Made available in DSpace on 2014-10-09T12:55:16Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:00Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:06/52973-9

Made available in DSpace on 2014-10-09T12:55:16Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:00Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A Prolactina humana hPRL é um hormônio protéico com 199 aminoácidos (MM ~ 23.000 Da) com um amplo espectro de atividades biológicas, sendo mais conhecido por estimular a lactação e regular o crescimento e diferenciação da glândula mamária. Além de quebra proteolítica, a maioria dos variantes de prolactina podem ser resultantes de outros processos pós-traducionais como polimerização, fosforilação, desamidação, sulfatação e glicosilação. Essa proteína contém apenas um sítio potencial de glicosilação por ligação à asparagina, localizada no aminoácido 31, que é parcialmente ocupado (10%) quando a proteína é sintetizada em células eucariotas. Apesar da atividade biológica in vitro da prolactina glicosilada (G-hPRL) ser muito menor (~4 vezes) quando comparada à não glicosilada, sua função fisiológica ainda não é bem definida e a porção de carboidrato parece ter um importante papel na biossíntese, secreção, atividade biológica, e clearance plasmático do hormônio. Com o objetivo de melhor caracterizar e estudar esta variante hormonal, foi realizada sua purificação em escala laboratorial a partir de células de ovário de hamster chinês (CHO) modificadas geneticamente, utilizando meio de cultura suplementado com cicloheximida, aumentando ~4 vezes sua concentração absoluta e ~10 vezes a razão entre a isoforma glicosilada e a não-glicosilada. A purificação da G-hPRL seguiu um processo simples e efetivo de duas etapas principais baseado em uma coluna de troca catiônica e uma coluna preparativa de exclusão molecular acoplada a um sistema de cromatografia líquida de excusão molecular de alta eficiência (HPSEC). A caracterização foi feita por HPLC de fase reversa (RP-HPLC) e exclusão molecular, SDS-PAGE, Western Blotting, espectometria de massa (MALDI-TOF) e um bioensaio in vitro utilizando células Nb2 e Ba/F3-LLP. Nossos resultados demostram que a cicloheximida pode ser uma importante ferramenta para aumentar a produção de prolactina glicosilada, facilitando a purificação e caracterização dessa isoforma. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:06/52973-9

Understanding the molecular mechanisms that govern productivity and growth of recombinant host cells is essential to devise informed approaches to increase commercial viability and availability of biopharmaceuticals. This work has focused on the roles of the mammalian target of rapamycin complex 1 (mTORC1) signalling pathway in CHO cells, the most widely used expression system in the biopharmaceutical industry. mTORC1 is a master regulator of cell growth, protein synthesis and metabolism in response to availability of nutrients, oxygen and growth factors. Therefore, it was hypothesised that increased activity of mTORC1 enhances growth and productivity of recombinant CHO cells. The study of a recombinant GS-CHO cell line in the serum-free suspension batch culture indicated a gradual decrease in the activity of mTORC1, as defined by the decreased extent of site-specific phosphorylation of two widely ascribed downstream target proteins (ribosomal protein S6 kinase 1 (S6K1) and 4E-BP1, an inhibitor of translation initiation). The decline in the activity of mTORC1 paralleled decreased growth rate, recombinant protein specific productivity and global protein translation. To further clarify the role of the mTOR pathway in cell growth and protein production, cells in batch culture were treated with rapamycin, a specific inhibitor of mTORC1. Treatment with rapamycin stalled the growth of the CHO cell line transiently, but recombinant protein specific productivity, longevity of batch culture, and final antibody titre were greater than control. Rapamycin addition produced discriminating effects on downstream signalling targets, implicating distinct roles for these targets in control of growth and protein synthesis. Engineering the mTORC1 pathway by overexpression of specific components of this pathway (S6K1 and Rheb) generated increased growth and extended viability. Greater proliferation was not associated with improved productivity suggesting highly proliferative phenotypes that prioritise cell growth over synthesis and secretion of recombinant antibody in the recombinant GS-CHO cells examined. Therefore, the engineering of mTORC1 pathway may be beneficial to increase robustness or adaptation to stressed conditions (such as serum- free suspension growth, low nutrition availability and hypoxia).

The increasing demand for recombinant therapeutic proteins coupled with advances in technologies allow research to develop approaches to improve the efficiency, yield, and quality of biopharmaceutical products from CHO cells. CHO DG44 cells used in this study were engineered to express erythropoeitin (EPO) as the model recombinant protein in a DHFR-based selection system. From a series of CHO-DG44 cell lines derived from a polyclonal population, one cell line expressed a notable change in growth phenotype during prolonged culture (10 weeks). This cell line (IJ4) exhibited prolonged growth, reached a greater density, and delayed cell death. The change in growth was reflected in an increased total yield of EPO, whilst the specific productivity of cell line IJ4 remained similar. The increased total yield of EPO presents a desirable goal for production and hence detailed ‘omics studies were performed to identify factors associated with better cell growth and survivability. Two different ‘omics analyses were performed (microarray transcriptomic and GC-MS metabolic profiling) to identify potential target genes and key metabolites associated with changes in growth profile. The -omics analyses identified a subset of genes (MMP20, PLA1A, POSTN, SLC46A3, and TOP2A), and a metabolic marker (farnesal) strongly associated with changes in cell growth and nutrient uptake. The use of complementary ‘omics approaches to identify molecular markers has allowed an integrated model to be built, which explains how CHO cell phenotype can adapt to long-term culture, and this defines molecular approaches for cell line screening and engineering.

The number of publications in the biomedical field increases exponentially, which makes the task of keeping up with current research more and more difficult. However, rapid advances in the field of Natural Language Processing (NLP) offer possible solutions to this problem. In this thesis we focus on investigating three main questions of importance for utilizing the field of NLP, or more specifically the two subfields Named Entity Recognition (NER) and Large Language Models (LLM), to help solve this problem. The questions are; comparing LLM performance to NER models on NER-tasks, the importance of normalization, and how the analysis is affected by the availability of data. We find for the first question that the two models offer a reasonably comparable performance for the specific task we are looking at. For the second question, we find that normalization plays a substantial role in improving the results for tasks involving data synthesis and analysis. Lastly, for the third question, we find that it is important to have access to full papers in most cases since important information can be hidden outside of the abstracts.

Electroporation is an efficient, low-toxic physical method which is used to deliver impermeant macromolecules such as genes and drugs into cells. Genetic modification of the cell is critical for many cell and gene therapy techniques. Common electroporation protocols can only handle small volumes of cell samples. Also, most of the conventional electroporation methods require expensive and sophisticated electro-pulsation equipment. In our lab, we have developed new electroporation methods conducted in microfluidic devices. In microfluidic-base electroporation, exogenous macromolecules can be delivered into cells continuously. Flow-through electroporation systems can overcome the issue of low sample volume limitation. In addition, in our method, electro-pulsation can be done by using a simple dc power supply, without the need for any extra equipment. Furthermore, our microfluidic chips are completely disposable and cheap to produce. We show that electroporation and electroporation-based gene delivery can be conducted employing tapered asymmetric curving channels. The size variation in the channel's cross-sectional area makes it possible to produce electric pulses of various parameters by using a dc power supply. We successfully delivered Enhanced Green Fluorescent Protein, EGFP, plasmid DNA into Chinese Hamster Ovary, CHO-K1, cells in our microfluidic chips. We show that the particles/cells undergo Dean flow in our asymmetric curving channels. We demonstrate that there are three main regimes for particle motion in our channels. At low flow rates (from 0 to ~75μl/min) cells do not focus and they randomly follow stream lines. However, as flow rate increases (~75 to 500μl/min), cells begin to focus into one line and they follow a single path throughout the micro-channel. When flow rate exceeds ~500μl/min, cells do not follow a single line and demonstrate more complex pattern. We show that the electric parameters affect the transfection efficiency and cell viability. Higher electric field intensity results in higher transfection efficiency. This is also true in the cases with longer electroporation duration time. In our experimental work, we executed flow-through electroporation for various duration times (t = 2 ms, 5 ms, and 7 ms), and at various electric field intensities (from 300 to 2200 V/cm) while we utilized different flow rates as well, i. e. 150 μl/min (focused flow) and 600 μl/min (complex flow). To explore the impact of individual electric pulse length and electric pulse number on electroporation results, we designed control channels with straight narrow sections. Cells experience different hydrodynamic forces in straight channels compared to curving channels. Flow pattern and cell focusing were also studied in control channels as well. Also, electroporation on CHO-K1 cells was successfully conducted in control channels. The hydrodynamic forces under the conditions we used do not appear to show substantial impact on transfection efficiency. / Master of Science

Nestas últimas décadas, investigações sobre compostos naturais, efetivos, não tóxicos, com potencial radioprotetor vêm despertando um grande interesse em consonância com a utilização crescente de vários tipos de radiação ionizante nas mais diversas finalidades. Entre eles a própolis uma resina coletada pelas abelhas (Apis mellifera), vem sendo apontada como promissora por apresentar uma série de características biológicas vantajosas, por exemplo, anti-inflamatória, antimicrobiana, antitumoral, imunomoduladora, antioxidante e também scavenger de radicais livres. O presente estudo teve como objetivo principal, averiguar efeito da própolis brasileira procedente de Rio Grande do Sul (AF 08) em células de ovário de hamster Chinês (CHO-K1) e em células tumorais de próstata (PC3), irradiadas com 60Co. Para tanto, foram utilizados três principais parâmetros interligados entre si: indução de micronúcleo, viabilidade celular e morte clonogênica. A escolha destes parâmetros se justifica pelo seu significado biológico, além do fato de serem prontamente observáveis e mensuráveis em células irradiadas. Os dados citogenéticos obtidos, mostraram um efeito radioprotetor da própolis (5 - 100 μg/ml) na indução de dano ao DNA em ambas as linhagens celulares, irradiadas com doses de 1 - 4 Gy. No entanto, o ensaio de citotoxicidade mostrou um efeito antiproliferativo pronunciado da própolis (50 - 400 μg/ml) em células PC3 irradiadas com 5 Gy. As curvas de sobrevida obtidas foram ajustadas satisfatoriamente pelo modelo linear-quadrático, cujo componente foi mais alto em células CHO-K1. Quanto à capacidade clonogênica, as células PC3 mostraram-se mais radiossensíveis que as células CHO-K1 nas doses mais altas da curva de sobrevida. A própolis, nas concentrações de 30 - 100 μg/ml, não influenciou no potencial clonogênico das células PC3, visto que, as curvas de sobrevida associadas ou não com a própolis, mostraram perfis similares, ao passo que o tratamento combinado em células CHO-K1 exibiu um efeito estimulador da proliferação. Os dados obtidos in vitro mostraram um potencial uso da própolis AF-08, como uma substância natural e não tóxica, na prevenção contra os efeitos danosos da radiação ionizante, nas doses e nas concentrações analisadas. / In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60Co radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 μg/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400 μg/ml) in PC3 cells irradiated with 5 Gy. The survival curves obtained were adequately fitted by a linear-quadratic model, where the coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 μg/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data obtained in vitro showed a potential use of propolis AF-08, a natural and nontoxic compound, in the prevention against the adverse effect of ionizing radiation, at the doses and concentrations here analyzed.

Nestas últimas décadas, investigações sobre compostos naturais, efetivos, não tóxicos, com potencial radioprotetor vêm despertando um grande interesse em consonância com a utilização crescente de vários tipos de radiação ionizante nas mais diversas finalidades. Entre eles a própolis uma resina coletada pelas abelhas (Apis mellifera), vem sendo apontada como promissora por apresentar uma série de características biológicas vantajosas, por exemplo, anti-inflamatória, antimicrobiana, antitumoral, imunomoduladora, antioxidante e também scavenger de radicais livres. O presente estudo teve como objetivo principal, averiguar efeito da própolis brasileira procedente de Rio Grande do Sul (AF 08) em células de ovário de hamster Chinês (CHO-K1) e em células tumorais de próstata (PC3), irradiadas com 60Co. Para tanto, foram utilizados três principais parâmetros interligados entre si: indução de micronúcleo, viabilidade celular e morte clonogênica. A escolha destes parâmetros se justifica pelo seu significado biológico, além do fato de serem prontamente observáveis e mensuráveis em células irradiadas. Os dados citogenéticos obtidos, mostraram um efeito radioprotetor da própolis (5 - 100 μg/ml) na indução de dano ao DNA em ambas as linhagens celulares, irradiadas com doses de 1 - 4 Gy. No entanto, o ensaio de citotoxicidade mostrou um efeito antiproliferativo pronunciado da própolis (50 - 400 μg/ml) em células PC3 irradiadas com 5 Gy. As curvas de sobrevida obtidas foram ajustadas satisfatoriamente pelo modelo linear-quadrático, cujo componente foi mais alto em células CHO-K1. Quanto à capacidade clonogênica, as células PC3 mostraram-se mais radiossensíveis que as células CHO-K1 nas doses mais altas da curva de sobrevida. A própolis, nas concentrações de 30 - 100 μg/ml, não influenciou no potencial clonogênico das células PC3, visto que, as curvas de sobrevida associadas ou não com a própolis, mostraram perfis similares, ao passo que o tratamento combinado em células CHO-K1 exibiu um efeito estimulador da proliferação. Os dados obtidos in vitro mostraram um potencial uso da própolis AF-08, como uma substância natural e não tóxica, na prevenção contra os efeitos danosos da radiação ionizante, nas doses e nas concentrações analisadas. / In the last decades, it has been given a great interest to investigations concerning natural, effective, nontoxic compounds with radioprotective potential together with the increasing utilization of different types of ionizing radiation for various applications. Among them propolis, a resinous compound produced by honeybees (Apis mellifera), has been considered quite promising, since it presents several advantageous biological characteristics, i. e., anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant and also free radical scavenging action. The purpose of the present study was to evaluate the effect of Brazilian propolis, collected in the State of Rio Grande do Sul, on Chinese hamster ovary (CHO-K1) and human prostate cancer (PC3) cells, irradiated with 60Co radiation. For this purpose, three interlinked parameters were analyzed: micronucleus induction, cell viability and clonogenic death. The choice of these parameters was justified by their biological significance, in addition to the fact that they are readily observable and measurable in irradiated cells. The cytogenetic data obtained showed a radioprotective effect of propolis (5-100 μg/ml) in the induction of DNA damage for both cell lines, irradiated with doses of 1 - 4 Gy. The cytotoxicity assay, however, showed a prominent antiproliferative effect of propolis (50 - 400 μg/ml) in PC3 cells irradiated with 5 Gy. The survival curves obtained were adequately fitted by a linear-quadratic model, where the coefficient was higher in CHO-K1 cells. Concerning the clonogenic capacity, PC3 cells were more radiosensitive than CHO-K1 cells at the higher doses of the survival curve. Propolis at the concentrations of 30 - 100 μg/ml, did not influence the clonogenic potential of PC3 cells, since the survival curves, associated or not with propolis, were found similar, although the combined treatment in CHO-K1 cells exhibited a stimulating proliferative effect. The data obtained in vitro showed a potential use of propolis AF-08, a natural and nontoxic compound, in the prevention against the adverse effect of ionizing radiation, at the doses and concentrations here analyzed.

The use of a channelization mechanism on model observers not only makes mimicking human visual behavior possible, but also reduces the amount of image data needed to estimate the model observer parameters. The channelized Hotelling observer (CHO) and channelized scanning linear observer (CSLO) have recently been used to assess CT image quality for detection tasks and combined detection/estimation tasks, respectively. Although the use of channels substantially reduces the amount of data required to compute image quality, the number of scans required for CT imaging is still not practical for routine use. It is our desire to further reduce the number of scans required to make CHO or CSLO an image quality tool for routine and frequent system validations and evaluations. This work explores different data-reduction schemes and designs an approach that requires only a few CT scans. Three different kinds of approaches are included in this study: a conventional CHO/CSLO technique with a large sample size, a conventional CHO/CSLO technique with fewer samples, and an approach that we will show requires fewer samples to mimic conventional performance with a large sample size. The mean value and standard deviation of areas under ROC/EROC curve were estimated using the well-validated shuffle approach. The results indicate that an 80% data reduction can be achieved without loss of accuracy. This substantial data reduction is a step toward a practical tool for routine-task-based QA/QC CT system assessment. (C) 2016 Society of Photo-Optical Instrumentation Engineers (SPIE)

Nowadays, more than half of the biotherapeutics are produced in mammalian cell lines as a result of correct protein folding and assembly as well as their faculty to bring about a variety of post-translational modifications. The widespread progression of biosimilars has moved the focus in mammalian cell-culture process development. Thereby, the modulation of quality attributes of recombinant therapeutic proteins has increasingly gained importance from early process development stages. Protein quality directly shapes the clinical efficacy and safety in vivo, and therefore, the control of the complex post-translational modifications, such as glycosylation (e.g. high mannose, fucosylation, galactosylation and sialylation), charge variants, aggregates and low-molecular-weight species formation, is pivotal for efficient receptor binding and for triggering the desired immune responses in patients. In the frame of biosimilar development, product quality modulation methods using the potential of the host cell line are particularly sought after to match the quality profile of the targeted reference medicinal product (RMP) as closely as possible. The environment the cell is dwelling in directly influences its metabolism and the resulting quality profile of the expressed protein. Thereby the cell culture medium plays a central role in upstream manufacturing. In this work, concentration adjustment of selected media components and supplementation with a variety of compounds was performed to alter various metabolic pathways, enzyme activities and in some cases the gene expression levels of Chinese Hamster Ovary (CHO) cells in culture. The supplementation of cell culture medium with the trisaccharide raffinose in fed-batch cultures entailed an increase of the abundance of high mannose glycans in two different CHO cell lines. Raffinose especially favored mannose 5 glycans. At the same time, it impaired cell culture performance, induced changes on the intracellular nucleotide levels and even varied the expression levels of glycosylation-related genes. Supplementation with a number of galactosyltransferase inhibiting compounds, in particular fluorinated galactose analogs (alpha- and beta-2F-peracetyl-galactose), consistently decreased the production of galactosylated monoclonal antibodies (mAb). By means of targeted addition during the culture rather than at the beginning, the inhibition was further increased, while limiting detrimental effects on both growth and productivity. High-throughput screening in 96-deepwell plates showed that spermine and L-ornithine also reduced the level of galactosylation. On the other hand, exploratory screening of a variety of potentially disulfide-bridge-reducing agents highlighted that the inherent low-molecular-species level of the proprietary platform cell culture process was likely due to favored reduction. This hypothesis was reinforced by the observation that supplementation of cysteine and N-acetylcysteine promoted fragmentation. Additionally, fragmentation decreased with higher protein expression. At that point, aiming to improve the efficiency in process development, a rational experimental design method was developed to identify and to define the optimal concentration range of quality modulating compounds by calling on a combination of high throughput fed-batch testing and multivariate data analysis. Seventeen medium supplements were tested in five parallel 96-deepwell plate experiments. The selection process of promising modulators for the follow-up experiment in shake tubes consisted in a three-step procedure, including principal component analysis, quantitative evaluation of their performance with respect to the specifications for biosimilarity and selection following a hierarchical order of decisions using a decision tree. The method resulted in a substantial improvement of the targeted glycosylation profile in only two experimental rounds. Subsequent development stages, namely validation and transfer to industrial-scale facilities require tight control of product quality. Accordingly, further mechanistic understanding of the underlying processes was acquired by non-targeted metabolomic profiling of a CHO cell line expressing a mAb cultured in four distinct process formats. Univariate analysis of intra- and extracellular metabolite and temporal glycosylation profiles provided insights in various pathways. The numerous of parameters were the main driver to carry out principal component analysis, and then, using the methodology of partial-least-square (PLS) projection on latent structures, a multivariate model was built to correlate the extracellular data with the distinct glycosylation profiles. The PLS observation model proved to be reliable and showed its great benefit for glycan pattern control in routine manufacturing, especially at large scale. Rather than relying on post-production interpretation of glycosylation results, glycosylation can be predicted in real-time based on the extracellular metabolite levels in the bioreactor. Finally, for the bioactivity assessment of the glycan differences between the biosimilar and the reference medicinal product (RMP), the health agencies may ask for in the drug registration process, extended ranges of glycan variants need to be generated so that the in vitro assays pick up the changes. The developed glycosylation modulator library enabled the generation of extreme glycosylation variants, including high mannose, afucosylated, galactosylated as well as sialic acid species of both a mAb and an antibody fusion molecule with three N-glycosylation sites. Moreover, to create increased variety, enzymatic glycoengineering was explored for galactosylation and sialylation. The glyco variants induced significant responses in the respective in vitro biological activity assays. The data of this work highlight the immense potential of cell culture medium optimization to adjust product quality. Medium and feed supplementation of a variety of compounds resulted in reproducible and important changes of the product quality profile of both mAbs and a fusion antibody. In addition to the intermediate modulation ranges that largely met the requirements for new-biological-entity and biosimilar development, medium supplementation even enabled quick and straightforward generation of extreme glycan variants suitable for biological activity testing. / Mehr als die Hälfte der Biotherapeutika werden heutzutage aufgrund korrekter Proteinfaltung und korrektem Zusammenbau in tierischen Zelllinien hergestellt, welche zudem die Fähigkeit besitzen, verschiedene posttranslationale Modifikationen zu bewerkstelligen, hergestellt. Der ausgeprägte Aufschwung von Biosimilars hat den Entwicklungsschwerpunkt von Zellkulturverfahren verlagert. Dabei hat die Modulierung der Qualitätsattribute von rekombinanten Proteinen bereits in frühen Entwicklungsstadien eine wichtige Bedeutung erlangt. Die Qualitätsattribute beeinflussen die klinische Wirksamkeit und die In-Vivo-Sicherheit direkt. Somit ist die Regulierung der posttranslationalen Modifikationen, einschließlich der Glykosylierung (mannosereiche, fukosylierte, galaktosylierte und sialylierte Glykane), der Ladungsvarianten, sowie die Bildung von Aggregaten und niedermolekularen Spezien, für effiziente Rezeptorbindung und das Auslösen der gewünschten Immunantwort in Patienten entscheidend. Im Rahmen der Biosimilarentwicklung werden Methoden zur Anpassung der Produktqualität innerhalb des Potentials der Wirtszelle gesucht, um sie möglichst genau dem Referenzarzneimittel anzugleichen. Die Umgebung, in der die Zelle verweilt, beeinflusst ihren Metabolismus und das resultierende Produktqualitätsprofil. Dabei spielen Medien eine zentrale Rolle in der Zellkultur. Im Rahmen dieser Doktorarbeit wurden durch Adjustierung von ausgewählten Medienbestandteilen und Ergänzung mit einer Vielfalt von Stoffen diverse Stoffwechselwege, Enzymaktivitäten und in einigen Fällen das Genexpressionsniveau von kultivierten Chinesischen Hamster-Ovarialzellen (CHO) verändert. Die Ergänzung von Zellkulturmedium mit Raffinose, ein Trisaccharid, führte zu einer Erhöhung des mannosereichen Glykosylierungsmusters in zwei unterschiedlichen CHO-Zelllinien. Raffinose begünstigte hauptsächlich Mannose-5-Spezien. Gleichzeitig wurde die Zellkulturleistung beeinträchtigt und zudem intrazelluläre Nukleotidkonzentrationen sowie das Expressionsniveau von Glykosylierungsgenen verändert. Ergänzung mit mehreren Inhibitoren der Galaktosyltransferase, insbesondere fluorierte Galaktosenachbildungen (Alpha- und Beta-2F-Peracetyl-Galaktose), verringerte stetig die Produktion von galaktosylierten monoklonalen Antikörpern (mAb). Durch gezielte Zugabe im Verlauf der Kultur, statt bereits am Anfang, wurde die Inhibition weiter erhöht, und dabei die Einwirkung auf das Zellwachstum und die Produktivität beschränkt. Ein Hochdurchsatz-Screening in 96-Deep-Well-Platten zeigte, dass Spermin und L-Ornithin auch das Ausmaß der Galaktosylierung reduzierte. Andererseits zeigten erste Nachforschungen anhand eines Screenings einer Auswahl von potenziellen Disulfidbrücken-Reduktionsmittel, dass wahrscheinlich begünstigte Reduktion das inhärente Niedermolekular-Speziesniveau des firmeneigenen Zellkulturplattformverfahrens verursacht. Die Hypothese wurde durch die Beigabe von Cystein und N-Acetylcystein bekräftigt. Diese Stoffe begünstigten die Fragmentierung, wohingegen sie bei höherer Proteinexpression abnahm. Mit dem Ziel die Entwicklungseffizienz zu steigern, wurde daraufhin zur Identifikation von qualitätsverändernden Stoffen und Bestimmung der optimalen Konzentrationsbereichen eine rationale Versuchsanordnungsmethode entwickelt. Dazu wurde eine Kombination von Hochdurchsatz-Fed-Batch-Tests und multivariater Datenanalyse herbeigezogen. Siebzehn Mediumergänzungsstoffe wurden in fünf parallelen 96-Deep-Well-Platten-Experimenten getestet. Das Auswahlverfahren von erfolgsversprechenden Modulatoren fürs Nachfolgeexperiment in Schüttelröhrchen umfasste drei Schritte: Hauptkomponentenanalyse, quantitative Evaluierung der Leistung der Modulatoren hinsichtlich der Biosimilaritätsspezifikationen und die Auswahl in Anlehnung an eine hierarchische Entscheidungsreihenfolge mit Hilfe eines Entscheidungsbaums. Die Methode führte in nur zwei Versuchsreihen zu einer erheblichen Annäherung an das gewünschte Glykosylierungsprofil. Anschließende Entwicklungsschritte (Validierung und Transfer in die großtechnische Anlage) erforden eine rigorose Kontrolle der Produktqualität. Demzufolge konnte dank der Non-Targeted Metabolomics Analyse von vier verschiedenen Herstellungsverfahren einer mAb exprimierenden CHO-Zelllinie weitere mechanistische Kenntnisse der zugrunde liegenden Vorgängen gewonnen werden. Univariate Analysen der intra- und extrazellulären Stoffwechselprodukte und die zeitliche Glykosylierungsprofile lieferten einen Einblick in verschiedene Stoffwechselwege. Die Vielzahl von Parametern führte dazu, nach dem Prinzip der Hauptkomponentenanalyse vorzugehen, und dann anhand der Partial Least Squares (PLS)-Projektion auf latente Strukturen ein multivariates Modell zu erstellen, das die extrazellulären Daten mit den individuellen Glykosylierungsprofilen korreliert. Das PLS Beobachtungsmodell stellte sich als verlässlich heraus und zeigte seinen außerordentlichen Nutzen zur Regulierung der Glykanen in der Routineherstellung, insbesondere in der Großanlage. Anstatt sich auf Glykosylierungsresultate nach dem Ende der Produktion zu verlassen, kann die Glykosylierung, basierend auf den Niveaus der extrazellulären Stoffwechselprodukte im Bioreaktor, in Echtzeit vorausgesagt werden. Schließlich können im Rahmen des Arzneigenehmigungsverfahrens Gesundheitsbehörden verlangen, die Glykanunterschiede zwischen dem Biosimilar und dem Referenzarzneimittel zu untersuchen. Damit der biologische Test die Unterschiede nachweisen kann, muss eine erweiterte Palette von Glykanvarianten hergestellt werden. Die entwickelte Glykosylierungsmodulierungsbibliothek ermöglichte, extreme Varianten für mannosereiche, afukosylierte, galaktosylierte und sialylierte Glykane von mAb und einem Antikörperfusionsmolekül mit drei N-Glykosylierungsstellen zu generieren. Für erhöhte Variantenvielfalt wurde die enzymatische Glykoengineering Technologie für die Galaktosylierung und Sialylierung untersucht. Die Glykanvarianten erzeugten signifikante Antworten in der jeweiligen In-Vitro-Bestimmung der biologischen Aktivität. Die Ergebnisse unterstreichen das immense Potential von Zellkulturmediumoptimierung zur Anpassung der Produktqualität. Ergänzung des Mediums und der Nährstofflösung brachte reproduzierbare und beträchtliche Veränderungen der Produktqualität von mAb und eines Fusionsantikörpers hervor. Zusätzlich zu den intermediären Modulierungsbereichen, die mehr als ausreichend den Anforderungen für die Entwicklung von neuen biologischen Wirkstoffen und Biosimilars genügen, ermöglichte die Mediumergänzung auf schnelle und einfache Art und Weise selbst extreme Glykanvarianten zu bilden, die für die Bestimmung der biologischen Aktivität geeignet waren.