Innovating protein purification through ‘tried and true’ methods is the keynote of this meeting, which features case studies from experts sharing their experiences of successfully meeting the demands created by high productivity cell culture. Ways to optimize purification processes will be featured throughout the in-depth presentations, including insights into affinity tags, electrophoresis, centrifugation, filtration, chromatography, and implementing operational excellence programs to streamline processes. Purification across various expression systems will also be explored, including more traditional mammalian systems, as well as ‘specialized’ systems including E. coli, baculovirus, and yeast.
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MONDAY, JANUARY 9
7:30 am Conference Registration and Morning Coffee
8:55 Chairperson’s Opening Remarks
Richard R. Burgess, Ph.D., Professor, Oncology, McArdle Lab for Cancer Research, School of Medicine, University of Wisconsin, Madison
» KEYNOTE PRESENTATION
9:10 Adaptation of Precipitation Technologies for Improved Downstream Efficiency for Monoclonal Antibody Production
Alahari Arunakumari, Ph.D., President, AHA Bioconsultants LLC - Biography
Flexible platform development strategies and innovation and improvement in existing technologies are constantly advancing manufacturing processes. Mammalian cell culture production dominates mAb manufacturing today, and will remain so for some time to come due to production technology advancements and established regulatory successes. The current trend of putting extra effort towards matching up downstream capabilities is much needed to alleviate the bottlenecks to process large upstream output. Some of the productivity enhancement methods in downstream capabilities in mAb production will be discussed in this context.
9:50 Process Development of Primary Recovery Steps for a High Cell Density Product Stream
Jessica Molek, Ph.D., Principal Scientist, Downstream Process Development, GlaxoSmithKline - Biography
Removal of cells and cell debris using centrifugation followed by depth filtration are often the first steps in primary recovery of expressed proteins from a microbial fermentation broth. In expression systems containing high volumes of solids, optimization of both centrifugation and depth filtration is necessary, for scalable economically-feasible production. The case study presented here describes the optimization of both the centrifugation and depth filtration processing steps and focuses on how the two steps are coupled effectively. Differences in performance between the batch and continuous centrifugation are discussed and scale-up options are summarized. Depth filtration trains are analyzed using resistance curves of each filter as a function of the throughput of each depth filter. The effects of filter ratio and feed flow rate on the overall capacity of the filter train are used to better understand the filtration system. Using the results of the bench scale experiments along with appropriate scaling parameters, a successful large-scale clarification was performed.
10:20 Networking Coffee Break
10:45 Expression and in vivo Ulp1-Mediated Cleavage of SUMO Fusion Proteins
Albert J. Courey, Ph.D., Professor, Chemistry & Biochemistry, UCLA - Biography
After expressing a protein as a fusion to SUMO, it is often desirable to cleave the SUMO off of the fusion protein using a SUMO-specific protease such as Ulp1. To facilitate such processing, we have constructed a dual expression vector encoding two fusion proteins: one consisting of SUMO fused to Ulp1 and a second consisting of SUMO fused to a His-tagged protein of interest. The SUMO-Ulp1 cleaves both itself and the other SUMO fusion protein in the bacterial cells prior to lysis, and the proteins retain solubility after cleavage.
11:15 Analysis of Cell Signaling Pathways with Affinity Purification-Mass Spectrometry
Alexey Veraksa, Ph.D., Assistant Professor, Biology Department, University of Massachusetts Boston
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.
11:45 New HyperStable Split Fluorescent Protein Technology for Efficient Protein Tagging, Detection, and Purification
Geoffrey S. Waldo, Ph.D., Team Leader, Biosciences, Los Alamos National Lab
GFP, RFP and ‘SNAP’ are convenient, genetically encoded tags for labeling proteins, but are bulky and can perturb protein behavior or cause misfolding and aggregation. We now report a new split fluorescent protein that is orthogonal to the original GFP. We describe its characterization and validation in protein applications. This unusually stable protein remains fluorescent in 7 M GndHCl when reconstituted from its two fragments. We also describe new color variants of our original split GFP.
We describe the new applications that are possible using these orthogonal tags, including multiplex tagging, enhanced FRET experiments without causing protein misfolding, host-pathogen interactions, and tracking protein complex formation in living cells. These tools should also enable new TAP MS applications and library screens for proteins with improved solubility and stability. Mix and match tools for improving protein crystallization are another area hinted at.
12:15 pm Close of Morning Session
12:30 Luncheon Presentation I
Proteomic Filter Aided Sample Preparation with the Vivacon® 500
Michael Vagell, Applications Specialist, Sartorius Laboratory Products and Services
Filter Aided Sample Preparation (FASP) allows gel-free processing of biological samples solubilized with detergents for proteomic analysis by mass spectrometry. In FASP, detergents are removed by ultrafiltration and after protein digestion, the peptides are separated from undigested material. Horizontal ultrafiltration devices, such as the Vivacon® 500 filtration units with nominal molecular weight cut-offs of 30 kDa and 50 kDa are suitable for FASP.
1:00 Luncheon Presentation II (Sponsorship Opportunity Available)
2:00 Chairperson’s Remarks
Albert J. Courey, Ph.D., Professor, Chemistry & Biochemistry, UCLA
2:05 Capture and Resolution of mAb Charge Variants with Cation Exchangers - Advances and Perspectives
Giorgio Carta, Ph.D., Lawrence R. Quarles Professor, Department of Chemical Engineering, University of Virginia - Biography
In a side-by-side study, we compare the properties and performance of traditional macroporous and newly developed polymer-grafted matrices. Fundamental insights about the mechanisms that are responsible for the process performance of these materials are obtained through macroscopic, microscopic, and modeling studies. The results provide a rational basis for the optimal selection of stationary phases and for chromatographic process design, and valuable clues to direct the development of future, more effective matrices.
2:35 Multi-Dimensional Chromatography for Monitoring Quality Attributes of Monoclonal Antibodies in Cell Culture Process
Methal Albarghouthi, Ph.D., Senior Scientist, Analytical Biochemistry, MedImmune, LLC
A multi-dimensional chromatography system was developed as a potential process analytical technology (PAT) to monitor antibody product quality during cell culture. The system employs protein A chromatography for antibody purification from cell culture medium followed by ion exchange chromatography for analysis of charge heterogeneity or RP-HPLC for analysis of fragmentation. The system is fully automated and provides fast sample analysis.
3:05 Quantification of a PEGylated Peptide Drug Candidate in Human Plasma Using Immuno-Affinity Purification with an Anti-PEG Antibody Followed by LC/LC-MS/MS
John T. Mehl, Ph.D., Senior Research Investigator, Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb - Biography
In the absence of a reliable ELISA method, two dimensional HPLC was coupled on-line to mass spectrometry for the quantification of a PEGylated peptide drug candidate. Protein precipitation followed by trypsin digestion to produce a surrogate peptide was used for sample preparation of clinical serum samples. To further refine the method and to increase the analytical selectivity for PEGylated peptide over possible drug metabolites, an immuno-affinity sample preparation utilizing anti-PEG antibody with on-bead digestion was developed. This presentation will highlight challenges and important considerations for the quantification of PEGylated peptide drugs using mass spectrometry.
3:35 One-STrEP-tag: The high affinity Strep-tag® for Protein Purification
Thomas Schmidt, Ph.D., COO, IBA GmbH
The success of tag based protein purification depends on the properties (e.g. specifity, affinity) of the used affinity tag. The widely used Strep-tag®II provides highly pure and functional proteins from crude lysates of any host. A further development of Strep-tagII is the One-STrEP-tag which combines its high specificity and mild conditions with higher affinity thereby enabling efficient purification in batch or directly from culture supernatants. Furthermore, it tolerates diverse buffer conditions and additives (high salt, detergents, reducing agents, metal ions and chelating agents) making it a universal tag for varying protein properties, particularly for protein complexes in protein interaction analysis.
3:50 Networking Refreshment Break
4:15 Insight into the Mechanism of Solubility Enhancement by E. coli Maltose-Binding Protein
David S. Waugh, Ph.D., Chief, Protein Engineering, Senior Investigator, Macromolecular Crystallography Lab, Center for Cancer Research, NCI NIH - Biography
To investigate the mechanism of solubility enhancement by maltose-binding protein (MBP), we have compared the properties of MBP fusion proteins refolded in vitro with those of the corresponding fusion proteins purified under native conditions. We selected 5 passenger proteins for these experiments with measurable activities that can be used to monitor their folding: glyceraldehyde 3-phosphate dehydrogenase (G3PDH), dihydrofolate reductase (DHFR), green fluorescent protein (GFP), human dual specificity phosphatase 14 (DUSP14) and TEV protease. Using Gateway recombinational cloning, these passengers were fused to 3 different N-terminal tags: His6, His6-MBP and His6-GST. The 15 fusion proteins were purified under denaturing conditions by immobilized metal affinity chromatography (IMAC) on Ni-NTA resin and then refolded by rapid dilution. In all cases we recovered far more soluble MBP fusion protein than soluble GST- or His-tagged protein after refolding. Hence, we can reproduce the solubilizing activity of MBP in a simple in vitro system, indicating that no additional factors are required to mediate this effect. However, we found that the vast majority of the passenger proteins precipitated after TEV protease digestion of the refolded MBP fusion proteins, suggesting that they were not properly folded. We also assayed both the soluble fusion proteins and the TEV digestion products for enzymatic activity (or fluorescence in the case of GFP).
Little or no activity was detected in every instance except one: the TEV protease fusion proteins were quite active. A strikingly different result was obtained when the MBP fusion proteins were purified from E. coli under native (non-denaturing) conditions. Substantial activity was detected for all of the passenger proteins except G3PDH, both prior to and after cleavage of the fusion proteins by TEV protease. Taken together, these results indicate that, in general, the ability of MBP to promote the solubility of its fusion partners in vitro does not also result in their proper folding under these circumstances. We conclude that some additional factor must be involved in the folding process in vivo.
4:45 Ultra Pure Functional Protein Recovery with a Novel High Affinity EF Hand Fusion Protein System
David O’Connell, Ph.D., Senior Scientist, School of Medicine, University College Dublin - Biography
We have developed an EF hand domain peptide pair that bind with very high affinity (<100pM). We have engineered a new cloning vector with one hand attached to the protein of interest. We achieve massively pure preparations upon purification with the second hand immobilized on silica nanoparticles. We show concrete proof principle data with scFv fusions, purification in calcium, release in EDTA and increased affinity on the np surface.
5:15 Expression and Purification of Novel Viral Fusion Proteins for Drug Target Discovery
David Bienvenue, Ph.D., Associate Director, VLST Corporation - Biography
A case study will be presented that describes VLST’s efforts to generate over 2,000 viral fusion proteins with the goal of identifying their binding partners using a novel bioinformatics/proteomics platform. The host proteins identified by VLST’s discovery platform represent key immunomodulatory pathways targeted by the virus, and are being exploited to develop novel biotherapeutics to treat autoimmune and inflammatory illnesses.
5:45 Welcome Reception in the Exhibit Hall with Poster Viewing
7:00 Close of Day
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Links to Companion Meetings
Higher Throughput Protein Purification
Protein Aggregation and Emerging Analytical Tools