Overview

Tuesday, March 22

7:15    Continental Breakfast in Exhibit Hall

8:00     Chairperson's Opening Remarks
Dr. Shekar Ganesa, Associate Director, BioAnalytical Development, Genzyme Corporation 

Emerging Science and Technologies

8:40    Selective Inhibition of Glycan Synthesis as a Therapeutic Approach to Disease
Dr. Jamey D. Marth, Abaron Bioscience, Inc.
The biosynthetic steps in carbohydrate (or glycan) formation are regulated by a large family of enzymes comprising 1% of the mammalian genome. Remarkable advances have been made during the past decade in defining the various and yet unique physiologic and disease roles that different glycosyltransferase enzymes play in both humans and mice, with evidence for therapeutic activities associated with their inhibition in inflammation, autoimmune diseases (SLE, lupus), stroke, heart attack, diabetes, and cancer. The development of orally-available small molecule inhibitors of specific glycosyltransferases is underway to provide an efficient method of blocking glycan-dependent processes that contribute to widespread and presently intractable human disease states.

9:40    Coffee Break, Poster and Exhibit Viewing

10:30    A Facile and Efficient Synthesis of Sialyl Lewisa Hexasaccharide Blood Group Antigen
Dr. Steven Sucheck, Group Leader, Medicinal Chemistry, Optimer Pharmaceuticals
Sialic acid glycosides are known to occur in wide variety of biological materials in the form of gangliosides and complex oligosaccharides attached to proteins. The blood group antigen Sialyl Lewisa is expressed on a wide variety of carcinomas and its presence is frequently associated with a poor clinical prognosis; therefore, Sialyl Lewisa is potentially a novel target for vaccine-based anti tumor therapy. Herein, we report and an efficient synthesis of this structure, which has remained a difficult challenge and serves as a valuable alternative to isolation from natural sources. In collaboration with: Om Srivastava, Geeta Srivastava, Sulan Yao, David Rabuka, Chang-Hsing Liang and Yoshitaka Ichikawa

11:00    Recent Developments in Carbohydrate Synthesis: Enabling Commercialization of the Carbohydrate Molecule Class
Dr. John Pena, President, Ancora Pharmaceuticals, Inc. 
The carbohydrate molecule class demonstrates the potential to introduce viable drug products into the marketplace. However, a significant impediment to both academic and industry carbohydrate-based R&D programs is the limited availability of defined structures, curtailing the implementation of R&D efforts utilizing carbohydrates. A synthetic capability for carbohydrate material represents an optimal solution to the access barrier. Recent advances in carbohydrate synthesis and the ability to enable drug development will be discussed.

11:30    Selected Oral Poster Presentations

Analytical Approaches / Glycosylation
of Recombinant Proteins

1:10    Towards the Production of Conjugated Vaccines in Escherichia coli
Dr. Michael Wacker, Department: Biology, ETH Zurich
Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. We engineered E. coli cells in a way that two different pathways, C. jejuni protein N-glycosylation and E. coli lipopolysaccharide biosynthesis converge at the step in which the oligosaccharyl transferase PglB, the key enzyme of the bacterial N-glycosylation system, transfers O polysaccharide from its lipid carrier to an acceptor protein. The relaxed specificity of the PglB could be valuable for production in vivo of O polysaccharides-protein conjugates for use as antibacterial vaccines.

1:40    Modulating Bioactivity of Cytokines by Altering Carbohydrate Content
Dr. Steve Elliott, Research Director, Amgen Inc.
Attached carbohydrate can affect many properties of proteins including solubility, stability, antigenicity and biological activity. Altering the content and composition of carbohydrate chains can modulate these properties. The presentation will discuss approaches to enhance properties of cytokines by adding new carbohydrate chains to recombinant protein therapeutics. 

2:10    Prediction of Glycosylation Sites
Dr. Karin Julenius, Assistant Professor, Medical Biochemistry & Biophysics, Karolinska Institute
We have developed a newpredictor for mucin-type O-glycosylation sites that correctly predicts 76% of the glycosylated residues and 93% of the non-glycosylated residues. The prediction server is available at www.cbs.dtu.dk/services along with other glycosylation prediction servers including one for N-glycosylation sites and one for Yin-Yang sites (commercial licenses are available). The prediction servers can be used as a test before deciding on an experimental study or if experiments are not possible/too expensive.

2:40    Technology Workshop Presentation

Sponsored by

Characterisation of Complex Oligosaccharides Using a MALDI QIT TOF Mass Spectrometer Dr. Chris Sutton, Shimadzu Biotech, Manchester, UK Oligosaccharides, cleaved from glycoproteins (by hydrazinolysis or enzymatically), were characterised using the MALDI QIT (quadrupole ion trap) TOF mass spectrometer. DHB (2,5-dihydroxybenzoic acid) was used as the preferred matrix for ionising oligosaccharides. High mannose, biantennary and triantennary oligosaccharides were analysed using MS, MS2, MS3 and MS4 modes. Intact oligosaccharides were analysed in MS mode using a cooling gas to prevent fragmentation. Individual precursor ions were isolated in the trap, subjected to fragmentation with Argon, to provide MS2 data. Product ions were selected for further fragmentation, which was achieved by increasing the energy for collisionally induced dissociation. In MS mode, the singly charged sodium adduct form of these molecules was detected, which is typical of the analysis on conventional MALDI mass spectrometers. In MS2 mode, high mannose oligosaccharides readily lost the core N-acetylglucosamine residues, whilst MS3 and MS4 modes were used to sequentially fragment the product ion corresponding to the residual branched mannose oligomer. In MS2 mode analysis of biantennary and triantennary structures also fragmented losing disaccharide units, such as the galactose -N-acetylglucosamine units that define each antennary branch, or core fucosylated-N-acetylglucosamine units. MS3 of selected MS2 products ions could be used to differentiate fragments generated from either the reducing or non-reducing ends. Cross-ring cleavages were also observed during fragmentation in MS2, and the relevant product ions could be used to differentiate branched structures by further fragmentation in MS3 mode. The MALDI QIT TOF mass spectrometer, therefore, provided a unique opportunity to dissect complex branched oligosaccharides using MSn and acquire as much fragmentation data as possible for structural and sequence interpretation. In collaboration with Rachel Martin.

3:10    Refreshment Break, Poster and Exhibit Viewing

3:40    An Analytical Platform for the Characterization of Glycosylation in Monoclonal Antibodies
Dr. Amy Que, Senior Scientist, ARD Global Biologics, Pfizer Inc.
Monoclonal antibodies (Mabs) have been developed as therapeutic proteins. Glycosylation may play important roles in the biological functions of the proteins such as bioactivity, immunogenicity, stability and the clearance rate. This presentation will introduce an analytical technology platform for the characterization of glycosylation in Mabs.

4:10    Monitoring and Controlling of Glycosylation in Bioprocess
Dr. Gary N. Rogers, Principle Scientist, Analytical Sciences Process, Amgen Inc. 
The strategies in monitoring and controlling the glycosylation quality and efficiency have been a great focus in the recent bioprocess development for human therapeutics. One of the direct outcomes from an optimized bioprocess with well understood process analytical controls is reduced cost of manufacturing. This presentation will cover the analytical methods and techniques applied in the various stages of bioprocess development for glycoproteins.

4:40    Glycoprofiling Strategies to Support Production of Recombinant Therapeutic Proteins
Dr. Shekar Ganesa, Associate Director, BioAnalytical Development, Genzyme Corporation 
Glycan analysis has become increasingly important for characterization of therapeutic glycoproteins. Due to the important role carbohydrates play, and the advances in the analytical technologies, regulatory authorities are ever-increasing their requirements needed for a well characterized biological product. A variety of methods (electrophoretic, chromatographic and mass spectrometric) exist to monitor the relative abundances of the various glycoforms. For many of these methods, information about identity and structure are determined by comparison to pure standards, which frequently are both expensive and difficult to acquire. This presentation will compare and contrast the strengths of various approaches and discuss how they augment or supplant current practices.

5:10    End of Conference