Aggregates and Subvisible Particles
Interest in protein aggregates was intensified when patients treated with therapeutic proteins became nonresponders. “Various researchers began to realize that maybe protein aggregates were the problem,” explained John Carpenter, Ph.D., professor of pharmaceutical biotechnology at the University of Colorado School of Pharmacy. “We realized the sweet spot for aggregates was in the subvisible range—probably around 10 microns.”
This is an important number, because all parenteral products have to be assayed for particles greater than 10 microns.
There is also interest in particles less than 10 microns as they may provide interesting quality product attributes that are not being analyzed nor reported in FDA filings. This has given rise to several issues: what is the smallest particle to worry about? is it a parameter that can be controlled? and are these particles an important issue for patient safety?
“The answer is, we’re not sure. If nothing else, it’s a product-quality issue and should be addressed.”
Although there is a lot of analytical work being done on protein particles, there is no definitive answer as to the best method. While particle counting is not difficult, it does have technical challenges that depend on factors like sample handling, and user experience.
Microflow imaging is becoming a popular method to study protein particles. In this technique, a microscope takes digital pictures of a field as fluid flows through it. It counts and averages the number of particles, which provides a distribution of the number of particles versus diameter.
A recent innovation by Graham Milne of Amgen uses video analysis on spun vials or syringes. The video of the particle behavior (sinking or floating), with their size and shape, enables noninvasive counting. This is helpful since all products require 100% visual inspection upon manufacture.
“This method may allow people to have quantitative data across the visible and subvisible, and may eliminate the arbitrary absolute that if you can see it, you can’t use it. By looking at the subvisible particles, companies gain valuable data that helps them to understand visible particles in the context of everything,” Dr. Carpenter noted.
Over the next year, there will be several in vitro and animal-based assays to analyze various aggregates and degraded proteins, he reported.
“In therapeutic proteins, we always worry about chemical modifications and particles are part of that.” However, he says, “it will be awhile before we really understand how to measure and control particles in products as a quality attribute. I don’t see anyone setting hard specifications for particles smaller than 10 microns for a while.”