Advances in Cell-based Therapeutic Process Development

The prospect of substituting damaged or diseased tissue by cell-based therapeutics has created much excitement in the last decades, since cell-based therapeutics open up new possibilities in patient treatment towards regenerative medicine. However, the first generation of cell-based therapeutics could not live up to market expectations and some were even withdrawn due to low reimbursement levels. While these products might be considered as market failures, identifying the obstacles early cell-based products were facing represents a major opportunity to improve the market performance of future products. In general, the inherent complexity of cell-based products in combination with limited experience represents a major obstacle translating a possible candidate from bench to bedside. A deeper insight was gained by the retrospective evaluation of the translational challenges where they could be further classified in three categories: (1) pre-market challenges, (2) manufacturing challenges and (3) post-market challenges. A fundamental step towards commercialization is the process development (industrial scale and grade) which affects all three translational challenge categories. Thus, looking at the process development associated translational challenges is important. Currently, process development for cell-based therapeutics is often done using a heuristic approach, however, a more directed and systematic approach would be highly beneficial in order to overcome the process development associated challenges. Such an approach would allow for gaining process knowledge and improve process performance and, thus, improve market performance. Effective process development for cell-based therapeutics is extremely important for product commercialization and could be achieved by implementing general bioprocess development tools and strategies. Hence, this doctoral thesis addresses the question if bioprocess development tools and strategies from the pharmaceutical industry can also be used for cell-based products. While process development requirements differ between unit operations and development stages, a general obstacle is to deal with limited material and time resources. As a consequence, most of the applied tools follow the principles of miniaturization, parallelization and automation. In order to demonstrate the power and versatility of bioprocess development tools, the objective of this thesis is the development and implementation of tools for different cell-based therapeutic manufacturing unit operations. The first part of the thesis focuses on the unit operation cell separation, more specific, the usage of cell partitioning in aqueous two-phase systems (ATPS) as cell separation method. Here, the usage of a high-throughput screening (HTS) platform is used to investigate the influence of the critical parameters polymer molecular weight and tie-line length on project. Here, batch data (cell partitioning in ATPS) is transferred into a microfluidic flow-through mode. The main scope of this project is the evaluation of 3D printing as a process development tool, since it allows for a fast and cheap realization of tailored process equipment. Whereas the second part of the thesis is focusing on cell formulation unit operations, namely, cell cryoperservation and bioprinting. A major clinical cell cryopreservation topic is the cryomedia formulation, since it needs to be toxin- and xenogen-free. Thus, the focus of the second part of this thesis is the development of a tool box to predict cryomedia performance to shorten development time. Additionally, an analytical strategy, allowing for evaluation of bioprinting process parameter on critical cell parameter, is developed. The evaluation of cell partitioning in ATPS as separation method in industrial scale cell downstream processing is the first central topic of this thesis. Cell partitioning in ATPS represents a promising alternative cell separation method to tackle the bottleneck condemned by limited availability of industrial scale methods, since it is scalable and facilitates a label-free separation. Up to now, the usage of cell partitioning in ATPS is limited due to its complex nature and not fully described partitioning theory. Thus, a time intensive and empirical screening is necessary in order to find optimal process parameter for each cell separation problem. First, a previously developed HTS platform (batch mode) is used for the investigation of critical ATPS parameter on cell partitioning behavior of five model cell lines aiming for a systematic and automated screening. The previously developed HTS platform was extended by a cell barcoding strategy allowing for a multiplexed high-throughput cell analysis in order to speed up the analytical strategy. Followed by a case study investigating the influence of the molecular weight and tie-line length on the resolution of the five model cell-lines in PEG-dextran ATPS. Both factors have an influence on cell partitioning behavior. The highest resolution of the five model cell lines could be achieved using low molecular weight PEG in combination with high molecular weight dextran. Additionally, a countercurrent distribution model was applied to calculate the theoretical purity and yield for the described separation issue. By applying the screening conditions to the countercurrent distribution model, an isolation of four of the five model cell lines is theoretically possible with high purity (> 99.9 %) and yield. A technology transfer of batch HTS cell partitioning data into a microfluidic flow-through mode is described in a subsequent project. Such a technology transfer is normally associated with high investment costs. Aiming for a flexible process development approach with low financial risk, the usage of 3D printing technologies as process development tool is proposed in this study. In general, 3D printing technologies allow for the layer-by-layer manufacturing of 3D objects. The advantage of those technologies is the relatively cheap and fast realization of complex geometries. Thus, they represent a promising and versatile tool for process development, since they enable a rapid manufacturing and implementation of tailored equipment. In this project, 3D printing was used to manufacture tailored devices complementing standard laboratory equipment for the realization of a cheap and flexible microfluidic experimental set up. The following tailored equipment was designed and 3D printed for the realization of the experimental set up: • replication master for microfluidic device manufacturing • camera mount, using the existing camera shaft, allowing for implementation of a small high performance camera • manual tailor-made fractionator with optimal installation space enabling the integration on the microscope table • frame for the microfluidic device: the frame consists of three parts, the bottom plate, an outlet- and inlet bridge. The in- and outlet tubing were connected with the microfluidic device and stabilized via the bridges using fittings. Implementing these tailored equipment parts, analysis of cell partitioning in ATPS in a microfluidic flow-through set up was possible. Cell suspension, top and bottom phase are pumped individually into the microfluidic device using three pumps. The cell suspension is focused at the interphase of the top and bottom phase allowing for cell partitioning. Simultaneously, a video of the microfluidic channel near the outlet is recorded. Using the developed snap shot analytic of the video material, an automatic estimation of cells in top and bottom phase is performed. Additionally, live cell rate was determined via flow cytometry by analyzing pooled samples. In order to provide a systematic process development and performance evaluation, the technology transfer case study was performed using the DMAIC (Define, Measure, Analyse, Improve and Control) framework. Using this DMAIC framework a successful implementation of the developed microfluidic process set up was performed. Additionally, two main factors influencing cell partitioning in the flow-through mode were identified, namely, cell load and fluid velocity. Process development of different cell formulation unit operations is the focus of the second part of this thesis. Depending on the manufacturing strategy of cell-based therapeutics, long-time storage of the cell product needs to be performed. Usually, the unit operation cryopreservation is performed for this purpose, since it allows for stocking of living material while maintaining critical cell characteristics (no genetic or metabolic alterations). While cell cryopreservation is a well described topic in literature, cell cryopreservation process development of cell-based therapeutics is currently rather time consuming and follows an empirical approach. Aiming for a streamlined and more systematic cell cryopreservation process development, a video-based tool for the characterization of the freezing and thawing behavior was developed in the first formulation project. Freezing and thawing profiles are critical process parameter influenced by many factors including e.g. cryomedia formulation and scale up (working volume and container geometry). In order to enable rapid process development for cryopreservation despite the high number of process variable, fast and directed analytical tools are required. To evaluate the performance and flexibility of the video-based tool, a cryopreservation case study was performed with the _-model cell line INS-1E. Here, the freezing and thawing behavior of two working volumes (1 mL and 2 mL) were analyzed. Additionally, cell recovery and proliferation were evaluated. As expected, a delay in freezing and thawing behavior due to scale up was detected, resulting in a decrease of process performance determined by live cell recovery. While a high live cell recovery (0.94 (±0.14) %) was achieved with 1 mL working volume, a decrease to 0.61 (±0.05) % could be observed for the 2 mL working volume. These findings are in good agreement with expectations, confirming the tool performance. The objective of the consecutive second cryopreservation project is the development of a tool box allowing for the prediction of cryomedia process performance by characterization of media properties, freeze/thaw behavior (using the previously developed video-based tool) and media toxicity. Using good manufacturing practice (GMP) compliant cryomedia is an essential requirement for the cryopreservation of cell-based therapeutics, meaning it needs to be chemically defined without the addition of animal-derived or toxic compounds. For this reason, using the well described and often used cryoprotectants dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) is not an option. Supplementing a cryomedia with cryoprotectant agents is, however, crucial for cryopreservation process performance since they protect cells from severe stress, e.g. non-physiological osmotic pressure conditions, during freezing and thawing. Hence, development of an effective and GMP compliant cryomedia formulation is necessary for cell-based therapeutics when long-term storage is part of the manufacturing strategy. In this project, the usage of the developed predictive tool box for cryomedia formulation screening is proposed. The project comprised a case study evaluating the developed predictive cryomedia screening tool box including two commercial, DMSO-free cryomedia, one negative (without any cryoprotectant) and one positive (supplemented with DMSO and FBS as cryoprotectants) cryomedia example. The case study was performed with the _-model cell line INS-1E. Additionally, a conventional cryomedia performance assessment was done in order to evaluate the predictive tool box. Using the tool box the commercial Biofreeze® media was classified as unsuitable for the beta-model cell line INS-1E due to media toxicity, while the second commercial cryomedia CryoSOfreeTM was classified as possible candidate. These findings were confirmed by the conventional screening proving the power and usefulness of the tool box. Thus, implementing the predictive tool box will facilitates a fast pre-selection of possible candidates with low sample volume and cell number, shortening the overall process development time for the development of cryomedia formulations. The focus of the third cell formulation project within this thesis is bioprinting. More precisely the development of an analytical strategy for cell-based bioprinting applications. Bioprinting is a relatively new field, however, it is believed to have a huge impact on regenerative medicine and tissue engineering, since it enables the manufacturing of artificial 3D tissue. Great efforts are being made in this field to move from academic research towards pharmaceutical industry or clinical applications. A critical hurdle to overcome this transition process is the development of a robust and well-known process, while maintaining critical cell characteristics (CCC). In order to gain process knowledge, a systematic and more directed process development approach for 3D bioprinting applications is required which includes the monitoring of CCC. Depending on the application, the critical cell characteristics may vary. However, universal CCCs are cell viability and proliferation. Flow cytometry represents a highly flexible, powerful and often used cell analysis method capable of analyzing multiplex CCC issues in parallel and might also be useful in the field of 3D bioprinting. However, flow cytometry analysis can only be performed with cell suspensions and needs, therefore, a destruction of the cell-laden 3D-structure prior analysis. Hence, the objective of this project is the development of a flow cytometry-based analytical strategy as a tool for 3D bioprinting research. The development of this anayltical strategy was conducted using a model process with the _-model cell line INS-1E, a commercially available alginate-based bioink and a direct dispensing system as 3D bioprinting method. The destructive strategy enables the evaluation of cell viability and proliferation. By using a flow cytometer set up including an autosampler, the strategy enables an automated high-throughput screening. Following the development of the strategy, an evaluation of the process steps was performed including: suspension of cells in bioink, 3D printing and cross-linking of the alginate scaffold after printing. The evaluation showed that each individual process step (using the selected process parameters) had a negative influence on cell viability and must therefore be carefully monitored. This highlights the importance of process optimization in 3D bioprinting and the usefulness of the flow cytometry-based analytical strategy. The presented strategy has a great potential as a cell characterization tool for 3D bioprinting and can even be extended to a multiplex CCC analysis. Thus, the developed tool may contribute to a more directed process development in the field of 3D bioprinting. In summary, several process development tools and strategies for different process units were developed within this doctoral thesis, demonstrating the applicability of general bioprocess development tools for cell-based products. The presented methods facilitating a more directed and systematic process development approach for cell-based therapeutics. These powerful tools represent an opportunity to streamline process development time, tackle translational hurdles and, ultimately, improve process understanding aiming for a QbD approach in the cell therapy industry

The Legal Impact of COVID-19 on Women's International Human Rights: Analyzing the #NiUnaMenos Movement in Latin America

Fabiola Gretzinger graduated from the University of Minnesota Law School in May 2022, with concentrations in International Law and Human Rights. Throughout her professional and academic career, Fabiola has focused on international women’s rights, with an emphasis on reproductive health and gender-based violence. Currently, Fabiola is a legal fellow with the Legal Strategies, Innovation and Research team at the Center for Reproductive Rights.This paper details the history and successes of the #NiUnaMenos movement, and subsequently the Marea Verde (Green Wave) movement, throughout Latin America. After the COVID-19 pandemic, there has been a massive increase in domestic violence and gender-based violence rates throughout the region. Similarly, the United States just stripped women of their constitutional right to an abortion in Dobbs v. Jackson Women’s Health Organization. This evidences how important this movement is throughout Latin America, and for the rest of the world, to understand how social, political, and legal movements can continue to protect women’s rights. Comparing successes and setbacks both before and after COVID-19, this paper establishes how governments and movements alike should proceed to ensure women’s rights are strengthened and protected.La

Local Governance in Alberta: Principles, Options and Recommendations

Municipalities in Alberta have faced challenges for some time. Many of these challenges have emerged at both local and regional scales, and include slower growth and aging populations, constrained finances, a shifting economic base and the impact of the COVID-19 pandemic (AUMA 2020). In response, Alberta Municipalities, an organization that advocates on behalf of more than 250 urban municipalities in the province, has commissioned several reports to address these concerns. We were tasked to assess the current state of Alberta’s local governance model and investigate if changes to government structure might offer some remedy to the deficiencies uncovered in our assessment. Accordingly, the report aims to do the following: 1) provide conceptualtools to understand local government structure; 2) impart a set of principles to guide strategic efforts; 3) evaluate existing regional governance in Alberta; and 4) offer several restructuring suggestions for Alberta Municipalities to consider in consultation with municipalities and to advocate for the province to act on those they wish to pursue. The key governance concepts of viability and legitimacy underlie this report. A government becomes viable when a critical mass of population and other antecedents are present to catalyze development. Local governments become legitimate when they can take justifiable actions in a legal manner and have active support from their citizens. To improve the viability and legitimacy of local governance, structural reforms must be guided by a set of principles. This report proposes five such principles: efficiency, capacity, accountability, accessibility and responsiveness. To supplement viability and legitimacy, we also invoked the additional concepts of fragmentation and its counterpart, consolidation. Together, these latter two concepts support a tiered, spatial and authority structure, which can work as an analytical tool to discuss and evaluate the local government models in Alberta and elsewhere in Canada. Collectively, these concepts enable us to identify and compare the number of local government units within regions to better understand how the spatial distribution of governance may uphold the five principles of good governance. The tiered structure of a municipal government, whether it is horizontal or vertical, reveals the extent of spatial geography it serves and the distribution of authority and service responsibility between and among tiered units. We find that Alberta’s primarily horizontally fragmented governance arrangement, which includes over three hundred urban and rural municipalities, provides an accountable, accessible and responsive system. Such a system, however, lacks efficiency and capacity. Our analysis suggests that no one-size-fits-all model will work for Alberta. However, strengthening the current fragmented governance model in Alberta through intermunicipal collaboration frameworks, growth management boards and regional service commissions can bring about meaningful improvements. More disruptive options — such as amalgamation, regional districts or a two-tiered governance structure — also offer some benefits, but they should be scrutinized against the particular context of the area for which they are considered. The report presents a series of recommendations that Alberta Municipalities can pursue with the Government of Alberta. The overarching suggestion is that the province mandate dispute-resolution mechanisms to resolve all intermunicipal challenges, including annexations. This would minimize intermunicipal frictions and foster cooperation to improve municipal viability, while also improving the fairness and the legitimacy of the governance system. To achieve this, the current dispute resolution mechanisms must be strengthened and expanded. The other recommendations are divided into three categories, as they pertain to three distinct geographies of the province: metropolitan regions, regions outside of census metropolitan areas and small and remote urban municipalities

Virtual Reality as Tool for Bioprinting Quality Inspection: A Proof of Principle

As virtual reality (VR) has drastically evolved over the past few years, the field of applications of VR flourished way beyond the gaming industry. While commercial VR solutions might be available, there is a need to develop a workflow for specific applications. Bioprinting represents such an example. Here, complex 3D data is generated and needs to be visualized in the context of quality control. We demonstrate that the transfer to a commercially available VR software is possible by introducing an optimized workflow. In the present work, we developed a workflow for the visualization of the critical quality attribute (cQA) cell distribution in bioprinted (extrusion-based) samples in VR. The cQA cell distribution is directly influenced by the pre-processing step mixing of cell material in the bioink. Magnetic Resonance Imaging (MRI) was used as an analytical tool to generate spatially resolved 2.5 and 3D data of the bioprinted objects. A sample with poor quality in respect of the cQA cell distribution was identified as its inhomogeneous cell distribution could be displayed spatially resolved in VR. The described workflow facilitates the usage of VR as a tool for quality inspection in the field of bioprinting and represents a powerful tool for visualization of complex 3D MRI data

Magnetic resonance imaging as a tool for quality control in extrusion-based bioprinting

Bioprinting is gaining importance for the manufacturing of tailor-made hydrogel scaffolds in tissue engineering, pharmaceutical research and cell therapy. However, structure fidelity and geometric deviations of printed objects heavily influence mass transport and process reproducibility. Fast, three-dimensional and nondestructive quality control methods will be decisive for the approval in larger studies or industry. Magnetic resonance imaging (MRI) meets these requirements for characterizing heterogeneous soft materials with different properties. Complementary to the idea of decentralized 3D printing, magnetic resonance tomography is common in medicine, and image data processing tools can be transferred system-independently. In this study, a MRI measurement and image analysis protocol was evaluated to jointly assess the reproducibility of three different hydrogels and a reference material. Critical parameters for object quality, namely porosity, hole areas and deviations along the height of the scaffolds are discussed. Geometric deviations could be correlated to specific process parameters, anomalies of the ink or changes of ambient conditions. This strategy allows the systematic investigation of complex 3D objects as well as an implementation as a process control tool. Combined with the monitoring of metadata this approach might pave the way for future industrial applications of 3D printing in the field of biopharmaceutics

Image analysis as PAT-Tool for use in extrusion-based bioprinting

The technology of bioprinting is arousing a growing interest in biopharmaceutical research and industry. In order to accelerate process development in the field of bioprinting, image-based analysis methods are non-invasive, time- and cost-saving tools which are useable for printer characterization, bioink printability evaluation, and process optimization. Image processing can also be used for the study of reproducibility, since reliable production is important in the transition from research to industrial application, and more precisely to clinical studies. This study revolves around the establishment of an automated and image-based line analysis method for bioprinting applications which enables an easy comparison of 3D-printed lines. Diverse rheological properties of bioinks and the printing process affect the geometry of the resulting object. The line represents a simple geometry, where the influence of the rheological properties and printing parameters is directly apparent. Therefore, a method for line analysis was developed on the basis of image recognition. At first, the method is tested for several substances such as Nivea®, pure and colored Kolliphor solutions, and two commercially available hydrogel formulations which can be used as bioinks. These are Biogelx™-ink-RGD by Biogelx and Cellink® Bioink by Cellink. The examination of limitations showed that transparent materials such as Kolliphor-based solutions cannot be analyzed with the developed method whereas opaque materials such as Nivea® and both bioinks can be analyzed. In the course of process characterization, the method was used to investigate the shrinkage behavior for both bionks. With the help of the line analysis tool, a shrinkage behavior of both bioinks was demonstrated and thus, process time could be identified as a critical process parameter

Local Governance in Alberta: Principles, Options and Recommendations

Municipalities in Alberta have faced challenges for some time. Many of these challenges have emerged at both local and regional scales, and include slower growth and aging populations, constrained finances, a shifting economic base and the impact of the COVID-19 pandemic (AUMA 2020). In response, Alberta Municipalities, an organization that advocates on behalf of more than 250 urban municipalities in the province, has commissioned several reports to address these concerns. We were tasked to assess the current state of Alberta’s local governance model and investigate if changes to government structure might offer some remedy to the deficiencies uncovered in our assessment. Accordingly, the report aims to do the following: 1) provide conceptual tools to understand local government structure; 2) impart a set of principles to guide strategic efforts; 3) evaluate existing regional governance in Alberta; and 4) offer several restructuring suggestions for Alberta Municipalities to consider in consultation with municipalities and to advocate for the province to act on those they wish to pursue. The key governance concepts of viability and legitimacy underlie this report. A government becomes viable when a critical mass of population and other antecedents are present to catalyze development. Local governments become legitimate when they can take justifiable actions in a legal manner and have active support from their citizens. To improve the viability and legitimacy of local governance, structural reforms must be guided by a set of principles. This report proposes five such principles: efficiency, capacity, accountability, accessibility and responsiveness. To supplement viability and legitimacy, we also invoked the additional concepts of fragmentation and its counterpart, consolidation. Together, these latter two concepts support a tiered, spatial and authority structure, which can work as an analytical tool to discuss and evaluate the local government models in Alberta and elsewhere in Canada. Collectively, these concepts enable us to identify and compare the number of local government units within regions to better understand how the spatial distribution of governance may uphold the five principles of good governance. The tiered structure of a municipal government, whether it is horizontal or vertical, reveals the extent of spatial geography it serves and the distribution of authority and service responsibility between and among tiered units. We find that Alberta’s primarily horizontally fragmented governance arrangement, which includes over three hundred urban and rural municipalities, provides an accountable, accessible and responsive system. Such a system, however, lacks efficiency and capacity. Our analysis suggests that no one-size-fits-all model will work for Alberta. However, strengthening the current fragmented governance model in Alberta through intermunicipal collaboration frameworks, growth management boards and regional service commissions can bring about meaningful improvements. More disruptive options — such as amalgamation, regional districts or a two-tiered governance structure — also offer some benefits, but they should be scrutinized against the particular context of the area for which they are considered. The report presents a series of recommendations that Alberta Municipalities can pursue with the Government of Alberta. The overarching suggestion is that the province mandate dispute-resolution mechanisms to resolve all intermunicipal challenges, including annexations. This would minimize intermunicipal frictions and foster cooperation to improve municipal viability, while also improving the fairness and the legitimacy of the governance system. To achieve this, the current dispute resolution mechanisms must be strengthened and expanded. The other recommendations are divided into three categories, as they pertain to three distinct geographies of the province: metropolitan regions, regions outside of census metropolitan areas and small and remote urban municipalities

Structured Data Storage for Data-Driven Process Optimisation in Bioprinting

Bioprinting is a method to fabricate 3D models that mimic tissue. Future fields of application might be in pharmaceutical or medical context. As the number of applicants might vary between only one patient to manufacturing tissue for high-throughput drug screening, designing a process will necessitate a high degree of flexibility, robustness, as well as comprehensive monitoring. To enable quality by design process optimisation for future application, establishing systematic data storage routines suitable for automated analytical tools is highly desirable as a first step. This manuscript introduces a workflow for process design, documentation within an electronic lab notebook and monitoring to supervise the product quality over time or at different locations. Lab notes, analytical data and corresponding metadata are stored in a systematic hierarchy within the research data infrastructure Kadi4Mat, which allows for continuous, flexible data structuring and access management. To support the experimental and analytical workflow, additional features were implemented to enhance and build upon the functionality provided by Kadi4Mat, including browser-based file previews and a Python tool for the combined filtering and extraction of data. The structured research data management with Kadi4Mat enables retrospective data grouping and usage by process analytical technology tools connecting individual analysis software to machine-readable data exchange formats

The Anglo-Saxon migration and the formation of the Early English gene pool

The history of the British Isles and Ireland is characterized by multiple periods of major cultural change, including the influential transformation after the end of Roman rule, which precipitated shifts in language, settlement patterns and material culture1. The extent to which migration from continental Europe mediated these transitions is a matter of long-standing debate [2,3,4]. Here we study genome-wide ancient DNA from 460 medieval northwestern Europeans—including 278 individuals from England—alongside archaeological data, to infer contemporary population dynamics. We identify a substantial increase of continental northern European ancestry in early medieval England, which is closely related to the early medieval and present-day inhabitants of Germany and Denmark, implying large-scale substantial migration across the North Sea into Britain during the Early Middle Ages. As a result, the individuals who we analysed from eastern England derived up to 76% of their ancestry from the continental North Sea zone, albeit with substantial regional variation and heterogeneity within sites. We show that women with immigrant ancestry were more often furnished with grave goods than women with local ancestry, whereas men with weapons were as likely not to be of immigrant ancestry. A comparison with present-day Britain indicates that subsequent demographic events reduced the fraction of continental northern European ancestry while introducing further ancestry components into the English gene pool, including substantial southwestern European ancestry most closely related to that seen in Iron Age France [5,6]