Sartorius AG · XETR:SRT
Sartorius — Technical Presentation (2022)
55 slides from Sartorius's technical presentation, 2022
Slide 1: Simplifying Progress: Microsart ATMP kits
Simplifying Progress Microsart® ATMP Extraction Microsart® ATMP Bacteria & Fungi Microsart® ATMP Sterile Release 2022
Slide 2: ATMPs put microbiological QC to novel challenges
Why new methods? ATMPs put microbiological QC to novel challenges 5, 14 or 28 days of waiting is too long for ATMPs! 2 Digital-Tool “Cell-Based Therapies – Be on the Safe Side”
Slide 3: Nucleic acid techniques
Why new methods? Nucleic acid techniques DNA extraction Amplification of a small sample of target sequence ATMP DNA Detection in (CHO, HEK,…) real-time PCR cycler: Contamination? Yes/No Results within 3 hours! 3
Slide 4: Bacteria & fungi contamination detection
Sartorius Microsart® ATMP Sterile Release Bacteria & fungi contamination detection ▪ Real-time PCR allows detection of bacteria and fungi Microsart® ATMP Extraction ▪ In 3 h ▪ Down to 2.5-99 CFU/ml ▪ Validated combination ▪ In accordance with EP 5.1.6, USP 1223, EP 2.6.27, and USP 1071 Microsart® Mi
Slide 5: Workflow bacteria & fungi contamination detection
Sartorius Microsart® ATMP Sterile Release Workflow bacteria & fungi contamination detection ▪ DNA isolation using the Microsart® ATMP Extraction kit Microsart® ATMP Extraction ▪ Extraction protocol includes centrifugation step to remove free bacterial DNA ▪ Harsh extraction allows to isolate Fungi &
Slide 6: Technical background: DNA-based detection methods
Simplifying Progress Technical background DNA-based detection methods
Slide 7: DNA-based detection methods
Technical background DNA-based detection methods real-time PCR Conventional PCR SYBR Green I TaqMan® Probe 7
Slide 8: What is a conventional PCR?
Technical background What is a conventional PCR? 1st cycle denaturation annealing extension 5‘ 5‘ 3‘ 3‘ 5‘ dNTPs Primer 5‘ Polymerase 8
Slide 9: What is a conventional PCR?
Technical background What is a conventional PCR? 1st cycle 2nd cycle 3nd cycle nth cycle denaturation annealing extension 5‘ PCR product 5‘ 3‘ 3‘ 5‘ ~2n copies dNTPs Primer 5‘ Polymerase 9
Slide 10: A real-time PCR visualizes the reaction
Technical background A real-time PCR visualizes the reaction PCR product How does that work? cycle 12
Slide 11: Real-time PCR
Technical background Real-time PCR SYBR Green I 5‘ 3‘ 5‘ 3‘ 3‘ 5‘ 3‘ 5‘ The dye SYBR Green I binds to double stranded DNA! 13
Slide 12: Real-time PCR
Technical background SYBR Green I Real-time PCR 3rd cycle 2nd cycle 1st cycle PCR product cycle Unspecific binding of SYBR Green I can result in false-positive signals! 14
Slide 13: A TaqMan probe is more specific compared to SYBR Green I
Technical background A TaqManTM probe is more specific compared to SYBR Green I Reporter R Q Quencher Target nucleotide sequence TaqManTM Probe 15
Slide 14: TaqMan real-time PCR
Technical background TaqManTM real-time PCR TaqManTM probe is degraded during real-time PCR R Q R R Q R Q Q During elongation: Taq Polymerase functions: As long as the probe is complete • Polymerase hydrolyses probe no light signal can be detected • DNA amplification • Dye and quencher are separated
Slide 15: TaqMan real-time PCR
Technical background TaqMan® Probe TaqManTM real-time PCR 3rd cycle 2nd cycle 1st cycle PCR product cycle The specificity of TaqManTM system reduces false-positive results! 18
Slide 16: A duplex real-time PCR assay monitors PCR functionality
Technical background A duplex real-time PCR assay monitors PCR functionality Problem: What does no signal mean? No microbial contamination? FAM Q PCR inhibition? target-probe target-DNA Duplex assay Solution: = two independent real-time PCRs Include a second real-time PCR and in one run using differ
Slide 17: A duplex real-time PCR Analysis
Technical Background This analysis is done for both, the bacteria real-time PCR and A duplex real-time PCR Analysis the fungi real-time PCR FAM ROX PC NTC PC, NEC, samples Threshold Threshold NTC, NEC, samples There is no contamination in the samples, There is no PCR inhibition, because the internal
Slide 18: What is in the kits?
Sartorius Microsart® Portfolio What is in the kits? real-time PCR master mix Bacteria • Primer for bacterial DNA • Primer for control DNA • FAM probe for target DNA • ROX probe for control DNA • Taq polymerase • Buffer real-time PCR master mix Fungi Rehydration buffer • Primer for fungal DNA Interna
Slide 19: Hands on fungi & bacteria detection
Simplifying Progress Hands on fungi & bacteria detection
Slide 20: Microsart ATMP Extraction
Hands on fungi & bacteria detection Microsart® ATMP Extraction 23
Slide 21: Microsart ATMP Bacteria
Hands on fungi & bacteria detection Microsart® ATMP Bacteria 24
Slide 22: Microsart ATMP Fungi
Hands on fungi & bacteria detection Microsart® ATMP Fungi All identical, but the fungi master mix has an orange cap! 25
Slide 23: Microsart ATMP Sterile Release - Analysis
Hands on fungi & bacteria detection Microsart® ATMP Sterile Release - Analysis FAM PC 1. Set the baseline to level the curves 2. Set the threshold 3. Check if all controls are as expected Baseline → see next slide Threshold 4. Analyze your samples NTC, NEC, samples ROX Identical analysis workflow fo
Slide 24: Result interpretation
Hands on fungi & bacteria detection The result interpretation is identical for both, the bacteria real-time PCR and Result interpretation the fungi real-time PCR PC NTC Sample NEC Positive Control No Template Control Negative Extraction Control FAM Template Water DNA extracted from Templates added t
Slide 25: Tips and tricks
Hands on fungi & bacteria detection Tips and tricks Bacterial DNA is everywhere! ▪ Extensive cleaning with chlorine-based agents ▪ Avoid cleaning with ethanol → Ethanol precipitates DNA ▪ Spatial separation of DNA extraction process, master mix setup and positive control ▪ E.g. isolator/glovebox or
Slide 26: Sartorius validation support
Simplifying Progress Sartorius validation support
Slide 27: Validation reports, templates & testing proposals
Validation support Validation reports, templates & testing proposals ▪ Product Validation Reports ▪ Validation Template ▪ Validation proposal ▪ Microsart® ATMP Bacteria + ▪ Combined validation Template for ▪ Standard matrix specific validation Microsart® ATMP Extraction Microsart® ATMP Bacteria + ▪
Slide 28: Further support for your validation
Validation support Further support for your validation ▪ Microsart® Validation Standard (99 CFU/Vial) & Microsart® Calibration Reagents (108 GC/Vial for bacteria, 106 GC/Vial for fungi) Non-viable CFU ▪ Bacillus subtilis Standards! ▪ Pseudomonas aeruginosa ▪ Kocuria rhizophila | Micrococcus luteus ▪
Slide 29: Status quo regulatory landscape
Validation support Status quo regulatory landscape Microbiological QC-Release testing Method Mycoplasma Bacteria Fungi USP<63> | EP 2.6.7 USP<71> | EP 2.6.1 Classical testing 28 days Sterility testing 14 days USP<1071> real-time EP 2.6.7 EP 2.6.27 PCR-based (USP<1223>/EP 5.1.6) (USP<1223>/EP 5.1.6)
Slide 30: Validation overview
Validation support Validation overview Regulatory guidance for validation of rapid / alternativ methods: Specificity • PDA, TR 33 • USP <1223> • USP <1071> • EP 5.1.6 part 4-1-1 primary validation by supplier Equivalence • EP 2.6.27 • (USP<71>) • (EP2.6.1) Robustness & Sensitivity Ruggedness Includi
Slide 31: In silico prediction by sequence alignment and blast
Validation - Specificity In silico prediction by sequence alignment and blast 16S / 18S rDNA http://www.biochem.umd.edu/biochem/kahn/bchm465- https://www.ncbi.nlm.nih.gov/ 01/ribosome/16SrRNA.html 34
Slide 32: Detection range – Microsart ATMP Bacteria
Validation - Specificity Specificity Detection range – Microsart® ATMP Bacteria Primer Mismatches Species Strain No Forward primer Probe Reverse primer Bacillus subtilis ATCC 6633 0 0 0 Clostridium sporogenes ATCC 3584 0 0 0 Pseudomonas aeruginosa ATCC 9027 0 0 0 Staphylococcus aureus ATCC 6538 0 0
Slide 33: Detection range – Microsart ATMP Fungi
Validation - Specificity Specificity Detection range – Microsart® ATMP Fungi Accepting 2 primer mismatches, already 37 % of the fungi soil, air, plant debris are detected, including all species of clinical and bioprocess relevance. skin microbiome organic substances soil, decaying wood Primer Mismat
Slide 34: Matrix Effects
Validation - Specificity Specificity Matrix Effects Bacteria Fungi Results Results No Cq No Cq No Cq Hela 0/2 No Cq Hela No Cq No Cq 0/8 No Cq No Cq No Cq No Cq No Cq Vero 0/2 No Cq No Cq No Cq No Cq No Cq No Cq Vero No Cq No Cq 0/8 CHO-K1 0/2 No Cq No Cq No Cq No Cq No Cq No Cq RK13 0/2 CHO-K1 No C
Slide 35: Limit of detection – Microsart ATMP Bacteria/Fungi
Validation - Sensitivity Limit of detection – Microsart® ATMP Bacteria/Fungi EP 2.6.1 regulatory advice; USP<71> EP 2.6.27; user feedback Currently tested = 6 Recommended Extension = 20 Streptococcus pyogenes Bacteroides vulgatus Escherichia coli Pseudomonas protegens Bacillus cereus Colony Forming
Slide 36: Limit of detection
Validation - Sensitivity Sensitivity Limit of detection Example: Candida albicans CFU/ml Mean currently tested Run 1 31.32 31.90 32.78 32.80 32.20 Run 2 32.96 32.88 33.07 32.25 32.79 Run 3 32.29 32.81 36.03 32.09 33.30 recommended 99 24/24 Run 4 32.46 32.14 32.29 32.33 32.30 extension Run 5 32.32 32
Slide 37: Limit of detection
Validation - Sensitivity Sensitivity Limit of detection Species Strain Acceptance LOD95 Species Strain Acceptance LOD95 No criterion (CFU/mL) No criterion (GC/PCR) Bacillus subtilis ATCC 6633 23/24 25 Bacteroides fragilis ATCC 25285 8/8 10 Clostridium sporogenes ATCC 19404 23/24 25 Enterobacter cloa
Slide 38: Device comparability
Validation – Robustness and Ruggedness Robustness & Device comparability Ruggedness Qiagen Rotor-Gene 6000 BioRad CFX96 touch 0,20 0,15 Norm. Fluoro. 0,10 0,05 Threshold 0,00 5 10 15 20 25 30 35 40 45 Cycle Agilent Mx3005p Thermo Fischer ABI Prism 7500 Spiking 99 CFU/ml of the species with the highe
Slide 39: False-positives due to free microbial DNA?
Validation – Robustness and Ruggedness Robustness & False-positives due to free microbial DNA? Ruggedness E. coli DNA spiked into DMEM DNA extraction Real-time PCR failed passed 4 3 10 GC/mL 10 GC/mL 100 GC/mL 10 GC/mL 0 GC/mL Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 39.00 No Cq N
Slide 40: False-positives due to cell culture medium?
Validation – Robustness and Ruggedness Robustness & False-positives due to cell culture medium? Ruggedness Bacteria Fungi Negative Results % Negative results Negative Results % Negative results 8/8 8/8 8/8 8/8 8/8 8/8 8/8 8/8 8/8 8/8 DMEM 7/8 8/8 98,95 % 100 % + 5% FCS 8/8 8/8 8/8 8/8 8/8 8/8 DMEM*
Slide 41: False-positives due to cell culture medium?
Validation – Robustness and Ruggedness Robustness & False-positives due to cell culture medium? Ruggedness Negative ROX™ % Negative Fungi detection Results results DMEM 8/8 8/8 Correct 100% Fetal bovine serum (FBS) 8/8 8/8 Correct 100% DMEM high Glucose, GlutaMAX 8/8 8/8 Correct 100% DMEM w/o Na-Pyr
Slide 42: Tolerance of eukaryotic cell background
Validation – Robustness and Ruggedness Robustness & Tolerance of eukaryotic cell background Ruggedness Aspergillus brasiliensis 99 CFU (FAM™) 106 cells/ml 105 cells/ml 37.54 31.26 33.72 32.10 Hela 35.24 31.84 33.34 32.07 4/4 4/4 33.30 31.43 32.78 31.39 Vero 34.45 32.76 33.48 31.60 4/4 4/4 33.65 32.4
Slide 43: Application Notes
Validation support Application Notes Rapid, real-time PCR-based detection of microbial contaminations in high cell density Jurkat-, HPBMC-and CHO-cultures using Microsart® ATMP kits In this study, we assessed the detection capability of Microsart® ATMP Extraction, combined with Microsart® ATMP Bacte
Slide 44: Tolerance of eukaryotic cell background
Validation – Robustness and Ruggedness Robustness & Tolerance of eukaryotic cell background Ruggedness HPBMC cells PC NEC NVS 15 PVS 20 PVS 20 V2 PVS 25 PVS 25 V2 NTC Successful detection of 99 CFU P. aeruginosa in up to 25 million HPBMC cells. Medium: Lympho One T-Cell Expansion HPBMC: human periph
Slide 45: Tolerance of eukaryotic cell background
Validation – Robustness and Ruggedness Robustness & Tolerance of eukaryotic cell background Ruggedness Jurkat cells PC NEC NVS 10 PVS 10 PVS 15 PVS 20 PVS 25 PVS 30 PVS 40 NTC Succesful detection of 99 CFU Kocuria rhizophila in up to 25 million Jurkat cells. Medium: RPMI 1640 Jurkat: an immortalized
Slide 46: Tolerance of eukaryotic cell background
Validation – Robustness and Ruggedness Robustness & Tolerance of eukaryotic cell background Ruggedness Jurkat cells PC NEC NVS 10 PVS 10 PVS 15 PVS 20 PVS 25 PVS 30 PVS 40 NTC Successful detection of 50 CFU C. albicans in up to 20 million Jurkat cells. Medium: RPMI 1640 Jurkat: an immortalized human
Slide 47: Equivalence with compendial culture method
Equivalence Equivalence Equivalence with compendial culture method Sartorius ? = real-time PCR-based detection classical sterility test according to EP 2.6.1. and USP<71> 2x LOD95 LOD95 ½ LOD95 all 6 mandatory species recommended extension • Bacillus subtilis • Streptococcus pyogenes • Clostridium s
Slide 48: Equivalence with compendial culture method
Application Note Equivalence Equivalence with compendial culture method Microsart® ATMP Bacteria Compendial culture method (External) 2x LOD95 LOD95 LOD95/2 2x LOD95 LOD95 LOD95/2 33.16 34.23 35.47 Bacillus subtilis B. subtilis B. subtilis B. subtilis 33.23 34.32 34.38 35.42 35.77 36.56 Staphylococc
Slide 49: Equivalence with compendial culture method
Application Note Equivalence Equivalence with compendial culture method Microsart® ATMP Fungi Compendial culture method (External) 2x LOD95 LOD95 LOD95/2 2x LOD95 LOD95 LOD95/2 32.25 32.27 32.96 Candida albicans C. albicans C. albicans Negative 31.94 32.12 33.96 34.38 37.06 34.94 Aspergillus brasili
Slide 50: Validation overview
Validation support Validation overview ▪ Sensitivity ▪ LOD95 - limit of detection Specificity ▪ Specificity ▪ Sequence alingment ▪ Sample matrix effects/cross reactivity ▪ Specificity of PCR with genomic DNA ▪ Comparison with compendial method ▪ Robustness Equivalence ▪ Spiked cell culture samples ▪
Slide 51: Coming soon! Customer validation data
Simplifying Progress Coming soon! Customer validation data Service Lab Labor LS
Slide 52: RESEARCH
RESEARCH 55
Slide 53: In-process contamination control of bacteria & fungi
Microsart® RESEARCH kits In-process contamination control of bacteria & fungi ▪ Key advantages ▪ Very robust towards inhibitors ▪ No prior DNA extraction required ▪ Internal control DNA included in real-time PCR master mix 2 µl ▪ One step preparation ATMP 23 µl Detection in →Quick ‘n’ Dirty for proc
Slide 54: In-process contamination control of bacteria & fungi
Microsart® RESEARCH kits In-process contamination control of bacteria & fungi Microsart® RESEARCH Microsart® RESEARCH Bacteria Fungi 57
Slide 55: Thank you.
Thank you. PCR@Sartorius.com