Stepping Up Transient Expression - ELRIGelrig.org/downloads/airpt13/henry-chiou-life-technologies.pdfStepping Up Transient Expression: ... Post Transfection Treatment: ... Harvest

1 | © 2013 Life Technologies

ELRIG: Advances in Recombinant Protein Technology Alderley Park UK 19-20 Nov 2013

Stepping Up Transient Expression: The Expi293™Expression System

Henry C Chiou, Ph.D.Gibco Cell Biology SystemsLife Technologies


Protein Expression from Mammalian Cells

5 to 7 days

High EfficiencyTransfection Expression

ProteinProduction

Transient Expression

Months

Transfection

Isolation,Adaptation,Banking & Expansion

ProteinProduction

Expression,Selection &

Sub-culturing

Classic Stable Expression


The Need: Keep fast turnaround, get MORE PROTEIN

5 to 7 days

High EfficiencyTransfection Expression

ProteinProduction

Transient Expression

Months

Transfection

Isolation,Adaptation,Banking & Expansion

ProteinProduction

Expression,Selection &

Sub-culturing

Classic Stable Expression


Published high density transient protocol forincreased yield

600mg/L at Day 7

Protocol:Cell prep:

Expand cells in growth medium

Concentrate cells → 20x106/ml transferred into transfection compatible medium

Transfection:PEI + 37.5% IgG HC, 37.5% IgG LC, 10% p18h, 10% p21h, 5%

aFGF(2.5X more DNA/cell than standard protocol)(1.7X more PEI/cell than standard protocol)

Post Transfection Treatment:

3-4 hours post-transfection → dilute culture with growth medium to 4x106/ml

Add medium supplements

7 days to reach 600mg/L Human IgG, 14 days to 1 gm/L

Backliwal et al., Biotechnol Bioeng 2007

Protocol is requires spin down of cells, multiple plasmids, proportionately higher

DNA and transfection reagent per cell. Is not readily scalable


Strategy to Increase Transient Protein Yields

++

+ +++

+

+

++

++ ++

++

+

+

++

+ +++

+

+

+

++

+ +++

+

+

+

3-10X Yield

Expi293 Innovations:• Culture medium that achieves 3-fold higher cell density

• Cells that express more protein per cell

• Transfection reagent that effectively delivers DNA into high cell density cultures

• System plugs directly into users current 5-7 day workflow

More Cells Per mL High EfficiencyTransfection More ProteinHigher Productivity

Per Cell


• Chemically defined, protein-free, AOF, serum free medium• Supports viable cell densities up to 18 x 106 cells/ml• High culture viabilities• Single medium solution for cell growth and transient expression

•Transfection compatible formulation•Supports high expression yields

Expi293 Medium

Medium 2

Medium 3

Medium 4

Expi293™ Expression Medium: A high density growth & transfection medium

Unless otherwise stated, all data shown was generated at Life Technologies.


0

200

400

600

800

1000

1200

hIgG

(ug/

ml)

hIgG titer (passage-to-passage)

d5

d6

d7

Adapted 293F cellshigh yield Expi293F™ cells Bank 2

Day

Bank 1

• high yield cells have higher viability, cell density, and cell size vs. other 293 cells

• Significant increase in protein production

Derived from FreeStyle™ 293F cells

Adapted into Expi293™ Expression Medium

Culture viability

95

95.5

96

96.5

97

97.5

98

98.5

FreeStyle293F

Kentaro-adapted

293F

High Yield 293F

perc

ent v

iabl

e ce

lls

Expi-

Culture Viability

Cell size

16

16.5

17

17.5

18

18.5

FreeStyle293F

Kentaro-adapted293F

High Yield 293F

mic

rons

Expi-

Cell Size

VCD

0

1

2

3

4

5

6

FreeStyle293F

Kentaro-adapted

293F

High Yield 293F

cells

/ml

(x10

^6)

Expi-

VCD

Expi293F™ cells: higher per cell productivity


ExpiFectamine™ 293 Transfection Performance

0

10

20

30

40

50

60

70

80

90

30 mL 1 mL Neg

% G

FP-P

osit

ive

Cells

Transfection Efficiency(day 3 pt)

Transfection Enhancers(added 18-24 hr pt)

(125 mL shake flask) (24-well plate)

• ExpiFectamine™ 293 achieves high efficiency transfection of high density cultures

• Enhancers combine to maximize yields

0

1

2

3

4

Fold

Cha

nge

In E

xpre

ssio

nEnhancer 1 Enhancer 2

−−

+−

−+

++


Human IgG

0

200

400

600

800

1000

1200

1ml 30ml 1000ml

ug/m

l hIg

G

ExpiFectamine™ 293 Transfection: Yield and scalability

EPO

0

1

2

3

4

5

6

7

8

9

FS 1ml

Expi 1ml

FS 30ml

Expi30ml

FS 1L

Expi 1L

EPO

(yie

ld re

lativ

e to

1m

l sca

le F

S st

anda

rd)

1ml 30ml 1000ml

FreeStyle™ 293Expi293™

B2 Adrenergic Receptor VLPs

0

20

40

60

80

100

120

FS 30ml Expi 30ml FS 1L Expi 1L

VLP

yie

ld (u

g/m

l of c

ultu

re)

30ml 1000ml

Cripto

0

200

400

600

800

1000

1200

FS 1ml Expi 1ml FS 30ml Expi 30ml FS 1L Expi 1L

prot

ein

mg/

L

1ml 30ml 1000ml

Human IgG

1 ml = 24 well plate30 ml = 125 mL shake flask1 L = 3 L shake flask

1ml 30ml 1000ml


Results at 8L Wave Reactor Scale

FreeStyle™ 293

Expi293™


External Results

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22 Labs, 98 Proteins,average 4.6-fold increase

Fold

Yie

ld I

ncre

ase

• 87% demonstrated increased expression in Expi293™ vs users current system

• 4.6X average increase for all proteins (n=98)

• 4.0X average increase for mAbs (n=54) highest level – 826 mg/L

• 5.3X average increase for non-mAbs (n=44 ) highest level – 790 mg/L


96 well plate (square well)2 mL deep well format700 µl fill volume3 mm-throw shaker1000-1250 rpm

0.0E+00

5.0E+06

1.0E+07

1.5E+07

2.0E+07

2.5E+07

3.0E+07

6 well 12well 96well

GFP

TU

/mL

plate format

Lentivirus (from pLenti6.3-EmGFP) produced in microplate format using Expi293™

96 well optimization

0100200300400500600

190 rpm(0.75 inch

throw)

700 rpm 1000 rpm 1250 rpm

IgG

yie

ld (

mg/

L)

96 deep well optimization

(19 mmthrow)

(3 mm throw)

Adaptation of Expi293™ to microplate formatsMost plates only require adjustments in shaker rotational speeds.

EppendorfMixMate

Benchmark Orbi-Shaker MP


Protein Functionality: equivalent to FreeStyle™ 293

0 5 10 15 20 25 300

10

20

30

40

Total BoundSpecific BoundNon-Specific Bound

BmaxKd

Total Bound31.411.143

Specific Bound30.081.069

[3H] DHA (nM)

Rad

ioac

tivity

Bou

nd (p

mol

/mg)

0 5 10 15 20 25 300

10

20

30

40

Total BoundSpecific BoundNon-Specific Bound

BmaxKd

Total Bound30.010.7543

Specific Bound29.141.057

[3H] DHA (nM)

Rad

ioac

tivity

Bou

nd (p

mol

/mg)

Expi293™ FreeStyle™

Beta2 adrenergic receptor VLPs challenged with increasing 3[H] DHA antagonist in a saturation binding experiment +/-100uM cold propanolol.0.5ug VLP protein per reaction

12 25 50 100 200ng60504025

12 25 50 100 200ng

Expi293™ FreeStyle™

IgG functionality: Abs show equivalent reactivity against target

Equivalent IgG glycosylation profiles:

GPCR:Comparable and physiological antagonist binding activity

pmol ligand/mg proteinnM

pmol ligand/mg proteinnM

Expi293™

3xyield

>10xyield

Expi293™

FreeStyle™

[Erythropoeitin] ng/mL

Rela

tive

Lum

ines

cenc

e Un

its (R

LU)

0.0001 0.01 1 100 10000

50000

100000

150000

200000

Expi293

Freestyle293

EPO Activity:Measured by TF-1 cell-based proliferation assay

5xyield


Clone GOI into pEF6 TOPO® Expression

Vector

MembranePro™

Reagent+

Add MembranePro™

Precipitation Mix

Re-suspend MembranePro™ particles (50-500ug protein, enough for up to

1,000 ligand binding assay data points)

Lipofectamine™ 2000 Transfection Reagent

Decant culture medium with MembranePro™ particles (48hrs post-transfection)

Precipitate for 18hrs at 4ºC

and centrifuge

Add transfection

mix to

HEK-293FT Cells

+

Expression of Membrane Proteins onVLPs – MembranePRO


Clone GOI into pEF6 TOPO® Expression

Vector

MembranePro™

Reagent+

Add MembranePro™

Precipitation Mix

Re-suspend MembranePro™ particles (50-500ug protein, enough for up to

1,000 ligand binding assay data points)

ExpiFectamine™ 293 Transfection Reagent

Decant culture medium with MembranePro™ particles (48hrs post-transfection)

Precipitate for 18hrs at 4ºC

and centrifuge

Add transfection

mix to

Expi293F™ Cells

+

Expression of Membrane Proteins onVLPs – MembranePRO


Scalable lentivirus production in Expi293™

0.0E+00

2.0E+07

4.0E+07

6.0E+07

6 well 12well 96well

Tite

r TU

/mL

plate format

Lentivirus from pLenti6.3-EmGFP produced in microplate format

Expi293™ protocol for 30ml suspension culture production of lentivirus:

9ug pLenti construct27ug Virapower™ packaging mixin 4ml Opti-MEM I

180ul Expifectamine™ 293in 4ml Opti-MEM I

20 minutes RT.Add to 22ml Expi293™ cells(3x10^6 vcd/ml).Shake 125rpm, 8% CO2, 37oC.Next day: add 150ul of Enhancer 1

1.5ml of Enhancer 2Harvest 24-48hrs later.

0.0E+00

2.0E+06

4.0E+06

6.0E+06

8.0E+06

1.0E+07

1.2E+07

1.4E+07

7.3TOPO 7.3GW

GFP

TU

/ml

AdherentLenti Yield


Acknowledgements

Expi293™ Development team:Carlsbad, CASanjay VasuIsabel CisnerosJeff RogersJingqiu LiDan BezdekStacey WinterToby JordanGabriella JaharisMaggie Abo-HebeishAlda ChangSahil NockRik BasiHenry Chiou

Frederick, MDMeredith JonesShyam KumarJon ZmudaAlice FanJim SanfordDana MeentsBrandon HarrisDianna FoxBryan Poltilove

Grand Island, NYChao Yan LiuRichard Del MaestroChang ChoiBryan GreeneBrian BurginBob KenersonDave Dugan


THANK YOU!

For more information on the Expi293™ transient expression system please go to:

www.lifetechnologies.com/expi293

Documents

Stepping Up Transient Expression - ELRIGelrig.org/downloads/airpt13/henry-chiou-life-technologies.pdfStepping Up Transient Expression: ... Post Transfection Treatment: ... Harvest