Sartorius AG · XETR:SRT
Sartorius — Technical Presentation (2022)
46 slides from Sartorius's technical presentation, 2022
Slide 1: Simplifying Progress: Microsart PCR Portfolio Overview 2022
Simplifying Progress Microsart® PCR Portfolio Overview 2022
Slide 2: Product overview
Sartorius Microsart® PCR Portfolio Product overview ▪ Validated solutions for QC release ▪ Microsart® ATMP Sterile Release ▪ Microsart® ATMP Mycoplasma & Microsart® AMP Extraction ▪ Microsart® AMP Mycoplasma & Microsart® AMP Extraction (special application) ▪ Non-validated solutions for in-process c
Slide 3: Why new methods? 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! 3 Digital-Tool “Cell-Based Therapies – Be on the Safe Side”
Slide 4: Why new methods? 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! 4
Slide 5: Validated solutions for QC sterile release
Sartorius Microsart® PCR Portfolio Validated solutions for QC sterile release Bacteria and Fungi Detection Mycoplasma Detection Microsart® ATMP Extraction Microsart® ATMP Bacteria Microsart® AMP Extraction Microsart® ATMP Fungi Microsart® ATMP Mycoplasma or Microsart® ATMP Sterile Release 5
Slide 6: Mycoplasma contamination detection
Sartorius Microsart® AMP Extraction & Microsart® ATMP Mycoplasma Mycoplasma contamination detection ▪ Real-time PCR allows detection of Mycoplasma ▪ In 3 h ▪ Down to 5-10 CFU/ml ▪ Validated combination Microsart® AMP Extraction ▪ In accordance with EP 2.6.7 and USP 63 ▪ Support ▪ Product Validation
Slide 7: Workflow Mycoplasma contamination detection
Sartorius Microsart® AMP Extraction & Microsart® ATMP Mycoplasma Workflow Mycoplasma contamination detection ▪ DNA isolation using the column-based Microsart® AMP Extraction kit ▪ Columns allow to isolate DNA from the whole ATMP sample (supernatant & cells) ▪ Real-time PCR using the Microsart® ATMP
Slide 8: 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 9: 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 10: Technical background: DNA-based detection methods
Simplifying Progress Technical background DNA-based detection methods
Slide 11: DNA-based detection methods
Technical background DNA-based detection methods real-time PCR Conventional PCR SYBR Green I TaqMan® Probe 11
Slide 12: 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 12
Slide 13: 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 13
Slide 14: A real-time PCR visualizes the reaction
Technical background A real-time PCR visualizes the reaction PCR product How does that work? cycle 16
Slide 15: Real-time PCR: SYBR Green I
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! 17
Slide 16: Real-time PCR: SYBR Green I
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! 18
Slide 17: 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 19
Slide 18: 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 19: 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! 22
Slide 20: 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 21: A duplex real-time PCR Analysis
Technical Background A duplex real-time PCR Analysis 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 because only the positive control is control DNA was detected in all reactions. detect
Slide 22: What is in the kits?
Sartorius Microsart® Portfolio What is in the kits? real-time PCR master mix • Primer for target DNA • Primer for control DNA • FAM probe for target DNA • ROX probe for control DNA • Taq polymerase • Buffer Rehydration buffer Internal Control DNA Positive Control DNA Ultrapure Water 25
Slide 23: Mycoplasma, bacteria & fungi contamination detection
Sartorius Microsart® Portfolio Mycoplasma, bacteria & fungi contamination detection Microsart® ATMP Mycoplasma Microsart® ATMP Bacteria Microsart® ATMP Fungi ▪ FAM probe: ▪ FAM probe: ▪ FAM probe: ▪ Targets Mycoplasma ▪ Targets Bacteria ▪ Targets Fungi ▪ PCR Mix with red cap ▪ PCR Mix with red cap ▪
Slide 24: Product Validation
Product Validation : LRTORILS
Slide 25: Validation reports, templates & testing proposals
Validation support Validation reports, templates & testing proposals ▪ Product Validation Reports ▪ Validation Templates ▪ Validation Proposals ▪ Microsart® ATMP Bacteria + ▪ Combined validation Template for ▪ Standard matrix specific validation Microsart® ATMP Extraction Microsart® ATMP Bacteria +
Slide 26: Further support for your validation
Validation support Non-viable CFU Standards! Further support for your validation ▪ Microsart® Validation Standard (10 CFU/Vial) & ▪ Microsart® Validation Standard (99 CFU/Vial) & Microsart® Calibration Reagents (108 GC/Vial) Microsart® Calibration Reagents ▪ Mycoplasma arginini (108 GC/Vial for bact
Slide 27: 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 28: Validation overview
Validation support Validation overview Regulatory guidance for validation of the Regulatory guidance for validation of Microsart® ATMP Bacteria/Fungi kit: the Microsart® ATMP Mycoplasma kit: Specificity • PDA, TR 33 ▪ EP 2.6.7 (Mycoplasma) ▪ EP 2.6.21 (NAT) • USP <1223> ▪ ICH Q2B • USP <1071> • EP 5
Slide 29: 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 32
Slide 30: Detection range – Microsart ATMP Mycoplasma
Validation - Specificity Specificity Detection range – Microsart® ATMP Mycoplasma # Primer/Probe Genus Mismatches 11 5 6 0 32 Acholeplasma Acceptance criterion: 1 25 ≤ 3 nucleotides mismatch Mycoplasma of primers and probe 2 Ureaplasma 3 42 99 At least 110 species are detectable based on sequence al
Slide 31: Detection range – Microsart ATMP Bacteria
Validation - Specificity Specificity Detection range – Microsart® ATMP Bacteria Primer Mismatches 0 1 2 3 Bacteria 48.8 % 69.4 % 85.7 % 94.7 % Archaea n.a n.a 0.1% 40.4 % Eukaryotes 0% 0% 0.1% 0.3 % Accepting 3 primer mismatches, 94.7 % of the bacteria are detected 34
Slide 32: Detection range – Microsart ATMP Fungi
Validation - Specificity Specificity Detection range – Microsart® ATMP Fungi soil, air, plant debris skin microbiome organic substances soil, decaying wood Accepting 2 primer mismatches, already 37 % of the fungi are detected, including all species of clinical and bioprocess relevance. 35
Slide 33: Matrix Effects
Validation - Specificity Specificity Matrix Effects ▪ Media ▪ Typical cell culture media ▪ Typical additives for cell culture media FYI: ▪ Typical buffers Application Note ▪ Cell lines challenging all Microsart® ATMP Kits with high cell ▪ Vero-B4 concentrations ▪ Per. C6 ▪ RK 13 ▪ CHO-K1 ▪ Hela ▪ Ju
Slide 34: Limit of detection – Microsart ATMP Mycoplasma
Validation - Sensitivity Limit of detection – Microsart® ATMP Mycoplasma EP 2.6.7 USP/EP required = 9 Mycoplasma arginini Mycoplasma orale Mycoplasma gallisepticum Mycoplasma pneumoniae Mycoplasma synoviae Mycoplasma fermentans Mycoplasma hyorhinis Acholeplasma laidlawii Spiroplasma citri Limit of d
Slide 35: Limit of detection – Microsart ATMP Mycoplasma
Validation - Sensitivity Sensitivity Limit of detection – Microsart® ATMP Mycoplasma Species (CFU-based) Acceptance LOD95 criterion (CFU/mL) Mycoplasma arginini 23/24 10 Mycoplasma orale 23/24 10 Mycoplasma gallisepticum 23/24 10 Mycoplasma pneumoniae 23/24 ≤5 Mycoplasma synoviae 23/24 10 Mycoplasma
Slide 36: 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 37: Limit of detection – Microsart ATMP Bacteria/Fungi
Validation - Sensitivity Sensitivity Limit of detection – Microsart® ATMP Bacteria/Fungi Species Strain Acceptance LOD95 No criterion (CFU/mL) recommended Bacillus subtilis ATCC 6633 23/24 25 currently tested extension Clostridium sporogenes ATCC 19404 23/24 25 Pseudomonas aeruginosa ATCC 9027 23/24
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: 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 40: 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 41: 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 42: 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 43: RESEARCH
RESEARCH LRTORILS
Slide 44: In-process contamination control of Mycoplasma, bacteria & fungi
Microsart® RESEARCH kits In-process contamination control of Mycoplasma, 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’ Di
Slide 45: In-process contamination control of Mycoplasma, bacteria & fungi
Microsart® RESEARCH kits In-process contamination control of Mycoplasma, bacteria & fungi Microsart® RESEARCH Microsart® RESEARCH Microsart® RESEARCH Mycoplasma Bacteria Fungi 48
Slide 46: Thank you.
Thank you. PCR@Sartorius.com