Standardizing Cell Banking Practices for Research and ... · •Media evaluation International Stem...
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Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
Standardizing Cell Banking Practices for
Research and Clinical Applications
Derek J. Hei, [email protected]
Technical Director
Waisman Clinical Biomanufacturing Facility
University of Wisconsin - Madison
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
Stem Cell Research at the
University of Wisconsin - Madison
UW Stem Cell and Regenerative Medicine Center
– Basic and translational research through to clinical trials
– Human ES cells, iPS cells, Neural Progenitor Cells, Mesenchymal Stem Cells
WiCell Research Institute
– Core support services for UW stem cell investigators
– Derivation of new cell lines (hESC, iPS cells)
– Hosted National Stem Cell Bank with UW
Waisman Clinical Biomanufacturing Facility
– Development support for translational researchers
– Clinical production and QC testing under cGMPS
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
Technical hurdles -• Culture methods – animal-free
• Genetic stability of cell lines
• Cryopreservation methods
• Selective differentiation methods
• Residual undifferentiated cells
• Rejection of transplanted cells
Establish well-characterized cell banks
Develop standardized Quality Control test methods
Develop processes for large-scale clinical production
Compliance with FDA regulations (current Good Manufacturing Practice guidelines)
Moving HESC Therapies to the Clinic – The Challenges
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
Setting Standards for hESC Research
Cell lines (reference standards)
Cell culture methods – undifferentiated hESCs
Differentiation methods
Cell characterization and Quality Control methods
Regulatory issues for clinical applications
Potential impact –
– Positive – allows comparison across studies and between labs
– Negative – may drive science toward early, sub-optimal
methods and technologies
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
The NSCB mission –• Bank and distribute NIH approved hESC lines
• Characterization of hESC lines
• Technology transfer and technical support for research community
Restricted to banking NIH approved hESC lines• Prior to July 2009 – 21 lines (Cellartis, ESI, Novocell, Technion, UCSF, WiCell)
• Genetically modified human ES cell lines
• NIH Guidelines for Human Stem Cell Research – July 2009
NSCB data moved to WiscBank (www.wiscbank.com)
New cell lines – motivation to deposit with national banking
system?
The future of hESC banking in the U.S. remains unclear
National Stem Cell Bank Program
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
The NSCB Banking Process
MCBs – full QC testing including complete adventitious agent panel
Distribution Lots – basic QC testing panel -• Identity (STR)
• Karyotype (G-band)
• microbial/fungal contamination, mycoplasma
• hESC marker expression (flow cytometry)
Characterization Studies – impact of culture on genetic stability,
gene expression, differentiation potential
www.nationalstemcellbank.org
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
Master Cell Bank Characterization
Identity – Short Tandem Repeat (STR) - PowerPlex 1.2, HLA typing, SNP testing
Bacterial/fungal contamination, mycoplasma
Karyotype – G-band
Flow cytometry – SSEA-1/3/4; Oct-3/4, TRA-1-60/81
Post thaw recovery (count/viability)
Growth characteristics – doubling time
Comparative Genome Hybridization
Gene Expression Profiling
Adventitious agent testing – human, murine, bovine, porcine (ICH)
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
NSCB/WiscBank Website –Cell ordering, COAs, Technical Support
www.nationalstemcellbank.org
www.wiscbank.com
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
NSCB/WiscBank Website – Technical Support
www.wiscbank.org
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
HESC Characterization Studies
Performed on Distribution Lots to provide information on typical hESC growth characteristics
Testing at 5 passage intervals to P = +20 from distribution lot
Characterization tests -
Growth characteristics
HESC marker expression by multi-color flow cytometry
(Kathy Schell, UW Cancer Center)
Karyotype by G-banding (Karen Montgomery, WiCell)
Gene expression profiling (Nimblegen)
Comparative Genome Hybridization (Nimblegen)
Differentiation potential (UW investigators)
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
NSCB Quality Control –Comparative Genome
Hybridization
Chromosome 12
NimbleGen 385K whole genome array
Complementary to G-Band
Resolution: G-Band = 2-10 Mb; CGH = 4-20 kB
Sensitivity: G-Band = 5-10%, CGH > 40-60%
Utilize opposite sex hESC as reference (H1 male, H9 female)
Identify Copy Number Variants (CNVs) and cross-check with normal CNV database (Database of Genomic Variants, http://projects.tcag.ca/variation)
Verify findings using FISH analysis
Data – WiscBank website (www.wiscbank.com), GEO (www.ncbi.nlm.nih.gov/geo)
Karen Montgomery - WiCell
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
International Collaborations for hESC Banking
Stem cell registries –• hESCReg – European hESC Registry
• UMass International Stem Cell Registry
• Compile databases of critical information – publications, cell
characterization data…
International Stem Cell Initiative• Cell line characterization
• Media evaluation
International Stem Cell Banking Initiative (ISCF)• Int. Stem Cell Forum/ Glyn Stacey – U.K. Stem Cell Bank
• Consensus guidance for supply of hESC for research (Stem Cell Rev
and Rep (2009) 5:301-314)
• Consensus guidance for banking of clinical-grade hESCs – under
development
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
Cell Banking Standards for Clinical Production
U.S. Food and Drug Administration –• Current Good Manufacturing Practice (cGMP) guidelines – 21 CFR
210, 211, 610
• Human Cellular and Tissue-Based Products (HCT/Ps) – 21 CFR
1271 (May 2005)
Donor eligibility
Current Good Tissue Practice guidelines
• Guidance documents – future?
EMEA – Guideline on human cell-based medicinal products
(EMEA/CHMP/410869/2006)
International Conference on Harmonisation• Harmonization for U.S. FDA, EMEA, Japan
• Viral Safety Evaluation for Cells of Human or Animal Origin (Q5A)
• Derivation and Characterisation of Cell Substrates (Q5D)
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
Human ES Cell Banking for
Clinical Applications
Waisman Clinical Biomanufacturing Facility-WiCell
Regulatory compliance
• Current Good Manufacturing Practice (cGMP) guidelines
• HCT/Ps, Good Tissue Practice guidelines (21 CFR 1271)
• Full cGMP batch records for media and MCB production
• Quality Control Procedures – full documentation, assay qualification
• IND support – WCBF DMF, cell line DMF (future)
Key technical issues
• Scalable cell bank production without the use of murine feeders
• Address potential pathogen contamination issues from prior derivation and
culture practices
• Aseptic processing
• Differentiation control
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
Human ES Cell Banking for
Clinical ApplicationsGoals
• Address key technical issues
• Establish reference standard cell banks for clinical applications
• Shorten timeline and cost from discovery to clinical trials
Current cell banks -• WA01, WA09 MCBs produced to date
• Full adventitious agent testing (ICH Q5A guidelines) including testing for
human, murine, bovine and porcine pathogens
• Provide portion of MCB and produce WCB for each investigator
Feeder independent culture – cGMP mTeSR1 media and growth
factors (FGF-2) produced by WCBF
Future –feeder independent derivation of new cell lines
Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison
WCBF, NSCB, WID Seed Grant, and PACT Teams
WiCell Research InstituteRobert Drape
Tenneille Ludwig
Jessica Martin
Dan Felkner /NSCB Banking Team
Karen Montgomery
Jeff Jones
Erik Forsberg
University of WisconsinJamie Thomson, Guokai Chen
Tim Kamp, Jianhua Zhang, Chad Koonce
Jon Odorico, Torey Browning, Carly Kibbe
Igor Slukvin, Jessica Dias
Su-Chun Zhang, Ben Thiede
Kathy Schell, Erik Puffer
Sean Palecek, Samira Azarin
Waisman Clinical Biomanufacturing Facility
Julie JohnsonCarol EmlerDiana DrierLisa Marie ByrneJohn WelpTim SparksLaurie Larson….
PACTPeiman Hematti Tim HackerAmish RavalMarlowe Eldridge