Conclusions - BioProcess Consultants | Performance ...
Transcript of Conclusions - BioProcess Consultants | Performance ...
Poster Draft IBC-Boston: October 26-29, 2015
IBC_poster(r2)_01OCT15i.doc 2 10/2/2015
Conclusions
VCD Profiles
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Time (days)
VCD
(vc/
cc)
PD.021 PD.022 5L-1.15L-1.2 5L-2.1 5L-2.2min vcd max vcd 50L SUB2000L SUB (E1) 2000L SUB (E2) 2000L SUB (GMP1)
Titer Profiles
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Tite
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L)
Min titar Max Titer PD.021PD.022 5L-1.1 5L-1.25L-2.1 5L-2.2 50L SUB2000L SUB (E1) 2000L SUB (E2) 2000L SUB (GMP1)
Osmo Profiles
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Osm
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Osm
o/kg
)
5L-1.1 5L-1.2 5L-2.15L-2.2 min osmo PD.02250L SUB 2000L SUB (E1) 2000L SUB (E2)2000L SUB (GMP1)
"Target" pH profile
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Time (days)
pH
5L-1.1 5L-1.2 5L-2.15L-2.2 PD.021 PD.022Min pH Max pH 50L SUB2000L SUB (E1) 2000L SUB (E2) 2000L SUB (GMP1)
Lac residual profiles
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Time (days)
Lact
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ol/L
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5L-1.1 5L-1.2 5L-2.15L-2.2 Min Lac Max Lac50L SUB 2000L SUB (E1) 2000L SUB (E2)2000L SUB (GMP1)
Glucose Residual
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Time (day)
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cose
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ol/L
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Min Gluc Max Gluc 5L-1.15L-1.2 5L-2.1 5L-2.250L SUB 2000L SUB (E1) 2000L SUB (E2)2000L SUB (GMP1)
Figure-2: Performance targets met Equivalence achieved
7.9031.41.82000L7.9430.62.250L7.8030.81.95L7.9530.01.8PDIVCD (ave)SPR (ave)Titer (ave)Scale
Final: Titer (g/L), SPR (pg/vc/d), and IVCD (cell-day/L)
7.9031.41.82000L7.9430.62.250L7.8030.81.95L7.9530.01.8PDIVCD (ave)SPR (ave)Titer (ave)Scale
Final: Titer (g/L), SPR (pg/vc/d), and IVCD (cell-day/L)
Poster Draft IBC-Boston: October 26-29, 2015
IBC_poster(r2)_01OCT15i.doc 3 10/2/2015
Performance Simulation Methodology Bioreactor & Process Characterization Figure-3: All vessel, agitation, and sparge design details that impact performance (18) are considered. Process characterization includes; cell growth, cOUR, RQ, Qgluc, pH, SPR, volume expansion, and MB for all growth phases (Confidential).
• Working volume range 1000-2000L• Aspect ratio & Ic
• at 2000L: 1.49 (Ic = 3.2xDi)• at 1000L: 0.75 (Ic = 1.0xDi)• Headspace: 1575-575 (L)• Dt: 47.00”
• Agitation• Di = 15.67”: Di/Dt: 0.33• impeller zone fraction 0.013xVw• impeller 45o pitched blade (3): solidity 55%• Max RPM: 75• No baffles:19.6o angled, (one baffle equiv.)• Bottom spacing (Si): 1.0xDi• System Np = 2.1(1.24)• Max Power: 20.0 W/m3 at max Vw
41.0 W/m3 at min Vw• Sparger
• MIC: (2) 20-40µ sintered, 57.4 cm2 (0.015 vvm Max)• MAC: (3) 6.4 mm OP (0.12 vvm Max)• MAC (2) 582 µ DHS (1380), (0.485 vvm MAX)• Impeller spacing (Sg): 4-7”, gas dispersion impact
Di
Dt
HL min
35.1”, 1xDi
HL max
70.4”
Si1.0xDi
Sg4-7”
20-40 µ (MIC), 2x6.4 mm OP (MAC), 2x
Ic3.1xDi
0.6 mm DHS (690) (MAC), 2x
2000L SUB
0.800.822.0App. 45o 3b, Solidity 93%5L SDM
0.800.642.145o 3b, Solidity 55%2kL SUB
0.800.802.145o 3b, Solidity 55%50L SUB
6785 SD Impeller data: “Np drives performance”
0.82
FlowNq
0.80
ShearKm
2.0App. 45o 3b, Solidity 93%PD 3L
PowerNp
System detail
0.800.822.0App. 45o 3b, Solidity 93%5L SDM
0.800.642.145o 3b, Solidity 55%2kL SUB
0.800.802.145o 3b, Solidity 55%50L SUB
6785 SD Impeller data: “Np drives performance”
0.82
FlowNq
0.80
ShearKm
2.0App. 45o 3b, Solidity 93%PD 3L
PowerNp
System detailP (Hp) = Np N3 Di5 ρ /gc Np is a measured value
Flow (Q) = Nq N Di3 Nq is a measured value
Shear = Km N Di Km is measured (or calc)
P/V (W/m3) = P(Hp)*1000/Vw *746 (watts/Hp)
KLA = C * (P/V)α (Vs)β KLACO2 = 0.89*KLA
Poster Draft IBC-Boston: October 26-29, 2015
IBC_poster(r2)_01OCT15i.doc 5 10/2/2015
Simulation modeling Flow sheet Figure-5: All necessary performance elements of the Bioprocess are included in the Simulation Building Blocks. These are applied to the specific bioreactor and Process selected from the Data Bases for a simulation run. The responses are driven by the operating conditions entered (initiate simulation). The response output selection is based on the project goals. In this project, initial efforts focused on the CPP acceptable range to identify the scale-up basis that best achieves performance and avoids SU obstacles. Efforts then shifted to defining the full set of operating recommendation for each bioreactor involved in the transfer (SU equivalence). Future efforts may focus on Design Space, or robustness and optimization outputs.
Bioreactor Data Base- all design parameters - all bioreactors
Process Data Base- Historical data sets- Performance Target development
Initiate Simulation- Starting Conditions & setpoints- Operating strategies (User preferred or alternate)
Simulation Building Blocks- Hydrodynamics - MT (O2/CO2)- Mixing - HT- Dispersion & settling - Cellular kinetics- Sizing Modules - Substrate kinetics- Shear (physical, interfacial, boundary) - MB
Simulation Output Selection- SU equivalence - SD equivalence - Tech Transfer - Design Space- Troubleshooting - Process Optimization- CPP acceptable range - Platform Development- Robustness testing - Data mining- CPP auditing (response to input & visa versa)- Sensitivity analysis (Obstacle to opportunity)
Team Work- Provides a common framework
for decision making.
- Provides rapid response to complex issues with verification.
- Promotes discussion with all stakeholders.
- Addresses SU Obstacles in advance or “on the fly”.
- Integrates with other technologies (CFD, MVA, PC, ME)
Poster Draft IBC-Boston: October 26-29, 2015
IBC_poster(r2)_01OCT15i.doc 4 10/2/2015
Operating Conditions and Sensitivities Figure-4: The Scale-up basis for this tech transfer is “=P/V”, established by the Max P/V capability of the 2000L SUB. The sparge strategy utilizes (2) spargers; sintered as the OTR driver (O2 only), and DHS for CTR. SU criteria reveal the potential scale-up obstacles, at each scale, that must be managed. Typical sensitivity limits for several commonly encountered performance sensitivities (i.e. growth or cell loss impacts) are shown in the “operating notes” column.
Process Demand OTR = OUR (constant DO)2.02.02.02.0Process OUR (mmol/L/h)
< 40 m/s, CHO safe0.60.61.1Spg exit velocity (m/s)
< 0.5% to assure minimal cell loss0.150.260.05%0.66%Interfacial/foam (-%vc/d)
≤ 60s (homogeneity, base addition)106421715Blend Time (s)
> 1.5 can lead to bubble column low OTE1.58-2.901.58-2.760.75-1.360.92-1.79Impeller clearance (Di units)
> 1 needed to assure uniform dispersion1.351.960.991.29Min Mix metric
> 20 (or cell diameter), CHO safe22333435Turbulent Eddy scale (µ)
< 25, CHO safe12.78.37.16.3IZ shear rate (1/s)
SU basis: 2KL SUB power limitation.18-1218-1218-1218-12P/V (W/m3)
Scale-up Criteria: SU Obstacle avoidance
100.5000.0350.050Overlay air sparge (slpm)
Fixed (CTR driver) (reduce spg reflux risk)10-150.1500.050MAC air sparge (slpm)
On demand: OTR driver000.300MAC O2 sparge (slpm)
On demand: OTR driver3.00.2000.012MIC O2 sparge (slpm)
Basis: “=P/V” SU ( at 2KL SUB max P/V)61135225200Agitation (rpm)
Operating Recommendation
Operating notes & Performance metrics2kL SUB50L SUB5L SDM3L SDMparameter
Bioreactor Operating Recommendations and Scale-up Criteria
Process Demand OTR = OUR (constant DO)2.02.02.02.0Process OUR (mmol/L/h)
< 40 m/s, CHO safe0.60.61.1Spg exit velocity (m/s)
< 0.5% to assure minimal cell loss0.150.260.05%0.66%Interfacial/foam (-%vc/d)
≤ 60s (homogeneity, base addition)106421715Blend Time (s)
> 1.5 can lead to bubble column low OTE1.58-2.901.58-2.760.75-1.360.92-1.79Impeller clearance (Di units)
> 1 needed to assure uniform dispersion1.351.960.991.29Min Mix metric
> 20 (or cell diameter), CHO safe22333435Turbulent Eddy scale (µ)
< 25, CHO safe12.78.37.16.3IZ shear rate (1/s)
SU basis: 2KL SUB power limitation.18-1218-1218-1218-12P/V (W/m3)
Scale-up Criteria: SU Obstacle avoidance
100.5000.0350.050Overlay air sparge (slpm)
Fixed (CTR driver) (reduce spg reflux risk)10-150.1500.050MAC air sparge (slpm)
On demand: OTR driver000.300MAC O2 sparge (slpm)
On demand: OTR driver3.00.2000.012MIC O2 sparge (slpm)
Basis: “=P/V” SU ( at 2KL SUB max P/V)61135225200Agitation (rpm)
Operating Recommendation
Operating notes & Performance metrics2kL SUB50L SUB5L SDM3L SDMparameter
Bioreactor Operating Recommendations and Scale-up Criteria
Equipment CharacterizationPower, Flow, Shear
Process requirementsVCD, Titer, Sensitivity limits
P/V NqKm
KLA (O2)Shear sensitivity
- impeller-cell- fluid-cell- cell-cell
OTR requirements- Min KLA- Vs (vvm) range- sparge composition- interfacial shear- foam
CTR requirements- Min KLA (CO2)- stripping sparge regmt.
Uniform mixing regmt- pH accep. range- DO accep. range- metabolites
Sparger selection
Poster Draft IBC-Boston: October 26-29, 2015
IBC_poster(r2)_01OCT15i.doc 4 10/2/2015
Operating Conditions and Sensitivities Figure-4: The Scale-up basis for this tech transfer is “=P/V”, established by the Max P/V capability of the 2000L SUB. The sparge strategy utilizes (2) spargers; sintered as the OTR driver (O2 only), and DHS for CTR. SU criteria reveal the potential scale-up obstacles, at each scale, that must be managed. Typical sensitivity limits for several commonly encountered performance sensitivities (i.e. growth or cell loss impacts) are shown in the “operating notes” column.
Process Demand OTR = OUR (constant DO)2.02.02.02.0Process OUR (mmol/L/h)
< 40 m/s, CHO safe0.60.61.1Spg exit velocity (m/s)
< 0.5% to assure minimal cell loss0.150.260.05%0.66%Interfacial/foam (-%vc/d)
≤ 60s (homogeneity, base addition)106421715Blend Time (s)
> 1.5 can lead to bubble column low OTE1.58-2.901.58-2.760.75-1.360.92-1.79Impeller clearance (Di units)
> 1 needed to assure uniform dispersion1.351.960.991.29Min Mix metric
> 20 (or cell diameter), CHO safe22333435Turbulent Eddy scale (µ)
< 25, CHO safe12.78.37.16.3IZ shear rate (1/s)
SU basis: 2KL SUB power limitation.18-1218-1218-1218-12P/V (W/m3)
Scale-up Criteria: SU Obstacle avoidance
100.5000.0350.050Overlay air sparge (slpm)
Fixed (CTR driver) (reduce spg reflux risk)10-150.1500.050MAC air sparge (slpm)
On demand: OTR driver000.300MAC O2 sparge (slpm)
On demand: OTR driver3.00.2000.012MIC O2 sparge (slpm)
Basis: “=P/V” SU ( at 2KL SUB max P/V)61135225200Agitation (rpm)
Operating Recommendation
Operating notes & Performance metrics2kL SUB50L SUB5L SDM3L SDMparameter
Bioreactor Operating Recommendations and Scale-up Criteria
Process Demand OTR = OUR (constant DO)2.02.02.02.0Process OUR (mmol/L/h)
< 40 m/s, CHO safe0.60.61.1Spg exit velocity (m/s)
< 0.5% to assure minimal cell loss0.150.260.05%0.66%Interfacial/foam (-%vc/d)
≤ 60s (homogeneity, base addition)106421715Blend Time (s)
> 1.5 can lead to bubble column low OTE1.58-2.901.58-2.760.75-1.360.92-1.79Impeller clearance (Di units)
> 1 needed to assure uniform dispersion1.351.960.991.29Min Mix metric
> 20 (or cell diameter), CHO safe22333435Turbulent Eddy scale (µ)
< 25, CHO safe12.78.37.16.3IZ shear rate (1/s)
SU basis: 2KL SUB power limitation.18-1218-1218-1218-12P/V (W/m3)
Scale-up Criteria: SU Obstacle avoidance
100.5000.0350.050Overlay air sparge (slpm)
Fixed (CTR driver) (reduce spg reflux risk)10-150.1500.050MAC air sparge (slpm)
On demand: OTR driver000.300MAC O2 sparge (slpm)
On demand: OTR driver3.00.2000.012MIC O2 sparge (slpm)
Basis: “=P/V” SU ( at 2KL SUB max P/V)61135225200Agitation (rpm)
Operating Recommendation
Operating notes & Performance metrics2kL SUB50L SUB5L SDM3L SDMparameter
Bioreactor Operating Recommendations and Scale-up Criteria
Equipment CharacterizationPower, Flow, Shear
Process requirementsVCD, Titer, Sensitivity limits
P/V NqKm
KLA (O2)Shear sensitivity- impeller-cell- fluid-cell- cell-cell
OTR requirements- Min KLA- Vs (vvm) range- sparge composition- interfacial shear- foam
CTR requirements- Min KLA (CO2)- stripping sparge regmt.
Uniform mixing regmt- pH accep. range- DO accep. range- metabolites
Sparger selection
Poster Draft IBC-Boston: October 26-29, 2015
IBC_poster(r2)_01OCT15i.doc 6 10/2/2015
Eliminating Scale-up Obstacles At the 2000L Scale Scale-up challenges in the LSM platform centered around the impeller clearance and blend time. Potential impacts on sparge OTE (coalescence) and pH control, specifically base addition (homogeneity) were considered in advance.
2000L O2 sparge Profiles
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2000L SUB (E1) 2000L SUB (E2)2000L SUB (GMP1) MIC-O2 sparge-SIMimpeller clearance (E2,1840L-har)
2000L dCO2 profiles
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3L Min dCO2 3L Max dCO22000L SUB (E1) 2000L SUB (E2)2000L SUB (GMP1) dCO2 SIM (MAC= 10/20 slpm)
Challenge #1 Figure-6: An impact of the impeller clearance on gas dispersion and coalescence with the use of dual spargers (MIC/MAC) in the LSM was anticipated. Available KLA data was used without adjustment for the first run (E1). The sintered (MIC) sparge rate increased sharply when the impeller clearance reached 2.0 (day-8), and as the MAC sparge rate was increased from 10 slpm to 20 slpm as planned starting day-7. The increase in MIC was due to coalescence, not demand. This was also reflected in the dCO2 profile. The E1 dCO2 was lower than projected and below the acceptable range. To compensate the 10/20 slpm MAC sparge recommendation was changed to 10/15 slpm for (E2) and GMP runs. Both the dCO2 and MIC O2 sparge profiles responded to the adjustment as expected. There was no impact on 2000L performance.
Poster Draft IBC-Boston: October 26-29, 2015
IBC_poster(r2)_01OCT15i.doc 7 10/2/2015
2000L Base addition
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5L-1.1 5L-1.2 5L2.15L-2.2 2000L SUB (E1) 2000L SUB (E2)2000L SUB (GMP1)
Challenge #2 Figure-7: In preparation for the long blend time and surface mixing, actions by the team focused on: 1) tight control of the base feed adjustment to pH, and 2) limiting the adjustment time to 1.5x Blend time. While the LSM volumetric base addition (ml/L) was 3.5 times that required in the SDM, there was no performance impact. The pH was managed within the acceptable range and Osmolality while higher then the SDM values were well within the acceptable range for the Process. Opportunities for Improvement Discussion for future homogeneity improvement identified: 1) limiting the working volume to eliminate the Blend time and surface mixing challenge experienced here, and 2) Adjustments to the system drive assembly.
Poster Draft IBC-Boston: October 26-29, 2015
IBC_poster(r2)_01OCT15i.doc 8 10/2/2015
Eliminating Scale-up Obstacles At the 3L Scale
3L & 5L SDM VCD Profiles
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max vcd min vcd PD.021 PD.0225L-1.1 5L-1.2 5L-2.1 5L-2.2
Figure-8: The 3L platform exhibits a slightly lower “apparent” growth rate compared to the 5L platform. The growth responses (VCD profiles) match the interfacial cell loss response for the 2 systems. The highest % cell loss (day 0-1) is likely due to the CO2 sparge for pH control. The cell loss a peak demand (day 5-8) results from the P/V used in the tech transfer which requires a higher Vs. This sensitivity in the 3L platform was not a factor in achieving large scale performance equivalence for this tech transfer.
SDM Interfacial Loss potential comparison
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oss
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3L Interfacial Loss (%vc/d) 5L Interfacial loss (%vc/d)
Poster Draft IBC-Boston: October 26-29, 2015
IBC_poster(r2)_01OCT15i.doc 9 10/2/2015
OBJECTIVE: MINIMIZE Interfacial shear
Cell loss per sparge bubble
Bioreactor volume (Vw) Total
sparge rate
Sparge bubble rate
Bubble coalescence
Liquid height
O2 sparge rate (slpm)
CO2 sparge Rate (pH)
sparge air CAP
DO setpoint
Sparge air rate (slpm)
Process additions
Baffle factor
Sparge air composition
Sparge OTR
Impeller spacing
pH control
Vs
Sparge orifice diameter
Surface tension
Bubble size
Liquid density
OUR Overlay OTR
Sparge KLA
O2 Diving force
CL
Head pressure Cellular
OUR VCD Ov
Comp.
H KLA surface
P/V
Inoculum density
System Np
Cell loss impacts
RPM Impeller diameter
Growth rate
a/V ratio Impeller
type
Interfacial Shear Sensitivity Analysis
Impact of a +10% change in selected control variables on interfacial cell loss
-15 -10 -5 0 5 10 15
bioreactor volume
surface tension
Inoculum density
sparge orifice
slpm
head pressure
RPM
Np
% Change in Interfacial cell loss impact
Reduction in interfacial shear Increase in interfacial shear
Opportunities for Improvement The modeling approach used for the tech transfer can be used to identify options for mitigating the interfacial loss. We can start by tracing the interfacial response back to all equipment and Process inputs. Nine inputs have been selected as possible candidates. A sensitivity analysis of the 9 inputs suggests the mitigation options for evaluation. 1) Increasing the sparge orifice size has a 1/1 impact on the cell loss response. Using a step increase from the current 20µm orifice to a 50µm will both verify the source of the low “apparent” growth rate, and utilize a orifice size consistent with the MIC sparger used in the LSM platform. 2) The total gas sparge (slpm) also has a 1/1 impact on the interfacial response. Increasing the agitation (P/V) will reduce the sparge rate (Vs). Moving the CO2 sparge to overlay may be needed to reduce the day 0-1 cell loss.