Recombinant protein therapeutics is a growing market within the human medical biotechnology industry. The majority of all approved biopharmaceuticals are protein based and includes for example blood factors, anticoagulants, hormones, vaccine and monoclonal antibodies. Some of these protein based drugs are glycoproteins which require special carbohydrate structures attached to certain amino acids for correct folding, biological activity and/or stability in the circulation. The biosynthesis and covalent attachment of these oligosaccharides to the polypeptide core, the glycosyaltion, is species and tissue specific. Eukaryotic cells can attach the glycoproteins either to the side chain of aspargine (N-linked) or through the side chains of threonine or serine (O- linked). Controlling the synthesis of these carbohydrate structures, the glycosylation, is of prime concern when developing therapeutic glycoproteins and today mammalian cell culture systems are the preferred systems due to their ability to perform human-like glycosylation. However, mammalian systems are often hampered by disadvantages such as long production times, low protein titres, product heterogeneity and viral containment issues. These factors complicate large scale production of therapeutic glycoproteins and consequently there is a continual search for alternative expression systems with improved performance. The methylotrophic yeast Pichia pastoris (P. pastoris) has a number of attractive characteristics for heterologous protein production, including the ability to perform post-translational modifications, such as N- and O- linked glycosylation, and secrete large amounts of recombinant protein. Recombinant protein antigens glycosylated by P. pastoris have shown enhanced immunogenicity compared to their non-glycosylated counterparts. The high mannose content of yeast derived N- and O-glycans is proposed to target the recombinant antigens to immunoregulatory, mannose specific receptors which upon binding promotes the enhanced immune responses. These findings suggest P. pastoris as a platform for production of recombinant vaccines. However, structural and functional characterizations of the glycans involved are poor, specifically for O-glycans. PSGL-1/mIgG2b, is a chimeric mucin-like protein with the potential to carry 106 O-glycans and six N-glycans, and AGP-1/mIgG2b is a chimeric protein with the potential to carry twelve N-glycans. In the context of mannose specific receptor targeted vaccines, glycoproteins with this type of glycosylation expressed by P. pastoris have not been studied before. The objective of this study was to develop bioprocesses for efficient production of PSGL-1/mIgG2b and AGP-1/mIgG2b The main purpose of this was to supply enough material for characterisation and functional studies of PSGL-1/mIgG2b and AGP-1/mIgG2b in the context of binding three mannose specific receptors, MR, DC-SIGN and MBL, but also to identify important bioprocess parameters for large scale production of the recombinant glycoproteins. Methanol feed, pH and certain media components were found to be critical for productivity and homogeneity of the recombinant proteins. During the course of this study a bioprocess which improved productivity from 10 mg/L to around 200 mg/L for PSGL- 1/mIgG2b and from 3.5 mg/L to 21 mg/L for AGP-1/mIgG2b along with significantly reduced proteolytic activity was developed. To relate a certain glycan structure with biological activity, characterization of the PSGL-1/mIgG2b O-glycans in combination with binding studies to the recombinant mannose specific receptors MR, DC-SIGN and MBL was conducted. Biacore analysis revealed high affinity binding of both PSGL-1/mIgG2b and AGP-1/mIgG2b to all receptors. MS of O-glycans released from PSGL-1/mIgG2b indicated Hex2-9 structures. For fast, on-line optimization of recombinant protein production an optimization system based on the intrinsic fluorescence of the green fluorescent protein (GFP) was developed. Recombinant strains of P. pastoris secreting the GFP fusion protein PSGL-1/mIgG2b/GFP were generated and the fluorescence system was applied to follow on-line fluorescence under various induction conditions. Subsequently, P. pastoris secreting PSGL- 1/mIgG2b was induced under the same conditions. Correlations between the on- line fluorescence and the secreted amount of PSGL-1/mIgG2b were investigated. It was concluded that the on-line system had the potential to reflect the translational rate of PSGL-1/mIgG2b/GFP. However, due to different secretion properties of PSGL-1/mIgG2b/GFP and PSGL-1/mIgG2b in combination with potential genetic instability no correlations were found. The system may still have a value for recombinant proteins expressed intracellularly. / Godkänd; 2008; 20080829 (ysko)

The present thesis demonstrates an integrated approach to the development of a potential active pharmaceutical ingredient, eritadenine, a cholesterol reducing compound originating from the shiitake mushroom (Lentinus edodes). The main areas covered in the thesis are a method for quantification of eritadenine, production of eritadenine by submerged cultivation of fungal mycelia and investigation of the influence of process parameters on mycelial growth and production, and finally solid state characterizations of eritadenine. The usage of the fungus as a source of eritadenine requires an analytical tool for quantification of the compound. An HPLC method was hence developed for identification and quantification of eritadenine, using chemically synthesized eritadenine as a reference. The amount of eritadenine in fruit bodies of selected strains of shiitake was determined and with the method developed in this study, eritadenine concentrations up to ten times higher than previously reported were detected. Since both fruit bodies and mycelia of shiitake have been shown to contain eritadenine submerged cultivation of shiitake mycelia was investigated as an alternative source for this compound. The mycelia were cultivated in various submerged conditions, both in shake flasks and in bioreactors. It was found that both the mycelia and the culture media contained eritadenine, of which the major part was detected in the culture media. While the biomass concentrations were higher in shake flasks, the eritadenine concentrations were considerably higher in the bioreactors, which were assigned to morphological variations. In an attempt to improve the mycelial growth and eritadenine production, a growth promotive substance in the form of a water extract of DDGS, a by-product from drygrind ethanol facilities, was added to the culture media. It was demonstrated that an amendment of the cultivation media with this extract caused a considerable growth promotive effect on shiitake mycelia in bioreactor cultivations, along with enhanced eritadenine production. If eritadenine will be used as a pharmaceutical agent, understanding about the solid state chemistry of the compound is required. Raman spectroscopy is a valuable technique for investigation of structural properties; hence, a Raman reference spectrum with line assignments for the solid state of synthetic eritadenine was established. To further investigate the solid state chemistry of eritadenine, its synthetic analogue was slowly crystallized from water and different ethanol concentrations, at different temperatures. Solids formed from slow cooling of either water or aqueous ethanol showed crystallinity. No polymorphism was detected, irrespective of solvent system or temperature. However, dissimilar thermal behaviours were observed, deducing crystals derived from water as dihydrates and crystals derived from aqueous ethanol as 2.5 hydrates. / Godkänd; 2009; 20090824 (joen); DISPUTATION Ämnesområde: Biokemisk och kemisk processteknik/Biochemical and Chemical Engineering Opponent: Professor Allan Myerson, Illinois Inst of Technology, USA Ordförande: Professor Kris Berglund, Luleå tekniska universitet Tid: Tisdag den 29 september 2009, kl 13.00 Plats: C 305, Luleå tekniska universitet

Cardiovascular diseases are among the main causes of death in our society and there is a strong correlation between enhanced blood cholesterol levels and the development of such diseases. The popular edible fungus, shiitake mushroom (Lentinus edodes), has been shown to produce a blood cholesterol lowering compound designated eritadenine, and the hypocholesterolemic action of this compound has been quite extensively examined in rats. Eritadenine is suggested to accelerate the removal of blood cholesterol either by stimulating tissue uptake or by inhibiting tissue release; there are no indications of this compound inhibiting the biosynthesis of cholesterol. If shiitake mushrooms are to be used as a source for a potential cholesterol reducing product, it is of great importance to determine the content of eritadenine in the mushrooms as accurately as possible. Hence, in paper I methanol extraction was used to recover as much as possible of the hypocholesterolemic agent from the fungal cells. In order to analyse the target compound, a reliable and reproducible HPLC method for separation, identification and quantification of eritadenine was developed. The amounts of eritadenine in fruit bodies of four commercially cultivated shiitake mushrooms were determined, and the mushrooms under investigation exhibited up to ten times higher levels of eritadenine (3.17-6.33 mg/g dry mushrooms) than previously reported. Not only the fruit bodies of shiitake, but also its mycelia contain eritadenine. Growing fruit bodies of shiitake is a fairly demanding and time consuming process. Hence, in search for a source of eritadenine, submerged (liquid) cultivation of shiitake mycelia could be an alternative. The reason why shiitake mushrooms synthesize eritadenine is yet not clarified; i.e. the function of this secondary metabolite and the growth conditions that favour its production are not elucidated. In addition, like other filamentous fungi, shiitake exhibits different hyphal morphologies in submerged cultures depending on cultivation conditions such as medium composition, temperature, pH, inoculum concentration, dissolved oxygen and shear. The fungal metabolism and hence production of secondary metabolites is in turn affected by the morphology, as have been shown in several studies on filamentous fungi. Submerged cultivation of shiitake mycelia offers a convenient way to change the cultivating conditions in order to improve eritadenine yield and productivity. The study in paper II focused on cultivation of mycelia at different conditions, both in shake flasks and in bioreactors, to investigate the effect of pH and stirring rate on production of eritadenine. The shiitake mycelia were found to produce eritadenine, and the compound of interest was found in both the fungal cells and the growth media. The major part (90-99%) was found in the culture medium, which offers a facilitated downstream processing if large scale production of the compound is to be conducted. The mycelial morphology in the shake flask cultures were macroscopic aggregates, pellets, and the specific productivity of eritadenine was relatively low; 6.56 mg/g dry cell weight (DCW). In the bioreactor cultivations, the mycelia grew as freely dispersed filaments, showing a higher specific productivity than in the shake flasks, ranging between 26.00- 39.58 mg/g DCW. This indicates the influence of morphology on eritadenine production. The biomass yield in shake flasks and bioreactors was in parity; 0.45 g in the shake flasks and 0.25- 0.62 g in the bioreactors. A stirring rate of 50 rpm in the bioreactors was preferable for eritadenine production, whereas for biomass production it was 250 rpm, indicating the influence of agitation on both growth and productivity. The pH did not have any major impact on growth, whereas the specific productivity in the bioreactors was higher when pH was uncontrolled than controlled at 5.7. / Godkänd; 2007; 20070515 (ysko)

An important issue in pharmaceutical industry is to develop processes to purify proteins. Thereare many purification methods that can be used for this purpose, but all methods have differentconstraints. A large interest in affinity chromatography methods can be seen but since theaffinity tag sometimes must be removed, a new self-cleaving affinity chromatography methoddeveloped by Cytiva has the potential of competing with existing chromatography methods. Inthis project two processes, one with conventional purification methods and one with the newself-cleaving affinity chromatography method are developed and compared. A protein calleddesigned ankyrin repeat protein, DARPin, is used as the protein of interest since this proteincan be used as a scaffold protein where a large variety of applications have been investigated. During the project, many different chromatography and other purification methods areevaluated. In the conventional process, after performed experiments, a strong anion exchange chromatography is selected as the first step. The second purification step is chosen to be heat-treatment because of the high melting point of the DARPin. In the last step hydrophobic interaction chromatography is used. This results in a purity of 72-97%, a recovery of 20% and ayield of 0,232 mg DARPin/g cell paste. The development was made in 11 weeks. This can becompared with the second process, where the first step is also strong anion exchangechromatography. The new self-cleaving affinity chromatography was used as a second stepgiving a purity of 89-100%, a recovery of 12% and a yield of 0,231 mg DARPin/g cell paste. Thisdevelopment was made in 5 weeks. The project shows that a process with the new self-cleaving chromatography method gives afinal sample that is purer and that this process can be developed in a much shorter time. Thesame amount of protein can also be obtained even though a lower recovery can be seen.

This report was conducted at the request of Testa Center to compile the essential requirements and challenges faced by the biomanufacturing industry. The gathered information will serve as a guide for Testa Center's future objectives in accelerating technological advancements in bioproduction. Based on insights provided by 14 companies involved in various stages of biological production, a comprehensive conclusion was reached. A bioproduction process was defined to encompass cell optimization, creation of starter cell cultures, cell cultivation/fermentation in bioreactors, product harvest, clarification, capture, and polishing, with the latter three stages focused on purification. The companies were contacted through surveys and personal interviews, which were tailored to suit each interviewee while aligning with the survey questions. Data obtained was subsequently categorized, highlighting the areas in need of significant development, where frequency of mentioned bottlenecks within each category determined its level of importance. According to the companies, the three most critical areas requiring attention were purification, continuous processing, and single-use technologies. Consequently, these areas were given thorough consideration during the analysis of individual company responses. However, all bottlenecks raised by company representatives were included, along with relevant background information to ensure a comprehensive understanding of each issue. The remaining areas of need, in descending order of significance, were classified as "miscellaneous" (bottlenecks not assignable to a specific area), "scalability", "cooperation and communication", "cell optimization and gene-editing", "other material and equipment issues", "high costs", "screening and assaying", "harvest", "bioreactors", and "virtual modeling". The expressed needs varied depending on each company's unique biological production process. By addressing these identified challenges and focusing on the crucial areas, Testa Center can effectively contribute to the acceleration of technological advancements in bioproduction.

Sustainable use of forest and agricultural resources will play an important role for solving urgent global challenges such as the enhanced green house effect and increasing demand for fossil fuels. The development of processes where lignocellulosic biomass can be refined to several different end-products in the same plant, i.e. a biorefinary, will be important in the development towards a more sustainable society where fossil fuels are replaced. To be able to compete with fossil resources, an efficient production of biomass based products is required in order to maximize overall process economics and to minimize negative environmental impact. One solution to increase profitability for forest biomass based plants can be production of value added derivatives produced through fermentation of sugars from hemicelluloses, extracted from lignocellulosic material. The first part of this thesis investigate the impact of hemicellulose pre-extraction on birch Kraft pulp properties. White liquor and water extractions of hemicelluloses from birch wood chips were performed under conditions compatible with Kraft pulping. The chips from select extractions were subject to subsequent Kraft pulping and the refined pulps were made into hand sheets. Several metrics for hand sheet strength properties were compared with a reference pulp made without an extraction step. This work also includes a demonstration of enzymatic hydrolysis and biological conversion of extracted xylan to succinic acid, a metabolite with the potential of a platform chemical. The study demonstrated that white liquor can be utilized to extract xylan from birch wood chips prior to Kraft cooking without decreasing the pulp yield and paper strength properties, while simultaneously impregnating cooking alkali into the wood chips. Alkaline conditions tested above pH 10 significantly degraded xylan and very low concentrations of xylose were obtained using any of the alkaline extractions. Water extractions resulted in the highest final concentration of xylose, 29.1 g/L; yielding fermentable liquor, but were found to negatively impact some pulp properties including decreases in compression strength, bursting strength, tensile strength and tensile stiffness while exhibiting minimal impact on elongation and slight improvement in tearing strength index. Since hot water extractions gave fermentable liquors, the next study was to integrate the production of green chemicals via hot water hemicellulose extraction of birch wood into a small-scale combined heat and power plant, in this case an externally fired gas turbine. The results show that the extracted wood chips would serve very well as a fuel for combustion and gasification processes due to the relatively high heating value. Most important, the extracted wood chips had low ash content and significantly lower concentrations of alkali metals. In addition a fermentable stream with a xylose concentration of 65 g/L was produced.The second part of this thesis was to optimise the production of the dicarboxylic acid, succinic acid, which can be produced via bioconversion as a renewable building block molecule for production of biodegradable solvents and polyesters. In this study the E. coli strain AFP184, which can ferment both five and six carbon sugars with a limited production of other organic acids was used. Earlier work using a high initial sugar concentration resulted in volumetric productivities of almost 3 g/L h, which is above estimated values for economically feasible production, and final succinic acid concentration was around 40 g/L. To further increase succinic acid concentrations, fermentations using NH4OH, NaOH, KOH, K2CO3, and Na2CO3 as neutralising agents were performed and compared. It was shown that substantial improvements could be made by using alkali bases to neutralise the fermentations. The highest concentrations and productivities were achieved when Na2CO3 was used, 77 g/L and 3 g/L h, respectively. A gradual decrease in succinate productivity was observed during the fermentations, which was shown to be due to succinate accumulation in the broth and not as a result of the addition of neutralising agent or the subsequent increase in osmolarity.

Butanol, butyric acid and butyraldehyde are important 4-carbon oxychemicals typically generated from petro-chemical sources. All have significant markets in the food industry either for direct use as flavorings or as chemical feedstocks for generating butyric acid and butyraldehyde derived flavour compounds. Strong consumer sentiment against the consumption of petro-chemical derived products yields a demand for butyrate and butyraldehyde generated through all-natural methods. The bacterial fermentation production of butyric acid as well as bioconversion of butanol to butyraldehyde by yeast is presented in this work as a means of producing these products naturally. This thesis demonstrates the fermentation production of butyric acid with the Gram-positive anaerobic bacteria Clostridia tyrobutyricum. The organism consumes monomeric hexoses and pentoses to generate the carboxylic acids lactate, acetate and butyrate. The fermentations undertaken in this thesis were performed with either glucose or xylose as the primary carbon source in minimal media. Butyric acid studies were performed under anaerobic conditions as batch fermentations with lag, log and stationary phase growth being monitored by the optical density of the fermentation broth. Samples were drawn throughout the fermentations and HPLC analysis was performed to determine sugar consumption and butyric acid production over time. Another element expounded in this thesis is the potential use of the economical and renewable resource hot water extracted (HWE) hemicellose as a substrate for Clostridial fermentation. HWE hemicellose is produced as a waste stream from the pulp and paper industry and is converted to fermentable xylose with the concomitant release of acetic acid from the acetyl groups on the xylan backbone. With the presence of such a high concentration of acetic acid, microbial inhibition occurs and the productivity of xylose fermentation to butyric acid is diminished with the increased lag phase. C. tyrobutyricum xylose fermentation studies were performed with synthetic media challenging the fermentation with up to 26.3 g/L acetic acid to gain an understanding of the effects of acetic acid inhibition. Once the acetic acid induced lag phase growth was characterized this work was furthered by adapting a strain of C. tyrobutyricum to 26.3 g/L acetic acid conditions and demonstrating that this pre-adaptation could drastically reduce the acetic acid induced lag phase of a batch fermentation. From this set of studies, it is noted that the presence of acetic acid in the media increases carbon efficiency of the fermentation as during stationary growth C. tyrobutyricum re-uptakes free acetic acid from the environment and converts it into butyric acid. This thesis also demonstrates the bioconversion of butanol to butyraldehyde by the methylotrophic yeast Pichia pastoris. P. pastoris were grown to high cell densities under glycerol feed and then induced to produce the endogenous alcohol oxidase (AOX) enzyme by beginning the culture on methanol consumption after a short starvation period. AOX converts short chain aliphatic alcohols to the corresponding aldehyde with the utilization of oxygen. The AOX enzyme is inhibited by the final product butyraldehyde so studies were performed utilizing alternative amine based pH buffering systems which also aid the bioconversion by binding free butyraldehyde as a Schiff-base. By binding the butyraldehyde longer bioconversions were observed. For these conversions, AOX activity was monitored with an absorbance based enzyme assay and the butanol substrate and butyraldehyde product were determined by gas chromatography.

No description available.

Användning av etanol som drivmedel och en efterfråga på gröna kemikalier driver utvecklingen av bioetanol framåt. Etanolpiloten, SEKAB, i Örnsköldsvik är en av få anläggningar i världen med kompetens och kunskap att producera bioetanol baserat på lignocellulosa. På senare tid har det dock uppstått problem vid etanolframställningen på grund av att en del jästodlingar blivit kontaminerade av bakterier vilket lett till ett sämre utbyte av biomassa och etanol. Det huvudsakliga syftet med detta examensarbete var att ta reda på orsaken till dessa misslyckade jästodlingar.   Examensarbetet delades upp i två huvudsakliga problemområden. Förutom orsaken till de kontaminerade odlingarna studerades även funktionen hos en ny jäststam, Saccaromyces cerevisiae torrjäst, i syfte att undersöka om det finns bättre alternativ till den jäststam som används i etanopiloten i nuläget.   En specialstudie av rengöringen av odlingstankar och ledningar i etanolpiloten utfördes i syfte att kartlägga var i utrustningen som infektionsrisken är som störst. Försöken påvisade att det huvudsakliga problemet kan lokaliseras till den största jästodlingstanken. Där befinner sig jästen under en längre tid i en miljö som är gynnsam för tillväxt av både jäst och bakterier. En annan orsak till de infekterade odlingarna är att rengöringen av utrustningen inte har skett på rätt sätt, samt att temperaturen hos tvättkemikalierna har varit för låg. En viktig slutsats är därför att bättre rutiner vid hanteringen av jästodlingsutrustningen samt att större noggrannhet i samband med rengöringen bör eftersträvas.   En bidragande orsak till de infekterade odlingarna kan också härröra från uppodlingsprocessen av ympjäst som i dagens läge sker på laboratorium. Genom att använda en stam av S. cerevisiae som köps in i frystorkad form kan flera steg i jästodlingsprocessen elimineras. Det både förkortar odlingsprocessen och minskar infektionsrisken. S. cerevisiae torrjäst undersöktes både i laboratorium och i etanolpiloten. Tre olika odlingsskalor användes, skakflaskor (250 ml), labfermentorer (3 l) och pilotskala (10m3). Försöken påvisar höga utbyten av både biomassa och etanol. För att kunna hålla nere produktionskostnaderna för etanolframställningen är det viktigt att jästen som används går att odla på det hydrolysat som produceras vid förbehandlingen av råvaran. Försök i pilotskala visar på lovande resultat vid uppodling av S. cerevisiae torrjäst när hela 70 % av sockerkällan kommer från hydrolysat. Ytterligare utvärdering och optimering av odlingsprocessen samt en ekonomisk jämförelse mellan de tillgängliga jäststammarna krävs dock innan S. cerevisiae torrjäst eventuellt kan användas kontinuerligt i pilotskala.

Screening of expression constructs suitable for protein pharmaceuticals is often done in batch cultivations. But the production of the recombinant protein is made during fed-batch cultivations. The two types of cultivations are different and therefore may good expression constructs that grow poorly in batch cultivations but good in fed-batch cultivations be rejected. Therefore would it be desirable to develop a fed-batch method that can be used in shake flasks. Biosilta has developed a method where starch is broken down into glucose by an enzyme creating fed-batch conditions. This method has been tried out and analyzed during this project. It is shown that the cells grown under these conditions can be glucose limited. However, at a later stage of the cultivation the cells produce a large amount of acetate and pH is not stable. The system builds on a booster tablet which content is unknown. If the booster is not added to the cultivations the cells stop growing, this indicates that there is some other limitation than just glucose. It is also seen that the amount of protein that is produced during this fed-batch mimic cultivation is much lower than that is produced during normal batch cultivations. I would therefore not recommend EnBase as a screening method. / Screening av nya rekombinanta proteiner som ska användas till läkemedel sker oftast i batch-odling. Men själva odlingen av proteinläkemedlet sker sedan under fed-batch förhållanden. Dessa två typer av odling är olika och då cellerna växer olika kan detta leda till att fel, eller den inte mest lämpade kandidaten väljs. Därför vore det önskvärt att ta fram en fed-batch liknande metod i skakkolvar. Biosilta har tagit fram en metod där ett enzym bryter ned stärkelse till glukos som påminner om fed-batch. Denna metod har testats och undersökts i detta examensarbete. Det har visat sig att cellerna som växer under dessa förhållanden är begränsade på glukos men producerar stora mängder ättiksyra under den senare fasen av odlingen och att pH varierar mycket. Systemet bygger mycket på att en booster-tablett tillsätts, vad denna tablett innehåller är okänt. Men om tabletten inte tillsätts slutar cellerna att växa, detta tyder på att det finns någon mer begränsning än glukos. Det visade sig även att protein produktionen blev mycket lägre än vid odling i batch-fas. Det skulle av anledning av ovanstående inte vara bra att använda sig av EnBase som en screening metod.