Recently HER3, member of the epidermal growth factor receptor family (EGFR), has been found to play a crucial role in the development of resistance towards inhibitors that are given to patients with HER1- and HER2-driven cancers. As HER3 is up-regulated or over-activated in several types of human cancers, it is of outmost importance that new innovative drugs target its oncologic activity. The Affibody® Molecule Z08698 inhibits the heregulin induced signalling of HER3 with high affinity (KD~50 pM). As the Affibody® Molecule is small, has high solubility and outstanding folding kinetics, an effective penetration of tumour tissue is suggested together with a rationalized manufacturing process. Further coupling to an albumin binding domain (ABD) expands the plasma half-life of the molecule, hence increasing the molecule's potential of serving as a therapeutic. A process development for production of Z08698-VDGS-ABD094 has been established, where the molecule is efficiently produced in the E. coli host strain BL21(DE3), through a T7 based expression system. Cultivations were performed with a fed-batch fermentation process and the conditions were further optimized in order to obtain highest expression, while avoiding undesirable modifications like gluconoylations. By employing Design of experiments in combination with multivariate data analysis, a production process resulting in ~3.5 g product/ l culture could be verified. Moreover, thermolysis was evaluated as a suitable method for cell disruption, enabling an easy and cost-effective manufacturing process of the ABD fused Affibody® Molecule.

Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for allknown forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes infood industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, anumber of techniques, all collectively referred to as ‘protein engineering’, are performed in thelaboratory.Traditionally, methods involving ‘rational design’, where a few alterations are introduced atspecific protein locations to hopefully result in expected improvements have been applied.However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, fromwhich rare clones showing enhanced properties can be isolated have contributed greatly to thefield of protein engineering.In the present thesis, different protein traits of biotechnological importance have beenaddressed for improvements by the use of such methods, in which there is a crucial need tomaintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterialcell-membrane compartmental confinement.In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genesencoding variants showing an enhanced soluble expression of the enzyme frequently used inbiotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein(GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles,was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay(PCA), based on the affinity driven structural complementation of a genetically split β-lactamase enzyme was used to identify variants having desired target protein binding abilities,including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to thehuman cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection. / QC 20100729

I detta projekt har uttrycket av Affibodymolekyler i bioreaktorer av olika volymetriska skalor jämförts för att fastställa om ett tillförlitligt samband mellan de olika bioreaktorernas prestanda kan etableras för att möjliggöra utvecklingen av en nedskalad produktionsmodell för Affibodymolekyler. Baslinjen för jämförelsen i denna studie har varit en enliters- stirred tank reactor (STR) som de andra (mindre) bioreaktorernas prestanda jämfördes med. Jämförelsen av prestanda gjordes genom odling och uttryck av sex olika Affibodymolekyler i replikat i varje bioreaktorstorlek. Prestandan i detta fall hänvisar till produktionen av Affibodymolekyler (mg/L Cellodling) under odlingen, som fastställdes genom ett protokoll för proteinrening av lösligt intracellulärt protein genom affinitetskromatografi och kvantifiering genom absorbans vid 280 nm. De sex olika Affibodymolekyler som har studerats i detta projekt hade tidigare visat sig ha olika uttrycksnivåer, och har här jämförts med varandra i de olika bioreaktorskalorna. De två olika bioreaktorstorlekarna som bedömdes var en 300 mL skakkolv med 50 mL arbetsvolym och en mikrotiterplatta (MTP) med 3 mL arbetsvolym. Dessutom innebar studien en bedömning av två system för en långsam frisättning av kolkälla i odlingarna, ett i varje nedskalad bioreaktorstorlek. Detta utfördes för att delvis efterlikna den kontrollerade fed-batch-kulturen i STR, där koltillförsel kontrollerades med hjälp av en feedprofil. Resultaten visade att proteinuttrycket av metoderna med en långsam frisättning av kolkällor överensstämde närmast med proteinuttrycket i STR. Anpassningen av dessa resultat mot proteinuttrycket hos STR gav i en linjär regression ett R2 på 99,69 % i 3 ml MTP och 97,46 % i 50 ml skakkolven. Slutsatserna som drogs var att SMFP08003 FeedPlate var den bästa kandidaten för en nedskalad modellering av uppströmsprocessen för produktion av Affibodymolekyler. Samt att faktorn att använda en fed-batch-process istället för en batch-process har en större inverkan på proteinuttrycket än skalan av processerna. / In this project the expression of Affibody® molecules in bioreactors of different volumetric scales has been compared, to determine if a reliable relation between the performance of the different bioreactors can be established to allow for the development of a scale-down Affibody® molecule production protocol. The baseline of comparison in this study has been a one litre Stirred Tank Reactor (STR) to which the other (smaller) bioreactors' performance were compared. The performance comparison was achieved by the cultivation and subsequent expression of six different Affibody® molecules in replicates in each bioreactor size. Performance in this case refers to Affibody® molecule production (mg/L culture) during the cultivation, which is assessed by a protocol of protein purification of soluble intracellular protein by affinity chromatography and quantification by 280 nm absorbance. The six different Affibody® molecules studied in this project had previously been found to have different expression levels, and were in this project compared to each other in the different bioreactor scales. The two different bioreactor sizes which were assessed were a 300 mL shake flask with 50 mL working volume and a 3 mL working volume microtiter plate (MTP). In addition, the study involved an assessment of the use of two systems for a slow carbon source release in the cultivations, one in each scale-down bioreactor size. This was performed to partly mimic the controlled feed systems in STRs. The results showed that the protein expression of the methods with a slow carbon source release corresponded most closely with the protein expression in the STR. The fit of these results onto the protein expression of the STR yielded a R2 of 99.69% in the 3 mL MTP and 97.46% in the 50 mL shake flask. The conclusions drawn were that SMFP08003 FeedPlate was the best candidate for scale-down modelling of the upstream process for production of Affibody® Molecules and that the factor of using a fed-batch process instead of a batch process has a larger impact on the protein expression than the scale of either process.