INTRODUCTION / BACKGROUND

The BioFire® Mycoplasma Panel is a closed and fully-automated “lab in a pouch” system that provides highly sensitive and broad detection of Mollicutes with less than five minutes of hands-on time. The disposable reagent pouch integrates nucleic acid purification and nested multiplex real-time PCR with less than one hour of run time and requires minimal training and skill to operate.

14 different PCR assays are included in every run to cover the wide genetic diversity of the Mollicutes class, including species of the genera Acholeplasma, Entomoplasma, Mesoplasma, Mycoplasma, Phytoplasma, Spiroplasma, and Ureaplasma. Three internal controls ensure the reliability of results by monitoring all aspects of pouch function, from sample extraction through amplicon detection.

Designed to meet compendial testing standards, Limit of Detection (LoD) studies have been performed with a particular focus on species listed by the United States, European, and Japanese pharmacopeias using 0.2 mL and 10 mL sample volumes. The LoD for 10 mL samples is ≤ 10 CFU/mL for all mycoplasmas tested and only requires a few simple pre-processing steps by centrifugation. Direct testing of 0.2 mL samples provides detection of ≤ 33 CFU/mL and is intended to facilitate more frequent surveillance of raw materials, cell banks, early product intermediates, and biologics with limited volume availability such as cell therapies.

BIOFIRE® FILMARRAY® SYSTEM

The FilmArray is a ‘lab-in-a-pouch’ medium-scale fluid manipulation system, performed in a self-contained, disposable, thin-film plastic pouch. The BioFire FilmArray platform processes a single sample, from nucleic acid purification to result, in a fully automated manner.

Testing requires minimal pre-processing of sample. The sample is loaded into the BioFire Mycoplasma Panel using a filter-injection vial. The user enters the sample and pouch type (using a barcode reader) into the software and initiates a run.

The BioFire Mycoplasma Panel has a fitment containing all necessary freeze-dried reagents, with plungers that push liquids into the film portion of the pouch. This portion consists of stations for cell lysis (blister C), magnetic-bead based nucleic acid purification (D & E), reverse transcription and first-stage multiplex PCR (F & G), and an array of 102 second-stage nested PCR reactions (I).

PCR primers are dried into the wells of the array and each primer set amplifies a unique product of the first-stage multiplex PCR. The second-stage PCR product is detected in a melting analysis using a fluorescent double-stranded DNA binding dye, LCGreen® Plus.

BioFire Defense, LLC is a cGMP-compliant manufacturing facility, and the BioFire Mycoplasma Detection system is designed to meet 21 CFR Part 11 requirements. BioFire Defense, a bioMérieux company.

Figure 1. BioFire FilmArray Instrument and FilmArray Pouch Diagram

SAMPLE PRE-PROCESSING METHODSTwo sample testing methods are being evaluated in BioFire analytical method qualification studies. Direct testing of 0.2 mL samples provides improved ease-of-use for screening raw materials and product intermediates, or for testing products with limited volume availability. Test volumes of 10 mL were also evaluated for improved sensitivity and only require a few simple pre-processing steps by centrifugation.

RESULTS

Organism StocksHighly viable Mollicutes reference stocks that display a low ratio of genome copies to colony forming units were sourced from the American Type Culture Collection (ATCC) and European Directorate for the Quality of Medicines (EDQM) for BioFire validation studies (Table 1).

Table 1. Mollicutes Organism Stocks - Genome Copies Compared to Colony Forming Units (CFU)

Mollicute Species Source / Cat # Ratio of Genome Copies a to CFU

Mycoplasma orale ATCC / 23714-TTR 0.75

Mycoplasma salivarium ATCC / 23064-TTR 1.0

Mycoplasma hyorhinis ATTC / 17981-TTR 1.4

Mycoplasma arginini ATCC / 23838-TTR 2.4

Mycoplasma synoviae EDQM / Y0000689 2.9

Mycoplasma pneumoniae ATCC / 15531-TTR 8.4

Mycoplasma fermentans ATCC / 19989-TTR 8.6

Acholeplasma laidlawii ATCC / 23206-TTR 13

Mycoplasma gallisepticum ATCC / 19610-TTR 21

Spiroplasma citri b ATCC / 27556 230

a Genome Copies were quantified by the supplier using PicoGreen® for all stocks except M. synoviae and S. citri, which were quantified in-house using commercially available qPCR kits specifically designed for each species.b S. citri stock titer was provided in color changing units (CCU).

Limit of Detection (LoD) - 10 mL and 0.2 mL Sample VolumesLoD was determined for 10 compendial mycoplasmas in Phosphate Buffered Saline (PBS) and for 3 species in Chinese Hamster Ovary (CHO) cell suspension (5.0E+06 CHO cells per mL) using 10 mL and 0.2 mL sample volumes (Table 2 and Table 3, respectively).

Table 2. Limit of Detection (LOD) – 10 mL Sample Volume

Mollicute Species

Limit of Detection – 10 mL(CFU/mL)

PBS CHO(5.0E+06 cells/mL)

M. orale 0.1 Not Tested

M. pneumoniae 1 0.3

M. fermentans 3 Not Tested

M. gallisepticum 3 Not Tested

M. arginini 3 Not Tested

A. laidlawii 3 3

M. hyorhinis 3 Not Tested

M. synoviae 3 Not Tested

M. salivarium 10 3

S. citri* 0.01 CCU/mL Not Tested

*S. citri stock was titered in color changing units (CCU) per mL.

Table 3. Limit of Detection (LoD) – 0.2 mL Sample Volume

Mollicute Species

Limit of Detection – 0.2 mL(CFU/mL)

PBS CHO (5.0E+06 cells/mL)

M. orale 1 Not Tested

M. pneumoniae 10 10

M. gallisepticum 10 Not Tested

A. laidlawii 33 10

M. arginini 33 Not Tested

M. fermentans 33 Not Tested

M. hyorhinis 33 Not Tested

M. salivarium 33 33

M. synoviae 33 Not Tested

S. citri* 0.1 CCU/mL Not Tested

*S. citri stock was titered in color changing units (CCU) per mL.

The LoD values for the compendial mycoplasmas were less than 10 CFU/mL for 9 mycoplasmas, and 10 CFU/mL for M. salivarium. Therefore, M. salivarium was selected for LoD evaluation in CHO cells as a ‘worst case’ scenario as it displayed the highest LoD in PBS. Mycoplasma detection sensitivity in CHO cells was equivalent or better than in PBS alone. Note: A study of comparability to the broth/agar and cell indicator methods will also be executed.

RESULTS CONTINUED

InclusivityThe BioFire Mycoplasma Panel is designed to cover the broad genetic diversity of the Mollicutes class, including species of the genera:

• Acholeplasma

• Entomoplasma

• Mesoplasma

• Mycoplasma

• Phytoplasma

• Spiroplasma

• Ureaplasma

Table 4. Detection of Additional Mycoplasma Species at 10 CFU/mL

OrganismTest

Concentration (CFU/mL)

Sample Volume Detection Rate

M. bovis 10 10 mL 3/3

M. hominis 10 0.2 mL 3/3

M. penetrans 10 0.2 mL 3/3

ExclusivityOrganisms from the Bacilli and Clostridia class, closely related to the Mollicutes class, were selected for evaluation.

The organisms that were evaluated, the test results, and the concentrations (10% stock) are shown in Table 5.

Table 5. Summary of FilmArray Mycoplasma Panel Exclusivity Evaluation - 0.2 mL

Organism Class OrganismTest

Concentration (CFU/mL)

Mycoplasma Test Result

Bacilli

Bacillus cereus 9.2E+05 Not Detected

Bacillus thuringiensis 3.9E+06 Not Detected

Lactobacillus plantarum 1.8E+08 Not Detected

Streptococcus pyogenes 9.0E+07 Not Detected

Clostridia Clostridium perfringens 1.2E+08 Not Detected

Evaluation of Interference A range of test substances were evaluated for possible interference by assessing unspiked substances (blank) and detection of 5 representative mycoplasmas at 3xLoD using 0.2 mL direct testing.

Mollicute species spiked in potentially interfering substances:

• A. laidlawii • M. fermentans • M. orale • M. pneumoniae • S. citri

All species were detected at 3xLoD in the substances listed below (Table 6). No interference with internal controls was observed.

Table 6. Substances Evaluated by 0.2 mL Direct Testing

Culture Media and Serum Antimicrobials Cell Lines Disinfectants

DMEM Gentamicin CHO Ethanol

Fetal Bovine Serum Penicillin SP2/0 Isopropanol

Horse Serum Neomycin SF9 Phenolic acid

FRIIS Broth Polymyxin B HEK293

Tryptic Soy Broth Streptomycin Vero

Grace’s Insect Medium Ciprofloxacin Enzymes Other

Cryoprotectants Plasmocin Trypsin EDTA E. coli

DMSO Amphotericin B Collagenase S. cerevisiae

Glycerol Thermolysin

CONCLUSIONS

The BioFire Mycoplasma Detection System can facilitate screening at various stages of the manufacturing process for low-level mycoplasma contamination in a wide range of possible sample types. With less than 5 minutes of hands-on time and a fully automated test system, more frequent testing near the site of manufacture will be possible to improve surveillance for potential mycoplasma contamination events.

Key Features of the BioFire Mycoplasma SystemEasy: Less than five minutes of hands-on time; one test panel per sample.

Fast: Instrument run time in under 1 hour.

Broad detection: Covers genetic diversity across the Mollicutes class.

Sensitive: Detection of compendial mycoplasmas at ≤10 CFU/mL.

Closed System: Automated sample extraction, nested multiplex PCR, results reporting, and reduced contamination risk.

Sample Volume Flexibility: Simple sample pre-processing allows sample volumes ranging from 0.2 mL to 10 mL, or even higher.

Room temperature stable: Store reagents at 18-30°C.

Compact instrument: 25.4 x 39.3 x 16.5 cm (10 x 15.5 x 6.5 in).

Software: 21 CFR 11 compliant.

ACKNOWLEDGMENTS

• Members of the Research and Development team at BioFire Defense, LLC critically involved in the development the BioFire Mycoplasma Panel, including: Angela Baird, Lydia Hall, Brian Jones, Jeff Nicholes, Mark Poritz, and Jesse Shelley.

• Lex Border, Brett Smith, and many others at BioFire Defense, LLC for their support.

• Lori Daane, Sandra Gay, and many others at bioMérieux for project support.

• Johanna Dimino and the R&D Raw Materials-Antigen group at bioMérieux, Marcy, France for providing CHO cell suspensions.

• bioMérieux group for funding this effort.

• BioFire Diagnostics, LLC for software development support.

Performance Evaluation of a Rapid, Fully Automated Mycoplasma Detection System WILLIAM E. BARRY 1, CORIKE TOXOPEUS 1, JESSICA BROWN 1, LINDSEY KORNOWSKE 1, CYNTHIA D. ANDJELIC 1, SYLVANIE CASSARD 2, FÉLIX A MONTERO JULIAN 2, MARIANNE KIM 1, CYNTHIA L. PHILLIPS 1

1 BIOFIRE DEFENSE, LLC, SALT LAKE CITY, UTAH, USA | 2 BIOMÉRIEUX INDUSTRY HEALTHCARE BUSINESS, MARCY L ’ETOILE, FRANCE

2019 PDA Cell & Gene Therapy Conference – Long Beach, CA

Pouch Process

RNA Process Control

DNA Process Control

PCR2 Control

Lysis –

Nucleic Acid Extraction –

Reverse Transcription – –

PCR1 (Multiplex) –

PCR2 (In Array)

Amplicon Detection

The BioFire Mycoplasma Panel has Internal Controls to verify proper function.

HYDRATION SOLUTIONSAMPLE MIXBA

C

D

E

F

G

H I

A. Fitment with freeze-dried reagents

B. Plungers - deliver reagents to blisters

C. Sample lysis and bead collection

D. Wash

E. Magnetic bead collection blister

F. ElutionG. Multiplex Outer PCR blisterH. Dilution blisterI. Inner Nested PCR array

Figure 2. Sample Pre-Processing Methods Evaluated in Analytical Qualification Studies