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.
In general, the manufacturer relies strongly on its research and development group for a comprehensive program that assesses
its drug product aggregates during characterization, manufacturing, and storage of the drug product. In addition, regulatory
agencies encourage implementing aggregate specifications, and have seen a surge in relevant information provided by manufacturers
that is based on sound analysis of their product aggregates. By the time of license application, a manufacturer should be
capable of providing a data-supported justification for its specifications for aggregate levels.
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,
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.
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.
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.