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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