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Consistently producing proteins of high quality demands a robust purification process, especially when scaling up to large quantities. Purifying and recovering proteins is typically viewed as a time-consuming bottleneck. The Purification & Recovery meeting will explore the latest trends and tools through case studies presented by leaders in the development of biologics who will pass on their experience and data to help you overcome the challenges and increase the quality and quantity of purified protein. Innovations in technology and protocols will be presented through talks that are based on science but display the nuances of art and innovation required for the purification step.
Recommended Pre-Conference Short Course*
Sunday, January 10
(SC4) Specialized Protein Expression Systems
*Separate registration required
Monday, January 11
7:30 am - 8:45 am Registration and Morning Coffee
8:45 Chairperson’s Opening Remarks
Pim Hermans, Head, Ligand Discovery, BAC BV
9:00 OPENING KEYNOTE PRESENTATION
Case Study: Evaluating a Series of Novel Conformases/Foldases for Their Ability to Enhance Heterologous Gene Expression in E.coli
Andrew Fosberry, Ph.D., Research Investigator, Biological Reagents & Assay Development, GlaxoSmithKline
One of the major problems associated with expression of heterologous proteins in E.coli, is the inability of many over-expressed proteins to reach a native conformation and their tendency to accumulate as insoluble aggregates (inclusion bodies). We have recently evaluated the effect of co-expression of a number of novel Conformases/Foldases (El-Gewely, patents pending) with a series of therapeutic targets in E. coli. In my talk I will report our findings and discuss some examples where this novel approach has helped us to progress targets for drug discovery.
9:50 Refolding, Purification, and Characterization of the Ectodomain Complex of the CGRP Receptor
Norzehan Abdul-Manan, Ph.D., Structural Biology, Vertex Pharmaceuticals, Inc.
Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. The CGRP receptor (CGRP-R) is comprised of a heterodimer of two membrane proteins: the calcitonin receptor-like receptor (CRLR), and receptor activity-modifying protein 1 (RAMP1). In this study, we present a reductionist approach for the expression, purification and refolding of a functional heterodimeric CGRP ligand binding complex comprised of the CRLR and RAMP1 ECDs. In addition, we present preliminary data on cloning and expression of the ECDs of other family members of B1 GPCR family using a similar reductionist strategy.
10:20 Networking Coffee Break
10:45 Expression and Purification of Human TRPV1 in Baculovirus-Infected Insect Cells for Structural Studies
Alla Korepanova, Ph.D., Senior Researcher, Department of Structural Biology, GPRD, Abbott Laboratories
TRPV1 is a ligand-gated cation channel that is involved in acute thermal nociception and neurogenic inflammation. By using the GP67 signal peptide, high levels of full-length human TRPV1 was expressed in High Five insect cells using the baculovirus expression system. The functional activity of the expressed TRPV1 was confirmed by whole-cell ligand-gated ion flux recordings in the presence of capsaicin and low pH and via specific ligand binding to the isolated cellular membranes. Efficient solubilization and purification protocols have resulted in milligram amounts of detergent-solubilized channel at 80-90% purity.
11:15 Purification of Baculovirus Produced Proteins for Vaccine Application
Indresh Srivastava, Ph.D., Associate Director, Vaccines Research, Protein Biochemistry, Novartis Vaccines & Diagnostics, Inc.
11:45 Purification of Recombinant Protein from Transgenic Tobacco - Development of Non-Chromatographic Based Processes
Chenming (Mike) Zhang, Ph.D., Associate Professor, Biological Systems Engineering, Virginia Polytechnic Institute & State University
Tobacco has proven to be a promising alternative for the production of recombinant therapeutic proteins and offers numerous advantages over other plants as a host system. However, the recovery and purification steps needed to obtain a protein at high recovery and purity have not been well investigated. In this study, a process was developed to purify a model acidic protein, recombinant beta-glucuronidase (rGUS), from transgenic tobacco leaf tissue, with polyelectrolyte precipitation as the anchor process and chromatographic techniques as the polishing steps. The recombinant protein could be recovered to near homogeneity by this process.
12:15pm Close of Morning Session
12:30 Luncheon Presentation
High-Productivity Ion Exchange Sorbents Offer Distinctive Selectivity Characteristics
Warren Schwartz, Ph.D., Sr. Technical Director, Pall Life Sciences
2:00 Chairperson’s Remarks
Andrew Zydney, Ph.D., Head, Chemical Engineering, College of Engineering, Pennsylvania State University
2:05 Model Assisted Scale-Up of Industrial Purification Processes
Jörg Thömmes, Ph.D., Director, Purification Development, Biogen Idec
2:35 Development of a “Chromatography-Array” System for High-Throughput Screening and Optimization of Affinity Ligands
Masa Iwakura, Ph.D., Principal Researcher, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST)
We have recently developed a novel type of array system, namely a “chromatography-array” system, which includes all the new types of components, such as array cell, protein spot and immobilization, detection of ligand–protein binding, and elution by protein absorption signals at in situ, data analysis, and so on. By using our developed system, we have successfully created several affinity ligand proteins for IgG purification with high affinity at neutral pH, and efficient elution profiles at mild acidic condition as high as pH 5.0. We believe by using our system that ligand development studies will be greatly accelerated not only for monoclonal antibodies but also for other therapeutic proteins.
3:05 Parallel Micro-Scale Purification for Lead ID and Process Development/Optimization
William Gillette, Ph.D., Senior Scientist, Protein Expression Lab, SAIC- Frederick, NCI
We have adopted a parallel micro-scale purification platform to screen for purification leads as well as for method development. Scale up from the micro-scale (microliter) to bench top (milliliter) has proven predictive both qualitatively and quantitatively and the speed of the process allows on-the-fly troubleshooting. Our experience has been primarily with IMAC, IEX and ProteinA/G resins. With total control over chromatographic parameters, we can adjust the system to screen for hits from low level expression systems such as HEK293 cells. In addition, more elaborate purification screens can be performed such as on-column protease digestion for affinity tag removal.
3:35 Poster Presentation -- #202 – Automated Parallel Chromatographic Separations in Process Development
Juergen Friedle, Ph.D., Senior Vice President, Innovations, Atoll GmbH
3:50 Networking Refreshment Break
4:15 Multiple Routes for Production and Purification of Fab Fragments in Biopharmaceutical Discovery Research Yonghong Zhao, Ph.D., Research Scientist, MDT, Centocor R&D, Inc.
Fab (fragment with the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. We use different routes of Fab production and purification to enable a variety of research and development efforts. The purified Fab fragments were characterized by various analytic methods, and some have been used in crystallization experiments in which diffraction quality crystals suitable for X-ray crystallographic analysis were produced successfully.
4:45 Strategies for Streamlining the Development of Downstream Process for Enzyme Therapeutics
Elsie DiBella, Ph.D., Associate Director, Purification Process Development, Shire Human Genetic Therapies
Downstream process development for therapeutic enzymes can be both complex and resource consuming. These proteins are not amenable to the typical platform process ideas as used in therapeutic antibody development. There is no one characteristic that uniquely defines therapeutic enzymes, resulting in chromatography steps with low binding capacities and less than ideal resolution of product from host cell proteins. However, the ideas of a platform strategy are beneficial to therapeutic enzyme downstream process development. Application of the platform strategy to a specific therapeutic enzyme will be discussed.
5:15 High Throughput Protein A Wellplate Purification Method for Monoclonal Antibodies
Judy Chou, Ph.D., Senior Group Leader, Oceanside PR&D, Genentech, Inc.
5:45 Welcoming Reception in the Exhibit Hall
7:00 Close of Day
Links to Companion Meetings