Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fed-batch operation for the production of t-PA using Chinese Hamster Ovary (CHO) cells was optimized using serial and parallel experimentation. The feed, an isotonic concentrate, was improved to obtain 2- to 2.5-fold increases in integrated viable cell days versus batch. With a low glucose inoculum train, the viability index was further increased up to 4.5-fold. Hydrolysates were substituted for the amino acid portion of the concentrate with no significant change in fed-batch results. The concentrate addition rate was based on a constant 4 pmol/cell.day glucose uptake rate that maintained a relatively constant glucose concentration (approximately 3 mM). Increased viable cell indices did not lead to concomitant increases in t-PA concentrations compared to batch. The fed-batch concentrate and feeding strategy were shown to be effective in hybridoma culture, where a 4-fold increase in viable cell index yielded a 4-fold increase in antibody concentration. The half-life of t-PA decreased from 43 to 15 days with decreasing cell viability (from 92% to 71%), but this was not sufficient to explain the apparent t-PA threshold. Instead, the CHO results were explained by a reduction in t-PA production at higher extracellular t-PA concentrations that limited the fed-batch maximum at 35 mg/L for the cell line investigated. Analysis of both the total and t-PA mRNA levels revealed no response to increasing extracellular t-PA concentrations upon exogenous additions. Instead, intracellular t-PA levels were increased, revealing a possible secretory pathway limitation. A new reactor configuration was developed using an acoustic filter to retain the cells in the reactor while an ultrafiltration module stripped t-PA from the clarified medium before the permeate was returned to the reactor. By adding this harvesting step, the t-PA fed-batch production was increased over 2-fold, up to a yield of 80 mg/L.
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PMID:Increased t-PA yields using ultrafiltration of an inhibitory product from CHO fed-batch culture. 1102 71

A Chinese Hamster Ovary cell line, CHO1-15(500), producing recombinant human tissue type plasminogen activator (tPA) via the dihydrofolate reductase (DHFR) amplification system, was studied in batch culture. In this system both DHFR and tPA are under the control of the strong constitutive viral SV40 early promoter. Employing the cumulative viable cell-hour approach, the specific productivity of tPA had maxima in the lag (0.065 pg cell(-1 )h(-1)) and early decline (0.040 pg cell(-1 )h(-1)) population growth phases. The viable population was assigned into four subpopulations (G1, S, G2/M phase, and Apoptotic cells) using flow cytometric analysis. As expected, intracellular DHFR was maximally expressed during the S cell cycle phase. The production of tPA, however, was found to be a direct linear function of the G1 phase, with a subpopulation specific productivity of 0.080 pg c-h(-1). Productivity maxima in the lag and early decline corroborate the flow cytometric data, indicative that this recombinant tPA production occurs primarily in the G1 cell cycle phase, not the S phase. This suggests that endogenous regulatory mechanisms are important controlling influences on the production of recombinant tPA in this ovarian cell line. Productivity from recombinant cell lines cannot be inferred from either the plasmid construct or the host cell alone. Elucidation of the relationship between expression of recombinant protein and the cell cycle phases of the host cell is a major component of the characterization of the animal cell production system. This information facilitates rational process design, including operating mode, modelling and control, and medium formulation.
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PMID:Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line. 1900 65

Directed control of cell metabolism by a modification of the physicochemical conditions (presence of Na-butyrate and modification of the temperature) was used to modulate the productivity of human recombinant tissular plasminogen activator (t-PA) expressed under control of SV40 promoter in Chinese Hamster Ovary (CHO) cell lines. We showed that both by adding Na-butyrate or lowering temperature from 37 degrees C to 32 degrees C there is an increase in the amount of t-PA excreted, while cell growth is significantly reduced. The treatments also increased the intracellular amount of t-PA. We measured the distribution of cell cycle phases by cytometry and used a modification of the equations of Kromenaker and Srienc (1991, 1994 a, b) to analyse the intracellular t-PA production rate in the different cell cycle phases. Intracellular t-PA was shown to accumulate in G1 phase in all conditions (at 37 degrees C, at 32 degrees C and in presence of butyrate). Moreover, we have shown that the distribution of the time cells treated by butyrate are maintained in the G1cell cycle phase is significantly increased. t-PA produced in the different cell culture conditions tested was analysed by zymogram and western blotting: neither butyrate, neither the shift of temperature changed significantly the overall quality of the protein. The N-glycan patterns of recombinant human t-PA was also analysed with carbohydrate-specific lectins. Butyrate caused a transitory increase in N-linked complex high-mannose oligosaccharides without any effect on the sialic acid content of t-PA.
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PMID:Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis. 1900 17

The effects of Na-butyrate on the physiological behaviour and on the specific productivity of recombinant tissue plasminogen activator (t-PA) Chinese Hamster Ovary (CHO) cells were characterized. Batch cultures were performed in a 3.5-L bioreactor. Na-butyrate was added either at the mid-exponential growth phase (48 h) or at the end of the exponential growth phase (74 h). The cultures with Na-butyrate showed higher net specific productivity of t-PA and lower final cell density and viability. Maximum specific productivity of t-PA for all cultures coincided with the early plateau phase (84 h). The cell's specific oxygen uptake rate (qO2) increased after the Na-butyrate addition and remained higher than that of the controlled culture. Triphosphate nucleotides, ADP, AMP and UDP-sugars all increased after 84 h in the cultures with Na-butyrate, showing different behaviours when Na-butyrate was added at 48 h or 74 h. Na-butyrate did not affect the cell's adenylate energy charge until the cell's viability started to decrease (156-168 h). The controlled culture and the culture with Na-butyrate addition, showed at 74 h, similar time trends as for purine and nucleotide ratios ((ATP+GTP)/(UTP+CTP) and UTP/ATP) with clear shifts in behaviour at 84 h and 168 h. However, the addition of Na-butyrate at 48 h resulted in damped variations of purine and nucleotide ratios in comparison to both the control culture and the culture with Na-butyrate addition at 74 h.
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PMID:Na-butyrate sustains energetic states of metabolism and t-PA productivity of CHO cells. 1961 65

An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncatedmutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncatedmutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.
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PMID:Combined TGE-SGE expression of novel PAI-1-resistant t-PA in CHO DG44 cells using orbitally shaking disposable bioreactors. 2221 Jun 17