The booming field of biologics places ever greater demands on protein production. Whether for research or to create biotherapeutics, scientists must purify protein following expression and scale-up. To streamline processes, researchers need to integrate
upstream and downstream phases, and reduce the number of steps required to achieve pure, properly-folded protein. A plethora of technologies and tools are available to support purification, but which are the best? As protein-based molecules become
more complicated, they pose greater challenges for purification. Bispecifics, ADCs, fusion and membrane proteins present purification obstacles that may not have been tackled before. And aggregation continues to be a nagging issue waiting to trip
up an earnest researcher. This conference presents the tricks and technologies used by purification leaders who have refined techniques that remove headaches from protein processing. Experts will address ‘traditional’ technologies as well
as new approaches, and will touch on issues of scale, as well as purifying protein in various expression systems; all with an eye to streamline purification while ensuring quality.
Final Agenda
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THURSDAY 15 NOVEMBER
13:00 Registration (Foyer C)
13:15 Dessert Break in the Exhibit Hall with Poster Viewing (Pavilion 1)
14:00 Chairperson’s Opening Remarks
Dorota Antos, PhD, Professor, Chemical and Process Engineering, Rzeszow University of Technology
14:05 KEYNOTE PRESENTATION: How Problems of Protein Purification Are Being Addressed across Structural Laboratories in Europe: Insights from the European Research Infrastructure Consortium
Ray Owens,
PhD, Professor, Molecular Biology and Head, Oxford Protein Production Facility, University of Oxford
Technology developments to streamline the production of proteins for structural biology have been largely driven by the demands of structural proteomics. However, the purification of increasingly complex proteins and protein assemblies has challenged
traditional high-throughput structural proteomic workflows. The response of a number of academic centres in Instruct, a distributed European Research Infrastructure Consortium (www.structuralbiology.eu) will be reviewed and common trends highlighted.
14:35 Simplification of Purification Strategies for Mammalian Proteins from Single Targets to High-Throughput Projects Using an Enhanced Biochemical Code
David O’Connell, PhD, Lecturer, Biotherapeutics, Biomolecular & Biomedical Science, University College Dublin
We are investigating the sequences of amino acids and their associated post-translational modifications that confer superior transport characteristics upon secreted proteins to move through and across gradients. With one eye on creating new protein
engineering design principles, we are aiming to understand the elements of encoded behaviour of proteins including IgG, cytokines, interferons and the very many proteins that make up our secretome using a high-throughput expression and interrogation
model in HEK293. Establishing high-throughput expression, purification and transport assays will be described.
15:05 Overcoming Limitations of Conventional Tag Systems – Strep-Tactin®XT Applications
Dennis Karthaus, MSc, Group Leader, Cell Culture Sciences, IBA Lifesciences
The Strep-Tactin®XT:Twin-Strep-tag®-purification system enables protein purification at high yields and purity under physiological conditions. Providing the highest binding affinity among all affinity tag systems, the technology fulfills the
demands of mammalian expression systems (e.g. Expi) and is well suited for downstream applications like SPR.
15:35 Networking Refreshment Break (Foyer D)
16:00 HTP Production of a New Human PDZome Allows Quantitative PDZ-Peptide Interaction Screening Through HTP Holdup Assay
Renaud Vincentelli, PhD,
Head, IBIsA Facility for High-Throughput Cloning, Expression and Purification of Proteins, Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS-AMU UMR
Using our custom E. coli HTP protein production pipeline (Saez et al, JOVE 2014), we could produce soluble the 266 human PDZ domains. To characterize in detail, the HPV E6 - PDZome binding interaction profile, we automated the Hold-up assay
(Vincentelli et al, Nature Methods 2015) a quantitative and versatile in vitro protein-protein interaction assay. The protocols and some new results on this application will be detailed during the seminar.
16:30 Protein Purification and Detection with Nanobodies
Ulrich Rothbauer, PhD, Professor, Pharmaceutical Biotechnology, Natural and Medical Sciences Institute, University of Tübingen
Nanobodies are attractive tools for protein purification, detection and analysis as they are small, highly stable, and are easily producible thereby offering an unlimited supply of consistent binding molecules. Recent advances in identification of
target-specific nanobodies from synthetic gene libraries makes these tools broadly available. Here, we present our latest progress in nanobody generation, functionalization and application for protein purification and detection.
17:00 End of Day
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17:30 Dinner Short Course Registration* (Foyer C)
17:30 – 20:30 Dinner Short Courses
Recommended Short Course*
SC9: Optimising Protein Purification Strategies in Advance: Getting Your Plan Right - View Detailed Agenda
David O’Connell, PhD, Lecturer, Biotherapeutics, Biomolecular & Biomedical Science, University College Dublin
This course addresses creating an effective strategy for purifying protein before beginning a purification project. What are key considerations before you launch an expression campaign? Which host should you select and why (Bacterial/Insect/Mammalian)?
We will examine ways to reduce complexity in your strategy in order to efficiently increase productivity. We will also discuss how to establish redundant steps that support and guide protein purification.
*Separate registration required.
FRIDAY 16 NOVEMBER
08:00 Registration (Foyer C)and Morning Coffee (Foyer D)
08:30 Chairperson’s Remarks
Maximilian Hartl, PhD, Senior Scientist, Roche Pharma Research & Early Development (pRED), Large Molecule Research, Roche Innovation Center Munich
08:35 Structural Biology of Membrane Proteins: Expression Tricks, and to Solubilize or Not to Solubilize
Dirk
Linke, PhD, Professor, Genetics and Evolutionary Biology, Biosciences, University of Oslo
In recent work, we have designed expression systems for bacterial outer membrane and surface proteins. These can be used to express and purify, e.g., important vaccine candidates for bacterial diseases, but also for studying membrane protein structure
directly in the membrane with solid-state NMR methods.
09:05 Adaptive Automated Membrane Protein Purification Using AkTA Avant
Jonas
Lee, PhD, Scientist, Protein Technologies, Amgen, Inc.
Transmembrane proteins are key targets in drug discovery. However, they are difficult to purify due to complex buffer requirements to solubilize. We use various high-throughput methods to screen for best detergent conditions followed by innovative
methods to purify multiple targets in different buffer conditions automatically.
09:35 Nanoparticles to Stabilize Membrane Proteins in a Lipid Environment
André Heuer, PhD, Senior Scientist,
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. Here we present the latest results on a novel system to stabilize membrane proteins
using Salipro nanoparticles. Salipro nanoparticles stabilize membrane proteins in a lipid environment that allows them to work in detergent-free buffer systems.
10:05 Networking Coffee Break (Foyer D)
10:35 Potential of Centrifugal Partition Chromatography for the Separation of Proteins
Mirjana Minceva, PhD, Assistant Professor, Biothermodynamics, Life and Food Sciences Weihenstephan, Technische Universität München
Centrifugal partition chromatography (CPC) is a solid support-free chromatography method in which both stationary and mobile phase are liquid. This technology combines advantages of liquid-liquid extraction and chromatography, such as high loading
capacity, high recovery and high resolution. Moreover, as a result of the liquid nature of the stationary phase, problems associated with column packing are avoided. CPC can be used with aqueous two-phase systems (ATPS) providing a mild environment
for the separation of proteins. In this talk, the potential of CPC will be demonstrated for the separation of model mixture of proteins.
11:05 Tailor Made Affinity Adsorbents for Selective Capture and Recovery
Ana Cecília Roque, PhD, Principal Investigator
and Associate Professor, Sciences and Technology, University Nova of Lisbon
Biological and chemical libraries containing a high diversity or designed to fit a target biopharmaceutical, are powerful tools to develop robust peptidomimetics based on different scaffold molecules. The scaffold molecules range from small synthetic
ligands, to artificial β-hairpin peptides and small protein domains produced chemically. We study the potential of these scaffold affinity reagents to find binding partners against several targets (e.g. recombinant proteins, phosphorylated
peptides, and virus-like particles), and to develop mild and selective affinity-based purification processes.
11:35 Membrane Device Technology Improves Productivity of Antibody Purification
William Barrett, PhD, Chromatography, Gore & Associates
Protein capture has been identified as the number one bottleneck in the overall purification process of antibodies. The GORE™ Protein Capture Device with Protein A utilizes a unique membrane which provides high binding capacity at extremely
short residence times. Productivity gains have been realized through the use of these devices in labs across the world. Results from current lab scale size devices and new test data will be presented on the next generation larger sizes.
12:05 Problem-Solving Breakout Discussions (Foyer E&F)
Application of Structural and Biophysical Methods in the Development of Biopharmaceuticals
Moderator: Ray Owens, PhD, Professor, Molecular Biology and Head, Oxford Protein Production Facility, University of Oxford
- What are the best methods for assessing the impact of post-translational modifications on higher order structure of biopharmaceuticals?
- Does structure determination by either X-ray crystallography or cryo-electron microscopy have a role to play in the development of biopharmaceuticals?
- What properties, that can be analysed by biophysical methods, are relevant to the stability, and/or aggregation propensity of biopharmaceuticals?
Challenges with Protein L -- Let’s Exchange Experiences and Ideas
Moderator: André Heuer, PhD, Senior Scientist, Salipro Biotech AB
- Is protein L the way to go to capture Fabs?
- What about recombinant Fabs from microbials?
- How stable is the ligand? And how pure is the product?
13:00 Chairperson’s Remarks
Ulrich Rothbauer, PhD, Professor, Pharmaceutical Biotechnology, Natural and Medical Sciences Institute, University of Tübingen
13:05 Process Development of the Antibody-Drug Conjugate (ADC) SYD985 – A Case Study
Xiaonan Li, PhD, Senior Scientist, Downstream Processing, Synthon Biopharmaceuticals BV
SYD985 is an antibody-drug conjugate (ADC) based on trastuzumab and a cleavable linker-duocarmycin payload. A case study will be presented in which a hydrophobic interaction chromatography (HIC) purification process was developed allowing
removal of the undesired antibody species together with unbound linker-drug. It was possible to elute the product (SYD985) using mild conditions without requirement for any organic solvent. The HIC purification step was scaled up demonstrating
consistency and robustness.
13:35 New Formats, New Experiences - DSP Feedback for Carefully Selecting a Molecule for Development
Maximilian Hartl, PhD, Senior Scientist, Roche Pharma Research & Early Development (pRED), Large Molecule Research, Roche Innovation Center Munich
Biopharmaceuticals evolved from copying natural molecules to tailor-made, highly engineered drugs with disease specific action modes. The impressive ideas of our molecule designers often result in promising early in vivo data that challenge technical-scale drug development. In this talk, we show examples of non-predicted challenges we faced during purification of novel drugs. We present solutions and feedback from purification for the selection process
of next-generation drugs.
14:05 The Effect of New Stabilizers in Downstream mAb Process Intermediates
Irina Ramos, PhD, Downstream Process Scientist, MedImmune,
Inc.
The stability of downstream process intermediates is extremely important to define fit-to-plant parameters and keep product quality attributes within the acceptance criteria. Here we present the impact of adding small novel organic molecules,
designed to stabilize a protein’s molecular structure, in the context of important downstream steps. In this work, we used a monoclonal antibody (mAb) that showed (1) aggregation during viral inactivation at low pH conditions
and (2) low flux throughput during viral filtration. Both steps are traditionally part of the downstream process platform used in mAbs as dedicated virus reduction steps and contribute to the virus safety for mAb-based medicines. Our
analysis identifies novel protein stabilizers that can significantly improve mAb process intermediates stability and manufacturing throughput.
14:35 Pitfalls in Design and Scaling Up Protein Chromatography
Dorota Antos, PhD, Professor, Chemical and Process Engineering, Rzeszow University of Technology
Because of the complexity of thermodynamic, kinetic, and hydrodynamic effects accompanying protein chromatography, design and scaling up of the process is often impossible without understanding underlying adsorption mechanism. Specific
effects that can occur in protein chromatography will be discussed. The phenomena of band deformation due to mass transport resistances, protein unfolding, sample solvent effect, and dispersion in extra column volumes will be illustrated.
The solvent gradient and temperature mediated separations will be considered.
15:05 Monoclonal Antibody Reduction and Re-Oxidation by Copper Sulfate during Manufacturing and Impact on Product Quality
Sanjay Nilapwar, PhD, Senior Research Investigator, Large Molecule Purification Development, Incyte Corporation
Significant disulfide bond reduction of an IgG1 monoclonal antibody was observed during the late stage of the CHO cell culture in manufacturing, leading to the batch failure due to significant amount of low molecular weight species and
aggregates. Two methods of copper sulfate spiking were investigated to prevent and reverse the product disulfide bond reduction during manufacturing. Both methods could re-oxidize the reduced product and prevent further reduction throughout
the manufacturing process.
15:35 End of Summit
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