This master thesis explores the launch process of new pharmaceuticals in Sweden. The path of a pharmaceutical from idea to innovation is a long and arduous process with only few new products actually reaching the patients in the end. Seeing as the drug development is also an expensive process, it is of importance that the products that get approval meet their expected revenue. New pharmaceuticals can also be life changing for the patient, and thus it is important that once approval is received the patients gain access to the new treatments. This study focuses on the post regulatory approval processes in Sweden, as well as activities carried out by the companies that affect the adoption of a new product. By utilizing a qualitative study, this thesis aims to describe the internal and external factors that affect the pharmaceutical launch process in Sweden. As well as exploring what future initiatives and possible changes that might affect it. Ten interviews with different company representatives as well as six interviews with governmental and regional stakeholders were analysed using grounded theory to answer what factors affect the adoption of new pharmaceuticals. Factors that were found to be important were: Utilisation of cross-functional teams, clear and simple strategy that includes the whole organisation, communicating with national and regional authorities, and get feedback from these, communicate with patient representatives and organisations as well as developing utility services for the product. From a couple of these factors a trend towards the servicification of the pharmaceutical industry was discovered.

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This master thesis report aims to highlight the importance ofinterorganizational relationships between experienced serviceproviders and emerging biopharmaceutical (EBP) companies within theSwedish healthcare industry. A shift in innovation strategiesregarding new pharmaceutical- and medical device products hasprompted a paradigm shift within a complex industry wherecollaborations between organisations has become increasinglycrucial. With a better understanding of how these companiesoperates, increased collaboration efforts could result in a fasterand more precise product development with new products reaching themarket improving the health for people around the world. In order toallow experienced service providers to enhance services towards EBPcompanies, a fundamental understanding of how decision makers withinthese EBP companies prefer to conduct relationships is needed. Wehave examined relationship preferences of EBP companies byconducting a qualitative case study through 14 interviews withdecision makers combined with a quantitative conjoint analysis.Eight factors was identified as important for when EBP companiesdecide to engage with a service provider: cost behavior,professional competence, adaptability, communication, personalrelationship, stability, EBP insight and size. The factorsadaptability, personal relationship, cost and size were used in theconjoint analysis to determine their relative importance which showthat adaptability and cost behavior was of the largest importance.With descriptions of each factor, we have provided a meaningfulguide to action of how to address these factors as a serviceprovider. The relationships is largely investigated as relationshipsbetween contract research organizations (as service providers) andEBP companies, but we have created a framework applicable forservice providers within the healthcare industry in general.

This project aimed to find a method for producing potential probe targets for identification of ESBL (Extended Spectrum Beta Lactamase) genes in bacteria. ESBLs are a type of enzymes responsible for antibiotic resistance in many bacteria. The result we developed was a semi-automated pipeline that utilises several Perl scripts to download gene sequences, identify sequence subgroups based on sequence similarity, find common target sequences among them and screen the target sequences against a background database. These target sequences should work with padlock probes and therefore had specific requirements regarding length and highest number of allowed mismatches. This report includes descriptions of the scripts and ideas for future improvements, as well as an ethical analysis about aspects relevant to research on antibiotic resistance.

The purpose of this project was to identify and evaluate nucleotide sequences enhancing translation of proteins in Chinese hamster ovary (CHO) cells. Candidate sequences were placed in the 5´-untranslated region (UTR) or 3´ UTR respectively and evaluated in a CHO-based expression system with a fluorescent Fc-fusion protein as a model protein.Five plasmid vectors were constructed, two of which designed to have a randomized nucleotide library in their 5´ and 3´ UTR respectively, and three of which designed to hold varying repeats of a known enhancing translation (ET) sequence in their 5´ or 3´ UTR. The plasmid constructs were transfected into CHO cells and the protein expression was analyzed both by fluorescence intensity in single cells using flow cytometry and in bulk by monoclonal antibody titer analysis based on Protein A affinity.The main result is that both flow cytometry and titer analysis indicate that insertion of five repeats of the ET in the 5´UTR has a negative effect on protein expression as compared to the control which had no ET repeats. Results related to the insertion of three ETs in the 5´ UTR were ambiguous. The titer analysis indicated that it had a negative effect on the protein expression compared to the control which had no ET repeats, whereas the flow cytometry results suggest that the effect is negligible. Transfection of library plasmids was unsuccessful; hence no library expression analysis results were achieved. Due to the time constraints of the project, the reason for the unsuccessful transfection of library plasmids was not investigated, but the LTX transfection method is stated as a highly plausible cause.Based on the outcome of this study, two recommendations for future work are suggested. The first one is to continue the focus on UTR sequences in terms of library screening, and to improve the method of transfecting library plasmid constructs into CHO cells using lipofection. The second suggestion for further studies is to test different UTR sequence lengths without involving potential ETs, to rule out the effect and positions of the ETs and investigate the expressional effect of UTR length solely.

Chronic obstructive pulmonary disease (COPD) is a high mortality disease, second to stroke and ischemic heart disease. This non-curable disease progressively exacerbates, leading to high personal and societal economic impact, reduced quality of life and often death. General treatment plans for COPD risk mistreating the individuals’ condition. To be effective, the treatment should be individualized following the practices of precision medicine. The aim of this thesis was to develop a data driven algorithm and system with visualization to assess individual COPD risk. With MRI body composition profile measurements, it is possible to accurately assess propensity of a multitude of metabolic conditions, such as coronary heart disease and type 2 diabetes.  The algorithm and system has been developed using Wolfram Language and R within the Wolfram Mathematica framework. The algorithm calculates individualized virtual control groups metabolically similar to the patient’s body composition and spirometric profile. Using UK Biobank data, our tool was used to assess patient COPD propensity using an individual-specific virtual control group with AUROC 0.778 (female) and 0.758 (men). Additionally, the tool was used to identify new body composition profiles related to COPD and associated comorbid conditions.

Our increasing understanding of disease mechanisms coupled with technological advances has facilitated the generation of pharmaceutical proteins, which are able to address yet unmet medical needs. Diseases that were fatal in the past can now be treated with novel biological medications improving and prolonging life for many patients. Pharmaceutical protein production is, however, a complex undertaking, which is by no means problem-free. The demand for more complex proteins and the realization of the importance of post-translational modifications have led to an increasing use of mammalian cells for protein expression. Despite improvements in design and production, the costs required for the development of pharmaceutical proteins still are far greater than those for conventional, small molecule drugs. To render such treatments affordable for healthcare suppliers and assist in the implementation of precision medicine, further progress is needed. In five papers this thesis describes strategies and methods that can help to advance the development and manufacturing of pharmaceutical proteins. Two platforms for antibody engineering have been developed and evaluated, one of which allows for efficient screening of antibody libraries whilst the second enables the straightforward generation of bispecific antibodies. Moreover, a method for epitope mapping has been devised and applied to map the therapeutic antibody eculizumab’s epitope on its target protein. In a second step it was shown how this epitope information can be used to stratify patients and, thus, contribute to the realization of precision medicine. The fourth project focuses on the cell line development process during pharmaceutical protein production. A platform is described combining split-GFP and fluorescence-activated droplet sorting, which allows for the efficient selection of highly secreting cells from a heterogeneous cell pool. In an accompanying study, the split-GFP probe was improved to enable shorter assay times and increased sensitivity, desirable characteristics for high-throughput screening of cell pools. In summary, this thesis provides tools to improve design, development and production of future pharmaceutical proteins and as a result, it makes a contribution to the goal of implementing precision medicine through the generation of more cost-effective biopharmaceuticals for well-characterized patient groups. / <p>QC 20170828</p>

Immunoassays are widely used for detection of antigens in a wide range of applications including assays in pharmaceutical development. Immunoassays are continuously improved in many aspects including automatization, miniaturization and extending the dynamic range. The need to measure low abundance molecules are challenging and the need to improve the sensitivity is desired. The Gyrolab technology is a miniaturized immunoassay performed in an automated system covering a broad concentration range. In order to  extend the sensitivity, the technology is combined with Binding Oligo Ladder Detection (BOLD) amplification. The technology behind BOLD or Exazym ® utilizes a DNA primer, a polymerase, and a template (RNA) to generate a ladder-like modified DNA strand. Antibodies with affinity for the polymerized DNA:RNA hybrid strand (duplex) conjugated with reporter molecules are introduced to the system, resulting in an increased number of signal-generating molecules associated with each bound analyte molecule. In this thesis, the development of an ultra-sensitive immunoassay is pursued by applying Exazym ® add-on reagents to the Gyrolab platform, comparing performance with the standard Gyrolab sandwich assay and other commercially available high-performing TNF-α assays. The work includes characterization of a wide range of reaction variables involved in the BOLD signal amplification process including hybridization, polymerization, and detection of a synthetic oligonucleotide. The breakthrough involves the introduction of Allophycocyanin (APC) as a fluorescent conjugate, significantly improving sensitivity and signal-to-noise ratios. The BOLD amplified sensitivity for the TNF-α assay approaches levels seen in ultra-sensitive biomarker assays like Erenna ® and Simoa®. Exazym® technology on the Gyrolab platform allows highly sensitive biomarker assays with minimal sample volume and a 1–2-hour run-time. The study marks substantial progress in achieving ultra-sensitive biomarker assays on the Gyrolab platform through BOLD signal amplification. The use of APC-conjugated detection reagents holds promise for future optimization studies.

This report reviews the field of bispecific antibodies, artificially engineered antibodies thathave the ability to bind two or more different antigen simultaneously. Historical as well asrecently developed techniques are demonstrated, together with formats in preclinical andclinical development. We studied the field with the future needs of the developers in mind,when it comes to the processes and tools that can be offered by GE Healthcare BiosciencesAB. The development of bispecific antibodies gave rise to new challenges and product-relatedimpurities, which are handled by various methods. We argue for, based on the formats inclinical and preclinical development, that the methods already used to purify monospecificantibodies remain the most successful methods for the purification of bispecific antibodies.This, together with the design strategies that resolve the initial bottle-necks, ensures that theneeds of the developers are met to the same extent as for monoclonal antibodies. The methodsand formats demonstrated here do not represent all that are available or under trial.

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly used classes of drugs. They target the cyclooxygenases (COX) 1 and 2 to reduce the physiological responses of pain, fever, and inflammation. Due to their role in inducing angiogenesis, COX proteins have also been identified as targets in cancer therapies. In this thesis, I describe computational protocols of molecular docking, molecular dynamics simulations and free energy calculations. These methods were used in this thesis to determine structure-activity relationships of a diverse set of NSAIDs in binding to their target proteins COX-1 and 2. Binding affinities were calculated and used to predict the binding modes. Based on combinations of molecular dynamics simulations and free energy calculations, binding mechanisms of sub-classes of NSAIDs were also proposed. Two stable conformations of COX were probed to understand how they affect inhibitor affinities. Finally, a brief discussion on selectivity towards either COX isoform is discussed. These results will be useful in future de novo design and testing of third-generation NSAIDs.