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Automated microbial detec/on for TGA regulated cell therapy services -‐ points to consider Dominic Wall PhD FFSc (RCPA), CSO Cell Therapies Pty Ltd Chair Legal and Regulatory CommiAee-‐ ISCT ANZ region OperaFons Director-‐ Pathology & CBCT Peter MacCallum Cancer Centre
Cell Therapies • A large academic facility supported by a
commercial operation • Majority owned/controlled by the academic centre • Manufacturing TGA licences
§ 149827 Stem Cells -2001 § MI-2009-LI-05411-3 Mesoblast- Mesenchymal
Precursor Cells- 2010 § MI-2011-LI-02539-3 Prima Biomed Immunotherapy
Phase 3 trial licence 2012 § MIC CAR-T cell therapies for DLBCL & ALL
• Trial CMO and other commercial & academic activity
• Consulting, trial and product approvals • Product and process development • Major focus on Asian market collaboration • 19 TGA OMQ audits over 11 years
Product and producFon experFse
Page 3
Cellular therapy categories • Autologous and allogeneic • Cell replacement • Cell vaccines • Gene modificaFon • Immunotherapy (mulFple anFgen types)
• Stem cell therapy
Cell types • Blood stem cells • CAR-‐T • Chondrocytes • DendriFc cells • MAK cells • Mesenchymal cells • Mononuclear cells • PancreaFc islet cells • Vectors (LVV, RVV)
Disease areas • Breast cancer • Diabetes • HepaFFs C • Lung cancer • Melanoma • Myeloma • OsteoarthriFs • Ovarian cancer
Consul/ng and tech transfer linkages • Germany • Indonesia • Japan • Malaysia • US
Potential risks of bone marrow cell transplantation into
infarcted heartsMartin Breitbach et al
Blood 2007 110: 4 1362-9
Inappropriate differentiation?
Malignant transformation?
Unexpected toxicity Q1 – micro contamination?
Regulators concerned about product risks
Cell therapies oWen cause severe toxicity
Lightly regulated products have been associated with paFent harm due to contaminaFon
• Rapid Availability of novel therapies, versus • NO markeFng approval or manufacturing oversight • Risk and harm to paFents and markets
RelaFve complexity Cells vs drugs
EukaryoFc cell therapies 50,000nm
Viral vectors 50nm
TherapeuFc anFbodies 5nm
Small molecule 0.5nm
Extraordinary market acFvity in cell therapy-‐ ALL, DLBCL, FL, CLL and others
Solid tumour control is the next T cell target
Medicinals n No collecFons/donors n Large lots n high throughput n Terminal sterilizaFon n Automated n control of starFng materiel n Stable protocol n unknown recipient n Fixed volumes/inoculums n Control of soluFons
Cells and Tissue n Donors and collecFons n single product lots, high value batches n low throughput n ParFal closed system, no term sterile n TradiFonally labour intensive n limited control of starFng materiel n Evolving research based protocols, n Known recipients n Variable volumes n OWen IV drugs/subclinical sepsis
Inherent challenges for cell & Fssue products
The legal framework
What is regulated?
• Not fresh organs, ART, fresh HPC, diagnosFc Fssues
• Not biological medicines (proteins, pepFdes, vaccines without living human cells), blood/components
• Things that are excluded • Things that are not biologicals
§ TherapeuFc Goods (Things that are not Biologicals) DeterminaFon No. 1 of 2011
www.tga.gov.au
Regulated or not
• HPC-‐A-‐ not captured in Biologics except in specific circumstances § A strategic decision in 2001 to seek licensing § High volume acFvity encompassing other licensable acFviFes
• Autologous Chondrocytes-‐ biologics Class 3 • Autologous mesenchymal precursor cells-‐ biologics Class 3
• GMO CAR-‐T cells-‐ Biologics Class 4 • Trial manufacturing CTN vs CTX-‐ medicines(?)
What is the funcFon of the tesFng?
• Release criteria or not • Product safety • Manufacturing control • For informaFon only • For management of donor/recipient
Other relevant quesFons • Compendial methods, if mandatory…
§ EP vs USP § Which markets/regulators?
• Sterility vs Micro contaminaFon • Appropriate method
§ AnFbioFcs § Risk matrix § Fresh product release? § TesFng at which point? § Post release QC? § Which media/method
• Relevant technical standards
HPC-‐A
• Culture Neg Result is not a release criteria • Not primarily about sterility • Not principally for individual product safety • Primarily for process control • Primarily to inform the clinician • To support clinical care
HPC-‐A
• IniFally TGA accepted raFonale for an unlicensed micro service
• Required that any method is validated • Required that certain materials are controlled • AWer publicaFon of EP 2.3.23 (Human HaematopoieFc Stem Cells) TGA mandated a path to licensed tesFng (even though this was an informaFonal Chapter)
When releasing a culture posiFve product
• Means the ID of posiFve culture is now within scope of TGA license § TGA licensed vendors? § Scope of compliance-‐ Vitek? AnFmicrobial suscepFbility?
• ExcepFonal release mechanisms • TGA consent required for product release
Page 17
ExpectaFons HPC-‐A v HPC-‐M M
• reported rates of microbial contaminaFon of HPC-‐M harvests range from 2 to 42%
• Rowley et al (1988), Lazarus et al (1991), Schwella et al (1998)
A • in contrast, contaminaFon rates of HPC-‐A appear to be somewhat
reduced-‐ 16 fold lower (Berz et al 2007) • FDA claim 7 avoidable deaths a year (2001) • Prince (1995) reported few if any sepFc complicaFons if culture pos is
for commensals/skin flora
HPC-A
• Prince et al (1995) reported a 0% contaminaFon rate in 551 cryopreserved autologous HPC-‐A units, as opposed to a 2.2% HPC-‐M contaminaFon rate
• Espinosa et al (1996) reported a 0.2% cryopreserved HPC-‐A contaminaFon rate in 1040 PBSC collecFons
• Padley et al (1996) reported a 0.5% (3/576) HPC-‐A contaminaFon rate in cryopreserved harvests
• Larrea et al (2004) reported a 7.2% (62/851) HPC-‐A contaminaFon rate, although only 3/851 deemed to be process related
• Hirji et al (2003) reported a contaminaFon outbreak in 34 products by an AcFnomycete (Microbacterium)
• Klein et al (2006) 1 reported death from Pseudomonas cepacia despite prophylaxis
• Padley et al (2007) 1.6% with 69 culture pos infusions, 23 with prophylaxis-‐ no impact on survival, 1 bacteremia
Conclusion from literature
• Reported contaminaFon rates of HPC-‐A: 0 to 7.2%
• Most organisms isolated are skin contaminants, also gram negaFve water borne orgs feature consistently
• infusion of contaminated product seldom results in serious complicaFons, regardless of immunosuppression, with or without prophylaxis
• Outbreak management, and product control warrants knowledge of whether there is culture posiFvity
HPC-A Processing • Apheresis bag well mixed • Transfer 1ml of Apheresis collection into
the side bulb for microbiology testing • Sterile weld Apheresis Bag to a Triple dry
pack
• Conventional lab environments • Only limited steps performed in a BSC
§ Volume adjustment § Cryoprotectant addition
Microbiology Testing • Three microbiology samples
§ Original Apheresis bag: Ø 1ml sample into the bulb
attached to the apheresis bag
§ Cryoprotectant Mixture: Ø 1ml sample into a white top
tube (no additives) from the laboratory prepared cryoprotectant (DMSO + Saline + Plasma)
§ Final HPC-A product Ø 2 X 2 mL of Final HPC-A
product added to a Adult FA+ & FN+ BacT/Alert bottles
Microbiology Testing
• All three samples despatched to our Microbiology laboratory
• BacT/Alert bottles are tested in automated BacT/Alert 3D, if positive →ID commences along with testing of apheresis bag and cryoprotectant mixture to identify the source of positivity
• Reference vials can also be tested
YEAR 2005 2006 2007 2008 2009 2010 2011 2012 2013
Collections (n) 408 403 321 352 465 405 361 369 277
Process (n) 0 0 0 0 1 0 1 0 1
Patient endogenous (n) 3 2 3 0 3 0 0 1 0
% Positive for Process (Limit < 1 %) 0 0 0 0 0.2 0 0.3 0 0.4
Assumed Process Related: 2009 – Corynebacterium jeikeium 2011 – Staphylococcus epidermidis 2013 – Staphylococcus hominis
Patient Related (Endogenous derived): 2005-2013: Staphylococcus epidermidis Staphylococcus aureus Staphylococcus capitis Micrococcus luteus Achromobacter xyosoxidans
HPC-A culture positives
Microbial Contamination QC limit
• Prior to applicaFon for TGA license, comprehensive validaFon of our exisFng procedure undertaken
• Re: microbial contaminaFon monitoring – a thorough review of literature & assessment of previous HPC-‐A contaminaFon rates at Peter Mac allowed us to determine a trigger point for acceptable process-‐related microbial contaminaFon which was accepted at audit.
< 1%
Quality Control Monitoring
Process Microbiology
0
1
2
Date
% P
ositi
ve
0
1
2
Num
ber P
ositi
ve
(summarised monthly)
limit (<1%)
number positive
% positive(12 month average)
pro jected trend
Outsourced BacT/alert
Insourced TGA licensed BacT/alert
PF FA+/FN+ FA/FN
1ml 2x 2ml 2x 0.5ml 2x 1ml
FA/FN
5 days 7 days
1x 35°C
Initial (pre validation) algorithm
• 1mL of Sample-3 added to a Paediatric PF BacT-Alert bottles
• Samples-1 & -2 provided in a white top tube
• All 3 samples despatched to our micro service provider
• Sample-3 tested, if positive → ID commences along with testing of samples-1 & -2 to identify the source of positivity
ValidaFon of Microbial DetecFon TGA required another extensive validation of our micro detection system to “demonstrate the acceptability and suitability of the methods used for the HPC-A” in the presence of the cryoprotectant
- Representative product sample (cell numbers etc)
- Albumex
- Cryoprotectant
Which technical standards?
-‐ EP 2.3.23 Human HaematopoieFc Stem Cells
-‐ Examine….Microbiological control
So defence of result availability only, not culture negaFvity invalid
But…”product may be released before availability of result”
EP 2.6.27 Microbiological control of cellular products
EP 2.6.27 vs 2.6.1
• 2.6.1 Sterility § Thioglycollate broth 30-‐35°C § Soya-‐bean casein medium 20-‐25°C § 14 days § Growth promoFon for each media batch <100CFU § Suitable for both membrane filtraFon & direct inoculaFon
§ “if the material being tested renders the media turbid…transfer aWer 14 days…”
§ Can source alternaFve method if…
2.6.27 Microbiological control of cellular products
§ “preferable to the test for steriliFty for certain cellular products” § “may be manual or automated” § Growth promoFon test-‐ 7 days, 10 & 100 CFU § Aerobic and anaerobic media § Method validaFon § InoculaFon of sample immediately or aWer storage at 5C
Ø V>10 ml 1% product volume Ø V1-‐10 ml 100uL Ø V<1 ml-‐ NA
§ 7 auto or 14 days manual § If pos , genus & species and anFbiogram
First ValidaFon 8 micro-organisms to be tested:
Escherichia coli, ATCC 8739 – Gram neg facultative anaerobe Pseudomonas aeruginosa, ATCC 9027 – Gram neg aerobe Staphylococcus aureus, ATCC 6533 – Gram pos facultative anaerobe Candida albicans, ATCC 10231 – yeast Corynebacterium pseudodiphtheriticum, ATCC 10700 - Gram pos aerobe Bacteroides fragilis, ATCC 25285 – Gram neg obligate anaerobe Aspergillus fumigatus, ATCC 96918- fungus Clostridium sporogenes, ATCC 11437 (not C. perfringens)- Gram pos
obligate anaerobe
5 Bac-T-Alert bottles to be tested – Paediatric PF
– Adult Aerobic FA
– Adult Anaerobic FN
– Adult Aerobic BPA
– Adult Anaerobic BPN
2 organism concentrations – 10 CFU/ bottle
– 100 CFU/ bottle
Duplicates, neg controls
Minimum 200 boAles to be tested
• Deceased & consented paFent HPC-‐A (containing 10% DMSO) thawed
• 1mL of micro-‐organism added to 4mL of HPC-‐A
• Bac-‐T-‐Alert boAle inoculated with 1mL of mix
• Inoculated boAles sent to our micro service provider for tesFng
First ValidaFon -‐ Inoculum at Silliker
BoAles tested for 1st validaFon
• Paediatric PF • Adult Aerobic FA • Adult Anaerobic FN • Adult Aerobic BPA • Adult Anaerobic BPN • …which boAles and why?
New boAles • FA+ and FN+ • iFA+ and iFN+ • Clinical use versus Industrial use • Products idenFcal but documentaFon differences • Clinical relevance vs pharmacopeia organisms • CoA and Product Inserts Clinical vs industrial • Cost and stock availability • Length of noFce for product variaFons • ValidaFon pathways-‐ same or different?
Page 34
Current batch growth promoFon • Aerobic Medium Bacillus sub8lis BioBalls (30cfu each) Pseudomonas aeruginosa BioBalls (30cfu each) Candida albicans BioBalls (30cfu each) Staphylococcus aureus BioBalls (30cfu each) Aspergillus niger BioBalls (30cfu each) • Anaerobic medium Bacteroides fragilis BioBalls (30cfu each) Clostridium sporogenes BioBalls (30cfu each)
Page 35
1st ValidaFon-‐ results E. coli CBCT No:
CBCT 03063.2
Date performed: 07/02/06
Paediatric PF
Adult Aerobic FA
Adult Anaerobic
FN
Adult Aerobic
BPA
Adult Anaerobic
BPN
Expected Result as per VP043.001 Negative Negative Negative Negative Negative
Actual Results Negative Negative Negative Negative Negative Negative Control
Acceptable / Unacceptable Acceptable Acceptable Acceptable Acceptable Acceptable
Target cfu/bottle
Expected Result as per VP043.001
for E. coli
Positive Positive Positive or Negative Positive Positive or
Negative
Actual Results
Set 1 Positive Positive Positive Positive Positive
Set 2 Positive Positive Positive Positive Positive 10
Acceptable / Unacceptable Acceptable Acceptable Acceptable Acceptable Acceptable
Target cfu/bottle
Expected Result as per VP043.001
for E. coli
Positive Positive Positive or Negative Positive Positive or
Negative
Actual Results
Set 1 Positive Positive Positive Positive Positive
Set 2 Positive Positive Positive Positive Positive 100
Acceptable / Unacceptable Acceptable Acceptable Acceptable Acceptable Acceptable
Concentration of Stock
Target Actual
100 cfu/ml mean 114 cfu/ml
1000 cfu/ml mean 1160 cfu/ml
Verify that isolate ID matches
• 2 CFU inoculums • Inoculum prep offsite • InoculaFon onsite • BacT/Alert offsite
1st ValidaFon results • All organisms typed as expected from CoA and protocol
v Escherichia coli-‐ all pos v Pseudomonas aeruginosa, all pos except Anaerobic FN v Staphylococcus aureus, all pos v Candida albicans, all pos except Anaerobic FN and 1 BPN (10 CFU) v Corynebacterium pseudodiphtheri8cum, all pos except Anaerobic FN & BPN v Bacteroides fragilis, only Anaerobic FN & BPN ( not paediatric PF) v Aspergillus fumigatus, all pos except Anaerobic FN & BPN v Clostridium sporogenes, only Anaerobic FN & BPN ( not paediatric PF)
§ Paediatric boAles not suitable for obligate anaerobes § PaFent/Product populaFon-‐ F vs B boAles § Selected adult FA and FN with 1 ml inoculum (0.5 each) § Extended with a small study for Albumex 20 § BP & PF not suitable for our paFent derived products
Method ValidaFon 2014 • FA/FN to FA+/FN+ migraFon & updated to new inoculum volumes • PaFent material (mobilised with high dose Rx (T-‐ICE) and G-‐CSF 10mcg/kg) • WCC of HPC-‐A 198.58 x 106/mL, (≤ 220 x 106/ml) • Ph. Eur. Monograph 2.3.23 & 2.6.27 & clinical relevance of microorganisms • Freeze dried BioBall re-‐hydrated 1.1ml of saline • 210µl of the rehydrated organism into 14ml of the HPC-‐A product • PosiFve control = 210µl of the rehydrated organism into 14ml of sterile
saline. • Average 10 cfu • Inoculate 2mls into each (higher than required to address product volume
range) so 2x2mls= 4mls • V> 10ml, 1% of product-‐ products under 200ml • Confirm ID, inoculum CFU
Page 38
2014 FA+/FN+ QC test pre validaFon
Page 39
C. sporogenes-‐ iniFal neg result caused by lab error with BioBall inoculaFon
Organisms for 2014 validaFon
Page 40
2014 validaFon typical result
Page 41
Final 2014 report Organism Actual FA+ Actual FN+ Expect FA+ Expect FN+ Accept
FA+ Accept FN+
Days to +
Aspergillus brasiliensis Positive Negative Positive Negative or Positive Yes Yes
2.49
Bacillus subtilis Positive Positive Positive Positive or Negative Yes Yes
0.64, 1.60
Candida albicans Positive Negative Positive Positive or Negative Yes Yes
1.60
Escherichia coli Positive Positive Positive Positive or Negative Yes Yes
0.63, 0.62
Propionibacterium acnes Negative Positive Positive or Negative Positive Yes Yes
4.82
Pseudomonas aeruginosa Positive Negative Positive Negative Yes Yes
0.74
Staphylococcus aureus Positive Positive Positive Positive or Negative Yes Yes
0.65, 0.83
Streptococcus pyogenes Positive Positive Positive Positive or Negative Yes Yes
0.58, 0.68
Page 42
No growth was detected in the posiFve control anaerobic FN Plus boAle for P.acnes. Discussions with the manufacturer of the bioballs (Biomerieux), incorrect rehydraFng fluid used for the re-‐suspension of the organism as 0.9% NaCl does not support the growth of P. acnes due to the organism’s fasFdiousness
• In 10 years have never seen an HPC-‐A product that was culture posiFve post 3 days • ValidaFon all posiFve within 5 days, excluding P. acnes , all within 3 days
Other tesFng issues
• Control of boAles • Contractual requirements
§ No subcontracFng § WarranFes for validaFon and method suitability § NoFficaFon requirements § ID /anFbiograms
Further quesFons
• Inoculum size-‐ what is the issue-‐ diluFon of media vs CFU sensiFvity
• What is the meaning of a final product culture pos & iniFal product culture neg result?
Ø Always proof of process contaminaFon? Ø CFU detecFon threshold/ random sampling
• IniFally tried to transfer service to a TGA licensed vendor, was insourced
Always an automated assay?
• Used Adult FA and FN boAles in a TGA licensed service
• Failed validaFon • DMEM/F12 including 1% penicillin/streptomycin and 1% amphotericin B
• Use membrane filtraFon for these products
Conclusion
• What is actually process related? • How to manage fresh product release • QC monitoring and relevant correcFve acFons • EssenFal to validate with representaFve samples • TGA licensed micro, or not, or how? • Media vs product tesFng (sterility vs contaminaFon) • Sample/lot size/inoculum (Trials, USP, EP, FDA PTC) • 5, 7 or 14 days
Page 47
New faciliFes 2016/17
A
B
C
D
U
Peter Mac’s (and CTPL’s) new home • 10 clean rooms fully PIC/S compliant • Most steps in grade A & B zones • SubstanFal amounts of in-‐process tesFng and PD in grade C • Scale-‐up and validaFon areas • SegregaFon of EM tesFng • Biosafety Levels 2 and 3
48
A
B
C
D
U
Nextcell -‐ Adelaide, SA • 2 clean rooms • same quality system • Serve CTM@CRC • Leased from UniSA • Commissioned 2015
Dominic Wall PhD Chief ScienFfic Officer
t +61 3 9656 1069 m +61 417 301 356 e [email protected] www.celltherapies.com.au
Further enquiries…
