Conclusions - BioProcess Consultants | Performance ...

Poster Draft IBC-Boston: October 26-29, 2015

IBC_poster(r2)_01OCT15i.doc 2 10/2/2015

Conclusions

VCD Profiles

0.0E+00

2.0E+06

4.0E+06

6.0E+06

8.0E+06

1.0E+07

1.2E+07

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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

0.0

0.5

1.0

1.5

2.0

2.5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Time (days)

Tite

r (g/

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

300

320

340

360

380

400

420

440

460

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Time (days)

Osm

o (m

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

6.6

6.7

6.8

6.9

7.0

7.1

7.2

7.3

7.4

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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

0

10

20

30

40

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Time (days)

Lact

ate

(mm

ol/L

)

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

0

10

20

30

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Time (day)

Glu

cose

(mm

ol/L

)

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)



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



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)



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



















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



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.





































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



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

0.00

0.50

1.00

1.50

2.00

2.50

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Time (d)

spar

ge ra

te (s

lpm

)

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

Impe

ller c

lear

ence

(Di u

nits

)

2000L SUB (E1) 2000L SUB (E2)2000L SUB (GMP1) MIC-O2 sparge-SIMimpeller clearance (E2,1840L-har)

2000L dCO2 profiles

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Time (days)

dCO

2 (m

mH

g)

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.



2000L Base addition

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Time (days)

1N c

arbo

nate

add

ition

(ml/L

)

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.



Eliminating Scale-up Obstacles At the 3L Scale

3L & 5L SDM VCD Profiles

0.E+00

2.E+06

4.E+06

6.E+06

8.E+06

1.E+07

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Time (days)

VCD

(vc/

cc)

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

0.0%

0.1%

0.2%

0.3%

0.4%

0.5%

0.6%

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Time (days)

Inte

rfac

ial l

oss

(%vc

/d)

3L Interfacial Loss (%vc/d) 5L Interfacial loss (%vc/d)



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.

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