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D 56 132 Volume 14 September 2010 9-10 A erosol albedometer [page 26] Ba cteria – Fighter Against Cancer [page 20] A daptability and Ergonomy [page 36] Don't be afraid of dirty jobs Metrohm – the tougher IC system Loaded with particles? Presence of proteins? Filtration impossible? Metrohm Inline Sample Preparation (MISP) is the solution. Your Metrohm IC system gets the job done. Always! Metrohm IC – built for heavy duty, built to last. Ecknauer+Schoch ASW www.professional-ic.com • Editorial Regeneration and Cell-Cell Signaling Dear Reader, try to see your body as a social organism formed by several billion individuals (cells). The way these cells organize, control and maintain their integrity as a functioning body, is a highly sophisticated communication network, established by only a handful of signaling molecules. This sounds as if the system should be simple but it isn’t. Each of the handful of signaling molecules organize a large set of jobs. For example, the family of secreted wnt-factors is involved in the development of three dimensional body plans, cell movement during gastrulation, patterning of the central nervous system, and they are also involved in the occurrence of polyposis cancer of the colon. The key are multiple layers of signal modulations by hundreds of interacting molecules and crosslinks between different signaling pathways. Already at a very early stage in the development of an embryo, no matter if it will become a morphologically complex body like a human, or a very simple one like a Hydra (cnidaria) polyp, the fate of every newly born cell is controlled by communication between the surrounding and the new one. Already beginning from the fourth cleavage, a sea urchin embryo (belonging to a taxonomic group closely related to humans) produces cells with different fates. At this point some cells become “master” cells that secrete signals which are detected by other cells. Through the signals the “master” cell gives advice, and the other cells do what they were told to do. Such “master” cells with the capacity to instruct other cells are organized in signaling centers at defined regions in a developing embryo. Ethel Browne Harvey proved this in 1909 by transplanting the tip of a Hydra head to the body wall of a second Hydra. The second Hydra developed a new (secondary) head at the closest point to the tip of the first. Some factors secreted by the tip of the head induced the cells of the body wall of the second animal to form an ectopic head. One of these “head inducing factors” was later identified to be wnt 3. But such “master” cells are also widely distributed in each and every tissue in the body in an adult organism, ready to advise the surrounding cells managing a regeneration process in the case of damage. Regeneration is a process that shares many aspects with embryonic development. It is also a process in which the communication between cells is crucial for their “correct” behavior. And again some “master” cells control the behavior of the cells which repair the injured area. The signaling factors involved in regeneration are the same like in normal development. In the regeneration process of Hydra after cutting off the complete head shows the expression of wnt 3. At the basis of the evolution, animals had much more capabilities for regeneration. The larger, higher evolved and more complex an organism is; the smaller are its regeneration capacities, leading to the assumption that regeneration capacities are “purposely” down-regulated in higher organisms, maybe to prevent communication problems between cells leading to cancer, or for other yet unknown reasons. Hydra can be completely dissolved in single cells. If some of these cells are remixed and brought together (by mild centrifugation) the cell-clumps reorganize themselves and built up new completely intact animals (Hobmayer et. al. Nature 2000). In sea urchins you can perform the same experiment with embryos up to the mid blastula stage. Comparable experiments can also be performed with a variety of cell types of tissues of higher animals like chicken retinae or even human cancer cells. In the case of higher organisms it is necessary to add signaling substances and growth factors to the dissoluted cells, which then somehow (at least partial) mimic the signaling environment of a developing organism leading to processes closely related with the embryonic development and regenerative processes. These substances are also used by Prof. Augustinus Bader for his research on wound healing and regeneration. At least on a theoretical level it should be possible to induce regeneration far above the normal (strongly down-regulated)level in our bodies by adding signaling molecules and growth factors that are responsible for the instruction of cells in normal development and regeneration. Sincerely, Dr. Arne Kusserow Editor-in-Chief Dissociated and reaggregated Hydra cells. Emerging heads of de novo patterned polyps are clearly visible. The expression domains of the wnt3-gene are visualized in blue (in situ hybridization. Image by Bert Hobmayer/University of Innsbruck; Nature 2000). Sea urchin embryo at 32 cell stage. In this stage some cells at the vegetal pole are already “master” cells that organize the cell fate of the other cells. G.I.T. Laboratory Journal 9-10/2010 ▪ 3 RESEARCH & DEVELOPMENT C ontents • EVENTS Regenerative Medicine Focus on Regenerative Medicine 5 The Past, the Present, and the Future JIB 2010 7 Advances in Metabolic Profiling 2010 8 PA R T I C L E M E A S U R E M E N T Mass Spec Europe 8 Aerosol Albedometer Lab-on-a-Chip World Congress 12 10 R. M. Nerem, Georgia Institute of Technology, Atlanta, Georgia, USA 26 A Tool for Measuring Optical Scattering and Extinction of Dispersed Aerosols Prof. J. Thompson, Texas Tech University, USA Munich to Become the Capital of Biotech Dealmaking in November 12 The ELRIG.de Meeting 2010 13 LabAutomation2011 14 BIOPROCESSING Lost in Translation 30 Very Early Process Development for Biopharmaceuticals Dr. S. Hellwig, Fraunhofer IME, Germany MAGAZINE World Congress on Preventive and Regenerative Medicine MATERIAL SCIENCES 6 Hanover, Germany, October 5–7, 2010 Prof. Dr. A. Bader, WCRM, Germany Simple Solutions Quantification Study of Drug Delivery by Nanocarriers 32 A Cell Mass Spectrometry Approach 9 W.-Ping Peng et al., National Dong Hwa University, Taiwan Simplicity is the ultimate sophistication P. Praet, GIT VERLAG S creenin g News 18 APPLICATION NOTE Reproducible Cell Assays P harmace u tics & D r u g D iscover y Bacteria – Fighters Against Cancer 22 Consistency of Cryopreservation is a Necessity 20 Rolf O. Ehrhardt, MD, PhD and Brian Schryver, BioCision, LLC, Larkspur, USA Bacteria as Promising Tools for Cancer Therapy P H A R M A C E U T I C S & D R UG D I S C O V E R Y Dr. S. Weiß and Dr. S. Leschner, Helmholtz Centre for Infection Research, Germany Enzyme-Amplified Array Sensing of Proteins 24 Identification of Low Protein Concentrations T echnolo g y & I nstr u mentation Prof. V.M. Rotello, University of Massachusetts Amherst, USA COVER STORY On-line Oxygen Monitoring in Cell Culture Adaptability and Ergonomy Effects of Mitochondrial Modulators on O2 Dynamics of Mammalian Cells 36 Built by Your Needs Lynn S.G. and LaPres J. J., Michigan State University, East Lansing, MI, USA K. Ansmann, Olympus Europa, Hamburg, Germany News ADVERTORIALS 38 High Energy Efficiency and Safety 39 J. Feddern, Siemens Building Technologies Division, Zug, Switzerland Dynamic Image Analysis Beats Laser Diffraction Nanoparticles in Liquids J. Westermann, Retsch Technology, Haan, Germany Count, Size and Visualize White Giant or White Dwarf? 42 A. Malloy, NanoSight Limited, Wiltshire, UK Particle Size Distribution Measurements of TiO2 Dr. Markus Ortlieb, Shimadzu, Düsseldorf, Germany Transfer of USP-based HPLC Methods for Pantoprazole Sodium to UPLC 44 20-fold increase in productivity A.H. Schmidt, Steiner & Co., Berlin, Germany 4 ▪ G.I.T. Laboratory Journal 9-10/2010 48 The Dow Europe Laboratory in Horgen APPLICATION NOTES Particle Size Distributions 46 P rod u cts 51, 53, 54 I nde x / I mprint Inside back Cover 52 • Events Focus on regenerative medicine © Michael Radtke/Flickr.com The 5th World Congress for Preventive and Regenerative Medicine (WRCM) from the 5th to the 7th of October, will be a part of the conference program of Biotechnica for the first time in 2010. More than 500 scientists and clinical experts from all over the world will be discussing new possibilities for treating hitherto incurable or intractable diseases using regenerative therapies. The program reflects the broad range of scientific approaches in the field of regenerative medicine. Due to the considerable synergies between the two fields, the link with preventive medicine has been highlighted this year for the first time as the second main theme. The other main subject focuses range from tissue culture to the use of regenerative medicine in clinics, anti-ageing medicine, and stem cell research. Stem cells are considered to be the main hope for the treatment of illnesses that are difficult to cure. Since legal regulations govern research work in the field of regenerative medicine, ethical and legal issues are also on the agenda. The event will take place in the Convention Centre at the Hanover exhibition centre. It is being organized by Professor Augustinus Bader who holds the chair of cell technologies and applied stem cell technology at the University of Leipzig. The congress is rounded off by a poster exhibition and a special presentation on the subject of preventive and regenerative medicine in the exhibition hall. Specialists such as Professor William Haseltine and Professor Madjid Samii will contribute as keynote speakers. Other speakers are the pioneer of tracheal transplantation, Professor Paolo Macchiarini, and Professor Aubrey de Grey who will present his theories on the reversing of ageing processes. Professor Jörg Wiltfang will report on clinical pioneering work in the field of bone regeneration in oral and maxillofacial surgery. Numerous other scientists have confirmed that they will be attending, including international experts from the fields of medicine, technology and biology, as well as experts from the pharmaceutical industry. www.messe.de Melting Point Fast and Accurate M-560/565 - Simple and straightforward determination of melting and boiling points. ■ ■ ■ ■ ■ ■ Failsafe fully guided calibration Video play and replay function on instrument MeltingPoint Monitor Software: easy handling of results and methods Sample Loader M-569 – World Novelty: reproducible results due to homogenous packing IQ/OQ available: a must in regulated environment According to the Pharmacopoeia requirements, e.g. Ph. Eur. 6.1.2 2.60, USP XXI, JP BÜCHI Labortechnik AG 9230 Flawil / Switzerland T +41 71 394 63 63 www.buchi.com Quality in your hands Magazine • World Congress on Preventive and Regenerative Medicine Hanover, Germany, October 5–7, 2010 Biotechnica strongly developed its scientific program during the past few years. This year more than 10 different international congresses will take place in Hanover during Biotechnica. G.I.T. Laboratory Journal talked to the Initiator of the WCPRM, Prof. Dr. Augustinus Bader. Prof. Dr. Augustinus Bader, Initiator of the WCPRM G.I.T. Laboratory Journal: Prof. Bader, this occasion marks the fifth World Congress that has been initiated by you. And it is the first time Hanover is hosting the event. What were the reasons to choose Hanover as the venue? A. Bader: This year, the fifth World Congress for Preventive and Regenerative Medicine (WCRPM) is entering into a strategic alliance with Biotechnica. The decision to combine the two was driven by the fact that the topics complement each other. Biotechnology has delivered significant anticipatory services for the clinical feasibility of regenerative and preventative approaches. However, especially the clinical sector is still not very familiar with these potentials and, at this time, the biotechnological corporate structures that are already established have only been linked with the potential users of these technologies in very few cases. Through interaction the connections between biotechnology and medical uses are to be significantly intensified and expanded far beyond to what they have been to date. Consequently, the fifth World Congress also is distinctly different from the event that has typically taken place in this discipline, which could be dedicated primarily to the aspect of fundamental research or materials development in these segments. The focus of the Congress is on prevention and regeneration. Which are the concrete core topics participants will be able to gain insights into? A. Bader: In the discipline of regenerative technologies the key topics depicted will be stem cell therapy processes used to treat the tissue categories of bones, cartilage, liver, the nervous system and the skin as well as the muscles including the heart. Additional associated tissue areas will be examined in open discussion forums and ad-hoc presentations. 6 ▪ G.I.T. Laboratory Journal 9-10/2010 As far as the discipline of preventative medicine is concerned, topics will comprise sports medicine aspects, hormone therapy and antioxidant treatments. We are planning to combine these issues, which have already been established in prevention and age research, with questions arising from traditional pharmacology with regard to the prevention of the major diseases that affect large portions of the population – diabetes and cardiovascular diseases. Sports medicine aspects will demonstrate the holistic approach of the prevention and regeneration concept of this Congress. We are planning to present biotechnological procedures in the form of cross sectional platforms in both – analytics and diagnostics; but we will also offer insights into the product development sector for laboratory and clinical requirements. Numerous companies will present their solutions. Who would benefit the most from attending this Congress? A. Bader: The Congress is particularly interesting for individuals who are eager to learn about cross sectional functions and cross sectional topics with the objective of boosting the innovation potential for their own scopes of application. Extending their horizons allows scientists and medical experts, as well as the true practitioners – i.e. surgeons working in hospitals, colleagues working in preventative and sports medicine to participate in biotechnological stem cell therapy innovations. Opinion drivers from the discipline of medicine as well as from biotechnological fundamental research and stem cell technology will be in attendance. The Congress targets decision makers and young scientists, who will be offered a pertinent forum for the presentation of their results. In addition, the event will cover the relevant regulatory aspects, which will be presented by the EMA (European Medicine Agency) and rep- resentatives of the Paul Ehrlich Institute. Consequently, the event is also particularly interesting for industrial continued GMP and GLP technologies education applications. Pertinent practical training results are also possible due to the fact that cooperating exhibitors are engaged in this subject matter. How important is stem cell research within the scope of the innovative results and approaches? A. Bader: As far as its innovative force is concerned, stem cell research is one of the most powerful innovation drivers in the disciplines of medical research and application. Principled fundamental improvements can be anticipated in areas such as bone-cartilage regeneration, skin and liver regeneration, but also as far as the regeneration of the human nervous system is concerned. Based on the large tissue system of animal and human bodies partial solutions for smaller tissue constructions will already be possible with greater precision in certain scopes of application. Aspects of rapid prototyping, as well as a method called organ printing will be presented; as will biotechnological procedures or stem cell treatment methods in the body that trigger tissue regeneration from the tissue itself. The entire bandwidth of stem cell research, covering everything from cell therapy based on the bionic principle, embryonic cell types, fetal cell systems to traditional tissue engineering will be introduced. Would you like to give potential visitors some recommendations on presentations and speakers they should definitely not miss? A. Bader: Numerous key note speakers who are highly respected around the globe, for instance Professor Aubrey de Grey, who is one of the pri- • Magazine mary pioneers in age research, will come to the event. Prof. Dr. Machens of the Klinikum rechts der Isar and of the Technical University Munich will present a report on the treatment of burn trauma patients and initial results from clinical trials. Prof. Su, who hails from Taiwan, will talk about technologies available for the preparation of blood platelet concentrates. This is a technology that will certainly be compatible with numerous applications in a variety of tissue areas. Professor Cimen Karasu will make a presentation on preventative medicine; specifically on aspects related to the prevention of age related diseases and oxidative stress. Professor Dr. Halle will segue to the discipline of sports medicine. Contact Andreas Guntermann World Federation/ World Virtual Institute of Preventive & Regenerative Medicine (Pyramed) Hannover, Germany andreas.guntermann@regmed.net www.regmed.net © trixnbooze/Flickr.de JIB 2010 A major meeting place in laboratory medicine, the International Biology Days (Journées Internationales de Biologie – JIB) will be held at the CNIT, Paris la Défense in France from November 2nd to 5th, 2010 (the 2nd is only dedicated to the congress). In this time of reform of the profession, the event becomes a guide to changes and offers not only a paying session dedicated to the ISOEN 15 189 accreditation norm during the congress but also “Café Scientifique” slots that deal with current and pertinent issues. Revealing know-how, the JIB gathers the suppliers of medical biology laboratories (automated systems, analyzers, reagents, data processing, financing, services…). 200 exhibitors meet with more than 10,000 professional visitors (private and hospital biologists, technicians, biomedical engineers, researchers…) and show them their new products and services on an International, European or French sneak preview. The BioMI pavilion, Molecular Biology Initiatives, created in 2008, continues its development on a dedicated area within the exhibition. Creating bridges, the JIB event works in partnership with the Ensaama – Olivier de Serres school in order to create the “Tomorrow’s medical laboratory” (welcome desk and sample rooms). Applied art students participate in this contest and present their own projects on a privileged place. Finally, the scientific influence of medical biology grows on the congress (2-3-4-5 Nov. 2010) with the auspices of the IFCC and the support of the EFCC. The scientific committee offers a special theme “Health & Environment: Challenges for Laboratory Medicine” to the attendees, in order to better understand the mechanisms that identify direct links between environment and human health. The 2010 program also contains a technological innovations session and a roundtable on Nov. 4th entitled “Mapping the future of European Laboratory Medicine for Young Scientists”. Contact www.jib-sdbio.fr G.I.T. Laboratory Journal 9-10/2010 ▪ 7 Events • Advances in Metabolic Profiling 2010 Select Biosciences is proud to announce their 6th annual Advances in Metabolic Profiling conference, which will take place from 9th–10th November 2010 at the Sheraton Hotel, Florence, Italy. The conference has been designed to give expert guidance on this sector by gathering some of the most influential and experienced players in the field from Europe, America and across the globe. It will also provide the opportunity to network with other colleagues from all over the world. The agenda will include the following key subject areas appropriate to the latest in metabolic profiling: ▪▪ Clinical Applications of Metabolomics ▪▪ Fluxomics and Pathway Discovery ▪▪ Drug Discovery and Development ▪▪ Plant Metabolomics ▪▪ Nutrigenomics and Metabolomics ▪▪ Microbial Metabolomics – Novel Technologies for Metabolomics ▪▪ Bioinformatics and Data Handling for Metabolomics ▪▪ Data Fusion – Bringing Together Different Omic Technologies To guarantee a high attendance at this exciting event, the traditional low registration fees and group booking discounts will be maintained. Colocated with Mass Spec Europe, European Biomarkers Summit and Advances in Protein Crystallography, full conference passes include admission to all sessions and the exhibition, as well as conference documentation. The conferences division of Select Biosciences is focused on organizing specialist biomedical meetings. Experts from both academia and commerce are invited to present timely information from current research through to commercial implementation of new technologies. These events also provide a unique networking facility and the opportunity to reach a highly targeted scientific audience. Contact Kirit Shah Conference Producer k.shah@selectbiosciences.com Aaron Woodley Exhibition Manager a.woodley@selectbiosciences.com www.MetabolicProfiling.com Mass Spec Europe Select Biosciences will hold its 2nd annual Mass Spectrometry Europe conference, in Florence Italy on 9–10th November 2010, under the auspices of the Italian Chemical Society. It will be a two day conference, co-located with Advances in Metabolic Profiling, European Biomarkers Summit and Proteomics Europe. It will be focused on all areas of mass spectrometry, from fundamental to applied research in the areas of biology, chemistry, physics and other related disciplines. Mass spectrometry is considered an ideal technology for image analysis due to its high sensitivity and molecular specificity. It has allowed researchers to profile, identify, characterize and quantify low molecular-weight compounds easily and is unequivocally an essential tool for research in the 21st century. This conference aims to aid the advancement of this unique technology by providing a world class agenda of presentations from influential figures within the industry. It will allow the sharing of information and techniques, related to mass spectrometry, as well as giving the opportunity for delegates to network with others from all over the world. Confirmed Speakers include: ▪▪ John Vickerman, Professor, University of Manchester ▪▪ Gianluca Giorgi, President of the Mass Spec Division, Italian Chemical ▪▪ Frank Sobott, Group Leader, University of Oxford ▪▪ Pedro Cutillas, Head of Analytical Signaling Group, Barts and the Queen Mary Medical School ▪▪ Colin Creaser, Professor, University of Loughborough ▪▪ Sabine Becker, Head of Trace and Ultra Trace Analysis, Research Centre Juelich ▪▪ John Langley, Head of Mass Spectrometry, University of Southampton Contact Aaron Woodley a.woodley@selectbiosciences.com www.MassSpecEurope.com 8 ▪ G.I.T. Laboratory Journal 9-10/2010 • Magazine Simple Solutions Simplicity is the Ultimate Sophistication olution of modern medicine. It stands to reason “How Good Feelings Can Make You Healthy” in Science is not only about complex formulas, figthat experts from all over the world react with the last paragraph of the article), which was ures or theories. It’s mainly about the results, not some kind of mistrust to this and make their published just shortly. Clever marketing to climb how to get there. Albert Einstein already knew: opinion public. up the bestseller rankings? ”When the solution is simple, God is answerThe result was a chivvy after Bader. But it is But no matter who is responsible for the aring.” But simple solutions are not accepted by obvious that he is not the one to blame. Werner ticle in the “Süddeutsche Zeitung”, he definitely everybody. If one finds such a “god-given” anBartens is the author of the above-named article achieved his objective: Everybody talks about swer, envy and animosity are often the conseand therefore responsible for the content. Bader this issue. quence. Prof. Augustinius Bader from the Universeems to be pushed in the role of a victim, while sity of Leipzig still adheres to this idea of simBartens is getting publicity for his new Book plicity and got to know what it means to do so. Philipp Praet, GIT VERLAG (There is a reference to his new Book about With his new stem cell derived product he gets ready to change the medical Industry – As simple as possible. What you need to produce it are stem cells from your body, taken from your blood or your bones, cytokines, growth factors and some other factors. All mixed together to result in a crème – called Sanamander, or in an injectable solution. The crème can be used in several wounds such as flash For more than 40 years we have been producing „Quality built on burns, big lacerations or even paraTradition” in our plant in Germany. Our product groups stand for plegia. Just coat your, e.g. burned skin detailed experience in every-day use, ongoing technical advances with the crème or inject the solution as well as toughest demands on material, functionality and design. into the injured tissue and let your Users in most varying research, standard and special labs profit body do the remaining work. The clue from the variety, precision and reliability of all GFL products. about this product is simple: Tell your A vested quality demand in accordance with international standards is documented for all GFL laboratory products with the body that the damage can be fixed certification to DIN EN ISO 9001: 2008. by its own self-healing power; and A tight-knit web of agencies and distributors worldwide ensures your body will exactly do so. a local presence to customers. GFL Laboratory equipment is used Sounds like the best medical inin more than 150 countries worldwide. vention for decades, if only the story Deep Freezers is reliable. During the last months, Water Baths Bader’s new invention, especially Shaking Water Baths some articles concerning this topic, Water Stills aroused a huge wave of disgust. Bad Incubators er was attacked from various sides Shakers that the results aren’t proved by clinical trials. A fact that Bader himself never disclaimed. Clinical trials are long-lasting and expensive issues. However, in his own understanding, Sanamander is not a “miracle cure”, as some press releases named it, nature is the only miracle. “We just figured out, how the self-healing process of our body is working, that’s it.”, Bader states. At this point there is a very simple way to prove whether the crème can support wound healing or not. Wait until clinical trials are performed. Time will prove the concept, – or not. But if Bader is such a humble man, where does all the disgust come from? The stumbling block is an article written by Werner Bartens and published in the “Süddeutsche ZeiGFL Gesellschaft für Labortechnik mbH · Schulze-Delitzsch-Strasse 4 · 30938 Burgwedel / Germany tung” in Germany. Bartens, not Bader Phone +49 (0)5139 / 99 58 - 0 · Fax +49 (0)5139 / 99 58 21 · E-Mail: info@GFL.de · www.GFL.de describes Baders research as the rev- GFL at the ANALYTICA in Munich / Germany, 23 -26 March 2010: Hall B1 / Booth 231/330 Quality built on Tradition G.I.T. Laboratory Journal 9-10/2010 ▪ 9 Magazine • Regenerative Medicine The Past, the Present, and the Future Regenerative medicine is a rapidly growing, multidisciplinary field that seeks to replace, repair, and/or enhance biological function in tissues and organ that has been lost due to congenital abnormalities, injury, disease, or aging. In this the goal is to harness the intrinsic biological ability of the human body. The basic concept of using a more biologic approach in the development of medical implants and related treatments goes back to the first half of the 20th century; however, the modern era only began a quarter century ago. Robert M. Nerem, Ph.D. Professor Emeritus at the Georgia Institute of Technology in Atlanta, Georgia, USA Neuronal stem cells from the brain with migrating precursor cells. (Credits: University of Tuebingen, ZRM/Institute of Anatomy) The initial focus was on replacement tissues, i.e. developing substitute tissues outside of the body for implantation into the body, with skin substitutes being some of the first targets. Although in the 1990s these had moved into commercial development, by the beginning of this decade commercial activity had for the most part encountered financial difficulties. Even while commercialization in this area was going through its “ups and downs,” the science was moving ahead. In the 1990s stem cell technology began to emerge and the focus on replacement evolved to include repair and regeneration with the result is that today there are a variety of approaches that are being pursued. The biological complexity of many if not most of the tissues and 10 ▪ G.I.T. Laboratory Journal 9-10/2010 organs of interest demands a broad range of approaches. Furthermore, this complexity suggests that repair and/or regeneration may be the more advantageous strategy. In this it must be recognized that the functional characteristics of a cell is orchestrated by a “symphony of signals.” This “symphony” is made up of soluble molecules, the substrate and extracellular matrix to which the cell adheres or is surrounded, cell-cell contact, and the physical forces, i.e. the mechanical environment in which a cell resides. The interest in stem cells is because a major issue is cell source, and there are a variety of types of stem cells. It is the embryonic stem cell (ESC) that has the ability to produce every cell type in the human body. Derived from embryos created through in vitro fertilization, for some such cells raise ethical issues. Then there are the various adult stem cells, with the most common being those that are bone marrow-derived such as mesenchymal stem cells (MSCs). A more recent and exciting advance was the demonstration that a somatic cell like a skin cell could be reprogrammed to create what is called an induced pluripotent stem (iPS) cell. Seemingly like ESCs, there is still much that we have to learn about iPS cells, and it may well turn out that iPS cells, though similar to ESCs, have their own distinct characteristics. Finally, there are a variety of progenitor cells in the human body. These have limited potency. One example is the endothelial progenitor cell that circulates in blood, homing in so as to repair vascular injury. Which of these different types of stem/progenitor cells will be important will very much depend on the particular tissue or organ and whether the approach is one of replacement, repair, or even regeneration. As exciting as the advances in the biology of stem cells and progenitor cells has been, regenerative medicine is more than stem cells. This is because for a strategy to be successful will require that one delivers the cells and/or the necessary signals, i.e. those that will orchestrate the desired cell function, at the right place and at the right time. This “symphony of signals” could be provided by the cells employed in the particular strategy used; however, if we actually understood the signals required and how and when to deliver them, in many cases the approach might be acellular in nature. What is now becoming intriguing to many in this field; however, is the concept of regeneration. The regenerative processes observed in species such as the newt and salamander have been replaced in the human by processes of in- • Magazine flammation and scar tissue formation. For the human, the extent to which we can jump start the regenerative process and supply the appropriate ingredients will dictate our success in achieving tissue and organ regeneration. For now it seems that the regeneration of adult human tissue is in the realm of science fiction; however, there is much that can be learned from the developmental process and thus from developmental biology, even though the geometric scale and the time scale will be very different in the adult than the developing embryo. It is clear, however, that no single approach will solve all problems; rather, each tissue and each pathologic condition is likely to require a different approach to obtain optimal results. Furthermore, if stem cells are to be used, whether they are ESCs, MSCs, iPS cells, or some other type of cell, there will be a need for the development of processing systems that will allow for the expansion of cells and their differentiation with the quality control necessary for their use in clinical therapies. The clinical translation of regenerative medicine, however, will require more than scientific discovery and the advancement of the relevant technologies. Regulatory agencies must become educated in non-traditional approaches and develop appropriate guidelines for safe and effective delivery of regenerative medicine strategies. Third party payers must come on board quickly to sustain promising approaches and reward regenerative medicine strategies that have the potential to significantly affect health care. Although the industry appears to have turned the corner, these regulatory and reimbursement issues will need to be addressed if this industry is to thrive. Furthermore, only then can the therapies developed through regenerative medicine be available in the widest possible way. With the ever accelerating advances in the science and technology, regenerative medicine has the potential of truly living up to the promise of delivering therapies for diseases, injuries, and disorders where currently patients have no options. As we look to the future, what are some of the advances that can be envisioned? To start with, there will be in vitro models of tissues and organs fabricated from human cells and used in drug development and toxicity testing. There will be blood cells derived from stem cells and expanded in vitro, thus reducing the need for blood donors. One can also envision an insulin-secreting, glucose responsive bioartificial pancreas, and this may not be that far away. For children born with a congenital heart defect, there will be a tissue engineered heart valve fabricated of living cells and one that grows with the child as the child grows. There also will be cell-based therapies for the repair of the wall of the heart following a heart attack. And finally, perhaps the real “holy grail” is the repair, even regeneration, of the central nervous systems. Acknowledgement The author is an Institute Professor Emeritus at the Georgia Institute of Technology in Atlanta, Georgia, U.S.A. He serves as the Director of the Georgia Tech/Emory Center (GTEC) for Regenerative Medicine, a unique interinstitutional research center bridging from the basic biology to the enabling engineering technologies to the clinical application. The author also is a Distinguished Visiting Professor in the World Class University Project at Chonbuk National University in Jeonju, Korea, and while there he authored this article. Professor Nerem will also present the plenary opening lecture of the BioStar 2010 – 4th Congress on Regenerative Biology and Medicine, 13.–15. 10. 2010 in Stuttgart, Germany. Contact Robert M. Nerem Parker H. Petit Institute for Bioengineering and Bioscience Georgia Institute of Technology Atlanta, Georgia USA ! "" # # %& '() **(+!, "$# G.I.T. Laboratory Journal 9-10/2010 ▪ 11 Events • Lab-on-a-Chip World Congress er will be running “Microfabrication Technologies for Microfluidic Devices”. Also at this year’s meeting we are pleased to announce that there will be a free workshop which is being run by Aushon. Full details will be announced on the website soon. Select Biosciences is pleased to announce their 2nd annual Lab-on-aChip World Congress. This year’s event is being held in the fabulous location of La Jolla, San Diego, CA, USA - Thursday 28th and Friday 29th October 2010. The congress is being co-located with Microarray World Congress, Molecular Diagnostics World Con- gress and Single Cell Analysis Summit. All delegates will have access to all four meetings and the large combined exhibition, ensuring a very cost effective trip. The conference will focus on Point of care diagnostics, Microfabrication/Engineering and Life science applications, and we have secured some fantastic speakers, along with a brilliant keynote speaker; Professor Jon Cooper from Glasgow University in Scotland. The conference can also boast two training courses, which will take place prior to the main event, on Thursday 27th October. Nicole Pamme will be running “Principles and Applications of Microfluidics in the Life Sciences” and Holger Beck- Contact Aaron Woodley Exhibition Manager a.woodley@selectbiosciences.com Sara Spencer Conference Producer s.spencer@selectbiosciences.com www.selectbiosciences.com Munich to Become the Capital of Biotech Dealmaking in November For the first time in ten years BIOEurope, Europe´s largest partnering event for the life science industry, returns to Munich. From November 15–17, 2010 Munich will be the center of dealmaking activity for the biotech industry. Munich is home to one of the largest and most vibrant biotech clusters in Europe. Featuring 180 life science companies, the Munich biocluster employs 8,800 people. The success of the region can be attributed to a rapidly developing commercial biotechnology industry, world-class scientific research institutions, excellent infrastructure, efficient access to capital, and highly qualified employees. The BIO-Europe event was last held in Munich in 2001, and in the intervening years it has grown into the premier biotechnology business exchange in Europe. Horst 12 ▪ G.I.T. Laboratory Journal 9-10/2010 Domdey, Managing Director, Bavarian Biotechnology Cluster, said he is looking forward to welcoming the event back to Munich. “We were just a baby biocluster, not even a cluster really, when this event was last held in Munich in 2001. It has been a busy time for us, launching successful companies, drugs that have been ap- proved, as well as new development programs.” The Munich life science industry today is made up of 43 pharmaceutical companies and 136 biotechnology companies, 8 of which have successfully been listed as public companies. Among the biotechs, 118 are SMEs between them employing 2,600 people largely dedicated to developing therapeutics and diagnostics. BIO-Europe is a conference that is designed to get partnerships started. Partnering today is a main engine of industry growth. For big pharma, partnering is the primary strategy for filling pipelines with new medicines. At BIO-Europe, attendees use partneringONE software to screen through the thousands of partnering opportunities, and automatically generate a calendar of meetings. This is proving to be the most effective way to meet with potential new partners. www.ebdgroup.com/bioeurope • events The ELRIG.de meeting 2010 Titrette ® Oral sessions will cover the topics: Design and quality of compound libraries, Storage, Logistics and Software. The speakers are experts in drug discovery and laboratory automation from the most important pharmaceutical companies like Mirek Jurzak/Ingo Kober (Merck-Serono), Thorsten Naumann (Sanofi-Aventis), Alexander Hillisch (Bayer Schering), Julien Grimont (Actelion), and Jerome Giovannoni (Novartis), from academic research institutes like Edgar Specker (FMP, Leibnitz-Institut für Molekulare Pharmakologie) and Heiko Zimmermann (Fraunhofer-Institut für Biomedizinische Technik IBMT), leading experts from the laboratory business; Doris Hafenbradl (Biofocus), Johannes Knob (Amgen), Ferry De Vugt (LabServices), Martin Frey, Hamilton, consultants: Dirk Schwammkrug (Logica) and contract research providers: Jean-Yves Ortholand (Edelris). © Michael Mertens/Flickr.com The ELRIG.de forum 2010, organized by ELRIG. de and G.I.T. Laboratory Journal will be held in the new venue “Darmstadtium” in the heart of Darmstadt, the “City of Science”. On November 25 in Darmstadt, Compound Management will be the core topic of this year’s meeting of the German division of the Laboratory Robotics Interest Group. During the past decade compound management developed towards a distinct discipline in industrial drug discovery. Compound management is the science of the storing and the management of very large collections of chemically or biologically active substances which are systematically tested for their pharmaceutical activity with high throughput screening assays. Compound libraries and their management therefore gained an enormous attention in the past years since the status of the individual compounds is crucial for the effective identification of possible drugs. In the past years compound management changed since a growing number of small biological substances entered the scene. Instrumentation that was originally developed for the storage and management of small chemical substances was adapted to the new compounds, thereby creating new challenges. www.elrig.de class A precision With the bottle-top burette Titrette®, you can titrate quickly and reliably with highest precision, within the error limits of Class A glass burettes. N Sensitive drop-wise titration N No switching between filling and titrating N Compact design Light and stable N Simple cleaning and maintenance right in the lab N PC interface (optional) for data transmission ! W NE Titrette® – the next generation of digital bottle-top burettes! BRAND GMBH + CO KG 97877 Wertheim (Germany) Tel.: +49 9342 808-0 www.brand.de · info@brand.de G.I.T. Laboratory Journal 9-10/2010 ▪ 13 Events • LabAutomation2011 Palm Springs Convention Center, Ca, USA, January 29–February 2, 2011 In January 2011, the LabAutomation Conference and Exhibition will celebrate 15 years of bringing science, technology and industry together. Throughout these years the laboratory automation community has experienced significant and positive changes. On July 1, 2010, the community experienced one of its most significant changes yet when two organizations, the Society for Biomolecular Sciences (SBS) and the Association for Laboratory Automation (ALA), united as Sections under the newly formed Society for Laboratory Automation and Screening (SLAS), an inclusive worldwide organization dedicated to advancing scientific research and discovery through laboratory automation and screening technology. LabAutomation2011 will be the perfect time to experience the positive impact that the SBS-ALA merger will have on both members and our professional community. The LabAutomation Conference and Exhibition will continue to deliver a first-class educational program and bring forth several new exciting offerings and opportunities. Presented by 14 ▪ G.I.T. Laboratory Journal 9-10/2010 the Laboratory Automation Section of SLAS, the event is a powerful platform for education, peer networking, and strengthening the laboratory automation community. It provides participants with direct access to the world’s top 100 podium presentations, short courses, numerous vendor -specific workshops, over 400 exhibit booths and many new product launches. Each year the event plays host to a line-up of influential, forward-thinking industry visionaries. In 2011, SLAS is proud to welcome Chad Mirkin, Ph.D., who in 2009 was asked by President Obama to participate as a member of the President‘s Council of Advisors on Science and Technology; John Butler, Ph.D., a Fellow and Group Leader of the National Institute of Standards and Technology; and Daryl Lund, Ph.D., Editor-inChief of Journal of Food Science, Institute of Food Technologists. LabAutomation2011 also boasts an extensive awards program that recognizes individuals who have contributed to laboratory automation and technology advancement. Among those awards are the Young Scientists Award, which provides travel to and lodging at LabAutomation2011 to students whose winning poster has been selected at another event, and the SLAS Innovation Award, which recognizes the work of those unique and special podium presentations at LabAutomation that are exceedingly innovative and contribute to the exploration of technologies in the laboratory. In the spirit of innovation, each year at LabAutomation dozens of new products are launched. To celebrate these breakthroughs and acknowledge the best of what‘s new, a team of experts will select up to three of the most promising new products launched on the exhibit floor for the official SLAS New Product Award (NPA) Designation. LabAutomation2011 participants will benefit greatly from the highest caliber of scientific presentations covering a variety of industries and scientific disciplines focusing on the following educational tracks: Detection and Separation, Micro- and Nanotechnologies, High-Throughput Technologies, Informatics, and Evolving Applications of Laboratory Automation, featuring Agriculture and Food. The Conference and Exhibition is a five-day event taking place January 29-February 2 at the Palm Springs Convention Center, Palm Springs, CA, USA. To help make the LabAutomation2011 experience affordable, participants may take advantage of the Smart-Savers Discount Program. This innovative cost-savings program offers discounts on everything from registration to hotel to travel, including: hotel rates as low as $139 per night, $200 off airfare, complimentary registration for academics and those unemployed, and more. For more detals and register, visit www.labautomation.org/LA11. www.labautomation.org/LA11 The revolution begins 1984 MDxQIAsymRGQ0910G6EU QIAGEN revolutionizes sample preparation by inventing the first kit for nucleic acid purification The discovery reduces time for the preparation of plasmid DNA from bacteria from 2–3 days to 2 hours. Sample & Assay Technologies The revolution continues — Enter a new era wi 2010 QIAsymphony RGQ — The power of flexibility The QIAsymphony RGQ introduces the power of flexibility MDxQIAsymRGQ0910G6EU for all your routine molecular testing! Q Bringing process safety and flexibility to the next level Q A complete series of systems Q Not just an instrument, but a foundation for the future! th QIAGEN today! Continue to find out more Sample & Assay Technologies Screening News • +++ News +++News +++ Expanded Collaboration Argenta, a service division of Galapagos, announced it has signed a contract extension with Genentech, a member of the Roche Group. The extended agreement expands the portfolio of projects which are in collaboration with Genentech and allows Genentech to place projects directly into Galapagos’ other service division, BioFocus. The collaboration dates back to 2005. The current agreement covers a number of drug discovery programs that utilize Argenta’s expertise in computer-aided drug design (CADD), medicinal chemistry, and in vitro biology and screening to discover new chemical entities acting against undisclosed drug. Under the announced amendment, BioFocus will provide Genentech with integrated medicinal chemistry, in vitro biology and ADME services. www.argentadiscovery.com www.biofocus.com Spin-Out Preclinical Oncology Services Limited (PRECOS) announced its official launch following its spin-out from the University of Nottingham. The newly formed company combines the innovation of one of the Country’s leading universities with the scientific background and expertise of multi-disciplinary scientists. This places the company in a position to deliver specialist services, backed by significant industry expertise and cutting-edge technology, to help both pharmaceutical and biotechnology companies develop new anti-cancer drugs. www.precos.co.uk Collaboration for the Development of Biomarkers Ariana Pharma announced that it has started a collaboration with the US Food and Drug Administration (FDA). Ariana Pharma is providing its KEM Biomarker technology to help enable FDA reviewers to analyze pharmacogenomic data combined with patient characteristics for biomarker signatures submitted through the FDA’s Voluntary Exploratory Data Submission (VXDS) program. This collaboration directly relates to the FDA’s desire to develop better tools for the analysis of genomic data in the context of the development of personalized medicine. This collaboration is intended to help the FDA systematically identify potential genomic “fingerprints” and develop recommendations for the analysis of genomic data prior to submission of biomarker signatures. www.arianapharma.com Monoclonal Antibodies GlaxoSmithKline (GSK) and Lonza announced that they have entered into a new agreement under which Lonza will support the ongoing development of GSK’s biopharmaceutical pipeline by supplying manufacturing capacity for five early stage monoclonal antibodies. Under the terms of the agreement, Lonza will initially manufacture clinical trial batches of five compounds currently in Phase 1 and 2 for GSK. The company will also provide access to flexible capacity to enable GSK to respond to future demand 18 ▪ G.I.T. Laboratory Journal 9-10/2010 dependent upon progression of molecules through late stage development and commercial launch. All other details of the agreement remain confidential. www.lonza.com www.gsk.com Protein Research Protagen, a specialist for in vitro diagnostics and GMP-compliant protein analysis announced a second round of finance totaling a volume of € 10.0 Mio. After closing a first round of € 3.7 Mio. in August 2009, the NRW. BANK, Dusseldorf, participated as new investor and joined the consortium of MIG Fonds, Munich, S-Capital Dortmund, S-Venture Capital Dortmund and KfW, Bonn, to finalize the actual round. Also the existing investors contributed significantly to the second closing. The capital will be used for the targeted expansion of the business unit Diagnostics and the clinical validation of proprietary diagnostic marker proteins. www.protagen.de JX-594 for the Treatment of Cancers Jennerex, a private clinical-stage biotherapeutics company focused on the development and commercialization of first-inclass targeted oncolytic products for cancer, and Transgene, a bio-pharmaceutical company specialized in the development of immunotherapeutic products, announced that they have entered into an exclusive partnership to develop and commercialize JX-594 for the treatment of solid tumors in Europe, the Commonwealth of Independent States (CIS) and the Middle East. The lead cancer biotherapeutic product, has shown anticancer activity and a welltolerated safety profile in Phase 1 and Phase 2 clinical trials. Objective tumor response has been demonstrated in a variety of cancers including liver, colon, kidney, lung and melanoma. www.transgene.fr www.jennerex.com Metastatic Breast Cancer Immutep announced the publication of a clinical research paper showing that its lead product, IMP321, given with first-line paclitaxel achieved clinical benefit in 90 % of metastatic breast carcinoma (MBC) patients. Correlations were observed with both the patients’ monocyte (i.e. the primary target cell for the drug) count before treatment and the degree of activation of monocytes during treatment. The study was an open-label fixed-dose-escalation trial carried out in three cancer centers in the Paris region. IMP321 induced both a sustained increase in the number and activation of APC (monocytes and dendritic cells) and an increase in the percentage of NK and long-lived cytotoxic effector-memory CD8 T cells. Clinical benefit was observed for 90 % of patients with only 3 progressors at 6 months. Also, the objective tumor response rate of 50 % compared favorably to the 25 % rate reported in the historical control group. www.immutep.com The power of flexibility QIAsymphony RGQ The most versatile system for all your molecular testing needs — today and tomorrow Q Unprecedented flexibility Q Workflow optimization and utility MDxQIAsymRGQ0910G6EU Q Maximized ease-of-use and convenience Q Designed for largest range of applications Experience a new era in molecular diagnostics today and tomorrow at www.qiagen.com/molecular-testing-revolution! Sample & Assay Technologies Pharmaceutics & Drug Discovery • Bacteria - Fighters Against Cancer Bacteria as Promising Tools for Cancer Therapy 200 years ago the observation was already described that tumors sometimes shrink when cancer patients undergo bacterial infections. This is the basis for bacteria‑mediated cancer therapy – an alternative therapeutic approach of growing interest. Several bacterial strains are able to colonize solid tumors after systemic administration; a few even induce tumor reduction. Apparently, the virulence of bacteria is crucial for an anti‑tumor effect. Now the major challenge is to tailor bacterial strains that combine safety with therapeutic efficacy. History At the beginning of the 19th century, Vaultier noticed that bacterial infections of cancer patients were associated with shrinkage of the tumors [1]. A first report of intentional treatment following this groundbreaking observation dates to the year 1868. The German physician W. Busch placed a woman with an inoperable sarcoma into a bed that had been occupied before by a patient suffering from erysipelas – a Streptococcus pyogenes infection. The woman successfully became infected and her tumor shrank. Unfortunately, she died of the infection nine days later [2]. The toxicity of bacteria could not be handled at that time. Therefore, similar attempts with different bacteria or bacterial products – like the well known Coley’s toxin – failed, despite some success regarding the anti-tumor effect. Nowadays, the situation has dramatically changed. Knowledge about hostpathogen interactions, the genome information of bacteria as well as state of the art molecular methods allow modulation of bacteria for particular purposes. Hence, the approach of using bacteria in tumor therapy presently undergoes a renaissance and is under intensive investigation. Different types of bacteria can colonize tumors Amongst the bacteria that are able to target and colonize tumors are obligate an- Dr. Siegfried Weiß and Dr. Sara Leschner, Helmholtz Centre for Infection Research, Braunschweig, Germany aerobics like Clostridia and Bifidobacteria but also facultative anaerobics like E.coli or Salmonella. As it is a characteristic of most solid tumors to have regions of low oxygen, it appears reasonable to use bacteria that can only grow under hypoxic conditions. This would avoid adverse effects on healthy tissues. However, it is just this specificity which limits the potential of obligate anaerobic bacteria. They will leave well-oxygenated tumor areas unharmed from which the cancer can regrow. In contrast, facultative anaerobic bacteria should have the potential to colonize all areas of a solid tumor and thus exhibit a stronger anti-tumor effect. Entering the tumor Although it could be shown for various bacterial strains that they can colonize tumors, it is still not absolutely clear how they manage to enter the cancerous tissue. This is a crucial point as an efficient anti-tumor effect demands sufficient colonization. Different scenarios of the entry process can be envisioned. One suggests an active mechanism where bacteria are chemoattracted by substances of quiescent or necrotic tumor cells [3]. Alternatively, we propose that bacteria are passively flushed into the tumor tissue through its leaky vasculature. Upon intravenous application of S. Typhimurium to tumor-bearing mice, a rapid and strong influx of blood into the tumor can be observed (fig. 1). At the place of this hemorrhage, a huge necrosis forms. Salmonella are colonizing this part and the region bordering the viable tumor rim. TNF-α was identified as one mediator that plays a crucial role in this process [4]. We consider this an impor- 20 ▪ G.I.T. Laboratory Journal 9-10/2010 • Pharmaceutics & Drug Discovery Fig. 1 Early events after an intravenous Salmonella infection of tumor bearing mice. Top: A subcutaneous CT26 colon carcinoma tumor in an uninfected mouse appears light and H&E stainings of paraffin sections show mostly viable (V) tumor tissue. Middle: Shortly after an i.v. infection with Salmonella, the tumor turns dark due to a strong erythrocyte infiltration peaking at 12 h post infection (p.i.). Bottom: The hemorrhage is cleared from the tumor by 24 h p.i. and in the core of the tumor a necrotic region (N) has developed. (Adapted from Leschner et al. PLoS ONE 4(8): e6692. doi:10.1371/journal.pone.0006692) tant finding. Clinical studies with an attenuated mutant strain of S. Typhimurium exhibiting a diminished potential to induce TNF-α showed only very poor tumor colonization in human cancer patients [5]. Thus, the challenging task is to generate mutant strains that are virulent enough to efficiently colonize tumors and at the same time attenuated enough to be safely administered. Controlled expression of therapeutic molecules Particular bacteria do have anti-tumor effects when administered systemically. Apart from this natural ability to cause tumor shrinkage, they can also be used as vectors to deliver therapeutic molecules directly into the tumor, thus enhancing killing of the cancerous cells. These molecules could be bacterial toxins, cytokines that activate an anti- tumor immune response or prodrug converting enzymes. Independent of which protein to be expressed, it is essential that the mode of expression can be controlled tightly to prevent adverse effects on healthy tissues. This could be achieved by the use of special promoters to drive expression, like inducible promoters. An example would be the E.coli promoter PBAD that can be induced by the sugar L-arabinose (fig. 2). Administration of this sugar to mice infected with bacteria that encode the therapeutic molecule under control of PBAD expression can be started at any defined time point [6]. Other possibilities are the use of in vivo inducible promoters. For instance, we have defined several promoters that respond to the special physiological conditions of the tumor tissue but are silent in other organs. Therefore, the expression of a therapeutic molecule can be rendered tu- Fig. 2 The inducible promoter PBAD allows to induce gene expression in tumor colonizing bacteria by addition of the sugar L-arabinose. Tumor bearing mice were infected with Salmonella that carry the bioluminescence operon of Photorhabdus luminescens under the control of the inducible promoter PBAD. The inducer L-arabinose was administered 24 h p.i. to the mice resulting in bioluminescence in the tumor, which can be visualized by an in vivo imaging system. Pictures show mice 0.5 h, 5 h and 24 h after L-arabinose application. mor specific. Using these control elements, the expression of the potentially toxic substances in healthy tissues should be prevented leaving them unharmed by the therapy. Conclusion Many different bacteria have demonstrated their potential to be used in cancer therapy as they are targeting solid tumors. However, the demands for an ideal anti-cancer bacterium include more than that. It has to be safe and efficient at the same time. A combination that appeared to be unsolvable when this therapy had been applied first more than 100 years ago. With today’s knowledge and the possibilities of molecular genetics to tailor bacteria to the very special purpose, a successful application of bacteria in cancer therapy appears to be in reach. References [1] Barbe S. et al.: J.Appl.Microbiol. 101:571-578 (2006) [2] Pawelek J.M. et al.: Lancet Oncol. 4:548-556 (2003) [3] Kasinskas R.W. et al.: Biotechnol.Bioeng. 94:710-721 (2006) [4] Leschner S. et al.: PLoS.One. 4:e6692 (2009) [5] Toso J.F. et al.: J.Clin.Oncol. 20:142-152 (2002) [6] Loessner H. et al.: Cell Microbiol. 9(6):1529-37 (2007) Authors Dr. Siegfried Weiß and Dr. Sara Leschner, Helmholtz Centre for Infection Research, Braunschweig, Germany Contact Dr. Sara Leschner Molecular Immunology Helmholtz Centre for Infection Research Braunschweig Germany sara.leschner@helmholtz-hzi.de www.helmholtz-hzi.de G.I.T. Laboratory Journal 9-10/2010 ▪ 21 Application note • Reproducible Cell Assays Consistency of Cryopreservation is a Necessity Cell Cryopreservation is a critical component of cell culture work. The cells which survive the thermodynamic journey from the warm temperature of the incubator to the –196°C environment of the liquid nitrogen storage tank are free from the influences of time. This capability provides the cell culturist with a means of taking a snapshot of the culture at a given time in its history. The challenge of cryogenic storage is, in a word, ice. Cells are approximately 70% water, and when chilled to below the freezing point, ice crystals will form in the cell interior, lethally disrupting the intracellular structures. Cryogenic storage methods are successful only because the process includes a reduction of the intracellular water content prior to freezing, and, with the added benefit of a cryoprotectant, is successful in sufficiently limiting ice crystal growth. As the freezing process initiates in the extracellular fluid space, the forming ice crystals exclude and concentrate the dissolved solutes. The degree of dehydration of the cell is a key parameter that is controlled by the rate of temperature decrease. If the rate of temperature reduction is too low, the cells will become dehydrated beyond the critical water content survival limit due to prolonged exposure to the exterior concentrated salt solution. In addition, the added time spent in the high salt concentration environment can have a deleterious effect on cell health through exposure to inappropriate pH, toxic ion levels and concentrated solute-induced cell surface protein denaturation. Conversely, should the rate of temperature reduction be too great, the cell interior will supercool and ice crystals will nucleate, initiating interior ice crystal growth while the interior water percentage is still dangerously high. The two opposing boundary conditions restrict the freezing rate associated with a peak of cell viability to a narrow range. The value for the optimal freezing rate may vary with cell type and is dependent upon both cell size and membrane permeability. Fortunately, for a large portion of cultured mammalian cell types, in the presence of common cryoprotectants such as DMSO, the optimal freezing rate will coincide with a value of -1°C/min, and any 22 ▪ G.I.T. Laboratory Journal 9-10/2010 means of reliably attaining this rate of freezing will be beneficial in the cryopreservation process. Control Crossways There are two main avenues for achieving a controlled rate of cell freezing. The first and most expensive one involves the use of microprocessor-controlled refrigeration systems that can be programmed to follow a pre-determined profile of temperature reduction. The second avenue leads to the use of passive freezing units, which exploit the consistent thermodynamic principles of temperature differentials and thermal conductivity. Starting with a reliable thermal sink such as a -80°C deep freezer or dry ice locker (-78°C), cell vials can be encased in a device that will, through an ap- propriate combination of thermal capacity and insulation, provide a freezing profile with the desired temperature reduction rate. Numerous other protocols for cell freezing include steps such as wrapping the vials in paper towels, cotton or tissue, or encasing the vials in recycled styrene foam tube racks. A common acceptable threshold for the success of these freezing methods is that sufficient cells be recovered alive upon thawing to repopulate a culture flask within a reasonable timeframe, while dismissing the fact that such methods can result in cell cultures populated by a sub- Fig. 1: High post-thaw cell viability. HUVEC cells were resuspended in freezing medium at a concentration of 2 x 106 cells per ml. 1 ml aliquots were portioned into 1.8 ml Corning cryovials and frozen at -1°C per minute in either a Biocision CoolCell or in an alcohol filled cell freezing unit. Five vials frozen by either method were rapidly thawed and resuspended in growth media. Live cell count were obtained by the trypan blue exclusion method • Application note archived samples. The negative impact upon archived samples due to repeated temperature cycling is avoided in diligent laboratory practice by assigning a common and remote region of the freezer to the cell freezing process. This practice, however, imposes a secondary concern in that busy laboratories can often require the freezing of samples generated by multiple researchers, and the combined heat from two or more alcohol freezing containers in the same location will significantly alter the temperature reduction profile of all containers present. Alcohol-filled freezing containers also require that the alcohol be changed every five uses as absorbed moisture and evaporation can alter the heat capacity of the system and thereby cause variance in the thermal profile. In addition to the cost, the alcohol replenishment results in continuous generation of contaminated solvent that must be removed through hazardous waste streams. In daily practice, tracking the number of use cycles requires vigilance and, as most alcohol freezing units are laboratory community property, the consistency in maintenance descends to the performance level of the least diligent lab member. Likewise, mistaken replacement of the alcohol with an alcohol other than the required isopropanol is a repeated error made by researchers unmindful of the fact that different alcohols have significantly different heat capacities and that switching alcohols will alter the freezing profile. CoolCell System set of the original culture. The selective influences imposed upon the frozen cell sample can result in a wide variation in cell function and, in the worst case, lead to unrepresentative cell performance, assay results, biomarker behavior or cellbased diagnostic parameters. Alcohol-Filled Systems The alcohol-filled systems rely on a large thermal mass and high heat transfer to slow the sample cooling rate to approximately –1°C/min. These insulation-free designs depend upon the thermal conduction limits of the alcohol and the heat transfer limits of the air inside the freezer to regulate the heat flow, in effect controlling temperature reduction by temporarily overwhelming the heat removal capacity of the freezing unit. The heat lost from the alcohol (250 mL) is approximately 10 x greater than the heat removal required for sample freezing, placing a greatly amplified thermal burden on the refrigeration system that has the potential to cause temperature fluctuations in locally stored A recent alternative to alcohol-filled freezing containers is found in the radiallysymmetric insulation solid-state core (SSC) based design of the BioCision CoolCell product, which takes advantage of the combination of precision insulation geometry and small solid core thermal ballast (eye catcher). The core has a total heat capacity that is approximately 7% of the alcohol-filled container system. The total heat capacity of a fully loaded unit is less than that of a typical freezer box of samples, therefore the freezing unit can be confidently placed next to previously archived samples without imposing a damaging thermal fluctuation. As the physical positioning of the insulation and the heat capacities of the insulation and solid core materials are unalterable, when placed into the constant temperature environment of a typical regulated deep freezer, the contained samples will experience very consistent freezing profiles (fig. 1). Moreover, the unit can be used repeatedly and indefinitely with no maintenance Fig. 2: Highly reproducible freezing profiles. 1 ml of cell freezing media was placed into12 Cryovials. A thermocouple probe was introduced into one vial in an axial orientation with the probe end at the center of the liquid volume. All vials were equilibrated to 20°C, then loaded into a Biocision CoolCell, placed into a -80°C freezer and internal vial temperature was recorded by a data logger at 10 s intervals. After a 4 h freezing cycle, the vials were removed, thawed and equilibrated to 20°C. The repeatability of the temperature profiles is shown with 5 consecutive freezing cycles beyond insuring that it is dry at the time of sample loading. This simple and widely used method allows researcher to perform cell cryopreservation with repeatability and uniformity in freezing rate and postthaw performance profiles (fig. 2). Conclusion In summary, cryopreservation of cultured cells is a proven and essential process. The preservation of samples of PBMCs, stem cells, patient cells, cell lines and other investigative cell material is of compromised value if a the method of cryopreservation imposes variable and unpredictable influences on the constituents of the emerging cell population. Precision-engineered insulation alcohol-free cell freezing containers such as CoolCell represent standardizable means of providing reproducible cell freezing profiles (figures 1 and 2). These devices can contribute greatly in assuring that valuable experimental assets are not only preserved, but provide consistent and meaningful results. References [1] Schryver B., and Ehrhardt, R.: Surprisingly Unscientific World of Preanalytical Sample Handling: A Simple Method for Standardization.“ American Biotechnology Laboratory, January 2010. Contact Rolf O. Ehrhardt, MD, PhD Brian Schryver BioCision LLC Larkspur, CA, USA Tel.: 001/888/478-2221 info@biocision.com www.biocision.com G.I.T. Laboratory Journal 9-10/2010 ▪ 23 Pharmaceutics and Drug Discovery • Enzyme-Amplified Array Sensing of Proteins Identification of Low Protein Concentrations Irregular protein concentration levels in biofluids provide an indicator for the early detection of cancer and other disease states, making protein sensing an important biomedical goal. In recent studies we developed a highly sensitive enzyme-nanoparticle sensor array that uses enzymatic amplification to detect and identify proteins at very low concentrations in both buffer solution and biofluids. Vincent M. Rotello, PhD, Professor, Department of Chemistry, University of Massachusetts Amherst Chemical Nose Approach in Sensing Most currently used methods for protein sensing are based on specific recognition between an immobilized capture agent, such as an antibody or a receptor, and the target protein. Sensory processes such as taste and smell, however, utilize “differential” binding events where the receptors bind through selective interactions rather than specific. In this “chemical nose” strategy, a sensor array is created with different receptors that are then trained to generate a response pattern. This strategy has been applied to a variety of small molecule analytes [1]. More recently, this method has been applied to proteins, albeit with relatively low sensitivity (1-350 µM) [2,3]. In our research we have focused on the creation of more sensitive sensors for proteins. Using the “chemical nose“ approach, we have developed gold nanoparticle sensor arrays for protein sensing, including nanoparticle-fluorescent polymer conjugates that could identify proteins in buffer at 4-215 nM[4] and an analogous GFP (green fluorescent protein) sensor for proteins in human serum that could detect and identify changes as small as 500 nM [5]. Enzyme-Amplified Array Sensing (EAAS) While the methods mentioned above are efficient biosensing systems, the sensitivity of these displacement assays is limited by the emissivity of the fluorescent species used. To enhance sensitiv- 24 ▪ G.I.T. Laboratory Journal 9-10/2010 Fig. 1: Molecular structures of the nanoparticles, and a schematic of the sensors comprised of β-gal and cationic nanoparticles. (a) Nanoparticles binding to β-gal inhibit the enzyme activity; (b) Molecular structures of the cationic gold nanoparticles (NP1-NP6); (c) Release of β-gal by the protein. ity, we applied enzymatic amplification to “chemical nose”-based sensing. Our enzyme-amplified array sensing (EAAS) system features three components: (a) β-galactosidase (β-gal) as the amplifying enzyme element; (b) cationic functionalized gold nanoparticles (~2 nm core diameter) as the receptors/ β-gal inhibitors, and (c) 4-methylumbelliferyl-β-Dgalactopyranoside (MUG) as the fluoro- • Pharmaceutics and Drug Discovery Fig. 2: Detection of proteins in phosphate buffer. (a) Fluorescence responses pattern ratio in phosphate buffer. Each value is an average of six parallel measurements. (b) Canonical score plot of the first three factors of the response patterns. genic substrate. The anionic enzyme β-gal (pI =4.6, Mw = 465 kDa) was chosen due to its stability under a wide range of temperature, pH, and ionic strength conditions, while our cationic gold nanoparticles were used to generate differential affinity required for sensing. In practice, electrostatic binding of the nanoparticle to the enzyme inhibits activity without denaturing the β-gal (fig. 1 (a)). Analyte proteins bind the nanoparticles to displace the enzyme and restores its activity (fig. 1 (c)) [6]. We first tested the EAAS system in phosphate buffer, using a sensor array comprised of six different functionalized nanoparticles (fig. 1 (b)) and β-gal. We chose nine biomedically relevant proteins having various size, surface charges, molecular weights, and isoelectric points to validate the methodology. The individual target proteins generated highly reproducible rates of fluorogenesis, developing distinguishable patterns from the six nanoparticles. The resulting data were analyzed through linear discriminant analysis and all nine proteins were readily iden- Fig. 3: Detection of proteins in desalted human urine. (a) Fluorescence responses patterns ratio in phosphate buffer. Each value is an average of six parallel measurements. (b) Canonical score plot of the first three factors of the response patterns. tified (fig. 2), with a limit of detection/ identification of 1 nM. To demonstrate the applicability of this methodology for detection in real-world biofluids, we first focused on human urine – a highly used clinical sample due to its availability and ease of collection [7]. However, it is a challenging biofluid for sensor design due to high overall protein concentrations (>1.5 μM, 0.150 g/L). Despite this challenging matrix, we successfully obtained reproducible fluorescence that allowed full discrimination of proteins spiked into the analyte solution at 1 nM concentration (fig. 3). In our future studies, we are developing platforms for the application of this methodology to clinical settings. Support from the NSF (DMI-531171, CHE-0808945, and DGE 0504485) and NIH (GM077173) is gratefully acknowledged. References [1] Goodey A. et al.: J. Am. Chem. Soc.123, (11), 2559–2570 (2001) [2] Baldini, L. et al.: J. Am. Chem. Soc. 126, (18), 5656–5657 (2004) [3] Wright, A. T. et al.: Angew. Chem. Int. Ed. 44, (39), 6375–6378 (2005) [4] You, C-C. et al.: Nat. Nanotechnol. 2, (5), 318–323 (2007) [5] De, M., et al.: Nat. Chem. 1, (6), 461–465 (2009) [6] Miranda, O. R. et al.: J. Am. Chem. Soc. 132, (14), 5285–5289 (2010) [7] Thongboonkerd, V.: Mol. BioSyst. 4, (8), 810–815 (2008) Authors Xiaoning Li, BS, PhD student; Brian Creran, AB, PhD student; Vincent M. Rotello, PhD, Professor, Department of Chemistry, University of Massachusetts Amherst Contact Rotello Research Group Department of Chemistry University of Massachusetts at Amherst Amherst, USA rotello@chem.umass.edu www.umass.edu/rotellogroup G.I.T. Laboratory Journal 9-10/2010 ▪ 25 Particle Measurement • Aerosol Albedometer A Tool for Measuring Optical Scattering and Extinction of Dispersed Aerosols Atmospheric particulate matter (aerosols) decrease visibility and are believed to affect climate by scattering and absorbing solar radiation aloft. Scattering by aerosols leads to an increase in planetary albedo (reflectivity) while light absorption can lead to warming of the atmosphere. In this work, an instrument to simultaneously measure aerosol scattering, extinction, and albedo is summarized. It is believed this method may find use in both field and laboratory studies of aerosol optics. Background and Rationale The attenuation of a beam of monochromatic light through an aerosol cloud can be modeled through the Beer-Lambert law relationship: (1) where bext is the extinction coefficient (here we use Mm–1 units) and z is the path length. In turn, the extinction coefficient is the sum of scattering and absorption coefficients, and single scatter albedo (ω) the ratio between the effects of scattering and extinction: (2) (3) The reduction in local visibility and net climate effect of the aerosol depends on a number of factors including aerosol scattering coefficient (bscat), extinction coefficient (bext), and the single scatter albedo (ω). Therefore, precisely measuring these variables on both lab generated aerosol mimics and genuine ambient aerosols is of considerable interest. Traditionally, scattering and extinction have been measured separately through nephelometry and long-path length optical loss/extinction measurements (transmissometry). A major step forward occurred in 2001 when Smith and Atkinson applied cavity ring-down spectroscopy (CRDS) to aerosol extinction measurements [1]. Soon thereafter additional groups reported aerosol extinction measurements based on CRDS [2,3]. CRDS provides the requisite sensitivity for the measurement within a portable, compact instrument package. Similarly, interest in integrating sphere (reciprocal) nephelometry by several groups [4, 5] has led to improvements in device performance. All nephelometers cannot collect light over all angles equally. This leads to an an- ▲ Image of wildfire smoke being transported southward from Quebec over the great lakes and northeastern United States. The image was acquired by the Moderate Resolution Imaging Spectroradiometer (MODIS) on the TERRA satellite on July 7, 2002. Visual inspection reveals the smoke plume appears very different from the other haze / clouds. This difference may be due to a difference in single scatter albedo. Image courtesy of Jesse Allen / NASA Earth Observatory. ◀ Fig. 1: Illustration of the albedometer. Extinction coefficient is measured through determining τ, the CRDS cell time constant. Scatter coefficient is determined by measuring intensity of scattered light on a second independent channel through the scattering photomultiplier. The photograph illustrates the interior of one-half of the sphere nephelometer. The transparent tube, diffuse reflectance material, interference filter, and arc shaped light baffle are apparent. 26 ▪ G.I.T. Laboratory Journal 9-10/2010 • Particle Measurement gular truncation. For commercial devices, the truncation angle is often 5–15°. The main technical advantage of integrating sphere nephelometry is this angle can be reduced to < 5°. Additionally, scattered light is collected over a solid angle of nearly 4π steradians – a condition that can help improve limits of detection. The albedometer directly builds upon these approaches by combining the technical advantages of CRDS with integrating sphere nephelometry. Device Function Figure 1 illustrates the experimental setup similar to that originally reported in our recent technical works [4,5]. Ambient air is drawn through either the aerosol inlet or an air filter. The filter can remove particles from the sample which provides a spectroscopic blank. The sample is then drawn through either the internal volume of a sphere nephelometer itself or a transparent tube placed within the sphere. The albedometer employs CRDS with a frequency doubled Nd:YAG laser to make extinction measurements at 532 nm. In CRDS, the rate of light attenuation is measured as a short pulse of light circulates in an optical resonator formed between two highly reflective mirrors (R > 0.999). After the light is introduced into the resonator, the beam is switched off and the light intensity then exponentially decays in time (first order) with a time constant τ. The time constant τ is the time required for the intensity to fall to 1 / e of its original value. Since mirror reflectivity is fixed, only light absorption and scattering by the sample (placed between the mirrors) leads to a change in rate of optical loss and a corresponding change in τ. The cavity time constant (τ) can then be linked to sample extinction coefficient through the equation shown in figure 1 if mirror reflectivity (R), distance between mirrors (L), and time required for light to make one “round-trip” through the cell (tr) is known. The effects of both particles and absorbing gases can lead to changes in τ. Experimentally, the effect of gases is subtracted by using filtered air as the spectroscopic blank. Ring down times on the order of 25–35 µs are often encountered for our system, offering detection limits for bext < 1 Mm–1. In the setup, ambient pressure and temperature are also monitored at the measurement cell outlet. This allows for corrections in Rayleigh scattering due to changes in air density to be accounted for. Sample relative humidity (RH) is also recorded since aerosol optical properties are known to change with RH. Simultaneously, on a second channel the device measures light scattered from the reflecting beam through use of an integrating sphere nephelometer and second photomultiplier tube. The interior of the 30.5 cm sphere we use is coated with a Lambertian diffuse reflectance material (also illustrated in fig. 1) with R > 0.95 for the visible region. The scatter channel signal also exhibits an exponential decay in time. For quantitative analysis, the scatter channel detector signal (ISCAT) is ratioed to the CRDS channel detector signal (ICRDS) at all points along the curve. Strawa et al. [6] have shown this ratio is linearly proportional to scattering coefficient (bscat) through a constant K’ that takes into account efficiency of light collection, detector electronic gains etc. The ISCAT / ICRDS ratio is then averaged for many points and this measurement related to scattering coefficient through calibration with gases of known Rayleigh scatter coefficient (often CO2 and R-134a is used). In the first generation design, aerosol filled the sphere. The second generation instrument contains the aerosol within a transparent glass tube to reduce sample volume and instrument response time. Differential reflection off the glass tube can bias light collection efficiency as a function of scatter angle. Figure 2 illustrates photographs and measured CRDS, scatter channel waveforms and ISCAT / ICRDS ratios for ambient aerosols sampled in Pasadena, CA. The photographs and data were taken roughly 6 hours apart and clearly show the change in aerosol loading that occurred over this time. For the figure on the left, dry PM2.5 aerosol extinction coefficient was measured to be 60–65 Mm–1. The much hazier photograph on the right illustrates a period of time when PM2.5 aerosol extinction coefficient was approximately 160 Mm-1. Viewing the data traces, one can clearly see the change in the rel- ������������� ������������� ��������� ���������������� �������������� ������������������������� ��������������������������������� ���������������������������� ���������������������������� ����������� ���������� � � �������������������������������� ������������������ ����������� �� ������������ � � � � � � � � � � � � � � � � � � � � � � � � � � �� ��������������������������� �������������������� Particle Measurement • Fig. 2: View towards the San Gabriel mountains from Pasadena, CA on June 8, 2010. Photos were taken about 6 hours apart. Insets show optical data collected with the albedometer at approximately the same time the photos were taken. The light green trace represents CRDS data (ICRDS) and the black trace is scatter channel measurements (Iscat). An increase in Iscat / ICRDS ratio (red trace) is clearly observed as the haze increases. A decrease in cavity time constant (τ) is observed for the thicker haze. The mountains are at a distance of approx. 8 km. ative size of the CRDS and scatter channel signals. The ISCAT / ICRDS ratio increases from 1.5 to 3.3 with increased aerosol loading. A decrease in ring-down time constant (τ) from 16.6 to 11.2 µs is also reflected in the data. S ample Data & Potential Technical Advantages There are several perceived advantages of this measurement platform. First, both scattering and extinction measurements are made on the exact same sample volume, simultaneously. This helps eliminate measurement uncertainty due to several different instruments being presented fundamentally different aerosol samples. Aerosol particles can sometimes be lost in tubing that connects different instruments. Furthermore, large differences in measurement volumes can lead to imprecision and / or errors when reporting albedo. Use of the integrating sphere design reduces truncation angle to approximately 3° on average. This increases the fraction of scattered light which is collected. Another unique feature of the design is that the scattering measurement is made during the CRDS transient as the beam circulates between the mirrors. Since the effective “time-constant” of the integrating sphere itself is only on the order of nanoseconds, any stray laser light introduced into the sphere during the initial pulse is quickly attenuated/removed. The CRDS beam continues to circulate for many microseconds, and since the scattering measurement occurs during this time period, only light scattered from the resonating beam is detected. This assures the measurement is free of the effects of laser light scattered off the walls or surfaces of the sphere. Conclusion The aerosol albedometer offers simultaneous, ensemble measurements of optical scattering and extinction on ambient aerosols. It is believed this platform may be of utility in making ambient measurements at fixed sites or on-board aircraft. The high sensitivity and rapid time response of the instrument may allow better understanding of the effect of aerosols on radiative transport in earth’s atmosphere. Future developments will focus on further reduction in device size and angular truncation. Additional measurement wavelengths can also be added. This research has been funded in part by the National Science Foundation (USA) under grants 1004114 and 634872 References [1] Smith J.D. and Atkinson D.B.: Analyst, 126, 1216 (2001) [2] Thompson J.E. et al.: Atmospheric Aerosol Measurements by Cavity Ringdown Turbidimetry. Aerosol Sci. and Tech., 37(3), 221–230 (2003) [3] Thompson, J.E. et al.: Monitoring Atmospheric Extinction Through Cavity Ringdown Turbidity” Anal. Chem., 74 (9), 1962–1967 (2002) [4] Varma R. et al.: Optics Lett. 28, 1007–1009 (2003) [5] Fukagawa S. et al.: Applied Optics 44, 3520–3526 (2005) [6] Thompson J.E. et al.: Optics Express 16(3), 2191–2205 (2008) [7] Dial K.D. et al.: Anal. Chem. ASAP, DOI:10.1021/ ac100617j. [8] Strawa A.W. et al.: J. Atmos. Ocean. Technol. 20, 454–465 (2003) Contact Prof. Jonathan Thompson Associate Professor Department of Chemistry & Biochemistry Texas Tech University, USA jon.thompson@ttu.edu DEGASSER® series 300, can be validated Semba Octave™, SMBC system in lab scale Aerosol Detektor NQAD™, new technologie: WCPC your individual partner in the LC • HPLC • GPC • SMBC • SFE/SCF ERC GmbH Otto-Hahn-Straße 28-30 · D-85521 Riemerling phone +49(89) 660 55 696 · fax +49(89) 608 24 826 partner@erc-hplc.de · www.erc-hplc.de 28 ▪ G.I.T. Laboratory Journal 9-10/2010 The best testimony to the innovative power of Merck, its reliability and close understanding of local and global markets is the sheer diversity of its products. The Merck portfolio currently encompasses more than 15,000 chemicals and reagents, active ingredients, test kits and analytical systems. Every day, new products join the fold, the result of purposeful research projects, specifically tailored to the needs of the customer. Naturally, each project meets Merck’s own high standards in terms of ultimate quality and reliability – which spells peace of mind for you and more time to concentrate on your work. www.merck-chemicals.com Does Merck Chemicals have something for stress? Yes, take your pick from nearly 15,000 products. Each designed to put you at ease – with ultimate purity, safety, reliability, and plenty of fresh prospects. The Barcelona® Chair by Knoll, Inc. That’s what’s in it for you. Merck Chemicals Bioprocessing • Lost in Translation Very Early Process Development for Biopharmaceuticals Today, scientists are searching for proteins to be used as biopharmaceuticals in the most remote corners of the plant and animal kingdoms. Drug candidates are cloned, trimmed and modified and heterologously expressed in a handful of relatively well-known host organisms. If things go extremely well for a new API (active pharmaceutical ingredient), it is expressed and milligrams can be purified, proof-of-principle can be shown, toxicity seems to be no issue and the need for a more useful amount for further characterization, usually along with first considerations of conducting a clinical trial, arises. In about a decade of upscaling and process development for a plethora of different API candidates at the Fraunhofer IME’s department for Integrated Production Platforms, we have noticed that at this point, many promising candidates and sanguine dreams (and business plans) die silently. This article tries to highlight a few of putative showstoppers in early process development for biopharmaceuticals and to point at the difficulties that will most likely occur as early as possible. Quick Success Versus the Search for a Sustainable Expression System The first pitfalls in process development occur at the very beginning. Unfortunately, the earlier a decision is made that is not far-sighted enough, the more damage will be done in later process stages. The work of years will be reset to the starting point because something in the process 30 ▪ G.I.T. Laboratory Journal 9-10/2010 simply turns out to be not feasible. Often, in an long and sometimes challenging process of generating an expression clone, decisions are made that are aimed at short-term objectives but cannot be adhered to in later phases. In other words, a quick success is – consciously or not – preferred to longterm sustainability. For example, commercially available bacterial expression kits that are optimized for high-expression levels are used in low-cell-density cultivations on complex media. The kits often feature modular solutions such as support for rare codons by additional plasmids, deletions and additions in the host strain that increase folding efficiency or stability, strong promotors that rely on specific genetic elements and environments and sometimes protein tags for purification or even detection of the foreign protein. These kits are designed to make life easier, but many elements or modules can cause severe problems when it comes to their usability in large-scale applications or regulatory compliance. Dr. Stehpan Hellwig, Head of manufacturing, Franunhofer IMEanufacturo The decision for an expression cassette or the use of a certain expression kit that promises quick success is not wrong in itself. The problem is that early achievements seduce to built on them and continue the easy way without asking how practicable it will be later. As we will see, things that make life easier by rendering careful design of the gene of interest, the expression cassette and the expression system unnecessary in the beginning might cause major discomfort later on. The Challenge of Scaling-Up One of the misunderstandings we’ve been faced frequently is the assumption that a bioreactor is basically a large shake-flask featuring a built-in autoclave. Processes that lead to a “fat band” in a shake-flask or a cell cultivation system do not always maintain their specific productivities when translated to a bioreactor. The term “specific productivity” describes the level of expression or the accumulation of a product per biomass or per cell. Expression kits are often tailored to low-cell density cultivations in very rich media. Under these conditions, optimal supply of nutrients or energy and accumulation of inhibitory metabolites is usually not a problem. In medium with high cell density fermentations, this may become limiting and decrease the specific productivity. Also, complex media compounds such as peptones or additives such as protease inhibitors can be unacceptable from the regulatory or economical point of view. As a result, actual productivities may be lower than the calculated numbers. This can often be fixed by intensive process development, but it needs resources and time to get there. Scaling-up a laboratory purification process to pilot scale usually includes major changes in the separation steps. Laboratory filtration steps sometimes rely on syringe filters and chroma- • Bioprocessing tography in gravity-driven columns. Also, “perform all steps on ice” is not an unusual direction in laboratory protocols, but neither very specific nor easy to in larger scales. Chromatography media suited for reasonable processing times and flow rates can differ fundamentally in terms of resolution and capacity from those used in gravity-flow approaches. Filtration processes such as ultra-filtration or diafiltration, when carried out in tangential flow devices can introduce thermal load to a process that may impair product integrity and cooling down some 50 liters of an intermediate in a carboy takes surprisingly long compared to 50 ml in a falcon tube. Assuming that a process aimed at the production of a clinical-grade API has been scaled up successfully, analytic results generated by the QC department are likely to hold some surprises. The purity of early-development protein preparations is usually overestimated - sometimes simply due to the detection limits of the method used. All of a sudden, with adequate overloading, bands of unknown identity pop above and below the familiar band. Also, the resolution of up-to date chromatography controllers will more often than not raise questions regarding the homogeneity of the main elution peak by unveiling shoulders or a distinct asymmetry that inevitably call for further investigation and explanation. IP issues Assuming once again that all technical obstacles can be adequately addressed and circumnavigated, there are legal issues that are often ignored until there is is a product, a market or some profit foreseeable. There’s always something more urgent or more interesting to take care of than analyzing which bits of somebody else’s intellectual property you have been using somewhere along the road. At this point, circumvention of a technology that has worked well for a protein is an extremely painful decision to be made. But it’s either that or negotiation of license fees. An easily obtained research license may turn out to be a costly mortgage on the future if not addressed in time. Time is Cash Time may be linear or not, but in biopharmaceutical process development it definitely has more dimensions than “time-to-market”. It may take half an hour to spin down 500 ml of cultivation broth, but in a pharmaceutical process, you’ll still end up with 2 days for the same process step if you account for buffer preparation, run time and cleaning-in-place. The same applies to other process steps in a very similar fashion. Cost of goods also make a remarkably non-linear impression on most researchers. A raw material or consumable order somewhere under 500 Euros or dollars passes through easily without raising any eyebrows and a scale-up factor of 100 is easily asked for. In fact we have been asked several times for a quote on the scale-up of laboratory protocols where the cost of buffers alone exceeded the customer’s budget expectations for the entire project. But these are relatively simple technical issues. More important are problems that cost time, no matter if there is enough money available. We’ve all heard the calculations of how many dollars of earnings are lost in a month’s delay of the launch date of a blockbuster. Such calculations may be rigged a little to impress the audience, but in fact, the further a product is down the pipeline, the more expensive and timeconsuming it becomes to clean up unsolved problems. Conclusion There are many points to consider in the early process development of biopharmaceuticals and even this very limited and superficial flashlight on the most easily made mistakes in the translation of a product from research to development may be disillusioning. The take-home-message is to involve know-how on the technical, regulatory and legal implications of biopharmaceuticals as early as possible. On the other side, the availability and accessibility of existing information and know-how as well as sophisticated technologies to efficiently produce and thoroughly analyze the biopharmaceuticals was never as abundant as today. Maybe it’s time to go back to one of the fundamental virtues of scientists – think twice before you pipet. Fraunhofer IME – Department for Integrated Production Platforms Fraunhofer IME is one of 5 Institutes of the Fraunhofer Life science alliance. Building on 20 years of experience of recombinant protein production with an emphasis on antibody-derived products and plant biotechnology, about 150 scientists are working on contract research and development projects focused on Pharmaceutical Product Development, Plant Biotechnology and Applied Ecology at the IME and the closelyattached institute for Molecular Biotechnology at the Aachen University. Dr. Stephan Hellwig is co-heading the Department of Integrated Production Platforms at the IME. The department does contract research in protein production process development and holds a manufacturing authorization for the GMP-compliant production of API for clinical phase one trials. Dr. Hellwig is Head of Manufacturing in the GMP facility. Contact Dr. Stephan Hellwig Fraunhofer IME Aachen, Germany Head of Department – Integrated Production Platform Stephan.hellwig@ime.fraunhofer.de G.I.T. Laboratory Journal 9-10/2010 ▪ 31 Material Sciences • Quantification Study of Drug Delivery by Nanocarriers A Cell Mass Spectrometry Approach Delivery of peptides, proteins, antibodies, vaccines and gene-based drugs by nanocarriers can greatly reduce drug resistance and achieve a therapeutic effect in humans or animals. To understand the drug delivery efficiency by nanocarriers, quantitative measurement is essential. We developed a cell mass spectrometry methodology that can quantify nanocarriers uptake into mammalian cells. The quantitative study will help examine the mechanisms of different types of sustained release formulations with nanocarriers including liposomes, drug loaded biodegradable microspheres, viral nanoparticles and drug polymer conjugates. nanocarriers is of importance in examining the efficiency of drug delivery. However, only a few studies have explored the quantitative measurement of the cellular uptake of nanocarriers during cell endocytosis and exocytosis process. Traditional Quantitation Approaches Dr. Wen-Ping Peng, Department of Physics, National Dong Hwa University, Hualien, Taiwan Dr. Alice L. Yu, Genomics Research Center, Academia Sinica, Taipei, Taiwan Dr. Chung-Hsuan Chen, Genomics Research Center, Academia Sinica, Taipei, Taiwan Nanocarriers Used in Drug Delivery The use of nanocarriers in drug delivery can enhance the intracellular concentrations of drugs. In general, nanocarriers bind specific receptors and enter the cancer cells via receptor-mediated endocytosis. With the help of nanocarriers, drug molecules can easily bypass the recognition of P-glycoprotein and thus reduce the drug resistance [1]. The concentration of nanocarriers uptake into cells is proportional to the drug concentration. Therefore, the quantification of 32 ▪ G.I.T. Laboratory Journal 9-10/2010 Inductively coupled plasma atomic emission spectroscopy (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS) are two major techniques for the quantification of internalized nanoparticle (NP) elemental composition and nanoparticle uptake [2,3]. ICP-AES and ICPMS have the advantages of sub-ppb detection limits, high precision and high accuracy but are confined to detection of elemental species, such as gold NPs [4]. Another technique is “mass barcode” in which the gold NPs are encoded with different functional groups and the uptake of multiple functionalized gold-NP cells is measured by using laser desorption/ionization mass spectrometry (LDI-MS) [5]. It is not widely adopted because the encoding process is tedious and time-consuming and the ionization efficiencies for various functional groups with different sizes of gold nanoparticles are different, which makes quantitative measurement somewhat uncertain. Cell Mass Spectrometry Approach We have employed cell mass spectrometry (CMS) technique to accurately measure cellular uptake of nanocarriers [6]. The CMS technique is unique in its ability to detect elemental species as well as other nanomaterials. It streamlined sample preparation and a rapid detection procedure. CMS provides an elegant way of weighing particles with diameters greater than a few micrometers, which cannot be achieved by commercial mass spectrometers as shown in figure 1a [7,8]. Figure 1b shows the four parts of CMS apparatus: a laser-induced acoustic desorption of microparticles ion source without a matrix, a lowfrequency quadrupole ion trap for the measurement of ultra-large m/z values, a pressure-controlled corona discharge to enhance the number of charges on a cell or microparticle, and a compact, low-noise charge detector for total-charge measurement. We used the CMS to quantify the uptake of NPs by living cells [6]. We first determined the weight range of living cells, e.g. the mouse leukemic monocyte/macrophage cell line Raw264.7, the human embryonic-carcinoma cell line NTERA2, and the human cervical-cancer cell line HeLa and plotted the weight distribution dia gram as shown in figure 1c. Next, we determined the uptake of NPs by measuring the mass difference for the target cells treated with and without gold NPs using CMS. Serial measurement of NP uptake by the living cells treated with gold NPs for various time intervals showed that NP uptaken by all the living cells reached a plateau after 15 hours. This proved the same for gold NPs in sizes ranged from 30 nm to 250 nm as shown in figure 1d. With the unique features of CMS, we found that the amount of gold NPs swallowed by each type of the living cells was the same as that determined by the ICP-MS. CMS required much fewer experimental steps as compared with the ICP-MS. Moreover, the total acquisition time spent by the CMS was 5 times shorter than ICPMS. More importantly, CMS could detect the exact amount of particles absorbed by each cell, instead of an average uptake per cell. Thus, it can provide detailed information regarding the heterogeneity in the particle uptake among each individual cell. The same experiments were carried out using nonmetal polystyrene NPs, and the result showed a similar trend in the uptake kinetics of gold NPs and of nonmetal polystyrene NPs. To the best of our knowledge, CMS is Power Up Your LCMS Capabilities 99.000 (125,00 0 USD*) €* Synthesis Confirmation/Structure Elucidation Reaction Monitoring Reaction Yield and Purity Determination Compound Library QC Compound Stability Testing Impurity Identification Product and Process QC Dramatically increase the productivity and data quality of your chemistry laboratory with this new, fully-integrated, LC/MS/MS system. Designed to far exceed the capabilities of traditional LCMS systems, the amaZon Powerpack LC/MS/MS system combines the high performance and proven reliability of the amaZon SL ion trap MS with a state of the art Dionex UltiMate 3000 UHPLC system - all available in one easy-to-use package at a premium value price. Don’t miss your opportunity to change the way you use LCMS. Visit www.bruker.com/powerpack for more information! *This promotional package is not available in all countries; please contact your local sales representative for details. This offer cannot be combined with other promotional offers, discounts or purchase agreements. The offer is valid until Dec. 31, 2010. For research use only. Not for use in diagnostic procedures. think forward Ion Trap MS Material Sciences • a) Fig. 2: Types of nanocarriers for drug delivery b) c) Nanodiamond+Proteins Gold NP+RNAs d) Fig. 1: (a) Range of size and mass of molecules analyzed by mass spectrometry. (b) CMS apparatus, including a quadrupole ion trap, a pulsed Nd:YAG laser, a He–Ne laser, a charge detector, and a CCD camera. The Nd:YAG laser induces cell desorption. The He–Ne laser illuminates the trapped cells so that they can be detected by the CCD camera. (c) Mass histogram analysis of NTERA2 cells. (d) Kinetics of the cellular uptake of 30 nm and 250 nm gold nanoparticles. efficiency, cellular uptake and cytotoxicity of nanocarriers of various size and surface properties. Conclusion the only device that can measure the uptake of nonmetal NPs by cells. Therefore it can be used for the measurement of drug uptake when drugs are carried by NPs such as virus and liposome. In contrast, ICP-MS cannot be used for measurement of the uptake of nonmetal NPs. Besides gold NPs and polystyrene NPs, CMS may also be useful for detecting the uptake of nanocarriers made of viruses, nanodaimonds, liposomes, carbon nanotubes, biodegradable polymers and micelles as shown in figure 2 [6]. Size Dependency and Surface Modifications of Nanocarriers Size and surface characteristics of nanocarriers determine if drugs can be effectively delivered to targeted tumor issue [9,10]. Figure 3 depicts a variety of targeted nanocarriers modified with different functional groups or drugs. Our CMS approach could help evaluate the drug targeting 34 ▪ G.I.T. Laboratory Journal 9-10/2010 So far, CMS technology offers the best approach to evaluate the cellular uptake of therapeutic nanocarriers for drug delivery in cancer cells. It is a rapid and accurate method for determining the quantity of gold NPs uptake into cells, and it can be used to determine the number of NPs taken up into each individual cell, whereas ICPMS provides only a mean uptake for all cells. Furthermore, CMS can be used to measure the cellular uptake not only of metal nanocarriers but also of nonmetal nanocarriers. In clinical use, CMS may help evaluate the drug delivery efficiency of nanocarriers of various sizes, shapes and surface properties and thus facilitate the development of optimal nanocarriers with the most efficient drug delivery capacity. References [1] Cho K. et al.: Clin Cancer Res 14, 1310–1316 (2008) Liposome+Prugs Virus+Antibodies Biodegrade Polymer+Drugs Fig. 3: Using nanocarriers as the base platform, a variety of tissue-specific ligands or other molecules may be attached on the particle surface. [2] Chithrani B. D. et al.: Nano Lett 6, 662–668 (2006) [3] Yang P. et al.: Bioconjugate Chem 16, 494–496 (2005) [4] Marquis B. J. et al.: Analyst 134, 425–439 (2009) [5] Zhu Z. -J. et al.: J Am Chem Soc 2008, 130, 14139–14143 (2008) [6] Lin H.-C. et al.: Angew Chem Int Ed 49, 3460–3464 (2010) [7] Peng, W.-P. et al.: Angew Chem Int Ed 119, 3865–3869 (2007) [8] Peng, W.-P. et al.: Mass Spectrom Rev. 23, 443–465 (2004) [9] Liu Y. et al.: Biomaterials 31, 330–338 (2010) [10]Singh P. et al.: Drug Dev Res 67, 23–41 (2003) Authors Dr. Wen-Ping Peng, Department of Physics, National Dong Hwa University, Hualien, Taiwan Dr. Alice L. Yu, Dr. Chung-Hsuan Chen, Genomics Research Center, Academia Sinica, Taipei, Taiwan Contact Dr. Wen-Ping Peng Department of Physics National Dong Hwa University Hualien, Taiwan pengw@mail.ndhu.edu.tw Cover story • Adaptability and Ergonomy Built by Your Needs As a core component to almost all clinical and research work, microscopy has a continuing and growing role within modern science and medicine. For example, even with the increasing prevalence of molecular tests, the microscopic view provides the unique opportunity to observe the physiology of a disease, or process. As a result, the quality of the diagnosis, or the progression of a research project, is reliant upon the resolution and clarity of the images produced by the microscope and its related imaging components. Consequently, both clinical and research laboratories require upright microscope systems which can effectively meet any needs. With a host of peerless features, the Olympus BX3 range of clinical and research microscopes establish a new standard in microscopy, enabling users to truly build a microscope system for their requirements. With the ability to adapt to any working environment, each user’s preferences are met by the new microscope concept. A workspace can therefore be completely tailored by each user to their own needs; from the positioning of hardware and controls through to personalizable software tools. Following on from the highly successful BX2 range, the BX3 portfolio consists of: the BX46 ergonomic screening microscope, the BX43 and BX system microscopes for clinical and research tasks with optional coded and automation modules, and the BX63 research system with advanced automation as standard. Future Proof and Adaptable to Any Workflow These microscopes are ideal for covering all your current requirements and protocols, as well as any future advances. Their modular nature ensures that they can facilitate multiple capabilities, including excellent fluorescence imaging. The full range of Olympus digital imaging cameras are available for the series, enabling every imaging requirement to be met. Furthermore, a series of motorization options, combined with the labSens or cellSens software packages, ensure that the user can automate as many, or as few, features as required. As a result, the optimal imaging system for every eventuality can be created with ease at any point in time, making your lab completely future-ready. As the basis of this concept in adaptability, a new level of flexibility allows this microscope to be ambidextrous, where many of the features are easily adaptable for left- or right-handed- 36 ▪ G.I.T. Laboratory Journal 9-10/2010 Instruments of the BX3 series ness. Building on this, users can completely define their working environment: from the microscope and imaging components to software workflows, GUIs and the positioning of the controls on the desktop. As such, the BX63 research system provides multiple options for managing the entire microscopy and imaging process. At the forefront of this concept, the programmable touch-panel (fig. 1) controls all components of the imaging system, enabling users to change the objective, mirror unit and observation method, as well as navigate and focus the sample. In addition, the research microscope can be fitted with a detachable remote control, providing the effect of traditional mechanical knobs for focus (Z) and stage position (X, Y).This also automates many of the optical adjustments required for changing between contrast methods, including condenser position; aperture stop; field stop; polarizer; fluorescence shutter; mirror turret position; and contrast method selected by the user. True Color LED The BX3 range features the novel, true-color LED illumination system, which has a unique wavelength profile, providing a color rendering index matching that of a halogen bulb with a daylight filter. This makes it ideal for high color clarity across the entire range of brightfield stains, a capability not offered with standard LED illumination systems. Furthermore, the true-color LED • Cover story scope is more flexible, adapting to the users individual posture and therefore resulting in less musculo-skeletal stress on the user. Advanced Automation Fig. 1: Programmable touch-panel Fluorescence Illumination system is controlled by the Light Intensity Manager (LIM), which automatically adjusts the intensity to user-defined levels on each objective change, thus removing the need for manual adjustment to increase the efficiency of each screen. In addition, users also benefit from the Olympus has introduced a new fluorescence illumination concept to both the research and clinical systems based around the unique fly-eye lens system (fig. 2), which provides homogeneity for fluorescence illumination and ease of use. Furthermore, the new 8 position filter turret not only provides the capacity for even the most comprehensive multi-color fluorescence studies, but also offers fast, tool-free mirror cube exchange (fig. 3). The cubes themselves are designed to maximize S/N by capturing >99% of stray light and using advanced multi-layer filters with steep cut-offs for highly defined wavelength selection. The BX43 and the BX53 enable users to build a system that suits their needs, with a host of optional automated components, including nosepiece, mirror turret, universal condenser, ND filter wheel and stage, allowing it to provide similar contrast management capabilities to the BX63. As a fully-motorized system, the BX63 is focused via the nosepiece, allowing the stage to be fixed for increased stability. Furthermore, the new ultrasonic Piezo-driven motorized stage uses highly accurate encoders which provide a continuous X, Y read-out, enabling the precise navigation to previously set coordinates at high speeds. Due to this motorization and X, Y readout, the stage can also be positioned by hand for rapid, gross sample alignment. Consultancy and Training Consultancy and training are essential functions in all applications within microscopy and Olympus is at the forefront of providing excellent efficiency in this area, offering customizable, dualobservation and multi-discussion units for laboratory conferencing. In addition, if fitted with a digital imaging camera and the labSens or cellSens software, the BX3 microscopes can essentially act as a netcam. Using standard TCP/ IP protocols the labSens ‘NetCam solution’ enables the transfer of live and stored images throughout the network for teaching, mentoring or supervision. Conclusion Fig. 2: Fly-eye lens system excellent life-span (approximately 20,000 hours) and minimal power consumption associated with LEDs, providing exceptionally low running costs and reduced downtime. Optically Superior As well as integrating the UIS2 optical components, the BX3 range has also been optically optimized to improve workflows. For example, the new wide-range condenser accommodates magnifications from 2–100 x without requiring a swing-top lens. This cuts out one of the common and time-consuming steps of microscope screening, offering substantial efficiency improvements. Fig. 3: Tool-free mirror cube exchange Setting New Standards in Ergonomy In addition to the established ‘ergo-tube’, the BX3 clinical microscopes are available with the world’s first tilting/telescopic/lifting observation tube that provides adjustment in three dimensions: eyepiece tilt –3° to +27º, tube extension (backwards/forwards) of 55 mm, and lifting of 45 mm. The BX46 also features an ultra-low fixed low-torque stage with ergo-grip controls, ensuring that the movements and force required by the operator are minimized. As their arms can remain on the desk at all times, even placing a sample on the stage requires very little effort. This further increases efficiency as the micro- The BX3 clinical and research microscopes form part of the broader range of highly versatile systems. Through the fully-customizable automation and motorization, in combination with the advanced camera and imaging software options, Olympus can create an imaging system tailor made for your individual needs. The unique modular concept allows users to configure their preferred workspace – whether this is a conventional set-up, or an automated system operated via the intuitive software and touch-screen controls. Olympus has therefore provided the ultimate level of flexibility. Contact Katja Ansmann Olympus Europa Marketing Communications Manager microscopy@olympus-europa.com www.microscopy.olympus.eu G.I.T. Laboratory Journal 9-10/2010 ▪ 37 Company News • Newsflow Safety Protocol Wyatt Technology offers a series of free of charge e-learning webinars on various aspects of protein and macromolecular characterization. Hosted by some of the company’s leading scientists, these educational platforms represent state-of-the-art in light scattering technology. 6 of the seminars are designed to optimize the usage of Wyatt instrumentation and three are focused on the scientific background of light scattering, and absolute macromolecular characterization. www.wyatt.com The CANopen Safety protocol (CiA 304) developed by CAN in Automation (CiA), international users’ and manufacturers group, is now published as an European standard and available as EN 50325–5 from the European Committee for Electrotechnical Standardization (CENELEC) and any National body. The CANopen Safety protocol is an addition to the CANopen protocol standardized in EN 50325–4, also known as CiA 301. CANopen Safety is designed to allow safety-related communication based on CAN according to IEC/EN 61508. The German TÜV has approved the protocol for use for systems requiring Safety Integrity Level 3 (SIL 3). www.can-cia.org Chemical Dispersants in Sea Water Paperless Lab Solutions The United States Environmental Protection Agency (EPA) has published two rapid screening methods for chemical dispersants in sea waters using Waters Acquity UPLC/Quattro Premier XE (UPLC/MS/MS). These analytical procedures were developed by Dr. Lawrence Zintek, National Organic Methods Development Expert, and Dr. David Schroeder, US EPA Region 5 Chicago Regional Laboratory (CRL) in collaboration with Dr. Johnson Mathew, Region 6 Houston Laboratory. These two methods specifically target the analysis of dipropylene glycol monobutyl ether (DPGBE), ethylene glycol monobutyl ether (EGBE), and dioctyl sulfosuccinate (DOSS) in sea water; three compounds used in the Gulf of Mexico in response to the oil leak. www.waters.com Thermo Fisher Scientific, announced that it has extended its reach across Central Europe by making Vialis the latest member of its Informatics Global Partner Alliance. Vialis, based in Switzerland, will provide local services and support for the full range of Thermo Scientific informatics solutions, including laboratory information management systems, chromatography data systems, electronic laboratory notebooks and spectroscopy software, as well as comprehensive laboratory automation and integration solutions. www.thermofisher.com www.vialis.ch. Free of Charge e-learning Seminars 3D Cell Culture 50 Years of Innovations Bruker was founded 50 Years ago, on September 7, 1960, in the suburbs of Karlsruhe, Germany. As a part of their 50th birthday, Bruker management team rang the Opening Bell at the NASDAQ Stock at the NASDAQ MarketSite in New York City’s Times Square. www.bruker.com Worldwide Distribution Agreement Dionex and EMD Millipore, the Life Science division of Merck KGaA of Germany, announced the signing of a worldwide distribution agreement for the EMD Millipore ICW-3000 water purification system for Dionex ion chromatography (IC) systems using RFIC-EG technology. Dionex is now able to sell this water purifier and consumables directly to customers globally. The system was designed by EMD Millipore specifically as an ultrapure water source for Dionex Reagent-Free IC systems with the powerful and convenient “Just Add Water” technology that eliminates the need for eluent preparation. The purification system’s easy installation with simple plumbing and control by the Dionex IC system make it very convenient to use. www.merck.de www.dionex.com www.milipore.com 38 ▪ G.I.T. Laboratory Journal 9-10/2010 reinnervate, a life sciences company driving the adoption of routine 3D cell culture, announced the commercial launch of ec23 a proprietary small molecule designed to mediate the controlled and reproducible differentiation of cells in culture. The molecule has multiple applications in academic and pharmaceutical research. ec23 is a synthetic retinoid based on all-trans retinoic acid (ATRA), which is known for its ability to modulate cell function and neural development. ATRA, as with other naturally occurring retinoids, is unstable and degrades readily when exposed to light (even low intensity light), which can result in cell culture heterogeneity and lack of reproducibility. The key advantage of the new molecule is that it is a more potent inducer of neurogenesis than ATRA and is also entirely stable. www.reinnervate.com • Application Note Particle Size Distributions Dynamic Image Analysis Beats Laser Diffraction in a Micron to Millimeter Range Laser diffraction is the most frequently used measurement technique for the analysis of particle size distributions in the range 1 micron to 1 mm in the context of quality control. Modern laser diffraction systems offer some convincing advantages such as short measurement times, easy operation and reproducible analysis results. However, they also have various disadvantages: Even if the instruments have been calibrated and validated, an absolute particle size measurement is not possible. Various round robin tests have shown that the analysis results depend strongly on the type of instrument and even on the particular model and software version. Principle of Dynamic Image Processing Measurements The particles move with the help of gravity, compressed air or dispersed in liquid through the measuring field. A light source illuminates them from one side while a camera takes their picture from the other side. The software evaluates the projections of the particles to determine the size distribution of all particles of the sample in a very short time. The maximum dynamic measuring range is substantially extended by using two aligned cameras. A high resolution camera detects small particles in a small measuring field. A camera with lower resolution but a wider measuring field simultaneously detects the larger particles, allowing for rapid measurement with good statistics. The laser diffraction method is further limited by the unsatisfactory detection for small outlier volume fractions (over-size and under-size) of approximately 2–3%, as well as the poor resolution of particles in the range from a few hundred microns to millimeters. Although laser diffraction systems are able to detect particles > 10 nanometers, only very few measuring channels are provided for particle sizes of approximately 1 mm. The resolution for these particles is rather poor. Thus, it is not possible to precisely resolve multimodal size distributions, as particles of a few hundred microns size difference are classified in the same size class. The complex, indirect measurement algorithm used by laser diffraction is like a “black box” for many users. The selection of the optimum evaluation parameters requires some experience; for the correct interpretation of the results it is often necessary to have some previous Jörg Westermann, Retsch Technology knowledge about the sample characteristics. Wrong assumptions and parameters lead to reproducible but inaccurate measurement results. Static laser light scattering is a rapid method, easy to carry out but difficult to evaluate. The ideal measurement method should directly detect the individual particle characteristics, for example by taking an image of the particle and calculating it directly. Now, such a direct measurement technology for fine powders > 1 micron is available with the new Camsizer XT (see Infobox). It uses the measurement technology of the ISO 13322-2 Dynamic Image Analysis standard, and beats laser diffraction with regards to resolution and detection limits by more than a factor 10. Until recently, dynamic image analysis was only established for the measurement of dry, pourable powders and granules in the size range above 30 µm. Thanks to an advanced computer and camera technology finer particles can now be displayed more sharply and evaluated in real time. The evaluation speed achieves 275 pictures per second, with up to a few 100 particles in each image. For the measuring range of 1 micron and above the image analysis method now also offers convincing benefits: As the particle images are taken directly with a camera of extremely high resolution, their size and shape can be accurately determined, even over a few orders of magnitude and consequently with a much higher resolution when compared with laser diffraction. The following application examples show the superiority of dynamic image analysis. Accurate Determination of Oversized Particles The laser light scattering method always detects a particle collective, i.e. the scattering signal is an average of many particles. Small amounts of undersized or oversized particles only cause a G.I.T. Laboratory Journal 9-10/2010 ▪ 39 Application Note • Fig. 1: Measurement of silicon carbide (abrasive) with a size distribution of 1–10 µm and a mean value of 5 µm (dry dispersion with compressed air). minor change in the light scattering pattern and therefore cannot be reliably detected with laser diffraction. Depending on the sample material, a volume fraction of 2–3% is considered as the absolute detection limit. The image analysis method, however, evaluates individual particles and detects, depending on the operation mode, every single particle of the sample. Only a few particles in the sample are enough for reliable detection, even if these particles amount to less than 0.01% of the entire sample volume. This opens up new perspectives for the characterization and ensures improved quality of the production monitoring process. Highly Precise Particle Size Measurement The laser diffraction method is based on the assumption that all particles are spherical. The real particle shape which deviates from the spherical shape changes the light scattering pattern; however, the software cannot transfer these changes to a particular distribution of size and shape. Although it is not possible to differentiate between the length and width of a particle, both parameters are included in the calculation of the “particle size”. As a result, the particle size distribution is often presented wider than it actually is and with a poorer resolution. If dynamic image processing is used for particle analysis, it is possible to determine the length, 40 ▪ G.I.T. Laboratory Journal 9-10/2010 width and equivalent diameter separately (see fig. 3). Thus it is possible to obtain various size distributions from one measurement, depending on which size definition is considered. Figure 5 shows the deviations between the actual particle shape and the ideal spherical shape the laser scattering method is based on. Spheres have a b/l ratio of 1.0. The majority of particles in the above example have a b/l ratio of < 0.9, i.e. they are clearly not spherical. Measuring the particle shape with digital image pro cessing thus leads to a more detailed knowledge of the sample quality. With dynamic image processing, just like with laser diffraction, the particles need to pass the field of view individually to ensure that each particle is analyzed individually. Agglomerates or particles which stick together give the impression of larger particle sizes. That is why both methods involve dispersion with compressed air or, alternatively, in liquid. The dispersion parameters have to be adjustable in a way that strong agglomerations can be separated without destroying the primary particles. Dynamic image processing provides information about the effectiveness of the dispersion tool as the particle projections are available as pictures at all times. For particles smaller than 1 micron, laser diffraction remains unrivaled. Image analysis with visible light encounters its physical limits here: as soon as the particle size comes down to the wave length of Fig. 2: Comparison of two different samples with different fractions of oversized particles. Sample 2 (red) contains 0.2 % more over-size at 20 µm. It is impossible to detect such small differences with laser diffraction. Fig. 3: Schematic representation of length (xL), width (xW) and equivalent diameter (xA). Fig. 4: The digital image processing method determines the size distribution with the help of the particle width (xW, red), the particle length (xL, green) and the equivalent diameter (xA, blue). The orange curve represents the results of laser diffraction. The results of image processing are more detailed with a better resolution. The accuracy of the image analysis results is confirmed impressively by sieve analysis and microscopy. Fig. 5: Shape analysis with digital image processing. The graphic shows the width-to-length ratio (w/l) of the sample represented in figure 4. 20 % of the particles are twice as long as they are broad, approx. 1 % are three times as long. the light, it is no longer possible to produce sharp pictures of them. Summary Dynamic image Processing is an established method for size and shape analysis of free flowing, dispersed particles with hundreds of customers and applications all over the world. With the help of the latest camera technology the method is now available also for particle sizes from 1 micron, to 3 mm, a size range which was previously covered exclusively by laser diffraction. The same advantages are now available for fine powders as well as for larger particles: reliable de- tection of over-size, high resolution and excellent reproducibility of the particle size results, information about particle shape, as well as easy operation, short measuring times, and an intuitive, simple measuring principle. Indirect methods with limited accuracy, such as laser diffraction but also complex optical methods with unreliable statistics, such as microscopy, become increasingly outdated. Kontakt Jörg Westermann Retsch Technology GmbH Haan, Germany j.westermann@retsch.com www.retsch.com NEW PARTICLE ANALYZER CAMSIZER XT Particle size and particle shape analysis of fine powders Q Digital image processing with patented two-camera-system (acc. to ISO 13322-2) Q Wide dynamic measuring range IURPNjPWRPP Q Reliable detection of smallest amounts of “undersize” and “oversize” Q Very short measurement time of 1 – 3 minutes Q Modular system X-Change for dry and wet dispersion Q Measurement results are 100% compatible to sieve analysis if required www.retsch.com/camsizerxt a : H a ll 9 · Visit us at Biotechnic www.gitverlag.com Booth B37 Phone: Fax: E-Mail: +49 (0) 21 29 / 55 61 - 0 +49 (0) 21 29 / 55 61 - 87 technology@retsch.com Ideal for: – metal and ore powders – pharmaceutical powders / granules / fine pellets – cement – detergents and enzymes – fertilizers – foodstuffs (pulverized and granulated) – plastic fibers / powders – refractory products – abrasives (mediumsize and small grit) and many more Application Note • White Giant or White Dwarf? Particle Size Distribution Measurements of TiO2 What have white tattoo ink, milk, toothpaste and lines on tennis courts in common? In all cases TiO2 (titanium dioxide) is used as a white pigment. It is the widest used white pigment due to its brightness and very high refractive index of 2.7. TiO2 is a naturally occurring mineral. Approximately four million tons of this pigment are consumed annually worldwide. In daily use, it can be found nearly everywhere - in paints, coatings, plastics, papers, inks, medicines as well as in toothpaste. It is applied even in the food industry as E171, e.g. for whitening of skimmed milk. Applications However, TiO2 has additional surprising features. It is a photocatalyst under ultraviolet (UV) light – and under visible light if it is doped with nitrogen ions or with metal oxides. The photocatalyst TiO2 captures ultraviolet light and forms activated oxygen from water or oxygen in the air. This process is similar to photosynthesis, in which chlorophyll captures sun- Dr. Markus Ortlieb, Product specialist Particle Size Analyzers, Shimadzu Europa light to turn water and carbon dioxide into oxygen and glucose. The activated oxygen formed is strong enough to oxidize and decompose organic materials and smelling gas. It is even strong enough to kill bacteria. Photocatalyst coating technology therefore adds advanced functions to building materials, for instance sterilizing, deodorizing and anti-fouling. The Graetzel cell (a type of chemical solar cell) uses the same effect. The cell is composed of a porous layer of nanoparticles of titanium dioxide, covered with a molecular dye that absorbs sunlight. The pigment-coated titanium dioxide is placed between two electrodes in an electrolyte solution. Sunlight is absorbed by the pigmentcoated titanium dioxide causing an electron to be injected into the conduction band of the semiconductor TiO2. These electrons travel through a wire from the anode to the cathode, creating an electrical current. In this way, energy from the sun is converted into electricity. Most of the materials used to make this cell are lowcost, easy to manufacture and flexible, allowing them to be integrated into a wide variety of objects and materials. However, TiO2 is produced in varying particle sizes, oil and water dispersible, and with varying coatings always depending on the application field and industry. Methods Measuring Small Particles – Laser Diffraction One method commonly used to measure particle size is laser diffraction (fig. 1). When light hits a particle, the resulting shadow image is not sharply defined due to the wave-like nature of light. It rather shows a light intensity pattern depending on the wavelength and the particle size. In order to simplify the analysis of this intensity pattern, a laser is used as a light source. In this way, only a single wavelength needs to be considered. The relationship between measured light intensity pattern and particle size can be derived from the so-called Mie theory. For sufficiently large particle diameters, the Fraunhofer approximation, which can be derived from Mie theory, can also be applied. Mie theory is able to describe the pattern of light and dark circles 42 ▪ G.I.T. Laboratory Journal 9-10/2010 • Application Note around the scattering center, whose width increases with decreasing particle size. Small particles generate a system of rings that are widely spaced. Rings at smaller distances originate from a large particle. Most laser diffraction particle size analyzers on the market are equipped with several light sources and/or detectors. Using this approach, a broad measuring range can be realized. Using such instruments is problematic since detectors or the light source have to be switched if the entire measurement range is to be covered. This causes further problems - switching needs time, recalibration is needed and the quality of measurement data is poor in the overlapping areas of the detectors. In the following measurements (fig. 1) a particle size analyzer was used with just a single light source, a single optical system and a single measurement theory of laser diffraction (Shimadzu SALD-7101). This setup features a perfect seamless and wide measuring range. There are no points of discontinuity over the entire measuring range. Figure 1 shows an example of a particle size distribution. This setup uses laser diffraction as the measurement principle. This method is perfectly suited for larger particles starting from 10 nm up to a range of a few millimeters. Fig. 1: Particle Size Distribution of TiO2: Average of ten consecutive measurements including standard deviation. Used Setup: Shimadzu SALD 7101 with batch cell. Measurement range: 10 nm – 300 µm Is TiO2 a White Giant or White Dwarf? Measuring Tiny Particles – Induced Grating Method With dynamic light scattering (DLS), the conventional method for measuring particles in the range of a few nanometers, the problem is that light scattered by particles decreases sharply for particle sizes of less than 100 nm. Furthermore, in the single nano region with particle sizes of less than 10 nm, physical restrictions make it difficult to detect scattered light. Measurement of particle sizes then becomes difficult. The IG method uses diffracted light instead of scattered light, and is free from these physical restrictions. Furthermore, it does not require input of the refractive index as a measurement condition. It allows simple measurement of nanoparticles with high sensitivity. The IG method uses specific electrodes dipped into the sample solution. If an electrical field is ap- plied to the electrodes, particles are “trapped” between the free spaces of the electrodes resulting in a diffraction pattern. With dielectrophoresis switched off, the particles diffuse back into solution and the detected diffracted light intensity decays. Based on the difference in diffusion velocities of large (slow) and small (fast) particles, the primary diffracted light will decrease at a faster or slower rate depending on particle size. This method enables stable measurements with excellent reproducibilities, particularly in the single nano range, as it is virtually resistant to contamination and even to the presence of small foreign particles. Special requirements of ambient air quality as well as sample filtration are therefore unnecessary. Measurements can also be carried out without any problems in many different solvents (fig. 2). Figure 2 shows an example of a particle size distribution measured with Shimadzu’s IG-1000. This setup uses a new measurement method called Induced Grating, developed by Shimadzu. It is a dedicated method for measuring nanoparticles. The measurement range starts at 0.5 nm and goes up to 200 nm. The critical range of measurements less than 10 nm is thereby extended. Both. TiO2 has a “giant” application field and is used as a white pigment or as a photocatalyst. Depending on the application field, the size range of the particles differs. Sometimes the particle diameter is in the range of nanometers, and sometimes in the range of micrometers. Sophisticated and advanced tools help in determining particle size distributions as accurately as possible. Fig. 2: Particle Size Distribution of TiO2: Average of three consecutive measurements including standard deviation. Used Setup: Shimadzu IG-1000. Measurement range: 0.5 nm – 200 nm. Measurement principle: Induced Grating. Contact Dr. Markus Ortlieb Shimadzu Europa GmbH Duisburg Germany shimadzu@shimdzu.eu www.shimadzu.eu G.I.T. Laboratory Journal 9-10/2010 ▪ 43 Application Note • Transfer of USP-based HPLC methods for pantoprazole sodium to UPLC 20-fold increase in productivity HPLC is a commonly used analytical method for assaying and purity controlling of active pharmaceutical ingredients („API’s“) in the pharmaceutical industry. Method transfer to the latest technologies can be timeconsuming and are therefore rarely performed for the improvement of validated methods. However, the transfer of established methods to a Pantoprazole sodium sesquihydrate (5-(difluoromethoxy)-2-[(3,4-dimethoxypyridin2-yl)methylsulfinyl]-3H-benzoimidazole, sodium salt, sesquihydrate) UPLC (ultra performance liquid chromatography) system can be worth the investment. In the reported case such an investment was rewarded with surprising savings in analysis time, operational costs and improved resolution. We demonstrate the successful method transfer for the analysis of pantoprazole sodium from the USP-recommended L1 column, run on a conventional HPLC system, to a sub 2 µm particle column on a UPLC system. With some small optimization changes, the final methodology reduced the an alysis run time from 55 min with HPLC to just 3 min with UPLC, resulting in a 20-fold increase in throughput and a remarkable reduction in solvent consumption and waste disposal costs! Pantoprazole sodium sesquihydrate is described in the USP and the European Pharmacopeia. The purity and assay testing for pantoprazole sodium is accomplished using high performance liquid chromatography (HPLC) with ultraviolet detection (UV) on L1-column in compliance with the USP 32—NF 27 monograph for pantoprazole sodium. 44 ▪ G.I.T. Laboratory Journal 9-10/2010 Standard solutions of pantoprazol sodium and related compounds A-F were prepared according to the USP procedure. All solutions were protected from light by use of amber glass ware and the autosampler’s sample compartments temperature was set to 4°C. LC System: Alliance 2695XE with PDA-detector 2996 and Empower 2 C/S-software (Waters) Column: XTerra RP18, 3.9x 150 mm, 5 µm (Waters) Mobile Phase A: 1.74 g/L dibasic potassium phosphate, adjusted with phosphoric acid (330 g/L) to a pH of 7.00 ±0.05 Mobile Phase B: Acetonitrile Gradient: 0–40 min 20 => 80% B (linear) 40–45 min 80 => 20% B (linear) 45–55 min 20% B (re-equilibration) Flow Rate: 1.0 mL/min Injection Volume: 20 µL Temperature: 40°C Detection: 290 nm Fig. 1: Original USP-method Introduction The active ingredient in Pantoprazole is Pantoprazole sodium (see eye catcher). It is commercially available as delayed-release 20 and 40 mg tablets and is functioning as a proton pump inhibitor: The 20 mg tablets are used for the treatment of patients with conditions caused by gastric acid secretion (reflux disease) and associated symptoms (heartburn, acid belches and pain on swallowing). Indications for the 40 mg dosis are the treatment of gastrointestinal diseases which require a reduction in acid secretion (different forms of ulcer). Experimental UPLC Conditions for Purity Testing – Geometrically Scaled Gradient LC System: Acquity UPLC system (Binary Solvent Manager, Sample Manager, Column Manager) with Acquity UPLC PDA eLambda-detector and Empower 2 C/S-software (Waters) Column: Acquity UPLC BEH C18, 2.1 x 50 mm, 1.7 µm (Waters) Mobile Phase A: 1.74 g/L dibasic potassium phosphate, adjusted with phosphoric acid (330 g/L) to a pH of 7.00 ±0.05 Mobile Phase B: Acetonitrile Gradient: 0–13 min 20 => 80% B (linear) 13–15 min 80 =>20% B (linear) 15–18 min 20% B (re-equilibration) Flow Rate: 0.29 mL/min Injection Volume: 1 µL Temperature: 40°C Detection: 290 nm Fig. 2: Geometrically scaled method • Application Note ceptance criteria of the USP monograph. A typical chromatogram is shown in figure 5. Conclusion UPLC Conditions for Purity Testing - Shortest Analysis Time Gradient: 0.00–6.55 min 20 => 80% B (linear) 6.55–7 .37 min 80 => 20% B (linear) 7.37 – 9.01 min 20% B (re-equilibration) Flow Rate: 0.803 mL/min Injection Volume: 1 µL Fig. 3: Shortest analysis time method UPLC Conditions for Purity Testing – Further Optimized Gradient: 0 .0–2.0 min 20 => 38% B (linear) 2.0–2.3 min 38 => 80% B (linear) 2.3–2.5 min 80 => 20% B (linear) 2.5–3.0 min 20% B (re-equilibration) Flow Rate: 0.8 mL/min Injection Volume: 1 µL The USP methods for assay and purity of the active pharmaceutical ingredient pantoprazole sodium, were successfully transferred from the USP-recommended L1 column on a conventional HPLC system, to a small particle 1.7 µm column on a UPLC system, while the system suitability criteria of the USP monograph are still met. With some small optimization changes the final methodology reduced the analysis run time from 55 min with HPLC to just 3 min with UPLC – that’s nearly a 20-fold increase in throughput and reduction in solvent consumption and waste disposal costs! Therefore, for our company, the investment in the ultra-performance LC technology is rewarded with significant savings in analysis time and operational costs while at the same time improving resolution. Fig. 4: Further optimized method Fig. 5: UPLChromatogram at 290 nm of a “real sample” of pantoprazole API HPLC Conditions for Purity Testing (fig. 1) (on the basis of the synthetic route, this test is recommended by the USP monograph when impurities C, D, E and F are potential related compounds) The HPLC separation of pantoprazole and impurities is shown in figure 1. The USP requirements for system suitability – the resolution between pantoprazole related compound E and D+F peaks is not less than 1.5 – are met. In addition, the tailing factor is not more than 2 and the relative standard deviation for replicate injections of the standard solution is not more than 5.0%. The current HPLC-based runtime for assay and purity is 55 min, with a retention time of 8.5 minutes for the active pharmaceutical ingredient pantoprazole. Method Transfer To meet the USP L1 column requirements an Acquity UPLC BEH C18 column, 2.1 x 50 mm, 1.7 µm was chosen. The HPLC parameters (gradient, flow rate, injection volume) were scaled down to UPLC by using the Waters UPLC Console Calculator (free download on www.waters. com). The calculator allows transferring isocratic or gradient LC methods thanks to fundamental Contact equations of chromatography. The program Alexander H. Schmidt takes into account the different system dwell Head of QC and Lab manager volumes, changes in column diameter and partiSteiner & Co., Deutsche Arzneimittelgesellschaft mbH cle size and calculates the conditions of „Geo& Co. KG, Berlin, Germany metrically scaled gradient“, „Maximum Peak info@steinerarznei-berlin.de capacity“ or of „Shortest Analysis Time“. www.steinerarznei-berlin.de For UPLC Conditions for Purity Testing - Geometrically Scaled Gradient see figure 2 and for ProductFurtherat the Shortest Analysis ion at m inform bra nd.co u Time on Equal Peak Cawww.vacu pacity see figure 3. In comparison to the HPLC method (runtime 55 min), the UPLC method – calculated with the shortest analysis time at equal peak capacity – has a runtime of only 9 min, with a retention time of 1.2 minutes for the active pharmaceutical ingredient pantoprazole (Fig. 3). Finally, the method New vacuum pumps ME 1 and ME 1C was further optimized to a total runtime of 3 Filtration is probably the most common application for vacuum in the laboratory. The new diaphragm min (Fig. 4). Tired of waiting for filtration? Let us help you speed it up. Application This optimized UPLC method was used to check for impurities in pantoprazole API, purchased from an Indian manufacture. All impurities were within the ac- pumps ME 1 and ME 1C offer a compact and high performance solution. With their easy-to-use functionality, they are perfect for both single and multiple filtrations. The ME 1C also offers outstanding chemical resistance. VACUUBRAND GMBH + CO KG Alfred-Zippe-Straße 4 ·97877 Wertheim ·Germany Tel.: +49 9342 808-0 · Fax: +49 9342 808-450 info@vacuubrand.de · www.vacuubrand.com G.I.T. Laboratory Journal 9-10/2010 ▪ 45 Application Note • On-line Oxygen Monitoring in Cell Culture Effects of Mitochondrial Modulators on O2 Dynamics of Mammalian Cells Real-time measurements of oxygen and pH in mammalian cell cultures are important to understand the metabolic dynamics in cell cultures. The observation of the time course of cellular oxygen consumption in response to environmental conditions or toxicological insults can be substantial for successful culture of mammalian cells. This type of monitoring could also be valuable for other applications such as tissue engineering and stem cell research. Non-invasive Oxygen Detection In the present work, oxygen levels in the media of cells plated at 4 different cell densities and varying oxygen tension (19% O2 (Fig. 2) and 7% O2 (Fig. 3)) were continuously monitored. In addition, the O2 consumption rates in response to mitochondrial modulators (Fig. 4) were also assessed. Mouse embryonic fibroblasts (MEFs) were used as a representative mammalian cell type, carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a metabolic uncoupler and antimycin was used as an electron transport chain inhibitor. The SDR SensorDish Reader was applied for on-line measurement of DO and pH in 24-well multidishes. Effects of Cell Density The first experiment was designed to investigate the effects of cell density on the cellular oxygen consumption rate and subsequent pO2 levels in the media. MEF cells were plated at three separate concentrations (100,000; 33,000; and 10,000 cells/cm2) along with a “media only” control. Measurements were made every 2 minutes for 10 hours at near atmospheric oxygen levels (approx. 19% O2). The oxygen profile (Fig. 2) clearly shows an effect of cell density on the O2 consumption level of each treatment. A lag period of < 1 hour is observed, presumably where the system is equilibrating to temperature and oxygen levels. After this period, all the cell concentrations show a decrease in pO2 levels from the “media only” control wells. Finally, during the course of the incubation, the deviation from the “media only” control wells is proportional to the number of cells initially plated, implying that individual cellular respiration is equivalent at all the cell densities tested. The second experiment was designed to investigate the effects of low ambient oxygen levels on cell density-induced observations in the first experiment. MEF cells were plated at three separate concentrations (100,000; 33,000; and 46 ▪ G.I.T. Laboratory Journal 9-10/2010 Cell Culture in Phase Contrast with Fluorescence (DAPI), friendly provided by Olympus 10,000 cells/cm2) along with a “media only” control and then placed in a 7% O2 atmosphere and monitored every 5 minutes for 10 hours. The oxygen profile confirmed the effect of cell density on the O2 consumption level of each treatment (Fig. 3). A short period of temperature equilibration (< 1 hour) is observed, after which the residual oxygen in the media is consumed by the cells in a density dependent fashion. Interestingly, even the “media only” is not fully equilibrated at the new oxygen levels at 7% O2 after 10 hours. However, the deviation from the “media only” control wells by each treatment is proportional to the number of cells plated. Effects of Mitochondrial Modulators The third experiment was designed to investigate the effects of specific mitochondrial modulators on the cellular oxygen consumption and subsequent pO2 levels in the media. MEF cells were plated at 33,000 cells / cm2 and allowed to adhere to the culture dish for 4 hours. The culture media was then replaced with the treatment specific media, containing either 1 µL / mL • Application Note Fig. 1: SDR SensorDish Reader for on-line monitoring of dissolved oxygen and pH in 24-well multidishes. Fig. 2: Average pO2 levels in the culture media of MEF cells plated at differing densities (100,000; 33,000; 10,000 and 0 cell/cm2) and exposed to approx. 19% O2; n = 3 for each treatment. dimethyl sulfoxide (DMSO, vehicle control); 10 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP); or 10 µM antimycin. Each well was then topped with 1 mL of mineral oil and the plate was incubated under standard culture conditions (5% CO2, 35% humidity; 37°C, and 19% O2 atmosphere) and monitored every 2 minutes for 10 hours. The oxygen profile shows a pronounced effect of treatment on the O2 consumption level of the cells (Fig. 4). Measurements from the 3 to 4 hour time points indicate that there was little variation in the treatments prior to receiving the dosed media. Within 30 minutes of dosing, observable differences emerged in the oxygen consumption curves between the treatments. DMSO, the vehicle control, caused little variation in the average O2 level in the MEF cells. A steady state between oxygen consumption and oxygen ingress was formed at about 17.5% O2. Antimycin, an inhibitor of Complex III of the electron transport chain, showed an observable decrease in the oxygen consumption rate of the cells, resulting in a steady state higher than the one with DMSO. Finally, treatment with CCCP, a mitochondrial uncoupler treatment resulted in an observable increase in cellular oxygen consumption. Materials and Methods The SDR SensorDish Reader (PreSens) (Fig. 1) was used for the noninvasive on-line monitoring of oxygen and pH in 24-well microtiter plates. Mouse embryonic fibroblast (MEF) cells were grown in 24-well multidishes with integrated oxygen sensors (OxoDishes). Cells were maintained in Dulbeco’s Modified Eagle Medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS), 1% peni- Fig. 3: Average pO2 levels in the culture media of MEF cells plated at differing densities (100,000; 33,000; 10,000 and 0 cells / cm2) and exposed to 7% O2; n = 3 for each treatment. Fig. 4: Average pO2 levels in the culture media of MEF cells exposed to Antimycin, DMSO and CCCP at the 4 hour time point ; n = 4 for each treatment. cillin (10,000 U/mL)/ streptomycin (10,000 µg/mL), 1% L-glutamine (200 mM), 1% non-essential amino acids (10 mM) and 1% 1 M HEPES (pH 7.8) under standard cell culture conditions (5% CO2, 35% humidity and 37°C). In all cases, cells were grown in 1 mL of media/well in a Coy O2 Controlled Glove Box. The oxygen concentration in the culture medium was monitored throughout the culture period at preset intervals using the SensorDish Reader software. when exposed to 7% O2. Therefore, preincubation of the medium at the desired oxygen level is recommended. Finally, CCCP was observed to increase cellular oxygen demand while antimycin was observed to decrease cellular oxygen demand compared to the vehicle control. The SDR SensorDish Reader allows for constant and rapid quantification of media oxygen levels which can be used as a measure of cellular oxygen demand and metabolic function. Conclusion We monitored media oxygen levels in mammalian cell cultures and showed a cell density dependent oxygen consumption. The density dependent differences observed at 19% O2 were also observed at 7% O2, a more physiologically relevant oxygen level. In addition, cell culture media with no cells took more than 10 hours to reach equilibrium Contact Lynn S.G. and LaPres J. J. Dept. of Biochemistry, Michigan State University East Lansing, MI, USA Sarina Arain PreSens Precision Sensing GmbH Regensburg, Germany sarina.arain@presens.de www.presens.de G.I.T. Laboratory Journal 9-10/2010 ▪ 47 Advertorial • ADVERTISEMENT High Energy Efficiency and Safety The Dow Europe Laboratory in Horgen Dow Chemical Company is one of the world‘s largest chemical companies headquartered in Midland (Michigan) in the United States. Dow is a worldwide leading R&D company for chemicals, plastics, and agricultural products. Sustainable activities are a fundamental part of corporate strategy and Dow sets an example at its own locations, including the European headquarters located in Horgen, near Zurich in Switzerland. The Siemens Building Technologies Division and Dow Chemical have enjoyed a close cooperation for many years, with many of Dow‘s R&D facilities using Siemens technologies within their laboratory operations. The Horgen facility, in addition to serving as the European corporate headquarters, is an important R&D location extended continuously for applied research and customer applications. Safety of staff and the environment are key, but energy efficiency at Horgen has also been a fundamental focus for a number of years. Thus, the new lab equipment they employ must not only meet existing standards but supersede them. mate be achieved within the buildings by adhering to business and ecological criteria. Siemens provides its laboratory solution as an answer to this challenge: a solution offering comprehensive building automation and control solutions along with specific functionality for the entire laboratory. The solution supports the requirements set forth in the European or Swiss standard EN14175 or SN EN14175 and is independent of the fume hood type. The solution combines control competency and quality, satisfying the highest demands of laboratory buildings while being user-friendly, reliable, extendable and flexible. Challenging Environment Automation of Laboratory Fume Hoods at DOW Chemical A laboratory is a challenging work environment, subject to stringent rules and regulations for workplace safety. However, in addition to fulfilling the primary duty of protecting laboratory staff from potential hazards, Dow‘s demanding requirements for the technical plants in the new laboratories also require that a comfortable cli- 48 ▪ G.I.T. Laboratory Journal 9-10/2010 The laboratory fume hood is an important protective piece of equipment in a laboratory environment, offering lab users a specially protected area to carry out experiments and tests. Targeted air flow or extraction ensures that neither contaminated air nor explosive concentrations exist in the working environment, while preventing uncontrolled return flow in the work area. To allow users to carry out experiments, the fume hood normally features a glass front sash that can be opened or closed, providing an effective safety barrier. When the front sash is closed, lab users are protected against contamination and exposure to toxic fumes or gases. To protect as well while the front sash is open, the volume flow extracted must prevent toxic gases from entering the room. Thus, fume hoods with constant flow are designed to extract the volume flow constantly to prepare for a possible opening of the front sash. This results in greater energy demand as well as unpleasant noise, particularly when the front sash is closed. Dynamic adjustment of the volume flow to the current position of the fume hood‘s front sash thus represents a more efficient and comfortable solution. Safety can be guaranteed at significantly lower volume flows when the front sash is closed, i.e. at a minimum opening of ca. 15 mm. Dow Europe employs exactly this type of technology. • Advertorial The fume hood controller, as part of the Siemens laboratory solution, uses specific sensors to acquire the front sash position linear and direct, allowing calculation of the effective opening of the front sash. The setpoint for volume flow is defined as per the opening of the front sash. The fast-acting control system can prevent toxic gases from entering the environment by dynamically adjusting the extracted volume flow when the front sash is opened. When closed, the volume flow is lowered again. Integrated Security The fume hood controller is supplied as a typetested laboratory fume hood extraction control unit with all components such as volume flow controller and measuring elements, actuator, and sensors. Intelligence is integrated in an autonomous automation station communicating via LON interface with room control and building automation and control. The fume hood controller constantly monitors the extracted volume flow and triggers a local warning in the event of any deviation from a pre-determined level along with a warning to the building automation and control system. In addition, users are notified under SN EN14175 when the front sash opening of 500 mm (still considered safe) is exceeded. The fume hood controller is designed to identify and signal wire breaks at the periphery, thus signaling interrupted communications. The application of the fume hood controller is based on type-tested functions customized locally via just a few parameters to meet individual project requirements: ▪▪ Manual fume hood extraction flow control using a fume hood operator unit by Siemens. Max 5 fume hood operating modes are available. ▪▪ Automatic fume hood extraction volume flow in dependence on front sash or slide window position ▪▪ Automatic calibration of pressure sensor ▪▪ Front sash position acquisition and alarming as per SN EN14175 ▪▪ Variable setpoint generation based on constant flow velocity, variable linear setpoint generation ▪▪ Supports strategies for simultaneous control and maintenance of minimum air exchange rate via fume hoods ▪▪ Monitoring for wire breaks ▪▪ Switching to emergency operation: Fire, emergency shutdown, purge, emergency underpressure, emergency overpressure ▪▪ Control of fume hood lighting via operator unit or by room occupancy ▪▪ Control of supporting flow fan ▪▪ Integration of switchable fume hood equipment ▪▪ Control via presence sensors ▪▪ Operating mode change based on room occupancy and scheduler program Siemens Total Building Solutions (TBS) Concept The laboratory solution from Siemens is based on the DESIGO building automation and control system and laboratory-specific applications. At Dow Europe in Horgen, efficient operations and fast alarm management/fault intervention is carried out via the management station, providing information from the building automation and control system on the state of all technical and mechanical installations. Thousands of data points for ventilation plants, laboratory rooms and fume hoods, along with hundreds of fire detectors, gas detectors, and supervision of critical doors are presented graphically on the management station in a user-friendly manner. Summary For many years, the management and R&D location Dow Chemical in Horgen has been setting the standard for safety and energy efficiency within one of the world‘s largest chemical companies. The decision in favor of the Siemens laboratory solution sets yet another standard toward dynamic control and monitoring of fume hoods, precise room control and efficient plant operations. Seamless integration in the location-wide building automation and control system is welcomed, especially by users, as it guarantees a high degree of safety, fast alarm/fault intervention, and continued plant optimization. The integrated solution approach by Siemens allowed for implementing Dow Europe‘s requirements from laboratory fume hoods to laboratory room control to primary plant control and comprehensive building automation and control system visualization from one supplier. Despite comprehensive technical requirements, the project was implemented within a short period of time and at a high standard of quality thanks to type-tested individual functionality and standardized communications interfaces. Fast customization to special needs by Dow Europe provided proof of the flexibility of the Siemens lab solution. Contact Jens Feddern Siemens Building Technologies Division, Zug, Switzerland jens.feddern@siemens.com www.siemens.com/lifesciences G.I.T. Laboratory Journal 9-10/2010 ▪ 49 ARE YOU ATTRACTED TO SCIENCE... Attend Pittcon—the ma gnet for scientific innovation. Network with thousands of the world’s leading scientific minds, get a hands-on look at the latest innovations, and experience all the compelling educational opportunities Pittcon has to offer. Science is the attraction, Pittcon is the Place. Let yourself be drawn. Atlanta, Geor gia, USA Mar ch 13 - 18 www.pittcon.or g • Products Particle Size Analysis System Beckman Coulter has extended the range of its LS 13 320 particle size analyzer to allow highresolution, reproducible measurement of samples from .017 to 2000 µm. The enhanced device also adds Rosin-Rammler and Folk & Ward Phi methods to its standard analytical capabilities. The instrument’s patented Tornado Dry Powder Dispersing System keeps sample particles intact, allowing true measurement of the original samples. Reproducibility is typically better than one percent. These features combine with the extended size range to provide high-accuracy/high-resolution detection without the risk of missing either the largest or the smallest particles in a sample. The analyzer differs from other laser-based instruments by virtue of its wide dynamic size range, number of detectors and sample measurement options. Reverse Fourier optics incorporated in a patented fiber-optic spatial filter system optimize light scattering measurement. Polarization Intensity Differential Scattering (PIDS) technology, a Beckman Coulter exclusive, furthers detection capabilities in pharmaceutical, plastics, food and beverage, and a variety of other applications. Vacuum Pumps for Filtration and Solid Phase Extraction In March 2010 Vacuubrand launched the diaphragm pumps ME 1 and ME 1C. They complete the range of compact vacuum pumps for applications like filtration and sample preparation in chemistry, microbiology, wastewater control and other analytical processes. The ultimate vacuum of 100 mbar obtains 90% of atmospheric pressure that is doing the work of forcing the media through the filter. For aqueous filtration, the ME 1 is the optimal choice; however, for more aggressive solvents, the ME 1C with its chemical resistance properties is the right solution. The new top mounted power switch and the space saving design offers an easy-to-use functionality even with gloves and requires minimal bench top space. An optional manual control valve with dial gauge enables variable fine adjustment of the pumping speed (max. 0.7 m³/h). Both pumps allow an almost maintenance-free use. New designed from the established technology of the three-stage model line MD 1 and MD 1C they stand out due to a proven long diaphragm life. Vacuubrand www.vacuubrand.com POCKET MICROSCOPE 30 or 100 magnification SPIRIG handy and self-illuminating Beckmann Coulter www.beckmancoulter.com www.spirig-30.com a: H Visit us at Biotechnic all 9 · Booth B37 www.gitverlag.com G.I.T. Laboratory Journal 9-10/2010 ▪ 51 Advertorial • Nanoparticles in Liquids Count, Size and Visualize With more and more manufacturing processes turning to the use of component materials having nanoscale dimensions, the need for characterization of nanoparticles becomes more critical. This applies from the fundamental understanding of how individual nanoparticles behave through processing to ensure reproducible performance of the finished product in use. This could apply to vaccine production, the development of drug delivery systems or to the production of inks and pigments. In all cases, the ability to count and size particles is required. With a new technique, nanoparticle tracking analysis, this is achieved in parallel with the real-time visualization of the particles. Nanoparticle Tracking Analysis (NTA) NTA is a light scattering method for nanoparticle analysis. It is being increasingly used for determining nanoparticle size through simulta- neously tracking and analyzing the trajectories described by a number of individual nanoparticles undergoing Brownian motion in a fluid. The technique is centred on a sample analysis module which comprises a small metal housing Fig. 1 52 ▪ G.I.T. Laboratory Journal 9-10/2010 containing a solid-state, singlemode laser diode (< 30 mW, 635 nm) configured to launch a finely focused beam through the sample of liquid containing a dilute suspension of nanoparticles placed directly above a specially designed optical flat. The sample chamber is approximately 250 μl in volume and 500 μm deep and the sample is introduced by syringe via a Luer port. The sample is allowed to thermally equilibrate for 20 seconds prior to analysis. The beam is caused to refract at the interface between the liquid sample and the optical element through which it is passed such that it describes a path which is close to parallel to the glass-sample interface, (Fig. 1) Particles resident in the beam (which is approximately 100 μm wide by 25 μm deep), are visualized by a conventional optical microscope aligned normally to the beam axis and which collects light scattered from each and every particle in the field of view. A video of typically 20-60 seconds duration is taken (30 frames per second) of the moving particles (Fig. 2a). The video is analyzed by a proprietary analysis program on a frame-byframe basis, each particle being identified and located automatically and its movement tracked. The thresholds for particle identification can be user adjusted, as can the gain and shutter speed settings of the camera, thus allowing the user to optimize the image for a particular sample type. The 8-bit video sequence can be automatically or user-adjusted in terms of image smoothing, background subtraction, setting of thresholds, removal of blurring etc. to allow particles of interest to be tracked without interference from stray flare or diffraction patterns which can occasionally occur with nonoptimum sample types. Particles diffusing into the scattering volume are identified and followed for the duration of the particle presence in the beam or until they diffuse to within a certain distance of an adjacent particle at which point tracking is • Advertorial sults shown as a particle size distribution plot (Fig. 2c). Looking ahead More than one hundred peer reviewed publications serve to illustrate the growing use of NTA to provide quantitative data leading to practical solutions for the characterization of nanoparticles. The ability to visualize individual particles counting each one separately provides exceptional confidence in the data being reported. ceased eliminating the possibility of analyzing particle trajectories which cross behind each other (Fig. 2b). Movements of individual particles are followed through the video sequence and the mean squared displacement determined for each particle. From these values, the diffusion coefficient and hence sphere-equivalent, hydrodynamic radius can be determined using the Stokes-Einstein equation and with the re- Sensitive Chemiluminescent Detection of Hydrogen Peroxide Lumigen HyPerBlu, a novel chemiluminescent substrate from Beckman Coulter enables direct detection of hydrogen peroxide. Combining a broad dynamic range and bright, sustained chemiluminescence, the ready-to-use reagent offers convenience for highthroughput screening laboratories. Reaction of the substrate with hydrogen peroxide rapidly generates sustained high-intensity luminescence for maximum sensitivity in solution assays. Direct detection increases accuracy and simplifies data analysis. When coupled with oxidases, the reagent also allows reliable, one-step, indirect detection of oxidases or their substrates. The reagent is provided as a single solution and does not require mixing. It is stable for one year when stored in an amber bottle at 2–8°C. Beckmann Coulter www.beckmancoulter.com Data Visualization Merck Millipore, the Life Science division of Merck KGaA, introduced the first software application for visualizing GPCR (G-protein coupled receptors), as well as kinase activity. The new Data Analysis and Report Tool (DART), which can be accessed through Merck Millipore’s Drug Discovery portal, creates an interactive map of target profiling assay results and enables drug researchers to make faster, more informed decisions. The application projects each compound’s activity profile onto a map depicting clusters of target protein families. This map provides scientists with graphical insight into cross-target interactions to help drive structure-activity relationship (SAR) studies. Data limits, sizing, colors, and target subclasses can be adjusted in seconds, thereby turning numerical data into a graphical display that can highlight biologically significant conclusions. Merck Millipore www.millipore.com Contact Andrew Malloy Head of Applications Science NanoSight Limited, Minton Park London Road, Amesbury, Wiltshire, UK www.nanosight.com Reporter Genes as Minicircle DNA Plasmid Factory offers a number of common reporter genes as minicircle. It consists almost only of the “gene of interest” – the reporter gene. Antibiotic resistance and the ori are removed. In order to compare the Minicircle with the classical plasmid, the company has developed the McBox. It is currently available as Luc (luciferase), GFP (Green Fluorescent Protein) and LacZ (ß-galactosidase). Custom made minicircle DNA and minicircle products with S/MAR elements are also available. Plasmid Factory web.plasmidfactory.com/en Plate Washer Tecan has developed the HydroSpeed plate washer, an advanced system for optimized washing of cells, beads and ELISAs in 96- and 384-well formats. It offers full control over critical wash parameters via an intuitive touchscreen interface, with drop-wise dispensing and tunable aspiration settings to help avoid loss of material and maximize assay efficiency. The system’s AntiClogging function is automatically rinsing and soaking the wash head when the system is idle between runs, and the instrument’s Easy X-change system allows rapid removal and replacement of wash heads for intense ultrasonic cleaning. The washer uses two magnets per well for magnetic bead washing, offering fast bead settling and high recovery rates, and can also be equipped with a vacuum filtration module for processing of non-magnetic beads. Tecan Trading info@tecan.com www.tecan.com/cell-protection G.I.T. Laboratory Journal 9-10/2010 ▪ 53 Products • Optical Emission Spectrometer SPE Concept Spectro has presented its Spectro Arcos 165 for the first time at Jaima Expo. The optical emission spectrometer with inductively coupled plasma (ICP-OES) records the elemental spectrum between 165 and 770 nanometer for every measurement; making it especially suited to challenging tasks in environmental analysis. The system rounds out the ICP-OES analyzer product series between the flagship Spectro Arcos and the entry-level Spectro Genesis. The engineering of the system “is based completely on the high-end components utilized in our flagship Arcos system and achieves exactly the same detection limits, the same precision and equally reproducible results,” explains Olaf Schulz, the manufacturer’s Product Manager for ICP-OES spectrometers. The optical system is the only difference between the models: While the Arcos records the entire spectrum starting at 130 nanometers for every measurement, the trimmed down Arcos 165 measures the wavelength range beginning at 165 nanometers. For sample preparation, cleaning and concentration of neutral, acidic and basic analytes from various matrices (e.g. urine, blood, tablets, food, water), Macherey-Nagel offers new Chromabond HR-Xpert – a professional SPE concept. Polymer-based RP- and mixed-mode ion exchange phases fulfill the demands of modern SPE phases. They provide an excellent enrichment of neutral, acidic and basic compounds. The spherical support polymer (PS/DVB) with optimized pore structure and high surface facilitates good reproducible, reliable and cost-efficient analysis with a broad spectrum of applications. The divers mixed-mode phases Chromabond HR-XC, HR-XCW, HRXA and HR-XAW provide the option of more aggressive washing procedures for matrix removal. Thus, cleaner samples and a protection of HPLC and GC instruments result. Spectro Analytical Instruments www.spectro.com Semi-Preparative HPLC Pump Cecil Instrument’s Adept HPLC range has been enhanced with a newly updated semi-preparative pump. The pump expands and streamlines the modularity of this range. Each pump has a range of 0.01 to 50 ml/minute and may be used isocratically or with others, to create a high pressure gradient. This makes for smooth, easy and reproducible transitions in scaling up from analytical to semi–preparative work. The use of semi-preparative pumps, with Autoquest autosamplers, column heater chillers, Wavequest UV/Visible ultra-fast scanning detectors, and fraction collectors makes for complete automation in the longterm, fast and reliable collection of fractions. As with all the Cecil modular components, the semi-preparative pump may be used with third party systems. Cecil Instruments www.cecilinstruments.com Vital Parameters Kept Under Surveillance Presens Precision Sensing has introduced the EOM-CO2mini. This electro-optical module is a solution for original equipment manufacturers (OEM) for customized monitoring devices. As for its small footprint, it can easily be integrated and, assembled according to ISO 9001:2008, it enables a precise and non-invasive CO2 measurement. The instrument extends the manufacturer’s scope of supply for OEMs. Customers from Scientific R & D as well as Biotech & Pharma can now monitor all parameters which are essential for life: O2, pH and CO2. PreSens Precision Sensing www.presens.de 54 ▪ G.I.T. Laboratory Journal 9-10/2010 Machery-Nagel www.mn-net.com Absorbance Reading BMG Labtech has introduced the Spectrostar Nano. This ultra-fast, full spectrum absorbance microplate reader with cuvette port is suited for all absorbance assays. Its rapid full spectrum analysis at a resolution of 1 nm allows for absorbance assays never before possible on a microplate reader. Using an ultrafast absorbance spectrometer, it can capture a full UV-Visible spectrum from 220 nm to 1.000 nm in less than 1 sec/well and measure sample volumes down to 2 µL. Use the built-in cuvette port for kinetic studies and quick experiments and measure all standard microplate formats up to 1536 wells. The microplate reader allows for single push button operation for basic commands, as well as specific assay protocol set up. The most common absorbance assays such as Elisas, DNA, RNA, Protein (Bradford, BCA, Lowry), cell growth, and beta-galactosidase can be performed with ease due to predefined protocols. BMG Labtech www.bmglabtech.com Giving pH Electrodes a Treat Metrohm has presented the “pHit kit”. It contains all that is needed to gently clean or regenerate pH electrodes: A step by step description of the cleaning procedure; 50 mL cleaning solution; 50 mL 3 M KCl electrolyte solution; 50 mL storage solution, and two electrode vessels. Metrohm www.metrohm.com • Company Index & Imprint Agilent Technologies Applied Biosystems. Beckman Coulter 8 Out Back Cover 51, 53 Biocision 22 BioCision 7 Helmholtz- Zentrum für Infektionsforschung IME Fraunhofer Inst. f. Molekularbiologie u. Angew.Ökologie 30 Kraeber 25 Macherey- Nagel 54 29 BMG Lab Technologies 54 Merck Brand Fabrik für Laborgeräte 13 Metrohm Bruker 33, 38 C- CIT 20 13 11 NanoSight 52 Cecil Instruments 54 National Dong Hua Univers. 32 CPC Colder. 31 Olympus Europa Holding David James Group 14 Phoenix MarCom 5, 27 Cover 22 Pittsburgh Conference on Analytical Chemistry 50 EBD 12 Plasmidfactory ERC 28 Presens 46, 54 9 Qiagen 15, 16, 19 GFL Ges. f. Labortechnik IMPRINT Segment Sales Manager Laboratory & Biotechnology Published by Dr. Katja Habermüller Tel.: +49 6151 80 90 208 Fax: -144 katja-carola.habermueller@wiley.com GIT VERLAG GmbH & Co. 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Manuscripts should be addressed to the editorial office. We assume no liability for unsolicited, submitted manuscripts. Reproduction, including excerpts, is permitted only with the permission of the editorial office and with citation of the source. Layout/Litho Media Consultants 39, 41 54, In Front Cover Molecular Sensing Deutsche Messe Retsch Oliver Haja (Layout) Elke Palzer (Cover Design, Litho) Ramona Rehbein (Litho) Reprints Dr. Stefanie Krauth Tel.: +49 6151 8090 191 stefanie.krauth@wiley.com Printed by Frotscher Druck Riedstrasse 8 · 64295 Darmstadt, Germany Circulation 27,000 copies, 6 times per annum Advertising price list from October 1st 2009 Subscription 2010 Six issues € 72.00, single copy € 14.50 plus postage. Pupils and students receive a discount of 50% at sight of a valid certificate. Subscription orders can be revoked within one week in writing. Dispatch complaints are possible only within four weeks after publishing date. Subscription cancellations are accepted six weeks before end of year. The publishing house is granted the exclusive right, with regard to space, time and content to use the works/ editorial contributions in unchanged or edited form for any and all purposes any number of times itself, or to transfer the rights for the use of other organizations in which it holds partnership interests, as well as to third parties. This right of use relates to print as well as electronic media, including the Internet, as well as databases/ data carriers of any kind. All names, designations or signs in this issue, whether referred to and/or shown, could be trade names of the respective owner. Printed in Germany ISSN 1611-6038 www.gitverlag.com The Path Is Clear Introducing the 7500 Fast Dx Real-Time PCR Instrument Forging a new path to clinical flexibility. The new 7500 Fast Dx Real-Time PCR Instrument from Applied Biosystems is a highly flexible, medium-throughput solution that may streamline your assay development and validation efforts—allowing you to work directly on an in vitro diagnostic platform. And the 7500 Fast Dx Real-Time PCR Instrument is flexible and open enough to integrate seamlessly into your workflow from single tube strips through 96-well plate setup. • Available for in vitro diagnostic use* – May help to minimize time and expense in obtaining assay clearance • Open software design – Full control over thermal cycling protocols • 96-well block format – Compatible with standard prep and setup instrumentation It’s the perfect combination of speed, flexibility, and productivity— the path is now clear. *For In Vitro Diagnostic Use Only. The 7500 Fast Dx meets the requirements of the In Vitro Diagnostic Medical Devices Directive (98/79/ EC). The 7500 Fast Dx is for distribution and use in specific European countries only. Not for use in the USA. ©2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. To learn more about the 7500 Fast Dx Real-Time PCR Instrument, visit www.appliedbiosystems.com/7500Dx