Starting with the first protein drug, insulin, in 1982, recombinant proteins have played a vital and growing role in the treatment of human disease.Yet inherent to the production of protein drugs is the possibility of aggregation, which may increase the risk of an immunogenic response to the dosed drug.
The expression, purification, formulation, and storage of protein therapeutics can result in the formation of dimers, trimers, and multimer aggregates that can range in size from the nanometer range all the way up to visible particles of 100 microns or larger. There is no single method capable of measuring and quantifying aggregates throughout this entire size range.
Although all biotherapeutics contain some level of aggregates, there has been no direct demonstration that aggregates are a major risk factor contributing to immunogenicity. This is the result of the complexities of the potential causes of an immunogenetic response and the inability to measure particulates.
Because protein product aggregates represent a potential risk factor for immunogenicity, industry and the FDA seek a method to accurately count and characterize these particles. The challenges faced in measuring and characterizing these protein aggregates were discussed at the recent “PepTalk: The Protein Science Week” conference.
“Levels of protein aggregates have long been mentioned as a critical quality attribute and have potential clinical consequences. Today we have size exclusion chromatography (SEC) to look at the increase in the dimers, trimers, and oligomers of proteins. On the other extreme we look at particles visible to the naked eye that are 80–100 microns or larger.
“Despite its drawbacks, light obscuration is the current accepted method for monitoring of subvisible particulates during lot release. So we are missing the gap between what we can detect with SEC and light obscuration and what the human eye can see,” said Danny Chou, Ph.D., chief scientific officer, NorthStar Bio.
“The quality of a protein pharmaceutical is defined by the analytical capability that is available. In the last few years the regulatory agencies have been making a push for the industry to assess subvisible particles (SVPs) and their relevance to product quality, as well as to evaluate the technologies that can be used to measure them.”
According to Dr. Chou, NorthStar Bio is the first company created to better understand SVPs. Functioning as a CRO, NorthStar Bio can characterize protein production samples using state-of-the-art microflow imaging, fluid imaging particle analysis, the Coulter principle, and other emerging technologies.
“These orthogonal technologies will be used to characterize SVPs in a sample, and to illustrate how to use the technology to develop protein formulations and decide which technology can better address regulatory concerns,” he continued.
“In five years we will have a more diverse set of analytical tools as well as a much better understanding of what type of particles we need to control to minimize immunogenicity in the wide range of protein product types.”
Subvisible particles can be quite heterogeneous and can come from a variety of sources, either externally from the production environment or from the protein itself.
“Ideal samples for detecting particles would consist of a clear, water-like fluid in which individual particles of high contrast and moderate buoyancy are effectively recognized,” said Tobias Frommknecht, formulation scientist, F. Hoffmann-La Roche.
“The color, refractive index, and opalescence of actual product samples can provide erroneous data.” To study the effect of matrix irregularities on light obscuration and microflow digital imaging (MDI), Frommknecht used a number of artificial matrices and particle standards. Polystyrene and glass particles and pseudo protein particles were added to opalescent, viscous, and colored matrices to access sizing and counting effects of the methods.
“FlowCam (Fluid Imaging Technologies) showed low counting and sizing ability overall, but it has the advantage of higher quality particle images and is really useful for differentiating silicone oil droplets from other particles,” he said. “MDI has not been validated for automated morphological analysis.” New particle standards mimicking protein particles will be needed to assess the limits of these technologies.
Sizing and counting by the light obscuration method was only slightly affected by the nonideal sample conditions. “Light obscuration remains the gold standard for quantification in a QC environment. Both light obscuration and MDI are used during formulation development to get the most information on our product.”