PEGS 2008 | Protein Scale-Up & Manufacturing

Wednesday, April 30

7:30 am - 5:00pm Registration Open

ADDRESSING CHALLENGES

8:30am Chairperson’s Opening Remarks

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Protein Scale-Up & Manufacturing Brochure

8:40 Refolding – A Challenge for Scale-Up?
Stefan R. Schmidt, Ph.D., Associate Director, Global Protein Sciences and Supply, AstraZeneca R&D
Protein expression in bacteria often results in the formation of inclusion bodies. Despite their negative image, inclusion bodies offer several benefits: protection from proteolytic degradation, simple protein enrichment and isolation, high-yield, cost-efficient production, and ability for isotopic labeling. However, the downside is the low recovery during refolding. We will present data from several medium-scale target protein production campaigns. We are comparing column refolding with traditional refolding by dialysis or rapid dilution with regard to efficiency and quality. The influence of typical parameters will be discussed, as well as opportunities and limitations for scale-up. The positive results demonstrate the power of this often neglected technology, and may convince other researchers of the usefulness of this approach.

9:10 Challenges in Scaling Up the Purification Process without Compromising the Purity, Recovery, Stability, Quality, and Potency
Indresh Srivastava, Ph.D., Associate Director, Vaccines Research, Novartis Vaccines & Diagnostics Inc.

9:40 Recombinant Erwinia chrysanthemi L=Asparagines Production
Richard Heath, Ph.D., Lab Director, Protein Science Division, St. Jude Children’s Research Hospital
Asparaginase is a protein therapeutic that has been used in the treatment of pediatric acute lymphoblastic leukemia (ALL) for over 50 years, and, more recently, is being incorporated in protocols for adult leukemias and other asparagine-requiring cancers. Three major products are available in the US with patients being switched from an E. coli preparation to the Erwinia protein upon development of an immune reaction. However, availability has been sporadic, and lack of alternate proteins has negatively impacted therapy. St. Jude, therefore, embarked upon a program of ensuring that this orphan drug is reliably available to firstly our own patients and secondly, through partnerships, that it is available to all children with ALL. We chose to target the Erwinia protein as this was unavailable at the time the project was initiated and had least commercial interest. It was decided to use recombinant technologies and express the protein in E. coli. A fed-batch fermentation protocol was established for optimal expression of the protein into the periplasm. A simple, two-step purification was developed to produce biologically active material in high yields and at low cost. The process was designed to be translatable to a cGMP facility, and such a transition is currently underway.

10:10 Coffee Break in the Exhibit Hall

INTO THE CLINIC

11:10 Implementation of a Flexible cGMP
Compliant Stability Program for Phase I, II and III Studies
Nanda Subbarao, Ph.D., Senior Consultant, Biologics Consulting Group, Inc.
An overview of the Stability Program as a product moves from phase I to commercial stage will be presented. Content of stability protocols, analytical methods, development and validation of analytical methods during the different phases will be addressed. The requirements for support systems such as equipment maintenance/ calibration program and training program during the different phases will also be discussed.

11:40 Improved and Cost Effective Biotherapeutics from Transgenic Avian; Preclinical and Clinical Studies
Yashwant Deo, Ph.D., President, AviGenics Inc.
AviGenics has developed a proprietary avian transgenesis technology to produce human-like glycosylated bio-therapeutics for cancer, infectious diseases and organ dysfunction.
This technology involves proprietary vectors and transgenesis procedures to facilitate commercially feasible levels of protein expression in avian eggs. Employing this technology, AviGenics is currently developing three bio-therapeutics targeted at disease conditions with urgent clinical needs. Preclinical data and recently completed, US-FDA approved human clinical trials for two of our products show that therapeutic proteins produced using AviGenics technology are well-tolerated and effective at clinically relevant doses. With the human-like glycosylation, these products are expected to offer an improved safety profile and better efficacy than the currently available products. Phase II clinical trial for one of these products is now in progress. A third product will enter clinical trials soon. The description of this avian transgenesis technology, as well as preclinical and clinical data, will be presented.

12:10 Luncheon Technology Workshop
Avecia’s pAVEway Expression System - 1 Year On
Andy Topping, PhD, R&D Director, Early Phase Programs, Avecia Biologics Inc.
Avecia’s innovative pAVEway E.coli expression system was launched 12 month ago. This system comprises 3 elements - vectors, strains and standardized fermentation conditions. The system allows tight repression, fine tuning of induction and rapid achievement of high product titres. In the past year the pAVEway™ system has been evaluated on many customer projects and real life data will be presented from projects undertaken in the past 12 months to demonstrate the utility of the system on multiple products, the improvement in titres seen and the rapid progression from cloning to high titre fermentation cultures. The session will include a review of the pAVEway™ system, the data generated to test the system prior to launch and comparison to the latest real-life examples.

Sponsored by

12:40 Luncheon Technology Workshop
(Sponsorship Available) or Lunch on Your Own

1:10 Break

OPTIMIZATION TOOLS

1:30 Chairperson’s Remarks
James Blackwell, Ph.D., Senior Consultant, BioProcess Technology Consultants, Inc.

1:35 Protein Optimization Using the GFP Toolbox
Geoffrey Waldo, Ph.D., TSM, Biosciences, Los Alamos National Laboratories
We have developed a suite of protein manipulation tools based on GFP reporters, fluorescent protein tagging peptide microdomains, and solubility detectors that allow researchers to engineer proteins to optimize their stability, folding, and solubility. Several case studies are presented to highlight the unique advantages of the platform. We show how this technology has been used to produce large amounts of soluble pathogen proteins for structural studies at LANL. We have taken unstable, insoluble, aggregation-prone proteins and have successfully engineered soluble, folded, active proteins by directed evolution using folding reporters based on GFP. Recently, we have produced all 6 domains for structural study of the largest polyketide synthetase from M. tb by our enhanced domain-trapping platform using split GFP. The split GFP has allowed us to screen constructs, mutants, for optimal versions of proteins. It also gives us the flexibility to follow protein expression and purification throughout the entire cloning, overproduction, and workup using a small, 15 amino acid tag from GFP.

2:05 Challenges in Scaling Up Newly Developed Microbial Manufacturing Processes
Susan Dana Jones, Ph.D., Vice President and Senior Consultant, BioProcess Technology Consultants, Inc.
Unlike antibody manufacturing processes today, microbial manufacturing processes are still developed individually and are very dependent on the unique structure and behavior of the target protein. Platform processes are usually not applicable and therefore the development timeline can be longer. In this talk, an approach to streamline development and scale up of a microbial manufacturing process will be described. The process was developed at the 5-10 L scale and parameters were fixed based on multiple process demonstration runs at that scale. The scale up to the 1000 L scale was seamless in most parameters due to the wealth of information obtained at the small scale. Some unit operations did not transfer perfectly and these will be described and reviewed for approaches that would increase the success rate of these steps. The overall program and GMP manufacturing were very successful, demonstrating that there is not a need for intermediate scales but that with rigorous development and testing at small scale, transfer directly to the large scale manufacturing plant can be performed. This approach will save time and money for companies that are developing unique products expressed in microbial systems.


2:35 Solutions Showcase I Ni-NTA / TAGZyme Technologies:　 A Case Study on Large-Scale Protein Manufacturing and Crystallization Frank Schaefer, Ph.D., Director R&D, Germany QIAGEN GmbH Moving from R&D to pharmaceutical development is a costly process. It is therefore of paramount importance to design a manufacturing process that combines robust and well-documented technological platforms. In the case of therapeutic recombinant proteins, the use of a scalable process and an established, proven purification technology are important cost factors. Another very important consideration is that therapeutic recombinant proteins designed for human administration should have a structure that is as similar to that of the authentic product as possible (i.e., the presence of affinity-tag or vector encoded amino acids should be avoided). The TAGZyme™ system allows the efficient and precise exoproteolytic removal of N-terminal tags from recombinant proteins. In combination with Ni-NTA technology, the TAGZyme system provides high-purity proteins free of vector-encoded amino acids for use in applications that demand recombinant reagents, an absence of non-specific cleavage, and a complete removal of all impurities from the target-protein preparation.　 We present a study on the efficient use of Ni-NTA and TAGZyme technologies for demanding applications, such as preparation of pharmaceuticals and protein crystallization. The human cytokine Interleukin 1 beta (IL-1) was expressed in E.coli and 2 gram purified as a 6xHis-tagged protein and the tag cleaved completely by TAGZyme exoprotease to obtain the native mature form of the protein. A comprehensive quality control was performed including gel filtration, light scattering, 2D gel electrophoresis, MS/MS, Edman degradation, and analyses of endotoxin, protease, and host cell protein content. In addition, the homogeneity of the IL-1 produced was investigated by protein crystallization and structural analysis. We obtained large crystals from conditions that differ significantly from the one reported previously and resolved the protein structure at 1.9 Å resolution.　　Our data show that the combination of Ni-NTA chromatography and TAGZyme tag cleavage delivers protein of high quality for use as a biopharmaceutical and for protein structural analyses.	Sponsored by:

2:50 Solutions Showcase II New Tools for Biopharmaceutical Process Development Anna Heijbel, Senior Product Manager, GE Healthcare Bio-Sciences-AB Process development is an important part of the biological drug development cycle. The development of robust, scalable, and regulatory-compliant biopharmaceutical processes requires significant time and resource investment. This session will focus on two new tools available to increase productivity and speed up the time-to-clinic. The following topics will be covered: • Development of a comprehensive process development platform • Scale up of chromatography steps • Screening of chromatographic conditions, either in manual or automated workflow • Method optimization and parameter screening • Two new tools for process development – HiScreen and PreDictor	Sponsored by:

3:05 Refreshment Break in the Exhibit Hall

OPTIMIZING THE PROCESS

3:50 Antibody Manufacturing Platforms Optimized for Speed & Quality
Ralf Ostendorp, Ph.D., Senior Director, R&D, MorphoSys AG
In the process of selecting customized therapeutic lead antibody candidates from large antibody libraries, various process challenges have to be met in terms of screening, manufacturing and analytics. The process design needs to ideally serve multiple requirements from the early research phase, where high-throughput methods for lead selection are required through early clinical development, where scaleable and robust methods need to provide high quality clinical trial material (and analytical data) within attractive time frames. This talk will address various technical strategies exemplified by case studies of typical research and therapeutic programs.

4:20 Process Analytical Technology within Talecris Biotherapeutics
Severin Butler, Engineering Manager, PAT, Talecris Biotherapeutics
This presentation will provide insight to companies without a PAT organization on some history of PAT, what a PAT organization looks like, how it operates and some example projects. I will specifically address: