Universität Stuttgart
Permanent URI for this communityhttps://elib.uni-stuttgart.de/handle/11682/1
Browse
44 results
Search Results
Item Open Access Population and single-cell based quantitative analysis of protein kinase D-mediated regulation of the cell cycle(2014) Räth, Sebastian; Pfizenmaier, Klaus (Prof. Dr.)The cell cycle consists of G1, G2, S, and M phase and is a tightly regulated process with various checkpoints to control order and length of the separate phases. A multitude of signal molecules and pathways are involved in this process. In cancer, cell cycle control is often changed and understanding of these changes may result in new therapeutic targets in the treatment of patients. Additionally, cell cycle control is of special interest in stem cells as important decisions of cell fate – to proliferate or to differentiate - are part of cell cycle control. The success of adult stem cell therapeutic applications is thus dependent on in-depth understanding of this regulation. The Fluorescent ubiquitination-based cell cycle indicator (Fucci) is a sophisticated technology, which can easily determine G1 and/or S/G2/M phases of the cell cycle. The technology analyzes living cells in a spatio-temporal manner using fusion proteins consisting of two distinct cell cycle proteins fused to two fluorophores - a dual color scheme of orange and green. The aim of this thesis was to characterize the influence of Protein kinase D (PKD) using this technology in cells with adult stem cell characteristics and an established human cancer cell line. At first, a characterization of primary human mesenchymal stromal cells (MSC) derived from umbilical cord (UC) and bone marrow (BM) was performed. Furthermore, murine bone marrow stromal cells (mBMSCs) were isolated and osteogenic differentiation was investigated in tissue culture and in vivo. Three out of seven independent cell isolates showed the ability to differentiate into osteocytes, adipocytes, and chondrocytes in vitro. In vitro multipotency of an established mBMSC line was maintained over 45 passages. The osteogenic differentiation of this cell line was confirmed by quantitative polymerase chain reaction (qPCR) analysis of specific markers such as osteocalcin and shown to be Runx2 dependent. Notably, the cell line, when transplanted subcutaneously into mice, possesses full skeletal stem cell characteristics in vivo in early and late passages, evident from bone tissue formation, induction of vascularization, and host derived hematopoiesis. This cell line provides, thus, a versatile tool to unravel the molecular mechanisms governing osteogenesis in vivo thereby aiding to improve current strategies in bone regenerative therapy. Consequently, multipotent mBMSC lines were established from transgenic Fucci mice. Single cell analysis of cell cycle progression was performed in these Fucci-mBMSCs and Fucci transgenic human HeLa cells. Specifically, the influence of protein kinase D (PKD) and the RAF/MEK/ERK pathway on progression through S/G2/M phase was investigated in detail. Inhibition of PKD but not of MEK resulted in a delay in progression through S/G2/M phase in HeLa cells and mBMSCs. Furthermore, MAPK pathway activation was quantitatively assessed during the synchronous progression of HeLa cells through S/G2/M and successfully used to develop a quantitative mathematical model describing this pathway. Taken together this study demonstrates the benefit of quantitative and single cell analysis in cells with stem cell characteristics and an established cell line to enlighten the role of PKD in cell cycle control and, on top of that, support the notion that PKD is a potential new target for cancer therapy.Item Open Access Combinations of costimulatory antibody-ligand fusion proteins for targeted cancer immunotherapy(2013) Hornig, Nora; Kontermann, Roland (Prof. Dr.)Combinatorial strategies are of emerging interest in cancer immunotherapy. Costimulation by individual members of the Ig- or TNF-superfamily have already revealed promising antitumor potential, thus prompting the exploration of their synergistic abilities in combinatorial approaches. Here, in order to avoid systemic side effects, costimulation was restricted to the tumor site by pursuing a targeted strategy with antibody-ligand fusion proteins composed of tumor antigen-directed antibodies and the extracellular domain of the costimulatory ligands B7 or 4-1BBL, respectively. Costimulatory activity was assessed in an experimental model system where tumor cells coexpressed the antigens fibroblast activation protein (FAP) and endoglin (EDG) and initial MHC-independent T cell activation and tumor-targeting was mediated by a bispecific antibody (scDbFAPxCD3). Combined costimulation with B7- and 4-1BBL-fusion proteins (B7-DbFAP, scFvEDG-4-1BBL) was shown to be superior to the individual effects in terms of cytokine release (IL-2/IFN-γ), proliferation and activation marker expression (CD25), leading to a T cell population with enhanced levels of an activation-experienced memory phenotype and with a higher capability for target cell killing. Furthermore, the model system was adapted for a time-shift costimulation setting. Here, enhanced T cell proliferation and granzyme B expression as well as reduced levels of PD-1 expression demonstrated the benefit of B7.1- and 4-1BBL-costimulation-assisted restimulation. Consequently, the antitumor activity of this combinatorial setting was confirmed in vivo in a lung metastasis mouse model. Finally, the combinatorial spectrum was expanded by the generation and subsequent incorporation of antibody-fusion proteins comprising the extracellular domains of the TNF-superfamily ligands Ox40L, LIGHT or GITRL. Here, advantages of combined costimulation with either B7.1- or 4-1BBL- fusion proteins were shown in terms of T cell proliferation and IFN-γ release. In summary, combinatorial approaches with tumor-directed costimulatory ligands in form of antibody-ligand fusion proteins were shown to be feasible, revealing a great potential for the modulation and enhancement of a T cell response. Thus, they appear to be a promising strategy in cancer immunotherapy that should be considered for further investigation.Item Open Access Multivalent antibody-scTRAIL fusion proteins for tumor therapy : impact of format and targeting(2017) Hutt, Meike; Kontermann, Roland (Prof. Dr.)Item Open Access Regulation of endocytic membrane trafficking by the GTPase-activating protein Deleted in Liver Cancer 3 (DLC3)(2015) Braun, Anja Catharina; Olayioye, Monilola (Prof. Dr.)Small GTPases of the Rho family are key regulators of the actin and microtubule cytoskeleton, whereby many cellular functions including cell migration, adhesion and polarity, as well as cell cycle progression are controlled. Increasing evidence suggests that Rho proteins are also critically involved in the regulation of membrane trafficking pathways within exocytosis and endocytosis. Although the molecular mechanisms are not well understood, Rho GTPases apparently have to govern and finely tune cytoskeletal remodeling, in order to support the formation, fusion and motility of transport carriers. However, the identity of their regulators, the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that ensure the balanced GTPase activation in space and time is largely elusive. The ‘Deleted in Liver Cancer’ (DLC1/2/3) proteins are a structurally conserved subfamily of RhoGAP proteins that act as negative regulators of Rho GTPases. In addition to the catalytically active GAP domain, all DLC proteins contain a sterile alpha motif (SAM) and steroidogenic acute regulatory protein-related lipid transfer (START) domain. Expression of the best studied member, DLC1, is frequently lost in various types of human cancers and a tumor suppressive function associated with its RhoGAP activity has been established in vivo. Although DLC3 was also observed to be downregulated in several cancer cell lines and primary tumors, the cellular functions of DLC3 are still poorly characterized. So far, GAP activity for RhoA has only been demonstrated in vitro and, associated with its localization at cell-cell contacts, a Rho-regulatory role in adherens junction stability was described. Thus, the aims of this thesis were to further investigate the subcellular localization of DLC3 and to shed light on the role of DLC3 in the regulation of Rho-mediated cellular processes, in particular endocytic membrane trafficking. This study provides convincing evidence that DLC3 is a functional, Rho-specific GAP protein in living cells and that its loss enhances perinuclear RhoA activity. DLC3 is recruited to Rab8-positive membrane tubules and required for the integrity of the Rab8 and Golgi compartments. Depletion of DLC3 impairs the transport of internalized transferrin to the endocytic recycling compartment, which is restored by the simultaneous downregulation of RhoA and RhoB. As a consequence, DLC3 loss interferes with epidermal growth factor receptor (EGFR) degradation and causes prolonged receptor signaling. Furthermore, it was found that DLC3-depleted cells show reduced surface N-cadherin levels, leading to decreased cell aggregation. Together, these findings identify DLC3 as a novel component of the endocytic trafficking machinery, wherein it maintains organelle integrity and regulates membrane transport via the control of local Rho activity.Item Open Access Die Rolle von XIAP und Caspase-8 bei der Progression und Resistenz humaner Melanomen(2012) Hörnle, Manuela; Scheurich, Peter (Prof. Dr.)Das maligne Melanom zählt auch heute noch zu den gefährlichsten und tödlichsten Krebserkrankungen weltweit. Eukaryote Zellen stellen ein vielschichtiges multifaktorielles System dar, in welchem die Summe aus pro- und anti-apoptotischen Proteinen im Kontext der zellulären Gesamtsituation über das Schicksal der einzelnen Zelle zugunsten des gesamten Organismus entscheidet. Dieser Entscheidung liegt ein komplexes und mannigfaltiges System zugrunde, in dem eine Vielzahl von Variablen und Faktoren moduliert und reguliert werden. Das Verständnis dieser zugrundeliegenden molekularen Mechanismen stellt das Fundament zur Bekämpfung dieser tödlichen Krankheit dar. Im Rahmen dieser Arbeit konnte gezeigt werden, dass der XIAP-Caspase-3-crosstalk das Schlüsselmoment der Entscheidung zwischen Leben und Sterben in humanen Melanomzellen darstellt. Initial TRAIL-resistente Melanomzellen können durch eine sublethale UVB-Bestrahlung für diese Rezeptor-vermittelte Apoptose sensitiviert werden. Dabei wird eine Beteiligung des intrinsischen mitochondrialen Apoptose¬wegs und den damit assoziierten pro-apoptotischen Faktoren nachgewiesen. Die Antagonisierung des anti-apoptotischen XIAP-Proteins führt zur vollständigen katalytischen Aktivierung der Effektor-Caspase-3 und resultiert final in einem positiven regulatorischen feedback-loop, in dem Caspase-3 durch proteolytische Spaltung die Degradierung des XIAP-Proteins initiiert. Zudem kann in der vorliegenden Arbeit eine weitere essentielle Funktion für Caspase-8 nachgewiesen werden. Diese vermeintlich ausschließlich pro-apoptotische Initiator-Caspase demonstriert eine fundamentale Bedeutung zur Aufrechterhaltung der zellulären Homöostase in humanen Melanomzellen. Neben ihrer etablierten Rolle bei der Initiierung der Rezeptor-vermittelten Apoptose zeigt sich diese Protease als essentiell für die Progression durch die S-Phase des Zellzyklus. Dabei kann von einer Beteiligung der Caspase-8-Prodomäne an der DNA-Reparatur sowohl während der regulären DNA-Replikation als auch nach DNA-Schaden ausgegangen werden. Die Abwesenheit von Caspase-8 führt zur Arretierung humaner Zellen in der S-Phase des Zellzyklus und resultiert in letzter Instanz in der Induktion des apoptotischen Zelltods über den intrinsischen mitochondrialen Signalweg.Item Open Access Serum albumin and its interaction with the neonatal Fc receptor (FcRn) : characterization of the albumin/FcRn-binding mechanism(2013) Färber-Schwarz, Aline; Kontermann, Roland (Prof. Dr.)In the past a lot of different biomolecular therapeutics were developed. A common problem using small molecules in therapy is their reduced serum concentration after short time period. The human body clears therapeutic molecules which have a size beneath the kidney clearance threshold in minutes to hours. The consequences are more frequent and high application doses. These facts demand the development of half-life prolonging strategies. plasma proteins, like IgG’s and albumin are recycled by the neonatal Fc receptor (FcRn) resulting in an extraordinary long circulating half-life which is used to prolong the serum circulation of small therapeutic proteins. Non-covalent binding or fusion to the Fc-part of IgG’s or albumin significantly improves the pharmacokinetic properties of small proteins. The aim of this study was to understand the interaction mechanism between albumin and the FcRn and to investigate the impact of point-mutations on the pharmacokinetic properties of albumin. A set of mouse serum albumin (MSA) mutants was generated and their pharmacokinetics were analyzed in vivo. Mutations of H464, E501, E505, D512, H510, S511, E531, H535, D565, T570 and E571 affect the in vivo half-life of albumin in mice. Even if these results are statistically not significant they still suggest the importance of the investigated amino acid residues for the albumin/FcRn binding. The highly conserved histidine residues H464, H510 and H535 play a major role in the albumin/FcRn interaction. In addition to the in vivo experiments a steered molecular dynamic (SMD) visualizes that the domain III of albumin removes as the last part of the protein from the receptor. Furthermore, the polar interactions beween MSA and the mouse FcRn were analyzed. Thus the amino acids S111 and L112 within the domain I and the amino acids E425, L466, T467 and P468 within the domain III of albumin were identified to be responsible for the FcRn interaction. Moreover, phage display was used to mature the affinity of MSA towards the mouse FcRn. A MSA library has been generated by site directed mutagenesis of five amino acids within the domain III of albumin. The selection of MSA variants with an increased affinity to the FcRn was not successful. Further approaches are required to mature the affinity of MSA towards the mouse FcRn.Item Open Access Phenocopy : a strategy to qualify chemical compounds during hit-to-lead and/or lead optimization(2010) Baum, Patrick; Kontermann, Roland (Prof. Dr.)A phenocopy is defined as an environmentally induced phenotype of one individual which is identical to the genotype determined phenotype of another individual. In the present work, the phenocopy phenomenon has been translated to the drug discovery process as phenotypes produced by the treatment of cellular systems with small interfering RNAs (siRNAs) or new chemical entities (NCE) may resemble environmentally induced phenotypic modifications. Various new chemical entities exerting inhibition of the kinase activity of Transforming Growth Factor Beta Receptor I (TGF-betaR1) were ranked by high-throughput RNA expression profiling. This chemical genomics approach was able to unravel both on-target effects (effects, caused by the inhibition of the drug target) and off-target effects (effects, caused by the interaction of the NCE with additional molecules). It resulted in a precise time-dependent insight into the TGF-beta biology (referred to as on-target signature) and allowed furthermore a comprehensive analysis of each NCE's off-target effects (re-ferred to as off-target signatures). Both signature types can support the drug discovery process. The on-target signature helps to characterize the mode of action of the drug target (TGF-betaR1) and thereby supports the target validation as well as the assay development process. Furthermore, the evaluation of off-target effects by the Phenocopy approach allows a more accurate and integrated view on the mode of action of the compounds, supplementing classical biological evaluation parameters such as potency and selectivity. The presented proof of concept study allowed the ranking of NCEs that were before indistinguishable solely based on potency and selectivity. According to the newly introduced criteria, several of the tested NCEs revealed liabilities at e.g. the induction of off-target effects and of induction of gene regulation inverse to the desired TGF-beta inhibition effect, at the induction of cell death, at acting as pro-inflammatory stimuli and as promoting cellular growth and at induction of cancer pathways. Ultimately, this approach has therefore the potential to become a novel method for ranking compounds during various drug discovery phases.Item Open Access Biomarker discovery and pathway activation profiling in breast cancer using protein microarrays(2013) Sonntag, Johanna Sigrid Nelly; Kontermann, Roland (Prof. Dr.)Breast cancer, the most frequent cancer entity among women, is nowadays widely recognized as a heterogeneous disease in terms of histopathology as well as on the molecular level. Over the last few years, gene expression profiling studies have improved our understanding of the underlying molecular mechanisms associated with the very heterogeneous outcomes of breast cancer patients. The existence of intrinsic molecular subtypes, which are linked to unique biological and prognostic features, was repeatedly demonstrated and points to the need of tailored therapy options. However on the functional level, breast cancer is not only a genomic but mainly a proteomic disease and gene expression profiling might provide only limited insights. Following the hypothesis that intrinsic biologic features of breast tumors affect prognosis and also therapy response, the general aim of this thesis was to further explore breast cancer heterogeneity with protein microarrays on the functional proteomics level. Around 70 – 80% of all breast cancer patients belong to the luminal intrinsic molecular subtype, characterized as a surrogate marker by overexpression of hormone receptors. An improved classification of this subtype is crucial for therapy decision as part of the patients are at higher risk of recurrence requiring chemo-endocrine treatment, whereas the other part is at lower risk and does not benefit from chemotherapy. However, accurate definition of low and high risk hormone receptor-positive breast cancer has remained a challenge so far. Thus, the first objective of this thesis was the identification of a robust and quantitative protein biomarker signature to facilitate risk classification of hormone receptor-positive breast cancer. To approach this aim reverse phase protein arrays were used to screen across over 120 breast cancer relevant proteins and a novel bioinformatics workflow for biomarker hit selection was applied. Using this approach, a biomarker signature consisting of caveolin-1, NDKA, RPS6, and Ki-67, was identified as most promising to distinguish between low and high risk hormone receptor-positive breast cancer. Since genomic and transcriptomic profiling alone cannot sufficiently predict protein pathway activation, it is important to explore and define the heterogeneity of hormone receptor-positive breast cancer on the proteome level. Especially as protein signaling pathways present the direct targets of new classes of therapeutics. Thus, the second thesis objective addressed the question of whether hormone receptor-positive breast cancer can be further categorized according to similar signaling pathway activation patterns and whether these patterns reflect common molecular mechanisms. Therefore, comprehensive protein pathway activation profiles of breast cancer specimens were obtained using reverse phase protein arrays. To complement this analysis, a microspot immunoassay was developed, which enabled the simultaneous quantification of eight different growth factors in tumor lysate as well as blood plasma of matching patient samples. Four subgroups were identified, based on differential expression of 90 cancer-relevant signaling proteins. Each subgroup showed unique characteristics which were also related to established clinicopathological features as well as growth factor expression. One subgroup, for example, was characterized by high expression levels of almost all analyzed proteins. In addition, VEGF tumor lysate levels were significantly higher in this subgroup and an enrichment of poorly differentiated tumors was observed underlining the aggressive phenotype. In contrast, another subgroup was characterized by weak signaling activity. Interestingly, this subgroup was mostly associated with invasive lobular carcinoma, the second most common histologic type of breast cancer, reflecting a link between histopathology and underlying molecular mechanisms. In summary, the reverse phase protein array based pathway activation profiling of hormone receptor-positive breast cancer, presented in this thesis, provides a comprehensive snapshot of the heterogeneity of this subtype on the proteomic level. Insights obtained can serve as basis to refine the concept of clinically relevant subtypes towards an improved definition of patient-tailored therapy options.Item Open Access A soluble TNF receptor 2 agonist as a new therapeutic approach to treat autoimmune and demyelinating diseases(2011) Fischer, Roman; Pfizenmaier, Klaus (Prof. Dr.)Tumor necrosis factor (TNF) exerts its biological functions via two distinct receptors. Whereas the TNF receptor (TNFR) 1 mainly mediates inflammatory responses, the TNFR2 is involved in tissue protection and regeneration. Accordingly, TNF variants selectively activating TNFR2 could potentially be useful as therapeutic regimen in a variety of diseases. In this study, there was developed a TNFR2-specific agonist, which may be a promising therapeutic in immune- and neurodegenerative diseases. In addition, the molecular mechanisms of TNFR2 signaling and turnover at the membrane were further unrevealed. Endocytosis is an important mechanism to regulate TNF signaling. In contrast to TNFR1, the relevance of receptor internalization for signaling as well as the fate and route of internalized TNFR2 is poorly understood. Upon generation of a human TNFR2-expressing mouse embryonic fibroblast cell line in a TNFR1-/-/TNFR2-/--background, I could demonstrate that TNFR2 was internalized together with its ligand and cytoplasmic binding partners. The internalization was dependent on a di-leucin motif in the cytoplasmic part of TNFR2 and the colocalization of the receptor-complex with clathrin suggested clathrin-mediated internalization of TNFR2. Internalization-defective TNFR2 mutants were capable to signal, i.e. activate NFkB, demonstrating that the di-leucin motif-dependent internalization is dispensable for this response. Therefore receptor internalization primarily seems to serve as a negative feed-back to limit TNF responses via TNFR2. Soluble recombinant TNF is a strong mediator of inflammation, predominantly through TNFR1 activation, as soluble TNF is not sufficient to activate TNFR2. In contrast, the membrane-bound form of TNF (memTNF) fully activates both TNFRs. Therefore, TNFR2-specific therapeutics need to comply with two basic requirements: mimicry of memTNF and, in order to avoid dose limiting severe inflammatory responses, receptor selectivity. As a basis for the construction of a memTNF-mimetic, TNFR2-selective TNF variant, a single-chain TNF (scTNF) molecule was used, that consists of three TNF monomers fused by short peptide linkers. Introducing two amino acid exchanges (D143N/A145R) into a scTNF variant resulted in the loss of TNFR1 affinity under retention of TNFR2 binding. To mimic memTNF, such a receptor-selective single-chain TNF (scTNFR2) was linked to the tenascin C (TNC) trimerization domain, resulting in stabilized TNC-scTNFR2 nonamers with respect to the TNF domains. In vitro TNC-scTNFR2 demonstrated memTNF-mimetic activity and exclusively activated TNFR2. TNC-scTNFR2-enhanced T cell activation was shown by the increased interleukin 2-dependent interferon gamma production. More revealing, TNC-scTNFR2 increased the number of regulatory FoxP3+/CD25+ T cells in cultures of human peripheral blood mononuclear cells, suggesting a potential role in downregulation of T cell immune responses. In cultures of primary astrocytes TNC-scTNFR2 induced the upregulation of ciliary neurotrophic factor, a neurotrophic factor, which enhances the formation of myelin. In addition, in in vitro cultures, TNC-scTNFR2 rescued differentiated neurons from hydrogen peroxide-induced cell death. First in vivo studies on the pharmacokinetic behavior and potential systemic responses in huTNFR2-transgenic mice revealed that compared to TNF, TNC-scTNFR2 has a dramatically extended plasma half-life, yet shows no signs of systemic toxicity and thus is well tolerated even at doses several fold above the MTD of wildtype TNF. These results warrant further studies on the therapeutic usefulness of TNC-scTNFR2 in appropriate animal models of autoimmune and neurodegenerative diseases.Item Open Access Prozessentwicklung zur Produktion und Reinigung eines PEGylierten Antikörperfragmentes(2019) Lindner, Robert; Kontermann, Roland (Prof. Dr.)Die vorliegende Arbeit beschreibt die Etablierung eines präparativen Herstellungsprozesses für ein PEGyliertes, humanisiertes Antikörperfragment (scFv) welches gegen den epidermalen Wachstumsfaktorrezeptor gerichtet ist. Dies beinhaltet die Optimierung der prokaryotischen Expression in Escherichia coli BL21 DE3 rha- mittels Hochzelldichtekultivierung. Durch Zugabe von Rhamnose erfolgt die Transkription des Zielproteingenes durch Aktivierung des rhaBAD Promotors. Um eine korrekte Faltung und Ausbildung der Disulfidbrücken im Zielprotein zu gewährleisten wird direkt nach der Translation mit Hilfe des pelB-Signalpeptides der Proteintransfer in den periplasmatischen Raum veranlasst. Durch Identifikation der optimalen Bedingungen war es möglich die spezifische Produktbildungsrate signifikant zu erhöhen und somit die volumetrische Ausbeute bis auf 0,73 g/L zu steigern. Das scFv, welches zum Zeitpunkt der Ernte in gelöster Form im Periplasma vorlag, konnte durch einen optimierten Extraktionsschritt vom Großteil der Wirtzellkomponenten getrennt werden. Hierfür wurde ein kombiniertes Verfahren entwickelt, welches einen Einfrier- und Auftauschritt mit einem osmotischen Schock und anschließender saurer Fällung von Wirtzellproteinen kombiniert. Dadurch konnten zwei Drittel des produzierten scFv-hu225 mit einer Reinheit von etwa 20 % für den nachfolgenden Reinigungsprozess bereitgestellt werden. Die weitere Reinigung des scFv bis hin zur pharmakologischen Qualität erfolgte, ohne Inkludierung von Diafiltrationsschritten, durch eine Kaskade von drei bis vier aufeinanderfolgenden Chromatographieschritten. Als innovativ gilt hierbei die Verwendung einer Mixed-Mode-Chromatographie als gleichzeitige Capturing- und Entsalzungsmethode. Dadurch konnte direkt im Anschluss eine Anionenaustauscher-Chromatographie zur Bindung von Endotoxinen und Nukleinsäuren zur Anwendung gebracht werden. Zur Verringerung des Wirtzellproteinanteiles wurde im Anschluss in orthogonaler Funktionsweise ein Kationenaustauscher verwendet. Als optionalen Schritt wurde eine Hydroxylapatit-Chromatographie in negativer Funktionsweise entwickelt. Da der gesamte Reinigungsprozess auch im scale-up möglichst ökonomisch und unter GMP umsetzbar sein sollte, wurde er so entwickelt, dass dieser innerhalb eines Tages durchgeführt werden konnte. Es wurde dabei eine Reinheit von über 99 % erreicht. Die volumetrische Ausbeute an scFv-hu225 lag mit einer Wiederfindung von über 50 % bei 0,38 g pro Liter Fermentation. Um die pharmazeutische Halbwertszeit des gereinigten Wirkstoffes zu verlängern wurde das Molekulargewicht, mittels der Kopplung von Polyethylenglycol (PEGylierung), erhöht. Die Reaktion erfolgte in diesem Fall ungerichtet durch Aldehyd-Chemie an primäre Amine. Im Rahmen dieser Arbeit wurden eine 5 kDa mit einer 30 kDa PEGylierung bezüglich der Prozessführung und dem in vivo Verhalten miteinander verglichen, wobei der Fokus auf einem mono-PEGylierten Produkt lag. Es stellte sich heraus, dass mit zunehmendem PEGylierungsgrad eine Trennung von mono-PEGylierten Zielprotein schwieriger und unwirtschaftlicher wird. Die Pharmakokinetik konnte nachweislich nur mit der 30 kDa PEGylierung signifikant erhöht werden.