1. Gabriela Franco Salinas Bas Leewis Rachel Pijls October 5th, 2012 BALANCE-project

2. 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

3. WP4 - Deliverables 3 #DescriptionDatePartners 4.1Report on Technology Transfer and qualified QC testsSep-12AMC, BP, PhC 4.2Qualified GMP Manufacturing process of bioreactor culturesSep-13AMC, PhC, UEDIN Selection and qualification of manufacturing process of monolayer cultureFeb-13 Identification secretome bioreactor culturesJul-13 Qualified manufacturing process of bioreactor culturesSep-13 4.3Specification of serum content utilization in the mediumNov-12AMC 4.4Report on preservation conditionsOct-13AMC, PhC Specifications of small bioreactor culturesMay-13 Specifications of large bioreactor culturesOct-13 4.5Report on stability of bioreactor culturesJul-14AMC, PhC, UEDIN Specification of maximum number of PDs related to full functionalityDec-13 Specification of shelf life for IMPDDec-13 Specification of stability in human ALF to determine treatment durationJul-14

4. 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

5. 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

6. 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

7. 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

8. 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

9. Status overview 9 #DeliverablesDateNew dateStatus 4.1Report on TechTransfer and qualified QC testsSep-12May-13In progress 4.2Qualified GMP Manufacturing process of bioreactor culturesSep-13In progress Selection and qualification of manufacturing process of monolayer culture Feb-13 In progress Identification secretome bioreactor culturesJul-13 Qualified manufacturing process of bioreactor culturesSep-13 4.3Specification of serum content utilization in the mediumNov-12In progress 4.4Report on preservation conditionsOct-13 Specifications of small bioreactor culturesMay-13 Specifications of large bioreactor culturesOct-13 4.5Report on stability of bioreactor culturesJul-14 Specification of maximum number of PDs related to full functionalityDec-13 Specification of shelf life for IMPDDec-13 Specification of stability in human ALF to determine treatment durationJul-14

10. BALANCE Sculpture from Hubertus von der Goltz; Photo from Corine van der Voort BALANCE AMC: contribution to WP4

11. 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

12. BALANCE Task 4.2: process development:hepatic functions in time Urea production 0.0 1.0 2.0 3.0 4.0 7142128 Culture day umol/h/BAL Ammonia elimination 0 5 10 15 20 25 7142128 Culture day umol/h/BAL Lactate elimination -5 0 5 10 15 20 7142128 Culture day umol/h/BAL Fresh cells Cryopreserved cells n=2

13. BALANCE Task 4.2: process development: cell leakage in time AST leakage 0.0 0.1 0.2 0.3 0.4 0.5 7142128 Culture day U/h/BAL LDH leakage 0.0 0.5 1.0 1.5 7142128 Culture day U/h/BAL n=2 <0.5% cell content

14. 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

15. 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 Total protein/well (ug)

16. BALANCE Task 4.3: Investigation on serum reduction: function Urea production ApoA-1 production Ammonia elimination 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0%1%2.50%5%10% % FBS Ammonia elimination (umol/h/mg protein) 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 0.020 0%1%2.50%5%10% % FBS Urea production (umol/h/mg protein) 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0%1%2.50%5%10% % FBS ApoA1 production (ug/h/mg protein) (n=4, 3 experiments)

17. 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 Relative expression Cyp3A4 0% 1% 2% 3% 4% 5% 0%1%2.50%5%10% %FBS Relative expression CPS 0% 2% 4% 6% 8% 10% 12% 14% 16% 18% 20% 0%1%2.50%5%10% %FBS Relative expression GS 0% 50% 100% 150% 200% 250% 300% 350% 0%1%2.50%5%10% %FBS Relative expression

18. 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 0510152025 Days of culturing Mg protein/25 cm2 P10Z 10% P10Z 5% P11Z 10% P11Z 5% Growth in T25 flasks Test of 2 passages in 5% and 10% FBS medium N=4/passage

19. 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