Biological medicines have transformed the treatment of numerous diseases thanks to their ability to act on specific therapeutic targets. However, their manufacturing and storage present challenges that are not typically encountered with chemically synthesised drugs. One of these challenges is the formation of protein aggregates, a phenomenon that can compromise product stability and alter its characteristics.
Monoclonal antibodies, recombinant proteins and certain therapeutic hormones are examples of medicines whose quality depends, among other factors, on maintaining an appropriate molecular structure throughout their lifecycle.
What are protein aggregates?
Protein aggregates form when two or more protein molecules associate with each other and form larger structures. These associations can be reversible or irreversible and may occur through non-covalent interactions or covalent bonds.
Under normal conditions, proteins have a three-dimensional conformation that is essential for their biological activity. When this structure is altered, regions that promote interactions between molecules and the subsequent formation of aggregates may become exposed.
Protein aggregates can be classified according to their size. They include soluble species, subvisible particles and visible aggregates, which may appear as turbidity or precipitates.
Their presence cannot always be detected through visual inspection. Therefore, analytical testing is essential for assessing the quality of biological medicines and characterising the different species present in a formulation.
Main causes of protein aggregation
The formation of protein aggregates can begin during manufacturing and continue during storage, transportation or preparation of the medicine prior to administration.
Factors that can promote this process include temperature variations, changes in pH and agitation conditions.
Exposure to elevated temperatures can cause conformational changes, while freeze-thaw cycles can generate local concentrations of proteins and excipients that promote their association. The surfaces with which the product comes into contact can also play a role. Air-liquid interfaces, packaging materials and certain components of drug delivery devices may contribute to protein destabilisation.
Protein concentration is another important variable. Highly concentrated formulations, which are common in some medicines administered subcutaneously, present specific challenges related to molecular interactions and viscosity.
In addition, chemical modifications such as oxidation or deamidation can affect the stability of certain proteins and promote aggregation, depending on the characteristics of each product.
How do protein aggregates affect medicine quality?
Aggregation is considered a critical quality attribute when it can influence the safety, efficacy or stability of a medicine.
One possible consequence is a loss of biological activity. If molecules undergo conformational changes or become associated into structures that hinder their interaction with the therapeutic target, the behaviour of the product may be altered.
Immunogenicity is also a concern. Certain aggregates may contribute to triggering unwanted immune responses, although the risk depends on multiple factors, including the type of protein, the characteristics of the particles and the conditions of administration.
Not all protein aggregates behave in the same way or necessarily present an equivalent clinical risk. Their assessment should therefore take into account the specific characteristics of each medicine and the conditions under which aggregation occurs.
The presence of particles can also affect the appearance of the medicine and compromise compliance with its quality specifications.
Analytical techniques for detecting and characterising protein aggregates
The control of protein aggregates requires a combination of methods capable of detecting species of different sizes.
Size-exclusion chromatography (SEC) separates components according to their hydrodynamic behaviour and is particularly useful for quantifying certain soluble high-molecular-weight species.
Dynamic light scattering (DLS) provides information on particle size distribution in solution and can be used as a complementary tool to investigate the presence of larger species.
Techniques such as flow microscopy and light obscuration can be used to study subvisible particles, depending on the characteristics of the formulation and the size range of interest.
No single technique provides a complete characterisation of protein aggregation. Method selection should take into account the limitations of each technique, the required sensitivity and the specific properties of the medicine.
Combining different techniques provides a more comprehensive view of the aggregation profile and contributes to the assessment of the quality and stability of biological medicines.
How to prevent the formation of protein aggregates
Preventing protein aggregates begins with the development of an appropriate formulation. The selection of pH, buffer systems and excipients can help maintain protein stability throughout the product lifecycle.
Manufacturing conditions must also be controlled, unnecessary mechanical stresses should be minimised, and compatibility between the product and its packaging should be assessed.
Stability studies help establish storage conditions and determine how aggregation evolves over time, following the principles established for biotechnological and biological products in the ICH Q5C guideline on stability testing.
Controlling variables such as temperature, agitation, transportation conditions and freeze-thaw processes is therefore essential for maintaining medicine quality.
Frequently asked questions
- What are protein aggregates? Protein aggregates are structures formed when two or more protein molecules associate with each other. They can be soluble, subvisible or visible and may form as a result of changes in protein structure or stability.
- Why do protein aggregates form in biological medicines? Protein aggregates can form during manufacturing, storage, transportation or preparation of the medicine. Factors such as temperature, pH, agitation, freeze-thaw cycles and contact with certain surfaces can promote their formation.
- How do protein aggregates affect medicine quality? Depending on their characteristics and concentration, protein aggregates can affect the stability or biological activity of a medicine and, in certain cases, contribute to unwanted immune responses. Their impact should be assessed based on the specific characteristics of each product.
- How are protein aggregates detected? The detection and characterisation of protein aggregates requires a combination of analytical techniques. These include size-exclusion chromatography (SEC), dynamic light scattering (DLS), flow microscopy and other techniques used to characterise subvisible particles.
- What is SEC and what is it used for when analysing protein aggregates? Size-exclusion chromatography (SEC) separates species present in a sample according to their hydrodynamic behaviour. It is particularly useful for detecting and quantifying certain soluble high-molecular-weight species associated with protein aggregation.
