Under the globalization trend¡Aeven the still growing industry of Taiwan Biopharmaceutical will meet the same developing rule ¡C Many of the Biotechnology or Pharmaceutical companies are not satisfied with the local market here in Taiwan¡Athey are not only trying efforts to make efficiency of the limited resources of Taiwan itself¡Abut have had take China market into consideration during make company business plan¡C There is no doubt that Taiwan¡¦s BioPharm insutry are moving their steps into mainland China, especially since 1990 when China slowly open the free market to the world capital ¡CMany of them have had achieved much success , yet many companies found difficulties and entry barriers much as well¡C So far¡Athere is still no any research or survey about how Taiwan BioPharmaceutical are doing in China? What were their strategy for the market¡Hwhat are the facing problems or challenge in China? What are the solutions to cope with ?...and so on¡C In this thesis¡AIt content both field study¡Bcompany interview and questionnaire survey to gather the first hand data from industry that could picture the real situation about the Taiwan BioPharmaceutical companies in China so far¡C Furthermore ,by analyze the data¡AIt could get the conclusions and findings that help the industry realized what are main problems for the China market and what could be the most successful strategy ,too¡C It shows ¡AChina is the future market for globe biopharmaceutical industry ¡ATaiwan companies need to build up the niche competency of its own self¡Ayet there still are many problems come form uncertain China government policy¡Bregulation and market game rules to face with ¡C

The main goal in biopharmaceutical production is achieving high volumetric productivity while maintaining product quality (i.e. glycosylation). The objectives of this project were to explore the use of dielectric analysis in the early detection of cell demise and to analyze the impact of nucleotide / nucleotide sugar precursor feedings in biopharmaceutical production and glycosylation. Measurements of changes in the polarizability of individual cells can be performed in a dielectrophoretic (DEP) cytometer designed at the University of Manitoba. In this instrument the trajectory of individual cells was tracked according to their polarizability and recorded as a force index (FI). The identified sub-populations from a batch bioreactor and apoptosis-induced cultures were correlated with the fluorescent markers of apoptosis analyzed in a flow cytometer. Discrete cell sub-populations were identified as cells passed through the various stages of apoptosis. In the batch and the starvation culture the early changes in the measured FI of cells correlated with the Annexin V fluorescent assay associated with early phase apoptosis. For the oligomycin and staurosporine cultures changes in the FI could be correlated to modifications in the mitochondrial metabolism linked with early apoptosis for both inducers. In fed-batch experiments 10 mM galactose alone or 20 mM galactose in combination with 1 mM uridine or 1 mM uridine + 8 μM MnCl2 was added to the basal and feed medium for two CHO cell lines to determine their impact on the biopharmaceutical production and the glycosylation process. The results showed that the addition of all three precursors combined increased UDP-Gal, which increased and maintained the galactosylation index during the bioprocess for CHO-EG2 and CHO-DP12 cultures by 25.4% and 37.9%, respectively, compared to the non-supplemented fed-batch culture. In both cell lines saturation was reached when a further increase in the UDP-Gal concentration did not increase the galactosylation. A negative impact on cell growth was observed with the uridine addition in the CHO-EG2 culture, which was linked to the CHO-EG2 cell line being DHFR-/-. This work presents a dielectric detection method to monitor early changes in the cell metabolism and information for shifting and maintaining galactosylation during biopharmaceutical production. / February 2016

The creation of stable mammalian cell lines for biopharmaceutical production often require several months, and is unfavourable for the rapid production of multiple drug candidates for screening in the early stages of development. Biopharmaceutical production by transient transfection provides a possible alternative of quickly producing these early stage drug candidates. The Epi-CHO transient expression system, which consists of a Chinese hamster ovary (CHO) cell line (CHO-T) expressing the murine polyomavirus Large T-Antigen (LT), emonstrated enhanced transient recombinant protein production. The aim of this study was to prolong transient recombinant protein prod.Jction of the Epi-CHO expression system by creating a CHO cell line expressing both LT and EBNA1 (ECHO-T). The pEBNA1-LT expression vector encoding LT and EBNA1 was constructed and transfected into CHO-K1. A total of 20 clones were isolated from the antibioticresistant pool and screened for the expression of functional LT and EBNA1. PCR analysis showed 16 of the 20 clones was positive for EBNA1 and LT DNA. Of the 16 clones, six were positive for EBNA1 and LT expression by RT-PCR. Detection of LT and EBNA1 by immunofluorescence showed positive staining for the P7-G3 clone. Western blotting suggested the P7-G3 clone was: positive for EBNA1, and clones P3-C7 and P7-E2 were positive for LT. A plasmid replication assay confirmed the expression of functional LT in all six clones. Plasmid maintenance assay confirmed clone P7-G3 as the ECHO-T clones to express functional EBNA1. The P7-G3 clone demonstrated prolonged and sustained transient recombinant protein expression when compared to CHO-T. The P7-G3 clone achieved sustained transient protein expression for 32 days in the absence of selection, the longest currently reported for CHO cells.

Orientador: Marcos Antônio de Oliveira / Resumo: L-asparaginases (ASNases) bacterianas são importantes biofármacos utilizados no tratamento de leucemia linfoide aguda (LLA), uma vez que este tipo tumoral é dependente da disponibilidade de asparagina (Asn) extracelular. As ASNases bacterianas são capazes de hidrolisar eficientemente Asn em ácido aspártico (Asp) e amônia (NH3), diminuindo a disponibilidade de Asn para células tumorais e induzindo apoptose. Comercialmente, indústrias farmacêuticas internacionais produzem ASNases de Escherichia coli e Erwinia chrysanthemi, entretanto, ASNase de nenhuma origem é produzida pelas indústrias farmacêuticas brasileiras. Adicionalmente, o tratamento com estas enzimas produz efeitos colaterais, entre eles imunogênicos, que estão relacionados com a alta massa molecular da enzima (140kDa) e neurológicos, atribuídos a atividade secundária de glutaminase (GLNase). Neste contexto, fontes alternativas destas enzimas e também a auto-suficiência em suas produções são importantes para mitigar os efeitos colaterais e evitar falhas no tratamento devido a flutuações internacionais de sua fabricação. Neste trabalho, realizamos a caracterização de uma ASNase de levedura, denominada de ASNaseM que compartilha elevada homologia (maior que 30% de identidade e 40% de similaridade) com as enzimas bacterianas utilizadas no tratamento da LLA e que possui todos os aminoácidos envolvidos na catálise conservados, sugerindo uma fonte alternativa potencial para o tratamento da LLA. Experimentos de cromatografia... (Resumo completo, clicar acesso eletrônico abaixo) / Bacterial L-asparaginases (ASNases) are important biopharmaceuticals used in the treatment of acute lymphoid leukemia (ALL), since this tumor type is dependent on the availability of extracellular asparagine (Asn). Bacterial ASNases are able to efficiently hydrolyze Asn in aspartic acid (Asp) and ammonia (NH3), decreasing the availability of Asn to tumor cells and inducing apoptosis. Commercially, international pharmaceutical industries produce ASNases from Escherichia coli and Erwinia chrysanthemi, however none ASNase is produced by the Brazilian pharmaceutical companies. Additionally, the treatment with these enzymes can produce side effects, among them immunogenic ones, that are related to the high molecular weight of the enzyme (140kDa) and neurological, attributed to the glutaminase secondary activity (GLNase), being glutamine (Gln) the most abundant amino acid in the bloodstream. In this context, alternative sources of these enzymes as also the self-sufficiency of the production are important to mitigate side effects and avoid treatment failures due to international fluctuations in their manufacture. In this work, we performed the characterization of a yeast ASNase, called ASNaseM, which shares high homology (higher than 30% identity and 40% similarity) with the bacterial enzymes used in the treatment of ALL, and which has all the amino acids involved in the catalysis conserved, suggesting a potential alternative source for the treatment of ALL. Molecular exclusion chro / Doutor

Mild hypothermic conditions (30ºC to 33ºC) have previously been shown to increase cell specific productivity (Qp) of recombinant proteins from mammalian cells. However, this is often associated with a lower growth rate which off-sets any potential advantage of higher product titres. This thesis describes the isolation of a novel population of Chinese Hamster Ovary (CHO) cells that have been adapted to low temperature growth by continuous subculture at low temperature for a duration of 400 days. This adapted cell population achieved a growth rate 2-fold greater than non-adapted cells under low temperature conditions (32ºC) while maintaining an elevated level of cell specific expression of recombinant beta-interferon. The volumetric titre of beta-interferon was enhanced by 70% in stationary cultures and by more than 2-fold by application of a temperature-shift strategy involving a growth to production phase. However, the low temperature-adapted cells were fragile and demonstrated an increased sensitivity to hydrodynamic stress in agitated cultures. This problem, caused by a weakened vimentin intermediate filament network, was resolved by the use of macroporous microcarriers which were demonstrated to entrap and protect the cold-adapted cells. Cold-adapted microcarrier cultures were able to achieve high cell densities (greater than 5x10^6 nuclei/mL) cultures under hypothermic conditions. This resulted in a 3-fold enhancement of volumetric titre of monomeric beta-interferon compared to the original control culture at 37ºC.

Mild hypothermic conditions (30ºC to 33ºC) have previously been shown to increase cell specific productivity (Qp) of recombinant proteins from mammalian cells. However, this is often associated with a lower growth rate which off-sets any potential advantage of higher product titres. This thesis describes the isolation of a novel population of Chinese Hamster Ovary (CHO) cells that have been adapted to low temperature growth by continuous subculture at low temperature for a duration of 400 days. This adapted cell population achieved a growth rate 2-fold greater than non-adapted cells under low temperature conditions (32ºC) while maintaining an elevated level of cell specific expression of recombinant beta-interferon. The volumetric titre of beta-interferon was enhanced by 70% in stationary cultures and by more than 2-fold by application of a temperature-shift strategy involving a growth to production phase. However, the low temperature-adapted cells were fragile and demonstrated an increased sensitivity to hydrodynamic stress in agitated cultures. This problem, caused by a weakened vimentin intermediate filament network, was resolved by the use of macroporous microcarriers which were demonstrated to entrap and protect the cold-adapted cells. Cold-adapted microcarrier cultures were able to achieve high cell densities (greater than 5x10^6 nuclei/mL) cultures under hypothermic conditions. This resulted in a 3-fold enhancement of volumetric titre of monomeric beta-interferon compared to the original control culture at 37ºC.

Šio darbo tikslas buvo ištirti hidrofilinių gelių pritaikymo galimybes transderminiam meloksikamo vartojimui. Gelių gamybos metu buvo taikomas temperatūros keitimo metodas. Visi hidrogeliai gaminami pagal pasirinktą 1,1 Pa•s ±15 proc. klampą. Meloksikamo mėginių analizė atliekama naudojant UV spektrofotometrijos metodą, puskiečių vaisto formų kokybiniam ir biofarmaciniam įvertinimui atliekami pH, dinaminės klampos, dalelių dydžio, gelių vienalytiškumo nustatymo testai, vaistinės medžiagos atsipalaidavimo tyrimai in vitro ir skvarbos į odą tyrimai ex vivo. Bandymo rezultatai parodė, kad meloksikamo hidrogelių, kuriuose vaistinė medžiaga įvesta tirpalo forma, pH siekė 6,24-6,70, suspensine forma – 5,45- 6,89. Atlikus klampos matavimą, paaiškėjo, kad įvedus meloksikamą į puskietę sistemą, jos klampa padidėja. Atpalaidavimo tyrimų in vitro metu buvo nustatyta, kad iš hidrogelio su natrio karboksimetilceliulioze (50-200 cP) per 120 min. atsipalaidavo 57,8 ± 4,5 proc. meloksikamo, su natrio karboksimetilceliulioze (200-400 cP) – 36,9± 2,2 proc., su metilceliulioze - 35,5 ±4,1 proc., su hidroksipropilmetilceliulioze – 33,4 ± 1,3 proc., su poloksameru 407 - 14,2 ± 2,3 proc. Iš hidrogelių, kuriuose meloksikamas įvestas suspensine forma, natrio karboksimetilceliuliozės (50-200 cP) sudėtyje turintis gelis atpalaidavo 20,3±4,7 proc. vaistinės medžiagos. Atlikus skvarbos tyrimą ex vivo, iš kontrolinio meloksikamo tirpalo prasiskverbė 0,96 proc. medžiagos, iš hidrogelio tikslaus... [toliau žr. visą tekstą] / The aim of this work was to investigate the application of hydrogels for meloxicam transdermal use. Gels were prepared using temperature mixing method. All types of gels were made according to established viscosity of 1,1 Pa•s ±15 %. The quantitative determination of meloxicam was performed by UV spectrofotometry method. Characterisation of prepared gels was evaluated by pH measurement, viscosity, particle size analysis, in vitro drug release and ex vivo skin permeation studies. Results showed that the pH of the prepared gel formulations was in the range of 6,24-6,70 in liquid form incorporated meloxicam and in the form of suspension 5,45-6,89. Viscosity measurement showed that meloxicam increases the viscosity of semisolid preparations. In vitro release studies showed that the hydrogel with sodium carboxymethylcellulose (50-200 cP) over 120 min relaxed 57,8 ± 4,5% of meloxicam, with sodium carboxymethylcellulose (200-400 cP) – 36,9± 2,2 %, with methylcellulose - 35,5 ± 4,1 %, with hydroxypropilmethylcellulose – 33,4 ± 1,3 %, with poloxamer 407 - 14,2 ± 2,3 %. Suspension type hydrogel of meloxicam with a sodium carboxymethyl cellulose (50-200 -cP) relaxed 20,3 ± 4,7 % drug substances. During the experiment were prepared similar to the skin pH meloxicam hydrogels. In vitro studies have shown that the incorporating meloxicam in gel by suspension do not provide sufficient drug substance release. Diffusion rate of drugs generally depends on the physical structure of the... [to full text]

Dans les trois essais de ma thèse, je prends une perspective processuelle sur la décision organisationnelle d'entrée sur le marché. Le regard processuel est très pertinent pour les entreprises dans les industries à forte intensité scientifique. Dans ce contexte empirique, une entrée sur le marché avec un produit est précédée de tentatives, parfois multiples, en recherche et développement (R & D), qui sont à la fois couteuses et empreintes d’incertitudes. Les tentatives par R&D d'avoir un produit sur un marché non seulement précèdent mais aussi sont plus fréquentes qu'une entrée réussie avec un produit. Pendant la période de tentatives d’entrée, une firme prend de multiples décisions stratégiques tel que, choix d’un marché et d'un collaborateur en R&D, choix d’abandonner R&D sur un marché mais remettre cette décision à plus tard et entrer le marché à nouveau. Ces types de décisions et ses effets sont des thèmes que j’étudie dans ma thèse. / In the three essays of my dissertation, I take a process perspective of market entry and study the drivers and outcomes of strategic decisions that firms make in their efforts towards an eventual product entry. The process perspective is highly pertinent for firms in science-based industries, such as, the biopharmaceutical. In this empirical context, actual product entry is preceded by multiple and frequent attempts in R&D that is not only costly but fraught with uncertainty. In the first chapter, I study the drivers of a firm’s decision to collaborate with a market incumbent upon new market entry. In the second chapter, I investigate the antecedents of a firm’s decision to re-enter a previously exited market. In the last chapter of my dissertation, I study the market-level drivers of a re-entering firm’s eventual R&D project performance in the market.

Magister Pharmaceuticae - MPharm / This study aimed to review the change management implemented in a Biopharmaceutical company in Cape Town in the light of existing literature on change management theory. Three main constructs were identified: process of change, readiness for change and climate of change. A quantitative pencil-and-paper survey were used to explore and describe employee experience of the change management process within a single department of a biopharmaceutical company in Cape Town. Cronbach alpha coefficient confirmed internal reliability (α = 0.94) of the questionnaire constructs. Employees across all ages reported average scores for all constructs (M ≥ 2.5 < 4), indicating a similar experience regardless of age. A medium-strong positive correlation (p < 0.01; r = 0.49) was observed between process of change and climate of change.

Masters of Science / This study aimed to review the change management implemented in a Biopharmaceutical company in Cape Town in the light of existing literature on change management theory. Three main constructs were identified: process of change, readiness for change and climate of change. A quantitative pencil-and-paper survey were used to explore and describe employee experience of the change management process within a single department of a biopharmaceutical company in Cape Town. Cronbach alpha coefficient confirmed internal reliability (α = 0.94) of the questionnaire constructs. Employees across all ages reported average scores for all constructs (M ≥ 2.5 < 4), indicating a similar experience regardless of age. A medium-strong positive correlation (p < 0.01; r = 0.49) was observed between process of change and climate of change. Based on the findings from the literature review and empirical research, recommendations were made to improve the change management processes and experience within biopharmaceutical companies. This study not only contributes to the body of knowledge on change management literature in the biopharmaceutical context, but also provides insight to a biopharmaceutical company to improve future change management practices.