QPCR in Biopharmaceuticals & Current Issues. Chaminda Salgado Head of PCR Services
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1 QPCR in Biopharmaceuticals & Current Issues Chaminda Salgado Head of PCR Services
2 Summary Biopharm lifecycle Candidate Plasmid Selection Cell Line Selection Cell Banking Process Support Potency/Stability testing Tox Studies Clinical Trials Post launch diagnostics Issues
3 A bit about me CBD Porton Down Scientific Officer Chemical & Biological Defence Gene Probes- Detection GlaxoSmithKline Principal Scientist BPCEDD (Biopharmaceutical Center of Excellence Drug Discovery) NDA Analytics Head of PCR Services
4 Candidate plasmid selection DNA vaccines, Proteins (mabs, dabs, fabs ) Promoter Humanisation Codon optimisation Selection using RT-QPCR based on the highest expressors (mrna transcripts)
5 Cell line selection mrna heavy mrna Antibody Antibody titre (mg/l) mrna light mrna Antibody Antibody titre (m g/l) H3 (0) 2H3 (60) 4H8(0) 4H8 (60) H3 (0) 2H3 (60) 4H8(0) 4H8 (60) 0 Choosing the right target, and transcript can correlate with translated protein
6 Cell Banking Virus testing Test Adventitious viruses Retroviruses Bovine Viruses Porcine Viruses Species Specific Viruses Method 28 day 4-cell line in vitro assay (incorporates 324K cells for detection of Minute Mouse Virus) In vivo assays (adult and suckling mice, guinea pigs and embryonated eggs) Extended S+L- focus assay on mink lung cells Reverse Transcriptase, QFPERT TEM Co-cultivation with a human cell line or other species In vitro assay for bovine viruses QPCR and/or in vitro assay for bovine polyomavirus In vitro assay for porcine viruses Hamster Antibody Production (HAP) test Lymphocytic choriomeningitis Virus (LCMV) challenge test
7 Process Validation Virus Clearance Validation Virus spike Orthogonal Process Steps Quantify Output Partitioning by selective absorption Inactivation Size Exclusion + Mechanical inactivation 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
8 Stability X Stable RNA Yeild transcript DNA Non genetic Phenotypic drift? Unstable No. of generations
9 rgdna Clearance 4.00 Std Curve Series1 Batch 01 Batch rgdna Levels (ug) Batch 04 Batch 05 Batch 06 Batch A B C D E F 25 Process Step Ct fg 10ng log10 Quantity (pg/ml)
10 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 Expression fold change Antibody conc [µg/ml] 5h incubation 3h incubation
11 QPCR Bioassay for DNA Vaccine 18 0 A DNA in cell ng tranfected Plasmid mrna in cell B C 0 A. Cell DNA compared to transfection load ng tranfected Plasmid DNA in cell 20 B. mrna compared to 0 transfection load mrna in cell C. mrna compared to DNA.
12 Cell line Stability/Selection Quantitative Gene Expression Candidate Selection Product Stability DNA copy No. Potency of candidate vectors following optimisation Potency (RT-QPCR) Real-time PCR in BPCEDD Master Cell Bank testing Virus Mycoplasma Product issues Replication incompetent reversion Sterility Process Validation Host cell DNA/RNA clearance Virus clearance Tox Bio distribution
13 Late stage and beyond In 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.
14 Issues
15 Standardisation Methods Sample preparation Targets used in normalisation for relative expression Absolute Quantitation- NO DNA Gold Standard!! OR Standardised means to quantify the QPCR Reference! Analysis Data has to be comparable- it s a regulatory must! Subjective threshold vs 2 nd derivative maxima algorithm Baseline normalisation Algorithms/methods for relative quantitation Realtime kinetic efficiency.
16 Sample 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
17 Replicates Software ignores blank/negative data! Required Replicates Log [Standard Copy Number] Poisson Distribution Model Copy Number Replicates Required
18 Raw data
19 Start baseline
20 Baselining
21 Baselining Individual baselining Critical for high throughput! Cp/Ct calling Threshold, Fit-Points/Threshold, 2 nd der max. Marketing gimmick vs truth
22 Efficiency correction??
23 Training Std Curve % Series1 Ct cycle difference = % log10 Quantity
24 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.
25 Please help! Put individual baselining & truthful cycle calling at the top of the list
26 Acknowledgments NDA Analytics Gerry Maxwell GlaxoSmithKline Jean Engela Anja Grohnert Phil Henwood Mike Aylott University of Sussex Kyle Morris Enigma Diagnostics Martin Lee
27 Backup
28 Technical 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
29 Experimental 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
30 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.
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