Do Not Drop: Mechanical Shock in Vials Causes Cavitation, Protein Aggregation, and Particle Formation

Abstract

Industry experience suggests that g-forces sustained when vials containing protein formulations are accidentally dropped can cause aggregation and particle formation. To study this phenomenon, a shock tower was used to apply controlled g-forces to glass vials containing formulations of two monoclonal antibodies and recombinant human growth hormone (rhGH). High-speed video analysis showed cavitation bubbles forming within 30 μs and subsequently collapsing in the formulations. As a result of echoing shock waves, bubbles collapsed and reappeared periodically over a millisecond timecourse. Fluid mechanics simulations showed low-pressure regions within the fluid where cavitation would be favored. A hydroxyphenylfluorescein assay determined that cavitation produced hydroxyl radicals. When mechanical shock was applied to vials containing protein formulations, gelatinous particles appeared on the vial walls. Size exclusion chromatographic analysis of the formulations after shock did not detect changes in monomer or soluble aggregate concentrations. However, subvisible particle counts determined by microflow image analysis increased. The mass of protein attached to the vial walls increased with increasing drop height. Both protein in bulk solution and protein that became attached to the vial walls after shock were analyzed by mass spectrometry. rhGH recovered from the vial walls in some samples revealed oxidation of Met and/or Trp residues.

Impact of Cavitation, High Shear Stress and Air/Liquid Interfaces on Protein Aggregation

“…In a previous study we showed that the influence of cavitation derived from a micro‐orifice reduced the monomeric content of human serum albumin (HSA) and enhanced dimeric HSA, whereas isolated shear rates of up to 10 8 s −1 had no influence on HSA aggregation behavior. Furthermore, we hypothesized that the main driving force for protein aggregation when cavitation occurs is most likely the increased vapor/liquid interface due to cavitation bubble formation and not the hydroxyl radical formation obtained from bubble collapse as previously described …”

A comparative analysis of heterogeneity in commercially available recombinant factor VIII products

“…Regulatory agencies place increased emphasis on the potential clinical implications of subvisible particles and recommend careful analysis to characterize the particles in therapeutic protein products . The precise mechanism by which subvisible particles form is unknown, but researchers consider freezing, oxidation, elevated temperatures, exposure to light, mechanical stress, stress during purification, production, storage and transport to be major contributing factors . Materials in contact with the product, such as the product vial components, can also affect particle formation .…”

“…11,12 The precise mechanism by which subvisible particles form is unknown, but researchers consider freezing, oxidation, elevated temperatures, exposure to light, mechanical stress, stress during purification, production, storage and transport to be major contributing factors. [13][14][15][16][17][18] Materials in contact with the product, such as the product vial components, can also affect particle formation. 17 Advances in analytical technologies have enabled quantification and examination of the morphology of these particles.…”

Shock-induced cavitation as a way of accelerating phenol oxidation in aqueous media

“…caused through cavitation phenomena, can be as well induced by shock-waves, as seen for example in under water explosions [25]. Moreover, Randolph and co-workers [26] have recently reported that the mechanical shock in vials, i.e.…”