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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
3
# 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
7
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
8
Status overview
9
# 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
BALANCE
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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
tal
pro
tein
/we
ll (
ug
)
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
rote
in)
0.0000.0020.0040.0060.0080.0100.0120.0140.0160.0180.020
0% 1% 2.50% 5% 10%% FBS
Ure
a p
rod
uct
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