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Gaining better understanding on agitational stresses applied to proteins for biopharmaceutical development

This application note was contributed by Ge Bai, Scientist, MedImmune LLC.

Agitation instruments and glass vial

Figure 1. Agitation instruments and glass vial

Agitation studies are a common and important part of biopharmaceutical development, yet the fundamental nature of the stresses involved and their impact on protein stability are not fully understood. Characterization of the agitation stress methods is critical to identifying protein degradation mechanisms or specific sensitivities. Stresses caused by shear, interfaces, cavitation, or other fluid and interfacial forces are difficult or impossible to measure with experimental methods. Recently, we conducted CFD simulations with FLOW-3D to model the hydrodynamics of liquid agitated in a 3-mL glass vial with different agitation instruments including a rotator, orbital shaker, magnetic stirrer, and vortex mixer (see Figure 1), at varying frequencies to identify and quantify stresses of potential importance to protein stability. The fluid properties of water at 25°C were used for these simulations.

The standard FLOW-3D code was customized so that potentially harmful stresses to proteins such as maximum system shear rate, volume-averaged shear rate, volume-averaged shear rate near air-liquid and solid-liquid interfaces, total shear, area of solid-liquid interface, and air-liquid interface regeneration rate can be numerically calculated and compared as additional outputs of the standard software package. We validated the CFD models by comparing the shape of the free surface of the fluid in the vial between simulation and experiment (Figure 2).

Comparison of the shape of fluid free surface between CFD simulation and experiment
Figure 2. Comparison of the shape of fluid free surface between CFD simulation and experiment for (A) orbital shaker at 300 rpm at steady state and (B) rotator at 35 rpm, 55° position.

Examples of stresses (shear rate and interface generation rate) and comparisons at the air-liquid and solid-liquid interfaces are shown in Figure 3 and in Figure 4. Overall, the vortex mixer provides the most intense stresses, while the magnetic stirrer presented locally intense shear to the hydrophobic stir bar surface. The rotator provides gentler fluid stresses, but the air-water interfacialarea and surface stresses are relatively high given its low rotational frequency. The orbital shaker provides intermediate-level stresses but with the advantage of a large stable platform for consistent vial-to-vial homogeneity.

Instantaneous shear rates near interfaces at maximum agitation frequencies
Figure 3. Instantaneous shear rates near interfaces at maximum agitation frequencies (A) orbital shaker, (B) magnetic stirrer, (C) vortex mixer and (D) rotator.
Air-liquid interface generation rates at maximum agitation frequencies
Figure 4. Air-liquid interface generation rates at maximum agitation frequencies (A) orbital shaker, (B) magnetic stirrer, (C) vortex mixer and (D) rotator.

We found that multiple stresses act concurrently on the liquid in a glass vial in each of the agitation methods described. These stresses varied for different methods and were often found to be strong functions of agitation frequency. We also determined that selecting the proper agitation method with known types and intensities of stresses can facilitate better understanding of the impact on protein degradation mechanisms. We concluded that CFD can be instrumental in characterizing fluid stresses in experimental systems and in validating their relevance to real world conditions.

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