Cellbased assays
VelaLabs offers analytical test methods to assess the in-vitro activity of biopharmaceuticals.
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Cell based assays
Biological activity is a critical quality attribute of biopharmaceutical products to ensure safety, efficacy and quality. Due to the complexity and heterogeneous functionalities of biopharmaceuticals, characterization of biological activity often requires analyte-specific assay(s).
Immunogenicity is an essential part of clinical studies related to Safety and Efficacy. The inhibitory potential of identified neutralizing anti-drug antibodies (NAbs) identified in patient samples is evaluated using cell-based assays. The analytical procedure is based on in-vitro functional assays that measure biological activity.
Our cell-based assays team offers state-of-the-art in vitro assays which are custom-designed for your analyte and aligned with your needs. Our scientific team supports you throughout the entire product development lifecycle - from early research to market release.
We support you in following stages of the analytical lifecycle:
- Analytical Method Development
- Method Optimization
- Method Qualification
- Method Validation
- Routine Use and On-going Monitoring
- Change Management
- Comparability
- Analytical Method Transfer
Potency and Biological Activity Assays
Potency testing
Potency or biological activity is an essential part of the characterization profile of a product. ICH Topic Q 6 B defines potency as "quantitative measure of biological activity based on the attribute of the product which is linked to the relevant biological properties", which is linked to the mode-of-action of the product. Depending on the product, one or multiple biological properties are relevant for characterization. Each assay is specifically designed for each product and its biological property. An established procedure to assess biological activity are cell culture-based biological assays. Single properties might be characterized with ligand and receptor binding assays.
A relevant, validated potency is required for drug substance and/or drug product
Detection of neutralizing antibodies (NAbs)
Therapeutic proteins, due to their larger size compared to small molecular drugs, have a greater potential for immune response. Unwanted immune responses can lead to serious side effects such as anaphylaxis. Another potential consequence is the loss of efficacy of the therapeutic protein due to neutralizing antibodies. Therefore, regulatory agencies require testing for anti-drug antibodies (ADA). The recommended testing strategy includes several steps. After detection of ADAs in screening and confirmation assays, testing for neutralizing antibodies (NAbs), should be conducted.
For testing of neutralizing antibodies (NAbs), a cell-based assay is recommended as it most closely mimics the in vivo mechanism. The assay is based on the principal that NAbs in the sample lead to a reduction in biological activity. Each assay is specifically designed for each product and its biological property. As a basis, an already established potency assay for the product can be used.
In the development of therapeutic antibodies targeting immune checkpoints, such as PD-1, preservation of the target cell population is a functional requirement. Pembrolizumab therapeutics, unlike IgG1 mAbs, utilize an IgG4 isotype to minimize the recruitment of the innate immune system. The negative assays provide the rigorous analytical validation confirming the lack of effector function. ADCC: Antibody –Dependent Cellular Cytotoxicity is evaluated in a reporter gene assay and mediated by CD16 - expressing cells. CDC: Complement-Dependent Cytotoxicity is evaluating cytotoxicity which is a result of a membrane attack complex (MAC) formation in the presence of exogenous complement (human serum).
Complement dependent cytotoxicity (CDC)
Complement dependent cytotoxicity is a primary mechanism of action (MoA) for many therapeutic monoclonal antibodies (mAbs). It is a part of the innate and adaptive immune system and has shown to be vital in cancer therapy. When specific antibodies bind an antigen on the cell membrane, the antigen-antibody complex activates the complement cascade leading to a "membrane attack complex" (MAC), resulting in lysis of the target cell. A CDC potency bioassay quantitatively measures the ability of a drug product to trigger this classical complement pathway.
In-vitro, CDC activity is initiated by the addition of human complement and antibody to target cells. The cytotoxic activity can be measured via cell viability dyes (ATP-based such as CellTiter-Glo) or fluorescent (such as Alamar Blue) or via membrane Integrity (LDH release)or via staining of dead cells and flow cytometry readout.
Antibody dependent cellular cytotoxicity (ADCC)
Antibody dependent cellular cytotoxicity is part of the innate and adaptive immune system and is mediated by immune cells. Specific antibodies bind to antigens represented on the cell membrane. The antigen-antibody complex is recognized by immune cells like natural killer cells (NK cells) or Peripheral blood mononuclear cells (PBMCs) via the Fc receptor (CD16 or FcγRIII). The release of IFN-γ and cytotoxic molecules such as perforin and granzymes lead to the lysis of the target cell.
In-vitro, ADCC activity is initiated by using target cells, representing the antigen, and effector cells (i.e. immune cells). Effector cells can be (i) NK cells or PBMCs isolated from human blood, (ii) an immortalized NK cell line or (iii) a genetically-modified reporter gene cell line. For (i) and (ii) ADCC can be measured via cell viability dyes or the staining of dead cells and flow cytometry readout. For (iii) ADCC can be measured via luminescence signal. The advantage of using genetically modified target and effector cells over PBMCS is ensuring standardization and reproducibility. While PBMCs which are derived from human donors introduce donor-to-donor variability, the reporter cell lines are clonal, stable and standardized, ensuring consistent performance across measurements, accuracy and precision. In addition, reporter cells are available in a thaw-and-use format allowing better schedule flexibility and faster set ups.
Antibody-dependent cellular phagocytosis (ADCP)
Antibody-dependent cellular phagocytosis is part of the innate immune system and provides a link to the adaptive immune system. ADCP is a mechanism of detection, internalization and degradation of target cells, which present non-self molecules. It also plays a major role in clearance of virus and virus infected cells. Phagocytes, like Macrophages, bind to Fc part of specific antibodies, opsonizing the cells presenting non-self molecules. Subsequently, the target cells are internalized and lysed.
In-vitro, ADCP is initiated by using target cells, representing the antigen, and effector cells (i.e. phagocytic cells). Effector cells can be (i) monocytes isolated from PBMCs and differentiated into macrophages or (ii) a genetically-modified reporter gene cell line. For (i) ADCP can be measured via flow cytometry readout. For (ii) ADCP can be measured via luminescence signal. Usage of genetically modified target and effector cells over PBMCS is ensuring standardization, reproducibility accuracy and precision.
Metabolic activity assay
Metabolic activity assay is a functional, cell-based bioassay designed to quantify the biological effectiveness of a drug by measuring its impact on a specific cellular biochemical process. Unlike a binding assay, metabolic assay also provides a quantitative measure of the drug´s ability to trigger downstream processes that occur subsequently to the drug-receptor binding. Common metabolic parameters that can be measured are: Nutrient uptake of glucose, amino acids or lipids into the cell, enzymatic conversion: how fast a certain substrate is processed by intracellular enzyme, secondary messengers such as changes in levels of molecules like cAMP or calcium and cellular respiration that evaluates intracellular ATP levels .
One example of a metabolic assay performed in - house is an Insulin dependent Glucose uptake in differentiated adipocytes. This assay measures the functional uptake of glucose across the cell membrane, in response to insulin agonists. For this assay, a differentiation protocol is applied to differentiate murine 3T3 MBX1 cells into adipocytes. Differentiated adipocytes serve as a good in vitro cell model for Glucose uptake assay. Cells are stimulated with dose range of insulin. Upon Insulin binding to its Insulin cognate receptors, intracellular signaling cascades such as the PI3K/Akt pathway are activated in adipocytes. These pathways result in Glut4 translocation from intracellular compartments to the cell membrane and a resulting Glucose influx. A dose response curve shows the correlation between the amount of the transported Glucose via Glut 4 transporters on the cell membranes of the adipocytes and the insulin dose.
Proliferation assays
A proliferation assay is a fundamental cell-based bioassay used to measure the ability of a drug substance, growth factor or cytokine to stimulate or to inhibit cell division over a specific period. Proliferation assay aim is to show whether cells are actively dividing. In the context of drug development and potency testing, these assays serve as the “functional readout” of a molecules biological activity.
The measurement of increase in cell number of a certain cell population can be achieved by measuring the following biological markers: 1. DNA synthesis following incorporation of labeled nucleotides (like BrDU) into a newly synthesized DNA during S phase of cell cycle. 2. Metabolic activity measuring the activity of mitochondrial enzymes (e.g LDH, ATP or reduction of tetrazolium salts like MTT) and 3. Direct cell counting using flow cytometry or counting device
Cytotoxicity assays
A cytotoxicity assay is a cell-based bioassay that measures the ability of a compound to damage or kill the cells. Depending on the compound this can either be desired (e.g., compounds targeting cancer cells) or a negative side effect. Therefore, in the context of drug development, a cytotoxicity assay can either be used to serve as the “functional readout” of a molecules biological activity or to show absence of cytotoxic side effects.
The measurement of damaged or dead cells can be achieved by several methods, for example by measuring the following biological markers:
- Detection of translocation of phosphatidylserine to the membrane surface via Annexin V in early apoptotic stage
- Activity of caspase enzymes in early to mid apoptotic stage
- Detection of DNA fragmentation via a TUNEL assay in late apoptotic stage
- LDH or G6PD is released due to cell membrane damage.
- Indirect through assessment of cell viability or proliferation or via cell count.
Protein expression
During the COVID-19 pandemic, (in vitro transcribed (IVT)) mRNA-based drugs gained significant attention, which accelerated their research and development for pharmaceutical applications. This is largely due to the potential of mRNA-based drugs to facilitate rapid responses to infectious diseases as well as adaptability for a wide range of pathological conditions.) In the context of drug development and potency testing, a protein expression assay serves as the “functional readout” of the mRNA-based drugs biological activity.
Cells are transfected with mRNA based drugs and subsequent protein expression can be measured for example by
- ELISA using supernatant or cell lysate
- In-Cell Western assay
“Classical” cell-based assays
Cell-based assays play a vital role in the characterization of biopharmaceutical products. In-vitro assays support in drug development and is required by international guidelines to ensure quality, safety and efficacy of a product.
Aim of in-vitro assays is to test the biological activity of a molecule on living cells to ideally mimic the situation in patients. The biological activity is not limited to the intended mode-of-action of the product, but can as well be complemented by negative assays. Cell-based assays are specifically designed for each product.
"Classical" Cell-based assays involve immortalized cell lines or primary cells. Analytical methods can be developed specifically for the needs of the biopharmaceutical product as well as the clinically relevant area.
Reporter gene assays (RGA)
Reporter gene assays (RGA) involve genetically modified cells expressing the molecule of interest and a downstream signaling, mostly luminescence. Advantages of RGA are a good variance and signal-to-noise ratio. For cases were cell lines expressing the target molecule are not available or show high variance in target molecule expression and for assays where time consuming isolation and/or differentiation of primary cells would be needed reporter gene assays are a valuable and time-saving alternative.
Multiple suppliers offer a broad portfolio of products and applications. In several cases, continuous cell models as well as ready-to-use kits are available, offering more flexibility.
An example of use for a reporter gene assay is measuring the potency of a drug to activate a specific cell signaling pathway. A short and easy protocol comprises seeding of reporter cells and overnight incubation, addition of serial dilutions of drug to the cells and further incubation until addition of detection reagent and measurement with a plate reader capable of luminescence detection.
Virus assays
Virus potency
Viral potency cell-based assay can be used to measure the relative potency of the viral vectors.
Mechanism of action involves infection of the engineered target cells, followed by peptide presentation. Antigen presentation and receptor binding result in the effector cells activation, followed by a signaling cascade and reporter activation, resulting in signal detection with a standard luminometer. Relative potency of a sample is calculated by parallel line analysis of the measured bioluminescence obtained from the test sample and a reference standard.
The objective of the viral relative potency assay is to reflect the functional infectivity rather than viral particle count alone.
Relative potency assays for viral vectors require product-specific design due the uniqueness of the viral products and their modes of action.
Virus Titer determination
The Foci Forming Unit (FFU) assay is an immunological technique which quantifies the specific non-lytic functional virus titer of a given sample. The assay relies on a direct viral infection and protein expression as the primary readout and is a critical method for both titer determination and safety assessment of viral vector preparations.
The objective of the assay is ...
- ... to measure the combined titer of all infectious particles (both replication competent and replication incompetent) by using GP-complementing cells.
- ... to quantify only the replication-competent viral particles by using non-complementing cells where replication-incompetent vectors are unable to form countable foci.
- ... identitiy testing: to confirm the absence of active viral titer in placebo materials.
PBMC Isolation
Peripheral Blood Mononuclear Cells (PBMCs) are blood cells with a single nucleus such as lymphocyte, monocytes and dendritic cells. PBMCs can be used as effector cells in ADCC assays and cytokine release assays and are a valuable tool in clinical research. Furthermore, PBMCs can be a starting point for isolation of specific cell types (e.g. natural killer cells).
PBMCs can be extracted from whole blood. A common separation technique is gradient centrifugation using hydrophilic polysaccharide (e.g., Ficoll) which separates the whole blood into layers of plasma, PBMCs and other cells (e.g., red blood cells). Isolated PBMCs can be used immediately or transferred to liquid nitrogen for long term storage.
Cell Services
Routine Cell Cultivation under GxP
Reliable results start with reliable cells. Our GxP-compliant cell cultivation ensures consistency, traceability, and top‑tier quality for research and biotechnological production. Following strict Good Practice (GxP) standards, we maintain, expand, and monitor cell lines under precisely controlled conditions. Every step - from cell handling and passaging to cryo-storage - follows standardized and documented procedures, using validated equipment and qualified reagents. Regular quality controls, including contamination testing (e.g., mycoplasma via isothermal PCR), and continuous evaluation of morphology and growth, guarantee that each cell line remains stable and traceable.
Our state-of-the-art BSL-2 laboratory offers a professional and sterile environment for the cultivation of a wide range of cell lines. With an in-house cell bank of over 80 established cell lines, we support projects under biosafety levels 1 and 2, adapting flexibly to each client’s specific requirements.
Our experienced scientific team combines technical excellence with flexibility, offering customized cell culture solutions for clients across the pharmaceutical and biotech industries.
Establishment of Cell Banks
The establishment of GxP-compliant cell banks is essential for reproducible and traceable cell-based research and production. Our standardized cell banking process ensures the long-term availability and genetic stability of high-quality cell lines.
We generate Master Cell Banks (MCB) from authenticated and well-characterized cell sources, cryopreserved under controlled conditions. From these, Working Cell Banks (WCB) are prepared for routine use, minimizing genetic drift.
Each cell bank is subjected to comprehensive quality control, including sterility testing, mycoplasma detection, and documentation according to regulatory requirements. This structured approach guarantees consistency, compliance, and reliability across all project stages.
Cell Line Characterization
Cell line characterization is fundamental to verifying the identity, purity, and functional integrity of cell cultures, ensuring reproducible outcomes in research and GxP-regulated biotechnological applications.
Our multi-parameter characterization includes assessment of morphology and growth dynamics, evaluation of expression profile and stability & purity (cell bank viability, functionality for its intended use, sterility testing, mycoplasma screening).
This rigorous approach delivers a complete, reliable cell line profile - powering consistent, compliant workflows in pharmaceutical and biotechnology projects.
Frequently asked questions about cell-based assays FAQs-CBA
FAQs about cell based assays
Why are potency and biological activity assays important?
These assays determine whether a biological drug produces its intended functional effect. Unlike purely analytical methods, they measure activity in a biological system and therefore reflect the mechanism of action more directly.
Applications include batch release, stability studies, biosimilar comparability, and process development for biologics such as antibodies, cytokines, and recombinant proteins.
What is measured during potency testing?
Potency testing evaluates the biological effectiveness of a drug product relative to a reference standard. It is a key quality attribute for biopharmaceuticals and required by regulatory authorities.
Typical applications include lot release, comparability studies, and stability testing using cell-based or biochemical assay formats.
Why are neutralizing antibodies analyzed?
Neutralizing antibodies can inhibit the activity of therapeutic biologics and reduce treatment efficacy. Their detection is therefore essential during immunogenicity assessment.
Cell-based assays are commonly used because they directly reflect the biological mechanism affected by the antibodies.
What is analyzed in a CDC assay?
CDC assays measure the ability of antibodies to induce complement-mediated target cell lysis. This mechanism is relevant for many therapeutic antibodies used in oncology and immunology.
The assay combines target cells, antibody, and complement serum to quantify cell destruction under controlled conditions.
What does a metabolic activity assay show?
These assays assess cell viability and physiological activity by measuring metabolic processes within living cells. They are widely used to evaluate toxicity, proliferation, and cellular responses.
Applications range from drug development to routine cell-based potency testing.
Why are proliferation assays performed?
Proliferation assays determine whether cells grow and divide under specific conditions. They help assess stimulatory or inhibitory effects of biologics, cytokines, or drug candidates.
They are commonly applied in immunology, oncology, and cell therapy research.
What is measured in cytotoxicity assays?
Cytotoxicity assays evaluate whether a substance damages or kills cells. This is important for assessing therapeutic effects as well as unwanted toxicity.
Different methods detect changes in membrane integrity, metabolism, or apoptosis-related pathways.
Why is protein expression analyzed?
Protein expression analysis determines whether cells produce a target protein and at what level. It supports cell line development, biomarker studies, and recombinant protein production.
Both intracellular and surface proteins can be investigated depending on the application.
What are classical cell-based assays used for?
These assays use living cells to evaluate biological effects such as signaling, proliferation, or cytotoxicity. They provide biologically relevant information beyond molecular binding data.
Applications include potency testing, antiviral assays, and receptor activation studies.
How do reporter gene assays function?
Reporter gene assays detect cellular activation through expression of a measurable reporter protein, often luminescent or fluorescent.
They are highly sensitive and commonly used for potency determination, signaling pathway analysis, and neutralization testing.
What can flow cytometry analyze?
Flow cytometry allows rapid multiparametric analysis of individual cells. Surface markers, viability, cell size, and intracellular proteins can all be measured simultaneously.
It is widely used in immunophenotyping, apoptosis studies, and cell characterization.
What are virus assays used for?
Virus assays evaluate infectivity, replication, or neutralization of viral particles. They are essential for vaccine development, antiviral testing, and gene therapy research.
Assay formats vary depending on the virus type and biological question.
Why is virus potency testing required?
Virus potency testing determines whether a viral product retains its intended biological activity. This is critical for vaccines and viral vectors.
The assay design depends on the mechanism of action and expected biological response.
What does virus titer determination measure?
Virus titer assays quantify the concentration of viral particles or infectious units in a sample. Accurate titers are essential for dose definition and process control.
Both infectious and total particle-based methods may be used.
Why are PBMCs isolated?
PBMC isolation provides immune cells for functional and immunological assays. These cells are frequently used in cytokine release, proliferation, and cytotoxicity studies.
High cell viability and gentle processing are important for reliable results.
What do VelaLabs' cell services include?
Cell services support the maintenance, expansion, and handling of mammalian cell cultures. This includes cultivation, cryopreservation, and contamination monitoring.
Such services help ensure reproducible experimental conditions and stable cell performance.
Why is GxP-compliant cell cultivation important?
GxP-compliant cultivation ensures traceability, consistency, and controlled handling of cell lines used in regulated studies.
Critical culture conditions and documentation are continuously monitored to support regulatory compliance.
Why are cell banks established?
Cell banks provide a consistent and well-characterized source of cells for long-term use. They reduce variability between experiments and production campaigns.
Master and working cell banks are typically stored under controlled cryogenic conditions.
What is included in cell line characterization?
Cell line characterization confirms identity, purity, and biological properties of cultured cells. This is essential for reliable research and regulated applications.
Testing may include authentication, growth behavior, marker expression, and contamination analysis.