BIOPHOTONICS IN BIOPHARMACEUTICAL APPLICATIONS

Biopharmaceutical products are composed of complex or ordinate combinations of proteins, lipids, sugars, and nucleic acids or living cells or tissues. Due to the intrinsic variability of biological systems and the complexity of the bioprocesses involved in the production of these products, new technologies are required to monitor and characterize, the production processes and the final products. Biophotonic techniques, particularly Raman and Infrared (IR) spectroscopy are rapid, robust, operator independent, non-destructive and label free, thus particularly suitable for these purposes. This dissertation first investigates the use of biophotonic techniques in research of Extracellular Vesicles (EVs). EVs act as intercellular messengers and therefore have considerable potential in drug delivery system, diagnostic biomarker, or therapeutic agent. Subsequently highlighting the need and potential of a new kind of time gated Raman spectrometer to be created. Raman and IR spectroscopy are methods able to characterize and assess the quality of EV suspensions with different degree of purity. These vibrational spectroscopic techniques show different intrinsic advantages, as they are label free and operator independent methods. Particularly, Raman might be the most suitable technology since it is less sensitive to water compared to IR. Raman spectroscopy reveals the information on the chemical composition and physical status of the analyte. However, it does not provide information of the analyte environment which the spectrofluorometer can gather. EVs studied by spectrofluorometer are required to be labelled with a fluorescent dye. Results obtained by fluorescence lifetime imaging spectroscopy underline that the cell uptake of the fluorescently labelled EVs is feasible. Attention must be paid to the efficacy of the labelling and further to the elimination of the unbound dye since the labelling may severely compromise the results or lead to wrong conclusions on EV functionality. The combined advantage of Raman spectroscopy and fluorescence decay are obtained by a previously in-house developed time resolved Raman spectrometer. Thanks to the peculiar sensor of the spectrometer, the width of the time gate can be modified which is used to separate the Raman signal from the fluorescence tail. The modifications can be done even in the data post-processing phase, to obtain the best possible Raman signal-to-noise ratios. The simultaneously detected Raman spectra and time-resolved fluorescence decay curves are used to study the diffusion of small molecular drugs in a hydrogel. The data reveal the chemical composition, physical status, and the interaction with the environment of the samples. Taken together the obtained results suggest that the quality of EV suspensions can be assessed by Raman spectroscopy and their cell uptake detected by fluorescence lifetime spectroscopy. Both Raman spectra and fluorescence decay can be measured simultaneously by a second generation of time-resolved Raman spectrometer.Biofarmaseuttiset tuotteet koostuvat proteiinien, lipidien, sokereiden ja nukleiinihappojen tai elävien solujen tai kudosten järjestäytyneistä tai monimutkaisista yhdistelmistä. Biologisten järjestelmien luontaisen vaihtelevuuden ja näiden tuotteiden valmistukseen liittyvien bioprosessien monimutkaisuuden vuoksi tarvitaan uusia teknologioita tuotantoprosessien ja lopputuotteiden seurantaa ja karakterisointia varten. Biofotoniset tekniikat, erityisesti Raman- ja Infrapuna (IR) -spektroskopia ovat nopeita, luotettavia, hellävaraisia, leimattomia ja käyttäjästä riippumattomia tekniikoita, jotka soveltuvat erityisen hyvin biofarmaseuttisten tuotteiden määritykseen. Tämä väitöskirjan alussa tutkitaan biofotonisten tekniikoiden käyttöä solunulkoisten vesikkeleiden (EV:iden) tutkimuksessa. EV:t toimivat solujenvälisessä viestinnässä ja siksi niillä on huomattava potentiaali lääkekehityksessä, diagnostisina biomarkkerina ja terapeuttisina tekijöinä. Myöhemmin tutkimuksessa korostetaan uudenlaisen aikaerotteisen Raman-spektrometrin tarvetta ja mahdollisuuksia biofarmaseuttisten tuotteiden määrityksessä. Raman- ja IR-spektroskopia ovat menetelmiä, joilla voidaan karakterisoida ja arvioida eri puhtausasteen omaavia EV-näytteiden laatua. Nämä värähtelyspektroskooppiset tekniikat ovat leimattomia ja käyttäjästä riippumattomia menetelmiä. Verrattuna IR-spektroskopiaan, Raman on luonteeltaan mahdollisesti käyttökelpoisin tekniikka EV:iden karakterisointiin, sillä veden läsnäolo näytteessä ei häiritse mittausta. Raman-spektroskopian avulla saadaan tietoa analyytin kemiallisesta koostumuksesta sekä fysikaalisesta tilasta ja spektrofluorometrin avulla saadaan kerättyä informaatiota analyytin ympäristöstä, mihin Raman ei puolestaan pysty. Spektrofluorometrillä tutkitut EV:t on leimattava fluoresoivalla väriaineella ja fluoresenssin eliniän kuvantamisspektroskopialla saadut tulokset korostavat, että fluoresenssileimattujen EV:iden soluunotto on mahdollista. Leimauksen tehokkuuteen ja sitoutumattoman väriaineen eliminointiin on kiinnitettävä huomiota, sillä virheellinen leimaus voi väärentää tuloksia tai johtaa virheellisiin johtopäätöksiin EV:iden funktionaalisuudesta. Talon sisällä rakennetun laitteiston avulla on saatu yhdistettyä Raman-spektroskopia ja fluoresenssin eliniän vaimeneminen. Spektrometrin omalaatuisen anturin ansiosta kuvantamisen aikaikkunaa pystytään muokkaamaan, minkä avulla Raman-signaali pystytään erottamaan fluoresenssisignaalista. Muutokset voidaan tehdä myös datan jälkikäsittelyvaiheessa parhaan mahdollisen Raman-signaali-kohinasuhteen saamiseksi. Pienmolekyylisten lääkkeiden diffuusion tutkimiseen hydrogeelissä voidaan käyttää samanaikaisesti havaittuja Raman-spektrejä ja aikaerotteisia fluoresenssin vaimenemiskäyriä. Kerätty aineisto paljastaa näytteiden kemiallisen koostumuksen, fysikaalisen tilan ja vuorovaikutuksen ympäristön kanssa. Yhdessä saadut tulokset viittaavat siihen, että EV-suspensioiden laatua voidaan arvioida Raman-spektroskopialla ja niiden soluunotto havaita fluoresenssielinaikaspektroskopialla, kun Raman-spektri ja fluoresenssin heikkeneminen voidaan mitata samanaikaisesti toisen sukupolven aikaerotteisella Raman-spektrometrillä

The Price of Progress: Funding and Financing Alzheimer\u27s Disease Drug Development

Introduction Advancing research and treatment for Alzheimer\u27s disease (AD) and the search for effective treatments depend on a complex financial ecosystem involving federal, state, industry, advocacy, venture capital, and philanthropy funding approaches. Methods We conducted an expert review of the literature pertaining to funding and financing of translational research and drug development for AD. Results The federal government is the largest public funder of research in AD. The National Institute on Aging, National Institute of Mental Health, National Institute of General Medical Sciences, and National Center for Advancing Translational Science all fund aspects of research in AD drug development. Non-National Institutes of Health federal funding comes from the National Science Foundation, Veterans Administration, Food and Drug Administration, and the Center for Medicare and Medicaid Services. Academic Medical Centers host much of the federally funded basic science research and are increasingly involved in drug development. Funding of the “Valley of Death” involves philanthropy and federal funding through small business programs and private equity from seed capital, angel investors, and venture capital companies. Advocacy groups fund both basic science and clinical trials. The Alzheimer Association is the advocacy organization with the largest research support portfolio relevant to AD drug development. Pharmaceutical companies are the largest supporters of biomedical research worldwide; companies are most interested in late stage de-risked drugs. Drugs progressing into phase II and III are candidates for pharmaceutical industry support through licensing, mergers and acquisitions, and co-development collaborations. Discussion Together, the funding and financing entities involved in supporting AD drug development comprise a complex, interactive, dynamic financial ecosystem. Funding source interaction is largely unstructured and available funding is insufficient to meet all demands for new therapies. Novel approaches to funding such as mega-funds have been proposed and more integration of component parts would assist in accelerating drug development

Opportunities for Improving the Drug Development Process: Results from a Survey of Industry and the FDA

In the United States, the Food and Drug Administration (FDA) agency is responsible for regulating the safety and efficacy of biopharmaceutical drug products. Furthermore, the FDA is tasked with speeding new medical innovations to market. These two missions create an inherent tension within the agency and between the agency and key stakeholders. Oftentimes, communications and interactions between regulated companies and the FDA suffer. The focus of this research is on the interactions between the FDA and the biopharmaceutical companies that perform drug R&D. To assess the current issues and state of communication and interaction between the FDA and industry, we carried out a survey of industry leadership in R&D and regulatory positions as well as senior leadership at the FDA who have responsibility for drug evaluation and oversight. Based on forty-nine industry and eight FDA interviews we conducted, we found that industry seeks additional structured and informal interactions with the FDA, especially during Phase II of development. Overall, industry placed greater value on additional communication than did the FDA. Furthermore, industry interviewees indicated that they were willing to pay PDUFA-like fees during clinical development to ensure that the FDA could hire additional, well-qualified staff to assist with protocol reviews and decision-making. Based on our survey and discussions, we uncovered several thematic opportunities to improve interactions between the FDA and industry and to reduce clinical development times: 1) develop metrics and goals at the FDA for clinical development times in exchange for PDUFA like fees; 2) establish an oversight board consisting of industry, agency officials, and premier external scientists (possibly at NIH or CDC) to evaluate and audit retrospectively completed and terminated drug projects; and 3) construct a knowledge database that can simultaneously protect proprietary data while allowing sponsor companies to understand safety issues and problems of previously developed/failed drug programs. While profound scientific and medical challenges face the FDA and industry, the first step to reducing development times and associated costs and facilitating innovation is to provide an efficient regulatory process that reduces unnecessary uncertainty and delays due to lack of communication and interaction.

KINETIC MODELING AND ITS APPLICATION IN THE BIOPHARMACEUTICAL INDUSTRY

It has been well recognized that biologics are efficient for cancer and immune disease treatment. Kinetic modeling is to mathematically model or to quantitatively illustrate how reactions occur in a biological or chemical process. A systematic study and understanding of kinetic model and its application in the biopharmaceutical industry are important for both scientific research and industrial technology development. This work consists of six chapters. First, a review of kinetic modeling and its application for cell culture was introduced. Second, the current status of biologics development and screening strategies of biomarkers and indications were discussed. Third, one experimental and kinetic modeling study for the temperature effects and temperature shift strategy development was presented. Forth, novel kinetic models were built up and applied to elucidate lactate dehydrogenase catalyzed reactions, which is a crucial metabolic process within tumor cells and Chinese hamster ovary cells. In the fifth chapter, strategies of biologics quality control via process development were briefly summarized. And finally, summary and outlook were made based on the above five chapters. Though kinetic modeling is not a FDA request tool, kinetic data are required for regulation approval of new drug discovery and process development. These data are applicable for a rapidly screening of the best biologics and the optimized manufacturing process with little extra cost via kinetic modeling. This work is potentially beneficial for speeding up and better understanding of the current biologics development in biopharmaceutical industry

Repairing the 'Broken Middle' of the Health Innovation Pathway:Exploring Diverse Practitioner Perspectives on the Emergence and Role of ‘Translational Medicine’

The emergence of Translational Medicine (TM) as a potential solution to health innovation challenges has gained currency in scientific, clinical and policy discourses. Using interview data from key professionals involved in TM, this article explores diverse practitioner definitions and the multiple meanings ascribed to TM in the context of a purportedly broken R&D system and promissory visions and expectations built around new life science. It also begins to address some of the transformative impacts of TM on the broader institutional landscape for life science innovation, particularly the changes in traditional institutional boundaries. I conclude that in light of the multiple framings of TM, it might best be conceived as an institutional mechanism or process for co-ordinating multiple actors and complex activities in the new collaborative research and development contexts now demanded of the life sciences

Novel Mass Spectrometry Methods for the Analysis of Covalent and Non-Covalent Protein Structures and Their Influence on the Functions of Therapeutic Proteins

Biotherapeutics consist of biopolymers (proteins, polysaccharides, DNA, and RNA) that are used to treat a wide range of conditions from cancer to autoimmune disease to enzyme replacement. In recent years biotherapeutics have experience tremendous growth due to advances in technology and our understanding of human biology. They are very important to modern medicine due to their ability to treat diseases which are unable to be treated with small molecule-based drugs. Unlike small molecule drugs which are synthetically produced, biotherapeutics are expressed inside cells. Produced biotherapeutics are not made up of a single homogenous population but instead a population of highly similar variants. The source of these variations are enzymatic and non-enzymatic post-translational modifications. By characterizing these modification, a profile is built that links the in vivo response of a drug to its modifications. The complexity and size of these biopolymers makes their characterization very challenging and demands the development of robust analytical techniques. Mass spectrometry- and liquid chromatography-base methods are an integral part of protein characterization. Mass spectrometry provides accurate mass measurements that are invaluable for confirming the identity of a protein and any modifications. Additionally, mass spectrometry is used to assess a protein’s higher order structure. Liquid chromatography is a very powerful tool that allows for different post-translational modified populations of a biotherapeutic sample to separate by their chemical or physical properties. Separated populations can further be characterized to identify and analyze their chemical or structural composition. The presented work utilized a blend of mass spectrometry and liquid chromatography methods to characterize proteins with biotherapeutic potential

Outlook Magazine, Winter 2018

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