Data analytics and soft sensors for single use bioprocessing

In the last decades, innovative research and engineering brought birth to a plethora of robust and mature process analytical devices. Nowadays, the possibilities to extract chemical-, physical- and biological data from single use processes are manifold and a great quantity of process data is collected on a routine basis. However, novel challenges in the field of data processing and information mining emerged: How can the maximum information content be extracted from the combination of process analyzers? How can big process data be handled and exploited efficiently? And ultimately: How can this information be translated in a business benefit for the manufacturers? Here, we demonstrate how these challenges can be addressed within the bioprocess lifecycle using innovative mechanistic methods. We present i) novel non-invasive soft sensors for real-time monitoring of single use processes, ii) information mining and process analysis based on the combination of mechanistic models and statistical tools and iii) efficient and scalable process control strategies. Financial support was provided by the Austrian research funding association (FFG) under the scope of the COMET program within the research network “Process Analytical Chemistry (PAC)” (contract # 825340). This programme is promoted by BMVIT, BMWFJ and the federal state of Upper Austria

Adaption durch Hybridisierung

Fortschreitender Klimawandel hat in den vergangenen Jahrzehnten zu einem drastischen Rückgang dauerhaft vergletscherter Gebiete geführt, sowohl an den polaren Eisschilden, als auch in den Hochgebirgen auf der ganzen Welt und eröffnet dadurch neue Gebiete, die von verschiedenen Organismen besiedelt werden. Der Rückzug von Gletschern bietet neuartige Lebensräume für die Besiedlung von Pionierpflanzen. Jenen Evolutionsprozessen, ausgelöst durch klimawandelbedingte Kolonisierungsdynamik und Sukzessionsprozesse, wurde bislang wenig Beachtung geschenkt. Tieflandarten werden demnach schneller in höhere Lagen verdrängt, als hochalpine Arten sich zurückziehen. Wenn keine postzygotischen Kreuzungsbarrieren vorhanden sind, können solche Kontakte von normalerweise ökologisch getrennten und vor allem in ihrer Höhenstufenpräferenz verschiedenen Arten zur Bildung von Hybriden führen, die sich leicht in einem neuen Habitat etablieren können. Diese Arbeit beschäftigt sich mit der Hybridisierung zwischen zwei Weidenarten (Salix helvetica und S. purpurea) am Vorfeld des Rhônegletschers (Schweiz). Chemische und ökologische Parameter wurden durch Bodenanalysen und durch eine Erhebung der Begleitvegetation bewertet. Darüber hinaus wurde unter Verwendung von Schöner’s D ein Modell zur Berechnung der Überlappung der ökologischen Nischen zwischen den Elternarten und den mutmaßlichen Hybriden erstellt. Die Ergebnisse weisen auf Hybridisierung in erheblichem Maße zwischen den beiden Elternarten hin. Es kommt dabei klar zum Vorschein, dass die Hybriden ihre eigene charakteristische Nische besetzen und dabei eine viel höhere Toleranz gegenüber Kälte, Trockenheit und niedrige pH-Werte im Boden aufweisen, als einer der beiden Eltern. Das legt nahe, dass eine erfolgreiche Etablierung der jungen Hybridpopulation auf längere Sicht zu erwarten ist, vor allem da sie sich an die standörtlichen Bedingungen besser anzupassen scheinen, als ihre Eltern. Diese Selektionsvorteile der Hybriden und die Etablierung von Folgegenerationen bergen daher ein Potential für die Bildung neuer Arten

Plasma lipid profiles discriminate bacterial from viral infection in febrile children

Fever is the most common reason that children present to Emergency Departments. Clinical signs and symptoms suggestive of bacterial infection are often non-specific, and there is no definitive test for the accurate diagnosis of infection. The 'omics' approaches to identifying biomarkers from the host-response to bacterial infection are promising. In this study, lipidomic analysis was carried out with plasma samples obtained from febrile children with confirmed bacterial infection (n = 20) and confirmed viral infection (n = 20). We show for the first time that bacterial and viral infection produces distinct profile in the host lipidome. Some species of glycerophosphoinositol, sphingomyelin, lysophosphatidylcholine and cholesterol sulfate were higher in the confirmed virus infected group, while some species of fatty acids, glycerophosphocholine, glycerophosphoserine, lactosylceramide and bilirubin were lower in the confirmed virus infected group when compared with confirmed bacterial infected group. A combination of three lipids achieved an area under the receiver operating characteristic (ROC) curve of 0.911 (95% CI 0.81 to 0.98). This pilot study demonstrates the potential of metabolic biomarkers to assist clinicians in distinguishing bacterial from viral infection in febrile children, to facilitate effective clinical management and to the limit inappropriate use of antibiotics

Platform-orientierte Prozessentwicklung durch neue kombinatorische Methoden für Biopharmazeutika

Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersBiopharmaceutical products emerged as the principal driver for innovation in the pharmaceutical industry. Although of high economic and social importance, the development of biopharmaceutical manufacturing processes is still driven by time-intensive empirical approaches, delaying the time-to-market of novel drugs and jeopardizing the economic competitiveness of manufacturing processes. A promising approach to speed up process development and to enable robust manufacturing is to build-up process knowledge to form well-characterized biopharmaceutical manufacturing platforms. This enables the efficient development of production processes for a broad spectrum of products while simultaneously drug product quality and economic manufacturing risks are reduced. Within this thesis, the development of a novel and highly versatile E. coli recombinant protein production platform is presented. The built-up of platform process knowledge is achieved using novel combinatory methods which combine cutting edge technologies such as first-principle soft-sensors, dynamic experimentation, mid infrared and dielectric spectroscopy as well multivariate data analysis and kinetic modeling. Major novelties include the presentation of highly automated methods for the extraction of strain specific parameters, information which is essential for science-based bioprocess design. Expression tuning on cellular level is demonstrated using solely process technological means, resulting in a high degree of processing flexibility for the intended pharmaceutical manufacturing platform. Furthermore, for the first time, generic control methods based on soft-sensors are presented, which allow controlling multiple physiological bioprocess parameters simultaneously and therefore result in a more robust manufacturing. This thesis can be considered a case study demonstrating how combinatory methods can be purposefully exploited for the fast development of an efficient bioprocessing platform. The methodological focus of this thesis allows leveraging the developed combinatory methods and the platform bioprocess to other biotechnological manufacturing tasks and will enable the development of more competitive and predictable bioprocesses.26

Quality by design for process understanding- multivariate studies on a recombinant E. coli expression system

Die vorliegende Arbeit zeigt exemplarisch einen "Quality by Design" Ansatz für ein rekombinantes Expressionssystem. Eine multivariate Studie mit den Faktoren Induktionstemperatur, Induktionszeit und Induktions-Fütterungsstrategie wurde an einem rhamnoseinduzierbaren E. coli Expressionssystem (rhaBAD Promoter) zur intrazellulären Produktion von Alkalischer Phosphatase durchgeführt. Die experimentelle Durchführung der notwendigen Fermentationen erfolgte in einem voll instrumentierten 10 Liter Bioreaktor. Als Hilfsmittel für die Versuchsplanung- und Durchführung diente Design of Experiments (DoE).Als Qualitätsattribut wurde die Biomasse spezifische Aktivität (U/g Biomass) definiert. Höchste spezifische Aktivitäten konnten bei einer Induktionstemperatur von 27.5°C und einer positiv exponentiellen Fütterungsstrategie nach 10 Stunden Induktion gefunden werden. Um Verständnis für dieses Verhalten zu erlangen, wurden die aufgenommenen Daten (Biomasse, extrazelluläres Protein, Daten aus Abgasanalyse und Waagensignale) in spezifische Raten und Ausbeuten prozessiert. Diese wiederum wurden mit den Parametern der multivariaten Studie multilinear regressiert. Dieser Ansatz lieferte Prozesswissen über das untersuchte System. Es konnten Hinweise auf eine Down-Regulierung des Expressionssystems durch den Prozessparameter Temperatur gefunden werden. Weiters korrelierte die spezifische Protein Abgabe Rate mit der Fütterungsstrategie. Bei einer höheren Fütterungsstrategie wurde weniger Proteinabgabe detektiert, was sich folglich positiv auf die intrazelluläre Aktivität des Zielproteins auswirkte. Weiters wurde das Medium im Verlauf der Fermentation mit Hilfe von ICP-OES untersucht, wodurch Hinweise auf eine Zinklimitation gefunden wurden.This contribution exemplifies a quality by design (QbD) approach for the characterization of a high density E. coli recombinant upstream process, investigating the multivariate interactions of three critical process parameters (CPPs) in respect to their impact on product quality using statistical experimental design and multivariate modelling.Intracellular production of alkaline phosphatase by means of the little studied positively regulated rhaBAD promoter was chosen as a model system. The multivariate study included the induction phase feeding strategy, expressed as the feed-exponent k (-0.007; 0; +0.007), induction temperature (20°C; 27.5°C; 35°C) as well as induction time (10 to 40 hours of induction) as presumptive critical process parameters (CPPs) under investigation. Based on a sequential approach for upstream design space development, the upstream critical quality attribute (CQA) was defined in respect to downstream demands (specific activity; U/gbiomass). Optimal operating conditions for the process were found at medium temperatures (27.5°C) and at a positive exponential feeding strategy. The result was the establishment of a knowledge space. The physiological impact of the critical process parameters on the culture was further evaluated by multi-linear regression of yields and specific rates with CPPs. This methodology revealed the protein release, expressed as the biomass specific protein release rate, to be correlating with the applied feeding strategy as well as product quality, making it critical for intracellular protein production. Lower induction temperatures increased the biomass yield, possibly due to a down regulation of recombinant protein expression. The biomass yield declined within induction time, indicating a rise in maintenance metabolism. This was found to go along with an increase in the carbon dioxide yield, independent of temperature and feeding strategy. The multivariate study gave evidence that specific activities are starting to decline after ten hours of induction, although SDS gel electrophoresis indicated that alkaline phosphatase levels were still increasing. Zinc, essential for folding and function of alkaline phosphatase (Torriani 1968), was monitored via ICP-OES, which gave evidence for a zinc limitation that explains the observed decrease in activities after 10 hours of induction.10

Tunable recombinant protein expression in E. coli: enabler for continuous processing?

The final publication is available at Springer via https://doi.org/10.1007/s00253-016-7550-4.Tuning of transcription is a powerful process technological tool for efficient recombinant protein production in Escherichia coli. Many challenges such as product toxicity, formation of inclusion bodies, cell death, and metabolic burden are associated with non-suitable (too high or too low) levels of recombinant protein expression. Tunable expression systems allow adjusting the recombinant protein expression using process technological means. This enables to exploit the cell’s metabolic capacities to a maximum. Within this article, we review genetic and process technological aspects of tunable expression systems in E. coli, providing a roadmap for the industrial exploitation of the reviewed technologies. We attempt to differentiate the term “expression tuning” from its inflationary use by providing a concise definition and highlight interesting fields of application for this versatile new technology. Dependent on the type of inducer (metabolizable or non-metabolizable), different process strategies are required in order to achieve tuning. To fully profit from the benefits of tunable systems, an independent control of growth rate and expression rate is indispensable. Being able to tackle problems such as long-term culture stability and constant product quality expression tuning is a promising enabler for continuous processing in biopharmaceutical production

Tunable recombinant protein expression in E. coli: promoter systems and genetic constraints

Tuning of transcription is a promising strategy to overcome challenges associated with a non-suitable expression rate like outgrowth of segregants, inclusion body formation, metabolic burden and inefficient translocation. By adjusting the expression rate—even on line—to purposeful levels higher product titres and more cost-efficient production processes can be achieved by enabling culture long-term stability and constant product quality. Some tunable systems are registered for patents or already commercially available. Within this contribution, we discuss the induction mechanisms of various Escherichia coli inherent promoter systems with respect to their tunability and review studies using these systems for expression tuning. According to the current level of knowledge, some promoter systems were successfully used for expression tuning, and in some cases, analytical evidence on single-cell level is still pending. However, only a few studies using tunable strains apply a suitable process control strategy. So far, expression tuning has only gathered little attention, but we anticipate that expression tuning harbours great potential for enabling and optimizing the production of a broad spectrum of products in E. coli.5015121

An Integrated Downstream Process Development Strategy along QbD Principles

The development, optimization, and analysis of downstream processes are challenged by a high number of potentially critical process parameters that need to be investigated using lab-scale experiments. These process parameters are spread across multiple unit operations and potentially show interactions across unit operations. In this contribution, we present a novel strategy for bioprocess development that considers the risk of parameter interactions across unit operations for efficient experimental design. A novel risk assessment tool (interaction matrix) is introduced to the Quality by Design (QbD) workflow. Using this tool, the risk of interaction across unit operations is rated. Subsequently, a design of experiments (DoE) across unit operations is conducted that has the power to reveal multivariate interdependencies. The power of the presented strategy is demonstrated for protein isolation steps of an inclusion body process, focusing on the quality attribute inclusion body purity. The concentration of Triton X-100 in the course of inclusion body (IB) purification was shown to interact with the g-number of the subsequent centrifugation step. The presented strategy targets a holistic view on the process and allows handling of a high number of experimental parameters across unit operations using minimal experimental effort. It is generically applicable for process development along QbD principles