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

Kumar, Ashok LU ; Bansal, V ; Nandakumar, K S ; Galaev, Igor LU ; Roychoudhury, PKR ; Holmdahl, Rikard LU and Mattiasson, Bo LU (2006) In Biotechnology and Bioengineering 93(4). p.636-646
Abstract
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... (More)
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 back-pressure 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. (c) 2006 Wiley Periodicals, Inc. (Less)
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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
IMAC, cell, scaffold, supermacroporous cryogels, integrated processing, urokinase
in
Biotechnology and Bioengineering
volume
93
issue
4
pages
636 - 646
publisher
John Wiley and Sons
external identifiers
  • wos:000235630600004
  • pmid:16435398
  • scopus:33644894497
  • pmid:16435398
ISSN
1097-0290
DOI
10.1002/bit.20719
language
English
LU publication?
yes
additional info
The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Medical Inflammation Research (013212019), Biotechnology (LTH) (011001037)
id
08c3de99-2789-4d27-b94d-a1b8ec5e9d66 (old id 417222)
date added to LUP
2016-04-01 12:18:54
date last changed
2021-06-16 03:54:51
@article{08c3de99-2789-4d27-b94d-a1b8ec5e9d66,
  abstract     = {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 back-pressure 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. (c) 2006 Wiley Periodicals, Inc.},
  author       = {Kumar, Ashok and Bansal, V and Nandakumar, K S and Galaev, Igor and Roychoudhury, PKR and Holmdahl, Rikard and Mattiasson, Bo},
  issn         = {1097-0290},
  language     = {eng},
  number       = {4},
  pages        = {636--646},
  publisher    = {John Wiley and Sons},
  series       = {Biotechnology and Bioengineering},
  title        = {Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices},
  url          = {http://dx.doi.org/10.1002/bit.20719},
  doi          = {10.1002/bit.20719},
  volume       = {93},
  year         = {2006},
}