Microscopy and light obscuration methods are routinely used for detecting and counting subvisible particles according to United States Pharmacopeia (USP) chapter <788>. Both tests apply to small and large volumes, but in general, multiple drug product vial samples are used
during this kind of testing. Normally, samples are first tested by the light obscuration method. If the sample fails the specified
limits, the microscopic assay method can then be used. However, the microscopic method can be the sole test if there is a
documented technical reason or interference from the product undergoing testing that would make the light obscuration method
unsuitable or the results invalid. Turbidimetry measures the opalescence or clarity of a solution against a reference standard.
A combination of light obscuration, turbidimetry, and DLS has been used to evaluate the time course of particle formation
in protein solutions.22
When these methods are correlated to the apparent globular size of the aggregate detected (Figure 1), there is a size gap
in the capability of methods used to detect and monitor small aggregates that range between a few nanometers and about 50
nm in diameter, and the methods used to detect and monitor large aggregates that range between a few micrometers and about
50 µm in diameter. This gap may pose a problem because the ability to detect, measure, and evaluate the fate of some small
aggregates as well as the precursors of larger aggregates may not be implemented in protein aggregate control strategies.
In fact, it has been pointed out that aggregates with apparent globular diameters around 0.5 µm are not routinely tracked
CONTROL OF AGGREGATES
Various approaches are taken by manufacturers to control aggregates in protein-based pharmaceuticals, which depend on the
nature and levels of the aggregate, and their potential impact on the safety, quality, and stability of the specific protein
product. Although a protein drug product may contain aggregates of different natures and sizes than can be controlled by proper
monitoring, some aggregates may require aggressive control strategies depending on their risk assessment and evaluation. In
some cases, controls are geared toward minimizing or inhibiting aggregate formation. Sometimes mutational changes can improve
the stability of a protein product susceptible to aggregation, such as during freeze–thaw.24
Control strategies may also include the addition of excipients. Such is the case for a recombinant human platelet activation
factor that showed aggregate formation by heteronucleation with silica particles on storage. The addition of surfactant pluronic
acid F68, or a change in the pH of the formulated drug product, reduced heteronucleation formation.25 In other cases, control strategies are oriented toward increased monitoring and re-evaluation of the limits and specifications
of the aggregate levels. This is the case with liquid Remicade, which showed higher turbidity than its lyophilized counterpart.
The measures taken were to add a turbidity specification limit, and to tighten monomer specification by gel filtration–HPLC.26
A robust program that studies product aggregates will have the capability to establish limits for product aggregate levels.
Whether aggregate levels are a stability indicator for the particular drug product or whether the aggregate mixture present
in the drug product is assigned as low risk, it is important to accumulate adequate data to support the conclusions. The rationale
for this is that the data accumulated during preclinical and clinical phases of product developmen, together with the study
of the degradation pathways of the protein, will contribute to a better understanding of the impact that aggregates will have
on drug product safety and quality.
There is no consensus on the maximum allowable limit for protein-based pharmaceutical aggregates because some proteins may
be largely stable and safe despite certain levels of aggregates, while for other proteins very small changes in aggregate
levels may profoundly affect protein stability, and even safety. By the time of license application, the manufacturer will
have collected sufficient data to justify a control strategy. These data include results from clinical lots, information from
in vivo and in vitro studies, the product stability profile, the type of aggregates, and their potential impact on product safety and quality.
Usually this background allows a formal analysis that can predict the expected level of aggregates during the complete lifecycle
of the product.