| 1. |
- Kumar, Ashok, et al.
(author)
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Affinity binding of cells to cryogel adsorbents with immobilized specific ligands : effect of ligand coupling and matrix architecture
- 2005
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In: Journal of Molecular Recognition. - : Wiley. - 0952-3499 .- 1099-1352. ; 18:1, s. 84-93
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Journal article (peer-reviewed)abstract
- The capture of human acute myeloid leukemia KG-1 cells expressing the CD34 surface antigen and the fractionation of human blood lymphocytes were evaluated on polyvinyl alcohol (PVA)-cryogel beads and dimethyl acrylamide (DMAAm) monolithic cryogel with immobilized protein A. The affinity ligand (protein A) was chemically coupled to the reactive PVA-cryogel beads and epoxy-derivatized monolithic cryogels through different immobilization techniques and the binding efficiency of the cell surface receptors specific antibody-labeled cells to the gels/beads was determined. The binding of cells to monolithic cryogel was higher (90-95%) compared with cryogel beads (76%). B-lymphocytes, which bound to the protein A-cryogel beads, were separated from T-lymphocytes with yields for the two cell types 74 and 85%, respectively. About 91% of the bound B-cells could be recovered without significantly impairing their viability. Our results show differences in the percentage of cell-binding to the immunosorbents caused by ligand density, flow shear forces and bond strength between the cells and the affinity surface once distinct chemical coupling of protein A, size of beads, sequence of antibody binding to protein A adsorbents, morphology and geometry of surface matrices were compared.
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| 2. |
- Kumar, Ashok, et al.
(author)
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Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices
- 2006
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In: Biotechnology and Bioengineering. - Hoboken, NJ : Wiley. - 1097-0290 .- 0006-3592. ; 93:4, s. 636-646
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Journal article (peer-reviewed)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.
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| 3. |
- Nilsang, Suthasinee, et al.
(author)
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Monoclonal antibody production using a new supermacroporous cryogel bioreactor
- 2007
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In: Biotechnology Progress. - Malden, MA : Wiley-Blackwell. - 1520-6033 .- 8756-7938. ; 23:4, s. 932-939
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Journal article (peer-reviewed)abstract
- A supermacroporous cryogel bioreactor has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibodies (mAb). Hybridoma clone M2139, secreting antibodies against J1 epitope (GERGAAGIAGPK; amino acids, 551-564) of collagen type II, are immobilized in the porous bed matrix of a cryogel column (10 mL bed volume). The cells got attached to the matrix within 48 h after inoculation and grew as a confluent sheet inside the cryogel matrix. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. Glucose consumption and lactic acid production were also monitored, and during the exponential phase (similar to 20 days), the hybridoma cell line consumed 0.75 mM day(-1) glucose, produced 2.48 mM day(-1) lactic acid, and produced 6.5 mu g mL(-1) day(-1) mAb during the exponential phase. The mAb concentration reached 130 mu g mL(-1) after continuous run of the cryogel column for 36 days. The yield of the mAb after purification was 67.5 mg L-1, which was three times greater than the mAb yield obtained from T-flask batch cultivation. Even after the exchange of medium reservoir, cells in the cryogel column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for 55 days without any contamination. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel column did not differ from antibodies purified from the cells grown in commercial CL-1000 culture flasks. Thus, supermacroporous cryogels can be useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside the porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice.
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