MabXpure for Innovative HCP Clearance Strategies andFlowthrough platform enabling technologies
Fabien Rousset, Ph.D., DAICEL Bioseparations, Chiral Technologies Europe, France
Mathieu Porte, Senior Scientist Bioproduction, PolyPlus Transfection, France

BioProcess International, Biotech Week, Boston, US
Thursday 12th Sept, 2019

13.000 employees , >$4B revenues (2018)
1919
1934
1966
1970
1988
1990
2016
2018

Reagents

Dainippon Celluloid Company
Creation of Fujifilm
Daicel Corporation
Daicel-Evonik
Daicel-UCB
Chiral Technologies (Pharma)
Daicel Bioseparations (Biopharma)
Daicel Life Sciences

Pharma Services

Chromatography

BioseparationsStandards

Excipients

Genomics

Intracell. Delivery

Outline
1

DAISEP MabXpure introduction

CS

Case Study with Polyplus Transfection

2

Addtional features

3

DAISEP MabXpure range

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SEC

DAISEP MabXpuremAb
HCPs

HCDNA

AEX

SEC for Size Exclusion Chromatography, AEX for Anion Exchange Chromatography

Small molecules
nucleic acids« To purify large quantities ofHCP and bioburden, at low
cost, and quickly, without
impacting mAb recovery, in a
SU flowthrough method »3

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Confocal Fluorescence Microscopy – SEC/AEX

GFP

DNA

IgG1

HCP
27kDa pI 5.8AEXDNA
100-300kDa pI 5.5
AEX-SECIgG
150kDa pI 8.3
SEC23

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Flexibility is keyC
2
3

DAISEP MabXpure as a filter aid

DAISEP MabXpure FT as a cartridge

MabXpure Evaluation

1C
2
3

Mathieu Porte
Senior Scientist - Bioproduction

Jonathan Havard
R&D research assistant - Bioproduction

Polyplus-transfection®

1
C

Innovative transfection solutions
to accelerate your research2
3

Wide range of products for research, processdevelopment, bioproduction and in vivo applications
18 years of expertise in transfection at your service
GMP grade reagents available
Tailored scientific, technical and regulatory support
High quality reagents made in France

www.polyplus-transfection.com

8

Product lines

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Transfection Solutions for R&D
Academic and University research laboratories, research
department of biotechs and pharmas
Labs performing day-to-day transfection experiments

23

Transfection Solutions for Direct Therapy
Reagents for direct administration to human
The reagent delivers the active drug

Transfection Solutions for Production of Biologics
Reagents for use in industrial process
To produce biotherapeutics at large scale (virus or protein)www.polyplus-transfection.com

9Bioproduction Product Porfolio

1C23

Protein
Production inSuspension cells

Virus production
For cell & gene
Therapy

FectoPRO®
FectoCHO®
Expression kitPEIpro®
PEIpro®-HQPEIpro®-GMP

www.polyplus-transfection.com

10Stable vs Transient Protein Production

1C
2

Stable clone establishement
Stable PolyclonalPool• 2 months

Single Clone
Selection

3
Screening for High
Producers• 1 month• 2 months

High quality proteins, very high yields

Bioprocess
Optimization

RecombinantProtein Production• 1 monthComplex process, time consuming

Transient Expression
Transfection andBioprocess Optimization

Recombinant ProteinProduction• 1 monthHigh quality proteins, fast and flexible process

www.polyplus-transfection.com

Limited protein yield

11

DSP Challenges for Transient Protein Production

1C

Transient antibody production:
High number of samples
Production of various antibodies (screening)
Small to mid-scale production2
3Problematic:
Antibodies have to be pre-purified in order to assess their biological activity
Host cell protein and DNA are difficult to remove contaminants and can reach up to400.000 ng/ml and 20.000 ng/ml respectively

Needs:
Efficient depletion of HCPs and DNA
Fast and flexible method
Not dependent on the protein to be purified

www.polyplus-transfection.com

12Seeding – Transfection – Cell Growth - Analyses

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3

Plasmides IgG1FectoPRO®
Clarification

ForteBIO BLItz

MabXpure
SDS PAGE

30 minELISA

J -1 / J0

Ensemencement

J0Transfectionwww.polyplus-transfection.com

J3

Production IgG1

J7

MabXpure - Analyses

13

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2
3

Expi293www.polyplus-transfection.com

CONFIDENTIAL

14

Expi293 – Residual HCP&DNA / IgG1 Recovery

1C
2

Day 7
100%

100%

100%

3

97%

97%
88%

80%
57%

60%
40%

www.polyplus-transfection.com

375.000 ng/mL
13.800 ng/mL
1.2 g/LHCP
DNAmAb52.500 ng/mL
Below LOQ
1.1 g/L

29%20%0%HCPDNAmAb

14%8%Feed

2%0%

Mix 1:20

Mix 1:10

Mix 1:5

HCPmAbDNA

CONFIDENTIAL

15Expi293 – SDS PAGE

1CDay 7

Day 3
LC

M1

M2

CFM1

M2

LC

M1

M2

CFM1

M2

SL

23

• Bio-rad Criterion system• Gel TGX 4-15%• Tampon Tris/Gly 0,1% SDS• Sample buffer Native All Blue
+ 2% SDS• Dilution ½ des échantillons• Migration 30’ à 200V
• Coloration Bleu Coomassie

www.polyplus-transfection.com

CONFIDENTIAL

161C23

ExpiCHO-S

www.polyplus-transfection.com

CONFIDENTIAL

17

ExpiCHO-S - Residual HCP&DNA / IgG1 Recovery

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Day 7
100%100%

3100%
92%

80%

90%

88%

68%

HCP
DNA
mAb

414.000 ng/mL
15.900 ng/mL
0.65 g/L

HCP
DNA
mAb

64.900 ng/mL
31 ng/mL
0.6 g/L

60%
40%

31%
16%

20%
4.0%

0%

www.polyplus-transfection.com

Feed

0.2%

0.3%

Mix 1:20

Mix 1:10

Mix 1:5

HCPmAbDNA

CONFIDENTIAL

18

ExpiCHO-S – SDS PAGE

1CDay 7Day 3
LC

M1

M2

CFM1

M2

LC

M1

M2CF M1M2SL23• Bio-rad Criterion system• Gel TGX 4-15%• Tampon Tris/Gly 0,1% SDS• Sample buffer Native All Blue+ 2% SDS• Dilution ½ des échantillons• Migration 30’ à 200V
• Coloration Bleu Coomassie

www.polyplus-transfection.com

CONFIDENTIAL

19

Conclusion

1
CMabXpure Evaluation:
High HCP and DNA depletion after Day 7 (higher for Day 3)
Excellent mAb recovery for Expi-CHO S and Expi-293 (from 88 to 97%)
Easy to use at lab scale

23Features:
Clear depletion observed with SDS-PAGE gel
Fast and flexible method to get purified mAb (time, mix ratio…)
Up to 1LRV for HCP with 1:5 mix ratio>2 LRV for DNA with 1:5 mix ratio

DAICEL MabXpure offers an easy and flowthrough pre-purification solutionwww.polyplus-transfection.com

20

ExpiCHO-S – 2D-gels

1C

Expi 293www.polyplus-transfection.com

ExpiCHO-S

2
321

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Optimized Clarification Platform « MabXpure + »2Cellulose-based depth filter sheet
Immobilized activated carbon
Mesopores *Feed 1:3Post ProA resin

 HCP: 800.000 ng/ml
HMW: 6%

 HCP: 200 ng/ml (<50 ppm)
 HMW: 0.5%

Feed 2:
 HCP: 200.000 ng/mL
 HMW: 8.8%

Feed 1:
 HCP: 17.000 ng/ml (98% depletion)
HMW: 0.6%

Feed 2:
HCP: <1.600ng/mL (>99% depletion)
HMW: 1%

DS criteria are obtained after
proAUsing « MabXpure + » platform

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3

pH of Feed

Titer g/L

CHO-K1
DNA 1.000 ng/mL
HCP 180 µg/mL
IgG 2 mg/mL
pIOVA 4.5
pIbIgG 5.4-8.5
pH vs pI

Conductivity

1100%

100%

99%

97%

100%

95%

77%

80%

C78%

80%

69%

70%

70%

68%

IgG Origins

50%

50%

40%

40%
30%

24%
17%

16%

20%30%
20%

23%
15%

20%

15%

15%

10%

10%

0%

0%

Feed

IgG1

IgG2

HCP

100%

IgG3

IgG4

IgG

90%
78%

80%

Mix ratio 1:20
DNA 500 ng/mL
HCP 70 µg/mL
IgG 2 mg/mL

Feed

Goat pH5 Rabbit pH5 Mouse pH5HCP

IgG

78%

70%

64%

60%

IgG types

50%
40%
27%

30%

27%
18%

20%
10%
0%

Feed

IgA 5HCPIgM 7.4

IgG

IgM 52
360%

60%

100%

95%90%90%

IgG subclasses

100%

MabXpure is working for all:- IgGs subclasses- IgGs origins- IgM & IgA

BovinepH6.7

1CDepletion and Recovery when MabXpure
In Pipette Tip
100%

92%

92%

2391%

90%
80%
70%
60%

The use of MabXpure inPipette tip format gives
reliable depletion
performances for very small
samples.

50%
40%
30%
20%
10%
0%

20%

20%

3%

0%

Tube/Rotation

18%

0%

200µL/0,25mL Pipette Tip

HCP

DNA

500µL/1mL Pipette Tip

mAb

CHO-K1, DNA 500 ng/mL, HCP 175 µg/mL, IgG 2 mg/mL

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Virus clearance MabXpure FT236

LRV Virus54MabXpure has the ability toremove viruses (yp to 5 LRV).
Potentially, clarification and/or
polishing using MabXpure canbe claimable

32
10
1:10

1:25

MVM

MuLV

1:50

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Endotoxin clearance MabXpure

23LOW

HIGH
30000

625000520000415000310000

25000

100

Feed

After
Depletion

MabXpure has theability to remove>2LRV for high andlow initialconcentrations*<0.05EU/mL
Feed

After
Depletion

1CAggregates clearance MabXpure FT

23Aggregates Post Protein A
3.5%

3.10%

3.0%
2.5%MabXpure helps to remove
aggregates after protein A.
MabXpure can also be used
for polishing applications

2.20%

2.0%
1.5%

1.20%

1.0%

0.60%

0.5%
0.0%
HerceptinHumira

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MabXpure is first truly Single-use FlowThrough Depletion chromatography technology• Host Cell Proteins – Up to 1LRV @ 400.000+ ng/mL (CHO and HEK cell lines)• DNA – 2LRV+ @ 15.000+ ng/mL• Endotoxin – 2LRV+ @ high and low concentration (25.000 EU/mL and 5 EU/mL)• Virus – 5LRV for MVM and XMuLV• Aggregates (HMW) – Up to 75% removal
With activated carbon, « MabXpure + » platform depletes 98%+ impurities from harvest feed Innovative high depletion technology for HCP and Bioburden Clearance for lab/process use
More data on Dynamic use of MabXpure for polishing of co-eluted HCP available at Booth 632

23

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DAISEP MabXPure
Porous Silica material
35µm
Multi-modal / Size Exclusion – Anion Exchange
Commercially available
BSE-TSE freeSlurry 50/50 v/v in 20% EtOH
Regulatory support file1DAISEP Spin XS & XLcentrifuge tube to work
under « Static mode »C2

MabXpure bulk

30,6mL

MabXpure FT 1 mL & 5 mL
ready to use cartridge

22mL

MabXpure FT 1500 mL
ready to use Resin FilterCartridge

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谢谢

GraciasThank you

DankeGrazieధనయ్�ా��ల�

спасибо
Merci

3

Mathieu PORTE
Jonathan HAVARD

Booth 528

Fanny COSTA
Alexandre MARGERIELoïc CRESTEYYasuto MORISHITABooth 632