An Approach to High Cell Density Fed-batch Experiments
- 1. An Approach to High Cell Density Fed-batch Experiments Developed for Our Mammalian Cell Culture Biotechnology Laboratory Course Delyan Rusev, MBS Class of 2010, Bioprocessing Focus Track, Keck Graduate Institute Interim Mammalian Cell Biotechnology Laboratory Instructor, Fall 2010 INTRODUCTION PROTOCOLS STUDENT DATA FOR BIOREACTORS The Mammalian Cell Culture Biotechnology course plays a significant role in the All experiments were conducted with DHFR- DG44 CHO cells adapted Bioprocessing curriculum at Keck Graduate Institute (KGI). The Amgen Bioprocessing 35 Viable Cell Density 7.7 pH Trend to 40mM Sodium L-Lactate (Sigma) in chemically defined OptiCHO Center (ABC) laboratories provide the students enrolled in the course with 30 7.5 medium (Invitrogen) supplemented with 8 mM Glutamine (Gibco) and VCD (106cells/ ml) 25 7.3 outstanding opportunities to work with state-of-the-art research equipment and 20 pH 7.1 15 10 6.9 starting osmolality of 350 mOsm. The feed consisted of Efficient Feed gain hands-on experience on various upstream and downstream aspects of the 5 0 6.7 6.5 B (Invitrogen), 1x CD CHO in powdered form (Gibco) and 16 mM bioprocessing field. Professor Matthew Croughan teaches the theoretical part of the 0 2 4 6 8 10 0 2 4 6 Culture Period (Days) 8 10 Glutamine (Gibco). The experimental conditions for shaker flasks and Culture Period (Days) course in close collaboration with Kirilynn Svay, senior bioprocessing engineer who bioreactors are shown in Table 3. covers the laboratory exercises with the students. I served as the interim lab instructor while Kirilynn was out on maternity leave. Lactate Trend Osmolality Table 3: Experimental conditions for shaker flasks and bioreactors 6 500 Osmolality (mOsm/kg) 5 450 Experimental Conditions Lactate (g/L) 4 400 3 350 Bioreactors: Shaker Flasks: PROJECT OVERVIEW 2 1 300 250 Initial Batch Volume: 1400 mL 60 mL 0 200 Seeding density: 0.3x10^6 cells/ mL 0.3x10^6 cells/ mL 0 2 4 6 8 10 0 2 4 6 8 10 Culture Period (Days) Culture Period (Days) Agitation: 250 – 300 RPM 130 RPM A novel fed-batch strategy to achieve 35 million cells/ml (one of the highest reported Day 0 - Day 5: CO2/Air – drilled tube sparger, Figure 1: Growth and metabolite data for bioreactors: The cell cultures grown in two bioreactors reached in the industry) developed by Nate Freund, a recent PhD graduate from KGI, was Aeration: O2 – sintered steel sparger; 8 % CO2 viable cell densities between 26 and 31 million cells/ mL (A). pH was well controlled at 7.1 with additions After Day 5 : O2/CO2 – drilled tube sparger, used as a basis for the laboratory experiments. of 1N Na2CO3 (B). A minimal amount of lactate was produced until day 4 and remained at a relatively Air – sintered steel sparger. DO% set-point: 50 % constant level until the end of the experiment (C). The osmolality of the cultures varied between 330-450 The strategy includes shaker flasks and bioreactor cultivation of a dihydrofolate mOsm/kg. (D) Temperature set-point: 37° C 37° C pH control: 1N Na2CO3 - reductase deficient (DHFR-) Chinese Hamster Ovary cell line, DG44 CHO (adapted to STUDENT DATA FOR SHAKER FLASKS pH set-point: 7.1 - high initial concentration of L - lactate in the batch media) with additions of various Culture duration: 10 days 10 days concentrated nutrient feeds. Custom CD-OptiCHO medium (Life Technologies) was pH trend used, wherein sodium chloride levels were reduced to maintain a starting osmolality Viable Cell Density 30.00 7.7 The growth parameters and the metabolite utilization of the cultures of ~315 upon lactate supplementation. An optimized sparging protocol utilizing two 25.00 7.5 VCD (106 cells/mL) 20.00 7.3 were monitored on a daily basis using ViCell analyzer (Beckman types of spargers (drilled tube and sintered steel) was incorporated in the bioreactor 15.00 7.1 pH 10.00 6.9 Coulter), Nova Biomedical 400 analyzer and Advanced Instruments experiments to meet the increased oxygen demand of the cultures during the 5.00 6.7 0.00 0.00 2.00 4.00 6.00 8.00 10.00 6.5 3250 Osmometer. exponential growth. 0 2 4 6 8 10 Process time (days) Process time (days) Lactate Trend Osmolality For the purpose of the laboratory part of the course, the original strategy was STUDENT DATA FOR BIOREACTORS: 6 500 Osmolality (mOsm/kg) 5 450 modified in terms of feed composition and feeding schedule to simplify the Lactate (g/L) 4 400 3 350 procedure and facilitate the students with their other academic tasks (Table 1&2). 2 1 300 SUMMARY 250 Also, standard CD-OptiCHO medium was used, as it was available at less expense. 0 0 2 4 6 8 10 200 0 2 4 6 8 10 Thus, starting osmolalities were higher. One goal of this experiment was to test the Process time (days) Process time (days) performance of this simplified and less expensive protocol for our course. The modified fed-batch strategy proved to be a successful tool for Figure 2: Growth and metabolite data for shaker flasks: The cell cultures grown in two 60 mL shaker flasks reached viable cell densities between 23 and 26 million cells/ mL (A). pH was not controlled with base achieving high cell density cultures in shaker flasks and bioreactors. The Table 1: The original fed-batch strategy includes multiple additions of various nutrient feeds throughout the addition but after day 3 was relatively stable in a very narrow range between 6.9 and 7.1(B). A minimal simplified protocol utilizes only one combined feed and it facilitates the cultivation. Feed volumes are shown as percent of culture volume. laboratory exercises performed in academic setting without a significant amount of lactate was produced until day 4 and was gradually consumed thereafter (C). The osmolality of Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 the cultures varied between 340-410 mOsm/kg. (D) deviation from the original fed-batch strategy developed by Nate Feed B/ 0.5x CD CHO 5% 5% 15% 5% Freund. Cell densities higher than 30 million cells per mL in bioreactors Feed B/ 20% 5% 5% INTEGRAL VIABLE CELL DENSITY (BIOREACTORS AND SHAKER FLASKS) and 26 million cells per mL in shaker flasks were successfully achieved. 1.0x CD CHO 200 mM Glutamine 2% 1% 2% 1% 500 g/L Glucose, 1% 1% 300 mM Glutamine Agitation Set Point Change: × 250 to 300 rpm Switch to O2 sparging × ACKNOWLEDGEMENTS Table 2: The modified fed-batch strategy includes the addition of only one combined nutrient feed throughout the cultivation. This project could not have been possible without the devoted work of: Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Feed B/ 1.0x CHO +16 mM 5%+5% 10% 10% 15% 7% Bioprocessing Students, Class of 2011 Glutamine Professor Matt Croughan Agitation Set Point Change: × Kirilynn Svay, Senior Bioprocessing Engineer 250 to 300 rpm Nate Freund, PhD Switch to O2 sparging × Figure 3: Integral viable cell densities calculated for bioreactors and shaker flasks using the trapezoidal Chris Warner, PhD candidate rule.