An Update on Cell-Based Technologies

View PDF

 

 

It’s always exciting to find out where the next meeting of the European Society for Animal Cell Technology will be. This venerable conference happens somewhere in Europe every other year. Recent sites have included Dublin, Ireland (2009); Dresden, Germany (2007); Harrogate, England (2005); Granada, Spain (2003); and Tylösand, Sweden (2001). This May, the gathering of animal cell culture scientists and engineers will convene in the palatial setting of the historic Hofburg Congress Center, formerly the Hapsburgs’ imperial residence in Vienna, Austria. ESACT meetings are famous for their beautiful locations and enjoyable social events. I for one will never forget the organized day outing at the magnificent Alhambra in Spain followed by a private classical guitar concert at the beautiful Huerta del Sello. But the biannual conference is far more than just an organized social event for industrial and academic cytologists from around the world.

The 1999 ESACT conference in Lugano, Switzerland featured Leonard Hayflick (an early pioneer in animal cell technology) providing a historic account of WI-38 and related human diploid cell substrates that were first considered “dangerous” for production of polio virus in the mid-20th century. By that year, most such fears had been finally put to rest, and goal-driven and pragmatic approaches to cultivated animal cells were under way. Improvement of yields and metabolic engineering were a major focus of the conference, and the idea of transient gene expression was first introduced as a session. Tissue engineering first appeared that year, as well. See the ESACT website (www.esact.org/meetings_esact.html) for reports and proceedings from several other of the 21 such conferences that have met so far.

Now the first decade of the 21st century is behind us — and five more ESACT meetings too. Product titers have improved exponentially since that 1999 conference, perhaps more than its attendees could have imagined.

Transient gene expression and tissue engineering are both seeing strong commercial potential. And new developments unforeseen then will be the talk of the meeting this year. A series of workshops beginning the conference on Sunday 15 May 2011 will focus on many practical aspects of using animal cells in biotherapeutic development and manufacturing. Recent advances will be discussed in Chinese hamster ovary (CHO) cell genomics, serum-free media, and cellular analysis and culture technologies, as well as new approaches in process design and development for familiar and new classes of products.

 

CHO Cells

 

A major achievement in animal cell technology was announced in January 2011: the complete sequencing of the CHO-K1 genome. Nearly three out of four therapeutic proteins are produced using CHO cells.

“Sequencing the CHO genome represents a major milestone in optimizing this widely used mammalian cell line both for pharmaceutical research and for production of therapeutic proteins,” said Bernhard Palsson, professor of bioengineering and adjunct professor of medicine at the University of California, San Diego. “This newly available knowledge will bring multiple benefits, including media optimization and improved cell growth, protein production, glycosylation, and cell line engineering. Ultimately, it brings genome-scale science to CHO-based production of biopharmaceuticals.”

CHO Genome Workshop: A workshop at ESACT 2011 will focus on the CHO genome’s impact. A website (www.CHOgenome.org) was recently founded as a community-based effort to combine and house CHO-related tools. Still under development, this site will include publicly available genomic, cDNA, and microRNA sequence information as well as other datasets to facilitate their exploitation in biological and bioprocess research. Organized by Mike Betenbaugh (professor of chemical engineering at Johns Hopkins University), Nicole Borth (professor of applied microbiology at Vienna’s Universität für Bodenkultur), and Kelvin Lee (professor of chemical engineering at the University of Delaware), this workshop will include presentations on current efforts of several international groups in sequencing CHO cell lines and the Chinese hamster itself. A discussion will follow on what the worldwide Scientific community needs regarding this genome.

CHO Platforms: Another workshop focuses on strategies for viral risk mitigation in CHO platform expression systems. Organized by Kevin Kayser and other scientists from the cell sciences and development group at SAFC, it will also feature speakers from Sartorius Stedim Biotech and Amgen. Sigma Aldrich is implementing zinc-finger nuclease technology to develop a new expression platform technology based on the CHO cell line that should reduce engineering development times and increase cell line stability and product titers. Media and feed formulations and protocols are being developed to create an industrially relevant “off-the-shelf” expression system. To meet modern industrial standards, chemically defined media need to be free of animal-derived components. A transparent and robust supply chain ensures continuity of supply and minimal risk of spongiform encephalopathy transmission.

Recent news events (from Genzyme and from Genentech/Roche, for example) emphasize the need for preventing manufacturing interruptions due to virus contamination. Consequently, regulators and industry are talking about supply-chain transparency. Companies need to build layers of protection into their biomanufacturing processes to systematically reduce the risk of all potential contamination sources. According to Kayser, a viral risk management strategy starts with cell line generation and continues with treatment strategies for all raw materials that go into upstream production, where the risk of viral replication in a host cell line is greatest.

SAFC and its strategic partner Sartorius Stedim Biotech will present four different strategies to reduce viral contamination risk from raw materials used in CHO cell culture. Their strategies address raw material sourcing, viral filtration, ultraviolet radiation, and pasteurization as preventive measures for treatment of cell culture raw materials. Finally, a case study will be presented on Amgen’s experiences in implementing a virus risk mitigation strategy for its current platform expression system.

 

“Software” Technologies

 

Culture media are vitally important to animal cell technology. And even as greater productivity is expected of cells in culture, so too are they now expected to adapt to feed sources that are increasingly different from their natural serum/plasma environment. The biopharmaceutical industry and its regulators are demanding more control over the ingredients of media and supplements than ever before: with ever-tightening restrictions from serum-free to protein-free to animal-component–free to chemically defined. So it’s no surprise to find workshops devoted to these issues at ESACT 2011.

Stem Cell Culture: Cell-based therapeutics using mesenchymal stem cells (MSCs) are emerging for treatment of many acute and degenerative human diseases. As discussed in our March 2011 topical supplement, for such therapies to reach their commercial potential, their associated manufacturing processes will need to be optimized for economy, robustness, and reproducibility (1,2,3). One step toward that lofty goal is control of culture media. A workshop organized by James W. Brooks (R&D manager at BD Advanced Bioprocessing) will present on BD Mosaic MSC SF high-performance medium, which can enhance MSC expansion with reduced culture times, associated labor, and medium amounts.

Supplements:Serum … hydrolysates … chemically defined …. Rethi
nking media supplementation requires identifying the actual molecules that drive growth and protein production. In a workshop organized by Elizabeth C. Dodson (R& D manager), BD Advanced Bioprocessing will report how biochemical deconstruction of hydrolysates coupled with high-resolution analysis were used to identify those molecules. Using design of experiments (DoE), BD developed a chemically defined media supplement that can substitute for yeast extract peptone in animal cell cultures.

Process Development: Development of optimal cell culture media formulas (growth media and feed solutions) is but one of three main parameters that form the foundation of successful upstream biomanufacturing processes. The other two are bioreactor technology and development of manufacturing protocols for consistent yields and product quality. Tom Fletcher (director of R&D cell culture at Irvine Scientific) has organized an ESACT 2011 workshop highlighting a rational approach to development of these processes.

“Optimization of cell culture media formulas can be accomplished using a variety of approaches,” Fletcher says, “each featuring certain strengths and weaknesses.” In his workshop, various methods and tools will be compared through case studies: media library screening, component heat mapping, metabolic profiling, DoE optimization of components individually and as groups, and metabolomics. “The most effective approach often involves combining several methods and tools chosen in combinations to fit particular project needs.” Media vendors can serve as partners to support innovation during cell culture manufacturing process development.

 

22nd ESACT SCIENTIFIC COMMITTEE

 

Nicole Borth VIBT-BOKU, Vienna, Austria

Mike Butler University of Manitoba, Kanada

Martin Fussenegger ETH Zürich, Switzerland

Josef Friedl Medical University, Vienna, Austria

Hansjörg Hauser HZI, Braunschweig, Germany

Roy Jefferis University of Birmingham, United Kingdom

Hermann Katinger VIBT-BOKU, Vienna, Austria

Otfried Kistner Baxter Innovations, Austria

Thomas von Zglinicki University of Newcastle, United Kingdom

Stable cell-line development is the first step of bioprocessing. A number of CHO-based platforms are widely marketed, but Life Technologies saw a need for an affordable research-use–only platform that could be available to a wide range of cell biologists. In a workshop titled “From Molecule to Market,” Peggy Lio (a senior process science fellow at Life Technologies) and Volker Sandig (chief Scientific officer for ProBioGen, a contract manufacturer) will present the Freedom CHO-S–based kit, which allows users to progress from transfection to stable clones in just 12–16 weeks. A lone scientist can perform the work, and the kit provides all necessary components. Lio says that cooptimized transfection, selection, and cloning are key to the optimized workflow. Monoclonal antibody (MAb) titers of ~1 g/L have been achieved with this platform in a simple, unoptimized, glucose-fed batch process — and up to 3 g/L with more complex nutrient feeds. Research-use rights are granted with purchase of the kit, and commercial licensing requires only a one-time fee.

In the same workshop, Steve Gorfien (Invitrogen’s director of bioproduction new products and PD-Direct media services) will focus on media and feed platforms. Cell line engineering advances have provided the industry with high-producing clones that also have high nutritional demands. According to Gorfien, depletion of critical nutrients in a production process can limit the potential of those cells, reducing titers and requiring inefficient, costly processes to compensate for media or feed deficiencies.

“Through integration of base and feed media development,” he says, “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 time consuming and are limited by the large number of possible component combinations and the need to integrate process parameters such as 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.” Gorfien will present case studies demonstrating the power of HT tools to evaluate broad design spaces, making possible rapid creation of chemically defined processes with multifold increases in titers while maintaining final product quality.

A subject of recent intense focus, the possibility of viral or mycoplasma contamination of mammalian cell culture during large-scale manufacturing of biological therapeutics has always been a concern. Because established tests take considerable time to perform, this kind of contaminant testing is often conducted only during cell banking and following final harvest. At the Life Technologies workshop, Michael T. Brewer (senior manager of technical marketing) will present a recently developed assay based on real-time PCR for 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,” Brewer says, “allowing for the earliest possible detection of a contamination event.” The importance of such rapid detection has been emphasized by public reports of viral contamination events that affected product quality and supplies.

 

Hardware Technologies

 

Analytical and platform technologies are changing the face of bioprocessing. “The process is the product” may no longer apply when both can be well characterized. As cell biologists focus on cytology, the vendors that serve them are advancing culture technologies to turn art into engineering. Two more ESACT workshops will provide diverse examples.

A Powerful Analytical Method: David Onions (chief scientific officer at Bioreliance) recently published an article on massively parallel sequencing for detection of adventitious agents in cell cultures (4). He has organized a workshop on the technique, at which he and his coauthor John Kolman (senior director and head of pharmacogenomics and global R&D) will speak.

According to Onions, 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 identifying viruses and other adventitious agents without having to know what you’re looking for. BioReliance has developed MP-Seq methods to detect free viruses in raw materials and fermentor samples.

“Our application of this technology has resulted in the discovery of a new parvovirus in bovine serum capable of infecting human cells,” Onions says, “and we have used this technology in the investigation of fermentor contaminations.” In some cases, the genomes of latent or transforming viruses may be present in cells that haven’t (yet) produced virus particles — but latency associated or transforming gene mRNAs are expressed. “We have developed a method to identify these latent viruses by sequencing the total transcriptome of a cell and applying an algorithm to identify viral-specific transcripts.” This process generates large amounts of data (~400 Mb), requiring a robust algorithm for analysis. “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.” Onions says this methodology can provide considerable reassurance that cell banks are free of unexpected contaminating agents. “We are also developing MP-Seq to provide rapid end points for 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.”

The MP-Seq technique has also been applied to identifying high-producing clones and validating the genetic stability of expression constructs, and Kolman’s presentation will highlight these applications. The method allows complete sequencing of construct genes with extraordinarily high coverage. In addition, it can resolve the flanking sequences of inserts and concatamer junction points. Transcript sequencing provides data not available from other types of sequencing, and MP-Seq shows rare variants that may be present within cells.

“We have coupled MP-Seq approaches with automated spectral karyotyping (SKY) methods to provide additional data on the genetic stability of the cell,” Kolman says. SKY can detect fine translocations that are missed by other karyotyping methods and can detect genetic instabilities that lie outside a target construct. “Perhaps the most exciting application of MP-Seq is in the developing field of clone selection,” Kolman adds.

Nucleotide arrays identify genes differentially expressed in high- and low-producing CHO cells, and resulting data have been used to modify media to optimize production (5). MP-Seq provides more data to support those arrays. “BioReliance has data sets on the transcriptome of multiple CHO clones,” says Kolman, “and is developing pathway analysis tools to identify signatures associated with high-producing cells.”

Single-Use Technology: Over the past decade, the available scale of single-use process solutions has increased enough to dramatically change many companies’ approach to making proteins from cell cultures. Many are adopting disposable bioreactors up to and beyond the 1,000-L scale. Christel Fenge (vice president of fermentation technology marketing and product management at SartoriusStedim Biotech) has organized an ESACT workshop called “Toward a Fully Single-Use Protein Production Facility.” Many biotherapeutics companies have already implemented hybrid and fully disposable production facilities for vaccine or MAb production. Process scalability is a key prerequisite for success: both when scaling up from bench-top to commercial-scale production bioreactors and scaling down for process validation. Ideally, says Fenge, disposable bioreactor designs should be as similar as possible at different scales to reduce the number of variables that can influence product quality and process performance.

With all the different disposable solutions used in GMP production, Fenge says that the vendor–user relationship has changed, as has the approach to validation for bioreactor bags and containers. “During the workshop,” she says, “we will discuss current status of single-use process solutions, challenges, and future outlooks.” In addition to presentations from her company on scale-up, DoE optimization of capture technology, leachables/extractables, and the state of the art in disposable technologies, Aziz Cayli (CEO of contract process developer CellCa) will report on high-titer MAb production in single-use bioreactors.

 

Systems Engineering

 

Promising Products: Antibodies are powerful biotherapeutics in themselves, and now they’re contributing to the success of other biologics. According to consultant Steven Chamow, the potential therapeutic value of many proteins — including enzymes, cell-surface receptors, cytokines, and peptides — can be aided by fusing such molecules to the Fc region of human immunoglobulin G. Of the 30 MAb products currently approved as human therapeutics in the United States to date, for example, four are Fc fusion proteins. Chamow says many more are in clinical testing. He has organized a workshop for ESACT 2011 on this growing class of therapeutics, with presenters from Selexis, Biogen Idec, Laureate Pharma, and Regeneron.

But bioprocessing isn’t only about proteins. Viral vectors are extensively used as delivery systems for gene and cell therapies, as oncotherapies, and as vectors for display or expression of antigens in different vaccination strategies. They are also are important tools in drug discovery. Amine Kamen (head of the animal cell technology group at the Canadian national research council’s Biotechnology Research Institute) and Otto Merten (head of the bioprocess development department at Genethon) have organized a workshop focused on recent advances in viral vector manufacturing.

Over many years, they explain, developments in cell culture technologies have been critical to enable mass production of viral vectors. This has helped facilitate preclinical and clinical trials for therapeutic applications. But progress reports have been confined to specialized conferences in these fields and results published in journals often not accessed by animal cell culture technologists. So the two put together a workshop to review the main technological advancements in cell-culture–based manufacturing of adenovirus and adenoassociated viruses, lentiviruses and retroviruses, baculoviruses and other enveloped vectors, and other vaccine vectors. In addition to Kamen and Merten, presenters from AMT Biopharma, Vivalis, and the Max-Planck-Institut für Dynamik Komplexer Technischer Systeme will address upstream and downstream processing technologies and critical developments of viral particle quantification and process intensification.

Quality by Design (QbD): No matter what type of cell culture system you’re using — or what you’re using it to make — the concept of QbD changes how you will approach process development. So William G. Whitford (senior manager of the bioprocess market for Thermo Scientific Cell Culture and BioProcessing) has organized a workshop to focus on the evolving demands for quickly and accurately improving the quantitative output from expression systems while also maintaining (and in some cases exceeding) the qualitative bioactivit y of expressed molecules.

QbD, process analytical technology (PAT), and platform imperatives are changing the bioprocess industry. Whitford says that bioprocess design now requires capabilities and capacities beyond the traditional facilities and personnel. “Producing high levels of quality product in a robust and flexible production process requires capabilities beyond even subject matter experts with access to a repertoire of reference formulations,” he explains. “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. Whitford will present case-studies illustrating such approaches are the fastest way to communicate the art and science of modern process design.

Joining him will be Karlheinz Landauer (director of R&D for Celonic) to describe fast-track process development using commercial feed solutions — a return to the all-impo
rtant subject of media. Rapid process development for high-yielding and robust processes is going to be essential to both new biological entities (NBEs) and biosimilars of the near future. To achieve those goals, Landauer explains, the industry needs platform technologies. “The prerequisite of such platform technologies,” he goes on, “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, and works within a proven design space.”

Media development for given cell lines can be part of such platform approaches. “Here the prerequisites are animal-component–free media and solutions, large-scale availability, low costs, and high lot-to-lot consistency.” Chemically defined media are becoming industrial standard for production of recombinant proteins. Hydrolysates and peptones are still widely used, says Landauer, but lot-to-lot variability for processes dependent on specific peptones makes for a production strategy that isn’t entirely controlled. Bioprocessors want to control all substances used to make their products while giving cells the vitamins, fatty acids, and trace elements that peptones provide.

“Media requirements for animal cells strongly depend on the recombinant product, the mode of fermentation, and on the cell line itself,” Landauer says. “The slightest changes in media composition may lead to changes in post-translational modifications.” Such differences could change the in vivo behavior of a biologic and even compromise development schedules and timelines. So chemically defined media are favorable for the future for biologics. This, according to Landauer, opens up the possibility for fast-track process development.

Cheryl Scott is senior technical editor of BioProcess International.

ESACT 2011

on Cell-Based Technologies

by Nicole Borth

Cell-Based Technologies is the scientific motto of the 2011 ESACT meeting in Vienna, Austria — highlighting the importance of cells as the central tool for all technologies and applications covered in the Scientific program. In previous and similar meetings, traditionally a “vertical” session organization has been chosen: following cell-line development, process optimization, and downstream processing complemented by specific sessions on different product categories. In line with this year′s motto, however, the members of the scientific committee have decided to use a more “horizontal” approach that puts the cell and different aspects of its functioning at the focus of our attention. Cellular properties and capabilities — such as stability, mechanisms of protein production, differentiation, and complex interactions — will be the focus of this conference. We’ve mixed approaches and topics that come from

  • basic science in pursuit of biological understanding
  • process engineering in search of technological advances
  • new products, both cell therapies and therapeutic proteins
  • case studies that reflect the challenges encountered by the community.

In the end, all of the above will contribute to better understanding and control of the cellular property highlighted within each session.

In total, the scientific committee received more than 370 abstracts for oral or poster presentations. So choosing oral presentations was not easy because it was necessary to free up additional time to enable participants to give due justice to the large number of posters that will be presented. The Scientific committee has worked on achieving a balanced program that highlights important topics while stressing that the science presented in the poster abstracts is also of high interest, so we look forward to lively discussions and interesting presentations in the poster sessions as well.

In view of the extensive participation in poster presentations, we developed a new procedure for poster prize selection that will be realized for the first time at this conference. Out of the contributions, 100 were chosen based on scientific content and informative detail according to their abstracts. These will be further reviewed by the poster committee, and 20 candidates will be selected to give short, five-minute presentations during a special session on Wednesday afternoon. Based on the posters and these presentations, the audience will then vote and determine who should receive this year′s poster prize.

 

Scientific Sessions

 

Cell Stability and Differentiation is chaired by Thomas von Zglinicki of the University of Newcastle in the United Kingdom. The use of cells both as producers or products depends on maintaining their phenotypic stability. Such stability is threatened by genetic and epigenetic mutation, cell aging, and differentiation. Selective pressure in cell culture can drive genetic/epigenetic drift and foster population heterogeneity. This session will cover population heterogeneity, genetic and epigenetic stability, differentiation and aging, and selection of cells with specific phenotypes. This applies both to new and primary cell lines as well as production cells. Introductory speakers include Fabrizio d′Adda di Fagagno of Italy on molecular mechanisms of cellular senescence and Johannes Grillari of Austria on MicroRNA-31, an inhibitor of osteogenic differentiation in mesenchymal stem cells.

Cells As Therapies and Stem Cells is cochaired by Hansjörg Hauser of HZI Braunschweig in Germany and Josef Friedl of the Medical University in Vienna, Austria. Cell-based therapies using cell-based medicinal products (CBMP) hold promise for treating diseases such as cancer, autoimmune disorders, and conditions resulting from infection, inflammation, and injury. New insights into the complex interactions between the different cellular components involved in such cases offer new possibilities to manipulate these orchestrated networks. Cell isolation, identification and expansion, specificity, cell-type, validated in-vitro assays, and genetic modifications are just a few examples of many important issues that must be considered for cell-based therapies. Obviously, “manufacturing” of cells has to follow the principles of good manufacturing practice (GMP) to ensure quality and safety. Bioprocessing primary cells for individual patients requires specific and innovative approaches to maintain phenotype and biological function.

Human embryonal stem cells hold great potential as testing substrates and for regenerative medicine. The direct reprogramming of somatic cells into pluripotent cells — namely the production of induced pluripotent stem (iPS) cells — overcomes ethical problems and presents a promising approach to deriving disease-specific cells. To reach that potential, a number of Scientific and technological problems have to be solved. Among those are methods of cell substrate derivation; cell engineering, cultivation, and expansion; medium composition; targeted differentiation; and several safet y aspects including prevention of residual undifferentiated embryonic stem cells (ESCs).

Introductory speakers for this session include

  • Nathalie Chaput of France on the critical role of IFNg for production of highly immunogenic dendritic cell– derived exosomes
  • Christopher Ramsborg of the United States on manufacturing process optimization strategies for autologous active cellular immunotherapies (ACIs)
  • Ilona Reischl of Austria on regulatory perspectives of ATMPs.

Cellular Mechanisms of Protein Synthesis, Processing, and Secretion is cochaired by Roy Jefferis of the University of Birmingham in the United Kingdom and Michael Butler of the University of Manitoba in Canada. Although recombinant human protein therapeutics are already established in clinical and commercial applications, there is
potential for virtually exponential growth. High-volume commercial production of large biomolecules represents a considerable challenge because fidelity of folding and posttranslational modifications (PTMs; e.g., glycosylation) are essential to their clinical efficacy. Currently these drugs are very expensive. The high cost of goods (CoG) can limit their availability to patients due to the strain it puts on national and private health budgets. Impressive increases in productivity have been achieved over recent years but are yet to be reflected in lowering CoG. Product fidelity can be compromised, however, in a productivity/quality “trade-off.” A comprehensive understanding of the multiple pathways that lead to secretion of human proteins from nonhuman mammalian cells is therefore essential and will offer opportunities for cell engineering that may further increase productivity and/or product fidelity.

This session will provide insights into protein translation and folding within the endoplasmic reticulum and PTMs to proteins passing through the Golgi apparatus. Presentors will discuss the fidelity of disulphide-bond formation and glycosylation as essential PTMs and their effects on protein function as well as analytical methods to assess product quality from clonal cell lines. Introductory speakers include Linda Hendershot of the United States on life-and-death decisions in quality control of immunoglobulin biosynthesis and Graham Warren of Austria on Golgi biogenesis in a protozoan parasite.

Biopharmaceutics and Vaccine Production is cochaired by Otfried Kistner and Hermann Katinger, both of Austria. Advances in bioprocessing and manufacturing involve translation of scientific know-why into technological know-how under the guidance of GMP and regulatory stipulations. This creates a more conservative than innovative momentum for manufacturing both new and biosimilar pharmaceuticals and vaccines. Many questions are still open relating to quality control, definition of safety, suitability and commensurability of required testing, and appropriate bioprocess schemes such as continuous/perfusion technology. The answers to those questions could act as guidance and support both for manufacturers and regulatory authorities. Presentations will include introductory talks by Mary Sliwkowski and John Aunins, both of the United States as well as a presentation on the identification and remediation of a recent vesivirus contamination in a CHO-based production process.

Understanding Complex Interactions and Cell Engineering is chaired by Martin Fussenegger of Switzerland. Metabolic engineering to program mammalian cells for achieving bioprocess and therapeutic goals is increasingly complex and multigene based. Such activity requires intelligent genetic design and sophisticated analysis technology to optimize engineering interventions in an iterative process. That provides insight into the metabolic actions and reactions of a cell. Introductory speakers for this session include Christopher Love of the United States on integrated single-cell analysis and Stefan Ludwig of Germany on influenza and host-cell signaling (from molecular mechanisms to novel antiviral approaches).

 

Social Program

 

The social program for 2011 was designed with the famous ESACT spirit in mind: a spirit that enables open communication and interchange of ideas while catering to your senses and humor. The traditional outing on Tuesday afternoon will take us to Schloss Hof, a baroque hunting palace near Vienna with beautifully laid-out and terraced gardens. After a short tour of the palace, relax on the terrace with drinks and snacks, take a walk through the gardens, and enjoy the entertainment of a baroque concert. After dinner, there will be ample opportunity to talk and laugh either with cocktails on the terrace, in the disco, or on a late-night tour through the underground tunnels and distillery of the palace — where you just might encounter the palace ghost.

 

 


 

The gala dinner on Wednesday evening will take place in the Prater Galeries, located in the center of the traditional Viennese “Wurstelprater,” a pre-Disneyland theme park that dates back to the late 19th century. After dinner, we will have ballroom dance teachers present to introduce you to the art of dancing, followed by ample opportunity to exercise your new skills.

Nicole Borth is a professor in the biotechnology department at the Universität für Bodenkultur, Muthgasse 18, 1190 Vienna, Austria; 43-1-47654-6232, fax 43-1-369-7615;nicole.borth@boku.ac.at.

The Exciting new CHo genome Era

by Michael Betenbaugh and Kelvin Lee

A new day is dawning on the biopharmaceutical community, and the landscape of mammalian biotechnology could likely be changed forever in the year 2011. The Chinese hamster (Cricetulus griseus) ovary (CHO) cell line was first isolated over 50 years ago and is now used extensively to produce biopharmaceutical proteins ($100 billion USD annual revenue). CHO cell lines also have been used in a wide range of biomedical research contributing to developments in pharmacology, toxicology, cancer biology, and numerous treatments (1). CHO cells are the dominant production cell line for biopharmaceuticals worldwide. Nearly 70% of all recombinant biotherapeutics are made using CHO cells (2,3). Biopharmaceutical production will continue to provide the pharmaceutical industry with tremendous growth over the next several years, enabling continued research into new medical treatments and the expansion of manufacturing facilities (3,4). CHO cells have become a dominant host system because of their ability to produce recombinant proteins with therapeutically acceptable glycosylation patterns and other posttranslational modifications as well as the presence of powerful gene amplification systems. But although affiliated technologies continue to progress at an unrelenting pace, advances in CHO cell innovation have stagnated in recent years. We believe this is about to change.

Despite the cell line’s scientific and economic importance, there is not yet a publicly available genome sequence for CHO cells. Previous efforts to develop genomic resources have used several methods: expressed sequence tag (EST) and transcriptome (5), microRNA (6,7), and low-coverage genome (8) sequencing. Even with the published map of the CHO mitochondrial genome (Genbank DQ390542) and the annotation of dozens of CHO genes and microRNAs in public databases and literature, the closest fully sequenced and annotated organisms are mouse and rat, both of which demonstrate significant differences from hamsters based on cDNA sequences (anecdotally investigators have observed about a 2–3% homology with those rodents). This limits the direct application of sequence-based molecular tools in bioengineering and cell-line development efforts.

 

Working Toward the Goal

 

Fortunately, parallel efforts are ongoing at multiple international centers of excellence in Asia, the United States, and Eu
rope to address this glaring def and bring the CHO community into the genome era. To help accommodate the rapid output of information that is likely to emerge, facilitate communication between groups, and provide a meetingplace for ongoing multipronged efforts, an international CHO community genome project is now under development at the web site www.CHOgenome.org. The goal is to develop a one-stop shop for the CHO community to learn about the latest genomic efforts and publicly available data similar to what is available for fly, mouse, yeast, and human genome communities.

Model organism databases (MODs) are an essential tool for the collection, curation, and dissemination of genetic data for research. Well-established MODs have been developed for several organisms including mouse (www.informatics.jax.org), rat (www.rgd.mcw.edu), Drosophila (www.flybase.org), and yeast (www.yeastgenome.org). As next-generation DNA sequencing technologies continue to improve, the time and cost of sequencing genomes are decreasing, which speeds up development of genome-scale data sets for nonmodel organisms.

Recent rapid advances that are about to emerge on the CHO genome front were vividly on display during a recent symposium, the CHO Genome Workshop, which was held as part of the Fifth International Conference on Genomics, 15–18 November 2010 in Shenzhen, China. The workshop opened with remarks from Kelvin Lee (giving a history of how the community came together) and Bernhard Palsson (providing context on the use of CHO cells in the biotechnology industry). Specific research talks were then led by a keynote address from Xu Xun of BGI (formerly the Beijing Genomics Institute), who provided the first-ever detailed description of the CHO-K1 genome and described many features of it. Takeshi Omasa provided an overview of physical mapping and karyotyping efforts, highlighting the dynamic nature of CHO chromosomes. Steve Quake discussed efforts to characterize the CHO exome using a variety of techniques and assembly of those data in the context of mouse data. Nicole Borth presented an analysis of CHO microRNA, including some possibly new microRNAs that have not been reported previously. Gyun-Min Lee gave a detailed overview of the proteomic methods used to characterize CHO cells and particular phenotypes of interest. Lars Nielsen provided a telling description of how genome-scale flux analysis models can be developed — something that is now possible with a CHO genome. Finally, Bernhard Palsson (who is working with BGI on a CHO genome sequencing project) elaborated on specific elements of the CHO-K1 genome, comparing it with mouse, rat, and human genome and identifying critical functional genes. Finally, we led a discussion of next steps going forward for the CHO genome community. These include the importance of sequencing the hamster and other cell lines and having a simple framework for the community to interact with these data. In addition to the groups mentioned above, a number of others are hard at work on CHO genome projects, including the long-term efforts of Wei-Shou Hu in collaboration with Miranda Yap as well as a new initiative emerging in the United Kingdom.

 

The Next Step

 

So, where do we go from here? Once the CHO genome sequence becomes available, a thousand follow-up questions emerge about how to organize the data and what additional experimental and computational tools can be brought to bear on the CHO genome. We envision www.CHOgenome.org playing a key facilitating role to inform and educate the scientific community on all these advances. For this community effort to generate significant benefits, a collaboration between research groups and those with bioinformatics expertise will be beneficial. To maintain community engagement and responsibility, relevant data will be stored in a publicly accessible database that will provide access to the information and provide the opportunity for people to participate in ongoing updates and to develop ancillary genome-scale tools. We envision the development of an organism database in the coming years to house and organize such tools. As the next step forward, the CHO genome community will come together for a special workshop at the upcoming ESACT conference in Vienna Austria, 15 May 2011.

Michael Betenbaugh is a professor of chemical engineering at Johns Hopkins University (1-410-516-5461; beten@jhu.edu), and Kelvin Lee is a professor of chemical engineering at the University of Delaware (1-302-831-8953, fax 1-302-831-1048; KHL@udel.edu).

REFERENCES

1.) James, D. 2011. Therapies of Tomorrow Require More Than Factories from the Past. BioProcess Int. 9.

 

2.) Shaw, R. 2011. Stem-Cell–Based Therapies: What’s in Development, Implications for Bioprocessing. BioProcess Int. 9.

 

3.) Brandenberger, R. 2011. Cell Therapy Bioprocessing: Integrating Process and Product Development for the Next Generation of Biotherapeutics. BioProcess Int. 9.

 

4.) Onions, D, and J. Kolman. 2010. Massively Parallel Sequencing, A New Method for Detecting Adventitious Agents. Biologicals.:377-380.

 

5.) Schaub, J. 2010. CHO Gene Expression Profiling in Biopharmaceutical Process Analysis and Design. Biotechnol. Bioeng. 105:431-438.

 

6.) Jayapal, KP. 2007. Recombinant Protein Therapeutics from CHO Cells: 20 Years and Counting. Chem. Eng. Progr. 103:40.

 

7.) Wurm, F. 2004. Production of Recombinant Protein Therapeutics in Cultivated Mammalian Cells. Nat. Biotechnol. 22:1393.

 

8.) Walsh, G. 2010. Biopharmaceutical Benchmarks 2010. Nat. Biotechnol. 28:917.

 

9.) Jacob, NM. 2009. Using Genomic Tools to Improve the Production of Biologics. Chem. Eng. Progr. 105:35.

 

10.) Kantardjieff, A. 2009. Developing Genomic Platforms for Chinese Hamster Ovary Cells. Biotechnol. Adv. 27:1028.

 

11.) Johnson, KC. 2011. Conserved MicroRNAs in Chinese Hamster Ovary Cell Lines. Biotechnol. Bioeng. 108:475.

 

12.) Hackl, M. 2011. Next-Generation Sequencing of the Chinese Hamster Ovary MicroRNA Transcriptome: Identification, Annotation, and Profiling of MicroRNAs As Targets for Cellular Engineering. J. Biotechnol. in press.

 

13.) Hammond, S. 2011. Genomic Sequencing and Analysis of a Chinese Hamster Ovary Cell Line Using Illumina Sequencing Technology. BMC Genomics 12:67.

Leave a Reply