Spelling suggestions: "subject:"human parathyroid hormone"" "subject:"human parathyroid mormone""
1 |
Chimeric and Recombinant Protein Reagents for Cellular Analysis and ImmunoassaysRauf, Femina January 2011 (has links)
Development of chimeric, recombinant peptides, proteins and enzymes expands the availability of protein/enzyme–based tools for cellular analysis and new assay platforms. Ideal protein reagents for cellular analysis must translocate into a variety of cells with minimum cell damage, retain stability and biological activity within the cell during analysis, and provide a reliable, measurable signal. This work focused on development, characterization and utilization of chimeric recombinant peptide, protein and enzyme reagents for cellular analysis and immunoassays. A cell-penetrating, fluorescent protein substrate (PKAS) was developed to monitor intracellular protein kinase A activity in cells without the need for cellular transfection. PKAS translocated into HeLa cells, βTC-3 cells and pancreatic islets with minimal toxicity. Upon cellular loading, glucose dependent phosphorylation of PKAS was observed in both βTC-3 and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 ± 6 %) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 ± 4 %) in βTC-3 cells was with 3 mM glucose indicating a left-shifted glucose sensitivity. A cell-penetrating luciferase chimera (Luc-TAT) and a cell-penetrating phospholipid nanoshell entrapped luciferase (Luc-PPN) was constructed to monitor dynamic changes in intracellular ATP levels in mammalian cells. Upon cellular loading, the activity of Luc-TAT and Luc-PPN was monitored with time. Luc-TAT lost approximately 50% activity within one hour, and decreased rapidly over time. In contrast Luc-PPNs retain approximately 95% activity in 1 hour and 77% after 12 hours showing longer biological lifetime. Luc-PPNs were able to detect dynamic ATP changes in intact HeLa cells in the presence of KCN and NaN3. The bioluminescence returned to background levels within 8-10 minutes after treatment with KCN, whereas NaN₃ showed ~ 40% reduction. Two novel recombinant human parathyroid hormone (hPTH) analogs hPTHEGFP and hPTH-Cys were prepared to develop immunoassays for PTH detection in clinical samples. Initial experiments show promise for these analogs for use in CZELIF based immunoassays. The analogs present a number of distinctive advantages for clinical assays and can be used to develop several immunoassay platforms.
|
2 |
ESTUDO DE METODOLOGIAS PARA AVALIAÇÃO DO HORMÔNIO DA PARATIREÓIDE HUMANO RECOMBINANTE / METHODOLOGY STUDY FOR THE EVALUATION OF RECOMBINANT HUMAN PARATHYROID HORMONEStamm, Fernanda Pavani 31 January 2013 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Human parathyroid hormone (hPTH) is a polypeptide with 84 amino acids and it is the
most important peptide regulator for calcium and phosphate ion homeostasis, maintaining
bone structure. Recombinant human parathyroid hormone, rhPTH (1-34), produced by DNA
technology in Escherichia coli contain the active amino-terminal fragment of the full length
hPTH and is currently being used worldwide for the treatment of osteoporosis at high risk of
fractures in postmenopausal women and men with osteoporosis primary or hypogonadal.
Reversed-phase liquid chromatography (RP-LC) and size exclusion liquid chromatography
(SE-LC) methods were developed and validated for the assessment of recombinant human
parathyroid hormone (rhPTH 1-34) in biopharmaceutical formulations. A gradient RP-LC
method was carried out on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d.), maintained
at 40 ºC. The mobile phase A consisted of 0.1 M sodium sulphate buffer, pH 2.3, and the
mobile phase B was acetonitrile. The SE-LC method was carried out on a BioSep-SEC-S
2000 column (300 mm x 7.8 mm i.d.), maintained at 25 ºC. The mobile phase consisted of 0.1
M phosphoric acid buffer, pH 2.5, run isocratically at a flow rate of 0.7 mL/min.
Cromatographic separation was obtained with retention times of 12.2 min, and 13.2 min, and
was linear over the concentration range of 1-250 μg/mL (r
2 = 0.9997) and 2-300 μg/mL (r
2 =
0.9993), respectively, for RP-LC and SE-LC, with photodiode array (PDA) detection at 214
nm. The limits of detection and quantitation were 0.44 and 1.47 μg/mL, respectively, for the
RP-LC and 0.79 and 2.63 μg/mL, for the SE-LC. Specificity was established in degradation
studies, which also showed that there was no interference of the excipients. Equally, the
accuracy was 100.49% and 100.22%, with bias lower than 1.12% and than 0.81%. Moreover,
the in vitro cytotoxicity test of related proteins and higher molecular weight forms showed
significant differences (p< 0.05) compared to intact molecule. The validated methods were
applied for the determination of rhPTH and related proteins in biotechnology-derived
products, giving lower mean differences of the estimated content/potencies of 0.64% and
1.31% for the RP-LC and SE-LC related to the in vivo bioassay, and of 0.98% and 0.31%
higher, compared to the in vitro cell culture assay, respectively. It is concluded that represents
a contribution to establish new alternatives to monitor stability, quality control and thereby
assure therapeutic efficacy of the biotechnology-derived medicine. / O hormônio da paratireóide humano (hPTH) é um polipeptídio constituído de 84
aminoácidos e tem função essencial como regulador da homeostase dos íons cálcio e fosfato,
e manutenção da estrutura óssea. O hormônio da paratireóide humano recombinante, rhPTH
(1-34), é produzido pela tecnologia do DNA em Escherichia coli, apresenta a sequência de
aminoácidos responsável pela porção biologicamente ativa do paratormônio natural e é usado
clinicamente para o tratamento da osteoporose de alto risco de fraturas em mulheres pósmenopausa
e de homens com osteoporose primária ou hipogonadal. No presente trabalho
foram desenvolvidos e validados métodos por cromatografia líquida em fase reversa (CL-FR)
e por exclusão molecular (CL-EM) para a avaliação de rhPTH (1-34) em formulações de
produtos biofarmacêuticos. No método por CL-FR, foi utilizada coluna Zorbax 300 SB C18
(150 mm x 4,6 mm d.i.), mantida a 40 ºC. A fase móvel A foi constituída por tampão sulfato
de sódio 0,1 M, pH 2,3, e a fase móvel B por acetonitrila, eluídas em gradiente. No método
por CL-EM foi utilizada coluna BioSep-SEC-S 2000 (300 mm x 7.8 mm d.i.), mantida a 25
ºC. A fase móvel foi contituída de tampão ácido fosfórico 0,1 M, pH 2,5, eluída em vazão
isocrática de 0,7 mL/min. Para ambos os métodos utilizou-se detector de arranjo de diodos
(DAD) a 214 nm. A separação cromatográfica foi obtida nos tempos de 12,2 e 13,2 min,
sendo linear na faixa de concentração de 1-250 μg/mL (r2 = 0,9997) e 1-300 μg/mL (r2 =
0,9993), respectivamente, para os métodos por CL-FR e CL-EM. Os limites de detecção e
quantificação foram 0,44 e 1,47 μg/mL, respectivamente, para o método por CL-FR e 0,79 e
2,63 por CL-EM. A especificidade foi avaliada em estudos de degradação, que também
demonstraram que não houve interferência dos excipientes. A exatidão foi 100,49 e 100,22,
com bias inferior a 1,12 e 0,81, respectivamente, para os métodos por CL-FR e CL-EM. Além
disso, realizou-se o teste de citotoxicidade in vitro das formas degradadas, as quais
apresentaram diferença significativa em relação a forma intacta (p<0,05). O métodos
propostos foram aplicados para avaliação da potência de rhPTH (1-34) e de proteínas
relacionadas em formulações biofarmacêuticas, e os resultados foram comparados com os
bioensaios, observando-se diferenças das médias de teor/potência 0,64% e 1,31% inferiores
para os métodos por CL-FR e CL-EM, em relação ao bioensaio in vivo, e 0,98% e 0,31%
superiores, respectivamente, em relação ao in vitro. Contribuíu-se assim para estabelecer
procedimentos que aprimoram o controle da qualidade, garantindo a segurança e eficácia
terapêutica do produto biotecnológico.
|
Page generated in 0.0604 seconds