Lactose represents approximately one third of the total solids in bovine milk. In the dairy industry, lactose is recovered from whey and whey permeates using a crystallisation process that involves both evaporation and a cooling stage. A good understanding of the lactose crystallisation kinetics enables both these processes to be operated at conditions that maximise the yield and minimise capital and processing costs. This study has looked at the nucleation and growth kinetics of the lactose crystallisation process. A model has been produced that can accurately predict the changing concentration profile as lactose is removed, via growth, from an industrial solution. This model incorporated the available literature information and expanded on it where required. The primary nucleation of alpha lactose monohydrate was investigated on the laboratory scale. The work identified the changing relationship, which occurs with increasing supersaturation, as lactose nucleation moves from being dominated by the heterogeneous mechanism to the homogenous mechanism. The absolute supersaturation at which the mechanism changes was found not to be affected by the solution temperature and agitation rate; however the presence of impurities lowered the supersaturation required for homogeneous nucleation. The effect of mixing on the primary nucleation rate was studied in a Rushton turbine agitated vessel and through a Venturi. Increasing the agitation rate increased the frequency of activated molecular collisions but the critical nucleus size remained constant. A strong correlation was found, for both mixing systems, between the nucleation rate and the frequency of vortex shedding.

Affibody® molecules are small therapeutic proteins which mimics antibody functionality. This is a report of several methods for increasing productivity and yield in recombinant production of Affibody® molecules. This literature study shows several steps in the production line which can be optimised, several novel methods for cultivating and harvesting cells and purication of proteins. There is also a section about validation of therapeutic protein production according to The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) are presented.

Free analyte is measured to be able to understand the pharmacological effects of a drug in the body, the binding to its ligand, and the effective drug level among other things. Thereby, it is important with correct measurements of free analyte, although it is not that easy to achieve since overestimations can occur. In this project, several immunoassays were developed for the bioanalytical methods Gyrolab and ELISA to measure free analyte, where the biotherapeutics Avastin® and Lucentis®, and the ligand VEGF were used as analytes. One difference between the methods is the short contact time of just a few seconds for Gyrolab compared to the sample incubation time of a couple of hours for ELISA. One difference between the antibodies is that Lucentis is an affinity-matured Fab region, and therefore, has a stronger affinity to VEGF compared to Avastin. When free Avastin was measured, the difference was significant, with ELISA estimating higher concentrations compared to Gyrolab. However, this was not the case for all assays. In some cases, this was probably due to differences between the methods that could not be seen. Otherwise, the results with no difference between the methods, when measuring free analyte with Lucentis as the drug, were expected due to the stronger affinity and longer halftime of dissociation. However, the difference with the longer sample incubation time for ELISA compared to the short contact time for Gyrolab seems to influence the measurement of free analyte, depending on the affinity of the components being measured.

Waste Stabilisation Ponds (WSPs) are characterised by low phosphorus (P) removal capacity. Heterotrophic bacteria are principal microbial agents in WSPs in addition to algae. As treatment proceeds in WSPs, algal growth increases and pH rises, this has lead to believe that P removal is mainly through sedimentation as organic P algal biomass and precipitation as inorganic P. In activated sludge treatment plants (AS), microbial P removal has been improved and is termed as enhanced biological phosphorus removal. There was a need to establish whether it was possible to enhance P removal in WSPs. A performance assessment of pond system at the University of Dare s Salaam (UDSM), Tanzania, has shown that 90% of the P removed was in the primary pond (facultative) and the rest in the maturation pond (aerobic). In these studies, a pure strain A. hydrophyla was isolated from an activated sludge wastewater treatment plant in Sweden. This plant has a train that functions with enhanced biological phosphorus removal. The strain was tested for P uptake in minimal media supplemented with glucose, succinate or acetate, grown aerobically and anaerobically/aerobically. This strain was able to take up P without having been subjected to the anaerobic phase. It was observed that P uptake was enhanced after the anaerobic phase with media supplemented with glucose, but not with succinate or acetate. Phosphorus uptake repeatedly followed the bacterial growth pattern with correlation coefficients of more than 95%. Therefore P removal has a direct correlation with bacterial growth. Two isolates Acinetobacter sp. (isolated from the primary facultative pond) and E .coli (isolated from the maturation pond) were obtained from a tropical WSP treatment system at the UDSM. They were subjected to aerobic P uptake experiment similar to those of A.hydrophyla. The uptake per unit absorbance of bacterial growth was found to be comparable to that of A.hydrophyla, isolated from AS. These results showed that heterotrophic activity is important in WSPs. It is possible to enhance P removal in these systems by designing the primary ponds for maximum heterotrophic activity and probably enrichment. / QC 20101119

Biomass is a valuable and limited resource that should be used efficiently. The potential of replacing fossil-based products with bio-based ones produced in biobased industrial systems is huge. One important aim of increasing the share of biobased products is to improve the sustainability of systems for production and consumption. Therefore, it is important to evaluate what solutions are available to improve the sustainability performance of bio-based industrial systems, and if they also bring negative impacts. The thesis focuses on assessing the role of biogas solutions in developing sustainable bio-based systems. Such assessments are often quite narrow in their scope and focus on quantitative environmental or economic aspects. This thesis aims at also including feasibility related aspects involving the contextual conditions that are assessed more qualitatively. Biogas solutions are identified as a versatile approach to treat organic materials which are generated in large volumes in bio-based industrial systems. The results show that biogas solutions in bio-based industrial systems (i) improve circular flows of energy and nutrients, (ii) are especially viable alternatives when the quality of the by-product streams become poorer, and (iii) may improve the profitability of the bio-based industrial system. To perform better assessments of these systems, it seems valuable to broaden the set of indicators assessed and include feasibility-related indicators, preferably through the involvement of relevant stakeholders as they contribute with different perspectives and can identify aspects that influence the sustainability in different areas. Future studies could benefit from applying those broader assessments on more cases to build on a more generalisable knowledge base.

With the advent of fluorescence microscopy and image analysis, quantitative information from images can be extracted and changes in cell morphology can be studied. Microscopy-based morphological profiling assays with multiplexed fluorescent dyes, like Cell Painting, can be used for this purpose. It has been shown that morphological profiles can be used to train AI models to classify images into different biological mechanisms. Hence, the goal of this project was to study the possibilities for Deep Learning models and Convolutional Neural Networks to distinguish between different classes of kinase inhibitors based on their morphological profiles. Three different Convolutional Neural Network architectures were used: ResNet50, MobileNetV2, and VGG16. They were trained with two different inputs and two different optimisers: Adam and SGD. Also, a comparison between the performances with and without Transfer Learning through ImageNet weights was executed. The results indicate that MobileNetV2 with Adam as an optimiser performed the best, with a micro average of 0.93 and higher ROC areas compared to the other models. The study also highlighted the importance of utilizing Transfer Learning.

En pilotanläggning för ultrafiltering testas nu i Norrvattens reningsprocess för att undersöka ifall den kan användas som en tredje mikrobiologisk barriär i reningsprocessen. Målet med detta projekt är att testa membranets kapacitet att filtrera bort viruspartiklar men även membranet generella reduktionsförmåga för andra mikrobiologiska och kemiska kontamineringar. För att hitta lämpliga kandidater att använda sig av för att mäta reduktionskapaciteten av membranet har en litteraturstudie samt experimentell testning av råvattnet genomförts. OD mätningar på bakteriekulturer samt plackbildandeenheter (PBE) har undersökt för att se om bakteriofager kan finnas i proven. Ungefär 9000 L av ingående och utgående vatten från ultrafilteringen har koncentrerats med hjälp av ett elektropositivt filter som senare har eluerats och ultracentrifugerats. Pellet från ultracentrifugeringen har testat för virusdetektion med hjälp av PCR, qPCR samt PBE. TOC och absorbansmätningar har också genomförts på ingående och utgående vatten från ultrafiltermembranet. Slutligen utfördes ett bänkskaleexperiment för att undersöka hur väl filtret reducerade MS2 fager i utgående vatten. Den inledande testningen visade att plantviruset PMMoV och Pseudomonas fager kan vara bra kandidater att använda sig av för att mäta virusreduktionen över ultrafiltermembranet. När elueringen från ultrafiltreringen testades indikerades en minskad DNA koncentrationen över ultrafiltermembranet med hjälp av Qubit-mätningar. Testningen visade även indikation på att PMMoV reduceras över membranet samt att Pseudomonas fager kan finnas i vattnet. TOC och absorbansmätningarna visade en konstant reduktion över membranet. I bänkskaleexperiment borde enlig teori alla fager stoppas av membranet eftersom viruset är större än porstorleken 20 nm, dock visade experimentell testning på att fager även fanns i utgående vatten från filteringen. Resultat av studien indikerar att mikrobiologiska och kemiska kontamineringar tas bort av membranet, dock för att bestämma den exakta virusreduktionen över membranet och ifall alla kontamineringar större än filters porstorlek (20 nm) tas bort kräver vidare testning.  E. coli fager, som i Livsmedelverket nya restriktioner används för att undersöka mikrobiologiska risker i vattenreningsprocesser, har också testats under studien på vattnet utan positiva utslag. Det kan därför vara av intresse att även undersöka andra fager, så som Pseudomonas fager för att kontrollera dem mikrobiologiska riskerna med vattenrening. / The present study was investigating the effectiveness of the ultrafiltration membrane as third biological barrier in Norrvattens drinking water treatment process, using a pilot scale model. This project aims to test the viral reduction capability of the membrane but also to remove other microbiological and chemical contaminants. To find suitable candidates for measuring the reduction capability, literature research has been performed as well as experimental testing of the raw water coming into the treatment plant and the backwash water from the membrane. Bacterial growth analysis using optical density (OD) measurements and plaque forming unit (PFU) has been performed to investigate the presence of bacteriophages. Approximately 9000 L of incoming and outgoing water from the ultrafiltration membrane has been concentrated using an electropositive membrane which then was eluted and ultracentrifuged. The pellet from ultracentrifugation has been tested for viral detection with PCR, qPCR and plating. TOC and absorbance measurement was also performed on the ingoing and outgoing water from the ultrafiltration pilot plant. Finally, a bench-scale experiment was performed using MS2-spiked water to investigate how well the filter reduced MS2 phages in the outgoing water.  The initial testing of the raw and backwash water showed that the plant virus Pepper Mild Mottle Virus (PMMoV) and Pseudomonas phages may be good candidates to use when evaluating the ultrafiltration membrane. When testing the eluate from the ultrafiltration pilot plant a reduction was seen in the starting DNA concentration when comparing the inlet and outlet water to the ultrafiltration pilot plant. The testing gave indications of a reduction of PMMoV and presence of Pseudomonas phages. The bench-scale experiment was hypothesized to stop all viral particles since according to theory the virus should be stopped by the membrane due to its pore size, but experimental testing indicated viruses in the outgoing water from the membrane as well. TOC and absorbance measurements showed a constant reduction over the membrane. The result of the study indicates that microbiological and chemical contaminants are removed by the filter, however, to determine the exact viral reduction potential of the filter and if all contaminant over the size of 20 nm is removed further testing is required.  No indications were seen for Escherichia coli (E. coli) phages in the water throughout the study, which in Livsmedelverket’s (The National Food Agency) new regulations is used for determining the microbiological risks in water treatment processes. It may be of interest to investigate the possibility to also look for other type of phages to determine the microbiological risks, for example Pseudomonas phages which has been seen in this study.

Antibodies have become indispensable tools in diagnostics, research and as therapeutics. There are several strategies to generate monoclonal antibodies (mAbs) in order to avoid the drawbacks of polyclonal antibodies (pAbs) for therapeutic use. Moreover, the growing interest in precision medicine requires a well-characterized target and antibody to predict the responsiveness of a treatment. This thesis describes the use of epitope information and display technologies to generate and characterize antibodies. In Paper I, we evaluated if the epitope information of a well-characterized pAb could be used to generate mAbs with retained binding characteristics. In Paper II, the epitope on the complement protein C5 towards Eculizumab was mapped with surface display, the results of which explained the non-responsiveness of Eculizumab treatment among a patient group due to a mutated C5 gene. With this in mind, we showed efficacy in treatment of the mutated C5 variants using a drug binding to another site on C5, suggesting that our approach can be used to guide treatment in precision medicine. In Paper III, a Gram-positive bacterial display platform was evaluated to complement existing platforms for selection of human scFv libraries. When combined with phage display, a thorough library screening and isolation of nano-molar binders was possible. In Paper IV, a solid phase method for directed mutagenesis was developed to generate functional affinity maturation libraries by simultaneous targeting of all six CDRs. The method was also used to create numerous individual mutants to map the paratope of the parent scFv. The paratope information was used to create directed libraries and deep sequencing of the affinity maturation libraries confirmed the viability of the combination approach. Taken together, precise epitope/paratope information together with display technologies have the potential to generate attractive therapeutic antibodies and direct treatment in precision medicine. / <p>QC 20170418</p>

Therapeutic proteins, a.k.a. biopharmaceuticals, are most commonly produced in expression systems derived from Chinese Hamstery Ovary (CHO) cells, thanks to great capacity of post-translational modifications like secretation, folding and glycosylation. The engineering of cells for regulation of protein expression has many options including knock-in and knock-out of genes, epigenetic studies or improvement of the expression casette of the protein of interest by e.g. promotor variants or modifications of the 5’ and 3’ untranslated region (UTR). The 3’-UTR is therefore a good optimization candidate for attempting to achieve increased expression of therapeutic proteins. The final aim of this study was to identify and design 3’-UTRs for improved expression of therapeutic proteins in HyClone™ CHO cells from GE Healthcare Bio-Sciences AB (GEHC). The impact goal is to increase the efficiency and lower the costs for pharmaceutical companies when producing biopharmaceuticals in the HyClone™ CHO cell line, leading to increased accessibility of monoclonal antibodies (mAbs) on the pharmaceutical market. The study was initiated with bioinformatic analysis of the CHO cell transcriptome from a set of RNA-seq data of HyClone™ CHO to find high expressing, context independent genes. The 3’-UTRs from the best candidate genes were used for construction of plasmids for expression of a Fc-eGFP fusion protein. Nine selected 3’-UTRs were designed, synthesized and cloned into a parent plasmid (pGE0520) creating nine plasmid variants (pGE0523-531). The constructed plasmids were used for evaluation with site directed integration (SDI) into the HyClone™ CHO cell line and expression analysis were performed by flow cytometry and antibody titer measurements from cells with successfully integrated plasmid sorted by fluorescence-activated cell sorting (FACS).   Result show a significant effect on protein expression when using different variants of 3’-UTRs. Two variants, pGE0524 and pGE0526, competing with the parent plasmid in expression levels and integration efficiency from SDI, making them candidates for further investigations against the parent plasmid. Results also show good correlation between flow cytometry data from pre- and post-sorting, which can make research for further 3’-UTRs more efficient by evaluations and prediction of expression levels before cell sorting.

Durante a produção industrial de etanol combustível, as linhagens comerciais de Saccharomyces cerevisiae introduzidas no processo são naturalmente substituídas por linhagens selvagens, cuja origem é desconhecida. Com os objetivos de entender melhor esta questão, de se saber mais sobre o habitat natural de S. cerevisiae, e também na busca de fenótipos de interesse para aplicações industriais, avaliamos neste estudo características fisiológicas e genéticas de 14 linhagens selvagens de S. cerevisiae isoladas de Mata Atlântica e Cerrado brasileiros. Cultivos foram realizados em condições padrão de laboratório, sem ou com estresses térmico, ácido ou etanólico e também em condições similares às usadas na indústria. Como principal resultado, a linhagem S. cerevisiae UFMG-CM-Y257 apresentou alta velocidade específica de crescimento (0,57 ± 0,02 h-1), alto rendimento em etanol (1,65 ± 0,02 mol etanol mol hexose-equivalente-1), alta produtividade em etanol (0,19 ± 0,00 mol L-1 h-1), alta tolerância ao ácido acético (10 g L-1) e à alta temperatura (40 oC). / During industrial fuel ethanol production, the commercial Saccharomyces cerevisiae strains introduced in the process are naturally substituted by wild strains, whose origin is unknown. With the aims of gaining insight into this issue, of getting a better understanding on the natural habitat of S. cerevisiae, and also of identifying industrially relevant phenotypes, we performed a physiological and genetic characterization of 14 indigenous S. cerevisiae strains, isolated from Atlantic Forest and Cerrado biomes in Brazil. Cultivations were performed under standard laboratory conditions, with or without stress (high temperature, acid or ethanol), and also under conditions mimicking the industrial environment. As a main result, we identified S. cerevisiae UFMG-CM-Y257 as a strain displaying high specific growth rate (0.57 ± 0.02 h-1), high ethanol yield (1.65 ± 0.02 mol ethanol mol hexose-equivalent-1), high ethanol productivity (0.19 ± 0.00 mol L-1 h-1), high acetic acid tolerance (10 g L-1) and high temperature tolerance (40 oC).