Measuring Protein Formulations with ARCHIMEDES
Therapeutic protein treatments can slow the progression of many human diseases, and can also replace critical proteins that are not produced endogenously. In the formulations used to deliver these treatments, however, aggregation of proteins can reduce their effectiveness1. It is therefore essential to monitor aggregation in the development, manufacture, storage, and delivery of therapeutic proteins and other biopharmaceutical formulations2.
To date, most accepted monitoring methods include manual microscopy, light obscuration, chromatography, and microfluidics coupled with automated imaging. These methods are suitable for some aggregation studies, but they are often challenged by protein aggregates that have widely variable transparency and shape, or high concentration. In addition, sub-visible and even submicron-scale aggregates are of growing R&D and quality control interest, but are not accessible to optical methods due to their limited resolution. Finally, protein formulations are often complicated by the presence of leachables such as silicone oil droplets and other foreign particulate matter introduced during manufacture and handling. An ideal analysis method would clearly distinguish such foreign matter from the aggregate content and analyze it independently.
ARCHIMEDES opens a new window on to protein formulations, allowing precise measurement of aggregates with sizes down 100 nm. It is immune to optical and shape variations and opalescence that can hinder optical methods. Its gentle fluidics do not disturb fragile aggregates, and can accommodate viscosities up to 50 cP without dilution. ARCHIMEDES’ sensors have extremely small, picoliter-scale volumes that consume as little as 100 µl of sample, and can accept aggregate concentrations up to ~5x108 per ml, allowing high-concentration formulations to be studied without dilution. The concentration is measured directly since each aggregate is counted, one by one.
The measurement below (left) shows the size distribution of submicron IgG protein aggregates in 4 µl of native sample. The measured concentration was 4x106 aggregates per ml for sizes >300 nm. Because the underlying measurements are of mass, the distribution can be cast as the number of base proteins that make up each aggregate, as is shown in the right plot.
ARCHIMEDES has successfully analyzed protein formulations from a number of major biotechnology companies. The plot at right shows measurements of the concentration of submicron aggregates for a range of samples. As can be seen, the aggregate concentrations for sizes >0.3 µm show a 10-fold increase through the sample series, which roughly corresponds to an aggregation cascade. Submicron aggregate concentration correlates with the formulation quality as defined by the manufacturer.
ARCHIMEDES can also clearly distinguish foreign matter from protein aggregates. For example, in the time trace below, downward-going peaks are caused by aggregates, whose density is greater than that of the suspension buffer. Also visible are upward-going peaks: these are caused by silicone oil droplets, whose density is less than that of the buffer so that their presence reduces the overall mass of the sensor and increases the sensor's resonant frequency. This difference in sign allows ARCHIMEDES to distinguish aggregates from oil droplets with complete certainty, and distributions can be produced for each population, as shown.
Studies with ARCHIMEDES have documented significant increases in submicron silicone oil droplet content from packaging during handling. For example, the plot below shows the increase in total submicron droplet content in parts per billion vs. material and shelf time. Significantly, the overall oil content at submicron sizes can in some cases dominate over larger droplets. Correlation between the presence of such leachables and the formation of aggregates can also be evaluated.
In sum, ARCHIMEDES enables quantitative analysis of subvisible and even submicron content in protein formulations. The information it provides can give valuable feedback on formulation quality and foreign content earlier in the aggregation process, and more quantitatively, than is possible with optical methods.
1 J. Carpenter, T.W. Randolph, W. Jiskoot. D. Crommelin, C. Middaugh, G. Winter, Y. Fan Y, S. Kirshner, D. Verthelyi, S. Kozlowski, K. Clouse, P. Swann, A. Rosenberg, B. Cherney, Overlooking sub-visible particles in therapeutic protein products: gaps that may compromise product quality. J Pharm Sci. 2008; 98 (4): 1201-1205.
2 <788> Particulate Matter in Injections. The United States Pharmacopeia - National Formulary, USP 29-NF 24 ed.: U.S. Pharmacopeia, 2006.