This presentation addresses the complexities and challenges associated with protein aggregation in pharmaceutical products. Ana Sara Cordeiro began by explaining that protein aggregates are highly complex and heterogeneous, varying in size from nanometres to micrometres. These aggregates could change or disappear during their lifecycle, making their evaluation difficult. They could be intrinsic or extrinsic in origin, adding to the complexity of their assessment.
Cordeiro highlighted the significant consequences of protein aggregation, which could compromise product stability and trigger immune responses, including anaphylaxis. She cited an example where a product was withdrawn after causing 49 anaphylaxis reactions and eight deaths, despite its physical-chemical stability remaining intact. This underscored the importance of understanding which aggregate types and quantities could generate an immune response.
She discussed the challenges of monitoring particle sizes, which ranged from 5 nanometres to 150 micrometres. Regulatory focus was on particles above 10 micrometres, but the FDA recommended monitoring smaller particles due to their potential to nucleate further aggregation and immune reactions. No single method could evaluate the entire size range, necessitating the use of multiple complementary techniques.
Cordeiro then described the various stresses proteins undergo during manufacturing, such as shear from pumps and filtration, mixing, and air interfaces, which could induce aggregation. Laboratory studies applied controlled shear, impulse stresses, stirring, heating, and filtration to evaluate their effects on aggregation. She noted that constant shear stress and stirring caused minimal or reduced particle formation, whereas impulse shear and heating significantly increased subvisible particle counts. Raman spectroscopy confirmed conformational changes with impulse and heat stresses.
She discussed the need for comprehensive monitoring of aggregates, especially subvisible and submicron particles, which could impact product shelf life and stability by agglomerating during storage and handling. Multiple orthogonal analytical methods were essential for confirming particle identity and behaviour over time. Cordeiro concluded by stressing the importance of using specific, tailor-made analytical tools adapted to the formulation's needs rather than the analytical method's requirements.
