SBIR-STTR Award

Progress in glycomics, the systematic study of the structure and function of glycans, lags far behind that of genomics and proteomics due to the more complex structures of glycans, whose biosynthesis is posttranslational and not template-driven. A major limitation in glycomics is the absence of powerful amplification technologies such as PCR, recombinant technology and automated chemical synthesis, which limits the availability of biologically relevant glycans for functional studies. To address this limitation, we developed a novel technology, oxidative release of natural glycans (ORNG), which uses simple oxidation by sodium hypochlorite (household bleach) to degrade peptides and nucleic acids and release bleach-resistant glycans from the glycoconjugates of natural sources such as animal/plant tissues. We have demonstrated that unlike current methods for releasing glycans, ORNG is a scalable process which has allowed us to generate unprecedented amounts of glycans from up to 20 kg of animal tissue. Thus, the absence of glycan amplification technology can be addressed by increasing the amount of starting material, which results in large quantities of released, biologically relevant glycans. Glycan vendors manufacture N-glycans using non- scalable methods resulting in limited amounts of material and high costs (20 µg packages for $200 to $500). Our scalable process will allow us to generate gram quantities of high-mannose, paucimannose, and complex- type N-glycans, and the economy realized by the large scale will allow us to provide mg quantities of N-glycans at <10% of current costs making these glycans available to non-specialized laboratories in mg quantities for functional studies. We have demonstrated the utility of our process for manufacturing mg quantities of N- glycans, which are not currently available in these quantities. However, ORNG is unique in that it releases all classes of glycans in a single, rapid reaction, and we have currently optimized it for release of N-glycans to demonstrate feasibility. Here, we are requesting funds to develop a streamlined “downstream process” for purification of all classes of glycans including glycosaminoglycans, O-linked glycans and glycosphingolipid- linked glycans from a single oxidation run. Vendors of O-linked glycans and glycosphingolipid-linked glycans generally manufacture them by chemical or chemo-enzymatic synthesis, which is expensive and limited to single products that are selected based on our limited information of what structures exist in nature. Since ORNG uses kg quantities of natural products as starting material, the resulting glycans will represent a combinatorial library of the source natural product containing not only the known, common glycans, but also minor glycans that have not been previously detected. This project will result in a robust manufacturing process for purified, natural N-, O-, GSL-linked glycans that we can offer at affordable prices. In addition, we will manufacture the corresponding shotgun glycan microarrays based on these “combinatorial” libraries that can be interrogated with biologically relevant GBPs to discover novel glycans with unique functions. 1

Public Health Relevance Statement:
Narrative Progress in glycomics, the systematic study of the structure and function of glycans, lags far behind that of genomics and proteomics due to lack of purified, biologically relevant glycans at quantities required for functional studies. Using its proprietary process for oxidative release of natural glycans (ORNG), NatGlycan, LLC has developed a commercial, scalable process to generate unprecedented quantities of all classes of glycans from natural sources in a single, rapid reaction which addresses the lack of amplification technology in this area. Here, we propose to develop a streamlined “downstream process” that will make natural, biologically relevant glycans available to the biomedical community at affordable prices based on economy of scale. 1

Project Terms:
Acids; Address; Agreement; Anabolism; animal tissue; Animals; Anions; Area; Austria; base; Binding; Binding Proteins; Biological; Biological Availability; Biotechnology; Budgets; Carbohydrates; chemical synthesis; Chemicals; Chickens; Collection; combinatorial; Communities; Complex; Complex Mixtures; Consumption; cost; Development; egg; Filtration; Funding; Genomics; Glycoconjugates; Glycosaminoglycans; Glycosphingolipids; Harvest; High Pressure Liquid Chromatography; Household; Kilogram; Label; Laboratories; Letters; Libraries; Link; Mannose; manufacturing process; Maps; Mass Spectrum Analysis; Methods; Minor; Natural Products; Nature; new technology; next generation sequencing; novel; Oligonucleotides; oxidation; Peptide Nucleic Acids; Phase; Plants; Polysaccharides; Preparation; prevent; Price; Procedures; Process; Production; protein aminoacid sequence; Proteomics; Protocols documentation; Publishing; Reaction; Reagent; Recombinants; Research; Resistance; Running; Sales; scale up; Services; Shotguns; Sodium Hypochlorite; Solid; Source; Soy Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; synthetic peptide; Techniques; Technology; Time; Tissues; Trisaccharides; Vendor; Work

During the first decade of the 21st century, NIH made significant investments in glycomics. The Consortium for Functional Glycomics (supported by NIGMS 2001-2012) led a major international effort using a microarray of 611 defined glycans to identify the protein-glycan interactions associated with normal physiology and disease (http://www.functionalglycomics.org/CFGparadigms/index.php/Welcome_to_the_CFG_Paradigm_Pages). This 611 glycan library transformed studies on protein-glycan interactions and revealed the importance of defining the human glycome, estimated to be in the range of 10,000 determinants. A microarray comprised of all possible glycans would allow us to detect and define all physiologically relevant glycan binding proteins (GBP), but the CFG library is now only 575 glycans making the production of large quantities of pure, defined glycans for functional studies critical for advancing glycomics; e.g, mgs of pure human milk oligosaccharides (HMO), available since the 1950’s supported research that led to our understanding of the benefits of HMO in breasfed infants, resulting in infant formula manufacturers introducing HMO into their products in 2014, based on clinical trials supporting their efficacy. Mammalian glycans,on the other hand, expressed on the surfaces of all cells, are generally available only in minute quantities; making them as available as HMO to the research community would rapidly advance understanding of their functions in animal cells. This is now possible using our proprietary technology, Oxidative Release of Natural Glycans (ORNG), which chemically releases glycans from glycoconjugates of any natural material using household bleach. NatGlycan, LLC (NGC) was established to provide biologically relevant natural glycans reliably, economically, and in large quantities. In Phase I we demonstrated the feasibility of producing high mannose N-glycans in mg quantities for sale to the glycoscience community for the first time. We believe the economy of scale will support reasonable prices. Sigma-Aldrich sells 20µg of Man9GlcNAc2 for $688, while NGC is selling 5 mg of Man9GlcNAc2-AEAB for $750. In Phase II of this project we will complete a manufacturing facility to produce 100’s of mg (purity >95%) of all high mannose N- glycans and derivatives that will be useful to the glycobiology community. In addition, we plan to launch highly purified N-glycans representing all of the structures expressed on human and recombinant IgG for use as QC standards in mAb manufacturing; currently an unmet need in the pharmaceutical industry. Since ORNG releases all classes of glycans simultaneously from natural products, we will rapidly expand our product offerings. We will purify the major O-glycans that are in great demand, but not currently available at reasonable cost. We anticipate that by the end of Phase II, we will be offering hundreds of N-glycans and O-glycans (purity >95%) and useful derivatives in multi-miligram amounts. Based on significant sales and orders after our initial product catalog launch, we anticipate being able to support our glycan manufacturing costs and increase profit margins as we increase manufacturing scale in the future.

Public Health Relevance Statement:
Narrative Since glycans are directly involved in the pathophysiology of most major diseases, NatGlycan, LLC is working to address the unmet needs for purified glycan standards and defined glycan reagents functional studies using its proprietary process for oxidative release of natural glycans (ORNG). Having demonstrated that ORNG is capable of supporting an industrial scale process, NatGlycan has successfully streamlined their downstream process and is now capable of manufacturing and selling 5 and 10 mg vials of pure (>95%) High Mannose N- Glycans, including isomers of Man7GlcNAc2 and Man8GlcNAc2, at prices that are compatible with research budgets. In Phase II of this project we will establish a manufacturing facility to expand our product offering of natural N- and O-linked glycans in previously unprecedented quantities and purities as we address our mission of making natural, biologically relevant glycans available to the biomedical community at affordable prices based on economy of scale. 1

Project Terms:
Acetonitriles; Address; Agreement; Aliquot; Animals; base; Binding Proteins; Biological; Biological Process; Budgets; Catalogs; Cattle; Cells; chemical release; Chemistry; Clinical Trials; Colostrum; commercialization; Communities; Complex; Contracts; cost; Development; Disease; Drug Industry; Egg White; Egg Yolk; Equipment; Equipment and supply inventories; Exhibits; experience; Functional disorder; Funding; Future; Glycobiology; Glycoconjugates; Glycolipids; Glycosaminoglycans; Goals; Heparin; High Pressure Liquid Chromatography; Household; Human; Human Milk; Hybrids; Immunoglobulin G; in vivo; Incubators; indexing; Industrialization; Industry; Infant; Infant formula; interest; International; Investments; Isomerism; Kilogram; Laboratories; Letters; Libraries; Link; Mannose; mannosyl(9)-N-acetylglucosamine2; Manufacturer Name; manufacturing facility; manufacturing process; meetings; method development; milligram; Mission; Monoclonal Antibodies; Mucins; National Institute of General Medical Sciences; Natural Products; Oligosaccharides; oxidation; Peptide Nucleic Acids; Pharmacologic Substance; Phase; Physiological; Physiology; Play; Polysaccharides; Powder dose form; Preparation; Price; Process; Production; Proteins; Protocols documentation; quality assurance; Quality Control; Reagent; Recombinants; Reproducibility; Research; Research Personnel; Research Support; Role; Sales; Sampling; Societies; Source; Structure; Surface; synthetic peptide; System; Technology; Time; United States National Institutes of Health; Vendor; Vial device; Water; Work