Higher Throughput Protein Purification
January 23-24, 2013
Day 1 | Day 2 | Download Pipeline 2 Brochure
High-Throughput Protein Processing is essential to quickly determining the best tests, protocols and materials needed to reach biologically active proteins of the highest purity. Automation and robotics are key elements of transforming the traditional protein-by-protein trial and error approach. Implementing single-use and disposable systems offers additional options to complement liquid handling systems. This meeting will cover the range of technologies and protocols used in HTP protein processing, such as screening, monitoring systems, data acquisition, test methods, parallel purification, and lab-on-a-chip. Experts will share their HTP case studies from a variety of lab types, covering varying scales of modeling and production. Optimizing purification conditions for challenging and novel proteins will also be addressed, along with a variety of expression platforms. Please fortify your knowledge of HTP by joining the comprehensive discussions aimed at reaching Higher Throughput Protein Purification.
TUESDAY, JANUARY 22
1:30pm Conference Registration
2:00 BuzZ Session A (Please visit our website for a listing of topics.)
2:45 Refreshment Break in the Exhibit Hall with Poster Awards
3:30 BuzZ Session B (Please visit our website for a listing of topics.)
4:15 End of Day
WEDNESDAY, JANUARY 23
7:30am Conference Registration and Morning Coffee
8:15 Chairperson’s Opening Remarks
Douglas MacDonald, M.S., Senior Scientist, Purification Process Development, Dendreon Corp.
» KEYNOTE PRESENTATION:
8:20 High-Throughput Cell-Based Studies and Protein Microarrays for Biomarker and Target Discovery
Joshua LaBaer, M.D., Ph.D., Virginia G. Piper Chair in Personalized Medicine, Director, The Biodesign Institute; Personalized Diagnostics, Arizona State University - Biography
We developed a novel form of protein microarray, called nucleic acid programmable protein array (NAPPA). In lieu of producing and printing purified proteins, this method substitutes the printing of cDNAs encoding the proteins. Thus, the resulting array is a DNA array that can be converted into a protein array by adding cell free protein synthesis machinery. This obviates the need to purify proteins, produces human proteins in a mammalian milieu, and avoids concerns about protein stability on the array.
9:00 High-Throughput Affinity Purification: Proteomics Profiling of the Poly (ADP-ribose) Related Response to DNA Damage Signaling
Guy Poirier, Ph.D., Canada Chair, Targeted Proteomics, Laval University and Chuq Research Center, Quebec - - Biography
Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. The most promising approach is to use PARG dead (which contains a macrodomain) with antibody purification.
9:30 Dendreon’s High Throughput Process Development Platform; a Case Study of a Second Generation Antigen Purification Process
Douglas MacDonald, M.S., Senior Scientist, Purification Process Development, Dendreon Corp. - Biography
A second generation purification process for a recombinant antigen for cancer cellular immunotherapy was developed. The goal was to establish an efficient, robust and high yielding process to reduce cost of goods. High Throughput Process Development was used to rapidly screen various resin modalities in 96-well filter plates, followed by scale up into RoboColumns. The optimized process was then further scaled up to allow a comparability assessment with the existing product.
10:00 Coffee Break in the Exhibit Hall with Poster Viewing
10:45 High-Throughput Production at the Macromolecular Therapeutic Development Facility: Expression and Purification
Ronald D. Seidel, Ph.D., Associate Director, Albert Einstein Protein Production Facility, Department of Biochemistry, Albert Einstein College of Medicine
The Macromolecular Therapeutics Development Facility at Albert Einstein College of Medicine (MTDF) leverages high-throughput (HTP) proteomic techniques for the development, analysis and production of protein-based therapeutics. Specifically, we have adopted high-throughput strategies as a function of the NYSGRC, one of four HT centers for the NIH Protein Structure Initiative (PSI), the Enzyme FunctionInitiative (EFI), the Immune function Network (IFN), and we further serve as the eukaryotic protein production facility for the Northeast BioDefense Center (NBC). Taken together, during the last 12 months, the MTDF has generated more than 11,000 constructs for more than 5000 protein targets, resulting in over 2500 soluble proteins distributed for functional and structural studies. We highlight our large-scale expression and purification process in bacterial, insect and mammalian systems.
11:15 Plate Based Purification Methods Applied to High-Throughput Expression Evaluation and Scouting of Buffer Conditions
W. Clay Brown, Ph.D., Scientific Director, High-Throughput Protein Lab, Center for Structural Biology, Life Sciences Institute, University of Michigan - Biography
The High-Throughput Protein lab at the University of Michigan carries out high-throughput expression trials using bacteria, baculovirus-infected insect cells and transiently transfected mammalian cells. These methods rely on a 96-well plate-based purification format. Recently, we have begun using the plate-based purification for identifying lysis buffer conditions for increased protein solubility as well as scouting conditions for improved purification. Data for trials with target proteins relevant to the study of human diseases will be presented.
11:45 Simulated Moving-Bed Chromatography for Insulin Purification: Design Methods and Simulation Tools to Achieve High Product Purity and High Yield
Nien-Hwa Linda Wang, Ph.D., Professor, Chemical Engineering, Purdue University - Biography
Batch chromatography has been widely used for analysis and purification of proteins because of its versatility, high selectivity, compact volume, and mild operating conditions. However, batch chromatography is less efficient than simulated moving-bed (SMB) chromatography for large-scale production. This presentation will explain the fundamental principles of SMB and how to apply standing-wave design method and simulation tools to develop a tandem SMB for insulin purification from a three component mixture. This process has a high product purity (>99.6%), and high yield (>99%). It has five times the column productivity and requires only 1/3 of the eluent compared to conventional batch chromatography. The issue of insulin residence time in SMB and the strategy of controlling the identity of insulin product from each feed batch will also be discussed.
12:15pm Close of Morning Session
12:30 Luncheon Presentation: Overcoming Biotherapeutic Process Development Challenges with the Simple WesternPeter A. Fung, Ph.D., Product Manager, Simple Western, ProteinSimpleCharge heterogeneity and apparent molecular weight are two commonly assessed parameters during biotherapeutic development. Peggy, the latest instrument in the Simple Western platform provides highly reproducible and fully automated charge- and size based characterization of protein therapeutics. Data generated on Peggy will be presented demonstrating the ability to identify, characterize and quantitate immunodetected products throughout biotherapeutic development.
2:00 Chairperson’s Remarks
Susanne Gräslund, Ph.D., Principal Investigator, Biotechnology, Structural Genomics Consortium, University of Toronto
» FEATURED PRESENTATION:
2:05 Bringing Industrial-Scale to the Evolution of Enzymes (to Produce Drugs)
Andrew Fosberry, Ph.D., Manager, Expression & Fermentation Sciences, Biological Reagents & Assay Development, PTS, GlaxoSmithKline Research & Development Limited
The manufacturing of pharmaceutical drugs and their precursors can potentially be made more efficient by the application of biocatalysts. This can reduce the cost of goods, reduce the carbon foot print associated with the manufacture, and vastly improve the sustainability of the process. Directed evolution can be utilised to alter an enzyme’s substrate profile and thus improve the function of that enzyme for a new substrate or reaction condition. We will give an industry insight into how we have developed our processes to tackle this challenge.
2:35 Experimental Design Clues from 3D-Structural Bioinformatics
Dietlind L. Gerloff, Ph.D., Assistant Professor, Biomolecular Engineering, School of Engineering, University of California, Santa Cruz - Biography
Limited yields of individual proteins in high (or low) throughput expression are often attributable to incorrect refolding in vitro. Improvement through mutation or truncation may be constrained by the interest in the target proteins, e.g., preserving antigenicity of surface proteins for vaccine development. When rational design is required, considering 3D-structural aspects can be beneficial alongside other optimization. Computational predictions and tools improve feasibility of incorporating structure in experimental design. I will review examples ranging from “classic” to “sophisticated” from our research and the literature.
3:05 A Comparative Analysis of Rapid Methods for Purification and Refolding of Recombinant Bovine Prion Protein for Biophysical Characterization
Eric M. Nicholson, Ph.D., Lead Scientist, Prion Diseases Research Project, National Animal Disease Center, USDA, Agriculture Research Service - Biography
Bacterially-produced recombinant prion protein (rPrP) is a common model system for evaluation of the properties of wild-type and mutant prion proteins by biochemical and biophysical approaches. Methods exist for the purification and refolding of untagged rPrP expressed as inclusion bodies in Escherichia coli. In order to perform a higher-throughput analysis of different rPrP proteins, an approach that produces high purity rPrP with a minimum of steps and high yield is desired. We directly compare rapid, small-scale purification approaches useful for higher-throughput studies.
3:35 POSTER HIGHLIGHT
A Novel Approach to Automated Large Scale Purification of Antibodies and Fc-Tagged Proteins – Poster #B202
Maciej Paluch, Research Associate, Protein Chemistry, Genentech, Inc.
3:50 Refreshment Break
4:15 Method Development at Micro-Scale and Validation at Scale-Up: A Case Study from a Core Service Lab
William Gillette, Ph.D., Senior Scientist, Protein Expression Lab, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research (FNLCR), NIH - Biography
The advent of higher-throughput approaches has greatly facilitated screening of expression samples. As this approach becomes more economical, it is useful to understand the limitations, in addition to the benefits, especially for the use on a diverse set of proteins. I will present a case study that was designed to determine how accurately the micro-scale platform in our lab was predicting scale-up results, both qualitatively and quantitatively.
4:45 Automated Methods for Protein Production, Protein-DNA and Protein-Protein Interactions
Renaud Vincentelli, Ph.D., Engineer & Head, Method Development, Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS/Marseille University - Biography
A protocol that allows the systematic screening of culture conditions and fusion-tags at a pace of hundreds of cultures per week has been initiated and validated. The statistics generated allowed the selection of the simplest protocol that gives the maximum soluble expression yield for the production of proteins upon scale-up, and in the microgram range at the screening scale. A new protein-DNA interaction assay (HTP SELEX) and a new protein-protein interaction assay (HTP HOLD-UP) have also been developed.
5:15 Close of Session
5:30-6:30 Networking Reception in the Exhibit Hall with Poster Viewing
6:30 Close of Day
Day 1 | Day 2 | Download Pipeline 2 Brochure