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Sensors in Bioprocess ControlHarry Lam
Department of Manufacturing SciencesGenentech, Inc.
February 28, 2000
Genentech, Inc.
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Harvard’s LawUnder the most rigorously controlled
conditions of pressure, temperature, volume, humidity, and other variables, the organism
will do as it darn well pleases
Translation …You put the organisms into the tanks and pray
Genentech, Inc.
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Presentation Outline
The needs for process controlIndustrial bioprocessesCell and its environmentMeasurementsSensor requirementsExamplesConclusion and future directionsAcknowledgments
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The Need for Process Control
To maintain consistent process performance (productivity, quality) throughout the development cycle (R&D to Manufacturing).
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Consistent Process Performance
R&DExperiment to ExperimentScale to Scale
ManufacturingThaw to ThawRun to RunCampaign to CampaignPlant to Plant
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Sensors for Bioprocess Control
Measurements for process or system analysis
Search for underlying functional relationshipsIn depth analysis of the interaction of the organisms with their environment
Provide capabilities for process controlSetting up and maintaining the optimum environmental conditions for growth and/or formation of product
Large-scale versus laboratory bioreactors
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Generic Process Flow Diagram for Protein Pharmaceutical
Inoculum Preparation
Medium Preparation
Raw Materials
Inoculum Fermentor
Production Fermentor CellsOxygen
Crude Product
Product ConcentrationPurification
Purified Product
Purified Product Formulation Sterile
FiltrationFilling & Freeze Drying
Final Product
Viral Inactivation
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Harvest
Harvest
PRODUCTION STAGE non-selective serum-free
Production MediumMCB WCB
PREQUALIFIED CELL AGE LIMIT (Master Cell Bank Thaw to Harvest)
Culture Fluid to Recovery
Culture Fluid to Recovery
Medium Exchange or Direct Transfer
Medium Exchange or Direct Transfer
TYPICAL LARGE-SCALE CELL CULTURE PROCESS
INOCULUM TRAIN non-selective
Inoculum Medium
SEED TRAIN
selective Seed Train Medium
with 2% FBS
(continuous passaging with methotrexate)
Nutrient Feeds
Nutrient Feeds
Wash Medium
Wash Medium
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Scaling Up From 2-L to 12,000-L
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Cell and Its EnvironmentCells are isolated from complex multicellular organisms
Homeostasis is maintained in these organisms by many specialized organs and tissues working synergistically.
Therefore mammalian cells do not have the capacity to maintain homeostasis by themselves
Cell culture is not a natural environment for mammalian cells.
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Measurements
BiologicalChemicalPhysical
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Biological Measurements
Cell DensityCell ViabilityCell SizeMorphologyCellular assaysMolecular/Genetic Assays
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Chemical Measurements
MediaCarbohydrates - e.g. glucose, galactoseComplex medium – protein hydrolysates, yeast extracts, etc.Amino acidsSaltsLipids - Linoleic AcidHormones, growth factors (serum, insulin)VitaminsTrace elements - e.g. metals (Fe, Mn, etc.)AntifoamF-68AntibioticsMethotrexate
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Chemical Measurements (cont.)
Product ConcentrationQuality
By-productsOrganic acids – acetate, lactate, etcProteinsAmmonia
Chemical environmentpHDissolved gases (dissolved O2, pO2, pCO2)Osmolality
Off-gasO2 (OUR)CO2 (CER)
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Physical Measurements
TemperatureAgitationPressureLevel (volume)WeightBroth densityViscosity
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Important Criteria for Sensors
Reliability, Accuracy, ReproducibilityLong-term Stability SpecificityResponse timeDynamic behavior Ability to be repeatedly cleaned and sterilizedEase of operationEase of maintenanceSizeCost
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Modes of Bioprocess Monitoring
Callis JB, Illman DL, Kowalski BR. Process Analytical Anal Chem 1987;59:624A-637A.
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Sampling Considerations
Representative sampleSample sizeSterility requirementsUtilities considerationsDisposal considerationsLiquid versus vapor samplesSample preservation
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Requirements for in Situ Sensors
Fully cleanable and sterilizableGood thermal stability and compressive strength, no temperature hysteresis
Long-term stability and accuracyFast responseNo flow dependenceNo interference
Air bubbles (O2 and CO2) or by microbesComplex media
No foulingLow maintenanceSmall size
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Sterile Sampling Designs
Sample locationSample withdrawal positionMethod of connectionfluid velocity profileContainment considerations
Sampler and container designMaterials of constructionProcess and sample variables
TemperaturePressureSlurry/two phasesviscosity
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Sampling Designs (Cont.)
Sterilization optionsPurging considerationsVenting considerationsContainmentDesign options
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Cell Density and Viability Measurements
Need to know the viable and nonviable cell density to evaluate growth rate, death rate and specific productivityDirect - Count the cells
Hemocytometer with Trypan Bluestaining - total viable and total nonviable cellsCoulter counting - total cell number
Indirect - Measure a factor which correlates with cell numberPacked Cell Volume (PCV) - correlation with cell numberOptical Density (OD)Dry weightTotal DNATotal proteinCellular enzymatic activityCellular metabolic activity
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CHO Cell Biomass Estimation via Oxygen Uptake Rate Measurement
0
2
4
6
8
10
12
0 10 20 30 40Viable Cell Density(1E5 vc/ml)
Oxy
gen
Upt
ake
Rat
e (%
sat/m
in) QO2 = 0.30 ± 0.03 mmol O2/E9 vc-hr
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Cellular Assays
Measure physiological or expressed parameters and will be useful in measuring “How” cell lines differ.
Bromodeoxyuridine (BrdU) cell cycleFluorescent methotrexate (F-Mtx) bindingSpecific productivityCell tracer
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Cell cycle analysis with anti-BrdU and flow cytometry
S Phase
G1 G2
BrdU-FITC
BrdU
-FIT
CNeg.
Pos.
Dual parameterhistogram
Single parameterhistogram
Y
Y
G2/M2n DNA
G11n DNA
S-phase1->2n DNA
YY
Y YY
Y Y Y
YY
YY Y
Anti-BrdUMAb
BrdUIncorporation
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Measurement of intracellular product
Cells are•Formaldehyde-fixed
•Detergent permeabilized
•Product is detected with FITC conjugated F(ab’)2
Neg control
Low producingcells
Normally producingcells
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Rapid Loss of Intracellular Product Over Time
7 days
15 days 57 days
C lineB lineA line
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Cell Culture pH Controlglucose, amino acids, vitamins, O2, …
----> Cells, CO2, Lactate, NH3, H2O, Product ...
Control ObjectivesMaintains desired pH with minimum osmolality riseEnsure consistent pH profiles from run to run
Typical Means of ControlUse acid source (CO2 gas) and base source (Na2CO3)Apply gap/deadband controller (± 0.03 pH units from setpoint)
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Control of Dissolved Gases in Cell CulturesDuring aerobic growth, cells require O2 and produce CO2Cells sensitive to extremes of dissolved gas concentrations
Hypoxia (<1% of air saturation?)Hyperoxia (>100% of air saturation?)CO2 required for synthesis/energy metabolism reactionsExcess CO2 can inhibit respiration reactions, change intracellular pHMinimum required levels unknownCO2 levels may influence product characteristicsControl of culture pH, dO2, dCO2, pressure all inter-related
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Theoretical Nutrient Depletion Times in E.coli Fermentation
OxygenAt 30% of air saturation, [O2] ~ 0.075 mMWith OUR = 5 mmoles/L-minDepletion Time = 1.8 seconds
GlucoseTo avoid acetate formation, [glucose] ~ Ks, glucKs, gluc ~ 20 µM = 3.6 mg/LDuring growth, glucose uptake rate ~ 2 mmoles/L-minDepletion Time ≤ 0.6 seconds
Typical mixing times = 12 to 50 seconds
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Galactosylation Control via Controlled Nutrient Feeding
NH3 can influence glycosylation efficiency by increasing intracellular pH
Reduce waste product accumulation (i.e., NH3) by controlled feeding of nutrients which generate NH3
Use on-line biomass estimation to generate feed profile
Process Controller
Nutrient Uptake Model:
Bioreactor
Nutrient Feed
In-situ viable biomass probe
(capacitance, OUR, etc.) Mass Balance
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Nutrient Feed Rate Based on Estimated Viable Cell Population
0
2
4
6
8
10
12
14
16
0 50 100 150 200Run Time (h)
Cap
acita
nce
(pF)
0
200
400
600
800
Total Fed (mL)
CapacitanceTotal Fed
2L Bioreactor
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Controlled Nutrient Feed Results in Improved Galactosylation
0123456789
10
0 50 100 150 200Run Time (h)
Glu
tam
ine
(mM
) Up-Front GlnFed-Batch GlnFed-Batch Gln
0123456789
10
0 50 100 150 200
Run Time (h)
Am
mon
ium
(mM
)
(2L Bioreactors)
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Controlled Nutrient Feed Results in Improved Galactosylation
0.30
0.40
0.50
0.60
0.70
0 50 100 150 200Run Time (h)
Gal
Con
tent
(mol
/HC
)
Up-Front GlnFed-Batch GlnFed-Batch Gln
0 50 100 150 200Run Time (h)
MA
b Ti
ter (
mg/
L)
Up-Front GlnFed-Batch GlnFed-Batch Gln
* No medium galactose present for these experiments *
(2L Bioreactors)
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Immediately Applicable But Still Missing...
On-line in situ glucose (and other critical metabolites) sensorsOn-line viability measurement for cell cultureSimpler (for manufacturing use) population assessment toolsSoft sensorsAnalysis tools for retrospective modeling, troubleshooting and optimization
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Conclusion and Future Directions
Improved sensor technology can provide the basis for new control strategiesIncrease our ability to run more consistent and more productive bioprocessesThe challenge is to develop sensors which can be readily implemented for more effective process control
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Acknowledgments
John FrenzCynthia HoyTom IhrigBob KissJim Swartz