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

Research output: Contribution to journalArticle

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.

Details

Authors
  • Ashok Kumar
  • V Bansal
  • K S Nandakumar
  • Igor Galaev
  • PKR Roychoudhury
  • Rikard Holmdahl
  • Bo Mattiasson
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Immunology in the medical area

Keywords

  • IMAC, cell, scaffold, supermacroporous cryogels, integrated processing, urokinase
Original languageEnglish
Pages (from-to)636-646
JournalBiotechnology and Bioengineering
Volume93
Issue number4
Publication statusPublished - 2006
Publication categoryResearch
Peer-reviewedYes

Bibliographic note

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)