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Overcoming Purification Challenges conference - Day 1


Overcoming Purification Challenges

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MONDAY, AUGUST 20


Protein purification has reached a plateau where favored steps, affinity tags, kits and protocols are widely accepted and used. Yet challenges remain, especially for novel and difficult proteins, such as membrane proteins. While cell culture processes have been refined to reach higher densities, greater demands are placed on purification to keep up with the booming biologics industry. High-throughput purification provides solutions leading to greater efficiencies while also posing additional challenges.

Emerging technologies present unrealized potential for monitoring processes, analyzing conditions, and identifying optimal strategies. Automation and robotics are revolutionizing protein industrialization. This meeting will explore purification challenges and how to overcome those challenges drawing on new technologies and innovative methods. Please join the discussion of next-generation strategies and technologies in the quest for purified proteins.

11:30 - 5:30 pm Main Conference Registration

 

CONQUERING CHALLENGES

1:00 Chairperson's Opening Remarks

David O’Connell, Ph.D., Senior Scientist, School of Medicine, University College Dublin

»1:10 Opening Keynote Presentation:

Addressing Challenges of Membrane Protein Expression, Purification and Stabilization

Lawrence-DeLucasLawrence J. De Lucas, Ph.D., Director, Center for Macromolecular Crystallography (CMC), University of Alabama, Birmingham - Biography 

This presentation will compare strategies using a variety of protein/peptide tags to provide an early quality assessment for protein expression, potential yield, purity and homogeneity of integral membrane proteins (IMPs). In addition, several examples will be presented for high-throughput self-interaction chromatography used as a method to improve membrane protein solubility and stability.

1:45  Expression and Purification of Diacylglycerol Acyltransferases

Heping Cao, Ph.D., Principal Research Scientist, Southern Regional Research Center, U.S. Department of Agriculture - Biography

Diacylglycerol acyltransferases (DGATs) are integral membrane proteins that catalyze the last and rate-limiting step of triacylglycerol biosynthesis in eukaryotic organisms. DGAT knockout mice are resistant to diet-induced obesity and lack milk secretion. DGAT genes have been identified from at least 83 organisms, but none of the enzymes from any organism had been expressed and purified from bacterial expression systems. We developed the first procedure for producing full-length recombinant DGAT proteins from any species using an E. coli expression system.

2:15  A Self-Cleaving Purification Tag System For Use In Mammalian Cell Culture

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

An attractive platform for the purification of recombinant proteins relies on self-cleaving affinity tags derived from engineered inteins. Unfortunately, this platform has not been generally applied to mammalian cell culture due to a variety of issues associated with control of the cleaving reactions. In this work, we report the development of a rationally engineered intein with enhanced controllability. Importantly, the mechanism of control is compatible with mammalian expression hosts, and preliminary data on expression and purification from CHO cell culture will be shown.

2:45  Refreshment Break

 

AFFINITY PURIFICATION

3:15  The Exploitation of S. cerevisiae: A Comprehensive Design of Affinity Tags and Overview of Microbial Applications

Carissa-YoungCarissa Young, Ph.D., Research Scientist, Delaware Biotechnology Institute, Department of Chemical and Biomolecular Engineering, University of Delaware - Biography

To improve the functional production of secreted or membrane proteins (e.g., ligand-binding yields indicative of active receptors), we have generated versatile yeast expression cassettes that incorporate numerous tags for identification and purification, and to assess protein interactions, protein structure/function, and intracellular localization. To reference selective organelles in S. cerevisiae, endogenous proteins were designed with codon-optimized fluorescent protein variant resulting in a valid assessment of heterologous protein subcellular localization. Furthermore, we have established methodologies to investigate the role of cellular quality control and its modulation during heterologous protein expression, focusing specifically on the unfolded protein response (UPR), autophagy, and ER associated degradation (ERAD) pathways.

3:45  Analyzing Cell Signaling Pathways with Affinity Purification-Mass Spectrometry

Alexey-VeraksaAlexey Veraksa, Ph.D., Assistant Professor, Biology, College of Science & Mathematics, University of Massachusetts, Boston - Biography

The study of cell communication mechanisms has been facilitated by the advances in protein complex purification and analysis. In my laboratory, we use affinity purification followed by mass spectrometry to map the structure of signaling networks, with follow-up functional studies in model systems. I will present the evolution of preferred tags used in my lab for affinity purification of protein complexes from higher eukaryotic cells and tissues, and will discuss the advantages and potential limitations of this approach.


4:15 Small-Group Breakout Discussions

This session provides the opportunity to discuss a focused topic with peers from around the world in an open, collegial setting. Select from the list of topics available and join a discussion to share ideas, gain insights, establish collaborations or commiserate about persistent challenges. The session includes a brief 'report-out' summary from each table. Afterwards, continue the discussion as you head into the lively exhibit hall for information about the latest technologies.

  • Bispecific Antibody Development: Challenges and Opportunities
    Moderator:Yun (Kenneth) Kang, Ph.D., Principal Scientist & Head, Purification Team, Bioprocess Sciences, ImClone Systems, a wholly-owned subsidiary of Eli Lilly and Company
    • Pros and cons of different bispecific formats
    • Strategies to improve the solubility, stability, and half-life as well as reduce toxicity for bispecific molecules
    • Development of cell culture/purification platform processes for bispecific antibodies
    • Formulation development challenges
    • Regulatory concerns
  • Cleaving Technologies for Purification Tag Removal
    Moderator:W. Wood, Ph.D., Associate Professor, Chemical & Biomolecular Engineering, The Ohio State University
    • Conventional proteolytic methods
    • Self-cleaving methods
    • Process and economic considerations
    • Issues related to expression systems and target proteins
  • Purification Testing: What is Needed?
    • What types of testing is needed to prove purity?
    • What is the role of Characterization Assays?
    • How pure is pure?
  • What are the Regulatory Concerns for Purifying Proteins?
    • What are the standards set by regulatory authorities such as the FDA?
    • What types of testing and data are required?
    • How complex do records need to be?
    • What would be a red flag to regulatory authorities?
  • How do Emerging Technologies Affect the Purification Process?
    • Modelling
    • Software
    • Biosensors
    • Molecular insights
    • Predicting outcomes
    • Data gathering

5:15 Breakout Group Summaries

5:30 Grand Opening Reception with Exhibit and Poster Viewing

7:00 End of Day



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