By Amanda Turner, Senior Product Manager for Custom Antibody Products at Bio-Rad and Laura Moriarty, Senior Marketing Manager at Bio-Rad
The global market for therapeutic antibodies represents a huge and ever-expanding opportunity for pharmaceutical developers, with the market for monoclonal antibody therapies alone being estimated to reach $300 billion by 2025.1 However, the value of these biological medicines far exceeds their monetary worth for the people living with serious and debilitating disease who have experienced their transformative effects. Innovations in protein engineering techniques have improved the efficacy, safety and tolerability of antibody-based treatments and revealed seemingly infinite possibilities for new therapeutic targets and mechanisms of action, well beyond those of the traditional monoclonal antibodies that are used most widely in clinical practice. Recent advances in this highly competitive arena have welcomed the development of antibody-mediated therapies, such as antibody-drug conjugates (ADCs) and bispecific monoclonal antibodies (bsMAbs), which have elevated expectations regarding the clinical possibilities for targeted biological medicines and brought hope for the treatment of diseases where few options were previously available.2-5
Although the technologies and techniques used to generate highly specific and novel biological molecules have been honed and improved over time, biopharmaceutical developers face a growing number of challenges as druggable targets and candidate molecules become ever more complex in structure and design. Failures at any stage in development lead to costly delays and preclinical/clinical data packages must meet increasingly stringent regulatory standards. Research and development (R&D) workstreams may be accelerated through the incorporation of high-throughput and automated platforms, but the standard of R&D output achieved remains critically dependent on the quality of the technologies and reagents used at each stage. Ideally, development strategies should embrace systems that offer sensitivity, specificity and flexibility to ensure efficient and timely transition through regulatory approval and beyond.
ADC and bsMAb therapies – applications in clinical practice
- ADC treatments enable a chemotherapy agent to be delivered/targeted directly to the cell type of choice
- Cytotoxic exposure is avoided in surrounding ‘healthy’ cells and tissues, reducing the likelihood of side effects typically associated with conventional cancer therapies
- BsMAbs contain two different antigen-binding sites within one molecule
- New mechanisms of action may be provided for immunotherapies:
- Tumour cell targeting in tandem with the induction of the body’s own T-lymphocytes to launch a directed attack on the cancer (e.g. bispecific T-cell engager [BiTE] antibodies)
- Modulation of the inflammatory response to reduce infusion-related reactions
- BsMAbs are more readily produced at a lower cost when compared with cell-based treatments such as chimeric antigen receptor cell therapy (CAR-T immunotherapy)
Embracing innovation at every stage of development
The development pathway for antibody-mediated treatments is a high-cost, lengthy and complicated journey (Figure 1). Scientific expertise and multifaceted laboratory processes underpin each and every step in the pathway and continue to be vital in ensuring the longevity of the final product after regulatory approval, with ongoing quality standards and patient safety in mind.
The challenges begin in the identification of new and unique druggable targets that may offer a competitive advantage above existing therapies. Scientists must then validate the target and develop a critical understanding of its properties, including how it might interact with any lead candidate antibodies, the stability of the drug-target complex, and the potential for off-site interactions that may influence the efficacy and safety of the final drug. The next generation of antibody-based medicines (including ADCs and bsMAbs) are more complex when compared with the existing biological treatments, and timescales are extremely pressurised. High failure rates can cause problems and delays when candidate molecules do not have the intrinsic biophysical parameters that enable a stable and well tolerated drug to be developed, or preclinical/clinical studies fail to prove therapeutic effectiveness. Studies in cellular or animal models may not translate appropriately to humans or accurately predict clinical efficacy. In addition, a suitable animal model might not be available for certain diseases, making preclinical work a greater challenge. Reproducible pharmacokinetic (PK) data are needed to support dosing decisions and to examine the drug’s behaviour within the body, from administration to elimination. Drug safety and clinical efficacy are the primary concerns for any new therapy in development and biopharmaceutical manufacturers are required to produce robust evidence throughout preclinical and clinical stages to meet rigorous regulatory standards. Immunogenicity is a leading safety concern for biologic medicines and data must demonstrate a low risk of developing anti-drug antibodies (ADA) following drug administration.
Stable, high-producing cell lines are fundamental when producing therapeutic antibodies at the scale needed to fulfil global demand. Optimisation of the production process, supported by careful evaluation and monitoring throughout the operation, ensures that top quality product is delivered at the final stage of regulatory approval when full scale-up for manufacture is required. Continual testing and quality control measures must be in place, as specified by good manufacturing practices (GMP), to ensure that standards are maintained over the long-term. Antibody therapies are produced in biological/cellular systems, rather than via conventional pharmaceutical manufacturing processes, and small molecular variations can be introduced over time as the manufacturing cell lines evolve. Ongoing monitoring of the product is therefore essential to verify that the efficacy and safety of the drug has not been compromised as a result of these changes.
The benefits of recombinant antibody generation: specificity, reliability and reproducibility
Whether selecting antibodies for initial candidate screening or for use in bioanalytical assays (e.g. PK and ADA assays during preclinical and clinical stages), in vitro antibody phage display technology offers multiple benefits, allowing rapid antibody generation alongside high levels of reproducibility and site specificity. Custom antibody services that use recombinant production provide a reliable long-term supply in Escherichia coli (E. coli) or mammalian cell lines. Unlike conventional animal-based systems for antibody generation, the full DNA sequence is known at the outset when using recombinant antibody libraries, the specificity is defined using in vitro guided selection strategies and the product is well characterised, meaning that a selective and sensitive assay can be designed with the confidence that availability will be assured and batch variation minimised. As highlighted in a report from the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM), non-animal-derived molecular antibody tools offer improvements over antibodies made using traditional methods of animal immunization, regarding precision, reproducibility and flexibility.6
Bridging and antigen capture enzyme-linked immunosorbent assays (ELISAs), also known as ligand binding assays, are widely implemented in PK and ADA analyses. Reliable and highly stable reagents are essential in ensuring reproducible results and trustworthy data that withstand regulators’ scrutiny. Anti-idiotypic antibodies, which are able to discriminate between the antibody drug and naturally occurring antibodies in patient serum, are used in PK bridging assays as both the capture and detection reagent. Access to anti-idiotypic antibodies with different binding modes is important when conducting and optimising the wide array of immunoassays required to support a regulatory submission. Recombinant monoclonal anti-idiotypic antibodies, developed using a combination of antibody phage display and automated high-throughput processes, can be rapidly produced in less than three months. Screening processes should have the ability to measure the off-rate and facilitate affinity ranking of the antibodies produced.
Recombinant engineered antibodies allow incorporation of both capture and detection technologies for faster and more consistent assay results with enhanced flexibility of assay design. A molecular tag may be genetically fused to the C-terminus of the recombinant antibody, where it becomes coupled with a highly specific ‘catcher’ molecule via a covalent isopeptide bond during mixing and incubation (e.g. Bio-Rad antibodies incorporating the SpyTag and SpyCatcher system; Figure 2).7 Such enabling technology permits rapid production of different antibody formats with detection labels incorporated in a site-specific manner, avoiding issues typically associated with conventional antibody conjugation methods (e.g. antigen binding site disruption). As this field continues to evolve, R&D teams will have the ability to easily conjugate their own antibodies to a wide range of labels, tailored to their current research needs, using off-the-shelf tag and catcher reagents.
Leveraging automation to streamline processes and improve efficiency
Once a therapeutic target has been identified, multiple analyses are required to screen for antibody hits before filtering to select lead candidates. Automation is critical in improving the speed and efficiency of therapeutic antibody development, while freeing scientists to focus on optimising study designs or conducting other important tasks. Automation also heightens the accuracy and reproducibility of data through the removal/minimisation of human error and the standardisation of conditions within a given system. Flow cytometry has become established as the gold standard process for antibody screening and characterisation, with high-throughput technologies permitting huge experimental volumes to be rapidly analysed with exceptional accuracy. Automated analysis, conducted continuously with unattended operation, provides researchers with the capability to process batches at remarkable rates; 96-well plates may be processed in less than 15 minutes and 384-well plates in under 50 minutes. Modern platforms (e.g. ZE5 Cell Analyzer), that incorporate up to five lasers, can support experiments using as many as 27 colours for greater flexibility of panel design, while having the sensitivity to detect rare events.
Ensuring long-term cell line stability
Selection of a genetically stable prolific cell line is critical in ensuring the enduring quality and safety of drug product and in securing the inbuilt capability to meet global demand for high volume production. Genome editing and cell line engineering techniques hold the promise of rapid cell line development. Unfortunately, detection of gene edits can be challenging using gel-based or sequencing methods and arduous clonal isolation and expansion processes remain in place. Difficulties in accurately assessing gene copy numbers can hinder the process and waste both time and resource. Newer technologies offer greater precision and reproducibility in confirming and quantifying gene edits. Innovative methodologies in cell line development, such as Droplet Digital PCR (ddPCR), generate precise and reproducible results while facilitating the absolute quantification needed for gene edit confirmation so that appropriate clones are promptly identified and taken forward.
Optimisation of protein purification processes
Purification and downstream processing workstreams aim to rapidly yield elevated concentrations of high-purity target protein, at the lowest possible cost, without compromising quality. Antibody purification from stable cell lines usually comprises a multistep workflow focusing on resin-based chromatography techniques to remove host DNA and proteins as well as various isoforms and aggregates of the target molecule. Ideally, resins should have excellent binding capacity and require minimal between-step dilutions with the ability to withstand high flow rates and varying pH conditions. Conventional Protein A resins offer the required purification and specificity properties, although they are expensive and can be associated with ligand stability issues. Ion exchange chromatography provides an effective alternative, efficiently clearing host cell protein and DNA while offering outstanding binding capacity and stability. Ion exchange resins may also be used during the intermediate polishing purification step to enhance binding capacity and remove impurities, closely related species, isoforms and aggregates. These resins maintain capacity under high flow rates and remain stable when exposed to a wide range of pH conditions. Mixed-mode chromatography resins, with the unique ability to combine different types of molecular interactions (e.g. affinity, hydrophobic, ion exchange) via a single-support matrix, are used in the final polishing step to effectively remove multiple product-related impurities. When mixed-mode resins are applied directly after the capture step, the intermediate polishing step may be removed in some cases.
Key considerations for effective therapeutic antibody development
- The quality of the final antibody product is dependent on the effectiveness, reliability and sensitivity of the scientific reagents used at each stage, from early development through to long-term manufacture
- Access to a wide variety of antibody reagents and formats enables greater flexibility of experimental design during the development pathway
- Antibody generation methods should ensure specificity, reliability and reproducibility (as provided by in vitro antibody phage display)
- Processes that appropriately leverage high-throughput automated technologies will be improve efficiency and accuracy across workstreams
- Genetically stable and prolific cell lines are required for reliable long-term production of therapeutic antibodies
- Resin-based protein purification techniques can be lengthy and expensive. Alternatives to conventional Protein A resins (e.g. ion exchange chromatography, mixed-mode chromatography) may provide time and cost efficiencies while enhancing product purity
Planning for success from the start of development
Newer antibody-based therapeutics, such as ADCs and bispecific antibodies, have inspired novel avenues for development and growth in the fiercely competitive area of biopharmaceuticals. Mounting confidence regarding the clinical use of antibody-based therapies and an evolving understanding of the true versatility of these treatments has prompted rising demand for antibody products against a wide range of therapeutic targets. To meet the challenges associated with developing such highly complex molecular products and avoid costly unnecessary delays along the increasingly demanding regulatory pathway, developers require high quality and reliable antibody reagents in multiple formats. Upfront planning and investment in the most relevant and appropriate technologies, cell lines and processes will streamline and optimise the pathway to reap the most rapid rewards and returns. Harnessing automation and innovations in recombinant antibody production provides greater accuracy, reliability, sustainability and efficiency from early development through to long-term manufacture. Systems that have inbuilt robustness and reproducibility at each step in the pathway provide the best chance of reaching timely regulatory approval, bringing us closer to the next ground-breaking treatment.
About the authors
Amanda Turner is Senior Product Manager for Custom Antibody Products at Bio-Rad. She is responsible for new product development and marketing for the Custom HuCAL Antibody Generation Service and the Anti-Biotherapeutic Antibody portfolio. She received an MA in Biochemistry from The University of Oxford, UK, and has held technical sales, business development, and marketing positions in several companies in the life science industry, before joining Bio-Rad in 2012.
Dr Laura Moriarty is Senior Marketing Manager at Bio-Rad, where she supports scientists working in translational research across all life sciences, from academia into pharma. Laura joined Bio-Rad in 2007 following a Post-Doctoral Fellowship at UCSF, San Francisco. Laura has an undergraduate degree and PhD in Biochemistry.
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