Virus Removal in Cell Culture Media

with PlanovaTM Nanofiltration

BioInnovation 2016

10 - 11 February 2016

Waldorf Astoria Hotel | Berlin | Germany

Konstantin AGOLLI

Technical Assistant

Asahi Kasei Bioprocess Europe

k.agolli@akbio.eu

www.ak-bio.com

3

Goal

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Goal

2

1) Discussion about virus removal in media: Technologies available

How much virus reduction required ?

How to decrease cost of nanofiltration ?

2) PlanovaTM filters: Specifications

Filtration mechanisms

3) Case studies: Filterability & Cell growth

Virus spiking filtration studies

Cost calculation

Content

Asahi Kasei Bioprocess

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Content

6

Sepacell™ Bioprocess Division

Blood purification Blood transfusion

Asahi Kasei Corporation

Holding company

Core operating companies

Chemicals & fibers

Asahi Kasei Chemicals

Asahi Kasei Fibers

Homes &

Construction Materials

Asahi Kasei Homes

Asahi Kasei

Construction Materials

Electronics

Asahi Kasei Microdevices

Asahi Kasei E-materials

Asahi Kasei Pharma

Asahi Kasei FibersAsahi Kasei Medical

ZOLL Medical

Health Care

DialysisTherapeutic

Apheresis

Foundation: 1922; HQ: Tokyo; Employees: 25 000; Turnover: 19 B$

Innovative leader in various kind of business fields

Asahi Kasei Group

PlanovaTM

Virus removal filters BioOptimalTM MF-SL

TFF microfilter

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… Asahi Kasei Bioprocess

Systems & Equipment

CellufineTM

Cellulose Beads

Chromatography media(made by JMC Corporation)

BioCradleMicrocarriers for Cell Culture

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… Asahi Kasei Bioprocess

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1) Discussion about virus removal in media: Technologies available

How much virus reduction required ?

How to decrease cost of nanofiltration ?

Content

10

Strict raw material testing & source control

Closed systems in the manufacturing process

Virus testing (traditional & new methods)

Etc…

Cell culture media ?

Despite a high level of Virus Safety Assurance, virus

contaminations occurred in the past

Current Approach To Virus Safety

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γ-irradiation

HTSTUV-treatment

Nanofiltration

Technologies for Virus Removal

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High volumes in short

time

Impact on media quality

Effectiveness on smallest virus ?

HTST: risk of precipitation

UV: Scalability ? Industrial reactor ?

Running costs ?

HTST

γ-irradiation

UV-treatment

Technologies for Virus Removal

13

Nanofiltration

Effective vs “all“ viruses

(known and unknown)

No impact on media

composition

Easy to implement

(like sterile filter)

Excellent & easy

scalability

With serum free media only

Performances can be affected

by some surfactants or lipids

Long filtration times required

Technologies for Virus Removal

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No regulatory requirement ➔ Production risk mitigation

“0” virus required ! ≠ DSP philosophy with LRV

In real life: “0” virus = below the limit of detection …

LRV still calculated according to the detection limit & virus titer

PlanovaTM: usually LRV > 4 log (TCID50/mL)

How Much Virus Reduction Required ?

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How To Decrease Cost Of Nanofiltration?

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Specific nanofilter for USP ? NO. Same virus as for DSP to remove !

“Low cost” nanofilter ? NO. High quality nanofilter required

Much higher flux ? NO. 1) Nano-pore limitation

2) Filterability of media

More filtration volume ? YES! Unlike in DSP, much higher L/m2

Longer filtration times required

How To Decrease Cost Of Nanofiltration?

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The only way to decrease the cost of the nanofiltration

is to filter longer !

How To Decrease Cost Of Nanofiltration?

2) PlanovaTM filters: Specifications

Filtration mechanisms

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Content

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PlanovaTM Nanofilters

20

Filter PlanovaTM N PlanovaTM BioEX

Hollow fibers cellulose PVDF

Pore size (nm) 15, 20, 35, (75) Parvovirus removal

Filtration surface (m²) 0.001 ➔ 4.0 0.0003 ➔ 4.0

Max TMP (kPa) 98 343

Water flux (LMH) 20N: 66 @ 98 kPa 170 @ 343 kPa

SIP/Autoclaving NO YES

315 mm

PlanovaTM Nanofilters

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How does it work?

Filtration Mechanism

Medium

+ Virus

Medium

Virus FreeMedium

Virus Free

Virus

Void

Single Layer

Capillary

Unique Membrane Structure

& Filtration Mechanisms

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Hollow fiber

Medium

+ Virus

Medium

Virus FreeMedium

Virus Free

Virus

Void

Single Layer

Capillary

3D network

Unique Membrane Structure

& Filtration Mechanisms

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Hollow fiber

Size Exclusion & Depth Filtration

Media Supply

P

P

P P

Sterile-filter

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Easy Setup

Bioreactor

Media Supply Nanofiltration Bioreactor

P

P

P P

Sterile-filter

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Easy Setup

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3) Case studies: Filterability & Cell growth

Virus spiking filtration studies

Cost calculation

Content

Filterability

Filterability & Flux depend on medium

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SERUM FREE MEDIA tested for CHO culturesA

V.

FL

UX

(L

MH

)

0

20

40

60

80

100

120

EXCELL302 CHO-S-SFMⅡ VP-SFM ×1 CD Opti ×１ CD Opti ×5

Filterability

Long filtration time required to achieve high filtration volumes (L/m2)

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Sigma EX-CELL 302 + Feed supplement

0

1000

2000

3000

4000

0 1000 2000 3000 4000 5000

Time (min)

Fit

rati

on

Vo

lum

e

(L/m

2)

3 days

Cell Growth

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10

5cell

s/m

L

No impact of BioEX on media quality after 7 days.

According to customers, no impact on media quality/composition even

after 20 days of filtration with BioEX or 20N.

0

10

20

30

40

50

60

BioEx + BioEX - BioEx + BioEX - BioEx + BioEX - BioEx + BioEX - BioEx +

Excell302 SFMII VP-SFM CD Opti CDOpti5

Day 0 to Day 7

Filterability & Cell Growth

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To remember !

Filterability depends on media & ingredients

Very high volume/m2 with serum free media, if

filtration time is long enough

No impact on the quality of the media and therefore

on the cell growth

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Are PlanovaTM nanofilters able to remove viruses

from contaminated media, while filtering such high

volumes during several days ?

Most Important Question

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Cell Culture medium

Nanofilter

PPV (Porcine Parvo Virus), 18-22 nm

Titer 20N 4.26 log (TCID50/mL)

(in medium) BioEX 5.57 log (TCID50/mL)

20N 10 cm2; N = 1

BioEX 3 cm2; N = 2

20N: 5000 L/m2

BioEX # 1: 10 000 L/m2 (non stop)

BioEX # 2: 5 000 L/m2 (3 h stop & restart)

CD-CHO (from GIBCO Invitrogen)

Filtration Volume

Virus

TMP 20N 98 kPa

BioEX 300 kPa

Virus Spiking Filtration with CD-CHO

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No impact of the virus

spiking on Filtration Volume

Consistency of the

performances

20N:

2 000 L/m² in 1 day

5 000 L/m² in 3 days

BioEX:

Same as 20N

10 000 L/m² in 7 days

Volume (L/m²) vs. Time

Virus Spiking Filtration with CD-CHO

0

2000

4000

6000

8000

10000

12000

0 50 100 150 200

Filte

rati

on

Vo

lum

e (

L/m

²)

Time (h)

BioEX-1

BioEX-2

20N

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No virus detected ( )

Difference in PPV LRV is

due to difference in initial

virus titer

0

1

2

3

4

5

6

0 2000 4000 6000 8000 10000

PP

V L

RV

(lo

g)

Filtration Volume (L/m²)

BioEX-1

BioEX-2

Planova 20N

Virus Spiking Filtration with CD-CHO

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Filtration

Volume (L/m2)10 442 752 1678 2399 3607 4997 5005 5005 5026 5046 5066 5086 5105

Time (h) 0 4 7 19 29 48 71 71 74 74 75 75 75 76

Flux (LMH) 122 108 94 77 72 65 60 62 - 63 63 63 63 63

PPV LRV ≥ 5 ≥ 5 ≥ 5 ≥ 5 ≥ 5 ≥ 5 ≥ 5 ≥ 5 - ≥ 5 ≥ 5 ≥ 5 ≥ 5 ≥ 5

Stop of 3 hours & restart with BioEX filter # 2 :

PPV LRV still > 5 log with no virus detected in the permeate

Same flux

3h stop

Virus Spiking Filtration with CD-CHO

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To remember !

Very high filtration volumes/m2 with virus spiked media

4 log PPV removal with no virus detected in filtrate…

….despite several days of filtration and very high filtered

volumes

Virus Spiking Filtration with CD-CHO

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Price of CD-CHO:

< 30 €/L

BioEX:

After 1.5 days, NF cost

< 10 % medium cost

20N:

After 1 day, NF cost

< 10 % medium cost

The longer the filtration

time, the more cost

effective !

Medium: CD-CHO

Volume: 10 000 L

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

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0 1 2 3 4 5 6 7 8

Filte

r co

st

(€/L

)

Filtration Time (days)

BioEX

20N

Medium: CD-CHO

Volume: 10 000 L

Cost Calculation:

Filter cost (€/L medium) vs. Filtration time (days)

Conclusion

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Content

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1) Virus removal nanofiltration with serum free media

2) High quality DSP nanofilter required because viruses are the same

3) Filtration of media is not always easy, flux decay is expected

4) High virus LRV for “ALL” viruses without impacting cell growth

5) Only way to decrease nanofiltration cost long filtration times, to

be able to achieve extremely high filtration volumes/m2

6) Removal of other contaminants: mycoplasma, phage, nanoparticles…

Conclusion

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Thank you to my colleagues:

Hongo-san

Mihara-san

Kederer-san

Dayani-san

Tanimoto-san

Kobayashi-san

Schrankler-san

Teitz-san

Martirene-san

Mathithas-san

Acknowledgements

OR

Goal

95

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どうもありがとうDomo Arigato!

(thank you very much)