Combined effect of shaking orbit and vial orientation on the agitation-induced aggregation of proteins

Orbital shaking in a glass vial is a commonly used forced degradation test to evaluate protein propensity for agitation-induced aggregation. Vial shaking in horizontal orientation has been widely recommended to maximize the air-liquid interface area while ensuring solution contact with the stopper. We evaluated the impact of shaking orbit diameter and frequency, and glass vial orientation (horizontal versus vertical) on the aggregation of three proteins prepared in surfactant-free formulation buffers. As soon as an orbit-specific frequency threshold was reached, an increase in turbidity was observed for the three proteins in vertical orientation only when using a 3 mm agitation orbit, and in horizontal orientation only when using a 30 mm agitation orbit. Orthogonal analyses confirmed turbidity was linked to protein aggregation. The most turbid samples had a visually more homogeneous appearance in vertical than in horizontal orientation, in line with the predicted dispersion of air and liquid phases obtained from computational fluid dynamics agitation simulations. Both shaking orbits were used to assess the performance of nonionic surfactants. We show that the propensity of a protein to aggregate in a vial agitated in horizontal or vertical orientation depends on the shaking orbit, and confirm that Brij® 58 and FM1000 prevent proteins from agitation-induced aggregation at lower concentrations than polysorbate 80.