Bioassays for Biologics

Bioassays are necessary to ensure the continued quality, safety, and efficacy of biopharmaceutical products, and also for the confirmation of biocomparability of innovator and biosimilar products. In turn, these assays must be reliable, standardized, and relevant to reflect the product’s mode of action.

Below we review technologies and assays which are appropriate to fulfill the testing needs for many different products. For more in-depth information about our experience with monoclonal antibody therapeutics and hormone and cytokine therapeutics, please access the technical sheets located in the Source℠, a portal of our technical, scientific, and educational resources.

Bioassay Technology

  • Flow Cytometry

    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
  • Time-Resolved Fluorescence

    The time-resolved fluorescence method 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.

Product-Specific Bioassays

  • Cytokines/Antiviral Compounds

    Compendial bioassays on various cytokine products (IFN-α, IFN-β) have been performed for more than a decade. 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).

    Compound Assay
    IFNα 2b Proliferation assay
    IFNβ 1a Proliferation assay
    IFNβ 1b Proliferation assay
  • Growth Factors & Hormones

    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).

    Compound Assay
    G-CSF; pegG-CSF Proliferation assay
    GM-CSF; pegGM-CSF Proliferation assay
    PTH and modified PTH

    Cell-based assay with cAMP ELISA

    HTRF cAMP assay (method of choice)
    EPO and modified EPO Proliferation assay
  • Monoclonal Antibodies

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

    Apoptosis/programmed cell death (PCD) is typically addressed by a reporter gene assay or by flow-cytometry-based assays.

    Name Assay Target Trade Name Innovator
    Rituximab
    • Epitope binding assay with flow cytometric readout
    • ADCC reporter bioassay
    • ADCP reporter bioassay
    • CDC assay with luminescence or flow cytometric readout
    • Apoptosis FACS assay (Annexin V)
    • ADCC assay with primary effector cells and LDH readout
    • ADCP assay with primary macrophages
    CD20 Mabthera, Rituxan
    Trastuzumab
    • ADCC reporter bioassay
    • Proliferation assay
    HER2 Herceptin
    Cetuximab
    • ADCC reporter bioassay
    • CDC luminescence based
    ERBB2 Erbitux
    Adalimumab
    • Competitive binding assay with flow cytometric readout
    • ADCC reporter bioassay
    • ADCP reporter Bioassay
    • Proliferation assay with luminscence readout
    • Annexin V apoptosis assay with flow cytometric readout
    • NFkB reporter bioassay
    • Caspase 3/apoptosis assay with luminscence readout
    TNF Humira
    Infliximab
    • Proliferation assay with luminscence readout
    TNF Remicade
    Etanercept
    • Caspase 3/apoptosis assay with luminscence readout, NFkB reporter
    TNF Enbrel
    Bevacizumab
    • VEGF reporter bioassay
    • Neutralization assay with primary HUVEC cells
    VEGF Avastin
    Ranibizumab
    • VEGF reporter bioassay
    • Neutralization assay with primary HUVEC cells
    VEGF Lucentis
    Palivizumab
    • ADCC reporter bioassay
    • ADCP reporter assay
    • CDC assay
    • Microneutralization assay
    RSV Synagis
    Ustekinumab
    • IL12/IL23 reporter bioassay
    IL12/IL23p40 Stelera
    Ipilimumab
    • CTLA4 reporter bioassay
    • ADCC reporter bioassay
    CTLA4 Yervoy
    Pembrolizumab, Nivolumab
    • PD1/PD1L reporter bioassay
    PD1/PD1-L Keytruda, Opdivo
    Secukinumab
    • HTRF bioassay with IL-6 readout
    IL-17A Cosentyx

Target-Specific Reporter Bioassays

For monoclonal antibody therapeutics that do not follow classical mechanism of action (MOA) pathways, target-specific reporter solutions are available; e.g., for antiVEGF antibody therapeutics.