Bio outsource mycoplasma_testing
- 1. Testing for Mycoplasma species using Polymerase Chain Reaction Techniques All regulatory guidelines specify that products manufactured using cells must be tested for the presence of Mycoplasma species. This includes all biologics and some vaccines. The testing is prescribed for master and working cell banks, virus seed lot and bulk harvests. Traditionally the tests for Mycoplasma have included cell culture or growth on media (broth and agar) however nucleic acid amplification techniques (NAT) may be used as an alternative to one or both of the other methods after suitable validation by certain guidelines. The reason for the acceptance of the NAT techniques by the European Pharmacopoeia (EP) is that they have recognised that some samples are difficult to test for Mycoplasma either due to cytotoxicity or due to the rapid turnaround of samples required for particular products. Polymerase Chain Reaction principal advantage is the sensitivity and specificity of these assays. The main disadvantage is the detection of SECTION 2.6.21 of the European Pharmacopoeia states non viable Mycoplasma sequences and these sequences that Nucleic acid amplification, such as PCR, techniques may be present in many of the reagents and culture may be used for detection of mycoplasmas. NAT indicate material used in the production process. This is because, the presence of a particular nucleic acid sequence and whilst the manufacturing of the reagents will have not necessarily the presence of viable mycoplasma. This eliminated the viable organisms, some DNA will remain presents some advantages and disadvantages; the due to it being very difficult to remove. A number of different techniques are available which can be applied to detect this presence and the EP does not specify a particular method but does outline the validation which is expected. The EP also indicates that commercial PCR detection kits will be suitable for use for release assays and specifies that certain elements of the validation may be carried out by the manufacturer to facilitate use of the assay commercially. The PCR technique may be used instead of the culture method and the indicator cell culture method after suitable validation as a release test, thus saving time and cost. BioOutsource Service Offering BioOutsource is pleased to offer the Applied Biosystems PrepSEQ™ Mycoplasma detection kit for use in detecting Mycoplasma to EP requirements. The PrepSEQ kit was
- 2. chosen as it has been shown to be compliant with the manufacturer and the specificity with contaminating validation criteria as specified by the EP Guidelines. The DNA has also been established. Using numerous kits technique starts with the extraction of DNA from the and samples, BioOutsource has also demonstrated the sample. This is a critical step and uses a lysis buffer as in robustness of the assay. standard DNA extraction techniques to release the DNA from the cells. This method can be applied to up to 10mL of sample, which is particularly useful for testing larger Assay Controls Included in volume samples such as bulk harvests. Following release, the Assay the DNA is captured on magnetic beads which reduces The assay includes an inhibition control which verifies the the cross reactivity and inhibitors in the assay. The PCR absence of inhibition to the PCR reaction. This control is technique is based on the TaqMan™ reaction conditions a DNA sequence which is unrelated to the Mycoplasma and also uses the SYBR Green which binds to double sequences detected and sample acceptance criteria stranded DNA. The temperature annealing profile can be specifies that this must be within an acceptable range for scrutinised should there be any positive reactions to verify the sample result to be valid. if the reactive sample is truly a mycoplasma contamination. BioOutsource assays offer, to all of our clients, our specific In our hands this assay has been shown to be suitable to Sample Extraction Control which is used to verify the detect Mycoplasma species in cell free samples as well as extraction efficiency of every extraction. We would typically cell harvest and cell bank preparation. expect to achieve < 70% of DNA recovered from the The speed at which the samples can be extracted and extraction control. assayed is particularly useful. This can be accomplished External controls. within 48 hours of sample receipt. This allows The positive control used in the assay is a specifically BioOutsource to offer a rapid turnaround to result in a designed plasmid which is tested in reaction buffer and matter of days rather than weeks for the traditional assays. is diluted to contain a defined number of target-sequence copies of 101 to 108. The negative control used is Validation of ABI MicroSEQ composed of buffer containing no Mycoplasma Mycoplasma Detection Assay target sequences. The EP specifies a large number of Mycoplasma species which should have validation data acquired before Replacing Existing Methods use. The Presept kit has shown by the manufacturer with PCR to achieve a sensitivity (<10 CFU/mL) of the assay To directly replace the traditional methods for Mycoplasma with A. laidlawii; M. fermentans; M. hyorhinis ; M. detection with PCR would require firstly a compatibility orale; M. pneumoniae ; M. gallisepticum; M. synoviae ; study and then a comparability study to be carried out. Mycoplasma arginini and Spiroplasma citri to comply with The EP suggests that this study should include mainly the EP guidelines and in our hands the assay performs. a comparison of the respective detection limits of the The Mycoplasma species tested in the validation have alternative method and traditional methods. The ABI been sourced from the EDQM where available. The assay Presept system has already been shown to be sensitive has also been validated to demonstrate the specificity of and specific to meet EP guidelines and therefore a the assay and has included include Escherichia, Bacillus, relatively small well designed study would be required Clostridium perfringens and sporogenes, Lactobacillus, to satisfy the guidelines. This study will include the Staphylococcus and Streptococcus. simultaneous testing of samples using both methods In our hands the assay has been qualified to achieve showing the detection limit of both methods using the same level of sensitivity as that specified by the samples of calibrated strains (EDQM). BioOutsource Ltd • Units 3/4 Technology Terrace • Todd Campus • West of Scotland Science Park • Glasgow • G20 0XA tel: +44 (0)141 946 4222 • email: info@biooutsource.com • web: www.biooutsource.com