How To Develop A Cell Line

(1)

1

Advances in The Development

of Biopharmaceuticals

The application of modern technologies and services to the development of Biopharmaceutical processes

Dr Jonathan H Dempsey Process Science Fellow Invitrogen

(2)

The New Challenges in Biopharmaceutical Development

Increased Numbers

(3)

3

Invitrogen Proprietary & Confidential

Potential Solutions to these Challenges

Automation

Platforms

(4)

Invitrogen’s Approach to These Challenges

• Develop lock and key products to shorten the development

cycle.

– Kits to Simplify Workflows.

– Innovative products to enhance development projects

• Offer outsourced services based upon new technologies and

products.

– Cell Line Development

– Medium and Process Development

• Develop a Platform For CHO Cell Lines • Invest in Automation

– ClonePixFL – SimcellTM

(5)

5

Invitrogen Proprietary & ConfidentialInvitrogen Proprietary & Confidential

5

Technology within the process

Cell & vector development Process development Scale-up and technology transfer Released banks Released drug product Assay development cGMP banking and testing Gene _{manufacturing}cGMP and testing

(6)

Examples of Our Approach

• Development of the Invitrogen CHO Platform and CLD kits. • Use of the Simcell for automated Process Development

(7)

7

The Invitrogen CHO Platform and

cell Line Development Service

(8)

Invitrogen CHO Platform

Molecular biology

Vector design and optimisation / Transfection kits GIBCO OptiCHO Antibody / Express Kit

Cell lines

Pre-adapted DG44 (Stable Expression), HEK293 and CHO-S (Transient Expression)

Dedicated serum-free cloning media

2xOptiCHO™ Cloning medium Custom cloning media

Dedicated CD Media

CDCHO / CD OptiCHO

Feeds for fed-batch

CHO CD EfficientFeeds™

(9)

9

OptiCHO Antibody Express Kit

• Contains

– all of the elements required to construct, clone and

produce the vectors

– Cell line

– Medium for transfection and selection ( wit and without

HT)

(10)

Process Flow

_Construct Vectors Tandem Transfect DG44 Selection with G418 / -_HT Amplification with MTX Screening Stability Lead Clone (s)

(11)

11

Case Study –

Generation of Cell Lines Expressing Recombinant IgG

• Serum and suspension adapted DG44 CHO • pOptivec vector tandem transfection

• OptiCHO Antibody Express Kit used for transfection and

cloning

• Serum free transfection and cloning using lipid based

transfection reagents.

(12)

Cell Line Development using OptiCHO Antibody Express

Antibody Expression from a DHFR DG44 CHO

0 50 100 150 200 250 300 C62 c202 c174 c207 C16 c210 c187 C80 c110 c159 c203 c201 c209 C122 C71 C121 c188 c185 C143 C102 C56 C117 c145 c175 C105 C103 C106 c161 C104 C2 C72 C41 C70 C109 c205 C144 c200 C79 Well ID A n ti b ody Ti tr e ( m g/ L)

Cell pool transfected, selected and amplified. Clones screened by FACS. Top 40 clones isolated.

(13)

13

Enhancing Screening by Process Optimisation

Chromos CHOK1 SV Line Expressing IgG

CD OptiCHO Fed Batch Chromos CHO K1 Clone 1

Time in Culture (days)

0 2 4 6 8 10 12 14

Viable Cell Dens

ity (cells/ml) 1e+6 1e+7 rIgG (m g/L) 0 1000 2000 3000 4000 CD OptiCHO

CD OptiCHO Fed batch 1 CD OptiCHO Fed batch 2 CD OptiCHO rIgG

CD OptiCHO Fed batch 1 rIgG CD OptiCHO Fed batch 2 rIgG

Two fold increase in Peak Cell Density

Five fold increase in rIgG

Viable Cell Density (cells/mL) IgG Co

ncentratio n (mg/L) 4000 3000 2000 1000 0 1x10^7 1x10^6 0 5 Fold improvement in IgG 2 Fold increase in cell density Unfed Batch Feed Protocol 1 Feed Protocol 2

(14)

Response of three cells lines to a Chemically Defined Cloning Medium. 0 2000 4000 6000 8000 10000 12000 14000 0 10 20 30 40 50 60 70 Clone 1 Clone 2 K1 R e la ti v e c e ll n u m b e r Media C e ll l_in e

Relative response to 55 panel plus 5 control media.

Response to Clone 1was used to rate the panel (medium resulting in the lowest to highest proliferation). The other two cell lines were rated based on the Clone 1 response. The two

(15)

15

Application of Simcell to Process

Development

(16)

The Simcell

TM

_{MicroBioreactor Array (MBA)}

• 6 MicroBioreactors per array • Working volume: 550 - 700 µL

• Proprietary gas permeable material • Has supported numerous mammalian

cell culture types

• Densities achieved have been up to

2e7 cells/mL

• Practical densities to ~1.2e7

• Air bubble traverses perimeter of MicroBioreactors

(17)

17

The SimCell™ System at Invitrogen

• Incubators

– Each holds up to 42 MicroBioreactor

Arrays

– T, CO2, O2 and agitation control • Sensing module

– Biomass

– pH

– DO / Glucose (in mid-2007) • Sampling module

– Sample removal to well plates

– “Sim-fed batch”, “Sim-pH” corrections • Dispensing module

– Eight pumps for experimental factors – Media / Feed delivery

– Cell Source delivery – pH adjustment

• Miniaturization enables larger, more thorough DoE

(18)

CHO – IgG Fed Batch Study

• 4-Factor 2-Level full factorial

• Cell line

• pH

• Feed type

• Temperature shift

(19)

19

CHO – IgG Fed Batch Study

Feed

Temp Shift

Batch Shake Flasks

0.00E+00 2.00E+06 4.00E+06 6.00E+06 8.00E+06 1.00E+07 1.20E+07 0 24 48 72 96 120 144 168 192 216 240 264 Hours T o tal C e ll s / m L Clone A Clone B

(20)

Increasing Titer via DoE

Design-Expert® Sof tware

Final Titer D- -1.000 D+ 1.000 X1 = B: Feed X2 = D: Temp Shif t Actual Factors A: Cell Line = Line A C: pH = 0.00 D: Temp Shift G-G-G 5X CD CHO Interaction B: Feed F inal T it e r 200 327.5 455 582.5 710

Design-Expert® Sof tware

Final Titer D- -1.000 D+ 1.000 X1 = B: Feed X2 = D: Temp Shif t Actual Factors A: Cell Line = Line B C: pH = 0.00 D: Temp Shift G-G-G 5X CD CHO Interaction B: Feed F inal T it e r 200 327.5 455 582.5 710

• 2-fold increase over batch titer

• Relatively simple, small design

(21)

21

Bench scale comparison

Cell Growth in Bioreactors and SimCell

0.0E+00 2.0E+06 4.0E+06 6.0E+06 8.0E+06 1.0E+07 1.2E+07 1.4E+07 0 24 48 72 96 120 144 168 192 216 240 264 Time (hrs) T o ta l C e lls /m L LEAN BrX TCD 37 - 33C RICH BrX TCD 37 - 33C LEAN SC 37 - 33C RICH SC 37 - 33C

• Two processes expected to yield differences were

simultaneously tested in bioreactors and the Simcell

(22)

Optimising IgG Productivity in SimCell™

IgG Production Comparison: Bioreactor, SimCell,

Flask

IgG Production in Three Systems

0 400 800 1200 1600 BRx BRx pH Shift SimCell SimCell pH shift Flask Run 1 Flask Run 2 IgG mg/L

(23)

23

Summary and Observations

• The rapidly evolving field of bioprocess development

present new challenges and opportunities.

• Applying the best ideas and technologies can engender

improvements and challenge the status quo

• A mix of services and products can leverage the best

experiences within companies and suppliers.

• Automation can rapidly enhance biopharmaceutical

(24)

Acknowledgements

• Invitrogen PD-Direct, Frederick, Maryland

• Invitrogen PD-Direct, Grand Island, New York State • Invitrogen Bioproduction Europe