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

COVID is a global health crisis, the virus infecting hundreds of millions and killing hundreds of thousands worldwide. The vaccines developed in rapid order may save millions of lives and are a wonder of modern science. But in rare instances the vaccines may cause safety issues due to undesirable responses to polyethylene glycol (PEG) and AdenoAssociated Virus (AAV). These immune responses to the vaccine nanoparticles or viral vectors could have implications beyond treating COVID and may prevent safe and efficacious delivery of life saving biotherapeutics and gene therapies. Are we immunizing hundreds of millions of people against PEGylated drugs and AAV gene therapies? 

Both the Moderna and Pfizer COVID vaccines contain PEG in their formulations to create stable nanoparticles containing viral mRNA. PEG is a common additive in food, cosmetics and pharmaceuticals. PEG, when conjugated to protein therapeutics enhances pharmaceutical kinetics, pharmacodynamics and can reduce immunogenicity. However, people can and do create anti-PEG antibodies. According to a publication cited below, there is a 22 – 25% occurrence of anti-PEG in healthy blood donors, compared with a very low 0.2% occurrence two decades earlier. This increase may be due to an improvement of the limit of detection of antibodies during the years and to greater exposure to PEG and PEG-containing compounds. These results raise obvious concerns regarding the efficacy of PEG-conjugated drugs for a subset of patients. (https://www.tandfonline.com/doi/full/10.1517/17425247.2012.720969). Anti-PEG antibodies can cause skin rashes and more severe allergic responses, including anaphylaxis. Anti-PEG antibodies may cause rapid clearance, poor uptake and decreased efficacy of PEGylated biotherapeutics. There are over a dozen PEGylated biotherapeutics on the market for many diseases (see below and cited above). Will a significant number of people getting the COVID vaccine develop anti-PEG antibodies? Will COVID vaccination status need to be considered when prescribing PEGylated drugs?

What about the impact of COVID vaccination on gene therapy drugs? Two adenoviral vector COVID-19 vaccines are now being used to protect people from COVID. The first, Ad5-nCoV from CanSino, uses a human adenovirus 5 (AAV5) vector; the second, AZD1222, licensed by AstraZeneca from University of Oxford, uses a chimpanzee adenovirus vector. At least seven adenovirus vector vaccines are in development for COVID-19. The AAV5 serotype, is used for gene therapy, and has double-digit pre-existing antibody prevalences that presents challenges to effective treatment.  AAVs are used for the few approved gene therapies and many more in development. Gene therapy may be the future of medicine and save millions with rare genetic diseases. But now AAVs are being leveraged to strongly activate both B and T cells against COVID-19 (https://www.biocentury.com/article/305392/aav-covid-19-vaccine-aims-for-the-sweet-spot-between-antibody-and-t-cell-immunity). Will gene therapy developers need to find ways to avoid these antibodies and T-cells once hundreds of millions of people have been immunized against COVID? Pre-existing antibodies to certain AAV serotypes are common and sometimes neutralizing antibodies develop. These neutralizing antibodies can be tested in functional bioassays. Clever ways to engineer AAVs or reduce the immune response to the gene therapy are being explored. This may be increasingly important once millions of people are immunized with AAV-SARS-CoV-2.

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 recent outbreak of Coronavirus in Wuhan China and subsequent spread across the globe is an example of how a viral infection can  cause a deadly systemic inflammatory response (SIRS).  SIRS is a serious medical condition caused by an overwhelming immune response to infection. Immune chemicals released into the blood to combat the infection trigger widespread inflammation, which leads to blood clots and leaky vessels. This results in impaired blood flow, which damages the body’s organs by depriving them of nutrients and oxygen. In severe cases, one or more organs fail. In the worst cases, blood pressure drops, the heart weakens and the patient spirals toward septic shock. Once this happens, multiple organs—lungs, kidneys, liver—may quickly fail and the patient can die (adapted from NIGMS website).

Human coronaviruses (hCoVs) can be divided into low pathogenic and highly pathogenic coronaviruses. The low pathogenic CoVs infect the upper respiratory tract and cause mild, cold-like respiratory illness. In contrast, highly pathogenic hCoVs such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) predominantly infect lower airways and cause fatal pneumonia and systemic inflammatory syndrome (SIRS). SARS and MERS had death rates of approximately 10%-35%. The mortality rate of the Wuhan Coronavirus may be approaching similar levels. Epidemiologists, researchers, clinicians, and governments are racing to find ways to contain and treat the virus. Severe pneumonia caused by pathogenic hCoVs is often associated with rapid virus replication, massive inflammatory cell infiltration and elevated pro-inflammatory cytokine/chemokine responses resulting in acute lung injury, and acute respiratory distress syndrome.  Anti-viral drugs that may reduce the replication of the virus are being evaluated. Treatments to reduce the out-of-control immune response may also be effective.

BioTether Sciences studies and develops plasmapheresis devices to selectively remove inflammatory cytokines and other targeted proteins from the blood. This affinity capture technique short circuits the damaging immune response, returns to body to homeostasis and therefore increases survival from SIRS/septic shock.

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.