Gabriela Franco SalinasBas LeewisRachel Pijls

October 5th, 2012

BALANCE-project

Contents

• WP 4 Deliverables • Progress work PhC

Technology transfer Selection manufacturing process for up-scaling

• Progress work AMC Process development: cryopreservation and thawing Investigation on serum-reduction

2

WP4 - Deliverables

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# Description Date Partners

4.1 Report on Technology Transfer and qualified QC tests Sep-12 AMC, BP, PhC

4.2 Qualified GMP Manufacturing process of bioreactor cultures Sep-13 AMC, PhC, UEDIN

Selection and qualification of manufacturing process of monolayer culture Feb-13

Identification secretome bioreactor cultures Jul-13

Qualified manufacturing process of bioreactor cultures Sep-13

4.3 Specification of serum content utilization in the medium Nov-12 AMC

4.4 Report on preservation conditions Oct-13 AMC, PhC

Specifications of small bioreactor cultures May-13

Specifications of large bioreactor cultures Oct-13

4.5 Report on stability of bioreactor cultures Jul-14 AMC, PhC, UEDIN

Specification of maximum number of PDs related to full functionality Dec-13

Specification of shelf life for IMPD Dec-13

Specification of stability in human ALF to determine treatment duration Jul-14

Task 4.1 Technology Transfer

Goal: Transfer the technology from AMC and BP to PhC and to comply to GMP standards

Main decisions•Technology Transfer divided in two parts

Culture and expansion of HepaRG cells Preparation of the BAL device

•Preparation of a Research Cell Bank (RCB) To adapt culture procedure to GMP To save time as MCB and WCB must be prepared in cleanroom

4

Task 4.1 Progress TechTransfer

Phase I: Information Exchange phase•TT list prepared by AMC and PhC; input BP in progress•TT protocol in draft•Risk assessment / GAP analysis in progress

Risk stability cell line•BP will provide passagenr. 9•MCB + WCB: 5-6 passages•Up-scaling: 6 passages

According to Biopredic: cells cannot be used after P20!

=> Project Strategy discussion 5

Final product:passage 20-21

Task 4.1 Progress TechTransfer

Phase II: Implementation phase•Demonstration run at AMC performed in Aug

Demonstration run at BP planned for Oct 22-23

•Writing GMP-documentation in progress Need QA department for review

•Verification run planned Performed at R&D lab To verify the documentation

•Qualification run planned Results in Research Cell Bank

6

TT protocol

Task 4.2 Progress up-scaling

Selection GMP cell culture system

•2D culture techniques Largest up-scaling technique available is Xpansion-180 of ATMI Need 2 systems in parallel to culture sufficient cells for 1 BAL

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Task 4.2 Progress up-scaling

Selection GMP cell culture system•3D culture techniques

Culture on hollow fibers or in suspension (e.g. on microcarriers) Easy up-scalable for further clinical phases / commercialization Cost estimation and risk management is performed for culture

on a microcarrier

=> Project strategy to be discussed

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Status overview

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# Deliverables Date New date Status

4.1 Report on TechTransfer and qualified QC tests Sep-12 May-13 In progress

4.2 Qualified GMP Manufacturing process of bioreactor cultures Sep-13 In progress

Selection and qualification of manufacturing process of monolayer culture

Feb-13 In progress

Identification secretome bioreactor cultures Jul-13

Qualified manufacturing process of bioreactor cultures Sep-13

4.3 Specification of serum content utilization in the medium Nov-12 In progress

4.4 Report on preservation conditions Oct-13

Specifications of small bioreactor cultures May-13

Specifications of large bioreactor cultures Oct-13

4.5 Report on stability of bioreactor cultures Jul-14

Specification of maximum number of PDs related to full functionality Dec-13

Specification of shelf life for IMPD Dec-13

Specification of stability in human ALF to determine treatment duration Jul-14

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BALANCEAMC: contribution to WP4

BALANCE

Task 4.2: process development: cryopreservation & thawing

• Cryopreservation & thawing of HepaRG cells prior BAL loading: improved logistics, standardization & low costs

• Test effects of freezing in 10% DMSO & thawing• Trypan blue exclusion test: 82±5% vitality after

thawing (100% in fresh isolate)(n=3)• Attachment in monolayer: maximal after 4.5 hrs• Test 3 pairs of cell isolates (750 million freshly isolated

cells and 920 million cryopreserved cells) in bioreactors after 4.5 hr attachment for max 5 weeks

BALANCE

Task 4.2: process development:hepatic functions in time

Urea production

0.0

1.0

2.0

3.0

4.0

7 14 21 28

Culture day

umol

/h/B

AL

Ammonia elimination

0

5

10

15

20

25

7 14 21 28

Culture day

umol

/h/B

AL

Lactate elimination

-5

0

5

10

15

20

7 14 21 28

Culture day

umol

/h/B

AL

Fresh cells

Cryopreserved cells

n=2

n=2

n=2n=2

n=2n=2 n=2

n=2

BALANCE

Task 4.2: process development: cell leakage in time

AST leakage

0.0

0.1

0.2

0.3

0.4

0.5

7 14 21 28

Culture day

U/h

/BA

L

LDH leakage

0.0

0.5

1.0

1.5

7 14 21 28

Culture day

U/h

/BA

L

n=2

n=2n=2

n=2

<0.5% cell content

BALANCE

• Cryopreservation of HepaRG cells seems to be feasible for BAL application, although maturation may be delayed

• Complete analysis: apolipoprotein synthesis, total protein&DNA content, mRNA levels, 6β-OH-testosterone production (CYP3A4), urea cycle activity (15N urea production from 15N ammonia)

• Investigate enhanced cryopreservation protocols: cryomax & differentiated cells

Task 4.2: process development : conclusions & to do

BALANCE

Task 4.3: Investigation on serum reduction: growth

• Culture low passage HepaRG in HepaRG media with different fetal bovine serum (FBS)% in 24 well plates (3 low passage lines; n=4/line)

• Test after 30 days

0

50

100

150

200

250

300

350

0% 1% 2.50% 5% 10%% FBS

To

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BALANCE

Task 4.3: Investigation on serum reduction: function

Urea production

ApoA-1 production

Ammonia elimination

0.000.020.040.060.080.100.120.140.160.18

0% 1% 2.50% 5% 10%% FBS

Am

mo

nia

elim

inat

ion

(u

mo

l/h/m

g p

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in)

0.0000.0020.0040.0060.0080.0100.0120.0140.0160.0180.020

0% 1% 2.50% 5% 10%% FBS

Ure

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ion

(u

mo

l/h/m

g p

rote

in)

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0% 1% 2.50% 5% 10%% FBS

Ap

oA

1 p

rod

uct

ion

(u

g/h

/mg

pro

tein

)

(n=4, 3 experiments)

BALANCE

Task 4.3: Investigation on serum reduction: mRNA

Transcript levels normalized for 18S, given as % of human liver (n=1)

Albumin

0%

10%

20%

30%

40%

50%

60%

70%

0% 1% 2.50% 5% 10%%FBS

Rel

ati

ve

ex

pre

ss

ion

Cyp3A4

0%

1%

2%

3%

4%

5%

0% 1% 2.50% 5% 10%%FBS

Rel

ati

ve

ex

pre

ss

ion

CPS

0%2%4%6%8%

10%12%14%16%18%20%

0% 1% 2.50% 5% 10%%FBS

Rel

ati

ve

ex

pre

ss

ion

GS

0%

50%

100%

150%

200%

250%

300%

350%

0% 1% 2.50% 5% 10%%FBS

Rel

ati

ve

ex

pre

ss

ion

BALANCE

Task 4.3: Investigation on serum reduction: growth curve

Growth curve HepaRG

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 5 10 15 20 25Days of culturing

Mg

pro

tein

/25

cm

2

P10Z 10%P10Z 5%P11Z 10%P11Z 5%

From day 1-13:population doubling time (days)

5% FBS 5.810% FBS 5.6

Growth in T25 flasksTest of 2 passages in 5% and 10% FBS mediumN=4/passage

BALANCE

• Growth and functionality in 5% FBS & 10% FBS medium comparable

• Test stability cultures during 10 passages on 2.5%,

5% and 10% media• Test serum free media (weaning step)• Near future: test low FBS or optimal serum free

medium on BAL cultures

Task 4.3: Investigation on serum reduction: conclusions & to do