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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: whoccblood@pei.de // whoccivd@pei.de

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 !!