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Chang H.N.,Korea Advanced Institute of Science and Technology | Kim N.-J.,Korea Advanced Institute of Science and Technology | Kim N.-J.,Samsung | Kang J.,Korea Advanced Institute of Science and Technology | And 9 more authors.
Bioprocess and Biosystems Engineering

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody. © 2010 Springer-Verlag. Source

Lee K.S.,Changwon National University | Lee K.S.,Binex Co. | Shim J.S.,Ajou University | Paik M.J.,Sunchon National University | And 4 more authors.
Biotechnology and Bioprocess Engineering

Human bone marrow-derived mesenchymal stem cells (hMSCs) are capable of self-renewal and differentiation into various tissue lineages, attracting attention as tools for use in cell therapy. However, hMSCs have very poor proliferative capacity and a short life span in culture. To overcome this problem, we expressed the T antigen of SV40 in hMSCs because it is known to have the ability to elevate the growth rate of various primary animal cells. We obtained several hMSCs lines (hMSCs-T) known for stable expression of T antigen. Cells expressing T antigen proliferated on the monolayer of hMSCs, forming high density foci. hMSCs-T showed changed morphology and improved growth rate and life span, and demonstrated preservation of the potential for differentiation into osteoblasts. In addition, hMSCs-T did not proliferate in soft agar culture, indicating that the cells did not transform into tumor cells. In order to evaluate metabolic change of amino acids in hMSCs-T compared to primary hMSCs, we investigated altered amino acids (AA) with gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode (GC-SIM-MS). A total of 14 AAs were positively measured. Results from the Student’s t-test on the hMSCs group mean of the hMSCs-T group showed significantly elevated levels of glycine, proline, pipecolic acid, aspartic acid, lysine and tryptophan, whereas valine, leucine and isoleucine as branched-chain amino acids (BCAAs), and phenylalanine showed a significant decrease. Altered AAs metabolic pattern in the hMSCs-T may explain the disturbance of AA metabolism related to the expression of SV40 T antigen in hMSCs. © 2015, The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg. Source

Kim J.C.,Sejong University | Kim J.C.,Binex Co. | Seong J.H.,Sejong University | Lee B.,PBS Biotech Inc. | And 3 more authors.
Biotechnology and Bioprocess Engineering

Single use culture systems are a tool in research and biotechnology manufacturing processes and are employed in mammalian cell-based manufacturing processes. Recently, we characterized a novel bioreactor system developed by PBS Biotech. The Pneumatic Bioreactor System™ (PBS) employs the Air-wheel™, which is a mixing device similar in structure to a water wheel but is driven by the buoyant force of gas bubbles. In this study, we investigated the physical properties of the PBS system, with which we performed biological tests. In 2 L PBS, the mixing times ranged from 6 (30 rpm, 0.175 vvm) to 15 sec (10 rpm, 0.025 vvm). The kLa value reached upto 7.66/h at 0.5 vvm, even without a microsparger, though this condition is not applicable for cell cultures. Also, when a 10 L PBS equipped with a microsparger was evaluated, a kLa value of upto approximately 20/h was obtained particularly in mild cell culture conditions. We performed cultivation of Chinese hamster ovary (CHO) cells in 2 and 10 L PBS prototypes. Results from the PBS were compared with those from an Erlenmeyer flask and conventional stirred tank type bioreactor (STR). The maximum cell density of 10.6 × 106 cells/mL obtained fromthe 2 L PBSwas about 2 times higher than that from the Erlenmeyer flask (5.6 × 106 cells/mL) andwas similar to the STR (9.7 × 106 cells/mL) when the CHO-S cells were cultured. These results support the general suitability of the PBS system using pneumatic mixing for suspension cell cultivation as a novel single-use bioreactor system. © 2013 The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg. Source

Lim J.I.,Yonsei University | Lim K.-J.,Yonsei University | Lim K.-J.,Binex Co. | Na Y.-C.,Korea Basic Science Institute | Lee Y.-K.,Denforus Co.
Journal of Bioscience and Bioengineering

Protease inhibitors have been usually isolated through a number of steps using various chromatographical methods, which are time consuming and tedious. In this report, an efficient and low-cost acrylamide affinity gel electrophoresis method for the detection and isolation of chymotrypsin inhibitor from a crude extract was studied. The affinity gel was obtained by immobilization of chymotrypsin on 5% (w/v) poly acrylamide-oleic acid gel, and the immobilized chymotrypsin showed high stability under varied concentrations of urea (0 to 8. M), pH (4 to 10) and temperature (30 to 80°C). The affinity gel made of immobilized chymotrypsin was applied to polyacrylamide affinity gel electrophoresis and reverse electrode electro-elution using a modified commercial electrophoresis kit. Polyacrylamide affinity gel electrophoresis method showed higher isolation efficiency for chymotrypsin inhibitor from Ganoderma lucidum crude extract than a chromatographical method. Specific activity and yield of chymotrypsin inhibitor increased around 2.3-folds and 1.4-folds, respectively, compared with a chromatographical method. Also, two isomers of the inhibitor could be isolated by this method. Therefore, this method can be applied for the detection and isolation of bio-active molecules as a fast and economical method. © 2010 The Society for Biotechnology, Japan. Source

Bae J.-H.,Pusan National University | Kim J.-Y.,Pusan National University | Kim M.-J.,Pusan National University | Chang S.-H.,Pusan National University | And 7 more authors.
Journal of Immunotherapy

It is known that treatments with heat shock, some anticancer drugs, and ionizing radiation increase the expression of heat-shock proteins (HSPs) and natural killer group 2D (NKG2D) ligands in tumor cells. The increased HSPs may make the tumor cells resistant to apoptosis and reduction of HSPs may make the tumor cells more susceptible to natural killer (NK)-cell mediated lysis of tumor cells. In this study, we investigated whether quercetin which has inhibitory activities against heat-shock factor, protein kinase C, nuclear factor-κB, and phosphatidyl inositol 3-kinase, can modulate the expression of NKG2D ligands and suppress the HSPs in tumor cells. The results of this study showed that quercetin significantly induced the expression of several NKG2D ligands including major histocompatibility complex class I-related chain B, UL16-binding protein 1, and UL16-binding protein 2 in K562, SNU1, and SNU-C4 cells. The quercetin-treated K562, SNU1, and SNU-C4 cells showed an enhanced susceptibility to NK-92 cells through induction of NKG2D ligands. This increased expression of NKG2D ligands seemed to be due to the inhibition of the nuclear factor-κB and phosphatidyl inositol 3-kinase pathways. The findings of this study suggest that the induced NKG2D ligands with the decrease of HSP70 protein by quercetin may provide an attractive strategy to improve the effectiveness of NK cell-based cancer immunotherapy. Copyright © 2010 by Lippincott Williams & Wilkins. Source

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