Cell-Based Therapies
Rubhu Biologics staff has expertise in the development and validation of bioanalytical assays to evaluate cell-based therapy products. One of the most critical factors in developing new cellular therapy and biologics products today is ensuring that bioanalytical assays are accurate, robust, and reproducible. The US Food and Drug Administration (FDA), United States Pharmacopeia (USP), the International Conference on Harmonization (ICH), and European Medicines Agency (EMA) have each recognized the importance of this to the biologics development process and have separately increased qualification and validation requirements in recent years.
in vitro Bioassays
Bioanalytical potency assays are operationally challenging due to their dependence on a biological substrate (e.g. animals, living cells, or functional complexes of target receptors). Because of multiple factors arising from these dependencies, they typically exhibit greater variability than do chemically-based tests. These are often cell-based assays, requiring special attention to cell banking and maintenance.
Rubhu Biologics has successfully developed and validated numerous potency assays for biologics as well as turned the art of cell culture into a creative and productive scientific strategy with primary and immortal cell lines.
Biologics are very complex as well as they have multiple modes of action, most of the biopharma companies are challenged with developing
assays that are biologically relevant for the analysis of these molecules. The ability of the assay to characterize and demonstrate biological activity is essential. Additionally, immunoassays also referred to as ligand-binding or activity-binding assays, are needed throughout the bioprocess development and production. Validation of these assays requires a meticulous assessment of accuracy, precision, range of quantification, dilutional linearity, parallelism, specificity and selectivity, ruggedness and robustness of the method, as well as potential matrix effect, sample, and solution stability, and more. Over the years Rubhu Biologics staff has acquired expertise in these bioanalytical tools and techniques and are continually evolving with scientific and regulatory experience in their applications.
Regulatory Assay Development Validation and Stability Testing: Our staff has experience in assay development, validation, and stability testing for biologics which have led to successful IND filings and product marketing applications in the US. Rapid screening assays have been developed using different technologies, which enhance accelerated stability studies.
Immunogenicity Studies
Immunogenicity studies to evaluate cell-based therapy products .
Unwanted immunogenicity is a significant issue affecting most biotherapeutic products. Anti-Drug Antibodies (ADA), also known as anti-therapeutic antibodies (ATA) can be associated with adverse reactions and can impact clinical efficacy and PK/PD profile. Neutralizing antibodies can bind to the active part of the biomolecule thereby blocking the therapeutic effect of the biomolecule, and may inhibit the activity of corresponding endogenous factors. There is a clear need to be able to detect these potential culprits early on and testing for its presence became a regulatory requirement. Prediction, minimizing, and assessment of immunogenicity are challenges facing the biotechnology industry.
The industry standard, regulatory acceptable testing strategy for ADA is step-wise: screening for total ADA, confirmation, and neutralizing activity testing. The screening step is a cut point assay set up to allow 5% false positives. The confirmation step, in which the binding of ADA is competed out by the addition of an excess of free drug, confirms the presence of specific antibodies and allows them to focus on true positives. These are usually tittered and evaluated for potential neutralizing activity using biological activity assays, which provide a functional biological system to assess if the antibodies detected by the immunoassay have the neutralizing capability. The common practice is to adapt the drug’s release potency assay for this purpose, but binding and even cell-free assays can be used when cell-based potency assays cannot be adapted for this purpose.
There is a critical need to understand the sensitivity and specificity of ADA assays. Sensitivity refers to the ability of an assay to detect very small concentrations of antibodies in a test sample.
A highly sensitive assay is more likely to detect low levels of specific antibodies and therefore has a low probability of producing false-negative results. Sensitivity is affected by many factors (a type of drug, the affinity of antibody, serum, methodology, technology, drug interference). Specificity refers to the ability of an assay to detect only those antibodies directed against the specific target protein. The specificity of each assay is a product of the biochemical mechanics of antibody binding to its ligand and the subsequent detection methodology used. A highly specific assay has a very low probability of measuring false positives resulting from non-specific binding.
One of the major problems to address when designing an ADA assay for a biologic is free drug interference. Biologics have long half-lives, resulting in high concentrations in circulation for a relatively long time. The ADAs form complexes with the free drug, thereby hindering detection.
To overcome this problem an appropriate study design is of paramount importance, Rubhu Biologics foresee it’s involvement early on in study designs and development of these assays may be in pre-clinical stages of drug development. Not less cardinal is the use of the best-fitted methodologies and technological platforms in assay design. Rubhu Biologics scientists have developed and validated immunoassays to evaluate immunogenicity and immunotoxicity of several biologics using technologies such as surface plasmon resonance (SPR), ELISA, RIA, immunoprecipitation, ELISPOT, flow cytometry-based assays (surface staining, intracellular cytokine/chemokine/nuclear proteins, apoptosis, calcium mobilization, telomere length analysis, cell division analysis using membrane labeled dyes), multiplexed bead array assays to detect cytokines/chemokines/kinases. In addition, we have several years of experience in futuristic assays to evaluate immune responses such as evaluation of T cell diversity using TCR vβ analysis and analysis of recent thymic emigrant T cells using TREC assay.
Immunohistochemistry Studies
- Tier I (Masson’s Trichrome, Iron, Periodic Acid Schiff, Gram, Alcian Blue, Luxol Fast Blue, Sudan Black, Oil Red O)
- Tier II (Acid-Fast-Bacteria, Verhoeff Van Gieson, Congo Red, Sirius Red, Steiner)
- Tier III – Ag Stains (Silver, PAMS, JMS, Bielchowski, Retic)
More information to come.
Operation Site
111 Riverbend Road
Athens, GA 30602