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May 15th several workshops will be held, Please register for the workshops you wish to attend, so we can estimate the size of room required. The list below will be constantly updated, so please recheck, especially before you register.
Workshop A: 8:30-10:00 Organised by Amine Kamen and Otto Merten
Viral vectors are extensively used as delivery systems for gene and cell therapies, oncotherapies and vectors for display or expression of antigens in different vaccination strategies. Also, viral vectors are important tools for acceleration of drug discovery. Over many years, developments in cell culture technologies have been critical to enable mass production of viral vectors and have greatly contributed in facilitating pre-clinical and clinical trials for therapeutic applications. However, progress reports are confined to specialized conferences in these fields and results are published in journals often not accessible by Animal Cell Culture Technologists. The purpose of this workshop is to revue the main technological advancements in the field of cell culture-based manufacturing of viral vectors including:
• Adenovirus and Adeno-Associated viruses • Lentiviruses and retroviruses • Baculoviruses and other enveloped vectors • Other vectors for vaccination
This workshop will address novel approaches in upstream and downstream processing as well as critical developments in quantification of viral particles, and process intensification. Short presentations will be delivered by experts from academia and industry to inform the cell culture community about key advancements in the field of viral vector manufacturing. We also wish to engage the audience in discussing the remaining challenges that cell culture technology can address in further advancements in Gene and Cell Therapy and Novel Vaccine development.
Speakers:
Large scale transfection for viral vector manufacturing Amine Kamen, BRI-NRC, Bioprocess Centre, CA Progress in lentiviral vector processing Otto Merten, Genethon, Evry, France AAV manufacturing using baculovirus system Andrew Bakker , AMT Biopharma, The Netherlands Cell culture derived- influenza virus processing Udo Reichl, Max-Planck-Institut für Dynamik komplexer technischer Systeme, Germany New cell line for viral vaccine manufacturing Majid Mehtali, VIVALIS, France
Workshop B:
Organised by Mike Betenbaugh, Nicole Borth and Kelvin Lee
During the past year, efforts to obtain useful sequence data information for CHO cells have increased significantly, thus stressing the necessity for easily accessible databases and sophisticated tools for analyses of biological data sets. The web site, www.CHOgenome.org was founded as a CHO community-based effort to establish a single site for combining and housing CHO related tools. This site will include publicly available genomic, cDNA and microRNA sequence information as well as other datasets in a format that facilitates their exploitation in biological and bioprocess research. The workshop will include presentations on the current efforts of several international groups in sequencing both CHO cell lines and the Chinese Hamster, followed by a discussion on the expanding needs of the worldwide scientific community with respect to the CHO genome and its use. Those interested in these efforts and in facilitating the use and future developments for this website are encouraged to attend and participate.
Workshop C: Fc Fusion Proteins: 14:00-15:45 Organised by Steven Chamow, Ph.D., S. & J. Chamow, Inc.
The potential therapeutic value of many proteins—including enzymes, cell-surface receptors, cytokines and peptides—can be realized by fusing these proteins to the Fc region of human immunoglobulin G. Of the 30 mAb products approved as human therapeutics in the USA to date, 4 are Fc fusion proteins, and many more are in clinical testing. Considerations in fusion protein design and production will be presented.
Speakers: Pierre-Alain Girod, Selexis Thomas Ryll, Biogen Idec Michiel Ultee, Laureate Pharma Kevin Bailey, Regeneron
________________________________________________________________________ Workshop 1: 8:30-10:00 Organised by David Onions, Bioreliance
Massively Parallel Sequencing (MP-Seq): a New Tool for Adventitious Agent Detection and Virus Discovery David Onions, Bioreliance Recent contaminations of manufacturing processes by porcine circovirus and vesiviruses have highlighted the need for broadly based and rapid methods to detect adventitious agents in cell banks, virus seeds and bulk product (drug substance). Massively parallel sequencing (MP-Seq) is a powerful new method for the identification of viruses and other adventitious agents, without prior knowledge of the nature of the agent. BioReliance have developed MP-Seq methods to detect free viruses in raw materials and fermenter samples. Our application of this technology has resulted in the discovery of a new parvovirus in bovine serum capable of infecting human cells and we have used this technology in the investigation of fermenter contaminations. In some cells, the genomes of latent or transforming viruses may be present in a cell but no virus particles are produced. However, latency associated or transforming gene mRNAs are expressed. We have developed a method to identify these latent viruses by sequencing the total transcriptome of the cell and applying an algorithm to identify the viral specific transcripts. Enormous amounts of data (~400Mb) are generated in this process and a robust algorithmic process is required to analyse the data. Using this method we have been able to identify a new retrovirus expressed in Vero cells and we have identified nodavirus and errantivrius contamination of insect cells. The definitive nature of the methodology provides considerable reassurance that cell banks are free of unexpected contaminating agents. We are also developing MP-Seq to provide rapid end points in in vitro virus detection assays. We have shown that we can detect virus infection as early as day 4 post infection with MP-Seq while conventional methods may require 14 or 28 days to reveal infection.
Reference: Onions D Kolman JMassively parallel sequencing, a new method for detecting adventitious agents. Biologicals. 2010 377-80. 2010
Application of Massively Parallel Sequencing (MP-Seq) to identify High Producing Clones and Validate the Genetic Stability of Expression Constructs John Kolman, Bioreliance
Application of Massively Parallel Sequencing (MP-Seq) methods to the analysis of genetic stability of cells has resulted in a quantum change in the quality of data. The complete sequence of the construct genes are obtained with extraordinarily high coverage. In addition the flanking sequences of inserts and the concatamer junction points are resolved. Sequencing of the transcripts also provides data not available from conventional Sanger sequencing where only a consensus sequence results. In contrast, MP-Seq provides the sequence of rare variants that may be present within the cell. We have coupled MP-Seq approaches with Automated Spectral Karyotyping (SKY) methods to provide additional data on the genetic stability of the cell. SKY is capable of detecting fine translocations that can be missed by traditional karyotyping methods and can give warning of genetic instability that lies outside of the target construct. Perhaps the most exciting application of MP-Seq is in the developing field of clone selection. Recently, nucleotide arrays have been used to identify genes differentially expressed in high and low producing CHO cells and the data has been used to modify media to optimise production (Schaub et al 2010). MP-Seq provides additional data above that available from arrays. BioReliance has data sets on the transcriptome of multiple CHO clones and is developing pathway analysis tools to identify signatures associated with high producing cells.
Reference : Schaub et al Biotechnology & Bioengineering. 2010 Feb 1;105(2):431-8. ________________________________________________________________________ Workshop 2: Using a Rational Approach 8:30-10:00 Organised by Irvine Scientific
Dr. Tom Fletcher, Director of R&D Cell Culture Successful upstream manufacturing processes for new biopharmaceutical products are dependent on two key parameters; the development of optimal cell culture media formulas (both growth media and feed solutions), and on the development of manufacturing protocols which result in consistent yields and product quality. Optimization of cell culture media formulas can be accomplished using a variety of approaches, each featuring certain strengths and weaknesses. Various methods and tools will be discussed and compared, including media library screening, component heat mapping, metabolic profiling, DoE optimization of components individually and as groups, and metabolomics, using data from actual case studies as examples. The most effective approach often involves combining several methods and tools chosen in combinations to fit particular project needs. The role of a media partner in supporting innovation during cell culture manufacturing process development will also be discussed.
Workshop 3: 10:15-11:45 Organised by Dr. James W. Brooks, PhD, R & D Manager, BD Advanced Bioprocessing
Cell based therapeutics using Mesenchymal Stem Cells (MSC) are emerging for the treatment of a wide range of acute and degenerative human diseases. BD Mosaic™ MSC SF is a unique, high performance medium, which, through several key features, enhances MSC expansion with reduced culture time, labor, and medium requirements.
Workshop 4: Evolution of Risk Mitigation 10:15 11:45 organised by SAFC® In order to continue to provide the industry with solutions for the future, SAFC as a member of Sigma Aldrich has ventured into new technology. The Corporation is developing new media and feed formulations to support industrially relevant platform expression systems. Historically, for a platform expression system to meet industrial standards, such as they are, product must be animal component free, use chemically defined media and have a supply chain that is transparent and robust to ensure continuity of supply and minimal TSE/BSE transmission risk. Speakers
Workshop 5: 12:30-14:00 Organised by Dr. Christel Fenge, Sartorius Stedim Biotech
During the last decade the advent of larger scale single use process solutions have changed our approach to making proteins from cell cultures dramatically. Driven by a growing pipeline of biopharmaceutical drugs in development and the cost pressure that the Pharma industry is experiencing, more and more companies are adopting single use bioreactors up to and beyond the 1000L scale. A similar trend can be observed in downstream processing although probably less matured yet. Membrane adsorption lends itself to single use applications with the potential of combining cell removal, clarification and capture into one unit operation. A number of companies have already taken the step and implemented hybrid or even single use production facilities for monoclonal antibodies or vaccine production. Saleability of the process and consistency of results from bench-top bioreactors used in process development and as scale-down models in process validation to commercial scale production bioreactors is a key prerequisite for successful drug development. Therefore, ideally the design of the single use bioreactors should be as close as possible to the full scale systems to reduce the number of variables that may influence product quality and process performance. Finally, with all this different disposable solutions used in GMP production, not only the vendor – user relationship has changed but also the approach to validation of single use equipment such as bioreactor bags and containers for process fluids.
During the workshop we will discuss current status of single-use process solutions, challenges and future outlook. In order to stimulate a lively discussion the following discussion primers will be prepared.
1) Current status of single-use process solutions Dr. Thorsten Peuker, Director Marketing and Sales Integrated Solutions, Sartorius Stedim Biotech
2) Characterisation and scale-up considerations of single-use bioreactors Dr. Gerhard Greller, Director Upstream Technology, Sartorius Stedim Biotech
3) High titer mAb production in single-use bioreactors Dr. Aziz Cayli, CEO of CellCa
4) DOE supported optimisation of membrane adsorption as a capture step Dr. Sybille Ebert, Teamleiterin Technologieentwicklung DSP, Rentschler Biotechnologie
5) Extractables and Leacheables – a concern? Heike Frankl, Director Validation Services Europe, Validation Services, Sartorius, Stedim Biotech
Workshop 6: 12:00-14:00 Organised by Life Technologies
Freedom™ CHO-S™ Platform: Gene to clone in 3-4 months achieving mAb titers of 1-3 g/L with no milestone or royalty payments Peggy Lio and Volker Sandig
Stable cell line development is a critical phase of biotherapeutic development. While several CHO based platforms are widely marketed they are costly to access even for research use only evaluation. To enable everyone to perform stable cell line development we have developed a CHO-S™ based kitted platform that allows the end user to go from transfection to stable clone in 3-4 months. Only one scientist is needed for the optimized workflows that are included in the kit. All components necessary to complete the workflow are included. Key to the optimized workflow is how transfection, selection and cloning have been co-optimized. mAb titers achieved with these platforms are ~1 g/L for an un-optimized simple glucose fed batch process and up to 3 g/L when more complex nutrient feeds are employed. Research use rights are granted upon kit purchase. Commercial licensing is unprecedented and requires only a onetime fee.
Media and Feed Platforms: Control of cell function by nutrient and process integration Steve Gorfien
Sustained growth of the biopharmaceutical market has created a need for high-titer processes to meet increasing demand and to reduce manufacturing costs. Advances in recombinant cell line engineering have resulted in high producing clones with high nutritional demands. Depletion of critical nutrients in a production process can limit the potential of these clones, causing reduced titers and resulting in inefficient, costly processes to compensate for media or feed deficiencies. Through integration of base and feed media development, we have addressed nutrient limitation issues in rCHO cultures and obtained substantial improvements in titer by sustaining specific productivity for extended periods of time. Such approaches are clearly effective, but are time consuming and are limited by the large number of possible component combinations and the need to integrate process parameters like pH, temperature, dissolved oxygen, agitation rate and time of feed addition with a balanced nutrient composition. We have applied high throughput (HT) tools that enable simultaneous evaluation of multiple nutrient and process variables in up to 420 simultaneous conditions. Case studies will be presented demonstrating the power of HT tools to evaluate broad design spaces making possible rapid creation of chemically defined processes with multi-fold increases in titers while maintaining final product quality.
Highly sensitive PCR based assays for In-Process testing: Rapid detection of cell culture contamination Michael T. Brewer
The possibility of viral or Mycoplasma contamination of mammalian cell culture during large scale manufacturing of biological therapeutics has always been a concern in the industry. Because the traditional adventitious agent or Mycoplasma tests take considerable time, contaminant testing is generally only conducted during cell banking and following final harvest. We have recently developed Real-Time PCR based assays that allow for rapid, highly sensitive detection of Mycoplasma, Mouse Minute Virus (MMV) and Vesivirus 2117. These rapid assays allow for testing for the presence of these agents at multiple points during the cell culture manufacturing process, allowing for the earliest possible detection of a contamination event. The importance of rapid detection using molecular based testing has been further highlighted recently in public reports of viral contamination events that affected both product supply and product quality. We will review the assay designs, sensitivity, sample preparation and proprietary discriminatory positive/extraction controls developed for these assays during our presentation.
Workshop 7: 14:15-15:45 Organised by Elizabeth C. Dodson, PhD, R & D Manager, BD Advanced Bioprocessing
Serum. Hydrolysates. Chemically-defined. Rethinking media supplementation requires the identification of molecules that drive growth and protein production. Biochemical deconstruction of hydrolysates coupled with high resolution analysis was used to identify these molecules. Using DoE, a chemically defined media supplementation has been developed to substitute for yeast extract peptone.
Workshop 8: How QbD is Changing Upstream Bioprocess Design 14:15-15:45 Organised by William G. Whitford, Thermo Scientific Cell Culture & BioProcessing
This workshop session will focus on the evolving demands of quickly and accurately improving the quantitative output from bioprocessing expression systems, while also maintaining (or in some cases increasing) the qualitative bioactivity of the expressed molecule. Examples of regulatory directives and process control technologies interfacing with bench-level cell culture optimization will be shown.
How QbD is Changing Upstream Bioprocess Design William G. Whitford, Sr. Manager, BioProcessing Market, Thermo Scientific Cell Culture & BioProcessing In light of QbD, PAT, and process platform imperatives, bioproduction process design now requires capabilities and capacity beyond essential facilities and personnel. The goals of producing high levels of quantity product in a robust and flexible production process require capabilities beyond even subject matter experts with access to a repertoire of reference formulations. In-house technologies now demanded include a full complement of cell and culture media analytics and HTS capabilities. In most cases, some level of product quality and attribute assays beyond simple product level quantitation is highly recommended. Process optimization toward highly regulated manufacturing can also draw upon such capabilities as regulatory certifications, quality management systems, and qualified raw materials sourcing. Modern demands for increased process understanding, CPP determination, and robust design space development virtually require access to qualified scale-up and technology transfer equipment and methods rather early on in process development. Increased demands in process implementation efficiency are best supported by early consideration and testing of appropriate product containment and transfer technologies. The case-studies presented here illustrating such approaches are the fastest way to communicate the art and science of modern process design.
Fast-track process development using commercial feed solutions Karlheinz Landauer, Director, R&D, Celonic AG, Basel
New biological entities, NBEs, as well as biosimilar development have high demands in rapid process development leading in high yielding, and robust processes. To achieve these necessities industry needs platform technologies for the development of high quality processes producing high quality recombinant products with stable features. The prerequisite of such platform technologies is the use of an excellent host cell line, which is safe in a regulatory point of view, proven to produce high amount of recombinant protein over many generations (stability) and works within a proven design space. Based on such cell lines media and feed-media development can also be based on a platform technology. Here the prerequisites are animal component free media and solutions; large scale availability, low costs and high lot to lot consistency. New industrial standard for production of recombinant proteins is the usage of chemically defined media. Albeit hydrolysates and peptones are still widely used, the issue of lot to lot variability and the dependency on the production process of a specific peptone and its features leads to a certain uncontrolled status within a production strategy. Therefore it is inevitable to control all substances used to produce the product, also “goodies” of conventional peptones, such as vitamins, fatty acids and trace elements. Media requirements for animal cells strongly depend on the recombinant product, the mode of fermentation and last but not least on the cell line itself. The slightest changes in the media composition may lead to changes in post-translational modifications. Such differences could change the behavior of a product in-vivo and may compromise timelines and whole development schedules. The use of chemically defined media in the processes are favorable and the future for biologics. In addition it helps to keep time lines and opens the possibility of a fast-track process development.
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On the morning of Sunday, 