2017 Archived Content
As demand for biological drugs increases, so does the need for purified protein. Whether for research or to create biotherapeutics, researchers must purify protein following expression and scale-up. The plethora of novel and complex protein-based
molecules pose greater challenges for purification professionals, including bispecifics and antibody-drug conjugates. Proteins’ tendency to aggregate is also a nagging problem, as is working with membrane proteins. The Protein Purification Technologies meeting explores the strategies and technologies employed to streamline steps and keep up with industry’s growing demands.
This conference also examines issues of scale and innovating ‘traditional’ technologies such as Protein A and chromatography. Purifying proteins in different expression systems will also be addressed including mammalian, bacterial, and
insect cells.
Final Agenda
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THURSDAY 16 NOVEMBER
12:30 Registration
13:00 Dessert Break in the Exhibit Hall with Poster Viewing
13:30 Chairperson’s Opening Remarks
Christopher H. Gray, Team Leader, Structural Biology, Drug Discovery Program, Cancer Research UK, The Beatson Institute
13:35 KEYNOTE PRESENTATION:
Protein Production Needs for Therapeutic Antibody Discovery and Development
Karin Felderer, Ph.D., Associate Director, Protein Production, Protein Sciences & CMC, MorphoSys
AG
Protein production needs for therapeutic antibody projects are manifold. They span a wide range of scales from small scale high-throughput for early in vitro characterization up to gram scale productions for
in vivo characterization and manufacturability assessment. In addition to antibodies and antibody fragments, various high quality antigens and tool proteins are required to facilitate successful projects. Case studies and strategies
will be presented covering this spectrum of applications.
14:05 Exploration of an Atypical ‘Platform’ mAb and Resolution of Its Non-Ideal Behaviour during Downstream Process Development
Karolina Les, Ph.D., Scientist II, Purification Process Sciences, Biopharmaceutical Development, MedImmune,
Ltd.
Efficient development of monoclonal antibodies (mAbs) is often founded on platform-based optimization of processes, allowing timely delivery of scalable and robust processes. While existing developability frameworks allow progression of best candidates
into development and typically assure a good platform fit, cases of low platform compliance are unavoidable. The talk will discuss reasons behind an atypical behaviour of an antibody during purification and its mitigations. The main challenges
included severe precipitation during purification and unusually high host cell protein (HCP) levels observed during early development.
14:35 Process Optimisation for Manufacturing of ScFv-Class Antibody with FOLDTEC®
Katrin Schweinitzer, Head, Downstream Processing Development, BioProcess Development,
Wacker Biotech GmbH
Wacker Biotech will present a case study for development and optimization of a scalable process employing microbial system to produce the scFv-class antibody for commercial application. Using its E. coli-based
FOLDTEC® technology, WACKER completely overhauled a process employed by customers to facilitate production on an industrial scale. WACKER’s proprietary refolding technology was able to produce the desired product in enhanced yields
and streamline the purification process with achieving higher purity than was hitherto possible.
15:05 Refreshment Break in the Exhibit Hall with Poster Viewing
15:50 Streamlining Antibody Purification: A Novel Approach by Caprylic Acid-Based Impurity Precipitation
Anja Trapp, M.Sc., Scientist, Bioprocessing Technology & Innovation, Rentschler Biopharma SE
Benefits of implementing caprylic acid- (CA-) based precipitation in antibody purification are presented. Combining the common low pH viral inactivation step with CA precipitation greatly improves HCP clearance in different mAb processes. We show
that CA precipitation can be applied as an excellent alternative to a polishing chromatography using case studies. The step constitutes a simple, robust and economic step in a mAb purification process resulting in increased purification performance
and higher safety of two-column processes.
16:20 A Microfluidic Toolbox for Screening and Downstream Process Optimization
M. Raquel Aires-Barros, Ph.D., Professor, Institute for Bioengineering and Biosciences
(IBB), Bioengineering, Instituto Superior Técnico, Universidade de Lisboa
Antibodies and other protein products are biopharmaceuticals of critical importance that need to be purified in a cost effective and efficient manner. Here, a methodology for the rapid screening of antibody extraction conditions using a microfluidic
channel-based toolbox is presented which can also be used for process development. A first microfluidic structure allows a simple negative-pressure driven rapid screening of up to 8 extraction conditions simultaneously, while a second microfluidic
structure allows the integration of multi-step extraction steps.
16:50 End of Day
17:00 Dinner Short Course Registration
17:30-20:30 Recommended Short Course*
SC7: Protein Purification Strategies
*Separate registration required
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FRIDAY 17 NOVEMBER
08:00 Registration and Morning Coffee
08:30 Chairperson’s Remarks
Anja Trapp, M.Sc., Scientist, Bioprocessing Technology & Innovation, Rentschler Biopharma SE
08:35 Do We Need to Purify Membrane Proteins? Expression Tricks and Solid-State NMR Spectroscopy in Native Membranes
Dirk Linke, Ph.D., Professor, Molecular Microbiology, Biosciences, University of Oslo
In recent work, we have developed E. coli expression strains that lack multiple of the most abundant outer membrane proteins. This enables us to perform solid-state NMR spectroscopy directly on native membranes
after overexpression of targets in 13C/15N labeling medium. Membrane protein purification becomes obsolete in this procedure, and the proteins can be studied in their native environment without risk of denaturation.
09:05 The Salipro Lipid Nanoparticle System for Detergent-Free Stabilization of Membrane Proteins
Jens Frauenfeld, Ph.D., CEO, Salipro Biotech AB
More than 60% of all current drugs target membrane proteins. However, membrane proteins are very unstable, which is a major challenge for the pharmaceutical industry. Salipro Biotech has developed a novel system to reconstitute membrane proteins
into self-assembling lipid nanoparticles. The Salipro system is applicable for various classes of membrane proteins (channels, receptors, transporters, etc). Salipro nanoparticles stabilize membrane proteins in a lipid environment and
allow working in detergent-free buffer systems.
09:35 SELECTED POSTER PRESENTATION
10:05 Coffee Break with Poster Viewing
10:35 Human Heparanase: Lessons Learned from a Self-Destructing Project
Mario Lebendiker, Ph.D., Head, Protein Purification Facility, Wolfson Center for
Applied Structural Biology, Hebrew University of Jerusalem
Our case study describes the challenges encountered in each step of crystallizing Human Heparanase (hHep), an endo-beta-D-glucuronidase. We needed to overcome irreproducible expression productivity of this “self-destructing” protein
in insect cells, followed by optimizing a purification strategy for this non-tagged target with conventional chromatography. We then faced batch-to-batch variability in crystallization, and finally, an inability to solve collected data
from crystals, until we produced a fusion protein construct with known structure to obtain phases.
11:05 Production of Human Aldehyde Oxidase (AO) in Baculovirus-Infected Insect Cells (BEVS)
Ciarán N. Cronin, Ph.D., Associate Research Fellow, Head, Parallel Protein Production Group,
and Group Leader, Gene-to-Structure, Pfizer Global R&D
Aldehyde oxidase (AO) is a key enzyme activity to consider during the development of small molecule human therapeutics. There are numerous literature examples of failed clinical drug trials due to a lack of preclinical testing of potential
drug exposure to AO activity. This presentation summarizes the numerous design, expression and purification experiments that were undertaken in order to optimize the production of recombinant human AO for deployment in Pfizer’s
screening cascade.
11:35 High-Throughput Knockout of Difficult-to-Remove and Troublesome CHO Host Cell Proteins to Create a Clean CHO Cell Line
Stefan Kol, Ph.D., Protein Biochemist, Novo Nordisk Foundation Center for Biosustainability, Technical
University of Denmark
The CHO Cell Line Engineering department is addressing the need to obtain high yields and quality of biopharmaceuticals produced in optimized CHO cells through genome-scale-based methodologies. Although many host cell protein (HCP) impurities
are effectively removed in downstream purification processes, a small population of HCPs are particularly challenging. Here, we present our efforts to identify and knock out several CHO HCPs and our characterization of the resulting
cell line.
12:05 Improve
Throughput and Productivity in Small-Scale Affinity Purification of mAbs
William Barrett, Ph.D., Chromatography, Gore & Associates
Protein purification can be a time-consuming process in drug discovery and research applications. Utilizing our understanding of fluoropolymer science, we’ve developed the GORE™ Protein Capture Device with Protein A. This column
offers higher binding capacity at short residence time, enabling faster processing times and potential for more highly concentrated elution pools.
12:35 Problem-Solving Breakout Discussions with a Light Snack in the Foyer (View All Breakout Discussions)
Automation to Accelerate Laboratory Protein Production
Moderator: Christopher H. Gray, Ph.D., Team Leader, Structural Biology, Drug Discovery Program, Cancer Research UK, The Beatson Institute
- Benefits and pitfalls of using increased automation in the protein production laboratory
- The cost of automation – when is it value for money
- The future of lab based protein purification – trends and directions
Production of Protein Complexes in Baculovirus-Infected Insect Cells
Moderator: Ciarán N. Cronin, Ph.D., Associate Research Fellow, Head, Parallel Protein Production Group, and Group Leader, Gene-to-Structure, Pfizer Global R&D
- Use of multiple viruses, or expression cloning vectors with more than one promoter
- Use of IRES sequences or 2A peptide-linked sequences
- Deployment of polycistronic vectors (MultiBac, OmniBac, biGBac)
Attendees with experience deploying a polycistronic insect cell expression system are particularly encouraged to participate.
13:35 Session Break
14:00 Chairperson’s Remarks
Jens Frauenfeld, Ph.D., CEO, Salipro Biotech AB
14:05 Novel Single-Column Simulated Moving-Bed Chromatography for Quasi-Continuous Purification of Biomolecules
José Paulo Mota, Ph.D., Professor, Chemical and Biochemical Engineering, LAQV-REQUIMTE,
Chemistry, Science and Technology, Universidade NOVA de Lisboa
14:35 Parallel Rapid Expression & Purification of Proteins for Crystallography (PREPX)
Michael Fairhead, Ph.D., PDRA, Structural Genomics Consortium, University of Oxford
Pipelines for high-throughput cloning and small-scale expression and purification are well established. However, subsequent scale-up and purification often involves only working with a few pipeline hits (usually the easiest to produce)
and these are not always suitable for crystallography. PREPX is a medium-throughput approach to evaluate larger numbers of clones (48 per week), and their suitability for producing diffraction quality crystals.
15:05 Expressed Protein Ligation: A New Paradigm as a Reagent Platform for Preclinical Drug Discovery
Rosalie Matico, Associate Fellow, Investigator, Protein Cellular and Structural Sciences,
GlaxoSmithKline plc
Expressed protein ligation (EPL) utilizes genetically engineered inteins to join two protein (peptide) fragments via a native peptide bond. Ligation occurs through an intein mediated C-terminal thioester on one protein fragment and an
N-terminal Cys-SH on a second protein or peptide fragment. We developed a reagent platform by harnessing (EPL) to specifically label proteins at the C-terminus with a cysteine-lysine dipeptide chemically modified at the lysine with
biotin, fluorescein, peglyation, etc., prior to ligation.
15:35 Optimization of Secreted Target Protein Yields by Introduction of in silico Designed Stabilizing Mutations
Svend Kjær, Ph.D., Deputy Head, Structural Biology Science Technology Platform, Francis Crick Institute
Protein stability is a fundamental and crucial parameter in a range of applications from crystallization to biotherapeutics. We present data of how a few stabilizing mutations, designed by the PROSS algorithm based on crystal structures
and multiple sequence alignments, increase the Tm of a misfolding-sensitive secreted protein by 20°C. We present data showing how protein expression in higher eukaryotic systems is significantly improved by the stabilization.
16:05 Tandem-Tag Vectors for Enhanced Soluble Yield, Expression Monitoring and Streamlined Purification
Christopher H. Gray, Team Leader, Structural Biology, Drug Discovery Program,
Cancer Research UK, The Beatson Institute
Our suite of novel, tandem-tagged Escherichia coli vectors offers multiple options for increased soluble expression, and highly efficient purification. Dual promoters allow the simultaneous expression
of an MBP-target fusion and a tag cleaving protease. Similarly, this in vivo cleavage system functions with GFP-target fusions allowing fluorescent tracking of recombinant expression during
culture. Finally, we have designed a set of tandem-affinity tagged vectors generating protein that can be highly purified in a single step.
16:35 End of Conference
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