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Using Flow Imaging Microscopy to Monitor Protein Degradation and Protein Agglomeration Formations in Pharmaceuticals

David Rhodes Protein Sciences Corporation using FlowCamProtein Sciences Corporation is a biotech firm based in Meriden, Conn. Their stated 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 FDA approval last year. Protein Sciences also supports investigators and product developers in academia, government, and the scientific community for disease research through their Research Antigens business. They also offer a product development and manufacturing service called GeneXpress, that produces vaccines, therapeutics and gene therapy products for clients and partners.

All of this big innovation means they have to understand very small particles.

The Challenge 

David Rhodes is in the formulation and analytical development group at Protein Sciences. In addition to testing and developing products, Rhodes and his group are responsible for investigations that involve the biochemical or biophysical analysis necessary to evaluate product stability.  Part of this process involves testing for sub-visible particles. 

Some methods of particle analysis are validated using conventional microscopy, however Rhodes wanted to compare the results to another method that looks at sub-visible particles utilizing dynamic imaging particle analysis. 

One of the common tests to determine the size and number of particles in injectable formulations is light obscuration. In this method, a sample passes through a narrow channel and interrupts a beam of light casting a shadow on a reactive surface - the larger the particle, the larger the shadow.  As the particle casts a shadow, the intensity and size of the shadow is measured.  It's an indirect measurement that provides count and estimated particle size.  However, it offers little insight on the nature of the particles, morphology or images. Microscopy alone offers its own set of challenges.  Using this method, a drop of the sample is placed on a slide and observe under a microscope while particles are manually counted.   

“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. 

The Solution 

One of the ways proteins can decompose is when they form aggregates.  It is 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 researching several 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. Additionally it can characterize protein 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 the better part of a day.”  Rhodes estimates that this translates to about a tenfold savings in laboratory costs for Protein Sciences. 

The Results 

The FlowCam is now used at Protein Sciences for routine testing for sub-visible particles in bulk drug substances and drug products. When they are introducing a new formulation or process conduct a stability study, they include the FlowCam 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.”



Learn more!

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Topics: Protein Therapeutics, FlowCam Technology, Biopharmaceutical Research