CELL CULTURE PROCESS DEVELOPMENT FOR ENZYME REPLACEMENT ... · What is the role of the Lysosome?...
Transcript of CELL CULTURE PROCESS DEVELOPMENT FOR ENZYME REPLACEMENT ... · What is the role of the Lysosome?...
CELL CULTURE PROCESS DEVELOPMENT FOR ENZYME REPLACEMENT THERAPY
Joe Peltier
Cell Culture Process Development
May 17, 2012
BioMarin at a Glance Global Biopharma Company Targeting Genetic and Serious Medical Disorders
• ~1000 Employees
• Headquarters: Novato, CA
• 20 miles north of the Golden Gate Bridge
• Therapeutic Strategy
• High unmet medical need
• Large impact in small patient populations
• First to market or best in class
• Four growing commercial products helping fund the pipeline
• Multiple new product opportunities
• Advancing clinical programs with several important data readouts in 2012
• New candidates in development
• Ongoing business development efforts
3
BioMarin’s Products and Pipeline
What is the role of the Lysosome? •Lysosomes – intracellular organelles that function as the cell’s garbage disposal and recycling system.
• Lysosomes break down cellular waste products (fats, carbohydrates, proteins, etc.) into simple compounds with specific enzymes
• Breakdown products return to the cytoplasm as new cell-building materials.
Lodish, et. al., Molecular Cell Biology, 5th Ed., 2004
Lysosomal Storage Diseases
•Lysosomal Storage Diseases (LSDs) - heritable disorders characterized by the accumulation of undigested macromolecules due to malfunction or absence of a specific enzyme
• 40+ separate diseases
• classified by the accumulated molecule - (lipids, sugars, etc.)
•Rare Unmet Medical Need
•MPS I (Aldurazyme®)—1:100,000
•MPS VI (Naglazyme®)—1:215,000
•MPS IVA—1:75,000
•Pompe Disease—1:40,000
•Replace the missing/defective enzyme with a functional version
Enzyme Replacement Therapy
Normal cell
Diseased cell
Corrected cell
Introduction of
the missing enzyme
through
the blood system
by IV
Once a week
Adding what is missing
via a receptor for lysosomal enzyme
Enzyme Replacement Therapy: Lysosomal Targeting
• M6P Receptor
• Binds lysosomal enzymes
• Targets them to the lysosome as it cycles from cell surface
• Natural targeting system for delivering drug where it’s needed
Lodish, et. al., Molecular
Cell Biology, 5th Ed., 2004
Mammalian Cell Culture Process Development
Clone
Evaluation
Media
Development
Process
Optimization
• Screen and
select the
highest
producing
and most
stable clone
• Develop
optimal
growth and
production
media for
each cell line
• Optimize
conditions for
biomanufacturing
process in a
“scale-down”
version
Scale Up
• Scale up process
for use in large
bioreactors for
production of
therapeutic
• Identify
target, isolate
gene, and
develop
expression
system
w/ Research Dept.
Expression
System
Development
w/ Manufacturing Dept.
Human Enzyme Production Process
Cell Line
Development and
Clone Evaluation
Cryovial Multi-Step Cell Expansion
Cell
Separation
Production Bioreactor
Purification &
Formulation
Time
Via
ble
Cel
l D
ensi
tyPerfusion
Fed-Batch
BatchContinuous
Modes of Bioreactor Operation
Higher maintained cell density results in higher titers (volumetric productivities) if the medium can handle the nutritional requirements.
Start
Viable Cell Density
Product Conc.
Nutrient Conc.
Viable Cell Density
Product Conc.
Nutrient Conc.
• High viable cell density
• High product concentration
• Continuous nutrient feeding
• Long product residence time
• High Metabolites concentration
• 7 - 14 days
• 20,000 L scale
Concentrated Feed
End
Fed-Batch Process
Suitable For:
• Stable Molecules
• High Productivity
• Low Product Inhibition
Concentrated Feed
Viable Cell Density
Product Conc.
Nutrient Conc.
Viable Cell Density
Product Conc.
Nutrient Conc.
• High viable cell density
• High product concentration
• Continuous nutrient feeding
• Short product residence time
• Low metabolites concentration
• 14 - 180 days
• 100 - 4000 L scale
Medium Feed, F,S 0
Spent Medium
& Products
F, Xh ,S,P
X,S,P
Purge
X,S,P
Medium Feed, F,S 0
Spent Medium
& Products
F, Xh ,S,P
X,S,P
Purge
X,S,P
Perfusion Cell Culture Process Suitable For:
• Stable & Unstable Molecules
• Low Productivity
• Product Inhibition
Cell Retention to Increase Cell Concentration
Cell Separation: Inclined Settler
• Gravity-driven cell separation (no moving parts)
• Multiple, parallel, inclined plates
• Slight density difference between cells and media generates countercurrent convective flows
Shen & Yanagimachi, Biotechnol Prog, 2011
ClearFluid
ClearFluid
Suspension
ConcentratedSediment Layer
L
b
Downward-facing Wall
Production in Perfusion Bioreactors (Mammalian)
Bioreactor
Media
Pump
Base
Pump Base
O2
MFC
CO2
Air
Sparge
Overlay DO
Probe
pH
Probe
Temp
Probe
Heating Blanket
Scale
Recycle
Pump
Acid Pump
Acid Media
Cell
Bleed
Chiller
Cell Bleed
Pump
Settler
pH-Adjusted
Harvest
Antifoam
Pump
Antifoam
Our Production Workhorse
Bioreactors are operated at constant pH, temperature, and DO levels. Other parameters are also measured off-line via analytical instruments. (glucose, lactate, gln, ammonia, LDH, dCO2, cell density & viability, & product titer).
Perfusion Process Development Goal: Produce safe & efficacious product in a cost-efficient manner
• Variables to Manipulate (Inputs):
• Media
• pH, Dissolved Oxygen, Temperature, Harvest pH
• Scale-Dependent: Bubble Size, Agitation Rate
• Perfusion: Cell Specific Perfusion Rate (CSPR), Cell Density (via Bleed Rate and Harvest Rate), Recirculation Temperature, Run Time
• Outputs to Monitor:
• Titer (product concentration)
• Product Quality: Activity, Glycosylation, Other Post-Translational Modifications
• Viability, Cell Density in Harvest Fluid, Cell Aggregation
• Metabolites: Glucose, Lactate, Glutamine, pCO2
Goal: Maximize Titer
• Assuming steady state
• in reality we include secondary terms to correct for imperfect control and cell bleed
Titer = qp•V•X/F
qp: Cell-specific productivity [g product/cell/time] (assumed constant)
V: Reactor volume (constant)
X: Cell density [cells/vol]
F: Perfusion flow rate [vol/time]
• Maximize Cell Density & Minimize Perfusion Rate Maximize Titer
• Also lowers media usage and therefore cost
• Must balance with nutritional requirements of cells
CSPR (nL/cell/d)
Productivity (g product/cell/d)
Optimization of Bioreactor Parameters (Perfusion)
Cell Specific Perfusion Rate (CSPR):
Perfusion Volume per Cell per Time
Perfusion Process Development: Background
• Product: Recombinant Human Enzyme Replacement Therapy
• Treatment for genetic lysosomal storage disorder
• Post-translational modification required for enzyme activity:
• Reaction occurs at active site
• Requires O2 as substrate
• Cell Line:
• GS-CHO expressing gene-of-interest
Perfusion Process Development
• Goal: Optimize culture conditions to improve productivity and product quality
• Important Process Parameters
• Dissolved Oxygen (DO)
• Known effects on cell growth and metabolism
• Substrate for active site post-translational modification
• pH
• Known effects on cell growth and metabolism
• Can also effect activity of lysosomal enyzmes
• Proteases
• Two-Factor Statistical Design of Experiments (DoE)
• First order and second order effects (including interactions)
Statistical Design of Experiments (DoE) Goal: Develop mathematical model to describe the experimental space
Presence of
Curvature
http://www.weibull.com/DOEWeb/estimating_regression_models_using_least_squares.htm
First Order Model
Second Order Model
Multifactorial Experimental Design
pH
DO
10% 25% 40%
7.0
6.9
6.8
Center
Point (2X)
Baseline (3X)
Design: pH & DO
pH 7.0, DO 10
pH 6.8, DO 10
pH 6.9, DO 25
pH 7.0, DO 40
pH 6.8, DO 40
pH 6.9, DO 10
pH 6.9, DO 25
• Drain and re-inoculate all reactors from baseline.
• Grow back up to steady state and collect harvest.
• Concentrate, Diafilter, Affinity Purify
• Blocking on reactor group for data analysis
Baseline:
pH 6.9, DO 40
pH 6.9, DO 40
Baseline:
pH 6.9, DO 40
Baseline Reactor: Cell Density and Viability
• Representative of all reactors throughout the experiment
Metabolic Rates: Glucose Consumption Average during steady state operation
• pH qGluc (p=0.004)
• DO qGluc (p=0.004)
Metabolic Rates: Lactate Production
• pH qLac (p=0.005)
• DO qLac (p=0.009)
Growth Rate
• No significant DO, pH, or interaction effects on growth rate (p>>0.05)
Baseline Reactor: Titer and Productivity
• Representative of all reactors throughout experiment
Specific Productivity
• No significant DO, pH, or interaction effects on qP (p>>0.05)
Product Quality: Post-Translational Modification
• Normalized to baseline
• Higher at low DO (p=0.034)
• ~20% increase from 40% to 10% DO
• pH and interaction effects not statistically significant (p>>0.05)
• Significant blocking effect: later harvests lower modification
• ~20% relative drop: 30 days
Specific Activity
• Normalized to baseline
• Specific activity higher at low DO (p=0.037)
• ~30% increase from 40% to 10% DO
• Minor DO quadratic effect (p=0.061)
• pH and interaction effects not statistically significant (p>>0.05)
Summary: DO & pH
Increasing
pH
Decreasing
DO
Increasing
Time (Block)
Growth Rate NC NC NC
qGluc
qLac
qP NC NC NC
Spec. Activity NC NC
Modification NC
• As expected: glucose/lactate metabolism
• Unexpected: active site modification/specific activity increase with decreasing DO
NC = No Statistically Significant Change
Dissolved Oxygen Effect: Potential Mechanism
• Paradoxically, high DO causes low active site modification and activity
• Counterintuitive because O2 is substrate for conversion
• Enzyme has free, unpaired cysteine
• Attaching fluorescent dye to this site rendered molecule inactive
• Peptide mapping shows adduct formation at the free cysteine
• Test effects of mild reduction
• To release adduct from cysteine Increased activity?
Dissolved Oxygen Effect: Potential Mechanism
• DTT (dithiothreitol): strong reducing agent
• Break intra-molecular disulfide bonds
• Renders molecule inactive
• TCEP (tris-carboxyethyl-phosphine): mild reducing agent
• Selectively reduces surface cysteine residues
• Improves activity of sample with high active site conversion
Modification* Specific
Activity*
#1 1.37 0.7
#2 1.00 1.0
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Act
ivit
y r
ela
tiv
e t
o r
efe
ren
ce
Concentration [mM]
TCEP
DTT
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0.00 1.00 2.00 3.00 4.00 5.00 6.00
Act
ivit
y r
ela
tiv
e t
o r
efe
ren
ce
Concentration [mM]
TCEP
DTT
#1
#2
*Normalized
Dissolved Oxygen Effect: Potential Mechanism
• Mild reduction improves activity
• May explain why high DO negatively impacts activity
• Oxidative environment likely causes adduct formation on free cysteine
• Molecular mechanism of adduct-induced inhibition is unknown
• Further investigation
• Mechanism of DO-induced inhibition of active site modification
Summary
• Perfusion Cell Culture
• Good for low productivity, unstable products
• Additional design variables
• e.g. perfusion rate, cell density, etc.
• Case Study
• Enzyme replacement therapy for lysosomal storage disorder
• 2-factor statistical design of experiments: pH & DO
• No effects on productivity
• But low DO improves product activity
• Counterintuitive: O2 is substrate for activation reaction
• High DO may cause adduct formation on free cysteine, inhibiting activity
• Mechanism for activation inhibition?
Acknowledgments
• Dr. Javier Femenia
• Dr. Erno Pungor
• Dr. Jim Michaels
• Dr. Michael Vellard
• Kelly Han
• Helai Asalati
• Maria Ng
• Jim Liu
• Julie Gardin
• Dr. Yvette Tang
• Sophie Xiao
• Dr. Vish Koppaka
• Sara Biancalana
• Kevin Do
• Jeannette Bustos
• Javier Lopez
• Gustavo Munoz
Thank You! Questions?
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