Made available in DSpace on 2014-10-09T12:28:48Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:12Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:28:48Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:12Z (GMT). No. of bitstreams: 0 / O Grupo de Hormônios do Centro de Biotecnologia do IPEN desenvolveu várias linhagens de células de ovário de hamster chinês (CHO) modificadas geneticamente e comprovadamente eficientes na expressão de proteínas heterólogas, dentre elas a prolactina humana (hPRL) e os análogos antagonistas de prolactina (S179D-hPRL e G129R-hPRL). No entanto, todas as linhagens para expressão são cultivadas em monocamadas e dependentes da presença de soro fetal bovino (SFB) no meio de cultivo para um crescimento eficiente. As células em suspensão apresentam um grande interesse industrial-farmacêutico, tanto pela facilidade de cultivo e ampliação de escala, como pela produtividade volumétrica. Desenvolvemos um protocolo para adaptação de células CHO para o crescimento em suspensão e também processos de produção em frascos spinners. Nesse trabalho foi realizada a adaptação das linhagens produtoras de hPRL; S179D-hPRL e G129R-hPRL para o crescimento em suspensão e em meio livre de SFB. Realizamos também a produção em escala laboratorial com as três linhagens adaptadas, assim como a correspondente purificação e caracterização de quatro proteínas heterólogas, incluindo a prolactina humana glicosilada (G-hPRL). / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

A positive correlation between the flux of TCA cycle and productivity of Chinese Hamster Ovary (CHO) cells has been reported. Earlier work in this laboratory revealed that supplementation with nutrients that enter the TCA cycle (combination of glucose (Glc), pyruvate (Py), aspartate (Asp), asparagine (Asn) and glutamate (Glu)) significantly increased maximum viable cell density and antibody production of recombinant CHO cells. Increased amounts of extracellular citrate was associated with feeding conditions. It was hypothesized that increased flux through the TCA cycle and related metabolism was linked to enhanced growth and/or productivity of CHO cells. Therefore, the aim of this thesis is to clarify these relationships to provide routes to improve the efficiency of CHO cells by nutrient supplementation and metabolic engineering. The relationship between growth, antibody production and metabolite profiles of CHO-LB01 cells was examined in response to individual supplementation with Asn, Asp, Glu, Py and β-hydroxybutyrate (HB). Feeding HB significantly increased antibody titre while Asn feeding increased maximum cell density but led to earlier cell death. Both nutrients increased the amounts of TCA cycle intermediates and decreased the amounts of lactate, glycerol, sorbitol and amino acids. Moreover, oxygen consumption rate was increased in the presence of Asn or HB. This finding inferred that increased production of the TCA cycle intermediates in cells fed Asn or HB correlated with enhanced flux of the TCA cycle leading to enhanced oxidative metabolism. Combination of Asn or HB with Glc further improved cell growth, increased antibody titre and enhanced metabolic responses to feeds (TCA cycle intermediates). Based on these results, inhibition of sorbitol production using chemical reagent (EPBC) and siRNA designed against Akr1b1 and overexpression of malate dehydrogenase II (MDH II) were undertaken in order to increased flow of carbon atoms to TCA cycle and/or increased flux in the TCA cycle, respectively. Inhibition of sorbitol production was achieved in the presence of EBPC but there was no improvement of cell culture performance and accumulation of TCA cycle intermediates remained the same. CHO cells transfected with exogenous Mdh2 did not show improved cell culture performance. Whilst stable clones exhibited variable MDH II expression at protein level (and antibody titre), overexpression of exogenous MDH II could not be confirmed by Western blot. One CHO-MDH II clone showed greater antibody titre and exhibited similar metabolite profiling with cells fed Asn or HB. This contrasted to the majority of clones that were low producers. Comparison by RNA-Seq transcriptomic profiling of high- and low-producing CHO-MDH II clones showed that the majority of differentially expressed genes were genes related to cytoskeleton-related element and cell signaling pathways. Overall, these results confirmed the relationship between increased the amount of TCA cycle intermediates and increased antibody production. Increased amount of TCA cycle intermediates could result in increased the flow of TCA cycle lead to enhance energy and antibody production. In addition, this work represents the first study on addition of HB offers a simple effective strategy to increase antibody production.

The irradiation of cells in culture is an essential part of many radiation biology experiments. Since these experiments necessarily involve the irradiation of cell culture vessels and nutrient medium, the possibility of effects due to the interactions of irradiated material with growing cells needed to be investigated. In the present study the micronucleus frequency in Chinese hamster ovary (CHO) cells as a function of such parameters as type of radiation, type of cell substrate, changes in cell environment, and time course of the effect were characterized. Observations of the persistence of micronucleus formation in irradiated CHO cells reveal that the number of cells containing micronuclei reaches its maximum within nine hours after irradiation and remain elevated for at least five days. The influence of the cell environment on micronucleus formation in CHO cells was examined by plating cells in preirradiated nutrient medium or on preirradiated cell culture vessels. In all experiments, pre-irradiation of the cell substrate (the culture dish or culture dish filled with medium) led to a significantly higher micronucleus frequency than when cells were plated on un-irradiated substrate. The difference is most pronounced at the lowest doses examined. These results suggest that methods of cell culture vessel sterilization and the composition of cell attachment surfaces could be confounding factors, particularly in the experiments which are intended to examine the response of cells exposed to low doses of ionizing radiation.

This PhD project explored the role of tricarboxylic acid (TCA) cycle enzymes in regulating Chinese hamster ovary (CHO) cell metabolism with respect to growth and recombinant protein expression. It was hypothesised that regulation of central metabolism can influence CHO cell productivity in terms of biomass and protein production. Thus the aim of these studies was to identify the key metabolic reactions of the TCA cycle associated with growth and protein expression in CHO cells. The gene expression of all TCA cycle genes was independently knocked-down using RNAi technology. The small interfering RNA (siRNA) mediated silencing of 11 TCA cycle genes significantly reduced cellular growth along with a decline in adenylate energy charges and an increase in catabolic reduction charges. The gene profiling of glucose and amino acid metabolism (not targeted by siRNA) suggested siRNA mediated knock-down of targeted TCA cycle genes led to cellular stress along with an enhanced rate of glycolysis leading to channelling of glucose for the generation of pyruvate. For the purpose of estimating intracellular metabolites, quenching and extraction method using ammonium bicarbonate and methanol was optimised to use with UCB CHO-K1 cell line and static transient siRNA transfections. A gas chromatography-mass spectrometry (GC-MS) analysis post-silencing of the aconitase gene, which catalyses the conversion of citrate to isocitrate in the TCA cycle, yielded higher MS peak intensities of at least four metabolites (gluconic acid, lysine, threonine and leucine) 72 h post-transfection in comparison to the controls. Transient knock-down of gene expression of seven TCA cycle genes in a recombinant stable cell line (expressing a rabbit monoclonal antibody) reduced cellular growth and altered the energy charges leading to a decline in antibody expression. Although silencing of the pyruvate dehydrogenase E1 gene, which is the component of the pyruvate dehydrogenase complex connecting glycolysis to the TCA cycle, did not affect cell viability, a reduction in antibody expression was recorded. Seven TCA cycle genes which demonstrated the most significant effect on cellular growth and energy charges were transiently over-expressed along with a monoclonal antibody in CHO-K1 cells with addition of their corresponding preceding intermediates. No differences in protein expression and cell specific productivity were observed compared to the control transfections. These results could be due to limitations of the effects of transient transfections for enhancing the metabolic activity of CHO cells. The aconitase gene demonstrated the most significant effect on CHO cell growth and proliferation in this study, therefore this gene was proposed as a novel selection marker for a metabolic selection system for the generation of recombinant therapeutics. This PhD project also established metabolite analysis tools and siRNA protocols for future metabolomic studies for investigating the intracellular CHO metabolism. The findings validated the hypothesis that TCA cycle plays an important role in CHO cell growth and recombinant protein production. The key metabolic genes affecting cellular growth and altering energy metabolism can be further explored for generation of an energy efficient CHO host-cell line (by over-expression of key TCA cycle genes) for enhanced recombinant protein production.

Transcriptional regulation is a determinant of stability of recombinant protein production in CHO cells. Fundamental studies of recombinant gene transcription in relation to chromatin environment and promoter regulation are important for CHO cell line development and selection. This study has developed a methodology based on a cell/vector system to study recombinant transcription and expression stability of different promoters and/or proteins in the similar genomic environment. The CHO-FRT mini-pools developed in this project were mini-pools of CHO-S cell lines containing Flp Recombination Target (FRT) sites with ß-galactosidase gene, under the influence of a SV40 promoter. Continuous culture of these mini-pools for 8 weeks using a robotic system demonstrated that 20% of the mini-pools studied revealed an unstable profile (with 30% loss of protein expression). Two of these mini-pools with different characteristics, CHO-FRT 1 (low producer/unstable) and CHO-FRT 108 (high producer/stable), were selected to be used on the study of influence of SV40 and CMV promoters in long-term recombinant expression. Genes encoding fluorescent proteins were integrated in a site-directed manner under the influence of SV40 or CMV promoters. A sub-clonal population of the top 10% yellow fluorescent protein (YFP) expressing cells of each mini-pool/promoter combination was selected by cell sorting and cultured for 4 weeks. During this period protein expression was monitored by flow cytometry and compared between both promoters. The results revealed that both SV40 and CMV promoters had an unstable expression with different degrees of instability and long-term expressing behaviours. For CMV, instability was considerably high displaying a long-term logarithmic loss of 50-80% of productivity while for SV40 the loss of productivity observed was only 40-45% with a linear behaviour during long-term culture. The vector system generated contained an MS2-RNA tag sequence cloned 3'- of the recombinant gene to track the recombinant mRNA by using the MS2/MCP-GFP system. This study showed the development of a protocol to measure the transcriptional output of recombinant promoters in CHO cells. The results showed background signal in CHO cells that requires further optimisation studies to allow the direct live cell image quantification of the transcriptional activity of recombinant promoters. Although not yet optimised, the successful combination of site-directed integration with recombinant mRNA tagging method has the potential to become a valuable tool to study the mechanisms of transcriptional activity and stability of transcription driven by different promoters in CHO cells.

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

The glycosylation pattern of a chimeric heavy chain antibody (EG2) produced from CHO cells was affected by the glucose concentration (0-25 mM) of cultures established at high density (>106 cells/ml) over 24 h. The resulting proportion of non-glycosylated Mab was directly correlated to the exposure time of cells to media depleted of glucose. Deprivation of glucose for the full 24 h resulted in a 52% non-glycosylated Mab fraction. Analysis of steady state levels of intracellular lipid-linked oligosaccharides (LLOs) showed that under glucose limitation there was a reduction in the amount of full length LLO (Glc3Man9GlcNac2), with a concomitant increase in the smaller mannosyl-glycans (Man2-5GlcNAc2). Glycan microheterogeneity was quantified by galactosylation and sialylation indices (GI and SI), which showed a direct correlation to the cell specific glucose uptake. These findings are important in relation to the low substrate that may occur in fed-batch cultures for Mab production.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.