We have cloned the cDNA encoding human GDP-mannose 4,6-dehydratase, the first enzyme in the pathway converting GDP-mannose to GDP-fucose. The message is expressed in all tissues and cell lines examined, and the cDNA complements Lec13, a Chinese Hamster Ovary cell line deficient in GDP-mannose 4,6-dehydratase activity. The human GDP-mannose 4,6-dehydratase polypeptide shares 61% identity with the enzyme from Escherichia coli, suggesting broad evolutionary conservation. Purified recombinant enzyme utilizes NADP ؉ as a cofactor and, like its E. coli counterpart, is inhibited by GDPfucose, suggesting that this aspect of regulation is also conserved. We have isolated the product of the dehydratase reaction, GDP-4-keto-6-deoxymannose, and confirmed its structure by electrospray ionization-mass spectrometry and high field NMR. Using purified recombinant human GDP-mannose 4,6-dehydratase and FX protein (GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase), we show that the two proteins alone are sufficient to convert GDP-mannose to GDP-fucose in vitro. This unequivocally demonstrates that the epimerase and reductase activities are on a single polypeptide. Finally, we show that the two homologous enzymes from E. coli are sufficient to carry out the same enzymatic pathway in bacteria.Fucose is found as a component of glycoconjugates such as glycoproteins and glycolipids in a wide range of species from humans to bacteria. For example, fucose is a component of the capsular polysaccharides and antigenic determinants of bacteria, while in mammals fucose is present in many glycoconjugates, the most widely known being the human blood group antigens. Fucose-containing glycoconjugates have been implicated as playing key roles in embryonic development in the mouse (1) and more recently in the regulation of the immune response, specifically as a crucial component of the selectin ligand sialyl Lewis X (reviewed in Refs. 1 and 2). In all cases, fucose is transferred from GDP-fucose to glycoconjugate acceptors by specific transferases. Thus, defects in GDP-fucose biosynthesis will affect all fucosylation within the cell. Recently, individuals deficient in the biosynthesis of GDP-fucose have been identified (3, 4) and suffer from the immune disorder leukocyte adhesion deficiency type II (LADII).1 These patients fail to synthesize fucosylated blood groups, and their leukocytes do not express the fucose containing carbohydrate sialyl Lewis X. The patient's leukocytes do not extravasate normally, which leads to recurrent infections.In his pioneering work in the early 1960s, Ginsberg (5, 6) elucidated the enzymatic pathway converting GDP-mannose to GDP-fucose. Later, Yurchenco and Atkinson (7) showed that this was the primary biosynthetic route to GDP-fucose. As shown in Fig. 1, GDP-mannose is converted to GDP-fucose by GDP-mannose 4,6-dehydratase via the oxidation of mannose at C-4 followed by the reduction of C-6 to a methyl group, yielding GDP-4-keto-6-deoxymannose. The reaction has been reported to proceed with transfer of a hydride fro...

Top-down electron capture dissociation (ECD) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was performed for structural analysis of an intact monoclonal antibody (IgG1kappa (κ) isotype, ~148 kDa). Simultaneous ECD for all charge states (42+ to 58+) generates more extensive cleavages than ECD for an isolated single charge state. The cleavages are mainly localized in the variable domains of both heavy and light chains, the respective regions between the variable and constant domains in both chains, the region between heavy-chain constant domains CH2 and CH3, and the disulfide bond (S-S)-linked heavy-chain constant domain CH3. The light chain yields mainly N-terminal fragment ions due to the protection of the interchain disulfide bond between light and heavy chain, and limited cleavage sites are observed in the variable domains for each chain, where the S-S spans the polypeptide backbone. Only a few cleavages in the S-S-linked light-chain constant domain, hinge region, and heavy-chain constant domains CH1 and CH2 are observed, leaving glycosylation uncharacterized. Top-down ECD with a custom-built 9.4 T FTICR mass spectrometer provides more extensive sequence coverage for structural characterization of IgG1κ than does top-down collision-induced dissociation (CID) and electron transfer dissociation (ETD) with hybrid quadrupole time-of-flight instruments and comparable sequence coverage for top-down ETD with orbitrap mass analyzers.

Abstract.Today, we are experiencing unprecedented growth and innovation within the pharmaceutical industry. Established protein therapeutic modalities, such as recombinant human proteins, monoclonal antibodies (mAbs), and fusion proteins, are being used to treat previously unmet medical needs. Novel therapies such as bispecific T cell engagers (BiTEs), chimeric antigen T cell receptors (CARTs), siRNA, and gene therapies are paving the path towards increasingly personalized medicine. This advancement of new indications and therapeutic modalities is paralleled by development of new analytical technologies and methods that provide enhanced information content in a more efficient manner. Recently, a liquid chromatography-mass spectrometry (LC-MS) multi-attribute method (MAM) has been developed and designed for improved simultaneous detection, identification, quantitation, and quality control (monitoring) of molecular attributes (Rogers et al. MAbs 7(5): 2015). Based on peptide mapping principles, this powerful tool represents a true advancement in testing methodology that can be utilized not only during product characterization, formulation development, stability testing, and development of the manufacturing process, but also as a platform quality control method in dispositioning clinical materials for both innovative biotherapeutics and biosimilars.KEY WORDS: biotherapeutic; mass spectrometry; multi-attribute method; quality by design.To date, multi-attribute method (MAM) has been applied to several early stage clinical programs with different classes of protein therapeutics and compared to current practices. This experience shows that the MAM technology can be utilized for different types of protein therapeutics delivering highly specific and quantitative information, which is invaluable during process development and essential for molecular characterization. Data has also been generated to support its use for release and stability in alignment with Quality by Design (QbD) principles. Utilizing recent advances in the technology, research, and development labs, in industry, has generated convincing data that MAM would be highly beneficial in a cGMP environment. This article will summarize the advantages of MAM relative to conventional product testing approaches. We recommend that MAM, unlike conventional purity methods, be used to specifically monitor and quantify molecular product quality attributes and product/process-related impurities. This increased specificity for attributes with increased relevance to safety and efficacy can lead to better product/process understanding, shorter process and product development timelines, and an improved control strategy by improving specificity of the measurement. MAM OVERVIEWReduced LC-MS-based peptide mapping methods have been used in academia and the industry for several decades. Biopharmaceutical companies are increasingly using quantitative LC-MS-based peptide mapping methods for clinical material characterization, as well as release and stability testing (1-7). Quantit...

No abstract

No abstract