Dataset Information

Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation.

ABSTRACT: Monoclonal antibodies (mAbs) have been developed as therapeutics, especially for the treatment of cancer, inflammation, and infectious diseases. Because the glycosylation of mAbs in the Fc region influences their interaction with effector cells that kill antibody-targeted cells, and the current method of antibody production is relatively expensive, efforts have been directed toward the development of alternative expressing systems capable of large-scale production of mAbs with desirable glycoforms. In this study, we demonstrate that the mAb trastuzumab expressed in glycoengineered P. pastoris can be remodeled through deglycosylation by endoglycosidases identified from the Carbohydrate Active Enzymes database and through transglycosylation using glycans with a stable leaving group to generate a homogeneous antibody designed to optimize the effector functions. The 10 newly identified recombinant bacterial endoglycosidases are complementary to existing endoglycosidases (EndoA, EndoH, EndoS), two of which can even accept sialylated tri- and tetraantennary glycans as substrates.

SUBMITTER: Liu CP

PROVIDER: S-EPMC5789950 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation.

Liu Chiu-Ping CP Tsai Tsung-I TI Cheng Ting T Shivatare Vidya S VS Wu Chung-Yi CY Wu Chung-Yi CY Wong Chi-Huey CH

Proceedings of the National Academy of Sciences of the United States of America 20180108 4

Monoclonal antibodies (mAbs) have been developed as therapeutics, especially for the treatment of cancer, inflammation, and infectious diseases. Because the glycosylation of mAbs in the Fc region influences their interaction with effector cells that kill antibody-targeted cells, and the current method of antibody production is relatively expensive, efforts have been directed toward the development of alternative expressing systems capable of large-scale production of mAbs with desirable glycofor ...[more]

PMID: 29311294

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Project description:The majority of mammalian proteins are glycosylated, with the glycans serving to modulate a wide range of biological activities. Variations in protein glycosylation can have dramatic effects on protein stability, immunogenicity, antibody effector function, pharmacological safety and potency, as well as serum half-life. The glycosylation of therapeutic biologicals is a critical quality attribute (CQA) that must be carefully monitored to ensure batch-to-batch consistency. Notably, many factors can affect the composition of the glycans during glycoprotein production, and variations in glycosylation are among the leading causes of pharmaceutical batch rejection. Currently, the characterization of protein glycosylation relies heavily on methods that employ chromatography and/or mass spectrometry, which require a high level of expertise, are time-consuming and costly and, because they are challenging to implement during in-process biologics production or during in vitro glycan modification, are generally performed only post-production. Here we report a simplified approach to assist in monitoring glycosylation features during glycoprotein engineering, that employs flow cytometry using fluorescent microspheres chemically coupled to high-specificity glycan binding reagents. In our GlycoSense method, a range of carbohydrate-sensing microspheres with distinct optical properties may be combined into a multiplex suspension array capable of detecting multiple orthogonal glycosylation features simultaneously, using commonplace instrumentation, without the need for glycan release. The GlycoSense method is not intended to replace more detailed post-production glycan profiling, but instead, to complement them by potentially providing a cost-effective, rapid, yet robust method for use at-line as a process analytic technology (PAT) in a biopharmaceutical workflow or at the research bench. The growing interest in using in vitro glycoengineering to generate glycoproteins with well-defined glycosylation, provides motivation to demonstrate the capabilities of the GlycoSense method, which we apply here to monitor changes in the protein glycosylation pattern (GlycoPrint) during the in vitro enzymatic modification of the glycans in model glycoproteins.

Dataset Information

Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation.

Publications

Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation.

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