What is Antibody Stability?

Several factors can affect an antibody’s stability, including protein structure and concentration, temperature, interfaces, light exposure, contaminants, and agitation. This is of particular importance when developing antibody therapeutics for their immunogenicity potential. The instabilities may be either chemical, physical, or a combination of both.¹

Chemical Instability: Oxidation, Deamination, and Fragmentation

The most prevalent form of chemical instability in antibodies is oxidation. This process can cause the formation of disulfide bonds, which leads to protein aggregation. Oxidation can occur at any stage of drug development, from production and formulation to storage and delivery. If not addressed, these aggregates can compromise the efficacy of the antibody, potentially leading to batch failures during product release. In the therapeutic context, such aggregates may trigger unwanted immune responses, posing risks to patient safety.²

Deamination and fragmentation are other common forms of chemical instability. Deamination typically involves the conversion of asparagine to aspartic acid, which can alter the protein’s structure and function. Fragmentation, on the other hand, breaks the antibody into smaller, often less functional parts, which can further exacerbate stability issues. These chemical changes can occur due to environmental factors such as temperature fluctuations or improper storage conditions, underscoring the importance of controlled environments during every stage of development and use. 

Physical Instability: Denaturation and Aggregation

Physical instabilities primarily involve denaturation and aggregation. Denaturation occurs when the protein loses its natural conformation, often due to changes in temperature, pH, or chemical environment. Once denatured, the antibody can lose its ability to bind its target antigen, rendering it ineffective. 

Protein aggregation, a significant concern in antibody therapeutics, is not only an indicator of instability but also a potential safety risk. Aggregated proteins can enhance the immunogenicity of the antibody, meaning the immune system might recognize the therapeutic antibody as foreign and mount an immune response against it. For therapeutic antibodies, this is particularly concerning as it could reduce the treatment's effectiveness and cause adverse reactions in patients.²

Stability of Antibody Derivatives: A Unique Challenge

As therapeutic and diagnostic applications evolve, smaller antibody derivatives, such as single-chain variable fragments (scFvs) and VHH antibodies, are becoming more popular due to their increased tissue penetration, faster clearance, and ease of production. These derivatives, however, can be less stable than their full-sized counterparts because they lack the rigid framework of the intact antibody. 

Protein stability issues in these derivatives are a challenge because the absence of stabilizing structures makes them more susceptible to denaturation, proteolysis, and aggregation. To address this, researchers and developers often employ several strategies: 

• Protein Engineering: Stability improvements can be achieved by sequence modification, such as swapping domains or grafting complementarity-determining regions (CDRs) onto more stable scaffolds. Insertion of additional disulfide bonds can also enhance structural stability. 

• PEGylation and Conjugation: Attaching polyethylene glycol (PEG) chains or conjugating the antibody derivative to other molecules can improve solubility and protect against enzymatic degradation. These modifications are commonly used to extend the half-life of the derivative in circulation and reduce immunogenicity. 

• Chemical Modification: Stabilizing the antibody derivative through chemical modification, such as introducing covalent modifications, can prevent unwanted degradation or aggregation.³

Optimizing Formulation for Stability

Devising a formulation to maximize protein stability and ensure a long shelf-life requires careful choice of buffers, additives and excipients. Common protein stabilizers include 5% (w/v) BSA, suitable pH, protease inhibitors, and a bacteriostatic agent: 

• Bovine Serum Albumin (BSA): Often used at 5% (w/v), BSA acts as a protein stabilizer by preventing the surface adsorption of antibodies, thereby reducing aggregation. 

• Protease Inhibitors: These prevent degradation by proteolytic enzymes, which could otherwise break down the antibody during storage or after administration. 

• Buffer Systems: Maintaining an appropriate pH level is crucial to avoid pH-induced denaturation or aggregation. Buffers such as phosphate-buffered saline (PBS) are frequently used. 

• Another important consideration is the addition of bacteriostatic agents, which prevent microbial contamination in formulations, especially for antibodies intended for therapeutic use. 

Proper Storage Conditions

The storage conditions of an antibody, particularly its temperature, container, and physical state (wet or dry), can impact its stability. Antibodies are typically stored frozen to slow degradation processes, but they must be handled carefully to avoid damage during freeze-thaw cycles. Slow and gentle thawing, as well as using small aliquots to minimize the number of freeze-thaw cycles, can prevent the formation of aggregates. 

When stored in a liquid state, antibodies may require specific buffers to maintain their activity, while dry formulations often exhibit better long-term stability. Lyophilization (freeze-drying) is commonly used for antibody preparations intended for long-term storage, as it reduces the risk of aggregation and degradation that can occur in solution. However, rehydration conditions need to be carefully controlled to avoid protein damage. 

Implications for Therapeutics and Diagnostics 

The stability of antibodies and their derivatives is a critical consideration for their development as therapeutics and diagnostic tools. In therapeutics, unstable antibodies can not only lose efficacy but also pose safety risks, particularly through unwanted immune responses. For diagnostics, unstable antibodies can lead to inconsistent results, reducing the reliability of tests. 

References: 

1. Basle, Y., Chennell, P., Tokhadze, N., Astier, A., & Sautou, V. (2020). Physicochemical Stability of Monoclonal Antibodies: A Review. Journal of Pharmaceutical Sciences, 109(1), 169–190. https://doi.org/10.1016/J.XPHS.2019.08.009

2. Wang, W. (2005). Protein aggregation and its inhibition in biopharmaceutics. International Journal of Pharmaceutics, 289(1-2), 1-30. https://doi.org/10.1016/j.ijpharm.2004.11.014

3. Ma, H., Ó’Fágáin, C., & O’Kennedy, R. (2020). Antibody stability: A key to performance - Analysis, influences and improvement. Biochimie, 177, 213-225. https://doi.org/10.1016/j.biochi.2020.08.019