Cell-Based Potency Assays: De-Risk Your Path To Patients With An Experienced CDMO
By Danny Rooney, QC Senior Manager
Source: FUJIFILM Diosynth Biotechnologies
Since the approval of the first monoclonal antibody (mAb) drug, muromonab-CD3, in 1986, this class of biologics has changed the therapeutic landscape, offering a highly targeted and precision approach to treat a wide spectrum of diseases. With over 100 approvals to date, antibody-based therapeutics have been developed for cancer, autoimmune disorders, rare genetic conditions, and more recently against viral pathogens like SARS-CoV-2 (1). Today, they are the fastest growing segment of the biopharmaceutical market, accounting for approximately one-fifth of new FDA-approved drugs annually (2).
While conventional IgG antibodies continue to dominate the market, emerging technologies like antibody drug conjugates (ADCs), bispecific antibodies (BsAbs), engineered antibody fragments, and Fc-fusion proteins have entered pre-clinical testing and clinical trials. These advancements offer novel mechanisms of action, refined pharmacokinetics, and improved tumor penetration, further broadening the scope of mAb
therapeutics (2).
The inherent complexity and diversity of therapeutic antibodies necessitate thorough characterization at every stage of development and manufacturing. By employing physical, structural, and biological analyses, characterization studies aid in defining the critical quality attributes (CQAs) of mAb drug candidates during early phase development. This comprehensive approach ensures the safety, potency, efficacy, and integrity of these biologics for human use, in accordance with international regulatory guidelines set forth by agencies like the FDA and EMA.
Regulatory Emphasis on Potency Testing
In recent years, regulatory agencies have placed increased emphasis on the development and implementation of an effective potency assay(s) for mAb products and biosimilars. According to ICH guideline Q6B, potency is the measurement of biological activity using a suitable quantitative biological assay (also called potency assay or bioassay) based on the attribute of the product which is linked to the relevant biological properties (3). These assays are expected to be specific, sensitive, reproducible, and capable of accurately measuring the intended biological activity relevant to the therapeutic effect of the antibody.
For early phase product development, a surrogate potency assay (i.e., ligand-binding assay) can also be used as surrogate assays for biological activity assessment. Biological potency assays are typically established during Phase II clinical development. In response to growing regulatory scrutiny, pharmaceutical companies and biotechnology firms are prioritizing earlier line-development of robust potency assays to mitigate risk and support regulatory IND submissions towards gaining market authorization.
Functional Assessment of Therapeutic Antibodies
Therapeutic antibodies exert their effects through various biological mechanisms, involving both antigen-binding (Fab) and crystallizable (Fc) fragments. Evaluating their biological functionality encompasses assessing a candidate molecule’s affinity for its target(s) and its ability to engage immune effector mechanisms, crucial for mediating anti-tumor or antipathogen responses (4,5).
The Fab regions of mAbs bind directly to target antigens and is mainly associated with binding specificity while the Fc portion is critical for the function of IgG at the cell level and for its metabolic fate. Additional effector functions can be activated through binding of the Fc regions with Fcγ receptors on immune effector cells like natural killer (NK) cells, macrophages, and neutrophils, or engage the C1q complement protein or neonatal Fc receptor. These include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) activity, and complement-dependent cytotoxicity (CDC) as illustrated in Figure 1 (5).
Before a candidate molecule can advance to clinical trials, a systematic analysis of its functional attributes, assessed using in vitro ligand-binding and cell-based potency assays and in vivo animal studies must be conducted. The data obtained from these analyses not only inform decisions about the molecule’s suitability for human use but also contribute to defining process set points and acceptable limits for critical quality attributes (CQAs) during later stages of Chemistry, Manufacturing, and Controls (CMC) development.
Ligand-binding kinetics
The biological activities as well as the pharmacokinetics of mAbs depend intrinsically on their ability to bind to specific target antigens with high affinity, which is why regulatory bodies often require binding assays during the approval process for mAb-based biologics. A stable and soluble antigen is used to represent the in vivo target of mAbs in ligand-binding assays.
ELISA and surface plasmon resonance (SPR)-based technology are two of the most popular methods for ligand-binding analysis (4). While enzyme-linked immunosorbent assays (ELISAs) are commonly used for binding activity testing due to their simplicity and cost-effectiveness, they lack the ability to provide kinetic, thermodynamic, and/or stoichiometric information. Surface plasmon resonance (SPR) offers a more sensitive approach for analyzing both Fab and Fc-based binding kinetics and dynamics in real-time with low sample consumption, providing valuable insights into affinity and avidity during early product characterization.
Cell-based potency analysis
Cell-based assays that reflect the biological activity in vitro are very important to understand the mechanism of action (MoA) and evaluate the product potency. These in vitro assays are designed to evaluate the downstream effector functions gained after Fcγ receptor or C1q binding including ADCC, ADCP, CDC or activation of neonatal Fc receptor (FcRn) binding activity (5). Additionally, products which promote agonistic signaling events (e.g. recombinant cytokines), or those that are developed to inhibit signal transduction through receptor blockade or ligand inhibition, may also be measured for potency within a cell-based format. The application of a cell-based model for such products enables a functional assessment of the molecule’s therapeutic potential, ultimately demonstrating the clinical relevance for the indication.
ADCC Assays: Traditionally, ADCC has been measured using primary effector cells, where the effector cell is isolated from peripheral blood mononuclear cells (PBMCs) from human donors. These assays gauge an antibody’s capability to induce the killing of target cells by immune cells. Target cells with a specific antigen are marked and incubated with the test antibody, then mixed with effector cells (purified NK cells or peripheral blood mononuclear cells). The antibody binds to target cells via its Fab region, while its Fc region interacts with Fc receptors on effector cells, triggering their cytotoxic activity. Target cell killing is quantified by measuring label release or assessing cell viability (classical pathway). More recently, the classical ADCC pathway has been augmented or replaced by the use of genetically engineered effector cell lines, where the effector cells are modified to express a stable reporter gene under the control of a response element. The application of a genetically engineered model negates the requirement for PBMCs from human donors, leading to improvements in lot-to-lot consistency, response range and inter-assay variability.
ADCP Assays: ADCP assays assess the ability of antibodies to induce phagocytosis of target cells. Like ADCC assays, target cells are labeled, and effector cells (usually macrophages) are added in the presence of the antibody. The antibody tags target cells for phagocytosis via Fc region binding to the Fc receptors on the surface of the phagocytes. Phagocytosis is quantified using flow cytometry by measuring the uptake of labeled target cells. Similar to the advancements in the ADCC assay format, the classical ADCP pathway has been enhanced by the use of genetically modified effector cells, such that phagocytosis can be measured as a function of reporter gene activity. The use of genetically engineered cells overcomes the limitations of primary cell cultures, such as lot-to-lot consistency from human donors and cell senescence, which can adversely impact the quality of the data generated and suitability of the method for cGMP batch release and stability determinations.
CDC Assays: These assays determine antibodies’ capacity to activate the complement cascade to induce target cell lysis. Target cells are labeled and sensitized with the test antibody prior to complement protein addition. The antibody binds to target cells via its Fab region, while its Fc region interacts with complement proteins, activating membrane attack complex (MAC) formation, and inducing target cell lysis. Target cell killing is quantified by measuring label release or assessing cell viability.
Establishing suitable effector-function assays can be challenging—it requires sourcing of target cell lines, primary human blood cells (effector cells) or genetically engineered effector cells, the cell culture expertise to isolate and/or differentiate relevant cell types, optimize assay conditions, and establish a sensitive and selective endpoint detection method. Given the specialized analytical methods and cell culture, method development and interpretation expertise required, many sponsor companies opt to outsource these activities to service partners.
Our Analytical Toolkit for Functional Characterization
Thorough characterization is key to unlocking the potential of antibody-based therapeutics. With FUJIFILM Diosynth Biotechnologies, companies gain access to a comprehensive toolkit for functional characterization of mAb candidates. Our Bioassay Department has established inhouse methodologies, access to primary cells as well as genetically engineered and characterised effector cells, and broad capabilities to execute established cGMP bioassays or develop customized potency assays that align with regulatory standards for different Fc modalities. Moreover, FDB’s Bioassay Department has the technical knowledge and expertise to design and develop custom potency assays including ELISAs for monoclonal antibodies, recombinant proteins and enzymatic products, ensuring that a suitable and phase-appropriate potency assay is available for cGMP activities.
- Routine cGMP analytics for lot release and
stability assessment - Custom potency bioassay development,
optimization and validation - Analytical cell banking to support cell-based
bioassays - Cell-based assays encompassing reporter gene,
ADCC, ADCP, cell death, and cell viability assays - Non-cell-based assays such as ELISA and
enzymatic activity assays
Additionally, our experts possess the necessary expertise to integrate data from orthogonal analyses like ligand-binding data from SPR, to provide a holistic understanding of the biological activity and therapeutic potential of a client’s antibody candidate. By leveraging our extensive capabilities and expertise, companies can confidently navigate the biopharmaceutical development landscape to bring innovative antibody-based treatments to patients worldwide.
Contact us to learn more about how we can support your needs.
References
- Mullard A. FDA approves 100th monoclonal antibody product. Nat Rev Drug Discov. 2021;20(7):491-495. doi:10.1038/d41573-021-00079-7
- Wang Z, Wang G, Lu H, Li H, Tang M, Tong A. Development of therapeutic antibodies for the treatment of diseases. Mol Biomed.2022;3(1):35. Published 2022 Nov 22. doi:10.1186/s43556-022-00100-4
- ICH Q6B (R2) – Test procedures and acceptance criteria for biotechnological/biological products, January 1999
- Alhazmi HA, Albratty M. Analytical Techniques for the Characterization and Quantification of Monoclonal Antibodies. Pharmaceuticals (Basel). 2023;16(2):291. Published 2023 Feb 14. doi:10.3390/ph16020291
- Wang X, An Z, Luo W, Xia N, Zhao Q. Molecular and functional analysis of monoclonal antibodies in support of biologics development. Protein Cell. 2018;9(1):74-85. doi:10.1007/s13238-017-0447-x
About the Author
Danny Rooney is part of the Quality Unit Leadership Team at the UK site and is responsible for overseeing potency assay method development, validation and leading cGMP laboratory services across the product life cycle. Augmenting quality by design concepts with biophysical data, Danny has evolved potency assay development studies, providing both effective and novel cell-based potency assay solutions for drug characterization, clinical stage development and CMC filings. He also supports risk management activities for cell lines and genetically modified organisms deployed within the organization. Danny joined FUJIFILM Diosynth Biotechnologies in 2012, and in parallel with his career he completed higher education studies at Teesside University, where he obtained a BSc in biotechnology and laboratory management with a focus on cell biology.
About FUJIFILM Diosynth Biotechnologies
FUJIFILM Diosynth Biotechnologies, a subsidiary of FUJIFILM Corporation, is a world-leading contract development and manufacturing organization (CDMO) for the development and manufacture of biologics, advanced therapies, and vaccines. The company operates a global network with major
locations in the Unites States of America, the United Kingdom and Denmark, offering end-to-end services including drug substance, drug product, and finished goods services. It is also building a new manufacturing site in Holly Springs, North Carolina, USA, scheduled to be operational in 2025.
FUJIFILM Diosynth Biotechnologies has over thirty years of experience in developing and manufacturing drug substance of recombinant proteins, monoclonal antibodies, vaccines, among other large molecules, viral products and medical countermeasures expressed in a wide array of microbial,
mammalian, and host/virus systems. We have drug product filling capabilities to support both clinical and commercial demands. Our finished goods services, supported by more than 15 years of experience, can accommodate commercial products for more than 65 countries around the world. The
company offers a comprehensive list of services from cell line development using its proprietary pAVEway™ microbial and Apollo™X cell line system to process development, analytical development, clinical and FDA-approved commercial manufacturing. For more information, go to: www.fujifilmdiosynth.com.