33 results about "Endoglycosidase" patented technology

The invention relates to an endonuclease S2 mutant, a preparation method of the endonuclease S2 mutant and application of the endonuclease S2 mutant in antibody glycosylation transformation. The glycosidase S2 mutant comprises a carbohydrate binding module (CBM) defined by M677-D843 in SEQ ID NO: 1, and the amino acid sequence of the glycosidase S2 mutant comprises SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

The invention relates to a method for preparing an antibody-coupled drug, the antibody-coupled drug is subjected to fixed-point coupling based on an antibody Fc region N-glycosylation site, and the method comprises the following steps: (1) providing a donor containing oxazoline oligosaccharide, an antibody containing a GlcNAc motif and immobilized endoglycosidase with glucoside transfer activity; (2) covalently linking an activated donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif under the catalytic action of glycosidase; and one-step coupling is realized. The invention also relates to an endonuclease fusion protein which comprises the endonuclease, a Halo tag and / or a His tag which are covalently linked. The invention also relates to an immobilized glycosidase fusion protein obtained by immobilizing the glycosidase fusion protein on a support, a preloaded column filled with the immobilized fusion protein, and a purification method of the immobilized glycosidase fusion protein for an antibody conjugate.

This disclosure relates to recombinant immunoglobulin endoglycosidases and uses in managing diseases or conditions associated with immune dysregulation or inflammation. In certain embodiments, the recombinant immunoglobulin endoglycosidases comprise sequences disclosed herein. In certain embodiments, this disclosure relates to pharmaceutical compositions and methods of treating or preventing an autoimmune disease or inflammatory condition comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase, or nucleic acids or vector encoding the same, as disclosed herein.

The disclosure provides pharmaceutical compositions comprising an anti-PD-1 antibody or an anti-PD-L1 antibody. In some aspects, the pharmaceutical compositions are formulated for subcutaneous delivery. In some aspects, the pharmaceutical compositions further comprise an endoglycosidase hydrolase enzyme. Other aspects of the present disclosure are directed to methods of subcutaneously delivering a pharmaceutical composition comprising an anti-PD-1 antibody or an anti-PD-L1 antibody.

The present disclosure provides a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is subjected to site-specific conjugation, based on an N-glycosylation site in an Fc region of an antibody, and the method includes the steps of: (1) providing a donor containing oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase with glycosyltransferase activity; (2) covalently attaching the activated donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif under the catalysis of the endoglycosidase. The present disclosure further relates to an endoglycosidase fusion protein, including the endoglycosidase, a Halo tag, and / or a His tag that are covalently attached. The present disclosure further relates to an immobilized endoglycosidase fusion protein obtained by immobilizing the endoglycosidase fusion protein on a support, and a prepacked column filled with the immobilized fusion protein, as well as use of the immobilized endoglycosidase fusion protein for purification of an antibody conjugate.

The disclosure provides pharmaceutical compositions comprising an antibody and at least two antioxidants. In some aspects, pharmaceutical composition is formulated for subcutaneous delivery. In some aspects, the pharmaceutical composition further comprises an endoglycosidase hydrolase enzyme. Other aspects of the present disclosure are directed to methods of subcutaneously delivering the pharmaceutical composition.

To provide a gum arabic seyal modified glycolytic enzyme useful for assimilating modified sugars of seyal gum arabic.SOLUTION: A gum arabic seyal modified glycolytic enzyme exhibits endoglycosidase activity and comprises one of following amino acid sequences (a)-(d): (a) a specific amino acid sequence; (b) an amino acid sequence having at least 90% of sequence identity to the specific amino acid sequence; (c) a specific amino acid sequence, different from (a) and (b); and (d) an amino acid sequence having at least 90% of sequence identity to (c).SELECTED DRAWING: Figure 1

Endoglycosidase-assisted peptide mapping workflow systems for mass spectrometry (MS) characterization of non-consensus N-glycosylation in monoclonal antibodies (mAbs) are disclosed. The feasibility of the workflow was demonstrated by an atypical glycosite located within an NPNNXN (SEQ ID NO: 1) sequence in a 25-residue tryptic peptide. With the aids of endoglycosidase treatment, the resulting truncated glycan structures improved peptide ionization efficiency in MS and hence facilitated reliable quantitation of glycosite occupancy. The remaining mono- / di-saccharides served as a large mass tag allowing differentiation between the glycopeptide and deamidated peptide, thus allowing for database searching for glycosite localization and automation of the data processing workflow. This workflow offers an efficient solution for characterizing non-consensus N-glycosylation for the development of therapeutic mAbs.

The present invention relates to a method for preparing protein molecules having an Fc region with site-specifically remodeled homogeneous glycan chains, comprising the following steps: a) providing a liquid comprising protein molecules having an Fc region with heterogeneous glycan chains expressed by eukaryotic cells before purification, wherein preferably, the protein molecule having the Fc region with the heterogeneous glycan chains is a Y-type antibody, an Fc fusion protein, or a heavy chain antibody; for example, the liquid comprising the protein molecules having the Fc region with the heterogeneous glycan chains expressed by the eukaryotic cells before purification is a cell supernatant; b) adding a disaccharide linker represented by formula I; and c) adding an endoglycosidase having an activity of specifically hydrolyzing heterogeneous glycan chains in glycoproteins and catalyzing a transglycosylation reaction to carry out a reaction, thereby obtaining a solution comprising protein molecules having an Fc region with site-specifically remodeled homogeneous glycan chains. The present invention further relates to a two-step method and a one-step method for preparing a protein molecule-drug conjugate having an Fc region with site-specifically remodeled homogeneous glycan chains using a liquid comprising the protein molecules having the Fc region with the heterogeneous glycan chains expressed by the eukaryotic cells before purification. The methods can be used in antibody glycoengineering and / or in the preparation of an antibody-drug conjugate.

A method for preparing an antibody-drug conjugate. The antibody-drug conjugate is formed by means of site-directed conjugation on the basis of an N-glycosylation site of an Fc region of an antibody. The method comprises: (1) providing a donor containing an oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase having a glycoside transfer activity; and (2) covalently linking the activated donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif by means of the catalytic action of the endoglycosidase; therefore, one-step conjugation is realized. The present invention also relates to an endoglycosidase fusion protein, which comprises a covalently linked endoglycosidase, a Halo tag, and / or a His tag. The present invention also relates to an immobilized endoglycosidase fusion protein obtained by means of immobilizing an endoglycosidase fusion protein on a support, a prepacked column filled with the immobilized fusion protein, and a method for purifying an antibody conjugate by using the immobilized endoglycosidase fusion protein.

The present disclosure relates to site-specific modification of antibodies at their Fc glycan site and subsequent conjugation with drugs and other entities to produce structurally well-defined antibody conjugates. The method is based on the discovery that N-azidoacetylglucosamine GlcNAz-derived sugar oxazolines can serve as substrates of endoglycosidases, such as Endo S2 and Endo S for enzymatic Fc glycan remodeling to site-specifically introduce an azide-tag in an antibody in a single step, followed by a click reaction to form site-specific antibody-drug conjugates and antibody-ligand conjugates

The disclosure provides pharmaceutical compositions comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody, an anti-LAG-3 antibody, and an endoglycosidase hydrolase enzyme. In some aspects, pharmaceutical composition is formulated for subcutaneous delivery. Other aspects of the present disclosure are directed to methods of subcutaneously delivering the pharmaceutical composition.

The disclosure provides pharmaceutical compositions comprising an antibody and at least two antioxidants. In some aspects, pharmaceutical composition is formulated for subcutaneous delivery. In some aspects, the pharmaceutical composition further comprises an endoglycosidase hydrolase enzyme. Other aspects of the present disclosure are directed to methods of subcutaneously delivering the pharmaceutical composition.

The disclosure provides pharmaceutical compositions comprising an anti-PD-1 antibody or an anti-PD-L1 antibody. In some aspects, the pharmaceutical compositions are formulated for subcutaneous delivery. In some aspects, the pharmaceutical compositions further comprise an endoglycosidase hydrolase enzyme. Other aspects of the present disclosure are directed to methods of subcutaneously delivering a pharmaceutical composition comprising an anti-PD-1 antibody or an anti-PD-L1 antibody.

The present disclosure provides a pharmaceutical composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody, an anti-LAG-3 antibody, and an endoglycosidase hydrolase enzyme. In some aspects, the pharmaceutical compositions are formulated for subcutaneous delivery. Other aspects of the disclosure relate to methods of subcutaneously delivering the pharmaceutical compositions.

The present disclosure relates to site-specific modification of antibodies at their Fc glycan sites, and subsequent conjugation to drugs and other entities, to produce structurally explicitly defined antibody conjugates. The method is based on the discovery that N-azidoacetylglucosamine GlcNAz-derived sugar oxazolines can be used as substrates for endoglycosidases such as Endo S2 and Endo S for enzymatic Fc glycan remodeling to site-specifically introduce the azide tag in the antibody in a single step, such as in a single step, for example, in the antibody. A click reaction is then performed to form a site-specific antibody-drug conjugate and an antibody-ligand conjugate.

The present invention describes a rapid and economical method for the deglycosylation of a glycoprotein. In this method, the glycoprotein is combined with an anionic surfactant, a reducing agent, and a non-ionic surfactant to obtain a stable denatured glycoprotein. Subsequently, an endoglycosidase is added to the denatured glycoprotein to cleave the N-linked glycans and thus obtain the deglycosylated protein. A rapid tool for evaluating the protein conformation by partial deglycosylation is also presented, in which the partially deglycosylated protein is analyzed by capillary electrophoresis (CE-SDS).

The present disclosure provides a method for preparing an antibody-drug conjugate, wherein the antibody-drug conjugate is subjected to site-specific conjugation, based on an N-glycosylation site in an Fc region of an antibody, and the method includes the steps of: (1) providing a donor containing oxazoline oligosaccharide, an antibody containing a GlcNAc motif, and an immobilized endoglycosidase with glycosyltransferase activity; (2) covalently attaching the activated donor containing the oxazoline oligosaccharide to the antibody containing the GlcNAc motif under the catalysis of the endoglycosidase. The present disclosure further relates to an endoglycosidase fusion protein, including the endoglycosidase, a Halo tag, and / or a His tag that are covalently attached. The present disclosure further relates to an immobilized endoglycosidase fusion protein obtained by immobilizing the endoglycosidase fusion protein on a support, and a prepacked column filled with the immobilized fusion protein, as well as use of the immobilized endoglycosidase fusion protein for purification of an antibody conjugate.

Methods of determining glycan pairing content of a protein composition are provided. In exemplary embodiments, the method comprises (a) treating a sample of the protein composition with (i) a mannosidase and (ii) a β1-4 endoglycosidase, to produce a mixture of components of the protein, wherein at least some of the components comprises one or more digested glycan structures; and (b) separating the components of the mixture based on molecular weight; and (c) quantifying the abundance of glycan pairs, optionally, quantifying (i) paired afucosylated glycans, (ii) unpaired afucosylated glycans, (iii) paired high mannose glycans, and / or (iv) unpaired high mannose glycans, of the mixture. In exemplary aspects, the method comprises quantifying the relative unpaired afucosylated (AF) glycan content and / or relative unpaired high mannose (HM) glycan content of the mixture.

A method for preparing a homogeneous antibody-drug conjugate and a related kit. The method comprises the following steps: 1) under a reaction condition 1, placing an antibody, a disaccharide linker, an endoglycosidase in a buffer solution for incubation to obtain an antibody intermediate; and 2) under a reaction condition 2, incubating a small molecule drug-linker and the antibody intermediate obtained in step 1) in a buffer solution to obtain an antibody-drug conjugate. The optimized method can provide a homogeneous antibody-drug conjugate, solving the technical problems in the prior art of inconsistent kinetic properties, poor stability, and a narrow therapeutic window caused by the heterogeneity of drug conjugates.

This invention belongs to the field of biotechnology and relates to the application of an endonuclease in the hydrolysis of high-mannose N-glycoproteins. The endonuclease can recognize and hydrolyze high-mannose N-glycoproteins; the endonuclease is Endo-LB, and its amino acid sequence is shown in SEQ ID NO.1; the hydrolysis temperature is 4–60℃, and the hydrolysis pH is 4.0–10.0. Studies have shown that Endo-LB has the activity of recognizing high-mannose N-glycoproteins, and structurally contains a MucBP domain, exhibiting broad temperature and pH adaptability.

This disclosure relates to recombinant immunoglobulin endoglycosidases. In certain embodiments, the recombinant immunoglobulin endoglycosidases comprise insertions and / or mutations disclosed herein. In certain embodiments, this disclosure relates to pharmaceutical compositions comprising recombinant immunoglobulin endoglycosidases disclosed herein and / or nucleic acids or vector encoding the same. In certain embodiments, this disclosure relates to pharmaceutical compositions. In certain embodiments, this disclosure relates to recombinant immunoglobulin G-specific endoglycosidase CU43 comprising a dipeptide FK insertion at position 240 wherein the dipeptide FK insertion is in reference to positions in the CP40 amino acid sequence.

The invention concerns fusion proteins, wherein two endoglycosidases are fused, possibly via a linker. The fusion enzymes according to the invention have structure (1): EndoX-(L)p-EndoY (1), wherein EndoX is an endoglycosidase, EndoY is an endoglycosidase distinct from EndoX, L is a linker and p is 0 or 1. Such fusion enzymes capable of trimming glycoproteins comprising at least two distinct glycoforms in a single step. The invention further concerns the use of the fusion enzyme according to the invention for trimming glycoproteins. In another aspect, the invention relates to the process of production of the fusion enzyme. In a further aspect, the inventions concerns a process for trimming glycoproteins, comprising trimming the glycoprotein with a fusion enzyme according to the invention, to obtain a trimmed glycoprotein.

The present disclosure provides a pharmaceutical composition comprising an anti-PD-1 antibody and / or an anti-PD-L1 antibody, an anti-LAG-3 antibody, and an endoglycosidase hydrolase. In some embodiments, the pharmaceutical composition is formulated for subcutaneous delivery. Other embodiments of the present disclosure relate to methods of subcutaneously delivering the pharmaceutical composition.

The present disclosure relates to site-specific modification of antibodies at their Fc glycan site and subsequent conjugation with drugs and other entities to produce structurally well-defined antibody conjugates. The method is based on the discovery that N-azidoacetylglucosamine GlcNAz-derived sugar oxazolines can serve as substrates of endoglycosidases, such as Endo S2 and Endo S for enzymatic Fc glycan remodeling to site-specifically introduce an azide-tag in an antibody in a single step, followed by a click reaction to form site-specific antibody-drug conjugates and antibody-ligand conjugates

The present invention discloses a method based on solid-phase fucosylated glycoprotein enrichment and fucosylation enzymatic digestion analysis, comprising the steps of: extracting proteins, then hydrolyzing the proteins to obtain polypeptides and glycopeptides, and then treating with endoglycosidase, including: ① treating the peptide segments with endoglycosidase, then oxidizing and binding the glycopeptides to hydrazide resin, and obtaining fucosylation sites or defucosylated glycopeptides after hydrolysis by fucosidase; ② treating the glycopeptides with endoglycosidase, and then obtaining intact fucosylated glycopeptides using hydrophilic interaction chromatography. The glycoprotein or enzymatically digested glycopeptides can also be enzymatically digested to obtain branched α1,3-fucosylated glycopeptides after oxidation and binding to hydrazide. Finally, mass spectrometry is used to analyze the fucosylated peptides and the peptides containing fucosylation sites. This method is of great significance for the discovery of fucosylated disease markers in diseased cells, clinical tissues and human body fluid specimens, as well as the research on early disease diagnosis and prognosis markers, etc.

The present invention relates to a pharmaceutical formulation with improved stability, comprising: an endoglycosidase hydrolase; a phosphate-based buffer; a stabilizer comprising sodium chloride, sodium edetate, and calcium chloride; and a nonionic surfactant.

The disclosure provides pharmaceutical compositions comprising an anti-PD-1 antibody or an anti-PD-L1 antibody. In some aspects, the pharmaceutical compositions are formulated for subcutaneous delivery. In some aspects, the pharmaceutical compositions further comprise an endoglycosidase hydrolase enzyme. Other aspects of the present disclosure are directed to methods of subcutaneously delivering a pharmaceutical composition comprising an anti-PD-1 antibody or an anti-PD-LI antibody.

This invention relates to a method and related kit for preparing homogeneous antibody-drug conjugates. The method includes the following steps: 1) under reaction conditions 1, incubating an antibody, a disaccharide linker, and a glycosidase in a buffer solution to obtain an antibody intermediate; 2) under reaction conditions 2, incubating a small molecule drug-linker with the antibody intermediate obtained in step 1) in a buffer solution to prepare the antibody-drug conjugate. The optimized method of this application provides a homogeneous antibody-drug conjugate, effectively solving the technical problems of inconsistent kinetic properties, poor stability, and narrow therapeutic window caused by the heterogeneity of the conjugated drug in existing antibody-drug conjugates.