who is for mycoplasma nat - nibsc - homepaul-ehrlich-institut who collaborating centre for quality...
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Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices
Project endorsed by Expert Committee on Biological Standardization
Geneva, 19 October 2010
Micha Nübling
Paul-Ehrlich-InstitutPaul-Ehrlich-Straße 51-59
63225 Langen
GERMANY
+49 (0) 6103 77 7010
+49 (0) 6103 77-1250
E-mail: [email protected] // [email protected]
Homepage: http://www.pei.de
WHO IS for Mycoplasma NAT
Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices
An initiative of
• Parenteral Drug Association (PDA) Task Force for Alternative
Mycoplasma Testing
(K. Brorson (CDER/FDA), T. Haemmerle (Baxter), D. Asarnow (Bayer) et al)
• NIBSC
• PEI
WHO IS for Mycoplasma NAT
Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices3
Rationale
● Bacteria class Mollicutes (trivial name: mycoplasmas) are unusual bacteria
• Absence of a cell wall• Small genome size (540 – 1,300 kbp)• Low G+C content• Monophyletic class includes three orders
– Mycoplasmatales– Entomoplasmatales– Acholeplasmatales
● Mollicutes are contaminants of biologic processes, e.g. cell cultures, leading to changes in cell metabolism / phenotype
Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices4
Rationale contd.
Mycoplasma testing regulations
● relied on culture (broth, agar) and/or indicator cell culture
• US FDA PTC
• Ph Eur
• Jap Ph
Limitations
• Testing time period of 28 days
• Complex conditions (different media; anaerobic, aerobic; multiple passages)
Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices5
Rationale contd.
Mycoplasma testing regulations
● (will) accept NAT testing methods as alternatives
• Ph Eur: replacement of cell culture possible after NAT validation and
comparability studies
• US Ph 63: alternative method possible
• Draft FDA NfG on Characterization and Qualification of Cell Substrates
• Jap Ph Section 14: example PCR method
Different PCR methods using 16S rRNA gene as target have been accepted by
the FDA, EMA and Jap Regulatory Authority
Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices6
Rationale contd.
Mycoplasma testing regulations
● Current Mycoplasma NAT testing methods are not standardized
C. Milne, 24/103/09
©2009 EDQM, Council of Europe, All rights reserved 7
Mycoplasma detection assays
StrainMean
CFU/ml
Mean
Copies/ml
by PCR
Lab B
Mean
Copies/ml
by PCR
Lab C
Vial Code
Estimated
CFU/prediluted
vial
Copies/ml
in prediluted
vial - Lab B
M. synoviae 1.86 • 107 7.27 • 107 2.4 • 109A 160 2514
D 1520 11789
M. hyorhinis 1.17 • 108 6.76 • 107 1.4 • 1010F 9800 3213
G 1010 403
M. orale 4.90 • 105 6.17 • 106 9.0 • 109J 290 11384
M 40 1734
M.
fermentans9.55 • 107 2.04 • 108 3.6 • 107
P 300 1135
S 3060 10992
A. laidlawii 2.45 • 106 1.00 • 108 3.6 • 1010X 1610 34718
Y 96 4447
No good/clear correlation between CFU and Genome Copy
Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices8
Anticipated Uses
● Validation of Mycoplasma NAT methods (e.g. LOD)
● Comparative assessment of NATs
● “Common language”
● Regulatory requirements for NAT assays
Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices9
Anticipated Users
● Biological medicines manufacturers
● Official medicines control laboratories (OMCLs)
● Research laboratories
● NAT tests manufacturers
● Diagnostic laboratories
Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices10
Source of Material
● Mycoplasma culture from Acholeplasma laidlawii
• Frequent contaminant of cell cultures: mammalian, avian, insect, fish, plant • Serum contaminant• (human pathogen)
• Requested for validation of culture and NAT methods and comparability studies (Eur Ph)
● Further Mycoplasma ssp are considered for the pilot study
• e.g. Mycoplasma hominis, M. arginini
Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices11
Proposed Studies
(1) Pilot study
● A feasibility study will be performed where participants will
evaluate a Mycoplasma panel
• different well-characterized Mycoplasma materials
• potential effect of lyophilization
● The results will provide information on assay performance and
the most suitable Mycoplasma material to develop into a WHO IS
Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices12
Proposed Studies contd.
(2) Collaborative study
● A larger collaborative study will follow for characterization of WHO IS candidate materials
● Full-genome sequencing of the WHO IS agent
Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices13
Comments received so far
• Commutability of Acholeplasma laidlawii to other Mycoplasma sp ?
• Commutability will be addressed for the most relevant sp (pilot
study)
• Conservation of 16S rRNA gene 83% (comp. to M hominis)
• 16S rRNA gene based NAT assays for different Mollicutes already in
place
• Diagnostic systems are species specific
• Future WHO reference panel for diagnostically relevant strains?
Paul-Ehrlich-Institut
WHO Collaborating Centre for Quality Assurance of Blood Products
and in vitro Diagnostic Devices14
Further comments / suggestions ??
• Thank you for your attention !!