Mycoplasma are an unusual type of bacteria that can infect animal, insect, and plant cells. They can be a very costly pest for biopharmaceutical researchers and manufacturers. These little organisms (0.2-2 micron) are the smallest and simplest free-living parasite. They can impact the safety and quality of cell-derived biopharmaceutical products. This puts patients at risk and could cause untimely and costly delays to manufacturing. To minimize these risks, routine testing for mycoplasma should be performed throughout the product research, development and manufacturing process.

There are several ways to test for mycoplasma.  These include direct culture, Hoechst DNA staining, PCR-based testing, and  hybridization-amplification by ELISA. For the production of biopharmaceuticals, direct culture-based approaches assess the presence of viable cells using indicator cell lines exposed to mycoplasma. However, this type of testing requires weeks and costs approximately $5000 per sample. An alternative approach to identifying mycoplasma contamination is through PCR-based or hybridization ELISA testing. The molecular-based PCR method or the hybridization-ELISA method is ideal for research laboratories. These methods are rapid, highly sensitive, specific, reliable, and cost-effective. The PCR-based method and hybridization method are designed to amplify or bind to the conserved 16S rRNA region of the mycoplasma genome. Eight species of mycoplasma are responsible for approximately 95% of cell culture contamination events and are detected using these techniques. There are numerous PCR based protocols (for example see ATCC.org kit offering) that use primers recognizing the 16S rRNA and can detect mycoplasma contamination by measuring a distinct PCR product. For the hybridization technique, (i.e. MycoProbe, Mycoplasma detection kit by R&D Systems) samples are hybridized in a microplate containing biotin labeled capture oligonucleotide probes and digoxigenin-labeled detection probes that map to the 16S rRNA sequence. The hybridization product is detected with anti-digoxigenin alkaline phosphatase.  PCR and hybridization techniques have been demonstrated to be extremely sensitive, comparable to direct culture, are rapid, and cost significantly less than direct culture (approximately $500 per sample).

Enhanced regulated testing with an overlay of quality assurance systems to comply with GMP requirements adds to the cost. For GMP lot release, Europe and the USA-FDA have established guidances to follow. For research and development, PCR and hybridization-ELISA techniques are ideal for routine, rapid screening. Cell lines can be tested every few months or screened immediately after receipt or before/after cryopreservation, therefore, allowing cell lines and biological products to be used after a short quarantine. BioTether Sciences performs both PCR based, and hybridization-ELISA based mycoplasma testing to support your research and development. This service is included free with cell based assay and stable cell line development projects placed at BioTether Sciences. We can also provide rapid turn-around and reporting on client supplied samples (cell line supernatant, cell pellets, reagents). Don’t let those little parasites derail your biopharmaceutical program!

Reference: Drexler HG, Uphoff CC. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology 39: 75-90, 2002. PMCID: PMC3463982

BioTether Sciences uses state-of-the-art instruments and technologies to support biomedical research and development.  Our new MesoScale Discovery QuickPlex instrument is sensitive, versatile, and robust. It is designed to support immunogenicity testing, pharmacokinetic analysis, and biomarker studies. The QuickPlex uses electrochemiluminescence assays (ECLA) to detect analytes at extremely low concentrations and across a broad dynamic range.

Electrochemiluminescent labels generate light when stimulated by electricity in the appropriate chemical environment. This reaction is incorporated into our immunoassays to provide the light signal used to measure important proteins and other biomedical molecules. This instrument platform is ideal for measuring antibodies in serum, biotherapeutic concentration in biological fluids and for the study of biomarkers.  BioTether Sciences recently transferred classic ELISA immunogenicity test methods onto the QuickPlex instrument. Assay sensitivity was improved, and the test is faster, easier, and uses less sample.  The MSD ECLA system can be used to study all areas of biomedical research, with hundreds of kits and antibodies available. (https://www.mesoscale.com/en/products_and_services/services/find_a_cro/biotether_sciences)

We can support your studies in the areas of oncology, cardiology, neurobiology, reproductive biology, and immunology. One great feature of the MSD platform is the use a cell friendly carbon electrode plate surface that allows immobilization of whole cells or membranes. Cell based assays to screen antibodies to cell surface antigens, receptor-ligand interactions, and functional antibody neutralization are just a few applications that could be developed. Many biomarker kits are available to quantitate cytokines, metabolites, intracellular signaling, growth factors and more. BioTether Sciences is skilled at developing novel methods, assay validation, and testing. We can create custom immunogenicity, biomarker, and pharmacokinetic assays to support your drug or device development program. We love to talk about the science and help design the best solution to advance your program. You provide the biotherapeutic or device material, we can do the rest. Antibody production and procurement, method development and troubleshooting, full GMP or GLP validation, clinical testing---we will be there for you!

The wonders of gene therapy using technologies to deliver or modify genes to treat inherited diseases, cancer, and other disorders is just becoming a reality. So far only a few such therapies have been approved. For example, Spark Therapeutics, Luxturna (voretigene neparvovec-rzyl) for an inherited form of blindness, and Zolgensma for spinal muscular atrophy are the only approved gene therapies in the USA for inherited genetic diseases. Many more of these life changing therapies are navigating clinical trials and some will be approved in the coming months and years. But many challenges confront gene therapy. The viral vectors used to deliver the gene therapy elicit an immune response or are met with an experienced immune system ready to stop the viral vectors before they can deliver the payload.

Gene therapies rely on adeno-associated virus (AAV), adeno virus, lentivirus, Herpes Virus, or another modified viral vector. In the case of AAV, approximately 70% of adults have antibodies and memory T cells that can bind and clear the vector. The viral capsid proteins are displayed by antigen presenting cells and rally an adaptive immune response.

Manufacturing high quality viral vectors requires specialized services. The gene therapy vectors should be assessed by immuno-assay, viral titer, infectious titer, host cell protein contaminants and stability. High quality gene therapies need to be well characterized and free of host proteins, degradation products, impurities or other manufacturing related liabilities that increase the risk of an immune response.  BioTether Sciences team has experience working with viral vectors and assessing immunogenicity risk.

Antibodies to AAV and other viral vectors can be detected by pre-screening clinical trial subjects and monitoring them during therapy Pre-existing antibodies or newly formed antibodies can be measured using an ELISA. Screening patients for viral antibodies may be helpful to design an effective clinical trial and improve safety. Tracking immunogenicity of gene therapy vectors during clinical trials and correlating the antibody response to efficacy and safety is important for a successful, FDA compliant, and safe clinical trial.