Protein Purification & Recovery
January 21-22, 2013
Day 1 | Day 2 | Download Pipeline 2 Brochure
The nature of proteins requires constant innovation of purification processes to streamline steps and ensure protein quality. Reducing the amount of protein lost between production and harvest is key, especially as cell culture titers increase, and greater demands are placed on purification efficiencies.New technologies and transforming traditional techniques will be presented in the “Protein Purification & Recovery” meeting, including affinity purification, centrifugation, filtration, chromatography, and cation and ion exchangers. In addition, operational excellence approaches, such as QbD and DoE, will be discussed as tools to help optimize processes. Purification across various expression systems will also be explored, including more traditional mammalian systems, as well as ‘specialized’ platforms including E. coli.
Please join protein purification experts for this strategy-sharing session focused on how to optimize Protein Purification & Recovery.
SUNDAY, JANUARY 20
4:00-8:00 pm Conference Registration
MONDAY, JANUARY 21
7:30 am Conference Registration and Morning Coffee
8:55 Chairperson’s Opening Remarks
» KEYNOTE PRESENTATION:
9:10 Moving Molecules from the Clinic to Manufacture: The Role of Rapid Bioprocess Development
Nigel Titchener-Hooker, Ph.D., Professor, Biochemical Engineering, University College London - Biography
Assessing a molecules ‘fit’ to manufacture has conventionally been deferred until such time that clinical promise makes the commitment to pilot-scale trials a viable risk. With the advent of high-throughput methods, and in particular scale-down models of bioprocesses, we can now envisage a degree of parallel process investigation as clinical trials progress. This talk will explore some of the advances in the field of rapid bioprocess development and will illustrate the nature of the insights gained by reference to industrial examples.
» FEATURED PRESENTATION:
9:50 An Engineered Self-Cleaving Tag System for Use with Any Expression Host
David W. Wood, Ph.D., Associate Professor, Chemical & Biomolecular Engineering, The Ohio State University - Biography
Intein-based self-cleaving affinity tag methods have been largely limited to E. coli expression systems, primarily due to premature cleavage in eukaryotic hosts and incompatibility with proteins containing disulfide bonds. We have recently developed a new engineered intein, with high affinity for zinc ion, which allows tight suppression of intein activity under conditions that are compatible with mammalian cell culture, and can be used effectively with any industry standard expression host.
10:20 Coffee Break
10:45 Challenges in the Purification and Characterization of Viral and Bacterial Antigens - Biography
Carlo Zambonelli, Ph.D., Lab Head, Protein Characterization, Novartis Vaccines & Diagnostics, Inc.
Early screening of HIV Env antigens from regional subtype C HIV acute phase isolates was initiated for selection of suitable vaccine candidates; all gp120s expressed in CHO cell lines were produced as a mixture of monomers and dimers. The dimers represent a misfolded species stabilized through the formation of an aberrant intermolecular disulfide bridge. We developed a purification scheme producing pure gp120 Env with a high monomer content. Initial purification protocol development was carried out using conditioned supernatant obtained from fed batch bioreactors producing starting material with relatively low target protein concentration; the purification scheme developed was adapted to the purification of culture media collected at day 7 of terminal batches from ongoing stability studies, with target antigen content as much as 10 fold higher. Further validation of the purification protocol will be carried out in consistency studies and the antigen obtained from stability and consistency studies will be subjected to biochemical and biophysical techniques.
11:15 Human Caspases in vitro: Expression, Purification and Kinetic Characterization
Heidi Roschitzki-Voser, Ph.D., Senior Scientist, Biochemistry Institute, University of Zurich - Biography
Caspases, a family of highly regulated cysteine peptidases play an essential role in cell death and inflammation. A number of strategies and protocols for the expression, purification and kinetic characterization of human caspases are described in the literature. I will discuss how we have systematically revised these protocols and present the optimized expression and purification procedures for caspase-1 to 9 and the improved assay conditions for their reproducible kinetic characterization.
11:45 Simplifying Protein Expression with Ligation-Free, Traceless and Tag-Switching Plasmids
Thomas Magliery, Ph.D., Assistant Professor, Chemistry & Biochemistry, The Ohio State University - Biography
Omics-type approaches and synthetic biology require simple, robust and high-throughput approaches to gene subcloning and protein expression and purification. Several recent plasmid vector systems for expression in E. coli make it easy to use ligation-free and recombinogenic cloning methods to install and switch traceless tags for purification and improved expression. An overview of some of the key recent advances will be followed by a description of the pHLIC plasmid system and applications to some difficult-to-express proteins.
12:15pm Close of Session
12:30 LUNCHEON PRESENTATION
Simplifying Clarification and Capture: Advances in Rhobust EBA Technology
Piet den Boer, Ph.D., Senior Scientist, DSM Biologics
Rhobust® is the Second Generation Expanded Bed Adsorption (EBA) technology that provides an elegant solution for direct product capture in the first downstream process unit operation. Centrifugation, depth-filtration and packed bed chromatography can be replaced by one unit operation, resulting in less preparation and process time, lower cost of goods and reduced investment costs. We will present the latest advances in Rhobust operating conditions and process scaling.
2:00 Chairperson’s Remarks
David W. Wood, Ph.D., Associate Professor, Chemical & Biomolecular Engineering, The Ohio State University
2:05 Recombinant Human Thrombin Produced in E. coli - A Case Study
Andreas Anton, Ph.D., Director, Contract Development, Scil Proteins GmbH - Biography
Scil developed a recombinant Thrombin manufactured using a new approach with E. coli. The development of the folding step for refolding of rhThrombin will be shown. The protein was produced in insoluble form in the cytoplasm of E. coli and could be folded to an active protein in a very effective procedure. The production was scaled up to 100 L pilot scale to produce sufficient amounts of the target protein for animal studies as well as formulation development.
2:35 Expression and Efficient Purification of Stable Isotope-Labeled, Functional Human Cannabinoid Receptor CB2
Alexei Yeliseev, Ph.D., Staff Scientist, LMBB, NIH/ NIAAA - Biography
The cannabinoid receptor CB2, a heptahelical G protein-coupled membrane receptor, has become an important pharmaceutical target. Structural studies on CB2 by nuclear magnetic resonance (NMR) spectroscopy and other biophysical techniques will contribute greatly to the development of novel specific ligands targeting this receptor. We developed protocols for incorporation of stable isotope-labeled amino acid residues into specific positions of CB2 protein. Labeling is performed through high density E. coli fermentation in minimal media.
3:05 POSTER HIGHLIGHT
Genetic Engineering of E. coli to Facilitate Downstream Purification – Poster #A210
Ellen Brune, Scientist, Chemical Engineering, University of Arkansas, Fayetteville
3:35 Sponsored Presentation (Opportunity Available)
3:50 Refreshment Break
4:15 How to Deal with Aggregation Problems of IDPS – ”Intrinsically Disordered Proteins”
Mario Lebendiker, Ph.D., Head, Protein Purification Facility, Wolfson Centre for Applied Structural Biology, The Hebrew University of Jerusalem - Biography
In this talk I will debate how we tackle the very difficult aggregation problems during the production of IDPs “Intrinsically disordered proteins”, or IDRs “Intrinsically disordered regions in proteins”. The main focus of my talk will be on protein solubility and stability issues during expression, purification and storage; type of factors that can prevent aggregation or promote solubilization, and the importance of the early elimination of aggregate conformations during protein purification.
4:45 Preparation of Uncooperative Proteins for NMR Studies: Human Galpha(i1) and Neurotrophin-4 as Case Studies
Karin Crowhurst, Ph.D., Assistant Professor, Chemistry and Biochemistry, California State University - Biography
Expression of human proteins in bacteria is a challenging but often necessary procedure for preparing affordable, isotopically labeled proteins for NMR spectroscopy. Recombinant human proteins are regularly expressed in inclusion bodies, isotopically labeled minimal medium often produces low protein yields, and the subsequent refolding and purification steps are frequently fraught with unexpected pitfalls. This talk presents the progression required to successfully express and purify Galpha(i1), and will briefly touch on the advances we have made in preparing neurotrophin-4 for NMR analysis.
5:15 Separation of Raft Associated Membrane Species Using a Patterned Supported Lipid Bilayer Extraction Platform
Susan Daniel, Ph.D., Assistant Professor, Chemical & Biomolecular Engineering, Cornell University - Biography
The handling and characterization of membrane-bound biomolecules is a significant bottle neck in the advancement of membrane glycomics and proteomics research. A prevailing hypothesis in cell membrane biology is the existence of lipid raft microdomains that organize specific glycolipid-protein interactions necessary for healthy biological function. However, this hypothesis has remained controversial because identifying species that associate with these domains is difficult with existing techniques. Here we present a biological membrane-based platform for the continuous, two-dimensional separation of lipid raft residents from non-raft species. This platform employs a two-dimensional (in plane) continuous extraction procedure to separate membrane-bound species based on their chemical affinity for a particular membrane composition.
5:45 – 7:00 Welcome Reception in the Exhibit Hall with Poster Viewing
7:00 Close of Day
Day 1 | Day 2 | Download Pipeline 2 Brochure