Measurement Technologies for up- and Downstream Bioprocessing

This book is devoted to new developments in measurement technologies for upstream and downstream bioprocessing. The recent advances in biotechnology and bioprocessing have generated a number of new biological products that require more qualified analytical technologies for diverse process analytical needs. These includes especially fast and sensitive measurement technology that, early in the process train, can inform on critical process parameters related to process economy and product quality and that can facilitate ambitions of designing efficient integrated end-to-end bioprocesses. This book covers these topics as well as analytical monitoring methods based either on real-time or in-line sensor technology, on simple and compact bioanalytical devices, or on the use of advanced data prediction methods

Application of metabolomic profiling and fingerprinting approaches to food fraud cases

[eng] Food fraud is an intentional and misleading act in food that generally does not comply with food law and is motivated by economic gain. It encompasses several fraudulent practices such as deception during manufacture, diversion into illicit supply chains, interventions with the food product, or misrepresentation. In this context, the coming to light of the horse meat scandal at the beginning of 2013 highlighted the shortcomings of the European system against food fraud, increasing concern and interest among European citizens and administrative bodies. Under these circumstances, in recent years, omics tools —comprising genomics, transcriptomics, proteomics, metabolomics, and elementomics/isotopollomics— have been applied to solve food fraud issues, along with biostatistics and chemometrics. In most cases, their application has relied on profiling (focusing on determining targeted secondary chemical markers) or fingerprinting approaches (based on the unspecific detection of instrumental responses without assuming any previous knowledge about the sample composition), overcoming the traditional targeted analysis. In particular, since a food product’s metabolome varies according to its biological nature and several external conditions (i.e., either from a natural or anthropogenic origin), metabolomics has shown excellent potential to assess several issues related to its authenticity and quality. Therefore, in this thesis, several metabolomic profiling and fingerprinting approaches were developed to address different food fraud cases. In this line, liquid chromatography coupled to low- or high-resolution mass spectrometry (LC–LRMS, LC–HRMS) was proposed for the targeted approaches. In contrast, non-targeted methods were based on liquid chromatography with ultraviolet detection (LC-UV) or fluorescence detection (LC-FLD), LC–HRMS, or direct mass spectrometry (MS)-based techniques. Furthermore, non-supervised and supervised chemometric techniques allowed sample assignation and classification. As a result, the proposed analytical methodologies were successfully applied to several food products —including paprika, nuts and seeds, hen eggs, vegetable oils, and red wine— guaranteeing their classification and authentication regarding the geographical origin, botanical origin, production system, or quality category.[cat] El frau alimentari és un acte intencionat i enganyós produït en els aliments que, generalment, no compleix amb la legislació alimentària i que està motivat per un benefici econòmic. La sortida a la llum de l’escàndol de la carn de cavall a principis del 2013 va posar de manifest les mancances del sistema europeu contra el frau alimentari, augmentant la preocupació i l’interès entre els ciutadans i els organismes administratius europeus. En aquestes circumstàncies, en els darrers anys, s’han aplicat eines òmiques —que inclouen la genòmica, la transcriptòmica, la proteòmica, la metabolòmica i l’elementòmica/isotopol·lòmica— per resoldre qüestions relacionades amb el frau alimentari, juntament amb bioestadística i quimiometria. En la majoria dels casos, la seva aplicació s’ha efectuat mitjançant estratègies basades en perfils (centrant-se en la determinació dirigida de marcadors químics secundaris) o empremtes dactilars (basades en la detecció inespecífica de respostes instrumental sense assumir cap coneixement previ sobre la composició de la mostra), superant l’anàlisi dirigida tradicional. En concret, com que el metaboloma d’un producte alimentari varia segons la seva naturalesa biològica i un seguit de condicions externes (siguin d’origen natural o antropogènic), la metabolòmica ha demostrat un excel·lent potencial per avaluar diverses qüestions relacionades amb la seva autenticitat i qualitat. En aquesta tesi, es van desenvolupar diverses estratègies de perfils i empremtes dactilars metabolòmiques per abordar alguns casos de frau alimentari. Així, es va proposar la cromatografia líquida acoblada a l’espectrometria de masses de baixa o alta resolució (LC–LRMS, LC–HRMS) per als enfocaments dirigits. En canvi, els mètodes no dirigits es van basar en la cromatografia líquida amb detecció ultraviolada (LC-UV) o fluorescent (LC-FLD), LC–HRMS o tècniques basades en l’espectrometria de masses (MS) directa. A més, tècniques quimiomètriques no supervisades i supervisades van permetre l’assignació i classificació de les mostres. Com a resultat, les metodologies analítiques proposades es van aplicar amb èxit a diferents productes alimentaris —incloent el pebre vermell, fruits secs i llavors, ous de gallina, olis vegetals i vi negre— garantint-ne la classificació i autenticació pel que fa a l’origen geogràfic, l’origen botànic, el sistema de producció o la categoria de qualitat

Biomarker der Inflammation

Ziel dieser Arbeit war die Standardisierung der in der Labordiagnostik der inflammatorischen Reaktion angewandten Methoden sowie die Identifikation neuer Biomarker. In einem ersten klinisch orientierten Teil wurden Vergleichsstudien verschiedener Hämatologie-Analysensysteme als Basissysteme für die Darstellung einer inflammatorischen Reaktion durchgeführt. Aufgrund der bisher nur sehr eingeschränkten Verfügbarkeit entsprechender Vergleichsdaten ist die im Rahmen dieser Arbeit gefundene variable Qualität der verschiedenen Systeme für die Entwicklung gerätespezifischer Algorithmen im Rahmen der klinischen Diagnostik von großer Bedeutung. In einem zweiten grundlagenorientierten Teil konnte ein potentieller Stellenwert von Markern des AA-Metabolismus in der diagnostischen Einordnung inflammatorischer Erkrankungen gezeigt werden. Fortführend wurde eine optimierte LC-MS/MS basierte Analysemethode für die Bestimmung von Eicosanoiden aus biologischen Flüssigkeiten entwickelt, die eine Prüfung des diagnostischen Stellenwertes in klinischen Studien mit größeren Patientenzahlen ermöglicht

Clinical Platelet Lipidomics in Targeted and Untargeted Approach by Liquid Chromatography Coupled to Mass Spectrometry

Platelets are small cellular components of blood with a primary role in hemostasis which in contrary are also responsible for a pathological condition called thrombosis that might results in cardiovascular disease (CAD) such as heart attack and stroke. During the hemostasis, lipids play important roles, especially the fatty acids and their derivatives such as oxylipins which are involved in platelets activation. Therefore, the analysis of platelets lipidomics is particularly interesting and the platelet lipidomic landscape might be considered as a powerful tool for diagnostic and prognostic biomarkers for CAD. The thesis is mainly divided into two parts. Part I involved in liquid chromatography and mass spectrometry (LC-MS) based analytical method developments including 1) the optimization of sample preparation procedure for large-scale clinical lipidomics, 2) the method development for targeted analysis of 3-OH-FAs in biological samples human plasma and platelets, 3) the method development for profiling of branched chain and straight chain saturated fatty acids in different types of biological samples including human plasma, platelets and Staphylococcus aureus, 4) the method development for targeted analysis of oxylipins with microLC coupled with MS and 5) the method development for chiral separation of oxylipins. Part II involved in the application of the developed methods to clinical lipidomics study of CAD patients including 1) investigation of the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome by targeted and untargeted lipidomics analysis, 2) investigation of the platelet lipidome by untargeted approach to highlight the significant changes between acute coronary syndrome (ACS) and chronic coronary syndrome (CCS) patients, 3) investigation of the platelet lipidome of CAD patients by untargeted lipidomics and highlighting significant changes between statin-treated and naïve patients. As a result, an advanced monophasic extraction protocol with methanol/methyl tert-butylether/ isopropanol, MeOH/MTBE/IPA (1.3:1:1, v/v/v) as extraction solvents, bead homogenizer for cell disruption and MeOH/MTBE (1:1, v/v) as reconstitution solvent which provides optimal cellular and subcellular extraction efficiencies for both polar (e.g. acylcarnitines) and apolar lipids (e.g. triglycerides TGs) and several targeted LC-MS methods with selected reaction monitoring (SRM) mode for fatty acids and their derivatives (3-OH-FAs and oxylipins) were reported. Further, the developed methods were successfully applied for the clinical platelets lipidomics studies and some conclusions were made: 1) platelet ACKR3/CXCR7 favors antiplatelet lipids over an atherothrombotic lipidome and regulates thromboinflammation which may offer a novel therapeutic strategy in CAD, 2) Lipids alteration was observed between acute coronary syndrome (ACS) and chronic coronary syndrome (CCS) patients and between CAD patients treated with statin and statin naïve patients

Biological availability and efficacy of folates from bioengineered rice

The basic concept of folate rice is to develop a means for rural areas to combat folate deficiency. Folate deficiency is of concern given the relation between folate status and birth defects, ranging in severity from mild back ache to stillbirth, due to a malformation of the neural tube. While western populations benefit from diverse and nutrient rich diets, even these populations are at risk for folate deficiency. One method to alleviate the health burden associated with folate deficiency is to add synthetic folic acid to commonly consumed food items such as breakfast cereals or bread. However, remote, mostly rural communities, do not have free access to folic acid fortification. As such, a rice variety with a high natural folate content that could be grown locally may serve to achieve an adequate folate status. However, since folate rice is a genetically engineered rice variety, public and governmental acceptance depends on scientific evidence that the folates contained within these rice grains are released into the bloodstream when consumed. This is because some genetically engineered food items have been criticized as a result of their limited influence on nutrient status. It is therefore of paramount importance to evaluate the impact of folate rice consumption on folate status. Given the need for strict control of nutrient intake and regulatory constraints, a rodent feeding trial was devised to evaluate the efficacy of folate rice as a dietary folate source. Due to the complexity of folate metabolism and body distribution, a long term study was performed including regular evaluation of folate status, folate related clinical parameters and general health. These results were compared between groups receiving no folate, a small amount of folate present in ‘normal’ rice, a larger amount of folate in either folate rice or folic acid fortified rice or ample folate as an optimal scenario. To quantify the biological outcome of folate rice consumption, an analytical method was developed to measure the amount of folates in 2 separate blood fractions, i.e. plasma and red blood cells. Given the low concentrations in which folates are present, a sensitive method using liquid chromatography coupled to tandem mass spectrometry was used. Since the term folate signifies several different molecules with different chemical and biological properties, the developed method required the measurement of individual folate species. Also, to exclude the influence of folates which might be present in the rodent diets used during the rodent trial, a separate method was developed to determine the folate concentration in the rodent diets used. Due to the necessity of folates for DNA-synthesis in white blood cells, the influence of folate concentration and speciation on the functioning of the immune system was investigated as well

High-Throughput Process Development in the Field of Protein Purification - Method Development, Application, and Characterization

High-throughput methods were developed for the quantification of mAb aggregate/monomer ratios, and the analysis of content, purity, and activity of the egg white protein avidin. Furthermore, by using high-throughput screenings, a new avidin purification process was developed using precipitation, aqueous two-phase extraction and mixed-mode chromatography. Finally, method-specific effects present in high-throughput column chromatography were evaluated using both experimental and simulation data