), and α-L-fucosidase C from Lactobacillus casei (AlfC), have been disclosed to remove core fucose from the innermost GlcNAc residues after removing the outer N-glycans on the Fc region. Due to the limited and low intrinsic function and activity of wild-type enzymes, there is a need to develop modified enzymes to efficiently cleave core fucose from N-glycans. Summary of the Invention

[0006] In one embodiment, this disclosure provides a mutant form of α-L-fucosidase for improving the enzymatic hydrolysis of fucose in vitro.

[0007] In one embodiment, this disclosure provides a mutant α-L-fucosidase comprising a polypeptide having at least 95% sequence identity with SEQ ID NO:1 and having a substitution mutation at amino acid position 247 (K247).

[0008] In one embodiment, this disclosure provides a mutant α-L-fucosidase comprising a polypeptide having at least 97% sequence identity with SEQ ID NO:1 and having a substitution mutation at amino acid position 247 (K247).

[0009] In one embodiment, this disclosure provides a mutant α-L-fucosidase comprising a polypeptide having at least 99% sequence identity with SEQ ID NO:1 and having a substitution mutation at amino acid position 247 (K247).

[0010] In one embodiment, this disclosure provides a mutant α-L-fucosidase comprising a polypeptide having the sequence of SEQ ID NO:1 and having a substitution mutation at amino acid position 247 (K247).

[0011] In some embodiments, the substitution mutation is selected from the group consisting of: K247A, K247C, K247D, K247E, K247F, K247G, K247I, K247L, K247M, K247N, K247P, K247Q, K247S, K247T, K247V, K247W, and K247Y.

[0012] In some embodiments, the mutant α-L-fucosidase has the sequence of any one of SEQ ID NO:2-18.

[0013] In some embodiments, the mutant α-L-fucosidase of this disclosure can be used to efficiently cleave the core fucose in natural glycoproteins without denaturing or degrading the glycoprotein.

[0014] In some embodiments, the fucosidases described herein can hydrolyze one or more α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucose. The fucose may be present in N- and / or O-linked polysaccharides within the glycoconjugate.

[0015] In some embodiments, the α-fucosidase is a recombinant Bacteroides α-fucosidase.

[0016] In some embodiments, the α-fucosidase exhibits an optimal pH of 6.5-7.5.

[0017] In some embodiments, this disclosure provides a composition comprising the mutant α-L-fucosidase described above to facilitate Fc glycoengineering of antibodies or Fc fusion proteins (such as therapeutic antibodies).

[0018] In some embodiments, the composition further comprises at least one glycosidase.

[0019] In some embodiments, the glycosidase may be an exoglycosidase.

[0020] In some embodiments, the glycosidase may be an endoglucosidase. The endoglucosidase includes, but is not limited to, endoglucosidase-β-N-acetylglucosidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof.

[0021] In some embodiments, the compositions are useful and efficient for the in vitro defucosylation of glycoconjugates. Specifically, the compositions described herein can be used for the in vitro core defucosylation of glycoproteins.

[0022] In some embodiments, the core defucosylation is core α-(1,6) defucosylation.

[0023] In some embodiments, the core defucosylation is core α-(1,3) defucosylation.

[0024] In some embodiments, the defucosylation can be performed without denaturing or degrading the glycoprotein.

[0025] In one embodiment, this disclosure provides a method for preparing defucosylated glycoconjugates in vitro, the method comprising contacting the glycoconjugate comprising one or more fucoses sequentially or simultaneously with an endoglucosidase and a mutant α-L-fucosidase as described above.

[0026] In some embodiments, the method includes the following sequential steps: (a) contacting the glycoconjugate with the endoglucosidase; and (b) contacting the glycoconjugate with a mutant α-L-fucosidase as described above.

[0027] In some embodiments, the method further includes step (c) terminating the reaction.

[0028] In some embodiments, step (c) is performed at approximately 65°C for 15-25 minutes.

[0029] In some embodiments, step (a) and / or step (b) are performed at approximately 37°C for 0.5-2 hours.

[0030] In some embodiments, the method includes the following sequential steps: (a) contacting the glycoconjugate with an endoglucosidase, the endoglucosidase comprising, but not limited to, endoglucosidase-β-N-acetylglucosidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof; and (b) contacting the glycoconjugate with the mutant α-fucosidase of the present invention described above. The glycoconjugate comprises one or more fucoses selected from α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucoses. The fucose may be present in N- and / or O-linked polysaccharides in the glycoconjugate.

[0031] In some embodiments, the method includes contacting a composition comprising the above-described mutant α-L-fucosidase and at least one glycosidase. The endoglycosidase comprises, but is not limited to, endo-β-N-acetylglucosidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof. The glycoconjugate comprises one or more fucoses selected from α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucoses. The fucose may be present in N- and / or O-linked glycans in the glycoconjugate. Detailed Implementation

[0032] For convenience, certain terms used in the context of this disclosure are collected herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0033] Unless the context clearly indicates otherwise, the singular forms “a”, “an” and “the” used herein include plural indicators.

[0034] When referring to polypeptides, the terms "substantially identical" or "substantially the same" mean that, when optimally aligned with another amino acid residue by appropriate polypeptide insertion or deletion, as measured by any known sequence identity algorithm (such as BLAST, ALIGN, or Megalign (DNASTAR) software), at least 95%, and more preferably at least 96%, 97%, 98%, or 99%, of the amino acid residues are substantially identical to the entire sequence of a reference polypeptide sequence. A polypeptide sequence substantially identical to a reference polypeptide sequence can exhibit the same function.

[0035] In this document, a range is expressed as from “about” one specific value and / or to “about” another specific value. When such a range is expressed, embodiments encompass a range from one specific value and / or to another specific value. Similarly, when a value is expressed as an approximation using the term “about,” it should be understood that the specific value forms another embodiment. It should be further understood that each endpoint of a range is valid relative to and independent of the other endpoint. As used herein, the term “about” means ±20%, preferably ±10%, and more preferably ±5%.

[0036] As used herein, the term "glycan" refers to either a polysaccharide or an oligosaccharide. Glycans can be homopolymers or heteropolymers of monosaccharide residues and can be linear or branched. Glycans can also be used to refer to the carbohydrate portion of glycoconjugates such as glycoproteins, glycolipids, or proteoglycans.

[0037] As used herein, the term "glycosidase" refers to an enzyme that catalyzes the hydrolysis of glycosidic linkages or glycosidic bonds.

[0038] As used herein, the term "glycoconjugate" refers to all molecules in which at least one sugar moiety is covalently linked to at least one other moiety, including but not limited to N-linked glycoproteins, O-linked glycoproteins, glycolipids, proteoglycans, etc.

[0039] As used herein, the term “N-linked glycoprotein” refers to a glycoprotein in which a glycan is attached to a nitrogen atom of a normal asparagine or arginine side chain.

[0040] As used herein, the term "O-linked glycoprotein" refers to a glycoprotein in which the glycan is attached to the hydroxyl oxygen of a normal serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side chain, or to the oxygen of a lipid such as ceramide.

[0041] A first aspect of the invention relates to a polypeptide fragment comprising a mutant form of α-L-fucosidase from Lactobacillus casei, the mutant form having a sequence substantially identical to that of polypeptide SEQ ID NO:1 and having a substitution mutation at position 247.

[0042] SEQ ID NO:1 refers to the sequence of α-fucosidase from Lactobacillus casei, with GeneBank accession number CAQ67984.1.

[0043] MNDNVAWFKQAKYGMMIHWGLYSLLAGEYRGESSSAYAEWIQSKFQIPNAEYGNLAT

[0044] AFNPLYFDAKKIVALAKQCGMQYLVVTTKHHDGFAMYHSKVDAYNVYDATPFHRDII

[0045] GELAEACQKAGLKFGLYYSQDLDWHDPNGGGYKSNDVETAGTTWDNSWDFPDEDQ

[0046] KNFDLLCFDNKILPQIKEIMSNYGDIATAWFDVPMTLSEAQSQTIYDTVRELQPNCLINSR

[0047] LGNGKYDFVSLGDNEIPKNKEDMNKTDVDYNEITGFKPSPLGLYETAGTINDSWGFSY

[0048] HDQNWKTPRTLYRYKQHLNDFGINYLLNVGLDPLGRVPMMAEENLLAAKALEDEANR (SEQ ID NO: 1).

[0049] In some embodiments, the substitution mutations described above are selected from the group consisting of: K247A, K247C, K247D, K247E, K247F, K247G, K247I, K247L, K247M, K247N, K247P, K247Q, K247S, K247T, K247V, K247W, and K247Y, preferably K247D and K247E.

[0050] In some embodiments, the substitution mutation described above is K247D, and the mutant α-L-fucosidase has the sequence

[0051] In some embodiments, the substitution mutation described above is K247E, and the mutant α-L-fucosidase has the sequence

[0052] In some embodiments, the substitution mutation described above is K247F, and the mutant α-L-fucosidase has the sequence

[0053]

[0054] In some embodiments, the substitution mutation described above is K247A, and the mutant α-L-fucosidase has the sequence

[0055]

[0056] In some embodiments, the substitution mutation described above is K247C, and the mutant α-L-fucosidase has the sequence

[0057]

[0058] In some embodiments, the substitution mutation described above is K247G, and the mutant α-L-fucosidase has the sequence

[0059]

[0060] In some embodiments, the substitution mutation described above is K247I, and the mutant α-L-fucosidase has the sequence

[0061]

[0062]

[0063] In some embodiments, the substitution mutation described above is K247L, and the mutant α-L-fucosidase has the sequence

[0064]

[0065] In some embodiments, the substitution mutation described above is K247M, and the mutant α-L-fucosidase has the sequence

[0066]

[0067] In some embodiments, the substitution mutation described above is K247N, and the mutant α-L-fucosidase has the sequence

[0068]

[0069] In some embodiments, the substitution mutation described above is K247P, and the mutant α-L-fucosidase has the sequence

[0070]

[0071]

[0072] In some embodiments, the substitution mutation described above is K247Q, and the mutant α-L-fucosidase has the sequence

[0073]

[0074] In some embodiments, the substitution mutation described above is K247S, and the mutant α-L-fucosidase has the sequence

[0075]

[0076] In some embodiments, the substitution mutation described above is K247T, and the mutant α-L-fucosidase has the sequence

[0077]

[0078] In some embodiments, the substitution mutation described above is K247V, and the mutant α-L-fucosidase has the sequence

[0079]

[0080]

[0081] In some embodiments, the substitution mutation described above is K247W, and the mutant α-L-fucosidase has the sequence

[0082]

[0083] In some embodiments, the substitution mutation described above is K247Y, and the mutant α-L-fucosidase has the sequence

[0084]

[0085] Mutant α-fucosidases can hydrolyze one or more α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucose. Fucoose can be present in N- and / or O-linked glycans within glycoconjugates. Fucoose can be core α-(1,3)-fucoose or core α-(1,6)-fucoose.

[0086] A second aspect of the invention relates to a composition comprising the above-described mutant α-fucosidase and at least one glycosidase.

[0087] In some embodiments, the glycosidase is an exoglycosidase.

[0088] In some embodiments, the glycosidase is an endoglucosidase. The endoglucosidase includes, but is not limited to, endoglucosidase-β-N-acetylglucosidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof.

[0089] In some embodiments, the endoglycosidase includes a mutated EndoS at position D233.

[0090] In some embodiments, the endoglycosidase is an EndoS2 containing mutations at positions T138, D226, T227 and / or T228.

[0091] In some embodiments, the endonuclease is an EndoS2 mutation selected from the group consisting of: T138D, T138E, T138F, T138H, T138K, T138L, T138M, T138N, T138Q, T138R, T138V, T138W, D182Q, D226Q, T227Q, and T228Q.

[0092] In some embodiments, the compositions are useful and efficient for the in vitro defucosylation of glycoconjugates. Specifically, the compositions described herein can be used for the in vitro core defucosylation of glycoproteins. Defucosylation can be performed without denaturing or degrading the function of the glycoprotein.

[0093] In some embodiments, the compositions of the present invention relate to the efficient hydrolysis of N-glycans on the Fc region of an antibody. As used herein, the term “N-glycan” refers to an N-linked oligosaccharide attached to an N-acetylglucosamine (GlcNAc) linked to an amide nitrogen of an asparagine residue in the Fc region of an antibody or a fragment thereof.

[0094] In some embodiments, the N-glycan is a high-mannose, hybrid, or complex type with or without a core fucose.

[0095] A third aspect of the invention relates to a method for preparing defucosylated glycoconjugates in vitro. The glycoconjugates may be treated sequentially or simultaneously with an endoglucosidase and a mutant α-fucosidase as described above.

[0096] In some embodiments, the method includes the steps of: (a) contacting the glycoconjugate with an endoglucosidase, said endoglucosidase comprising, but not limited to, endoglucosidase-β-N-acetylglucosidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof; and (b) contacting the glycoconjugate with the mutant α-fucosidase of the present invention described above. The glycoconjugate comprises one or more fucoses selected from α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucoses. Fucoose may be present in N- and / or O-linked glycans in the glycoconjugate.

[0097] In some embodiments, the method includes contacting a composition comprising the above-described mutant α-L-fucosidase and at least one glycosidase. The endoglycosidase comprises, but is not limited to, endo-β-N-acetylglucosidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof. The glycoconjugate comprises one or more fucoses selected from α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linked fucoses. Fucose may be present in N- and / or O-linked glycans in the glycoconjugate.

[0098] In some embodiments, the endoglycosidase is Streptococcus pyogenes endoglycosidase S2, wherein Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position D233, Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position T138, Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position D182, Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position D184, Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position D186, Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position D226, or Streptococcus pyogenes endoglycosidase S2 has a mutation at amino acid position T227.

[0099] In some embodiments, the endoglycosidase includes a mutated EndoS at position D233.

[0100] In some embodiments, the endoglycosidase is an EndoS2 containing mutations at positions T138, D226, T227 and / or T228.

[0101] In some embodiments, the endonuclease is an EndoS2 mutation selected from the group consisting of: T138D, T138E, T138F, T138H, T138K, T138L, T138M, T138N, T138Q, T138R, T138V, T138W, D182Q, D226Q, T227Q, and T228Q.

[0102] In some embodiments, the duration of the reaction mixture is set to at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, or 100 minutes, preferably less than 60 minutes. The reaction temperature is preferably room temperature, more preferably about 20°C, 25°C, 30°C, 35°C, 40°C, or 45°C, and most preferably about 37°C.

[0103] Example

[0104] Example 1. Protein expression construct

[0105] Wild-type α-fucosidase C (AlfC) was amplified from *Lactobacillus casei* BL23 genomic DNA (ATCC 393) by polymerase chain reaction (PCR) and cloned into pET47N, where a His tag fusion was performed at the N-terminus. Using the following forward primers and a plasmid serving as a template encoding wild-type AlfC, saturation mutagenesis was employed to produce substitutions of wild-type lysine at position 247 with each of the other 19 natural amino acids.

[0106] Table 1. Forward primers used for site-directed mutagenesis.

[0107]

[0108] All substitutions were performed using the following reverse primer: 5'–CGG GAT CTC GTT ATC GCC CAG–3' (SEQ ID NO:38). The PCR program for saturation mutagenesis was as follows: 16 cycles of 95°C for 3 min; 95°C for 45 s, 58°C for 50 s, 68°C for 8 min; and a final step of 72°C for 10 min. The mutations were confirmed by DNA sequencing. Other enzymes used in the study (such as EndoS or EndoS2) were cloned into pET28a, with His-tag fusion at the N-terminus.

[0109] Example 2. Protein Expression and Purification

[0110] The protein expression construct was transformed into BL21(DE3) cells (EMD Biosciences, San Diego, CA). Protein overexpression was induced for 20 hours at 16°C by 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG). Cells were homogenized using an autoclave, and the supernatant was collected after centrifugation. The collected supernatant was loaded into a Ni-NTA agarose column and washed with ten column volumes of wash buffer (Tris-HCl, pH 7.4, 200 mM sodium chloride, and 40 mM imidazole). Elution was performed with two column volumes of elution buffer (Tris-HCl, pH 7.4, 200 mM sodium chloride, and 300 mM imidazole). Protein purity was determined by SDS-PAGE and analyzed by… An Ultra-0.5 centrifuge filter (UFC5010BK, 10kDa cutoff) exchanges the protein buffer to the reaction buffer (50mM Tris-HCl, 50mM mannitol, 50mM sorbitol, pH 7.0).

[0111] Example 3. Core defucosylation of glycoproteins

[0112] The core defucosylation activity of mutant fucosidase was determined by analyzing the percentage of core fucose after enzyme treatment. Trastuzumab (Herceptin) was used in this study. (24 mg) was treated with EndoS2 (15.2 μg) at 37 °C for 1 hour, followed by reaction with AlfC fucosidase mutant (2.8 μg) at 37 °C for another 1 hour. The reaction was paused for 20 minutes by heat inactivation at 65 °C. Washed three times with ddH2O in an Ultrafiltration device. The supernatant was collected for quality analysis (Table 2). The percentage of core fucose on the N-glycan in the control group (enzyme-free) was approximately 87%. The defucosylation activity of the wild-type AlfC enzyme was approximately 19%. Mutating a negatively charged amino acid residue at K247 significantly increased the defucosylation activity to approximately 80%, and other mutants at K247, besides the positively charged residue, also showed enhanced defucosylation activity. The percentage of defucosylation was calculated as follows:

[0113]

[0114] Table 2. Analysis of the percentage of defucosylation.

[0115]

[0116]

[0117] “NF” represents the structure of the core GlcNAc-fucose, and “N” represents the structure of the core GlcNAc.

[0118] Although this disclosure has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications, and variations thereof will be apparent to those skilled in the art. All such alternatives, modifications, and variations are considered to fall within the scope of this disclosure.

[0119] References:

[0120] US11193155B2

[0121] Klontz EH, Li C, Kihn K, Fields JK, Beckett D, Snyder GA, Wintrode PL, Deredge D, Wang LX, Sundberg EJ. Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation. Nature Communications, Dec 4, 2020; 11(1):6204.

[0122] Li C, Zhu S, Ma C, Wang LX. Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins. Journal of the American Chemical Society, 25 October 2017; 139(42):15074-15087.

[0123] Osanjo G, Dion M, Drone J, Solleux C, Tran V, Rabiller C, Tellier C. Directed evolution of the alpha-L-fucosidase from Thermotoga maritima into an alpha-L-transfucosidase. Biochemistry. January 30, 2007; 46(4):1022-33.

[0124] US10415021

[0125] Sequence list translation

[0126] CHO PHARMA, INC. (CHO PHARMA, INC.)

[0127] FUCOSIDASE MUTANTS AND THE USE THEREOF Fucosidase Mutants and Their Uses

Claims

1. A mutant α-L-fucosidase having an amino acid sequence as shown in any one of SEQ ID NO: 2-18.

2. A method for preparing defucosylated sugar conjugates in vitro, the method comprising: A glycoconjugate comprising one or more fucoses is sequentially contacted with an endoglucosidase and the mutant α-L-fucosidase according to claim 1.

3. The method according to claim 2, comprising the following sequential steps: (a) contacting the glycoconjugate with the endoglucosidase; and (b) Contact the glycoconjugate with the mutant α-L-fucosidase according to claim 1.

4. The method according to claim 3, further comprising (c) terminating the reaction.

5. The method according to claim 4, wherein step (c) is performed at 65°C for 15-25 minutes.

6. The method according to any one of claims 3 to 5, wherein step (a) and / or step (b) are performed at 37°C for 0.5 to 2 hours.

7. The method according to any one of claims 3 to 5, wherein the endoglucosidase is endoglucosidase-β-N-acetylglucosidase (NAG), EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, EndoS2, and variants thereof.

8. The method of claim 7, wherein EndoS2 comprises a substitution mutation selected from the group consisting of: T138D, T138E, T138F, T138H, T138K, T138L, T138M, T138N, T138Q, T138R, T138V, T138W, D182Q, D226Q, T227Q, and T228Q.

9. The method according to any one of claims 2 to 5, wherein the fucose is α-(1,6) linked fucose.

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