Performance study of perfusion cultures for the production of single-chain urokinase-type plasminogen activator (scu-PA) in a 2.5 l spin-filter bioreactor

Jo, EC; Yun, JW; Jung, KH; Chung, Si Yin; Kim, Jung Hoe

doi:10.1007/pl00009024

Cited by 7 publications

(3 citation statements)

References 24 publications

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“…Thus, possible biotechnological configurations need to be designed and tested for simultaneous production and capture of urokinase from the bioreactors (Kadouri and Bohak, 1985). Solid microcarrier based perfusion bioreactors where reported previously for the production of urokinase (Jo et al, 1998). These however, lead to the formation of large heterogeneous cell aggregates that decrease the viable cell density and subsequently the urokinase production levels.…”

Section: Introductionmentioning

confidence: 99%

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

Kumar

Bansal

Nandakumar

et al. 2006

Biotechnol. Bioeng.

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An integrated cell cultivation and protein product separation process was developed using a new type of supermacroporous polyacrylamide gel, called cryogel (pAAm-cryogel) support matrix. Human fibrosarcoma HT1080 and human colon cancer HCT116 cell lines were used to secrete urokinase (an enzyme of immense therapeutic utility) into the culture medium. The secreted protein was isolated from the circulating medium using a chromatographic capture column. A pAAm cryogel support with covalently coupled gelatin (gelatin-pAAm cryogel) was used for the cultivation of anchorage dependent cells in the continuous cell culture mode in 5% carbon dioxide atmosphere. The cells were attached to the matrix within 4-6 h of inoculation and grew as a tissue sheet inside the cryogel matrix. Continuous urokinase secretion into the circulating medium was monitored as a parameter of growth and viability of cells inside the bioreactor. No morphological changes were observed in the cells eluted from the gelatin-cryogel support and re-cultured in T-flask. The gelatin-pAAm cryogel bioreactor was further connected to a pAAm cryogel column carrying Cu(II)-iminodiacetic acid (Cu(II)-IDA)-ligands (Cu(II)-IDA-pAAm cryogel), which had been optimized for the capture of urokinase from the conditioned medium of the cell lines. Thus an automated system was built, which integrated the features of a hollow fiber reactor with a chromatographic protein separation system. The urokinase was continuously captured by the Cu(II)-IDA-pAAm cryogel column and periodically recovered through elution cycles. The urokinase activity increased from 250 PU/mg in the culture fluid to 2,310 PU/mg after recovery from the capture column which gave about ninefold purification of the enzyme. Increased productivity was achieved by operating integrated bioreactor system continuously for 32 days under product inhibition free conditions during which no backpressure or culture contamination was observed. A total 152,600 Plough units of urokinase activity was recovered from 500 mL culture medium using 38 capture columns over a period of 32 days.

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Section: Introductionmentioning

confidence: 99%

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

Kumar

Bansal

Nandakumar

et al. 2006

Biotechnol. Bioeng.

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“…Additionally, the producer cells employed in viral vector production are mainly anchorage-dependent, such that the cells must adhere on a matrix surface to support cell growth. Microcarriers, initially invented to culture anchorage-dependent cells in a suspension culture (7), have been successful in producing viral vaccines (8)(9)(10)(11) and recombinant therapeutic proteins (12)(13)(14). Microcarriers have been recently reapplied to produce viral vectors used in gene therapy (15)(16)(17).…”

Section: Introductionmentioning

confidence: 99%

Production of Retrovirus and Adenovirus Vectors for Gene Therapy: A Comparative Study Using Microcarrier and Stationary Cell Culture

Huang

Liu

2002

Biotechnology Progress

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In gene therapy, retrovirus and adenovirus vectors are extensively used as gene-delivery vehicles and further large-scale processing of these viral vectors will be increasingly important. This study examined stationary and microcarrier cell culture systems with respect to the production of a retrovirus vector (encoding a monounit hammerhead ribozyme gene with an intron) and an adenovirus vector (encoding a reporter lacZ gene). Cytodex 1 and Cytodex 3 solid microcarriers were found to be able to provide good cell growth and high-titer vector production in suspension cultures. Porous microcarriers such as Cytopore 2 gave slightly lower but still efficient growth but produced significantly lower titers of retrovirus and adenovirus vector from the producer cells. The specific retrovirus production was not proportionally related to the specific growth rate of the producer cells. High MOI infection was essential for high-titer production of adenovirus vector in 293 cells. Hydrodynamic shear forces on microcarrier-grown cells increased the production yield for retrovirus vector but decreased for adenovirus vector. The cellular productivity was much more efficient for adenovirus vector produced in 293 cells as compared to the retrovirus vector produced in PA317-RCM1 cells. These findings can provide further insight into the feasibility of applying microcarrier cell culture technology to produce gene-therapy virus vectors.

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“…Urokinase is produced by cell culture in T‐flasks, roller bottles, spinner flasks, spin filter bioreactors, and hollow fiber bioreactors. Processes employing microcarrier‐based suspension cultures have enjoyed reasonable success 7–9. There is general agreement that high shear stress, low cell density, and product degradation are primary reasons for reduced reactor performance.…”

Section: Introductionmentioning

confidence: 99%

External modulation of HT‐1080 human fibrosarcoma cells improves urokinase production

Khaparde¹,

Roychoudhury²,

Gomes³

et al. 2008

Biotechnology Progress

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in Wiley InterScience (www.interscience.wiley.com).Urokinase was produced in a hollow fiber reactor using HT-1080 human fibrosarcoma cells. External modulation comprised replenishing of the medium in the extracapillary space, reducing the serum concentration in the extracapillary space from 10% to 2% and increasing flow rate of the circulating medium in the intracapillary space from 20 to 80 mL/min, each according to a specific protocol. More than sixfold increase was observed in the cumulative urokinase production for two and three medium replenishing modulations of the extracapillary space. After 15 days of continuous operation, the highest cumulative urokinase obtained was 1.63 Â 10 6 PU/mL. SDS-PAGE and zymogram study established that the urokinase obtained was in the high molecular weight range of 54 kDa. The effect of external modulation on cumulative urokinase production was visualized as trajectories with respect to the ratio of lactic acid production rate (LPR) to the glucose uptake rate (GUR). The collective external modulation data showed two separate physiological regions in the cumulative urokinase vs. LPR/GUR plane. The HT-1080 cells exhibited two distinct morphologies in these regions that may be related to acidosis and metastasis. These regions also correspond to low and high urokinase productivity.

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Performance study of perfusion cultures for the production of single-chain urokinase-type plasminogen activator (scu-PA) in a 2.5 l spin-filter bioreactor

Cited by 7 publications

References 24 publications

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

Production of Retrovirus and Adenovirus Vectors for Gene Therapy: A Comparative Study Using Microcarrier and Stationary Cell Culture

External modulation of HT‐1080 human fibrosarcoma cells improves urokinase production

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