Join BioTether Sciences for an engaging webinar discussing strategies and techniques

 to study the pharmacokinetics, bio-distribution, pharmacodynamics, immunogenicity, and the potency

of cell, gene, and protein therapeutics.

https://attendee.gotowebinar.com/recording/7058460459861473536

Primary human dermal fibroblasts are extremely helpful to support research and development of drugs and devices. Material obtained from the skin biopsy can be utilized in several investigative procedures such as examination of cellular morphology, immunofluorescence staining of biochemical markers,  enzymatic studies, and molecular biology. A fibroblast is a specific type of connective tissue cell that is found in skin. In genetics research, cultured fibroblasts are particularly important because they can be isolated and grown from a small biopsy from forearm skin cells. The cells can be examined for genotypes and grown and tested for disease associated phenotypes. Human dermal fibroblasts are useful to study extracellular matrix production and wound healing. They can also be used as model systems to study many aspects of cell physiology, reprogramming, and induced pluripotency. Among the various uses of a skin biopsy, the most frequent is the confirmation and testing for clinical diagnoses. More recently primary human skin fibroblasts are used to study CAS9-CRISPR mediated gene editing and knock-out. Gene therapies using lentivirus, AAV, and nanoparticles can be delivered to primary human fibroblasts.

In the specific area of inborn errors of metabolism, cell culture from skin biopsies are used for enzymatic measurements to provide a diagnosis. For example, primary fibroblasts are used for the definitive diagnosis and study of Mucopolysaccharidoses. A hallmark of these lysosomal storage diseases is the accumulation of glycosaminoglycans and deficiencies in the enzymes that process them. Many other inherited metabolic disorders can be studied using primary dermal fibroblasts.  Similarly, other genetic diseases affecting the skin, muscle, connective tissue, bone, and immune system may be diagnosed and studied using skin fibroblasts. Disorders such as Parkinson’s, cancer, heart disease may be studied using primary fibroblasts or cells derived from them.  

One exciting aspect of working with these cells is the ability to dedifferentiation fibroblasts to an artificial stem cell type by the induction of pluripotent cells (iPSCs). This can be accomplished by the forced expression of certain factors. The iPSCs can be then directed toward other cell lineages. The multipotent plasticity of these cells and the  ability to isolate them from a consenting individual patient for autologous treatment opens many avenues for research and therapeutic development.

Primary human dermal fibroblasts may be collected with little discomfort by taking a small (1-8mm) forearm skin biopsy punch. The aseptically collected tissue is then transported in cell culture media containing serum and antibiotics by overnight courier. Once in our laboratory, BioTether Sciences can culture, study, and preserve these cells. We have the cell culture and bioanalytical expertise to support your program and advance the research and development of your therapeutic (www.BioTether.com).

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

By Erik Foehr

BioTether Sciences

Last year saw the approval and marketing of the most expensive drug ever created. Zolgensma, by Novartis, is a one time gene therapy for the rare condition, spinal muscular atrophy. The therapy costs $2.125 million and may be used to treat children around 2 years of age stricken with the disease. Other new drugs with large price tags also added to the growing class of cell and gene therapies that are ‘The Most Expensivest’ drugs ever developed. Spark Therapeutics gene therapy drug for a rare form of blindness, is called Luxturna, and is priced at $425,000 per eye.  A few CAR-T therapies have been approved for cancer over the last couple years,  including Novartis Kymriah for non-Hodgkin Lymphoma.  CAR-T therapies are complex and cost nearly $500,000 for the treatment. 2020 is likely to see several more cell and gene therapy approvals ignite costs like a pile of cash on fire. Million dollar drugs may become commonplace someday.

Cell and Gene Therapy drugs have enormous potential to treat and even cure disease. For instance, Zolgensma, was shown to keep some of the treated children free of the devastating neuromuscular disease years after the therapy was administered. CAR-T therapies have demonstrated astonishing successes curing people of deadly cancers. The FDA projected that 10-20 gene and cell therapies will be approved per year by 2025. That would indicate 3-5 approvals this year, 5-10 next year and so on until cell and gene therapies become a significant part of the biopharmaceutical ecosystem. Hundreds of genetic diseases are being targeted for gene therapy treatment by small biotech start-ups to large multinational biopharmaceutical companies.

How will cell and gene therapies impact the biopharmaceutical and healthcare ecosystems? On the one hand fortunes will be made on the new discoveries, break-throughs, and mega-mergers. Intractable diseases, many rare genetic diseases, with no other treatment option may be ameliorated or even cured. This new class of therapy could improve and save many lives and may even reduce the long term cost burden on the healthcare system. But no one has a clear answer for how to pay for these new million dollar therapies. Cell and gene therapy drugs are being developed for rare diseases, with thousands, hundreds, or even a few dozen treatable patients in the USA. But some new cell and gene therapies may be used on a larger population of patients. Will insurance companies be able to absorb the costs of these million dollar drugs? Can the system find a solution to a low probability, but high impact claims?

These drugs are also extremely expensive to develop, manufacture, and test. CAR-T therapies require highly specialized manufacturing and quality testing systems. Similarly, gene therapies have shown great potential, but have significant challenges relating to vector delivery, immunogenicity, and other safety concerns. Cell and gene therapies are only a small fraction of the overall drug development spend with small molecule and biotherapeutics accounting for over 95% of development dollars. But this new class of drug is going to grow rapidly and will require significant expenditures on in-house and outsourced activities. The outsourced analytical chemistry services spend alone will reach into the hundreds of millions in 2020 and beyond. But that may just be the price to pay for the best, most effective, and expensivest drugs ever developed.