Protein Sciences Corporation is a biotech firm based in Meriden, Conn. Their mission is to “save lives and improve health by responding to the changing world through the creation of innovative vaccines and biopharmaceuticals.” Flublok – its next-generation, eggless vaccine – received Food and Drug Administration approval last year. Protein Sciences also provides products to investigators and product developers in academia, government, and the scientific community for disease research through their Research Antigens business. With GeneXpress, their product development and manufacturing services, they produce vaccines, therapeutics and gene therapy products for clients and partners.
All of this big innovation means you have to understand very small particles.
David Rhodes is in the formulation and analytical development group at Protein Sciences. In addition to testing and developing improved products, Rhodes and his group are responsible for investigations that involve the biochemical or biophysical analysis needed to evaluate the stability of their products, including testing for sub-visible particles.
There are methods of particle analysis that are validated using conventional microscopy, but Rhodes wanted to compare them to another method that looks at sub-visible particles utilizing dynamic imaging particle analysis.
One of the tests for determining the size and number of particles in injectable products is light obscuration. In this method, a sample goes through a narrow channel and the intensity of the light is diminished by the presence of the particles. The dark areas get counted, and the bigger the area, the bigger the particle. This method can count and estimate the size of particles, but offers little insight on the nature of the particles. It’s even more challenging to characterize particles by microscopy alone. Using this method one puts a drop of the sample on a slide to observe under a microscope and manually counts the particles.
“Neither of these methods is very informative,” said Rhodes. “For example, when we were characterizing samples known to contain particles, we weren’t able to adequately characterize the sub-visible particulate. We knew they were there, we could see a little something at the resolution limit of the dynamic light scattering experiment, but we didn't know how much of it was there, or how big it was or anything else.” With the light obscuration method, for example, the error bars can be big. It could be a 1 µm particle or it could be a 20 µm particle and you would not always be able to tell the difference. Rhodes was anxious to have something that worked a lot faster, saw a lot more and could quantitate a lot more easily.
One of the ways that proteins can potentially decompose is to form aggregated protein and it's important that investigators are able to characterize the progression of the potential degradation pathway in their product. Protein aggregates can easily remain undetected, especially if they are less than 20 µm, so it makes for difficult work.
After doing some research on the available instruments on the market, Rhodes determined that an imaging particle analyzer would provide the valuation of product quality he was looking for. He had seen the FlowCAM® dynamic imaging particle analysis system from Fluid Imaging Technologies displayed at conferences and was intrigued by what it could do with aggregated proteins.
The FlowCAM provides information on particle size, concentration and appearance, and it also can characterize agglomerates that are transparent and therefore not detected by light obscuration devices.
“We demoed a couple particle analyzers and we decided on the FlowCAM because of the flexibility in choosing objectives, detection of particles in the 5 µm range or less, and it provided real-time data analysis including particle count and shape,” said Rhodes.
With its VisualSpreadsheet® software, the FlowCAM can record over 30 different measurements per particle and can capture particle images at up to 22 frames per second allowing for high sampling efficiency and fast analysis times. It sorts and filters particle data and immediately displays all similar-type particles. “The testing process is a whole lot easier, quicker, and more informative, in some ways, than the USP <788> testing,” adds Rhodes. “It's a question of taking 15 minutes compared to taking a better part of a day.” Rhodes estimates that this equals about a tenfold savings in laboratory costs for Protein Sciences.
The FlowCAM is now used at Protein Sciences for routine testing for sub-visible particles in bulk drug substances and drug products. When they have a new formulation or process and want to do a stability study, they include the instrument as part of the testing at each point. This step is important when they want to characterize the progression of the potential degradation pathway in a product. “We’ll monitor at one week, two weeks, or a month to determine the progression of particle counts and particle size,” said Rhodes. “We get to see the particle morphology instead of just counting, and that really facilitates the debugging process if we have any issues in developing new products.”
Visit our FlowCAM Protein Aggregate Application page to find more resources, including videos, articles, and technical posters!