2016 Archived Content

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Cambridge Healthtech Institute’s 5th Annual
Higher-Throughput Protein Purification
Innovating Processes
January 21-22, 2016

High-throughput processes have transformed the traditional protein-by-protein trial-and-error approach for testing criteria and scaling up. In this leading meeting on Higher-Throughput Protein Purification, HTP will be explored in the quest to develop methods that ensure quality and translate to large scale. Automation, robotics and liquid handlers will be discussed, along with developing small-scale models that shed light on bioproduction. Case studies will be presented that illustrate how leaders in the field are integrating HTP approaches in order to reduce the time and effort needed to successfully establish parameters and achieve pure protein.

Please join this in-depth discussion of HTP protein processing.

Final Agenda

THURSDAY, JANUARY 21

7:45 am Conference Registration and Morning Coffee

8:15 Chairperson’s Opening Remarks

David Wood, Ph.D., Associate Professor, Chemical & Biomolecular Engineering, The Ohio State University

KEYNOTE PRESENTATION

8:20 Enhancing Biotherapeutic Discovery and Optimization Using High-Throughput Protein Platforms

Scott_Lesley Scott Lesley, Ph.D., Director, Protein Sciences and Biotherapeutics, The Genomics Institute of the Novartis Research Foundation & Scripps Research Institute

HT protein expression and purification permits experimental iteration to find optimal activities and biophysical characteristics. While each protein is inherently unique, generalized approaches for expression, screening and purification have largely overcome this challenge to standardization. Automation adds both throughput and consistency to the process while integrated analytical methods allow for rapid characterization and sample triage. These robust platforms have found great utility for biologics discovery, expression and optimization.

9:00 Characterization of the Poly(ADP-ribose) Proteome during DNA Damage

Guy_Poirier Guy G. Poirier, Ph.D., Professor, Biochemistry, and Canada Chair, tier1 in Proteomics and PROTEO, CHU de Québec Research Center, Laval University

We have developed a high throughput protein purification of poly(ADP-ribose) interacting proteome by using mass spectrometry hybrid instruments. More recently, we have established a new technology to analyze, on a proteome basis, sites of poly(ADP-ribosyl)ation on aspartic and glutamic acids on chromatin and on nuclear proteins. The identification of these sites modified by PARP-1 was achieved both by chemical and biochemical methods generating hydroxamic and ribose-P adducts.

9:30 A Fast, Automated, Quantitative, High Throughput Platform for Detailed Glycan Analysis and Monitoring of Biotherapeutic Production and Biomarkers by LC/MS/MS

Pauline_Rudd Pauline M. Rudd, Ph.D., Research Professor, Glycobiology, National Institute for Bioprocessing Research and Training (NIBRT)

Monitoring glycosylation remains a challenge for the production of recombinant therapeutics and biomarkers. A new robotic HT platform for releasing and fast labelling N-glycans provides a front end to high resolution LC/MS/MS. Experimental data bases to aid LC data interpretation are open source (http://glycobase.nibrt.ie/tools.html). Databases and software designed for Pharma are incorporated into Waters UNIFI 1.7. Monosaccharide sequence and linkage information (LC) is directly compared with on line MS/MS for confident structural assignment.

10:00 Coffee Break in the Exhibit Hall with Poster Viewing

11:00 The Use of High-Throughput Robotic Purification Platforms and Online, Real-Time Systems to Streamline Process Development

Susan_Callahan Susan Callahan, Scientist, Amgen, Inc.

Biopharmaceutical companies are moving towards high-throughput assays to meet analytical needs with accuracy, precision and speed. Many technological improvements/breakthroughs in robotics and instrumentation have been made recently which have allowed for enhancements in the analysis of various critical attributes. This presentation will focus on the evolution of traditional methods to the most recent breakthroughs in high-throughput purification, advanced analytical technologies and online real-time systems used to streamline process development.

11:30 Presentation to be Announced

12:00 pm Session Break

12:15 Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own

1:15 Ice Cream Break in the Exhibit Hall with Poster Viewing

2:00 Chairperson’s Remarks

Dietmar Reusch, Ph.D., Director, Development Characterization, Pharma Biotech Production and Development, Roche Diagnostics GmbH

2:05 The Application of Synthetic Biochemistry: HTP Approaches to Enhance the Sustainability, Cost Effectiveness and Safety Efficiency of Manufacture for Small Molecule APIs

Andrew_Fosberry Andrew Fosberry, Ph.D., Manager, Expression and Fermentation Sciences, GlaxoSmithKline

Traditionally, the enzymes and microbes available for screening against chemical transformations were limited in number, single sourced from a commercial supplier, and came with restricted freedom to operate. Due to these constraints, it was necessary to fit a chemical process to a specific enzyme. Combined with the recent improvements in molecular biology and enzyme evolution technologies, it is now possible and practical to find and modify an enzyme hit to suit a specified process.

2:35 High-Throughput Purification of Synthetic Peptides by Reversed Phase Chromatography

Mathias_Schaffrath Mathias Schaffrath, Ph.D., LGCR Chemistry, Library and Peptide Purification, Sanofi-Aventis Deutschland GmbH

The purification of large synthetic peptides (25-55 amino acids) is still a challenge. The unwanted by-products of these peptides are often peptides with only one wrong amino acid in the sequence. Therefore, the peptide and the by-products elute at the same time during the chromatographic separation. Chromatographic experience, thorough method development and scaling up is needed for successful separations. Partial automation of the process leads to a “really” high throughput purification.

3:05 High-Throughput Small-Scale Purification for Various Types of Biologics to Support Cell Line and Cell Culture Process Development
Shashi Prajapati, Ph.D., Senior Scientist, Cell Line Development-High Throughput Analytical Group, Biogen
High-throughput small-scale protein purification (SSP) is critical for product quality analysis in cell line and cell culture process development. We have developed three different types of SSP to purify different kinds of biologics including mAbs, non-mAbs and blood factor molecules. The mAbs and non-mAbs were purified using 96-deep well plate whereas mini Atoll Robo columns were used for blood factors purification. The mAbs and blood factor purification are based on one-step affinity chromatography whereas two-step ion-exchange chromatography was used for non-mAbs purification. We have established the automated HTP-SSP capabilities where we can purify up to 384 samples per day.

3:35 Refreshment Break in the Exhibit Hall with Poster Viewing

4:15 A Practical Self-Cleaving Tag System for Complex Glycoproteins

David_Wood David Wood, Ph.D., Associate Professor, Chemical & Biomolecular Engineering, The Ohio State University

Premature cleaving and thiol requirements have made self-cleaving intein affinity tag methods ineffective for complex mammalian glycoproteins. We have recently developed a re-engineered intein and resin combination for these targets, and have successfully used it to purify fully glycosylated and active human tPA and secreted alkaline phosphatase expressed in HEK293 cells. This approach provides all the power of Protein A affinity methods, but with applications to non-mAb targets.

4:45 High-Throughput Purification of Toxins in E. coli for the Development of Novel Therapeutics

Renaud_Vincentelli Renaud Vincentelli, Ph.D., Head, Protein Production, Structural Biology Facility, AFMB, CNRS University Aix Marseille

For the development of novel drugs, animal venoms constitute a library of millions of toxins which is unexplored due to the difficulty of getting these proteins correctly folded in E. coli. After modifying our custom HTP E. coli protein production pipeline (Saez NJ and Vincentelli R, Methods Mol Biol 2014), we could purify in a few months the majority of the 4000 recombinant toxins of the European FP7 VENOMICS project.

5:15 High-Throughput Production of Human Proteins for Structural Genomics and Generation of Affinity Reagents

Susanne_Graslund Susanne Gräslund, Ph.D., Principal Investigator, Structural Genomics Consortium, Medical Biochemistry and Biophysics, Karolinska Institute

The Structural Genomics Consortium has deposited more than 1700 structures in PDB. The focus on high-throughput protein production combined with a multi-construct approach has laid the foundation for this success. The methods used for cloning, expression screening in various expression hosts and parallel protein purification will be presented as well as some recent advances to increase throughput. Development areas like producing protein complexes and biotinylated antigens will also be included.

5:45 Close of Day

6:00-7:00 Reception in the Tiki Pavilion

FRIDAY, JANUARY 22

8:00 am Conference Registration and Morning Coffee

8:30 Chairperson’s Remarks

Jonas V. Schaefer, Ph.D., Head, High-Throughput Laboratory, University of Zurich

8:35 High-Throughput Protein Expression, Purification and Analysis for Next-Generation Binder Selection

Jonas_Schaefer Jonas Schaefer, Ph.D., Head, High-Throughput Binder Selection Facility, Biochemistry, University of Zurich

Expressing, screening and analyzing thousands of affinity reagent candidates for their properties remains one of the major bottleneck in binder generation. In my presentation, I will highlight our recent developments using a mixture of adapted and novel technologies to improve these steps, and thus to increase the speed and efficiency of our binder generation pipeline. In addition, innovative applications of our selected binders will be presented.

9:05 High-Throughput Techniques for Glycosylation Analysis of Therapeutic Antibodies

Dietmar_Reusch Dietmar Reusch, Ph.D., Director, Development Characterization, Pharma Biotech Production and Development, Roche Diagnostics GmbH

Several methods for glycoanalysis of antibodies are presented, including chromatographic, electrophoretic and mass spectrometry-based methods, with a focus on high-throughput techniques. We developed a method that is based on a DNA Sequencer. After APTS labelling, the glycans are detected with a fluorescence detector. The other method is based on glycopeptides. We compared the results for a therapeutic antibody obtained with the two high-throughput techniques with other methods for glycosylation analysis.

9:35 High-Throughput Mutagenesis and Molecular Dynamics Studies of Large Recombinant Protein Complexes

Robert_Weinzierl Robert O.J. Weinzierl, Ph.D., Reader, Molecular Biology, Life Sciences, Imperial College London

We have carried out a concerted experimental program focused on nanomechanically active portions of a multi-subunit RNA polymerase that are known to play a critical role in coordinating the translocation of nucleic acid substrates through the active site. Using a fully automated robotic in vitro reconstitution method, we tested the functional consequences of more than 600 site-directed mutants. We complement these approaches with atomistic molecular dynamics simulations.

10:05 Coffee Break with a Poster Pavilion

PepTalk is proud to support and recognize the protein scientists of tomorrow during the Poster Pavilion. This time has been set aside to view the Student Fellowship posters and interact with presenters one on one. This opportunity gives job seekers the chance to share their expertise with future/potential employers or develop contacts to further their research.

⊲ Featured Presentation
11:00 Enhanced Protein Purification Efficiency through Automation and Laboratory Design

Kenneth_Walker Kenneth Walker, Ph.D., Scientific Director, Biologics, Amgen, Inc.

In order to keep pace with the high-throughput cloning and expression needed to screen large panels of therapeutic candidates, we developed systems to substantially increase protein purification throughput through enhanced laboratory design and the use of novel chromatography instrument automation. With minimal user intervention, these systems can process a wide array of samples employing flexible, advanced chromatography methods that efficiently generate high-quality products.

11:30 Automated High-Throughput Protein Purification Workflows for Discovery of Novel Therapeutics

Avinash_Gill Avinash Gill, Ph.D., Scientific Manager, Antibody Engineering, Genentech, Inc., a member of the Roche Group

In combination with automated HTP workflows for antibody expression and purification, an efficient process has been put in place for making large numbers of purified antibody variants available to be screened in biological/functional assays, epitope identification and manufacturability evaluation. Expression and purification are carried out at 2 scales of cell culture – 1mL (deepwell blocks) and 30 mL (tubespin columns), to generate antibodies, antibody fragments and other therapeutic protein formats.

IT’S A WRAP:
PEPTALK 2016 CLOSING PLENARY PANEL DISCUSSION
Friday, January 22, 12:00 pm

Protein therapeutics is one of the fastest-growing global markets, driven by increasing adoption of protein- over non-protein drugs, growing funding for protein engineering and reduced drug discovery timelines and costs. As the science improves, so does the complexity of the R&D organization: it really does “take a village” to bring nextgeneration therapies to market and patients who need them. Ensuring product quality plus speed to market requires collective insights from experts working across the stages of protein science R&D – as embodied by panelists representing each PepTalk Pipeline topic.

They discuss: