The very forces critical to creating the three-dimensional structure of proteins are also responsible for unwanted and often dangerous protein aggregates in protein-based therapeutics. The presence of such aggregates can mean the discontinuation of potentially critical therapeutic products. The FDA has advised in its 2014 Guidance for Industry that "It is critical for manufacturers of therapeutic protein products to minimize protein aggregation to the extent possible,'' while acknowledging that the technology does not currently exist to accomplish this goal, particularly in the critical range from 500 nm to 10 microns.
Total Holographic Characterization® enables the detection and monitoring of protein aggregation.
Figure (A): From this typical plot of particle size, it is impossible to tell how many species of particles are in this sample.
Is it all silicone oil from vials and pipette tips? Is it potentially dangerous protein aggregates? Is it both? When particles of different species but similar sizes are mixed in one sample, a particle sizer alone is not enough to properly characterize the mixture.
Figure (B): THC provides users with an additional dimension of information: particle refractive index. The refractive index varies with particle composition and allows THC to tell particles apart even when they are the same size.
Figure B is a distribution of particle refractive indexes of the same sample as in Figure A. The right peak corresponds to silicone oil droplets and the left peak corresponds to protein aggregates.
Figure C combines the size density plot and the refractive index density plot, shown above, with a scatter plot of the same data set. In the scatter plot, the horizontal axis is the particle diameter in micrometers and the vertical axis is the particle refractive index. Each point represents a single particle analyzed with THC during the measurement. The color of each point represents the density of points in the region, with warmer colors (yellow) representing higher density regions and cooler colors (blue) representing lower density regions.
This figure summarizes data from nearly 4000 particles, about half of which are silicone oil droplets and half are protein aggregates.
The diameters of all particles, represented by all of the points on the scatter plot, create the distribution in (A). Similarly, the refractive indexes of all of the points create the distribution in (B).
The following size and index density distributions show the results of 16 independent THC measurements from xSight of a sample containing a mix of IgG aggregates and silicone oil in water. All of the curves show excellent reproducibility. In this sample, the concentration of IgG aggregates is 2.9 ± 0.3 x 105 particles/mL and that of oil droplets is 6.6 ± 1.0 x 105 particles/mL.
The results below show the effect of ejecting an IgG sample from a standard syringe with different force and duration. Faster ejection causes increased shear forces, which elute silicone oil from the surfaces of syringes and simultaneously result in higher protein aggregation. Size alone does not distinguish the protein aggregates from the silicone oil. Measurement of the index of refraction using Spheryx’s xSight enables the independent determination of the concentrations of both the silicone oil and the protein aggregates.
In this figure, the peaks on the left are silicone oil droplets and the peaks on the right are IgG aggregates. Since both sets of curves grow with higher ejection rate, the concentrations of silicone oil and IgG aggregates increase concurrently with higher shear forces.
By measuring particle refractive index, THC detects and identifies multiple contaminants even when they are the same size and mixed together in one formulation.
Below is an example of a sample with silicone oil droplets and air bubbles in a sucrose solution. xSight can easily distinguish air bubbles and silicone oil droplets by their index of refraction even though they are the same size and shape.
• protein aggregates
• silicone oil
• air bubbles
• rubber particles
• glass shards
• fatty acid particles
• material fibers
• metal particles
Protein aggregation occurs at almost every stage in the biopharmaceutical pipeline, from development, formulation, and manufacturing to storage and point of use. Thus, there is a pressing and as-yet unmet need for instrumentation that can assess the size distribution and morphology of protein aggregates in this size range. Spheryx’s xSight meets this need.
xSight’s use of Total Holographic Characterization® of protein aggregation provides the detailed data needed for product development, process control and quality assurance in the biopharmaceutical industry and will result in increased efficiency, safety and cost savings in the promising and rapidly growing area of protein-based therapeutics. Whether measuring the changes in aggregation behavior as a function of added excipients during formulation development, or monitoring product degradation over time, xSight delivers reliable biopharmaceutical product characterization.
xSight has the ability to detect and characterize protein aggregates in pharmaceutical formulations. This transformative technology can measure single clusters rapidly enough to be used for in-line characterization of pharmaceutical samples, and offers unprecedented precision and accuracy to monitor aggregation behavior and to distinguish aggregates from similarly sized contaminants. xSight detects contaminants, both particles and emulsion droplets, and delivers accurate, repeatable concentrations of multiple contaminants simultaneously. Spheryx’s xStream provides automated sample delivery from a standard 96-well plate to handle large volumes of samples with walk-away operation. xStream’s robotic sample-handling increases uniformity of sample delivery, and removes many sources of operator error or added contamination.
Contact us for more information on how Spheryx can help with your biologic products.