Understanding Monoclonal Antibody Stability and Strategies to Combat Aggregation

Monoclonal antibodies (mAbs) have transformed the therapeutic landscape, offering treatments for cancer, autoimmune disorders, and infectious diseases. Despite their success, aggregation remains a persistent challenge in their development and formulation. Aggregates not only reduce efficacy but also pose significant safety risks, including immunogenicity and adverse immune responses. Developing robust strategies to combat aggregation is essential for ensuring the long-term stability and effectiveness of mAbs.

Molecular Mechanisms of Protein Aggregation

Aggregation is driven by the destabilization of the antibody's native structure, exposing hydrophobic regions that promote self-association. This process typically follows three stages:

1. Conformational Change: Native monomers adopt aggregation-competent intermediates due to structural fluctuations.

2. Nucleation: Small aggregates form and act as seeds for further growth.

3. Aggregate Growth: Monomers and smaller aggregates combine into larger species.

Stress conditions such as temperature changes, pH shifts, agitation, and high ionic strength exacerbate aggregation. For example, thermal stress promotes unfolding, while agitation at air-water interfaces can lead to hydrophobically driven aggregation.¹

Accelerating Aggregation Assessment for Long-Term Stability

A recent study introduced a platform that uses temperature-dependent aggregation data to predict long-term stability, significantly reducing the time required for formulation optimization. Traditional stability studies rely on stress conditions (e.g., elevated temperatures) to accelerate aggregation. However, this approach often fails to accurately predict long-term behavior at storage temperatures.²

The novel platform uses kinetic and thermodynamic analysis to predict aggregate fractions for up to three years based on short-term data. By studying six antibodies across therapeutic areas like oncology, rheumatology, and osteoporosis, researchers demonstrated that this approach captures aggregation mechanisms across pharmaceutically relevant temperatures and concentrations.

Intrinsic and Extrinsic Factors Affecting Aggregation

Intrinsic Factors:

The sequence and structural properties of mAbs influence their aggregation propensity. Aggregation “hotspots,” often located in the Fab or Fc regions, are solvent-exposed residues prone to initiating self-association. Engineering efforts can mitigate these vulnerabilities, with computational tools like TANGO and CamSol identifying and optimizing aggregation-prone regions.

Extrinsic Factors:

• Temperature: Elevated temperatures destabilize antibodies, accelerating aggregation. High temperatures also reduce activation energy, favoring faster nucleation and growth events.

• pH and Ionic Strength: Extreme pH conditions destabilize antibodies, particularly in the CH2 domain, while ionic strength influences electrostatic interactions.

• Agitation and Freeze-Thaw Cycles: Mechanical stress at interfaces and repeated freeze-thaw cycles can generate aggregates, with hydrophobic interactions playing a key role.

Strategies to Combat Aggregation

1. Antibody Engineering:

• Sequence Modifications: Introducing charged residues in aggregation-prone regions reduces self-association.

• Computational Design: Tools like AGGRESCAN and SAP guide sequence optimization for improved solubility and reduced aggregation risks.

• Peptide and Polymer Tags: Additions like PEGylation or P17 peptide tags enhance stability and solubility, particularly for scFv-based therapeutics.

2. Formulation Optimization:

• Buffers: Histidine buffers are preferred for stabilizing pH near 6, minimizing aggregation.

• Excipients:

• Sugars (e.g., sucrose, trehalose): Stabilize proteins by reducing hydrophobic interactions.

• Amino Acids (e.g., arginine): Decrease protein-protein interactions and viscosity.

• Surfactants (e.g., polysorbates): Prevent aggregation at air-water interfaces.

• Temperature Management: Gradual pH adjustments during freeze-thaw cycles minimize aggregate formation.

3. High-Volume Delivery Systems:

Emerging technologies, such as on-body delivery systems (OBDS), support low-to-moderate concentration formulations, reducing risks associated with high-concentration mAbs. These systems are particularly advantageous for subcutaneous administration.

Aggregation remains a central challenge in monoclonal antibody development. However, with innovations in kinetic modeling, computational tools, and formulation strategies, the industry is well-equipped to tackle these obstacles, ensuring the stability, efficacy, and safety of next-generation antibody therapeutics.

Biointron provides antibody products for in vivo research at Abinvivo, where we have a wide range of Benchmark Positive Antibodies, Isotype Negative Antibodies, Anti-Mouse Antibodies, Bispecific Antibodies, and Antibody-Drug Conjugates. Contact us to find out more at info@biointron.com or +86 400-828-8830 / +1(732)790-8340.

References:

1. Pang, K. T., Yang, Y. S., Zhang, W., Ho, Y. S., Sormanni, P., Michaels, T. C., Walsh, I., & Chia, S. (2023). Understanding and controlling the molecular mechanisms of protein aggregation in mAb therapeutics. Biotechnology Advances, 67, 108192. https://doi.org/10.1016/j.biotechadv.2023.108192

2. Marko Bunc, San Hadži, Graf, C., Matjaž Bončina, & Lah, J. (2022). Aggregation Time Machine: A Platform for the Prediction and Optimization of Long-Term Antibody Stability Using Short-Term Kinetic Analysis. Journal of Medicinal Chemistry, 65(3), 2623–2632. https://doi.org/10.1021/acs.jmedchem.1c02010