Improving Single Use Bioreactor Design and Process Development: New Research Towards Intensifying Seed- Train and Scale-Up Methods Using 5:1 Turn-Down
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1 Improving Single Use Bioreactor Design and Process Development: New Research Towards Intensifying Seed- Train and Scale-Up Methods Using 5:1 Turn-Down Nephi Jones Advanced Technology R&D Manager BioProduction Single-Use Technologies Interphex NY, NY March 2017 The world leader in serving science
2 Outline Lab Efficiency Clinical Trial Assurance Bioprocessing Productivity Logistics & Transparency Safety & Quality Thermo Scientific TM S.U.B. capabilities Benefits and Challenges of low turn-down (5:1) operation S.U.B. modifications for 5:1 operation Mass Transfer Review/Results Cell Culture Performance Conclusions 2
3 Two S.U.B. Family Offerings Open Architecture Thermo Scientific S.U.B. Integrate with the control platform of your choice Scalability from 50 to 2000 L Turnkey Solutions Thermo Scientific HyPerforma S.U.B. TK State-of-the-art S.U.B. with integrated control choices DeltaV or PLC options Choice of leading controller platforms for Turnkey S.U.B. 3
4 Single-Use Provides Lower Environmental Impact Single-Use vs. Traditional Steel Systems An Environmental Lifecycle Assessment of Single-Use and Conventional Process Technology: Comprehensive Environmental Impacts. M Pietrzykowski et al. BioPharm International. 27(3) 01 Mar *GE Affiliated Authors Environmental Impact of Single-Use and Reusable Bioprocess Systems. Rawlings and Pora. BioProcess International. February *Pall Affiliated Authors 4
5 Advantages to 5:1 Improved utilization of floor space Fewer required vessels for seed train Improved utilization of floor space Potential to increases capacity of facility Lower Risk Fewer liquid transfers and sterile line connections Fewer required vessels Fewer sizes More standardized parts & flexibility Fewer required single-use components Simplifies ordering & inventory Homogeneous mix through drain during harvest Robust scale-up 5
6 5:1 Cell Culture Design Challenges Primary Challenges CO 2 Headspace Buildup Traditional overlay gassing ineffective Changes to Fluid Mixing Impeller oversized Impeller shaft position and adjustment Fluid deflection increases off reactor bottom Mixer shear zone proportionally larger Secondary Challenges Existing Sparge Design Frit too tall in 50/100 L (removed in all vessel sizes) DHS as primary DO control Temperature Control Water jacket recommended Bottom jacket required Feed strategy implementation Probe and overlay locations Upper probe belt for overlay gas Sensors position on 50/100/250 L Film choice important Surface area to volume ratio L&E Film platform 6
7 CO 2 Build-up in Headspace Lake Nyos Analogy Lake Nyos lies on the edge of an inactive volcano in Cameroon Magma pocket below lake leaks CO 2 into the lake leading to acidification, CO 2 blanket over lake surface In 1986, a landslide led to CO 2 on the surface of the lake pouring into nearby valleys suffocating 1700 people, 3500 livestock Future catastrophes mitigated by installing CO 2 degassing tubes into lake Photo source: U.S. Geological Survey 7
8 Thermo Scientific Cross Flow Sparger CO 2 builds in the headspace, heavier CO 2 blankets the liquid surface Increases dissolved CO 2 Dampens oxygen transfer, as headspace overlay gasses affect partial pressure of gas in solution Traditional top entry overlay sparger insufficient to mix headspace gas Gas velocity is too low at the liquid height Solution: Thermo Scientific patented Cross Flow Sparger Introduce gas just above the liquid height Improves mass transfer using headspace Use DHS to fine-tune DO and ph Run more like a rocking/stirred-tank reactor combo Reduces bubble sparging and associated foam damage to cells It is important to position the overlay gas entry point in close proximity to liquid surface when at 20% rated working volume. 8
9 Reactor Turndown Strategy Retrofit Kit Design Space Parameters (5:1 Turn-down) Agitation angle 19.6 to 16.5 Bottom sparge location maintained 20% liquid volume covers top of impeller Side entry cross flow sparge ~12 lpm 2:1 5:1 500L SUB (50, 100, 250, 500L S.U.B.s) Motor mount angle lowered (3.1 less) Drive shaft length increased (6-16 cm) 250L 250L 1000L SUB (1000, 2000L S.U.B.s) Pneumatic controlled agitator positioning Safety Interlocks (shaft loading, auto-locking up & down positions, agitation PIV scaling) Simple design for cgmp two pre-defined motor positions fix the agitator location High Standard impeller position Low 5:1 cell culture seeding or harvest 2,000L 5:1 9
10 Thermo Scientific Drilled Hole Sparger SUB scale-up strategy using DHS as primary sparge Performance less dependent on agitation Uniform, consistent bubble size independent of gas flow rate (0.1 VVM) Trivial gas entrance velocities Better bubble pre-distribution Less stable foam generation Target specific O 2 :CO 2 mass transfer rates (3:1) Flexible use of 4-gas strategy Provides oxygen delivery and effective CO 2 stripping both are important 10
11 What is mass transfer and k L a? Comes from simplified gas liquid film theory equation: N L = k L a(c Li C L ) k L is the transfer coefficient (1/m 2 /hr) a is the area of flux (m 2 ) C Li is the interface concentration (mg/l) C L is the liquid bulk concentration (mg/l) N L is the mass transfer rate (mg/l/hr) Driving factors Partial pressure difference (C Li C L ) Area available for transfer vs. volume (a) Gas bubble liquid film thickness (k L ) Gas bubble liquid film resistivity (k L ) Simplified Mass Transfer Diagram 11
12 Measuring O 2 and CO 2 Transfer % gas sat. Measure k L a via Dynamic Method Commonly used for measuring O 2 delivery performance Can assess CO 2 stripping as well Repeatable, consistent, inexpensive Test solution 1 g/l Poloxamer g/l HEPES buffer ph 7.25 at air saturation O 2 vs. CO 2 Considerations Solubility Operating partial pressure O2 20 CO Time CO 2 Stripping Data Vertically Mirrored On O * 2 Table 1. Driving Partial Pressure Delta Available to Strip CO 2 and Add O 2 in a Typical Animal Cell Culture Bioreactor (in atm assuming 1 atm ambient pressure) & Corresponding Ratio of Dissolved Gas in DI H 2 O at 37ºC Reactor Dissolved O 2 Setpoint CO 2 Stripping partial O 2 Delivery partial pressure delta Mass Ratio Dissolved Gas pressure delta with air sparging with air sparging with O 2 CO 2 O 2 30% air saturation % air saturation * Note: CO 2 sensor limits exceeded causing signal saturation near 20%. 12
13 Cross Flow Sparger CO 2 Stripping Performance k L a (1/hr) L S.U.B. CO 2 stripping rates of cross flow sparger VVM frit L/min 35 L/min 20 L/min 10 L/min Position above liquid surface (inches) 13
14 Oxygen Transfer, 250L 5:1 k k L L a a (1/hr) Consistent O 2 mass transfer between standard overlay and cross flow sparger Overlay/CFS = 14 L/min air 250 L 5:1, O 2, 40 W/m DHS flow (VVM) 250 L, CFS L, Standard L, No Overlay Overlay 250 L, No Overlay 14
15 CO 2 Stripping Improvements k L a (1/hr) L 5:1, CO 2 stripping, 40 W/m 3 Large increase in CO 2 stripping utilizing cross flow sparger, Overlay/CFS = 14 L/min air DHS flow (VVM) 250 L, CFS 250 L, Standard L, Overlay Standard L, No Overlay Overlay 250 L, No Overlay 250 L, No Overlay 15
16 System Scalability, CO 2 Stripping k L a (1/hr) CFS at L/m 2 /min air 5:1, CO 2 stripping, 40 W/m Gas flow (VVM) 50 L 100 L 250 L 500 L 1000 L 2000 L 16
17 Cell Culture Studies GIBCO TM Freedom TM CHO-S TM, mab producer GIBCO Dynamis TM AGT TM Medium Advanced Granulation Technology TM 0.1% GIBCO Anti-Clumping Agent Feeds EfficientFeed TM C+ AGT Supplement 2X concentration 15% constant feed (day 3-10) 45% Glucose constant feed as needed (<5 g/l) 17
18 S.U.B. Operation 20 W/m 3 agitation DHS as only sparger O 2 as primary gas; CO 2 /N 2 as needed CFS/Overlay ~50-70 L/m 2 /min Seed at 20% WV on D0 Feed to 85% WV on D2-3 with standard media Continue standard feed D5-12 Variable ph control ph 7.2 at start CO 2 stripping too high D0-D2/3 No ph control after D2/3 (no base, no CO 2 ) Maintains CO mmhg ph variable
19 Scalable Cell Culture Results VCD (E06 cells/ml) Viability (%) Feed to full volume: 100 L = D3 All others = D Time (Day) 50 L 100 L 250 L 500 L 1000 L 2000 L 19
20 Consistent Cell Culture Performance VCD (E06 cells/ml) Viability (%) Time (Day) 50 L seed 5:1 --> full vol 50 L seed 5:1 --> maintain 5:1 250 L seed 5:1 --> full vol 250 L seed full vol 20
21 Mix Through Drain, Harvested Bioreactor Normalized Biomass time motor speed reduction in 250 L at half volume to reflect 20 W/m 3 power input Time (hr) 50 L 250 L 50 L harvest at constant motor RPM through drain 250 L harvest included 1-time motor adjustment to reduce to 20 W/m 3 at half volume Motor stopped at 20% WV, biomass monitoring continued through harvest Maximum 10% cell density difference through harvest maintained 21
22 Cell Culture Strategies Option 1: Option 2: Option 3: Improved Seed Train for Multiple Production Vessels Optimized Floor Space for Single Production Run Intensified Seed Train for Reducing Production Run Time 750 ml Flask 750 ml Flask 750 ml Flask 50 L at 5:1 Volume 50 L at 5:1 Volume, then full volume 50 L at 5:1 Volume 50 L at Full Volume 2000 L at 5:1 Volume* 2000 L at Full Volume 1000 L at 5:1 Volume, then full volume 6 x 2000 L at Full Volume 50 L at Full Volume, Initiate Perfusion to 50E06 cells/ml 1 x 2000 L at Full Volume, 4X seed concentration 22
23 Thermo Scientific HyPerforma 5.1 Single-Use Bioreactor Improved scale volume 10 L-2000 L WV Retrofit kits available for all HyPerforma S.U.B.s Bottom-jacketed for improved heat transfer Drilled hole sparger for superior mass transfer scale-up Cross flow sparger for CO 2 stripping control Scalable mass transfer among vessel sizes Repeatable cell growth with no loss in performance Homogeneous mix through drain 23
24 Questions? Thank You Products for research use only. Not for use in diagnostic procedures Thermo Fisher Scientific Inc. All rights reserved. All 24 trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
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