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Product Validation Report

Venor®GeM qEP

Dated: 11.01.2018 Document Version: 2 Document ID: MB_VA05.02EN of 40

© Minerva Biolabs GmbH 2018 Confidential Information

Table of Contents 1 Introduction ................................................................................................................................ 1-3 2 Objective .................................................................................................................................... 2-4 3 Definitions and Abbreviations ........................................................................................................ 3-5 4 Responsibilities ........................................................................................................................... 4-6 5 Test Materials ............................................................................................................................. 5-7

5.1 Test System 5-7 5.2 Sample Matrix 5-7 5.3 Microorganisms and Eukaryotic Material 5-8 5.4 Equipment 5-10

6 Test Procedure .......................................................................................................................... 6-11 6.1 Manual DNA Extraction 6-11 6.2 Analytical procedures 6-12 6.3 Calculations 6-13 6.4 Reporting requirements 6-13

7 Study Results ........................................................................................................................... 7-14 7.1 Specificity 7-14

7.1.1 Sequence Alignment ................................................................................................... 7-14 7.1.2 Sample Matrix Cross Reactivity ..................................................................................... 7-17 7.1.3 Mollicutes Detection Range .......................................................................................... 7-18 7.1.4 Cross Reactivity .......................................................................................................... 7-21

7.2 LOD95 Detection Limit 7-29 7.3 Robustness 7-35

7.3.1 Matrix Effects ............................................................................................................. 7-35 7.3.2 Device compatibility .................................................................................................... 7-38

8 Conclusion ............................................................................................................................... 8-39 9 Reference Documents ............................................................................................................... 9-40


Venor®GeM qEP



1 Introduction

Mycoplasmas are important contaminants of biological products derived from cell lines in the biopharmaceutical industry affecting every parameter of a cell culture system. Contaminated cultures can result in production loss and unsafe products. Mycoplasmas are the smallest of the free-living organisms. Unlike viruses, mycoplasmas can reproduce outside of living cells. Many species within the genera Mycoplasma, Acholeplasma and Spiroplasma thrive as parasites in human, bird, plants and animal hosts. Some species can cause disease in humans. Such contaminations can arise from the contamination of the source cell lines themselves (cell substrates) or from adventitious introduction of mycoplasmas during production. Based on this, contamination risk guidelines and technical papers published guidance on mycoplasmas safety for the manufacture of biological products as for instance the European Pharmacopoeia, chapter 2.6.7., “Mycoplasmas” or Japanese Pharmacopoeia, 17th edition, chapter G3. The diagnostic kit Venor®GeM qEP detects Mollicutes (Mycoplasma, Acholeplasma, and Spiroplasma) contamination in cell cultures and other cell culture derived biologicals. The kit utilizes the polymerase chain reaction (PCR), established as the method of choice for high sensitivity. The kit includes a Primer/Probe/Nucleotide mix containing a FAM labelled probe specific for a broad range of different mycoplasma species. The internal amplification control prevents false negative results due to PCR inhibitors or improper DNA extraction. It is possible to add the Internal Control DNA directly to the sample prior to DNA extraction and analysis for verification of the complete process (DNA extraction and PCR reaction). If added to the PCR master mix directly, the Internal Control DNA acts as a PCR control only. The amplification of the control reaction is detectable at 560 nm (HEX channel). The mycoplasma-specific amplification is detectable at 520 nm (FAM channel). The kit contains dUTP instead of dTTP, providing an optional degradation of amplicons from previous analysis by use of uracil-N-glycosylase (UNG).


Venor®GeM qEP



2 Objective

We designed and completed a study to evaluate the Mycoplasma detection capability for the Mycoplasma Detection Kit Venor®GeM qEP for qPCR. Section 2.6.7 “Mycoplasmas” of the European Pharmacopoeia describes a protocol for validation of a kit as burden of the manufacturer. This chapter includes guidelines and specifications for relevant parameters like specificity, detection limit and robustness in comparison to the traditional culture method. We released product version 1 of Venor®GeM qEP for sale in early 2007. New results of our permanent product optimization efforts became available during that time. We considered these findings as relevant improvement for the performance of the kit and for the users in general. Product version 4 contains these modifications resulting an updated user’s protocol and product design. Product versions 2 and 3 were part of an internal performance study not released for sale. The product modifications required a full validation of all relevant performance aspects according to section 2.6.7 of the European Pharmacopoeia, section 2.6.21 of the EP “Nucleic acid amplification techniques” (NAT, PCR), and with respect to ICH guideline Q2B. The employed method obtains qualitative result only (positive/negative). Based on the requirements of the European Pharmacopoeia 2.6.21, it was not necessary to demonstrate compliance with all individual requirements of ICH Q2B. The validation plan considered the core requirements of validation in accordance with ICH Q2B in the context of their applicability to the qualitative nature of the test employed. The validation of the Venor® GeM qEP was completed with the determination of the sensitivity for Mycoplasma salivarium in order to fulfil the requirements of the Japanese Pharmacopoeia (JP) paragraph “Mycoplasma testing for cell substrates used for the Production of Biotechnological/Biological products” [6].

In detail, the new kit version comprises of the following improvements:

1. New primer/probe set to cover specificity for all relevant Mollicutes species to omit the necessity of

setting up three master mixes for an analysis 2. Polymerase-optimized buffer 3. Use of the Internal Control DNA as an extraction control 4. Updated protocol for qPCR cycler 5. Updated protocol for DNA extraction comprising a protease incubation step which might be required for

highly proteinogenic samples 6. Freeze-drying of all relevant components of the kit, including the critical ingredients polymerase,

nucleotides and probes in a substantially detectable pellet 7. Replacement of ROX as Internal Control Probe dye

Based on a risk assessment the product modifications may affect the relevant parameters specificity, detection limit, and robustness. This report does not include data from the validation report for product version 1. The validation takes advantage of the 10CFU standards to provide an additional correlation to the traditional culture method. This standard material predicates on vital colony counts titration using qualified culture media. As far as possible, the validation design requires a most characteristic application and challenging test setup.


Venor®GeM qEP



3 Definitions and Abbreviations

ATCC American Type Culture Collection CoA Certificate of Analysis DMEM Dulbecco´s modified Eagles medium DNA deoxyribonucleic acid ENC Extraction negative control EP European Pharmacopoeia FBS Foetal Bovine Serum g g-force (unit for measurement of rotation speed of centrifugation) IC internal amplification control ICH International Conference on Harmonization of Technical Requirements for Registration of

Pharmaceuticals for Human Use JP Japanese Pharmacopoeia LOD95 concentration, where 95 % of all samples were positive mg/ml milligram per millilitre N/A, n.a. not applicable N/M not measured nm nanometre N/P not provided NTC no template control OD260 optical density (at a wavelength of 260 nm) PBS phosphate buffered saline PC positive control PCR polymerase chain reaction CFU/ml colony-forming units per millilitre pH potentia hydrogenii RPMI Roswell Park Memorial Institute s second TE80 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA Tris tris (hydroxyl methyl amino methane)


Venor®GeM qEP



4 Responsibilities

The Product Development Department at Minerva Biolabs GmbH is responsible for developing the test protocol. The Quality Manager was responsible for reviewing the test protocol to ensure its accuracy, completeness and validity.

Test initiation was scheduled by the head of the Product Development Department at Minerva Biolabs after approval of the validation plan by signing the plan cover page and the necessary material for testing have been received.

Minerva Biolabs technicians executed the test protocol. Minerva Biolabs technicians were responsible for the execution of dedicated parts of the protocol.

The Product Development Team drafted the validation report and the Quality Manager reviewed and approved the document to ensure its validity. The signatures on the cover page closed the report.

No deviations, including test failures and protocol modifications, occurred during the execution of the test protocol.


Venor®GeM qEP



5 Test Materials

This chapter lists the test systems, product solutions and material used for the study. This document does not show the batch numbers of the products used. The result reports contain this information. They are available on request.

5.1 Test System

The test system used for the detection of mycoplasma during this study is as follows:

Table 1: Test System Information

System type Supplied by Cat. No. Storage Conditions

Venor®GeM qEP Minerva Biolabs GmbH 11-9100 2 – 8 °C

Proteinase K Minerva Biolabs GmbH 56-0002 ambient temperatures

Venor®GeM Sample Preparation Kit Minerva Biolabs GmbH 56-1200 ambient temperatures

5.2 Sample Matrix

The specificity testing used defined cell culture medium components as described in table 2. The robustness testing used DMEM medium containing 10 % (v/v) FBS as sample matrix for all spiking experiments.

Table 2: Matrix Formulation

Product Ingredient Manufacturer Cat. No. Storage Conditions

PBS Dulbecco Biochrom AG L1815 +2 – 8 °C

immune globulin, solubilized after ammonium sulphate precipitation n.a. n.a. < -20 °C

Donor horse serum Sigma Aldrich 12449c < -20 °C

Human serum albumin GE Health Care T1312 < -20 °C

Calf serum PAA Laboratories 11-5140 < -20 °C

Porcine serum GE Health Care T1303 < -20 °C

Tissue, homogenized n.a. n.a. < -20 °C

Mycoplasma broth Heipha 146251 +2 – 8 °C

Hayflick Bouillon acc. EP In house preparation n.a. +2 – 8 °C

Hayflick Agar acc. EP In house preparation n.a. +2 – 8 °C

DMEM medium Biochrom AG FG 0415 +2 – 8 °C


Venor®GeM qEP



5.3 Microorganisms and Eukaryotic Material

Table 3, 4 and 5 describe the microorganisms and eukaryotic material used for spiking or specificity testing during the study.

Table 3: Description of Mollicutes preparations – quantified by culture method

Species Natural Host Supplier Cat. No.

10CFU™ Sensitivity Standard Acholeplasma laidlawii ubiquitous

Minerva Biolabs

102-8003

10CFU™ Sensitivity Standard Mycoplasma fermentans human 102-6003

10CFU™ Sensitivity Standard Mycoplasma hyorhinis mammal 102-7003

10CFU™ Sensitivity Standard Mycoplasma orale human 102-2003

10CFU™ Sensitivity Standard Mycoplasma pneumoniae human 102-4003

10CFU™ Sensitivity Standard Mycoplasma gallisepticum bird 102-3003

10CFU™ Sensitivity Standard Mycoplasma synoviae mammal 102-5003

10CFU™ Sensitivity Standard Mycoplasma arginini mammal 102-1003

10CFU™ Sensitivity Standard Mycoplasma salivarium human 102-1103

10CFU™ Sensitivity Standard Spiroplasma citri plant 102-9003

Table 4: Description of Mollicutes DNA - quantified photometrical based on OD260 excitation

Species Natural Host Supplier Cat. No.

Acholeplasma laidlawii ubiquitous

Minerva Biolabs

51-0116

Mycoplasma fermentans human 51-0117

Mycoplasma hyorhinis mammal 51-0130

Mycoplasma orale human 51-0112

Mycoplasma pneumoniae human 51-0119

Mycoplasma gallisepticum bird 51-0115

Mycoplasma synoviae mammal 51-0124

Mycoplasma arginini mammal 51-0162

Mycoplasma genitalium human 51-0195

Mycoplasma hominis human 51-0111

Mycoplasma penetrans mammal 51-1746

Ureaplasma urealyticum human 51-0177

Table 5: Description of non-Mollicutes Bacterial Strains - quantified photometrical based on OD260 excitation Species Culture Collection No. Supplier Cat. No.

Acinetobacter baumanii DSM 30007

Minerva Biolabs

2127-30007

Bacillus cereus DSM 31 51-0031

Bacillus subtilis DSM 347 51-0010

Bordetella pertussis DSM 5571 51-5571

Campylobacter jejuni DSM 4668 2102-04688

Clostridium acetobutylicum DSM 792 51-0792

Clostridium perfringens DSM 756 2108-00756

Enterobacter aerogenes DSM 30053 51-0053

Enterococcus casseliflavus DSM 20680 2111-20680

Enterococcus faecalis DSM 20478 51-0478


Venor®GeM qEP



Species Culture Collection No. Supplier Cat. No.

Escherichia coli DSM 30083 51-0083

Geobacillus stearothermophilus DSM 5934 2131-05934

Lactobacillus acidophilus NCTC 1723 51-1723

Micrococcus luteus DSM 20030 51-0030

Pseudomonas aeruginosa DSM 50071 51-0071

Salmonella enterica DSM 17058 51-7058

Staphylococcus aureus DSM 20231 51-3361

Staphylococcus epidermidis DSM 20044 51-0044

Staphylococcus saprophyticus DSM 20229 2120-20229

Streptococcus dysgalactiae DSM 6176 2123-06176

Streptococcus mutans DSM 20523 2126-20523

Streptococcus pneumoniae DSM 20566 51-0566

Streptococcus sanguinis DSM 20567 2139-20068 Table 6: DNA of eukaryotic origin Species Family Origin Supplier No.

CHO-K1 ovary hamster DSMZ ACC 110

HELA cervix human DSMZ ACC 57

Vero-B4 kidney African green monkey DSMZ ACC 33

RK 13 kidney rabbit ATCC CCL 37 Mycoplasma tests according to the European Pharmacopoeia (EP) Chapter 2.6.7 require a sensitivity of 10 colony-forming units (CFU) per ml sample volume for NAT-based methods such as PCR in order to replace traditional culture methods. The sensitivity must accomplish 10 CFU/ml as part of the robustness testing for any particular sample matrix. Using vital mycoplasma is not acceptable for the majority of cell culture laboratories due to safety regulations. 10CFU™ sensitivity standards contain non-vital material and allow safe and reliable validation.

The mycoplasma grew in culture medium as described in EP 2.6.7, subsequently titrated and plated on Hayflick and Frey medium for CFU determination. The mycoplasma were harvested in the early logarithmic growth phase to ensure a high ratio of vital to non-vital mycoplasma and thus a low ratio of GU to CFU. All strains were obtained from official culture collections and cultivated in low passages.

In more details, all mycoplasma listed in Table 3 had been cultivated in broth according to EP 2.6.7 until a slight colour change of the phenol red indicator contained in either Frey or Hayflick medium became visible. The culture broth was divided into two portions: One portion was used for quantification of the mycoplasma. The broth was vortexed intensively prior titration to break up mycoplasma clusters. Two tenfold dilution series were prepared in culture broth. Of each dilution step two Hayflick/Frey agar plates were inoculated with 20 μl each, incubated at 37 °C (30 °C for Spiroplasma citri) and checked frequently for colony formation by microscope. Frequent counting was stopped at constant colony numbers and titre calculated as CFU/ml culture broth.

The second portion of the culture broth was heat inactivated (10 min, 95 °C), adjusted to 200 CFU/ml and filled in 50 μl aliquots before lyophilisation. All tubes were stored at 4-8°C until use.


Venor®GeM qEP



Table 7: Mycoplasma Cultivation Media Medium Manufacturer Cat. No. Frey Bouillon acc. EP MerckMillipore 146311 Frey-Agar acc. EP MerckMillipore 146006 Hayflick Bouillon acc. EP MerckMillipore 146452 Hayflick-Agar acc. EP MerckMillipore 146029

For the titration of the mycoplasma spike, the quality of the culture medium is of severe relevance for the subsequent spiking experiments. Each lot of the mycoplasma culture material was quality controlled with EDQM standards. Certificates of Analysis (CoA) were available for each lot.

5.4 Equipment

The following lab equipment was used for the study: Table 8: Lab Equipment Equipment Equipment-ID Manufacturer Brand qPCR cycler R 04 0843, ES72 Corbett Research RotorGene 6000 qPCR cycler 785BR11826, E36 BioRad CFX96 Pipettes for Master Mix setup 0.5-10 μl 10–100 μl 100–1000 μl

M25061D, E3 L10238D, E4 E4O10885D, E5

Eppendorf Eppendorf Eppendorf

Reference 2 Reference 2 Reference 2

Pipettes for DNA/sample handling 10-100 μl 100-1000 μl

L10224D, E13 O10970D, E14

Eppendorf Eppendorf

Reference 2 Reference 2

DNA Extraction Robot 307115.01.0105, L2-32 AJ Innuscreen Innupure C16

PCR Hood n.a. Minerva Biolabs Air filtered, individual, walk-in housing units

n.a.

Centrifuge CTG 015 F, E31 HEMA Medical Instruments

Hema 1424

The following consumables were used: Table 9: Reagents, materials and critical lab ware Article name Cat. No. Manufacturer / Supplier Micro tubes, 1.5 ml/0.5 ml 72.690.001 / 72.699.001 Sarstedt PCR Tubes Multiply - μStrip Pro low profile 72.991.103 Sarstedt 0.5-20 μL Biosphere Filter Tips 2-100 μl Biosphere Filter Tips 101-1000 μL Biosphere Filter Tips

70.1116.210 70.760.212 70.762.211

Sarstedt


Venor®GeM qEP



6 Test Procedure

Based on the results of different proficiency tests (data available from Minerva Biolabs on request) DNA extraction prior testing is strictly required for highest confidence and sensitivity. The design and performance of pre-analytical procedures are part of this study in respect of the intended use but cannot reflect the diversity of the sample material in total. The performance of the kit within the entire analytical process has to be demonstrated by the user. The templates for the PCR analysis are prepared by direct extracting the sample and subsequent PCR analysis.

6.1 Manual DNA Extraction

The Venor®GeM Sample Preparation Kit purifies genomic DNA from different sample matrices including cell culture samples. Mycoplasma lyse by a combination of a detergent and chaotropic salt. The lysate goes directly onto the spin columns. The DNA is selectively bound to the highly specified silica membrane. Two subsequent washes remove residual contaminants, like proteins, metabolites, dyes, detergent etc. Tris buffer elutes the DNA, which is ready-to-use.

The Internal Control DNA of Venor®GeM qEP can be useful to monitor the extraction process. 12 μl of the Internal Control DNA are required and directly added to 200 μl of sample. The sample is vortexed briefly prior extraction. The reaction mix for these samples does not require additional Internal Control DNA.

The isolation of the DNA was carried out according to the instruction manual provided with the kit. In detail:

Transfer 200 μl of sample material into a fresh 1.5 ml reaction tube.

Add 200 μl Conditioner, vortex for at least 10 sec, incubate at 70 °C for 10 min, and equilibrate for 20 min at room temperature.

Optional: add 10 μl Proteinase K per sample if the protein content is >10 mg/ml. Vortex briefly and incubate as described above.

Add 400 μl of binding buffer to the mixture. Vortex immediately and very thoroughly in order to prevent any precipitation of nucleic acids.

Take one spin column per sample from the kit and insert it into a collection tube. Mark the sample identification on the lid of the spin column. Fill the sample lysate into the spin column without moistening the rim of the spin column.

Centrifuge the system for 1 min at 10.000 x g (approx. 10,000 rpm with a bench top centrifuge). Discard the flow through from the collection tube and reassemble the spin column and the collection tube.

Add 500 μl of Buffer A1. Centrifuge the system for 1 min at 10,000 x g (approx. 10,000 rpm with a bench top centrifuge), discard the flow through and re-assemble the spin column.

Fill the spin column with 500 μl Buffer A2. Centrifuge the system for 1 min at 10.000 rpm (10,000 x g), take the spin column out of the collection tube, dump the containing Buffer A2, discard the flow through and re-assemble the spin column.

Centrifuge for 3 min at full speed (approx. 13.200 rpm) in order to remove the remaining Buffer A2.

Discard the collection tube containing the Buffer A2 and place the spin column into a new 1.5 ml reaction tube.

Pipette 60 μl of pre-heated Buffer E (70 °C) into the spin column directly onto the centre of the silica membrane. The complete membrane should get in touch with the Buffer E. Secure the sample storage tube and incubate for 2 min at room temperature.


Venor®GeM qEP



Centrifuge for 2 min at 8,000 x g.

The eluate contains the DNA and can be used directly for PCR or stored at +2 to 8 °C for a week. Long-term storage should be at <-18 °C.

6.2 Analytical procedures

The detection of mycoplasma DNA was carried out according to the up to date version of the instruction manual. In detail:

Rehydration of the Reagents: 1. Centrifuge tubes with lyophilized components (5 sec at maximum speed) 2. Add 390 μl of Rehydration Buffer to the Master Mixes (each) 3. Add appropriate amount of deionized, DNA-free water Positive Control DNA 300 μl Internal Control DNA 300 μl 3. Incubate for 5 minutes at room temperature 4. Vortex and centrifuge again

PCR Master Mix Setup: Total volume per reaction is 25 μl including 10 μl of sample. When setting up reactions, calculations include positive (PC) and negative controls (NC). Pipet master mix on ice into a 1.5 ml reaction tube and mix gently.

Pipetting scheme: for 1 reaction for 25 reactions Master Mix 15.0 μl 375.0 μl Internal Control DNA 1.0 μl 25.0 μl Aliquot 15 μl of master mix into each PCR reaction tube. After pipetting the negative control (10 μl of water or elution buffer of DNA extraction kit), the tube must be sealed before proceeding with the samples. Add 10 μl of sample to each PCR reaction tube. Seal the tubes completely before proceeding with the positive control (10 μl) in order to avoid cross contamination. Programming the qPCR cycler CFX96:

Program Step 1: Pre-incubation Setting Hold Hold Temperature 95°C Hold Time 3 min 0 sec Program Step 2: Amplification Setting Cycling Cycles 45 Denaturation 95 °C for 30 sec Annealing / Detection 55 °C for 30 sec Elongation 60 °C for 45 sec


Venor®GeM qEP



Programming the qPCR cycler Rotorgene 6000 (5-plex):

Cycle Cycle Point Hold @ 95°c, 3 min 0 secs

Cycling (45 repeats) Step 1 @ 95°c, hold 15 secs Step 2 @ 50°c, hold 20 secs Step 3 @ 60°c, hold 30 secs, acquiring to Cycling A (Green and Yellow)

Result Interpretation: The presence of mycoplasma in the sample is indicated by an increasing fluorescence signal in the mycoplasma FAM channel during PCR. Detection of Mollicutes FAM™ channel

Internal control HEX™ channel

Interpretation

positive (Ct < 40) irrelevant Mollicutes positive

negative (no Ct) negative (no Ct) PCR inhibition

negative (no Ct) positive (Ct < 40) Mollicutes negative

borderline (Ct > 40) positive (Ct < 40) result not valid, repeat process including DNA extraction

borderline (Ct > 40) negative (no Ct) PCR inhibition A successfully performed PCR without inhibition is indicated by an increasing fluorescence signal in the internal control channel, provided the Internal Control was added to the master mix or as extraction control to the master mix. Mycoplasma DNA and Internal Control DNA are competitors in PCR. Because of the very low concentration of Internal Control in the PCR mix, the signal strength in this channel is reduced with increasing mycoplasma DNA loads in the sample.

6.3 Calculations

None.

6.4 Reporting requirements

The reports generated by the qPCR machine will be printed in colour. All run information will be printed, including protocol, sample identification, internal amplification control curves and target curves and filed according to the chapter structure of the validation plan. Sample identification should contain information on the species, the contained concentration in CFU/ml or alternatively the type of control (PC for positive control and NTC for No Template Control).


Venor®GeM qEP



7 Study Results

The study conditions have to provide information on all relevant validation parameters requested by ICH Q2B, EP 2.6.7 and EP 2.6.21. As the requirement of the method is to provide a qualitative result only, the parameter linearity, range, accuracy and quantification limit are irrelevant.

7.1 Specificity

7.1.1 Sequence Alignment Procedure Acceptance Criterion Results Comparison of all primer sequences with the genomic database. Mollicutes sequence alignments were performed. Even though EP 2.6.7 for specificity determination does not recommend this technique, it provides additional information for species not available for testing.

Mollicutes species showing ≤ 3 nucleotides mismatch in the alignment of the primer and probe sequence with the 16S rRNA genome are considered specifically detectable.

At least 107 species are putatively detectable based on sequence alignment.

Species; Type Strain Primer Mismatches

Forward Primer Probe Reverse Primer

Acholeplasma equifetale (T); C112. 0 0 2 Acholeplasma granularum (T); BTS-39. 0 0 0 Acholeplasma hippikon (T); C1. 0 0 1 Acholeplasma laidlawii (T); PG8 ATCC 23206. 0 0 0 Acholeplasma oculi (T); 19L ATCC 27350. 0 0 1 Acholeplasma pleciae (T); ATCC 49582; PS-1. 0 0 0

Mycoplasma adleri (T); G145. 1 0 0 Mycoplasma agalactiae (T). 0 1 0 Mycoplasma agassizii (T). 0 0 2 Mycoplasma alkalescens (T); PG51. 0 0 1 Mycoplasma alligatoris (T); A21JP2(T). 0 0 2 Mycoplasma alvi (T); Isley. 0 1 2 Mycoplasma amphoriforme (T); A39. 0 1 2 Mycoplasma anatis (T); 1340(T). 0 0 2 Mycoplasma anseris (T); 1219(T). 0 0 1 Mycoplasma arginini (T); G230(T). 0 0 1 Mycoplasma arthritidis (T). 0 1 1 Mycoplasma auris (T); UIA. 0 0 1 Mycoplasma bovigenitalium (T). 1 1 0 Mycoplasma bovirhinis (T); PG43. 0 0 0 Mycoplasma bovis (T); Donetta (type strain); pMb16S. 0 0 0 Mycoplasma bovoculi (T); M165/69. 0 0 2 Mycoplasma buccale (T); CH20247(T). 0 0 1 Mycoplasma buteonis (T); BbT2g(T). 0 0 1 Mycoplasma californicum (T). 0 0 0 Mycoplasma canadense (T); 275c. 0 0 1 Mycoplasma canis (T); PG14. 0 0 2 Mycoplasma capricolum. 0 1 1


Venor®GeM qEP





Mycoplasma caviae (T); G122(T). 0 0 0 Mycoplasma citelli (T); RG-2C(T). 0 0 0 Mycoplasma cloacale (T); 383(T). 0 0 1 Mycoplasma columbinasale (T); 694(T). 0 0 0 Mycoplasma columbinum (T); MMP-1(T). 0 0 0 Mycoplasma columborale (T); MMP-4(T). 0 0 1 Mycoplasma cricetuli (T); CH(T). 0 1 2 Mycoplasma crocodyli (T); MP145(T). 0 0 2 Mycoplasma cynos (T); H831(T). 0 0 1 Mycoplasma edwardii; PG24. 0 0 2 Mycoplasma elephantis (T); E42(T). 0 0 1 Mycoplasma equigenitalium (T); T37(T). 0 0 1 Mycoplasma equirhinis (T); M432/72(T). 0 0 1 Mycoplasma falconis (T); H/T1(T). 0 0 0 Mycoplasma faucium (T); DC333(T). 0 0 0 Mycoplasma felifaucium (T); ATCC 43428. 1 0 0 Mycoplasma felis (T); ATCC 23391. 1 0 1 Mycoplasma fermentans (T). 0 0 0 Mycoplasma gallinaceum (T); DD. 0 0 0 Mycoplasma gallinarum (T); PG16. 1 0 0 Mycoplasma gallisepticum str. F; 1. 1 1 0 Mycoplasma gallopavonis (T); WR1(T). 0 0 1 Mycoplasma gateae (T); ATCC 23392. 0 0 1 Mycoplasma genitalium (T); G37. 0 1 2 Mycoplasma glycophilum (T); 486(T). 0 0 1 Mycoplasma gypis (T); B1/T1(T). 0 1 1 Mycoplasma hominis (T); PG21; ATCC 23114. 0 0 2 Mycoplasma hyopharyngis (T). 0 0 1 Mycoplasma hyorhinis (T); BTS7(T). 0 0 0 Mycoplasma hyosynoviae (T); S-16. 0 0 1 Mycoplasma iguanae (T); 2327. 0 1 1 Mycoplasma imitans (T); 4229. 0 1 0 Mycoplasma indiense (T); 3T(T). 0 0 1 Mycoplasma iners (T); PG30(T). 0 0 0 Mycoplasma iowae (T). 0 1 1 Mycoplasma lagogenitalium (T); 12MS(T). 0 1 2 Mycoplasma leonicaptivi (T); ATCC 49890. 1 0 1 Mycoplasma leopharyngis (T); ATCC 49889. 1 0 0 Mycoplasma lipofaciens (T); R171(T). 0 0 1 Mycoplasma lipophilum (T). 1 0 0 Mycoplasma maculosum (T); PG15(T). 0 0 0 Mycoplasma meleagridis (T); 17529. 0 0 0 Mycoplasma microti (T); IL371. 0 1 1 Mycoplasma moatsii (T); MK405(T). 0 0 0 Mycoplasma mobile (T). 0 0 1 Mycoplasma molare (T); H542. 0 1 2 Mycoplasma mucosicanis (T); type strain: 1642. 0 0 1 Mycoplasma muris (T). 0 1 2


Venor®GeM qEP





Mycoplasma mustelae (T); MX9(T). 0 0 0 Mycoplasma opalescens (T); MH5408(T). 1 0 0 Mycoplasma orale (T); NC10112; CH 19299; ATCC 23714. 0 0 1 Mycoplasma oxoniensis (T); 128(T). 0 0 0 Mycoplasma penetrans HF-2. 0 1 1 Mycoplasma phocae; CSL 4693. 0 1 2 Mycoplasma phocicerebrale (T); 1049; ATCC 49640. 0 0 2 Mycoplasma phocidae (T); 105; ATCC 33657. 0 0 2 Mycoplasma phocirhinis (T); 852; ATCC 49639. 0 0 0 Mycoplasma pirum (T). 1 1 2 Mycoplasma pneumoniae (T); ATCC 15531. 0 1 2 Mycoplasma primatum (T); HRC292(T). 0 0 0 Mycoplasma pullorum (T); CKK. 0 0 2 Mycoplasma pulmonis (T); PG34(T). 0 0 1 Mycoplasma salivarium (T); PG20(T). 0 0 1 Mycoplasma simbae (T); ATCC 49888. 0 0 1 Mycoplasma spermatophilum (T); AH159(T). 0 0 0 Mycoplasma sphenisci; UCMJ. 1 0 1 Mycoplasma spumans (T); PG13(T). 0 0 1 Mycoplasma sturni (T); UC/MF; p170/171. 0 0 0 Mycoplasma sualvi (T); Mayfield B(T). 0 0 0 Mycoplasma subdolum (T); TB(T). 0 0 1 Mycoplasma synoviae (T); WVU 1853; pMSk3-4 pMSF16S. 0 0 0 Mycoplasma testudineum (T); H3110. 1 0 1 Mycoplasma testudinis (T); ATCC 43263. 0 1 3 Mycoplasma verecundum (T); GIH(T). 0 0 0 Mycoplasma vulturii; Gb-V33. 0 0 3 Mycoplasma zalophidermidis; CSL 4779. 1 0 1

Ureaplasma diversum (T); A417. 1 1 3 Ureaplasma parvum (T); ATCC27815. 1 1 3 Ureaplasma urealyticum (T); ATCC27618. 1 1 3


Venor®GeM qEP



7.1.2 Sample Matrix Cross Reactivity Procedure Acceptance Criterion Results Testing of at least 9 different samples using the media components according to Table 2 and 5 to exclude the possibility of false-positive results. The internal amplification control was added to the sample matrix as extraction control.

All tested samples shall show a negative result.

passed 8 out of 9 samples were tested negative. One sample was inhibiting PCR.

No Sample Matrix Target IC Result

1 anti-human-T-lymphocyte immune globulin, rabbit, solubilized after ammonium sulphate precipitation No Ct 30.86 negative

2 Donor horse serum No Ct 31.20 negative

3 Human serum albumin No Ct No Ct inhibited

4 Calf serum No Ct 34.16 negative

5 Porcine serum No Ct 32.37 negative

6 Human placenta, homogenized No Ct 30,80 negative

7 Dulbecco’s MEM No Ct 30,80 negative

8 PBS Dulbecco No Ct 31.22 negative

9 Hayflick medium No Ct 31.53 negative

10 ENC No Ct 30.91 negative

11 PC 24.31 No Ct positive

12 NTC No Ct No Ct negative Target Internal Amplification Control

The protocol used for completing chapter 7.1.2 did not include a previous proteinase K treatment of the sample. Samples with a protein concentration above 10 mg/ml will clog the membrane of the spin columns provided in the Venor®GeM Sample Preparation Kit leading to an incomplete DNA extraction and purification. The DNA is extractable from serum albumin following this improved protocol (see chapter 7.3).


Venor®GeM qEP



7.1.3 Mollicutes Detection Range Procedure Acceptance Criterion Results All DNA extracts derived from Mollicutes listed in Table 4 were tested at a load of 0.1 ng/test. At least 3 replicates were tested for each sample.

All tested samples shall show a positive result.

passed

No. Species Ct Result 1

Acholeplasma laidlawii

24.98 positive 2 25.25 positive 3 25.08 positive 4 24.85 positive 5 PC 24.52 passed 6 NTC No Ct passed 7 NTC No Ct passed


Mycoplasma fermentans



Mycoplasma hyorhinis



Mycoplasma orale

23.82 positive

2 23.58 positive

3 23.79 positive

4 24.16 positive

5 PC 24.52 passed

6 NTC No Ct passed

7 NTC No Ct passed


Venor®GeM qEP




Mycoplasma pneumoniae



Mycoplasma gallisepticum



Mycoplasma synoviae



Mycoplasma arginini



Mycoplasma arthritidis



Venor®GeM qEP




Mycoplasma genitalium



Mycoplasma hominis



Mycoplasma penetrans



Spiroplasma citri



Ureaplasma urealyticum



Venor®GeM qEP



7.1.4 Cross Reactivity

Procedure Acceptance Criterion Results

All DNA extracts listed in Table 5 and 6 derived from microorganisms and cells were tested at a load of ≥ 0.1 ng/test for microorganisms and ≥ 30 ng for mammalian cells. At least 4 replicates were tested for each sample. The amplification products of positive tested samples were analysed by sequencing.

All tested samples shall show a negative result.

failed 3 out of 28 tested microorganisms/tissues showed cross reactivity: Bacillus cereus Staphylococcus epidermidis Bacillus subtilis

Species Results

Acinetobacter baumanii Negative

Bacillus cereus Positive

Bacillus subtilis Positive (1/4 replicates Ct value< 40)

Bordetella pertussis Negative

Campylobacter jejuni Negative

Clostridium acetobutylicum Negative

Clostridium perfringens Negative

Enterobacter aerogenes Negative

Enterococcus casseliflavus Negative

Enterococcus faecalis Negative

Escherichia coli Negative

Geobacillus stearothermophilus Negative

Lactobacillus acidophilus Negative

Micrococcus luteus Negative

Proteus mirabilis Negative

Pseudomonas aeruginosa Negative

Salmonella enterica Negative

Staphylococcus aureus Negative

Staphylococcus epidermidis Positive

Staphylococcus saprophyticus negative (Ct values > 40)

Streptococcus dysgalactiae Negative

Streptococcus mutans Negative

Streptococcus pneumoniae Negative

Streptococcus sanguinis Negative

CHO-K1 Negative

HELA Negative

Vero-B4 Negative

RK 13 Negative


Venor®GeM qEP



Microbial DNA in Detail

Sample Target IC Result Target IC

Acinetobacter baumanii

No Ct 31.07 negative



No Ct 31.26 negative NTC No Ct 31.46 passed NTC No Ct 31.40 passed PC 24.29 31.61 passed

Bacillus cereus

38.15 31.47 positive



36.05 31.29 positive NTC No Ct 31.22 passed NTC No Ct 31.51 passed PC 24.43 30.86 passed

Bacillus subtilis


43.82 31.35 negative



Bordetella pertussis





Campylobacter jejuni





Clostridium acetobutylicum






Venor®GeM qEP




Clostridium perfringens





Enterobacter aerogenes





Enterococcus casseliflavus





Enterococcus faecalis





Escherichia coli





Geobacillus stearothermophilus






Venor®GeM qEP




Lactobacillus acidophilus





Micrococcus luteus





Proteus mirabilis





Pseudomonas aeruginosa





Salmonella enterica





Staphylococcus aureus






Venor®GeM qEP




Staphylococcus epidermidis




33.16 31.17 positive NTC No Ct 31.21 passed NTC No Ct 32.04 passed PC 24.72 31.22 passed

Staphylococcus saprophyticus




41.09 31.47 negative NTC No Ct 31.46 passed NTC No Ct 31.40 passed PC 24.29 31.61 passed

Streptococcus dysgalactiae





Streptococcus mutans





Streptococcus pneumoniae





Streptococcus sanguinis






Venor®GeM qEP



Evaluating the Bacillus cereus PCR product: Sequence analysis confirmed a significant homology of the 16S rRNA gene of the Bacillus cereus genome with the PCR products. Venor®GeM qEP cross-reacts with Bacillus cereus DNA. Sequence alignment

A detection limit of 0.1 ng DNA/10 μl of sample volume per PCR was determined. Assuming a genome size of 5,411,809bp [12], the detection limit corresponds to 1.7x106 cells per ml of sample. Evaluating the Bacillus subtilis PCR product: Significant homology of the amplified PCR products to the 16S rRNA gene of Bacillus subtilis was confirmed by sequence analysis. Venor®GeM qEP cross-reacts with Bacillus subtilis DNA. Sequence alignment

A detection limit of 1 ng DNA/10 μl of sample volume per PCR was determined. Assuming a genome size of 4,214,814 bp [12], the detection limit corresponds to 2.2x107 cells per ml of sample.


Venor®GeM qEP



Evaluating the Staphylococcus epidermidis PCR product: Significant homology of the amplified PCR products to the 16S rRNA gene of Staphylococcus epidermidis was confirmed by sequence analysis. Venor®GeM qEP cross-reacts with Staphylococcus epidermidis DNA. Sequence alignment

A detection limit of 0.05 ng DNA/10 μl of sample volume per PCR was determined. Assuming a genome size of 2,499,279 bp [12], the detection limit corresponds to 1.8x106 cells per ml of sample.


Venor®GeM qEP



Mammalian DNA


CHO-K1 240 ng/PCR





NTC No Ct 31.46 passed


PC 24.29 31.61 passed

HELA 160 ng/PCR







PC 24.29 31.61 passed

Vero B4 50 ng/PCR







PC 24.29 31.61 passed

RK-13 270 ng/PCR







PC 24.29 31.61 passed


Venor®GeM qEP



7.2 LOD95 Detection Limit

The employed method obtains a qualitative result. Proof of linearity is not required. If however the concept of linearity extends the working range, the detection limit becomes extremely important. In practice, the detection limit is determined in the form of the positive threshold (i.e. the cut-off point in the form of the minimum number of amplified target sequences by volume positively detected in 95% of the sample series).

Procedure Acceptance Criterion Results The Mollicutes preparations according to chapter 5.3 were diluted in 1:10 dilution steps (one deviating dilution step for accurate adjustment of concentration) to prepare a suspension containing 200 CFU/ml. Subsequently dilution levels of 20, 10 and 5 CFU/ml were prepared in DMEM medium containing 10 % (v/v) FBS. Three individual dilution series were prepared at three different days and each dilution were analysed in 8 replicates resulting in 24 data points for each concentration. The results were confirmed testing 8 replicates for each 10CFU standard.

23 of 24 samples containing 10 CFU/ml must be positive for all species. All 8 replicates of the 10CFU Standards must show positive results.

Passed

Acholeplasma laidlawii


Acholeplasma laidlawii 10 CFU/ml





31.5 31.44 positive




NEC No Ct 31.52 passed



CFU/ml 20 10 5

run 1 run 2 run 3 run 1 run 2 run 3 run 1 run 2 run 3

Hit rate 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 7 / 8 5 / 8 7 / 8

Out of 24 24 24 19


Venor®GeM qEP



Mycoplasma pneumoniae


Mycoplasma pneumoniae 10 CFU/ml





35.4 31.46 positive







Mycoplasma fermentans


Mycoplasma fermentans 10 CFU/ml







31.78 31.5 positive





CFU/ml 20 10 5


Hit rate 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 7 / 8 8 / 8 8 / 8

Out of 24 24 24 23

CFU/ml 20 10 5


Hit rate 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 5 / 8 8 / 8

Out of 24 24 24 21


Venor®GeM qEP



Mycoplasma arginini


Mycoplasma arginini 10 CFU/ml












Mycoplasma hyorhinis


Mycoplasma hyorhinis 10 CFU/ml

36,16 30,82 positive








NEC No Ct 30,47 passed

NTC No Ct 30,48 passed

NTC No Ct 30,64 passed

CFU/ml 20 10 5


Hit rate 7 / 8 8 / 8 8 / 8 7 / 8 8 / 8 8 / 8 2 / 8 8 / 8 8 / 8

Out of 24 23 23 18

CFU/ml 20 10 5


Hit rate 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8

Out of 24 24 24 24


Venor®GeM qEP



Mycoplasma orale


Mycoplasma orale 10 CFU/ml












Mycoplasma gallisepticum


Mycoplasma gallisepticum 10 CFU/ml



35.73 31.6 positive



35.62 31.4 positive






CFU/ml 20 10 5


Hit rate 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 7 / 8 7 / 8

Out of 24 24 24 22

CFU/ml 20 10 5


Hit rate 7 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 6 / 8 6 / 8 7 / 8

Out of 24 23 24 19


Venor®GeM qEP



Mycoplasma synoviae


Mycoplasma synoviae 10 CFU/ml








35.94 31.6 positive




Mycoplasma salivarium


Mycoplasma salivarium 10 CFU/ml










NTC No Ct 33.,87 passed


CFU/ml 20 10 5


Hit rate 8 / 8 8 / 8 8 / 8 7 / 8 8 / 8 8 / 8 0 / 8 5 / 8 8 / 8

Out of 24 24 23 16

CFU/ml 20 10 5


Hit rate 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 7 / 8 5 / 8 8 / 8

Out of 24 24 24 20


Venor®GeM qEP



Spiroplasma citri


Spiroplasma citri 10 CFU/ml








35.5 31.37 positive




CFU/ml 20 10 5


Hit rate 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 8 / 8 7 / 8

Out of 24 24 24 23


Venor®GeM qEP



7.3 Robustness

The robustness testing of Venor®GeM qEP Mycoplasma requires the most relevant mycoplasma species from a risk-based point of view. Mycoplasma orale is such a relevant spike for robustness testing due to its high prevalence in cell cultures used in academic and biopharmaceutical industrial applications.

7.3.1 Matrix Effects Procedure Acceptance Criterion Results To demonstrate robustness, at least 20 mycoplasma negative samples (selected at random from samples submitted by customers for Mycoplasma detection) spiked with 10 CFU/ml of Mycoplasma orale should be tested. The internal amplification control was added to the sample matrix as extraction control. All samples were tested in duplicate.

All samples should be found positive.

passed PCR inhibiting samples were tested positive using the extraction protocol including proteinase K treatment.

Sample batch 1 − prepared according to protocol without proteinase K treatment of the samples

No Sample Code / Characteristics

Replicate 1 Replicate 2 Result

Target IC Target IC 1 Cell culture supernatant 36.31 33.42 35.01 33.55 positive

2 CHO 35.07 34.43 35.21 34.2 positive

3 ISF in HAT medium 35.05 34.11 35.68 34.28 positive

4 RPMI/10%FCS 35.04 32.62 35.37 32.73 positive

5 DMEM/10% FCS 34.6 32.65 35.02 33.05 positive

6 Interstitial fluid 34.34 33.72 35.09 34.06 positive


8 Tissue homogenate 34.66 32.95 33.88 33.06 positive

9 Cell extract 36.15 33.35 35.11 33.82 positive

10 Immune globulin No Ct No Ct No Ct No Ct inhibited

11 Extraction control No Ct 32.77 No Ct 32.69 negative

12 NTC No Ct No Ct No Ct No Ct negative

13 Positive control 24.06 No Ct 23.62 No Ct positive Target Internal Amplification Control


Venor®GeM qEP



Sample batch 2 − prepared according to protocol without proteinase K treatment of the samples



Target IC Target IC 1 Cryo cells 35.03 34.89 35.15 34.72 positive

2 Cell extract 35.04 36.01 35.40 36.15 positive

3 Cell precipitate 35.43 37.09 35.60 36.33 positive

4 Ammonium sulphate precipitate proteins

No Ct No Ct No Ct No Ct inhibited

5 ISF cells 34.36 34.36 34.04 34.98 positive


7 Cell culture supernatant 36.08 34.41 35.22 34.09 positive

8 Cell suspension 34.74 35.09 34.07 36.50 positive

9 Foetal calf serum No Ct No Ct No Ct No Ct inhibited


11 extraction control No Ct 33.04 No Ct 35.66 negative

12 NTC No Ct No Ct No Ct No Ct negative

13 Positive control 23.82 No Ct 23.68 No Ct positive Target Internal Amplification Control

The protocol used for extraction of sample batches 1 und 2 did not include a previous proteinase K treatment of the sample. High concentrated protein preparations clog the spin columns provided in the Venor®GeM Sample Preparation Kit leading to an incomplete DNA extraction and purification. Treatment with proteinase K during the lysis step results in successful DNA extraction from the inhibiting samples ammonium sulfate precipitate protein, immune globulin and foetal calf serum.


Venor®GeM qEP



Sample batch 3 − prepared according to protocol including a proteinase K treatment of the samples



Target IC Target IC 1 ISF cells in HAT medium 33.78 30.23 34.68 30.19 positive


3 Ammonium sulphate precipitate proteins

34.13 32.03 34.08 31.72 positive

4 Foetal calf serum 33.04 33.00 32.98 32.65 positive

5 Tissue homogenate 32.71 32.05 33.04 32.16 positive

6 immune globulin 34.19 33.30 33.98 33.25 positive

7 immune globulin 34.40 33.06 34.40 33.09 positive

8 extraction control No Ct 32.44 No Ct 33.21 negative

9 NTC No Ct No Ct No Ct No Ct negative Target Internal Amplification Control


Venor®GeM qEP



7.3.2 Device compatibility Procedure Acceptance Criterion Results As the test can basically be performed with any qPCR cycler capable of interpreting FAM and HEX signals performance of the test on these machines needs to be validated. As not all qPCR cycler commercially available are accessible for validation an air heating system was tested: Rotor-Gene 6000. To demonstrate device compatibility, DMEM medium containing 10 % (v/v) FBS were spiked with Mycoplasma pneumoniae, Mycoplasma fermentans and Acholeplasma laidlawii using 10CFUTM Standards.

All samples show a positive result.

Rotorgene 6000 passed

No Species Concentration Target IC Result

1

M. pneumoniae 10 CFU/ml


2 28,26 29,14 positive

3 28,61 29,39 positive

4 27,99 29,28 positive

5

A. laidlawii 10 CFU/ml


6 33,28 28,77 positive

7 32,63 29,21 positive

8 35,41 29,38 positive

9

M. fermentans 10 CFU/ml


10 32,55 29,13 positive

11 31,40 29,15 positive

12 31,86 29,44 positive

13 NEC n.a. No Ct 29,11 passed

14 PC n.a.

21,72 29,36 passed

15 21,67 28,92 passed

16

NTC n.a.

No Ct 29,32 passed

17 No Ct 29,61 passed




Venor®GeM qEP



8 Conclusion

The validation of Venor®GeM qEP Mycoplasma Detection Kit complied in full to the authorized validation protocol. The validation protocol reflects the method itself and variations expected by the diversity of samples from different customers during QC testing in the manufacturing process of biologicals. The report shows all relevant data, but can be subject to amendments of significant findings and customer submissions at any time.

Venor®GeM qEP was found as state-of-the-art product for mycoplasma detection. This product is featuring all criteria of the mycoplasma testing guidelines according to European Pharmacopoeia 2.6.7 as well as Japanese Pharmacopoeia, 17th edition, chapter G3. The test provides confident results with any kind of sample material occurring in the manufacturing process of biologicals during bulk harvest testing or final lot release, like cell culture media und supplements, cryo stocks, cell culture supernatants, cell suspensions, or antibody formulations.

The test system detects all the listed Mollicutes species, but many more show up positive as well. At least 107 different Acholeplasma, Mycoplasma, and Ureaplasma species are detectable based on the sequence alignment. This feature increases the chance to detect mycoplasmas, which are rare contaminants of cell cultures, or not described as contaminants so far or are not cultivable by using the traditional mycoplasma testing method.

Specificity testing showed that the kit detects Staphylococcus epidermidis with a detection limit of approximately 1.8x106 cells per ml of sample, which corresponds to 0.05 ng DNA/10 μl sample volume. The detection limit for Bacillus cereus, a species genetically very closely related to Mycoplasma but not addressed by the EP 2.6.7 for specificity testing, is approximately 1.7x106 cells per ml (0.1 ng DNA/10 μl of sample volume per PCR). The detection limit for Bacillus subtilis is as high as 2.2x107 cells per ml (1 ng DNA/10 μl of sample volume per PCR). The determined detection limits are easily visually detectable by naked eye as strong turbidity for all three species. Such titres have a significant impact on biopharmaceutical processes leading instantly to the correct interpretation of the test results. Venor®GeM qEP does not detect any DNA of eukaryotic origin even at concentrations higher than 30 ng/PCR.

Robustness is a key issue in evaluating the characteristics of a release test. Different authentic pharmaceutically relevant samples were analysed with Venor®GeM qEP. The species Mycoplasma orale was used for spiking, the most prominent Mycoplasma species in means of contamination from the environment. It showed the same sensitivity as Mycoplasma synoviae, Spiroplasma citri, Mycoplasma arginini, Mycoplasma gallisepticum, Mycoplasma salivarium and Mycoplasma pneumoniae. Mycoplasma orale was easily detectable at the required concentration of 10 CFU/ml even in complex sample matrices and multiple replicates. We postulate that Venor®GeM qEP detects all mycoplasma species listed in the EP 2.6.7 and in Japanese Pharmacopoeia, 17th edition, chapter G3 with same efficiency in biopharmaceutical samples.

Different cycler allow the application of the kit with same robustness. This study shows compliant results for the air-heated qPCR cycler RotorGene 6000 (former Corbett, now QIAGEN) and the block-heated qPCR cycler CFX96 (BioRad). Other brands show equal results (data not shown) assuming, that the brand of the qPCR cycler has no impact on the performance of the kit.

Additionally, the test system provides an Internal Amplification Control to monitor the entire process including DNA extraction by spiking the sample directly. The Internal Amplification Control DNA monitors the PCR reaction and the DNA extraction process with highest accuracy in typically incurring sample matrices. These results show the robustness of the Venor®GeM qEP Sample Preparation Kit for the preparation of different kind of sample material. It is noteworthy that pure serum samples require an additional proteinase K digestion.

We demonstrated that the product Venor®GeM qEP fully complies with the requirements of EP 2.6.7 [3] and with the Japanese Pharmacopoeia [6]. The system provides additional protocols, which fulfil even higher limits in respect of process control and sensitivity for the detection of Mycoplasma in any kind of sample material occurring in the manufacturing process of biologicals, like cell culture media und supplements, cryo stocks, cell culture supernatants, cell suspensions, antibody formulations, for bulk harvest testing or final lot release.


Venor®GeM qEP



9 Reference Documents

1. DIRECTIVE 2004/23/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 31 March 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells

2. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use (ICH). “Validation of Analytical Procedures: Methodology” Q2B. November 1996

3. European Pharmacopoeia 7th edition. Strasbourg, FR; European Directorate for the Quality of Medicines; 2010. 2.6.7 Mycoplasmas

4. European Pharmacopoeia 7th edition. Strasbourg, FR; European Directorate for the Quality of Medicines; 2010; 2.6.21 Nucleic Acid Amplification Techniques

5. US Pharmacopoeial Convention (USP). USP 33/NF 28 <63> Mycoplasma tests. Rockville, MD; 2010

6. Japanese Pharmacopoeia. 15th ed. Tokyo. JP: Ministry of Health. Labour and Welfare; 2006. Mycoplasma Testing for Cell Substrates used for the Production of Biotechnological/Biological Products.

7. United States Pharmacopoeia <1223> 32nd ed. Rockville. MD: The United States. Validation of Alternative Methods.

8. European Pharmacopoeia 7th edition. Strasbourg, FR; European Directorate for the Quality of Medicines; 2010. 5.1.6. Alternative Methods for Control of Microbial Quality

9. Haruo Suzuki, Tristan Lefébure, Paulina P Bitar and Michael J Stanhope; Comparative genomic analysis of the genus Staphylococcus including Staphylococcus aureus and its newly described sister species Staphylococcus simiae. BMC Genomics 2012. 13:38

10. Ribosomal RNA Operon Copy Number Database: http://rrndb.mmg.msu.edu/search.php

11. Parkhill J.; public database AL513382; Project: PRJNA236; Salmonella enterica subsp. enterica serovar Typhi str. CT18. complete complete genome

12. Zhang YQ. Ren SX. Li HL. Wang YX. Fu G. Yang J. Qin ZQ. Miao YG. Wang WY. Chen RS. Shen Y. Chen Z. Yuan ZH. Zhao GP. Qu D. Danchin A. Wen YM; http://www.straininfo.net/publications/77712;jsessionid=527857D35B14C8565319EBB4421B7E54; Mol Microbiol 49(6). 1577-1593. 2003

13. TIGR CMR