Cell-Based Bioassays

In addition to traditional cell-based bioassays, flow cytometry provides a fast, highly specific, and accurate quantitative readout tool, especially for complex heterogeneous samples. It allows simultaneous, multiparametric, and fast analysis of the physical and chemical characteristics on a single cell level in real-time (several thousand particles per second). Complex heterogeneous samples can be tested and multiple markers can be correlated.

Applications

• Mode of action assays for monoclonal antibody therapeutics
• Antigen, receptor or ligand density (e.g., binding assays and competitive binding assays)
• Multiplexing analyses of cytokines (CBA technology)
• Cell-based immunogenicity
• Intracellular protein expression
• Transgenic products in vivo (e.g., green fluorescent protein [GFP])
• Enzyme activity
• Phosphoprotein analysis
• Apoptosis/viability
• Cell cycle analyses
• Changes in intracellular pH, calcium and glutathione
• Various combinations (DNA/surface antigens, etc.)
• In-process quality control of primary cells

The time-resolved fluorescence method, which is used for cell-based bioassays, is based on fluorescence resonance energy transfer (FRET) in a microtiter plate. It is often used for third-generation anticancer and anti-inflammatory drugs, which tend to activate/act on specific phosphorylation pathways in the target cells. For proof of the mode of action of such drugs, the assay must reflect the effect on the phosphorylation of key mediators of the involved pathway.

Advantages of the method are low background, increased assay sensitivity, compared to classical approaches for the determination of phosphorylation (e.g., ELISA), fewer false-positive or false-negative results, and suitability for cell-based assays.

The homogeneous time-resolved fluorescence (HTRF) technology is an interesting approach that might be used as an alternative mode of action assay.

Compendial bioassays on various cytokine products (IFN-α, IFN-β) have been performed for almost two decades. These bioassays are based on the inhibitory activity of cytokines on the cytopathic effect of a virus on a susceptible cell line. All assays comply with the requirements of the European Pharmacopoeia and have been validated according to ICHQ2(R1).

The potency of human growth factors, such as EPO, GMCSF, and G-CSF, is measured with classical proliferation assays. These assays have been successfully applied on originators as well as first- and second-generation biosimilar products. If applicable, the assays comply with the requirements of the European Pharmacopoeia, and all assays have been validated according to ICHQ2(R1).

For parathyroid hormone (PTH), a cell-based assay is performed based on the determination of cyclic AMP (cAMP) release, detected by homogeneous time-resolved fluorescence (HTRF) or ELISA. The method has been validated according to ICHQ2(R1).

Antibody-dependent cell cytotoxicity (ADCC) is measured by using a surrogate reporter cell line as effector cell replacement. For characterization or biocomparability purposes, it is possible to run a primary ADCC assay where the activity is measured by LDH release using NK effector cells freshly isolated from peripheral blood mononuclear cells (PBMCs) or with a reporter-based bioassay. The target cell line is selected based on the product.

Antibody-dependent cellular phagocytosis (ADCP) is measured with a luminescence-based reporter bioassay or by flow cytometry with macrophages differentiated from monocytes isolated from PBMCs.

Complement-dependent cytotoxicity (CDC) is measured by a luminescence-based approach or by flow cytometry using a live/dead discrimination dye. An appropriate target cell line is marked by the antibody and attacked by the complement cascade.

Apoptosis/programmed cell death (PCD) is addressed by a pathway-specific assay (e.g., Caspase 3/7) or by monitoring (e.g., early death) via flow cytometry-based assays or via a reporter-based assay.

• Cell-based binding assays with different readouts (e.g., flow cytometry, ELISA)
• Proliferation assays
• Cell death assays
• Assays reflecting classical immunological pathways for mAb therapeutics (ADCC, ADCP, CDC)
• Surrogate reporter assays reflecting the MoA (mechanism of action) of a drug by activation of a relevant signaling pathway

A bioactivity assay should reflect the mechanism of action (MoA) of a drug. It measures the capability of a drug to induce a certain dose-dependent response within a relevant assay system compared to an appropriate reference item and a relative bioactivity is reported.

Cell-based potency bioassay testing determines the relative bioactivity/the ability of a test item to induce a certain response in a relevant test system compared to a reference standard.