World Congress on Chromatography September 21-23, 2016 Amsterdam, Netherlands

Biography:

Yukihiro Shoyama worked in MGH as a Post-doctor in 1975. During 1978 to 1991 he worked as an Associate Professor and as a full professor during 1991 to 2007 in Kyushu University. During these periods he managed as the director of Pharmacognosy department, the director of herbal garden and the dean ship (2004-2006). He moved to Faculty of Pharmaceutical Sciences, Nagasaki International University as a full professor from 2007. He had efforts for the President of Japanese Society of Pharmacognosy (2007-2008) and Vice Chairperson of Specialty Committee of TCM Pharmaceutical Chemistry of World Federation of Chinese Medicine Societies (2012-). His research interests are marihuana studies, monoclonal antibodies against natural product, biotechnology of medicinal plants and bioactive natural products.

Abstract:

We prepared many kinds of monoclonal antibodies (MAbs) against natural products and developed a new staining method using MAb named as Eastern blotting. Glycosides like ginsenoside were developed by TLC, and the TLC plate was covered by PVDF or PES membrane and blotted. The membrane was treated with NaIO₄, and then with carrier protein resulting in glycoside-carrier protein conjugates on membrane. In the case of non-glycoside natural product like aristolochic acids, the conjugate with carrier protein was synthesized by appropriate pathway on the membrane. Peroxidase labeled secondary MAb and then substrate were added, successively. Several ginsengs were analyzed to find out unknown ginsenosides in American and Japanese ginseng by this fingerprinting. Also we separated ginsenosides using affinity column combined MAb from the crude extract. The other bioactive glycosides like saikosaponins and glycyrrhizin can be stained and applied for fingerprinting analysis. Aristolochic acids having kidney toxicity in Alistolochia spp. were separated by TLC and then blotted onto a PES membrane by employing a modified carbodiimide method. The resulting membrane-bound aristolochic acids-protein conjugates can be stained by Eastern blotting and fingerprinting. The staining of aristolochic acid in mouse kidney tissues was succeeded. Moreover, we detected sequensed and determined a target protein against aristolochic acid in mouse kidney cell lines using anti-aristolochic acid MAb. These related results will be also discussed. 

Biography:

Abstract:

Biography:

Ahmed Mohamed Ali Hemdan has completed his PhD from Ain Shams University. He has published more 10 papers in reputed journals.

Abstract:

A reversed phase HPLC method with U.V detection has been developed and subsequently validated for the determination of some adulterants in illegal herbal medicines. Five Herbal medicines for weight loss and other one for erectile dysfunction were tested for the presence of possible adulterants. The weight loss products were found adulterated with the already withdrawn drug Sibutramine and with Phenolphthalein that is proved to cause tumors. On the other hand, Sildenafil, a contraindicated drug for patients with heart diseases, was found in the herbal products for erectile dysfunctions. The methods use Inertsil C18 column (250x4.6mm, 5µ) and a flow rate of 1mL/min. The mobile phase for quantitation of Sibutramine and Phenolphthalein was consisting of acetonitrile-potassium hydrogen phosphate buffer pH=3 adjusted by o-phosphoric acid (40/60 v/v) and detection at 223 nm. While the mobile phase for Sildenafil was acetonitrile-potassium hydrogen phosphate buffer pH=3.2 adjusted by o-phosphoric acid (50/50 v/v) and detection at 230 nm. The confirmation of the presence of the adulterants was done using LC-PDA and MASS spectrometry. For this, a complete investigation of any herbal medicine should be done through orthogonal analysis methods before use even if it is labeled with (100% herbal content). 

Biography:

Evelien Wynendaele has completed her PhD in Pharmaceutical Sciences in 2014 at Ghent University. She is currently working as a Post-doctoral fellow in the DruQuaR laboratory at the same university, under the supervision of Prof. Bart De Spiegeleer. She has published more than 35 papers in reputed journals. 

Abstract:

In pharmaceutical analysis, the use of mass spectrometry (MS) coupled to liquid chromatography in quality control has become very significant. This is observed in i. a. the European Pharmacopoeia, where this technique is already incorporated in different general texts as well as specific monographs. Here, often (GMP) single quad MS platforms can be used, characterized by acceptable low costs, easy-to-use smooth lab-integration, increased efficiency and providing additional detection possibilities over a traditional UV/VIS detector. Single quad mass spectrometers also work well as analytical tools for the detection of low-abundant genotoxic impurities and their basic confirmation. Moreover, also in drug development, this detector can be useful: low UV-detectable impurities, obtained during synthesis, formulation or after degradation, can be analyzed, even in complex matrices. In developmental quality control of complex mixtures (e.g. plant extracts), the UPLC-single quad MS platform was found to be of great use in transdermal research as well.

Biography:

Rafea Naffa is pursuing his PhD from Massey University, New Zealand. He has worked as Lecturer at University of Sharjah from a period of 2007-2014. Rafea Naffa completed his Masters in Chemistry from Hashemite University. His research interest includes Chemistry, Bio-Analytical Chemistry and Leather Chemistry.

Abstract:

The natural crosslinks between tropocollagen molecules in skin contribute to its physical properties of strength and flexibility. Despite advances in analytical techniques such as mass spectrometry, the methods used for their analysis and quantitation date back to the 70s through to the 90s, are challenging and time consuming. As a result, it is difficult to obtain standards to verify analyses of these compounds. We have developed methods to isolate highly pure, natural crosslinks from skin, and have verified their structures using mass spectrometry and NMR. Fragmentation studies of these crosslinks will enable the development of a method for label-free quantitation in skin hydrolysates. We have also developed a novel chromatographic analytical method for the simultaneous determination of natural crosslinks. Seven crosslinks were separated on Cogent Diamond Hydride HPLC column using isocratic and gradient conditions then detected by mass spectrometry without derivatization. Total run time of less than 10 minutes was achieved under isocratic conditions using water and acetonitrile. To the best of our knowledge, this is the first method in which histidinolysinonorleucine (HHL) and histidinohydroxymerodesmosine (HHMD) were separated and identified by the mass spectrometry. This technique was applied on skin, elastin and cartilage in which strong evidence suggested the presence of undocumented crosslinks. The developed method will be widely used for quantitative and qualitative analysis of natural crosslinks in biological samples as well as characterization of new crosslinks. 

Biography:

Britta Eggenreich has finished her studies of Pharmaceutical Science at the University of Vienna in the year 2011. She has worked for two years as a Pharmacist in Vienna. In 2014, she started her Doctoral thesis in the group of Biochemical Engineering at Vienna University of Technology. Currently, her main focus is the downstream development for inclusion bodies of a novel antibody fragment produced in E. coli.

Abstract:

The bacterium Escherichia coli, is a well-studied recombinant host organism with a plethora of applications in biotechnology. High valuable biopharmaceuticals, such as recombinant enzymes, antibody fragments and growth factors, are currently being produced in E. coli. These molecules are usually produced intra-cellularly which is why cell disruption is required as the first step in the downstream process. For that purpose high pressure homogenization is the system of choice since it is scalable and can be run in continuous mode. However, it is crucial to determine cell disruption efficiency to: Avoid product loss in intact cells, but also to avoid unnecessary long disruption cycles and thus harm the product. Usually, cell disruption efficiency is evaluated either by determination of colony forming units (CFUs) or photometric measurements of nucleic acids and protein content in the lysate. However, these methods are both characterized by disadvantages, as CFUs can only be counted on the next day, resulting in great time delay, and photometric measurements are affected by matrix effects. In this study, we implemented a novel online tool based on UV chromatogram fingerprints and chemometric techniques to monitor cell disruption efficiency. We used: 1) Measurement of the total protein content in the supernatant, 2) determination of CFUs and 3) flow cytometry as reference analytics to validate this novel tool. Finally, we performed a design of experiments study, where we changed the factors concentration of biomass per ml buffer, number of homogenization cycles and pressure during homogenization to analyze and optimize the unit operation high pressure homogenization for a recombinant E. coli strain producing a highly valuable antibody fragment. Summarizing, we could nicely demonstrate the power of the novel online tool, which will certainly facilitate the evaluation of this crucial unit operation in the future.

Biography:

Britta Eggenreich has finished her studies of Pharmaceutical Science at the University of Vienna in the year 2011. She has worked for two years as a Pharmacist in Vienna. In 2014, she started her Doctoral thesis in the group of Biochemical Engineering at Vienna University of Technology. Currently, her main focus is the downstream development for inclusion bodies of a novel antibody fragment produced in E. coli.

Abstract:

The bacterium Escherichia coli, is a well-studied recombinant host organism with a plethora of applications in biotechnology. High valuable biopharmaceuticals, such as recombinant enzymes, antibody fragments and growth factors, are currently being produced in E. coli. These molecules are usually produced intra-cellularly which is why cell disruption is required as the first step in the downstream process. For that purpose high pressure homogenization is the system of choice since it is scalable and can be run in continuous mode. However, it is crucial to determine cell disruption efficiency to: Avoid product loss in intact cells, but also to avoid unnecessary long disruption cycles and thus harm the product. Usually, cell disruption efficiency is evaluated either by determination of colony forming units (CFUs) or photometric measurements of nucleic acids and protein content in the lysate. However, these methods are both characterized by disadvantages, as CFUs can only be counted on the next day, resulting in great time delay, and photometric measurements are affected by matrix effects. In this study, we implemented a novel online tool based on UV chromatogram fingerprints and chemometric techniques to monitor cell disruption efficiency. We used: 1) Measurement of the total protein content in the supernatant, 2) determination of CFUs and 3) flow cytometry as reference analytics to validate this novel tool. Finally, we performed a design of experiments study, where we changed the factors concentration of biomass per ml buffer, number of homogenization cycles and pressure during homogenization to analyze and optimize the unit operation high pressure homogenization for a recombinant E. coli strain producing a highly valuable antibody fragment. Summarizing, we could nicely demonstrate the power of the novel online tool, which will certainly facilitate the evaluation of this crucial unit operation in the future.

Biography:

Filip Cuyckens is a Scientific Director & Fellow at Janssen R&D in Beerse, Belgium. He is responsible for Analytical Sciences in the Pharmacokinetics, Dynamics & Metabolism (PDM) department. Analytical Sciences PDM consists of Biotransformations, focusing on metabolite profiling and identification of discovery to late development compounds, and Discovery & Exploratory Bioanalysis, focusing on quantification of drug candidates, metabolites and biomarkers in biological matrices. Filip earned a pharmacist degree in 1998, a degree in industrial pharmacy in 2002 and a Ph.D. in pharmaceutical sciences in 2003.He has (co‑)authored more than 50 publications, is a member of the associate editorial board of Rapid Communications in Mass Spectrometry and board member of the Belgian Society for Mass Spectrometry.

Abstract:

Quantification of metabolites in the absence of an authentic standard is a challenging task. Metabolites are usually present in relatively low concentrations residing in a large background of endogenous compounds and their MS response factor can significantly differ from the parent molecule. The use of a radiotracer overcomes these challenges and, therefore, remains the method of choice for quantification of metabolites in complex matrices, but is not always available or cannot always be applied. Typically samples from first-in-human studies are not radioactive but still extremely valuable giving a first insight in human metabolism. Therefore, estimation of metabolite abundance in these samples is important and also recommended by regulatory guidelines (ICH M3).

An overview will be given of different established and novel approaches for the quantification of metabolites in in vitro and in vivo matrices in the absence of authentic standards. The following techniques will be discussed: radioactive detection¹, Accelerator Mass Spectrometry (AMS), UV detection, Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)^2-4 and Electrospray Ionization-Mass Spectrometry (ESI-MS) using matrix mixing⁵ or a ¹²C/¹⁴C isotope ratio approach⁶.

Depending on the circumstances (sample volume, sample matrix, compound structure, question to be answered, availability of a radiolabel, etc.) the right tool or combination of tools need to be selected since none of these techniques should be seen as the standard technique that suits all measurements. 

Biography:

Dr. Ip received his PhD degree in the Department of Biochemistry, The Hong Kong University of Science and Technology.  Currently, he is working as a Research Fellow in the School of Chinese Medicine, Faculty of Medicine, The Chinese University of Hong Kong.  He is also appointed as a member of Examination Committee, The Chinese Medicine Council of Hong Kong and a Technical Assessor of Hong Kong Accreditation Service. Dr. Ip has extensive research experience on herbal medicines. He has published about 100 papers in reputed journals.

Abstract:

Herbal medicines have used for thousands of years and they are recognized as safe and effective remedies for the prevention and treatment of diseases.  However, the herbal materials are usually obtained from natural sources and they are often criticized for their lack of quality control.  The quality of herbal drugs can be affected by many factors including source, geographic location, growing conditions, harvesting season, and processing procedures.   Therefore, it is necessary to develop regulatory standards to control the quality of herbal medicines.  The Hong Kong Chinese Materia Medica Standards (HKCMMS) project was launched in 2002 by Department of Health, HKSAR Government to develop standards for commonly used herbal drugs to ensure the safe use and the quality of the drugs.  Now, it has become widely recognized reference standards for Chinese medicine traders, laboratories in the field of Chinese medicine testing and certification, and some overseas organisations.   The HKCMMS project is under the guidance of the International Advisory Board which consists of international renowned experts.  The research works for the development of chromatographic methods are conducted by research teams from seven local and overseas research institutes and the National Institutes for Food and Drug Control, China.  Our research team had participated in the HKCMMS project since its beginning and contributed significantly to the successful implementation of the project.  Up to now, 236 monographs were published in 7 volumes of HKCMMS.  Among of them, the research works of 47 monographs were totally contributed by our research team. 

Biography:

Dong Wang is a PhD candidate in China Agricultural University, majoring in food biotechnology. She studied in College of Food Science and Nutritional Engineering of China Agricultural University from the year of 2007 until now. As the leader of the research group, she has studied raisins flavor, amino acid, sensory evaluation, browning mechanism and quality characteristic of raisins for 6 years and published 3 papers in reputed journals.

Abstract:

Raisins are a welcome dry fruit all around the world. They are either consumed directly or are used in cooking especially in desserts. The aroma of raisins affects consumers’ preferences. The aroma of raisin is determined by grape variety, drying method and storage environment. Over 100 volatile compounds were identified in different raisins by using gas chromatography-mass spectrometry (GC-MS) coupled with headspace solid-phase micro-extractions (SPME), the majority of raisin volatiles are derived from three sources: fresh grapes, the oxidative degradation of unsaturated fatty acids and the Millard reaction process. Most of the volatiles were existed in fresh grapes, while the furans (5-methyl furfural, 2-acetyl furan 2-pentyl furan) and pyrazines (2-ethyl-6-methyl pyrazine, 2, 6-diethyl pyrazine and 5-ethyl-2,3-dimethyl pyrazin) generated by Maillard reaction during grape drying, they contribute to characteristic “raisin” flavor, and determine the aroma characteristics of raisins. This program will study the evolutionary pattern of these volatiles responsible for the characteristic “raisin” flavor and their precusors (amino acids and monosaccharides) during grape drying by integrating GC-MS, HP-LC, model reaction and metabonomics technology. Then illustrate the generation source and reaction mechanism of the characteristic volatiles and dissect the influence of drying methods, storage packaging form and environment conditions on these volatiles. The results will provide strong academic supports and practical technology for manipulation of the flavor of raisins.

Biography:

Katsuhiro Maeda has received his BS (1993), MS (1995) and PhD (1998) degrees from Nagoya University. In 1998, he joined the Graduate School of Molecular Design and Engineering, Nagoya University, as an Assistant Professor and was promoted to an Associate Professor in 2002. He moved to Kanazawa University in 2008 and was appointed as a Full Professor in 2015. He has published more than 80 original papers in various reputed journals. 

Abstract:

Separation of enantiomers by high-performance liquid chromatography (HPLC) is an effective method both for analyzing enantiomer composition of chiral compounds and obtaining pure enantiomers. Although a large number of chiral stationary phases (CSPs) for HPLC have been developed, it is still a challenging issue to switch the elution order of enantiomers under identical chromatographic conditions. Recently, we have found that a polyacetylene derivative bearing 2,2’-biphenol-derived pendants can form a preferable helical conformation in response to the chirality of non-racemic guest compounds, such as 1-phenylethanol (PEA), in the solid state as well as in solution, and the induced preferred-handed macromolecular helicity can be maintained, that is memorized, even after complete removal of the chiral guests. By taking advantage of this unique feature, we have succeeded in developing an unprecedented switchable CSP for HPLC, in which the elution order of the enantiomers can be switched, which will be based on reversible switching and subsequent memory of the macromolecular helicity by the treatment with (R)- and (S)-PEA in the solid state. In order to improve the chiral recognition ability, we synthesized analogous polyacetylene derivatives with ester or carbamate groups as the effective interaction sites and investigated a relationship between the structures of the pendants and the recognition abilities of the polymers. Repetitive switching of the elution order of enantiomers based on the switching of the macromolecular helicity was achieved by immobilizing these polymers onto silica support.

Biography:

Michael Soll has completed his PhD in Biology in 1993 at RUB, Germany. Since more than 20 years, he is working in business development, marketing and sales of GC- and LC-MS based laboratory equipment.  Since 2014 he is representing Frontier Laboratories Japan in Europe as business development manager.

Abstract:

The promise of converting various biomass feed stocks directly to biofuels or value-added specialty chemicals using catalytic pyrolysis has precipitated a demand for a fast, reliable method to characterize newly developed catalysts. Catalytic pyrolysis is a three-step process: (1) the feedstock is first pyrolyzed (which is often referred to as “fast pyrolysis”), (2) the pyrolyzates flow through a catalyst bed and (3) the ‘products’ are identified and quantitated. The tandem micro-reactor GC/MS system integrates these three processes into a single bench-top instrument. The tandem catalytic reactor is designed for the rapid evaluation and characterization of catalysts in various atmospheres, at different temperatures and pressures. Three modes of operation allow different experiments to be conducted on the same equipment. The GC/MS can operate in an on-line MS mode for continuous analysis of gases from the catalyst bed. Alternatively, the GC/MS can operate in several integrated high resolution GC/MS modes for step-wise experiments on gases exiting the catalyst bed.  There is also a flash vaporization mode used for the pyrolysis of a solid sample. The system consists of an upper micro-furnace and lower micro-reactor each with independent temperature and reaction gas controls. The micro-furnace can accept solid or viscous liquid samples in a batch sampling mode with catalysis occurring under different conditions in the micro-reactor. The micro-reactor is designed to allow a quick change of the catalyst bed. Batch or continuous experiments can be performed with this system to evaluate both catalyst performance and to characterize catalysis products. Each capability outlined above will be illustrated using ethanol, wood flower and a variety of catalysts. By adding a newly developed Medium Pressure Flow Controller, the influence of reaction pressure against the rate of glycerine conversion using a palladium catalyst is also demonstrated.

Biography:

Vignesh Rajamanickam procured his Master of Science in pharmaceutical biotechnology from Hamburg University of applied sciences, Germany and, Bachelor of Technology in Biotechnology from Anna University, India. He started his PhD on March 2014 in Biochemical engineering from Vienna University of Technology (VUT), Austria. Currently, he is working as a project assistant for developing a novel PAT tool for bioprocess monitoring and control in Christian Doppler laboratory for mechanistic and physiological methods for improved bioprocesses, VUT, Austria.

Abstract:

The bacterium Escherichia coli is a well-studied recombinant host organism with a plethora of applications in biotechnology. High valuable biopharmaceuticals, such as antibody fragments and growth factors, are currently being produced in E. coli. However, the high metabolic burden during recombinant protein production can lead to cell death, consequent lysis and undesired product loss. Thus, fast and precise analyzers to monitor E. coli bioprocesses and to retrieve key process information, such as the optimal time point of harvest, are needed. However, such reliable monitoring tools are still scarce to date. In this study, we cultivated a recombinant E. coli strain producing a recombinant single chain antibody fragment (scFv) in the cytoplasm. In bioreactor cultivations we purposely triggered cell lysis by pH ramps. We developed a novel toolbox using UV chromatogram fingerprints and chemometric techniques to monitor these lysis events and used flow cytometry (FCM) as reference method to quantify viability offline. Furthermore, we tested the applicability of the novel toolbox for montiroing other E. coli bioprocesses. We are convinced that this toolbox will not only facilitate E. coli bioprocess monitoring, but will also allow enhanced process control in the future.