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Cell therapies and quality level in Cell Factories for Pharma
Vittoria Ardissone
BICB, Sfax, November 8, 2012
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Procelltech was born in 2009 and has established itsoffices and GMP laboratories (250 m2) in the BioindustryPark Silvano Fumero, close to University and internationalPharma companies.
Procelltech is member of bioPmed, the new Italianinnovation cluster dedicated to bio and medicaltechnologies that gathers around 60 companies, researchcentres and three academic institutions (Università di Torino,Università del Piemonte Orientale and the Politecnico diTorino)
Procelltech’s activities are aimed to sustain public andprivate institutes in clinical and R&D Research field and isrecognized in BioInItaly Reports (2010-2011) issued byEarns & Young and Assobiotech as one of the 320 ItalianCompanies performing R&D activities in the red biotechenvironment.
Procelltech: Who we are
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Presentation Outline
Regulatory perspective for Regenerative Medicine and
Advanced Therapy Medicinal Products (ATMPs)
The Good Manufacturing Practice
Procelltech GMP approach for ATMP Quality Control Validation
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How Stem Cells Are Changing the Way We Think About DiseaseTime Magazine_ Mar. 17, 2011
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Regenerative medicine is an emerging clinical disciplinethat aims to employ cellular medicines to restore thefunctions of damaged or defective tissues and organs(Trounson A., 2008)
Regenerative Medicine from science to therapies
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Regulatory perspective for Regenerative Medicine and
Advanced Therapy Medicinal Products (ATMPs)
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Regenerative Medicine and Advanced Therapy
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The field of regenerative medicine encompasses various areas of technology:
Gene therapySomatic cell therapyTissue engineering
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Advanced Therapy Medicinal products (ATMP)
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Manipulation
Cells can be associated with non-cellular components, biomolecules, chemicals or matrices
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CELLS AUTOLOGOUSCELLS
ALLOGENICCELLS
ATMP
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Regenerative Medicine models
a personalized medicine/point of contact model, in which cells are harvested from the same or a related individual and undergo minimal or extensive manipulation before being delivered to the recipient;
a banking model, akin to the existing umbilical-cord blood system and involving indeterminate periods of storage and minimal processing;
a manufacturing model, whereby cells are extensively manipulated and produced in a central facility and a single lot is used for a single or relatively large number of patients, as in existing drug delivery paradigms.
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Each model is likely to require distinct regulatory regimes, as determined in part by a range of pertinent issues concerning:
sources of material; manufacturing process shipping, storage and tracking; issues related to combining cell and gene therapy
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Regenerative Medicine models (II)Click t
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Regulatory framework
European MedicinalAgency (EMA)
Food and DrugAdministration (FDA)
Definition Advanced TherapyMedicinal Products(ATMPs)
Human Cells, Tissues, and Cellular andTissue-based Products (HCT/Ps)
Regulations Directive 2001/83/ECDirective 2002/98/EC Directive 2004/23/ECReg. (EC)N 1394/2007Directive 2009/120/EC
21 CFR 127121 CFR 600’s21 CFR 200’s21 CFR 300’s
current GoodManufacturing Practice (cGMP)
Directive 2003/94/ECEudralex vol. 4
21 CFR part 210-211
Hospital Exemption YES NO
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The Good Manufacturing Practice
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Eudralex Volume 4 (I)
Part I – Basic Requirements for Medicinal ProductsChapter 1 Quality Management (revision February 2008)Chapter 2 PersonnelChapter 3 Premise and EquipmentChapter 4 Documentation _Coming into operation 30 June 2011Chapter 5 ProductionChapter 6 Quality ControlChapter 7 Contract Manufacture and AnalysisChapter 8 Complaints and Product RecallChapter 9 Self Inspection
Part II – Basic Requirements for Active Substances used as Starting MaterialsBasic requirements for active substances used as starting materials
Part III - GMP related documentsSite Master FileQ9 Quality Risk ManagementQ10 Note for Guidance on Pharmaceutical Quality SystemMRA Batch Certificate19
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2012
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Eudralex Volume 4 (II)
Annex 1 Manufacture of Sterile Medicinal ProductsAnnex 2 Manufacture of Biological Medicinal Products for Human UseAnnex 3 Manufacture of RadiopharmaceuticalsAnnex 4 Manufacture of Veterinary Medicinal Products other than Immunological
Veterinary Medicinal ProductsAnnex 5 Manufacture of Immunological Veterinary Medicinal ProductsAnnex 6 Manufacture of Medicinal GasesAnnex 7 Manufacture of Herbal Medicinal ProductsAnnex 8 Sampling of Starting and Packaging MaterialsAnnex 9 Manufacture of Liquids, Creams and OintmentsAnnex 10 Manufacture of Pressurised Metered Dose Aerosol Preparations for InhalationAnnex 11 Computerized Systems (revision January 2011) Annex 12 Use of Ionising Radiation in the Manufacture of Medicinal ProductsAnnex 13 Manufacture of Investigational Medicinal ProductsAnnex 14 Manufacture of Products derived from Human Blood or Human Plasma (May
2011)Annex 15 Qualification and validationAnnex 16 Certification by a Qualified person and Batch ReleaseAnnex 17 Parametric ReleaseAnnex 19 Reference and Retention Samples
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Eudralex vol. 4_Annex 1Manufacture of Sterile Medicinal Products
The manufacture of sterile products should be carried out in cleanareas entry to which should be through airlocks for personneland/or for equipment and materials. Clean areas should bemaintained to an appropriate cleanliness standard and suppliedwith air which has passed through filters of an appropriateefficiency.The various operations of component preparation, productpreparation and filling should be carried out in separate areaswithin the clean area. Manufacturing operations are divided intotwo categories; firstly those where the product is terminallysterilized, and secondly those which are conducted aseptically atsome or all stages.Clean areas for the manufacture of sterile products are classifiedaccording to the required characteristics of the environment. Eachmanufacturing operation requires an appropriate environmentalcleanliness level in the operational state in order to minimize therisks of particulate or microbial contamination of the product ormaterials being handled.
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For the manufacture of sterile medicinal products 4 grades can be distinguished.
Grade A: The local zone for high risk operations, e.g. filling zone, stopper bowls, open ampoules and vials, making aseptic connections.Grade B: For aseptic preparation and filling, this is the background environment for the grade A zone.Grade C and D: Clean areas for carrying out less critical stages in the manufacture of sterile products.
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Eudralex vol. 4_Annex 1 (II)Manufacture of Sterile Medicinal Products
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The production of biological medicinal products involves biological processes and materials, such as cultivation of cells or extraction of material from living organisms. These biological processes may display inherent variability, so that the range and nature of by-products are variable.
Control of biological medicinal products usually involves biological analytical techniques which have a greater variability than physico-chemical determinations.
In-process controls therefore take on a great importance in the manufacture of biological medicinal products.
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Eudralex vol. 4_Annex 2Manufacture of Biological Medicinal Products for Human Use
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Manufacturing workflow for cell products
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17Ahrlund-Richter L et al, Cell Stem Cell, 2009
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Product testing must cover aspects of:
Safety: with regards to microbial contamination: sterility, endotoxinand MycoplasmaQuality: attributes such as viability, identity, purity and potency
The product is defined by release specifications:
Release specifications and Assay method
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Regulatory expectations for productcharacterization
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Procelltech GMP approach for ATMP Quality Control
Validation
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Quality System (ICH Q10)
Valid
atio
n (E
P, IC
H Q
2)
Ris
k A
sses
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t (IC
H
Q9)
Documents
Personnel
Facility
Instruments
In/Out material Cleaning
Trac
eabi
lity
Dat
a R
ecor
ds
Quality Controls
EudraLex Vol 4, Annex 2(EC) 1394/2007 and 2001/83/EC CPMP/BWP/2879/02, CPMP/BWP/3752/03, ICH Q5A(R1)
GMP approach for ATMP Quality Control ValidationPr
ocel
ltech
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Validation: definition
EU GMP Guideline
Validation: Establishment of evidence in accordance with therules of ”Good Manufacturing Practice” that procedures,processes, items of equipment, materials, operations orsystems do in fact result in the intended outcomes.
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GMP approach for ATMP Quality Control ValidationICH Q2 (EC, USA, JP)
New validation studies may be required when relevant changes inthe manufacturing process are being undertaken, or if theanalytical method is changed
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PROCEDURE IDENTIFICATIONTESTING FOR
IMPURITYQuant Detect
ASSAY-Dissolution
-Content/potency
Accuracy - + - +
Precision:RepeteabilityInterm. Precision
--
+ -+ -+ -
+++
Specificity + + + +
Detection Limit - - + -
Quantitation Limit - + - -
Linearity - + - +
Range - + - +
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Quality Controls for ATMP:In Process and Batch Release
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Test for: QC Assay Reference
Impurity Sterility (Rapid, 7 day test) 21 CFR 610.12, EP 2.6.27
Impurity Endotoxin level detection (LAL test) EMEA/CHMP/410869/2006 /USPC chapter 85, EP 2.6.14)
Impurity Mycoplasma detection (NAT, qPCR, Rapid) EP 2.6.7
Impurity Viral detection (NAT, qPCR and IVVT) EP 2.6.7
1) Identity DNA profiling EP 2.6.212) Identity Flow citometry staining EP 2.7.24
Identity Isoenzyme test ICH Q5D
Identity Karyotyping (G-banding) EP 5.2.4
Potency CFU-F assay EP 2.7.28
Potency Cell count and Viability EP 2.7.29
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GMP Validation:
IQ/OQ/PQ of ABI310 genetic analyzerMethod validation according to ICHQ2
Validation to analyze human stem cell line cultures accordingto ICHQ2 requirements for identity methods (interspecie-/intraspecie-specificity)
1) Method GMP validation:DNA profiling
Method:Multiplex PCR (AmpliFISTRIdentifier PCR Amplification kit,Applied Biosystem) for 15STRs+Amelogenin
Cells spotted on FTA® Classic Cards(Whatman)
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Aim To verify if the method is specific for human cell lines (3repetition of the test)
In-house Reference cellular preparation StemPro® HumanAdipose-Derived Stem Cell (Invitrogen) has been compared tothe following non-human cell lines and a bacterial preparation(*):
• MDBK (ATCC CCL 22, bovine origin)• ST (ATCC CRL-1746, porcine origin)• Staphilococcus aureus (ATCC 6538, aerobic bacteria)
For all 3 test repetitions, only with the StemPro® cellularpreparation a full STR profile was obtained
DNA profiling validationinterspecie-specificity
(*) Specificity controls have been chosen by analyzing possible non-human contaminants during in-housereference cellular preparation colture(Cell spotted on FTA cards at 100 cells/µl and Bacteria at 100 CFU/µl)
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Aim To verify if the method discriminates two different humancell lines (3 repetition of the test)
In-house Reference cellular preparation StemPro® HumanAdipose-Derived Stem Cell (Invitrogen) has been compared toMRC-5/ATCC CCL171 cells
For all 3 test repetitions, two different and full STR profileshave been obtained for both human cell lines: StemPro®and MRC-5
The DNA profiling method can identify a unique full STRprofile for each human cell line in analysis
DNA profiling validationintraspecie-specificity
Time to validate 0.75 year/1 FTE
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Immunophenotyping validation
GMP Validation:
IQ/OQ/PQ of FACSCanto II©Method validation according to ICHQ2
Phase 1 FACSCanto II© IQ/OP and PQ:
Phase 2 7 color immunophenotyping (IPT) validationStemPro® (Invitrogen) Mesenchymal Stem Cells (MSC) havebeen stained with monoclonal Antibodies (mAbs)
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Phase 1 FACSCanto II© PQ (I)
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PMT performance evaluation of MFI brightest beads:• BD™ Cytometer Setup and Tracking Beads• Rainbow Calibration Particles (Sperotech)
Levy-Jennings plots of beads MFI (single value +2SD)
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Phase 1 FACSCanto II© PQ (II)
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Steady and highly reproducible instrument performance
PMT performance evaluation of CST Voltage:• CST Performance vs Baseline ( voltage)
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Phase 2 7 color IPT validation (I)
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Specificity Assessment of mAbs ability to identify targeted MSCsurface antigens (Dominici M et al., Cytotherapy, 2006) byFluorescence Minus One (FMO) method
7 color staining of StemPro®
MSCFMO controls
MSC markers (CD73, CD105, CD29, CD44 and CD90): highly expressed on cell surface
Haematopoietic markers (CD34)
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Phase 2 7 color IPT validation (II)
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Repeatability 3 independent experiments with the in-house Reference cellular preparation StemPro®have beenperformed by a single operator
Time to validate 0.5 year/1 FTE
Results in line to literature dataHigh specificity and repeatability
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Analysis of human stem cell lines (I)
Use of the validated IPT platform to analyze different stem celllines:
• Hematopoietic Stem Cells (Prof Tarella, UNITO)• Bone Marrow Mesenchymal Stem Cells (Prof Camussi, UNITO)• Human Liver Stem Cells (Prof Camussi, UNITO)• Kidney Papillar Stem Cells (Prof Bussolati, UNITO)
Validity criteria: marker expression percentage of In-houseReference Preparation (StemPro® MSC cells) in accordance withproduct specification
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•MSC and HLSC display a typical MSCs phenotype•Kidney papillar SC do not fulfill all the criteria of classical MSC,CD105 and CD90 are almost absent•HSC express typical Hematopoietic cell markers (CD34 and CD45)and display some of the staminal markers like CD44, CD29 andCD105.
•StemPro® markers in accordance with product specification
Stem cell lines CD34 CD73 CD44 CD29 CD90 CD105
% +SEM % +SEM % +SEM % +SEM % +SEM % +SEMHLSC (n=2) 0.00+0.00 100.00+0.00 100.00+0.00 100.00+0.00 69.00+8.00 97.00+2.00Kidney papillary SC(n=2) 0.00+0.00 100.00+0.00 100.00+0.00 100.00+0.00 21.50+10.50 4.04+3.96
MSC (n=3) 0,07+0.10 100.00+0.00 100.00+0.00 100.00+0.00 100.00+0.00 83.33+1.50HSC (n=1) 99.00 3.00 100.00 97.00 42.00 86.00StemPro® (n=1) 1.00 100.00 100.00 99.00 100.00 93.00
Analysis of human stem cell lines (II)
Immunophenotyping Results
This validated immunophenotyping platform can guarantee precise and quick identification of stem cell lines
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Quality Control methods validation (EP/ICHQ2)
Conclusions
The validated DNA profiling and Immunophenotyping methods shown:
Steady and highly reproducible instrument performances
Results in line to literature data and reference standard
High specificity and repeatability
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Thank you for your attention!!!
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