32 results about "N linked glycans" patented technology

The present disclosure provides recombinantly expressed insulin polypeptides that comprise an N-linked glycan motif. The N-linked glycan motif is not present in wild-type insulins and enables the recombinant expression of glycosylated insulin polypeptides (e.g., in yeast cells). Based on results obtained with synthetic glycosylated insulin conjugates we predict that when these recombinant glycosylated insulin polypeptides are administered to a mammal, at least one pharmacokinetic or pharmacodynamic property of the glycosylated insulin polypeptide will be sensitive to serum concentrations of glucose (or an exogenous saccharide such as alpha-methyl mannose). Exemplary insulin polypeptides, polynucleotides encoding these insulin polypeptides, glycosylated insulin polypeptides, pharmaceutical formulations and sustained release formulations are provided in addition to methods of use and preparation.

The invention belongs to the field of biotechnology, and in particular relates to a method for fast and high throughput detection of N-linked glycans in a glycoprotein sample based on membrane separation. A hydrophobic membrane porous plate and a hydrophilic membrane porous plate are used to process the glycoprotein sample, the glycoprotein sample processing flux is improved, and steps are simplified. In addition, by changing the reaction conditions of PNGase F enzyme, and the sample processing time is successfully shortened. The detection time of the N-linked glycans in the glycoprotein sample can be shortened from 2 days of a traditional method to within 4-6 hours. In addition, the method successfully solves the problem that in traditional experiments only a purified glycoprotein sample can be used to carry out the experiments, and by use of the method, cell culture supernatant can be directly used for experiments. A strong support is provided for fast qualitative analysis of the N-linked glycans in the glycoprotein for screening of early cell lines.

The presently disclosed subject matter provides methods using mass spectrometry for direct profiling of N-linked glycans from a biological sample. In addition, the embodiments of the present invention also disclose novel methods, known as targeted analyte detection (TAD), for improving the detection limit of MALDI-MS. These methods take advantage of the carrier effect of the added standard analytes, which occurs due to the generic sigmoidal shape of the calibration curve. The functionality of TAD depends on the relative enhancement of sensitivity over the increase of the standard deviation at the analysis of target analytes with spiking in exogenous concentration. At certain ranges of exogenous concentration, the increment in the sensitivity overcomes the standard deviation, resulting in an improved LOD. Theoretically, exogenous concentrations approximately at 1 LODorig would generate the optimum LOD improvement. TAD is a cost-effective LOD improvement method, which is not limited to a certain group of analytes, or detection methods or instruments. It can be applied to enhance the detection of any analyte with different detection methods, provided that the analyte of interest can be extracted or is available in synthetic form.

The present invention relates recombinant human α1-antitrypsin (rhAAT) comprising N-linked glycans, wherein at least 10% of said N-linked glycans are tetra-antennary glycans; and the degree of capping with sialic acid on said N-linked glycans (Z/A) is at least 50%. The invention further relates to rhAAT for use as a medicament, in particular for use in the prevention and/or treatment of a disease associated with AAT deficiency, and/or a disease involving neutrophil-mediated tissue damage.

The present invention discloses mannosyl-glycoprotein endo-beta-N-acetylglucosamidase (E.C.3.2.1.96, endo-N-acetyl-beta-D-glucosaminidase acting on the di-N-acetylchitobiosyl part of N-linked glycans) from filamentous fungi such as Trichoderma reesei.

The present invention provides recombinant cells that contain a genetic modification to at least one mannosyl transferase gene. As a result of the modification the cells produce a glycoprotein or glycopeptide that has an N-linked glycan profile that is simplified or more easily humanized. The glycoprotein or glycopeptide can have at least 25% fewer high mannose structures on than the glycoprotein or glycopeptide produced by a reference cell. In some embodiments the modification is a deletion or disruption of a mannosyl transferase gene, which can be in an alg3 gene. Therefore, the proteins produced are more useful for the production of therapeutic glycoproteins than those produced by species having foreign or plant-like patterns of glycosylation. The invention also provides compositions of the glycoproteins or glycopeptides and methods of making them.

Disclosed herein are glycan-modified anti-CD4 monoclonal antibodies with N-linked glycans attached to the variable region. Expression vectors and cell lines useful for the production of such antibodies, and use of such antibodies for HIV prevention and therapy are also disclosed.

The invention provides a polynucleotide encoding an envelope protein comprising gp120 of human immunodeficiency virus-1 (HIV-1) having a mutation at amino acid residues 197-199 whereby a single N-linked glycan is removed. In a typical embodiment, the mutation replaces the asparagine (N) at residue 197 with another amino acid, such as glutamine (Q), creating an N197Q mutation. Because the N197 glycan is highly conserved among HIV-1 subtypes, this approach is applicable across HIV-1 isolates. The invention provides polypeptides, polynucleotides encoding the polypeptides, vectors, and recombinant viruses containing the polynucleotides, antigen-presenting cells (APCs) presenting the polypeptides, immune cells directed against HIV, and pharmaceutical compositions. The invention additionally provides methods, including methods for preventing and treating infection, for killing infected cells, for inhibiting viral replication, for enhancing secretion of antiviral and/or immunomodulatory lymphokines, and for enhancing production of neutralizing antibody.

Provided are compositions comprising one or more isoforms of an erythropoietin (“EPO”) comprising glycans linked thereto, wherein the glycans have Lewis x structures and on average at least six sialic acid moieties per EPO molecule. Further provided are methods for obtaining a composition comprising one or more isoforms of EPO comprising glycans linked thereto, wherein the glycans comprise on average at least six sialic acids per EPO molecule and from zero to two Lewis x structures, the method comprising: a) providing a eukaryotic cell containing a nucleic acid sequence encoding an adenoviral E1A protein in expressible format and a nucleic acid encoding EPO in expressible format, wherein the cell further contains a nucleic acid sequence encoding a sialyltransferase, e.g., an α-2,6-sialyltransferase or an α-2,3-sialyltransferase, under control of a heterologous promoter; b) culturing the cell in a serum-free culture medium and allowing expression of EPO in the cell; c) harvesting the expressed EPO from the cell and/or from the culture medium; and d) purifying and fractionating the EPO to obtain fractions that have an increased average sialic acid content of the N-linked glycans per EPO molecule, to obtain a composition comprising one or more isoforms of an EPO comprising glycans linked thereto, wherein the glycans comprise on average at least six sialic acids per EPO molecule and from zero to two Lewis x structures.

The present invention relates to the use of N-linked glycan profiles of blood or blood component proteins as biomarkers for diagnosing diabetes mellitus and for monitoring the efficacy of anti-diabetic therapy. Specifically, the present invention relates to detecting changes in the amounts of N-linked glycans as diagnostic biomarkers for diabetes mellitus and as indicators of the efficacy of anti-diabetic therapy over time.

A fixed-length alpha-numeric code for representing N-linked glycan structures commonly found in secreted glycoproteins from mammalian cell cultures. The code employs a pre-assigned alpha-numeric index to represent the monosaccharides attached in different branches to the core glycan structure. The present branch-centric representation allows visualization of the structure while the numerical nature of the code makes it machine readable. A difference operator can be defined to quantitatively differentiate between glycan structures for further analysis. The code can be incorporated in a retrievable format into an information management system. A method is also provided for representing the structure of at least a portion of an oligosaccharide, using the fixed-length alpha-numeric code.

Systems and methods for characterizing low molecular weight (LMW) protein drug product impurities are provided. One embodiment uses hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry analysis. After removal of the N-linked glycans from the protein drug product, for example an antibody drug product, the elution of LMW impurities from the HILIC column was determined by the size of the molecular weight species. In some embodiments, the HILIC separation is performed under denaturing conditions, making the detection of LMW forms using this method highly comparable to both SDS-PAGE and CE-SDS methods. LMW drug product impurities include, but are not limited to light chain, half antibody, H2L, H2, HL, HC, peptide backbone-truncated species, and combinations thereof.