Cell-Based Assays and Cellular Assays: These assays not only measure the quantitative effects of drugs but are also crucial for evaluating DNA/RNA/protein/small molecules/nanoparticles in biology assays. Our cell-based assays are strategically designed based on various factors such as disease states, proliferation, and cytotoxicity. They play a crucial role in cell-based assays in drug discovery, focusing on signaling pathways, gene expression, among other endpoints. MarinBio is set to assist you in your discovery, potency development through IND/BLA applications as well as PK studies.
Explore our deep-rooted expertise in GMP analytical assays and GLP PK assays by reviewing this groundbreaking research on the development of GIGA-2050, a recombinant hyperimmune globulin for SARS-CoV-2 treatment.
Whether you’ve already established a cell-based assay or cellular assays for transfer to MarinBio, or are looking to develop a customized cell-based bioassay, our extensive experience in cell-based assay screening and biology assays over 25 years enables us to provide highly efficient, rapid, and cost-effective solutions for the successful development, qualification, and/or validation of the assays you need. MarinBio also performs bioassays for lot release.
We are experts in the art and science of cell culture. Scroll down for more information on some of the different assays MarinBio has successfully designed and executed in client contracts.
SHSY5Y cells are differentiated into neural cells after seven days’ treatment with retinoic acid. The cells were treated with or without client drug for one additional day. Left Panel: control (no drug); Right Panel: with client drug. The observed morphological changes were a result of the drug treatment.
CTLL-2 cells were cultured with increasing concentrations of client drug for four days. Cell proliferation was measured with CellTiter-Glo (Promega, Wisconsin, USA). Application of the client drug led to cell proliferation with the two standards showing precise and identical results.
Cell-based assays (bioassays) measure functional effects of drugs to establish their biological relevance and physiological importance. MarinBio’s contract research and development service professionals are expert in designing customized or standardized bioassays and in the measurements of interactions of drugs (including small molecules, biologics, or device interactions) with cultured cells and primary cells. MarinBio’s Ph.D. scientists have successfully interfaced with the FDA on behalf of our clients, when requested.
Additionally, MarinBio specializes in in vitro cell-based assay testing services for clinical and pre-clinical studies, including method development, qualification, validation, transfer, lot-release testing, and potency testing according to guidelines from both FDA and International Council for Harmonization (ICH). MarinBio has extensive experience with cells and cell banks at both non-GMP/‑GLP and GMP/GLP levels of many tissue cell types and clones, in addition to custom development of cells, clones, or sub-clones. Our expertise has enabled us to produce over 200 different bioassays for our clients during the past 25 years. Some of the assays we perform are listed below (click on highlighted terms to link to our Glossary):
Potency bioassays, lot-release bioassays
A potency assay used for lot release is a GMP assay that measures the activity of a drug in response units per drug concentration. Typically, the assay is a cell-based bioassay in which increased concentrations of drug induce a sigmoid shaped response curve (stimulation or inhibition) of a biological event. The EC50 (or IC50) determination defines the half maximal effective (or inhibitory) concentration of the drug, and it is used to compare samples of unknown potency to reference standards tested simultaneously.
Clinical studies, pre-clinical studies, sample analyses
ADCC, antibody-dependent cellular cytotoxicity bioassays
Killing of a target cell by immune cells when directed by a specific antibody to the target. A positive aspect if the antibody is designed to kill cancer cells. A negative aspect if the antibody is designed for other functions. Measured by radioactive 51Cr release from the target or by fluorometric methods.
CDC, complement-dependent cytotoxicity bioassays
Complement Dependent Cytotoxicity (CDC) is an effector function of IgG and IgM antibodies. When they are bound to surface antigen of a tumor or other cell, complement protein in blood are triggered, resulting in target cell lysis. This is a desired characteristic for a cancer therapeutic, and undesirable for other antibody drug.
CTL, cytotoxic T lymphocyte killing bioassays
A key activity of cellular immunity in rejection of transplants, reactions to pathogens such as viruses and tumors, is the development of T lymphocytes that specifically kill target cells. These activated cells develop during in vivo exposure or by in vitro sensitization. The CTL assay consists of increasing number of sensitized lymphocytes cultured with a fixed number of tumor or other target cells that have been prelabeled with 51Cr or fluorometric methods. When these target cells are incubated with sensitized lymphocytes, the target cells are killed and the 51Cr or fluorescent compound is released into the supernatant and detected.
ADA, anti-drug antibody inhibiting drug effectiveness (AKA neutralization) bioassays
A drug may elicit an immune response in recipients. The ELISA is the typical method for detecting antibodies that bind to a drug. The Neutralizing Antibody Assay (NAb) measures whether the antibody blocks the activity of the drug. The assay usually is a bioassay in which the drug induces a biological response in a cell or serum sample and the antibody is tested for inhibition of that response.
Flow cytometry measures the physical and chemical characteristics of a population of cells or particles. A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam and the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so that light is first absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined, and the data gathered are processed by a computer.
MLR, mixed lymphocyte reaction bioassays
Mixed Lymphocyte Response (MLR) is a test used by to show the safety of a drug or implantable material. It is commonly used as part of the FDA clearance process. Blood lymphocytes of one or two donors are cultured or co-cultured. If there is a detectable mismatch, the lymphocytes will proliferate and demonstrate activation markers and new cytokine synthesis.
Cell enzyme bioassays
Marin Biologic is experienced in many types of assays, including those employing proteases, dehydrogenases, oxidases, phosphorylase, nucleic acid polymerase and modifying enzymes, and glycolytic enzyme assays. Colorimetric, fluorescent and radiometric formats are used. Substrate specificity, ED50, ID50, substrate and inhibitor kinetics, temperature dependence and sensitivity are factors in selecting an assay. Assays for glucose, glutamine, lactic acid, endotoxin, etc. are also performed.
Cell proliferation bioassays
For most studies, cell growth is measured by a homogeneous, vital dye method in which one of several choices of dye is added to cells in a 96 well plate at the conclusion of the study or at time point intervals and read directly in a plate reader. The dye is enzymatically changed in healthy cells so that development of color or fluorescence is measured using a different wavelength than the unaltered dye. The effects of adding a growth factor, an inhibitor, or a cytotoxic factor to cells is easily read. This procedure has very few steps, has minimal manipulation of cells, and allows for good reproducibility. CellTiterGlo® from Promega, is a very efficient fluorescent compound we use for these assays. Alternatively, uptake of 3H-thymidine is used specifically to assay DNA synthesis, or as a more sensitive assay of cell proliferation for slow growing cells.
Cell toxicity, apoptosis, and necrosis bioassays
Cell death occurs by lysis, necrosis, or apoptosis. Lysis is the destruction of the cell surface membrane such as by the action of an antibody and complement that makes holes in the membrane. Necrosis occurs through the action of toxic factors that act within the cell, such as irreversible inhibitors of protein, RNA or DNA synthesis, or mitotic poisons. Apoptosis is a programed cell death used by the body to remove damaged or unwanted cells and occurs during cytotoxic T cell killing and with some cancer chemotherapies. Apoptosis is characterized by early events such as expression of phosphotidylserine on the cell surface and fragmentation of the DNA, followed by loss of membrane integrity and mitochondrial function. The quantitative percentage of dying cells is determined microscopically or by flow cytometry using vital stains or DNA-binding dyes. High throughput measurement of cell death is performed by release of a label from cells prelabeled with a radiotracer, typically 51Cr, or a fluorescent or color marker. Alternatively, the fluorescent or colorimetric dye method described above is used.
Cell transfection and transient or stable gene transcription and protein expression bioassays
Transfection is the process of introducing nucleic acids (genes) into eukaryotic cells. The result may be to overexpress certain proteins, to correct a genetic defect, or to create a reporter cell that conveniently fluoresces when triggered by an agonist.
Drug combination bioassays
Drug delivery bioassays
Drugs may be presented to biological systems by themselves, as prodrugs that have to be metabolized to a form more readily taken up or in liposomes that facilitate transport across lipid membranes. Drugs may also be formulated in degradable polymers or other slow-release systems or attached to carriers to facilitate transport such as nanoparticles, to decrease clearance or to maintain circulating concentrations. Drug capture by a cell surface receptor is measured by localization (subcellular fractionation), or by its action of triggering a biological reaction (cell signaling, reporter gene assays, proliferation, cell death, metabolic assays). The concentration of drug in its initial formulation, and of free drug, internalized drug, and subsequently metabolized or degraded drug can be measured.
Drug metabolism bioassays
Drug subcellular fractionation and localization bioassays
Drug target pathway and phosphorylation bioassays
Gene expression, the activation of a specific gene to produce a messenger RNA that directs the synthesis of protein. Other gene expressions include RNA molecules from non-protein coding genes such as transfer RNA (tRNA) or small nuclear RNA (snRNA) genes. Gene expression can be studied by Real-time Quantitative Polymerase Chain Reaction (RT-qPCR).
Gene therapy corrects or modifies and existing gene or introduces a new gene to treat or prevent diseases.
Mechanism of action bioassays
Receptor, protein binding, and turnover bioassays
Exosomes are biological, vesicular nanostructures that transport information and bioactivity within and between cells. Exosomes can be isolated and their content studied.
Migration and wound healing bioassays
When you require an assay to prove your science and there is no commercial kit available, you need a custom assay. MarinBio’s extensive expertise enables rapid and methodical development of your assay for eventual validation. Typically, a custom assay can be developed from the inception to a completed bioassay in 5 or less experiments, while minimizing any effects of the matrix.
MarinBio expert at preparing and validating GMP/GLP or research level cell banks. The banks could include primary cells or cell lines for specific bioassays, as well as cell bank uses for production, isolation and characterization of genetically defined clonal cell populations and small-scale manufacturing of biologic products.
MarinBio professionals have vast experience with improving cell culture growth, freezing, serum-free culture, cloning for client bioassays, as well as transfection with DNA or RNA and/or liposomes (lipofection) for genetically variant cell lines as well as for gene or protein expression bioassays.
Our scientists have developed over 200 custom bioassays involving growth, toxicity, transfection, cell surface, cytokine or gene expression. We are your scientists; MarinBio will make your GMP potency lot release assay or drug screening assay.
Many companies planning an IND submission or in Phase I/II may only require a well-qualified assay, rather than a fully validated assay. You may or may not need GLP/GMP stringency and auditability. The expert MarinBio scientific team works with your scientists to qualify your assay for reproducibility, appropriate precision and accuracy, with validation in mind for the future.
To validate an assay, the precision, linearity, accuracy, robustness, system suitability and repeatability is measured to meet acceptance criteria. We in-source your assay or fully develop and validate an assay to meet the FDA GLP or GMP requirements.
When patient samples or drug product samples need analysis, MarinBio will map out a timeline and follow through with sample receipts, meeting your regulatory needs at all points. We perform your assay as a developed, qualified or validated bioassay, and promptly communicate the results and/or provide a Certificate of Testing for any or all of your pre-clinical or clinical phases.
Knowing the importance to the FDA of lot release potency testing data, our professional scientific team tests every manufactured lot with your assay and provides Certificates of Testing.
In order to determine shelf-life for a particular drug substance, stability tests are performed at pre-determined calendar intervals. MarinBio’s expert project coordinators communicate closely with you to anticipate the arrival of samples in order to be prepared for timely analysis and the submittal of data and Certificates of Testing.
The complexity of successful cell-based assay and bioanalysis demands advanced bioassay skills, cell culture expertise and knowledge of the bioassay parameters, as well as the FDA regulatory standards. The MarinBio senior Ph.D. scientific staff averages 15-25 or more years of professional experience in designing and developing cell-based assays, as well as interfacing on behalf of our clients with the FDA, when requested. We have developed and perfected a comprehensive, rapid, cost effective approach for successful cell-based assays, including client customized bioassays that are technically demanding and intricate.