Functional Assays

Functional assays are indispensable throughout all stages of drug development, to evaluate the efficacy, quality, and profile of drugs and their targets. These assays are crucial in determining the suitability of potential therapeutic interventions and guiding decisions regarding progression to subsequent developmental phases. NJ Bio specializes in providing integrated functional assay and assay development services aimed at facilitating in-depth mechanistic studies for a wide range of biopharmaceutical products, including antibody-drug conjugates (ADCs), monoclonal antibodies (mAbs), and small molecules. We support our clients’ drug development program by customizing assays to match the specific characteristics of the product being synthesized, based on its development stage, and the materials available. Our assays are optimized to maximize signal-to-noise and minimize variability, thus generating high-quality data. Digital imaging and spectroscopic techniques are used to analyze various crucial parameters like mechanism of action (MoA), antibody internalization, target localization, etc., based on the project requirements.

Functional Assay Service Options

NJ Bio’s comprehensive functional assay platform helps elucidate the involvement or function of a protein in cellular pathways or biological processes. This aids in establishing the desired functionality and quality profile of drug and drug targets before advancing to the next stages of development.

Cell-free assays play a crucial role in drug discovery and preclinical research by assessing drug efficacy through biochemical reactions or interactions, in a cost-efficient and timely manner. On the other hand, cell-based assays provide analytical measurements using a set of reagents to detect signals associated with biological processes, offering insights into compound potencies, cytotoxicity, permeability, and inhibition.

Our robust analytical platform supports a wide range of biochemical and cellular assay types for various biopharmaceutical applications. We utilize a variety of cancer cell lines expressing targeted surface antigens, as well as normal cell lines, to evaluate in vitro safety and efficacy of the compounds. Our assay development strategies prioritize efficiency and effectiveness, accelerating the optimization process to meet the requirements of each assay.

Target Validation

Target validation is a crucial step in confirming the involvement of specific targets in a disease process. To enhance this validation process for entities like ADCs, several in vitro assays are employed which measure the biological activity of the target. Our comprehensive suite of in vitro assays, including ligand binding assays, cytotoxicity assay and receptor density studies, offers robust methodologies for target validation of ADCs and related entities.

Ligand Binding assays (LBA)

Ligand binding assays detect and quantify various entities such as biotherapeutics, biomarkers, and anti-drug antibodies in biological matrices. They measure interactions between molecules or the binding of molecules to receptors, antibodies, and other complex molecules. At NJ Bio, we offer plate and cell-based approaches across various biological matrices, ensuring tailored solutions for our clients’ project requirements. With extensive expertise, we develop robust and specific assays, ensuring sensitivity, selectivity, and reproducibility.

Cytotoxicity IC50 Assay

For a highly cytotoxic agent, it is desirable that the IC50 value be ~10 nM or less.¹ The Cytotoxicity assay measures the cancer cell-killing activity or inhibitory concentration (IC50) of the therapeutic, under study. IC50 values and graded dose cytotoxicity curves are calculated to assess the efficacy of the ADCs and their potency in killing cancer cells, thus helping in the optimization of their therapeutic potential.

Receptor Density Studies

Receptor density studies determine the abundance of specific receptors on cell surfaces, crucial for understanding target interactions. Low expression of tumor-associated antigens can adversely affect ADC delivery to tumor target cells. An optimal density of ~10,000 copies/ cell has been suggested as the minimum requirement for effective biologic-mediated ADC internalization.² Analyzing mean density and statistical distribution among different cells, enhances the understanding and validation of target engagement.

mAb/ ADC Characterization

Characterization of mAbs and/ or ADCs presents numerous challenges due to their structural complexity and heterogeneity. Thus, the use of precise and robust analytical techniques and assays is essential to study these molecules, NJ Bio offers comprehensive ADC characterization services including stability assessment, receptor binding and PK Analysis.

ADC Stability Assay

Stability assays are crucial for assessing the ability of an ADC molecule to remain stable under physiological conditions, including chemical stability to prevent premature payload release, plasma stability to avoid off-target drug release and physical stability to ensure solubility and prevent aggregation. In vitro plasma stability analysis involves incubating ADC samples in plasma at 37°C, 5% CO₂, then measuring total and conjugated antibody levels using ELISA, along with quantifying free drug via LC-MS.

ASSAY FORMATS

• LC-MS methods enable the detection and characterization of ADC to evaluate its stability in plasma.
• The MSD® platform is based on electrochemiluminescence and provides excellent sensitivity, a large dynamic range, and flexibility.
• ELISA is used to check for plasma stability by measuring the amount of total antibody and conjugated antibody and calculating the degree of drug loss.

Receptor Binding EC₅₀

Receptor Binding or half-maximal effective concentrations (EC₅₀) assay determines the concentration needed for an ADC to bind to a specific receptor by 50%, providing insights into binding kinetics and potency. The binding activity of an ADC compared to untreated antibody is evaluated using flow cytometry.

ASSAY FORMATS

• Real-time label-free biosensor technologies based on biolayer interferometry (BLI) are widely used platforms that provide kinetic constants for the protein-protein interactions without the need of washing or labeling steps.
• The direct ligand-receptor interaction ELISA (direct LRA) is used to measure the receptor-ligand dissociation constant (KD), determining the receptor-ligand binding affinity.
• Flow cytometry is used to measure the expression of individual receptors on the surface of each cell and make real-time measurements of ligand-receptor interactions.

Fig.2: EC₅₀ determination: PE conjugated mAbs-1&2 were tested for receptor binding on the cell line of interest at the highest concentration of 150 ug/mL and 10-3X dilutions. NovoCyte 3000 was used to acquire data and GraphPad Prism was used to analyze data and determine the EC₅₀ values.

PK Analysis

PK analysis examines the kinetics of different components of an ADC, including their stability, binding affinity, and metabolism, which are crucial for predicting their behavior in vivo and optimizing their therapeutic efficacy. Multiple analytes are assessed to reveal PK characteristics, including total and conjugated antibodies, free and conjugated drugs, and catabolites. For total and conjugated antibodies, modified ELISA assays are employed, using either targeted antigens or anti-human monoclonal antibodies as capturing reagents, followed by detection with anti-human monoclonal antibodies. For free and conjugated drugs and catabolites, advanced LC-MS analysis is utilized. Affinity capture LC-MS/MS isolates conjugated drugs, while ELISA competition assays or sensitive LC-MS analysis quantifies free drugs and catabolites after protein content extraction and precipitation.

NJ Bio specializes in ADC pharmacokinetics (PK) analysis, offering diverse assay formats for different analytes. We provide various quantification assays, analyzing plasma/serum samples to determine ADC PK profiles.

Small Molecule Characterization

Small molecule characterization involves assessing the properties and effects of low molecular weight organic compounds, particularly in the context of drug discovery. Functional assays can provide quantitative data on the potency, efficacy, and cytotoxic effects of these small molecules.

Protein degradation DC50

The DC₅₀ assay is a crucial tool in characterizing small molecules by evaluating their ability to induce degradation of target proteins. By determining the concentration of a small molecule needed to degrade a target protein by 50%, this assay offers vital insights into the potency and efficacy of potential drug candidates. It helps researchers understand how small molecules interact with proteins, guiding the selection of lead compounds for further development.

Apoptosis cell cycle studies

Analyzing cell treated with varying concentrations of the small molecule provides quantitative data on the compound’s ability to induce cell cycle arrest, apoptosis, or cell death. This information aids in understanding the pharmacological properties of small molecules and their potential therapeutic applications.

Bystander Killing Assay

The bystander killing assay assesses cytotoxicity and cell death induction by small molecules not only in directly treated cells but also in neighboring cells through a bystander effect.

Antibody Internalization

The antibody internalization assay is a fundamental method used to measure antibody internalization by cells expressing antigen of interest. This assay provides valuable insights into antibody – cell surface receptor interactions and the potential of specific antibodies to deliver payloads into target tumor cells.

ASSAY FORMATS

Antibody/ADC internalization is assessed by Incucyte® Live-Cell Analysis System using Fabfluor-pH Antibody labeling dyes. The system enables real-time kinetic evaluation of internalized antibody/ ADC, and the graphs can be plotted with either pre-defined or user-defined metrics.

Fig.3: Time-course of Fabfluor-mAb#1/ #2 internalization: Graphs showing quantified values for internalized antibody as the total red object integrated intensity for each time point up to 48 hrs in (A) antigen-positive cell line and (B) antigen-negative cell line. The Incucyte® (S3) Live-Cell Analysis system was used to acquire images and Incucyte 2023A software was used to analyze this data.

Apoptosis cell cycle studies

Apoptosis cell cycle studies allow researchers to explore how therapeutic agents impact cell viability, cell cycle progression, and apoptosis in cultured cells, by observing changes in the cell cycle profile and apoptotic markers.

ASSAY FORMATS

• Flow-cytometric assays are used to measure caspase-3/7 activation or PS externalization.
• Apoptosis assays using Annexin V conjugated to a fluoroprobe have been optimized for detection of PS externalization and are most commonly measured by flow cytometry.
• The Incucyte® Live-Cell Analysis System enables real-time, automated apoptosis assays that measure multiple apoptotic pathways simultaneously and in real time using the mix-and-read Incucyte® Caspase-3/7 and Annexin V Dyes.

Bystander Killing

The bystander killing assay evaluates the capacity of therapeutic agents to induce cell death not only in directly targeted cells but also in neighboring bystander cells. In the bystander killing assay, cells expressing the target antigen are treated with mAbs or ADCs, and subsequently, the extent of cell death is assessed using vital dyes followed by flow cytometry analysis.

NJ Bio offers the following functional assays:

Our team follows standardized protocols and utilizes state-of-the-art instrumentation to ensure accuracy, precision, and reliability in data generation.

Can you customize functional assays to fit the unique requirements of the drug development project?

Yes, we develop tailored assay protocols to meet the unique research requirements of each project.

Do you provide support for data analysis and interpretation?

Yes, our team of experts offers comprehensive support for statistical data analysis and in-depth interpretation of assay results.