Provided herein are methods for assessing cell surface glycans, e.g., N-glycans, by assessing a sample of released surface glycans, and determining the presence, absence, or level of glycans present in the sample. Also provided are methods of assaying and/or evaluating a cell composition by assessing the cell surface glycan profile of the cell composition and comparing the profile to a reference sample. Methods for manufacturing and/or culturing a plurality of cell compositions having consistent surface glycan expression with low variability are also provided.

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.

Disclosed herein, are compositions and methods useful in expressing a functional NGLY1 protein in a subject by administration of an rAAV containing a transgene encoding NGLY1. Also disclosed herein are methods for treating an NGLY1 gene deficiency in a subject in need thereof.

A method for making a milk product (e.g. a yogurt) comprising adding an effective amount of an N-linked glycosidase and/or an O-linked glycosidase to milk.

Provided herein is an -fucosidase that can cleave a conjugate comprising an N-glycan and a label where the label is added by amine reactive chemistry. The -fucosidase also has an accelerated reaction time using Schiff base labeled N-glycans compared with BKF. A reaction mix, enzyme mix and kit comprising the -fucosidase are provided, as well as a method for analyzing glycoproteins. The -fucosidase finds particular use in analyzing the N-glycans of therapeutic glycoproteins.

Compositions and methods are provided for efficiently preparing a completely deglycosylated antibody where efficiency is measured in relative amounts of reagents in soluble or lyophilized form, and time and temperature of the reaction. Compositions and methods are also provided for separating substantially all N-linked glycans from a glycosylated antibody and for preserving functionality of the antibody. The methods are compatible with glycan labeling and protease digestion without the need for prior purification steps.

Provided are lectenz molecules, which are mutated carbohydrate processing enzymes that are catalytically inactive and that have had their substrate affinity increased by at least 1.2 fold. Further provided are methods for making and methods of using such lectenz. Additional mutated proteins following the lectenz approach are further provided.

A method for detecting a site which can be modified with an N-linked glycan chain and to which an N-linked glycan chain is actually linked in a glycoprotein; and a method for determining the state of an N-linked glycan chain addition at the site are provided. A glycoprotein having an N-linked glycan chain linked thereto is subjected to an N-linked glycan chain removal treatment with a peptide N-glycanase, subsequently a site capable of being modified with an N-linked glycan chain, in which an Asn residue has been changed to an Asp residue by the action of the peptide N-glycanase, is treated with an endo-type peptidase capable of recognizing an Asp residue to thereby produce peptide fragments, and subsequently the mass of the fragments is detected. In this manner, a site which can be modified with an N-linked glycan chain and to which an N-linked glycan chain is actually linked can be detected. Furthermore, the proportion or state of of the N-linked glycan chain addition at the site can be determined from the intensity of a signal generated upon the detection.

The carbohydrate processing enzyme PNGase F was catalytically inactivated through mutation. Additional mutations yielded a catalytically inactive carbohydrate-binding protein with lectin-like properties including high affinity and specificity N-linked glycans, O-linked glycans, or both.

N-glycosylation profiling of glycoprotein biotherapeutics is an essential step in each phase of product development in the biopharmaceutical industry. For example, during clone selection hundreds of clones should be analyzed quickly from limited amounts of samples. On the other hand, identification of disease related glycosylation alterations can serve as early indicators for various pathological conditions in the biomedical field. We describe an improved packed bed column PNGase F functionalized column reactor. The reactor may be packed into a pipette tip column. In some embodiments, a second column or mixed stationary phase may be packed into the column to capture and purify the cleaved glycan prior to analysis. Complete N-glycan removal can be obtained in 10 minutes from all major N-linked glycoprotein types. The approach can be readily applied to automated sample preparation systems, such as liquid handling robots.