Maximize success in biotherapeutic antibody development

The increasing application of antibodies for disease treatment has sparked a surge in demand for novel antibody formats, designed to enhance therapy efficacy and hit previously ‘undruggable’ targets. For those working in biotherapeutic antibody development, the process of generating a clinical lead molecule and optimizing candidates holds a central role, but there are many aspects to consider to reduce the risk of a failure later in the development program and to maximize success.

As biotherapeutic discovery and development processes become more integrated, the role of analytical technologies as guiding tools and decision-making aids is ever increasing¹. Biacore™ SPR systems by Cytiva for example are designed to deliver true, real-time, and label-free interaction analysis data, which plays a valuable role in biotherapeutic antibody discovery and development, finding widespread application throughout the entire development process, from candidate selection to clinical lead.

In this SelectScience^® article, we explore this journey to generating a suitable clinical lead molecule, assessing the impact of candidate optimization on the downstream translational potential of novel therapeutics.

Navigate the journey: Antibody generation to clinical lead

The landscape of biotherapeutic antibody development has undergone significant transformations, with a shift in focus from solely considering potency and functional aspects towards incorporating developability aspects early in the process. While factors like specificity, affinity, and kinetics for the molecular targets remain crucial, understanding and addressing developability aspects have become increasingly vital to mitigate risks and avoid failures in later stages of the development program and translational journey².

Developability studies encompass a range of investigations that aim to ensure the robustness and success of antibody development. They examine key areas of interest, such as the impact of post-translational modifications on the stability and conformation of antibodies^3,4. By studying these modifications, researchers can optimize the antibody’s attributes and ensure its desired functionality.

Another crucial area of investigation is the evaluation of aggregation and fragmentation tendencies of antibodies⁵. Understanding and mitigating these tendencies is essential to maintain the stability and efficacy of the antibody product. Researchers also must assess the solubility and solution stability of antibodies, which are critical for formulation and delivery considerations⁶. Ensuring that the antibody remains soluble and stable throughout its intended shelf-life is vital for its successful clinical application.

Furthermore, developability studies address immunogenicity and pharmacokinetic properties⁷. They aim to identify any potential immunogenic responses triggered by the antibody and assess the antibody’s absorption, distribution, metabolism, and excretion. These factors play a pivotal role in determining appropriate dosing regimens and optimizing the therapeutic potential of the antibody. By addressing these developability aspects, researchers can begin to answer fundamental questions about the feasibility of antibody candidates.

‘Developability’ assesses factors such as manufacturability, safety, bioavailability, and efficacy.

• Manufacturability refers to the ability to efficiently produce the antibody at a reasonable scale and with consistent quality, ensuring it meets the demands of clinical applications.

• Safety evaluations are crucial to identify any potential risks or adverse effects associated with the administration of the antibody to patients.

• Assessing the bioavailability and efficacy of the antibody helps determine its ability to reach its target site and exert the desired therapeutic effect.

Optimize antibody candidates: Efficient strategies for selection

Striving to support the biotherapeutic antibody development process, Biacore systems from Cytiva can play a vital role in multiple stages, ranging from screening candidates to antibody engineering and final development. These systems have consistently been utilized to assess the specificity of binding, characterize interactions between antibodies and antigens or Fc receptors, and provide guidance for achieving a clinical lead.

In developability studies, Biacore SPR systems have proved valuable in monitoring the effects of forced degradation on antigen and Fc gamma receptor binding, as well as evaluating pharmacokinetic properties associated with antibody half-life through binding to FcRn. Other recent advancements also include the use of binding mode-specific reagents to detect changes in antibody topography resulting from forced degradation⁸. By offering these capabilities, Biacore systems can contribute significantly to the comprehensive understanding and optimization of antibodies during their development journey.

Explore Biacore systems’ utility for:

• Interaction analysis
• Assay development
• Biotherapeutic characteristics
• Kinetic analysis
• Epitope binning experiments
• Efficacy prediction

Advance analysis: Accelerating antibody development

Biacore SPR systems offer several advantages for efficient interaction analysis with multiple target molecules. Through its flow system design, a single sample injection enables direct analysis. Active analysis is carried out in one dedicated measuring spot, while another spot serves as a reference. Depending on Biacore SPR system, one to eight active/reference pairs can be utilized.

Each system's sample throughput is closely tied to the number of microwell plates that can be handled in an automated run. Systems with higher throughput capabilities are particularly beneficial for antibody screening and large-scale epitope binning experiments. Additionally, specific features of certain systems make them well-suited for detailed characterization studies. Nevertheless, there is significant overlap among the different Biacore SPR systems, as they can be used for both screening and characterization purposes.

With a variety of sensor chips and ready-to-use protocols, assay development becomes rapid and convenient with Biacore SPR systems. The systems support antibody titer, concentration measurements and kinetic analysis of antibody-antigen interactions through sensor chips like Protein A, Protein G, Protein L, PrismA, and CM5. Specific capture kits are available for capturing different antibody subclasses and fragments. Biotinylated reagents can be easily captured using specific sensor chips or the Biotin CAPture Kit. These features enhance the efficiency and versatility of the systems in biotherapeutic antibody development and analysis.

Biotherapeutic characteristics: Gain deeper insights

Early assessment of expression levels, target specificity, and binding stability is crucial for clone selection, especially when dealing with a large number of samples from hybridoma cells or recombinant expression systems. ELISA-based methods are commonly utilized during screening stages to provide end-point results, despite the often crude and limited volume of the samples.

Biacore SPR systems enable the comprehensive monitoring and quantification of antibody-antigen interactions, with the added advantage of directly injecting samples from cell culture supernatants. The sequential injections of antibody and antigen provide valuable information about expression levels, interaction rates, stability, and even stoichiometric details, while further examination of the buffer flow phase can reveal the nature of the interaction (monophasic or biphasic).

When dealing with many sensorgrams, it is convenient to condense the results into plots of report points. These plots highlight regions of stability and enable rapid identification of binders with slow off-rate. By utilizing single injections of antibody and antigen, Biacore SPR systems provide high-content information, and the straightforward data analysis can be focused on the desired binding properties.

Enhance antibody selection and characterization: Epitope binning and kinetic insights

In antibody selection, antigen binding is an important criterion, but additional considerations include epitope specificity and the desired biological effect⁹. Epitope binning experiments on Biacore SPR systems allow for the identification and grouping of antibodies with similar epitope specificity. By testing selected antibodies against each other, a result matrix is generated, indicating shared epitopes and potential therapeutic or assay applications. To further understand epitopes, inhibition mapping can be performed by pre-incubating antibodies with antigen domains or peptides to observe any inhibition of antibody binding.

Kinetic data plays a crucial role in understanding the affinity, and stability of binding interactions. It provides insights into target occupancy, drug residence time, and potential off-target effects. Additionally, kinetic analysis aids in humanization and re-engineering efforts by assessing how structural changes impact binding properties. It is also utilized in developability assays to study the effects of forced degradation, estimate antibody half-life, and assess binding specificity through thermodynamic analysis. Biacore™ 8K and Biacore™ 8K+ SPR systems offer the capability to perform kinetic and concentration analysis simultaneously.

Improve efficacy prediction for candidate selection

The interaction between antibodies and the neonatal Fc-receptor, FcRn, plays a role in determining antibody clearance and half-life. Antibodies taken up into endosomes can be protected from degradation and recycled by binding to FcRn, which occurs at pH 6.0. The antibody is then released at pH 7.4 upon returning to the cell surface. Understanding and manipulating this interaction is important for enhancing antibody efficacy and optimizing dosage in antibody engineering projects.

The pH-dependence of antibody-FcRn interactions can be studied using Biacore SPR systems by capturing biotinylated FcRn to streptavidin and maintaining the running buffer at pH 6.0. Antibody binding and dissociation at different pH conditions can be monitored by injecting the antibody at pH 6.0 and immediately following it with buffer at pH 7.4, allowing for the investigation of both binding and release processes.

Conclusion

As early and late-stage antibody development processes become more integrated, analytical technologies that guide developers and support decision making become key tools in these workflows. Biacore SPR systems deliver the resolution, sensitivity, precision, throughput, ease-of use, and assay versatility required to develop a first candidate into a clinical lead with the critical quality binding attributes required for approved biologics.

The incorporation of developability studies early in the antibody development process is vital for optimizing overall success and reducing the risk of failures in later stages. By assessing post-translational modifications, aggregation tendencies, solubility, immunogenicity, and pharmacokinetic properties, researchers ensure the manufacturability, safety, bioavailability, and efficacy of antibody candidates. These studies provide valuable insights and inform decision-making processes, ultimately contributing to the development of robust and clinically viable antibody therapies.

References

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Brooks, B. D. Miles, A. R. and Abdiche, Y. N. High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge. Drug discovery today, 19(8), 1040–1044 (2014).