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Consistently producing proteins of high quality demands an efficient purification process, especially when scaling up to large quantities. The “Purification & Recovery” meeting will explore the latest trends and tools through case studies presented by leaders in the development of biologics who pass on their experience and information to help you with the art of protein purification.
The success of protein production primarily depends on the media conditions and streamlined processes that are necessary to ensure cell survival and growth. As yields continue to increase, demand grows exponentially. Highly producing clones, cell line selection and optimization are required to enable cell culture to meet the increasing demand for protein-based products. The day-long session focused on “Mammalian Cell Culture” explores the details that lead to higher yields within the context of Protein Production.
The number of biologics in development continues to rise with numerous new targets under investigation. This biologics boom puts pressure on existing manufacturing facilities stretching resources and capacity. The “Protein Production” meeting brings together experts who will share their knowledge and experiences of reaching ever higher yields. Their expertise will help you effectively plan for scale-up by addressing the unique issues associated with protein complexity.
Recommended Short Course
(SC6) Three-Phase Partitioning:
A Novel & Scalable Protein Mini-Prep
Please see page 3 for details
*Separate Registration Required
MAIN CONFERENCE
7:30am – 6:00pm Registration Open
7:30 Morning Coffee
SHARING KNOW HOW AND SOLUTIONS
Keynote Presentation
9:00 Novel Binding Proteins Mined from Tick Saliva
Maud Déruaz, Ph.D., Merck Serono Research Center
Blood sucking parasites such as ticks feed for extended periods on their hosts without eliciting an immune response. They secrete a battery of anti-coagulant, anti-pain and anti-inflammatory molecules in their saliva in order to remain undetected by the host. Chemokines direct the localized migration of leukocytes so their neutralization would be a good strategy for the tick. We have cloned three distinct chemokine binding proteins from a cDNA library constructed from tick salivary glands, which we have named
Evasins. As opposed to viral chemokine binding proteins which have very broad specificities, the Evasins are highly selective. We have characterized the recombinant proteins both in vitro and in vivo, where they show potent anti-inflammatory activities. The solution of their three dimensional structure has revealed that they possess novel folds of which no homologues exist in the pdb database. We believe that understanding the binding modalities of these extremely small proteins, smaller than the single chain camel antibodies or
nanobodies, will help us design better anti-inflammatory therapies.
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Featured Presentation
9:50 New Protein Diagnostic Methods from Brighter Ideas, Inc.
 William Ward, Ph.D., Associate Professor & Director,
CREBB, Rutgers University, and Founder and President, Brighter Ideas, Inc.
Dr. Bill Ward, early pioneer in GFP research and applications, Rutgers professor, and President of Brighter Ideas, Inc. will present the company’s newly patented (February, 2008), discriminating HTS assay for proteases and protease inhibitors called “GFP-on-a-String.” Using GFP as a reporter, the “GoaS” assay discriminates among proteases using a multi-channel battery of different
GFP-based substrates to effectively and precisely “fingerprint” the protease in question. Unlike FRET-based assays, the “GoaS” technology, an appearance assay, has extremely high sensitivity and remarkable signal-to-noise (6000 to 1 in prototype) as leaching of fluorescence from GFP cannot occur. Other Bii technologies include a newly developed GFP “dip stick” and a patented protein mini-prep kit, the latter being presented as a short course on January 11.
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10:20 Coffee Break
OVERCOMING CHALLENGES
10:45 Synergy of Co-Optimized Culture and Purification Processes for the Production of a Recombinant Protein Expressed at Low-Level
Dominique Rolland, Ph.D., Senior Research Scientist, Protein Biochemistry, bioMérieux France
The expression and purification of recombinant proteins from host cells incite the biotechnologist to solve a multitude of problems which could impact an industrial project. Among these, low level expression in particular can be seen to complicate the purification task. This case study concerns the low-level expression of a membrane protein of Toxoplasma gondii in the yeast Schizosaccharomyces pombe. To obtain a sufficient quantity for in vitro diagnostic purposes, optimized strategies for both expression and purification were implemented. The first step involved the simultaneous selection of the medium components for improving the yeast growth, and for considering the chromatographic behavior of peptones. Following this, the best yield fermentation mode was shown to be fed-batch. Secondly, the capture step on expanded bed chromatography was demonstrated to be more favorable than fixed bed ion exchange chromatography for the processing of large feed stock volumes. These improved upstream and downstream processes produced increments of twelve fold in productivity and two fold in purification factor, reduced process time, and thus decreased the economic costs by a factor 10.
11:15 Developing High Concentration Formulations of Peptides and Proteins: Use of Self-Interaction Chromatography
Mark Cornell Manning, Ph.D., Chief Scientific Officer, Legacy BioDesign LLC
Numerous studies have now demonstrated that protein-protein interactions affect a wide variety of important physical properties of peptides and proteins, including solubility, viscosity and aggregation rates. The extent of protein-protein interactions is reflected in the osmotic second virial coefficient (so-called B22 value). Traditionally, this parameter was determined using static light scattering
(SLS). However, this is a time-, labor-, and material-intensive approach. Furthermore, B22 values cannot be obtained for peptides by SLS due to their small size. Recently, the advent of self-interaction chromatography (SIC) has allowed B22 to be obtained directly for both peptides and proteins in a rapid fashion using conventional HPLC equipment. This approach allows a number of potential formulations to be screened in a relatively short period of time using little material. We have used SIC to identify solution conditions that provide sufficient solubility to allow development of high concentration formulations. This has been done for polypeptides ranging from 4 to 150 kD in size.
11:45 CaptureSelect Affinity Products in Proteomics Discovery, Bench Scale Purification and Bioprocess Applications
Laurens Siekstra, Ph.D., Chief Executive Office, BAC BV
The development of novel antibody formats, protein scaffolds and fusion proteins for the new generation of biotherapeutics requires efficient and standard purification tools to isolate these lead molecules but as well can subsequently be used in the larger scale purification of such compounds. In this presentation the CaptureSelect technology will be described, which has a proven track-record of solving these kind of purification challenges. Based on single domain antibody fragments produced in yeasts, it provides rapid development of highly stable, specific and easy to use affinity
ligands. Purification examples will be described for various biomolecules (e.g.
Fab, Factor VIII, Adeno associated virus), antibody classes (e.g., IgA, IgM, IgE), fusion proteins and non-antibody protein scaffolds using CaptureSelect affinity
ligands.
12:15 Close of Morning Session
12:30pm Luncheon Workshop (Sponsorship Available) or Lunch on Your Own
TAG TECHNOLOGY
2:30 Chairperson’s Remarks
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Featured Presentation
2:35 Recombinant Protein Purification Using Self-Cleaving Non-Chromatographic Bioseparation Tags
 David W. Wood, Ph.D., Assistant Professor, Department of Chemical Engineering, Princeton University
We have developed two novel non-chromatographic alternatives to conventional affinity-tag separations. These methods rely on self-cleaving self-aggregating tags that can be used in a variety of expression hosts to produce “self-purifying” recombinant proteins. In the first system, the target protein is fused to a self-cleaving
elastin-like peptide (ELP). This fusion can be purified via salt and temperature-dependent aggregation of the ELP tag, combined with sequential rounds of centrifugation and
resuspension. In the second system, the target protein is tagged with a protein that binds selectively to polyhydroxybutyrate
(PHB) and expressed in a host that is engineered to produce macroscopic PHB granules. The target binds to the granules via the tag, and is purified by simple centrifugation. In both cases, the tags are triggered to self-cleave after purification by a mild pH shift at room temperature, and can then be easily removed. These methods retain the power and generality of conventional affinity methods, but have the added advantages of a self-cleaving tag and a simple non-chromatographic separation. Recent results include genetic optimization of the ELP tag, as well as expression and purification of various test proteins in E.
coli, Pichia pastoris and CHO, with a variety of self-cleaving conventional and non-chromatographic affinity tags. Further, we have modified the self-cleaving moiety for use with Gateway® cloning, providing a means for very rapid development of new purification schemes with arbitrary protein targets.
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3:05 Novel Affinity Tag System Using Structurally Defined Antibody-Tag Interaction: Application to Single-Step Protein Purification
Junichi Takagi, Ph.D., Professor, Institute for Protein Research, Osaka University
We introduce a novel affinity tagging system that is suitable for the use in protein purification for structural as well as functional studies. Unique features of the mAb (clone P20.1) enabled the efficient capture of tagged proteins from the culture supernatants of transfected cells, and gentle elution by water-miscible organic solvents. Three dimensional information for antibody-tag interaction derived from the crystal structure of P20.1 Fab fragment in complex with the epitope peptide, along with the availability of single-chain Fv fragment of P20.1, would guide even further improvement of the system by protein engineering.
3:35 Element-Coded Affinity Tags
Claude Meares, Ph.D., Professor, Department of Chemistry,
University of California, Davis
4:05 Refreshment Break
DOWNSTREAM
INTO LARGER SCALE
4:30 RAPPTor® - A New Technology for High Throughput Downstream Development
Dorothee Ambrosius, Ph.D., Director, Downstream Development, Boehringer Ingelheim Pharma GmbH & Co.
Downstream processing is becoming a cost driver in the production of biopharmaceuticals since upstream technologies nowadays achieve very high product titers. Together with the demand for short development timelines a major challenge arises to develop both not only safe and robust but also highly economic downstream processes. Platform processes do address requirements to increase speed but often to the cost of yield and process productivity. Therefore we developed and implemented the RAPPTor® technology to address both parameters: development speed and process quality. With RAPPTor®, Boehringer Ingelheim’s Rapid Automated Protein Purification Technology, many conditions can be screened in parallel at a fraction of time and effort compared to conventional techniques. In early product development phases when little information is available about molecule or cell line properties, multiple binding and elution conditions can be rapidly screened and evaluated in parallel. The potential of RAPPTor® technology will be presented in two different case studies.
5:00 HPTFF for Non-Affinity Monoclonal Antibody Purification
Nuno Fontes, Ph.D., Senior Engineer, Late Stage Purification, Genentech Inc.
High performance tangential flow filtration (HPTFF) enables protein purification, concentration and formulation in one unit operation. Several applications have been demonstrated in the literature including the separation of monomers from
oligomers, an antigen binding fragment from a similar size impurity, protein variants differing at a single amino acid residue, and more recently, using real-world industrial feed streams, a 10-fold removal of E.coli host cell proteins and a 100-fold reduction in Chinese Hamster Ovary cell host cell proteins. This talk reviews recent applications and advances in HPTFF technology that have enabled its use for industrial scale monoclonal antibody purification, in particular as part of a non-affinity purification platform.
5:30 Welcoming Reception in the Exhibit Hall
7:00 Close of Day
Overview | Short
Courses | Day 1 |
Day 2 | Day 3 (Joint
Session) | Download Brochure
For more information, please contact:
Mary Ruberry, Conference Director
Phone: 781-972-5421
E-mail: mruberry@healthtech.com
For exhibit and sponsorship information, please contact:
Suzanne Carroll, Manager-Business
Development
Phone: 781-972-5452
E-mail: scarroll@healthtech.com
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