QPCR in Biopharmaceuticals & Current Issues · 2009. 4. 30. · Real-time PCR in BPCEDD Product...
Transcript of QPCR in Biopharmaceuticals & Current Issues · 2009. 4. 30. · Real-time PCR in BPCEDD Product...
QPCR in QPCR in Biopharmaceuticals & Biopharmaceuticals &
Current IssuesCurrent Issues
Chaminda SalgadoChaminda SalgadoHead of PCR ServicesHead of PCR Services
SummarySummary
Biopharm lifecycle● Candidate Plasmid Selection● Cell Line Selection● Cell Banking● Process Support● Potency/Stability testing● Tox Studies● Clinical Trials● Post launch diagnostics
Issues
A bit about me…A bit about me…
● 1996 CBD Porton Down Scientific OfficerChemical & Biological DefenceGene Probes- Detection
● 1999 GlaxoSmithKlinePrincipal ScientistBPCEDD (Biopharmaceutical Center of Excellence Drug Discovery)
● 2008 NDA AnalyticsHead of PCR Services
Candidate plasmid selectionDNA vaccines, Proteins (mAbs, dAbs, fAbs…)
● Promoter● Humanisation● Codon optimisation
Selection using RT-QPCR based on the highest expressors (mRNA transcripts)
Cell line selectionCell line selection
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2H3 (0) 2H3 (60) 4H8(0) 4H8 (60)
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heavy mRNAAntibody
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ody
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L)
light mRNAAntibody
Choosing the right target, and transcript can correlate with translated protein
Cell BankingCell Banking● Virus testing
Test Method28 day 4-cell line in vitro assay (incorporates 324K cells for detection
of Minute Mouse Virus)Adventitious viruses
In vivo assays (adult and suckling mice, guinea pigs and embryonated eggs)
Extended S+L- focus assay on mink lung cellsReverse Transcriptase, QFPERTTEMCo-cultivation with a human cell line or other speciesIn vitro assay for bovine virusesBovine VirusesQPCR and/or in vitro assay for bovine polyomavirus
Porcine Viruses In vitro assay for porcine viruses
Hamster Antibody Production (HAP) testSpecies Specific VirusesLymphocytic choriomeningitis Virus (LCMV) challenge test
Retroviruses
Process ValidationVirus Clearance Validation
● Partitioning by selective absorption● Inactivation● Size Exclusion + Mechanical inactivation
Virus spike Quantify OutputOrthogonal Process Steps
Robust Clearance (LRV > 4 log TCID50/ml):Low pH (inactivation of acid labile model virus)Nanofiltration (removal of small non- enveloped viruses)
Additional Clearance (LRV > 1 log TCID50/ml):Column’s (partitioning based on charge or functional ligand)Ion Exchange Filter
StabilityStability
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No. of generations
X Stable
Unstable
YeildRNA transcriptDNA
Non genetic Phenotypic drift?
rgDNA ClearancergDNA Clearance
Std Curve
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log10 Quantity (pg/ml)
Ct
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Process Step
rgD
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ls(u
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1fg – 10ng
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Antibody conc [µg/mL]
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essi
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hang
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5h incubation 3h incubation
QPCR Bioassay for protein based products
• Candidate Biomarkers involved in intracellular pathways can be identified using Arrays
• RT-QPCR subsequently used to validate and quantify expression of the biomarkers for potency and stability indicating assessment.
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ng tranfected Plasmid
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ng tranfected Plasmid
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ell
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B. mRNA compared to transfection load -20
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mRNA in cell
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QPCR Bioassay for DNA Vaccine
Real-time PCR in BPCEDD
Product Stability
• Potency (RT-QPCR)
Cell line Stability/Selection
• Quantitative Gene Expression
• DNA copy No.
Candidate Selection
• Potency of candidate vectors following optimisation
Process Validation
• Host cell DNA/RNA clearance
• Virus clearance
Master Cell Bank testing
• Virus
• Mycoplasma
Product issues
• Replication incompetent reversion
• Sterility
Tox
• Bio distribution
Late stage and beyondLate stage and beyondIn the Clinic● Transcriptomics to determine drug effects on gene regulation
After market● Companion Diagnostics – genetic profile to determine suitability
of a drug.● Health care costs will be driven down through improved prescribing and
compliance, and reduction in ADRs. ie. DxS:K-Ras, mutations in VKORC1, and warfarin.
Issues
StandardisationStandardisation
● Methods ● Sample preparation● Targets used in normalisation for relative expression● Absolute Quantitation- NO DNA Gold Standard!! OR
Standardised means to quantify the QPCR Reference!
● AnalysisData has to be comparable- it’s a regulatory must!
● Subjective threshold vs 2nd derivative maxima algorithm● Baseline normalisation● Algorithms/methods for relative quantitation● Realtime kinetic efficiency.
Sample preparationSample preparation
● Variable extraction efficiencies due to matrix● Variable extraction efficiencies due to load● NAT guidance 80% yield…relevance to QPCR?● Extraction technologies to standardise on % Yeild
as a measure of comparison
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1 10 100
Log [Standard Copy Number]
Req
uire
d R
eplic
ates
ReplicatesReplicates
CopyNumber
ReplicatesRequired
1 21
2 11
5 5
10 3
50 1
100 1
Poisson Distribution Model
• Software ignores blank/negative data !
Raw dataRaw data
Start baselineStart baseline
BaseliningBaselining
BaseliningBaselining
Cp/Ct calling
•Threshold, Fit-Points/Threshold, 2nd der max.
•Marketing gimmick vs truth
Individual baselining
•Critical for high throughput!
Efficiency correction?Efficiency correction?
?
TrainingTraining
Std Curve
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log10 Quantity
Ct
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-2.25140 %
100 %
6 cycle difference 26 = 64
What’s missing?What’s missing?
• Currently most of the available data is ignored in the final analysis
• Currently most base PCR efficiencies according to separate reactions
• A lot of potential data is lost due to fluorescent noise.
• More investment into realtime kinetic algorithms for both efficiency determination, QC criteria, and non subjective Cycle calling.
• Base lining algorithms.
• Investment into gated detection and time resolved chemistries.
Please help!Please help!
Put individual baselining & truthful cycle calling at the top of the list
AcknowledgmentsAcknowledgments
NDA Analytics● Gerry Maxwell
GlaxoSmithKline● Jean Engela● Anja Grohnert● Phil Henwood● Mike Aylott
University of Sussex● Kyle Morris
Enigma Diagnostics● Martin Lee
BackupBackup
Technical InnovationTechnical Innovation
● Reagents●Enzymes etc- speed and utility are constantly
improving.● Time delay chemistries
● Instrumentation●Speed + combined sample prep. (<30min from raw
sample)●Gated/time resolved detection●Non-subjective software algorithms
Experimental vs PoissonExperimental vs Poisson
● Both models agree that as copy number per volume is decreased, so variation is increased. At the low end due to poisson distribution.
● Increasing replicates at the lower end brings the observed average values closer to the expected values
What I’m trying to get at…What I’m trying to get at…
Applicable well beyond the realms of real time – any assay using a standard curve for absolute quant.
Replication of standard curve points (and where possible samples)
should be increased at lower copy numbers.
Reliability of abs quant below 100 copies should be questioned.