The only group of aggregates that has maximum allowable limits based on USP <788> (also European Pharmacopeia [Ph. Eur.] 2.9.19 and Ph.Eur. 2.9.20), is the group subvisible particles. Some manufacturers have initiated efforts to optimize alternative methods for evaluating subvisible particles to reduce the volume of sample required, and limitations that USP <788> may pose when analyzing high-concentration, highly viscous protein solutions using the recommended light obscuration and microscopy methods.²⁷ However, the USP testing for sub-visible particles is designed to mitigate the risk associated with the presence of extraneous particles in injectable solutions that might lead to blood-vessel occlusion and were not intended to address safety issues potentially associated with large protein aggregates. Visible particles have only qualitative specifications according to USP <788>, which indicates that the injectable sterile solution should be essentially free from particulate matter that can be observed with visual inspection. Because of the subjective nature of visual inspection, care must be taken by manufacturers when routine analysis of visible particles is performed.

REGULATORY GUIDANCE ON AGGREGATION

A specific regulatory guidance documents on biopharmaceutical protein aggregates is currently not available. The International Conference on Harmonization (ICH) has put forth guidelines that deal with drug product and drug substance impurities (ICH Q5C, Q6B, Q1AR). Stability guidances deal with the evaluation of impurities as part of the stability program during long-term, stress, accelerated, and photonic exposure (ICH Q1 series), and comparability guidances consider impurities during manufacturing changes (ICH Q5e). These guidances offer general guidelines for the evaluation of impurities, but do not specifically address aggregates as a separate issue in protein-based pharmaceutical manufacturing.

CONCLUSIONS

There are important considerations to be made when assessing aggregates. Aggregates in protein-based pharmaceuticals can be viewed as an evolving mixture of various types, large and small, that actively undergo transitional equilibrium states. At any given time, the population of such a mixture may reach a new equilibrium and advance toward even larger aggregate structures. The dynamics of a protein aggregate mixture are complex and require multiple analytical methods for detection, evaluation, and monitoring.

Formulation remains a critical aspect of protein stabilization, and an important step in protein aggregate minimization. In the same way that chaperones assist endogenous proteins to maintain their folded state, the right combination of excipients, pH, and temperature keeps the protein-based pharmaceutical from assuming aggregate-permissive states.

A successful assessment of aggregates in protein-based pharmaceuticals rests on a science-driven, risk-based program that evaluates the protein stability profile and determines the impact that a given aggregate mixture will have on the safety and efficacy of the drug product during its lifecycle. Knowledge of the potential degradation pathways that a protein will encounter in every environmental exposure during its shelf-life sets the basis for a well-characterized protein product. This knowledge should be linked to preclinical, clinical, and manufacturing experience to have a better understanding of what types and quantities of aggregates should be allowable.

DISCLAIMER

The views offered in this article represent the author's personal perspective on the issue and should not be construed as regulatory policy or guidance.

ACKNOWLEDGEMENTS

The author would like to thank Steve Kozlowski, Kathleen Clouse, Patrick Swann, Barry Cherney, and Chana Fuchs from the Office of Biotechnology Products at the Center for Drug Evaluation and Research, FDA, for their insightful comments.