Aggregation from Shear Stress and Surface Interaction: Molecule-Specific or Universal Phenomenon?
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Exposure to solid–liquid and air–water interfaces during production, freezing and thawing, shipment and storage of protein therapeutics may be a contributing factor in their degradation (e.g., aggregation, fragmentation) (1, 2). Surface exposure, particularly during manufacturing processes, often is accompanied by various degrees and durations of shear stresses originating from fluid flow and acting on proteins at interfaces. The magnitude and duration of shear rates depends on velocity gradients within each solution and varies significantly among manufacturing steps. On the low end, a shear rate of 50 s–1 (inverse seconds or Hertz) is applied to biotherapeutics during mixing processes typically lasting from minutes to hours. On the high end of the range, shear rates of up to tens of thousands of Hertz arise during filling (up to a million during high-pressure homogenization), but those usually are limited in duration to mere seconds (3). Additionally, proteins may be subjected to high shear rates when injected through thin needles (4).
No consensus yet has been reached on whether surface interaction, shear stress, a combination of those, or other events (e.g., cavitation) are causative for frequently observed protein aggregation (3, 5–7). Although Bee and colleagues found that the shear rates of manufacturing and injection are insufficient to induce protein aggregation or denaturation, Biddlecombe and colleagues have expressed an opposing opinion (4, 6). Using a rotating-disk device as a shear stress environment, they found that a shear rate of 30,000 s–1 can cause aggregation of an IgG4 as assessed by size-exclusion chromatography (SEC). However, the extensive solid–water interface could be a major contributor to protein degradation in their experimental set-up.
Previously, Perevozchikova and colleagues showed that a monoclonal antibody (MAb 1) in a citrate formulation adsorbed irreversibly to solid silicon oxide (SiOx) surfaces (8). Desorption induced by gentle buffer rinses over the SiOx layer resulted in formation of subvisible, micron-sized protein particles; however, no smaller oligomers were observed using multiple techniques.
Here, we extend this area of study by introducing high shear stress and evaluating the stability of the above-mentioned formulation (MAb 1 in citrate buffer) exposed to solid–water interfaces in the presence of a 20,000-s–1 shear rate, which resembles the maximum shear stresses encountered in biomanufacturing. We compare the degradation of MAb 1 in a citrate formulation to that of MAb 1 and MAb 2 in alternative histidine formulations to determine whether a common aggregation pathway exists for shear/surface-stressed antibodies. To test whether higher shear rates but shorter surface exposures would have the same detrimental effect on protein quality, we also subject those formulations to shear rates typical of injections.
Materials
We used three MAb formulations at 20 mg/mL in this study: MAb 1 in a citrate-based formulation and a histidine-based formulation and MAb 2 in a histidine-based formulation. To mimic the effects of manufacturing stress on biotherapeutic stability, we subjected both MAbs to the shear rate of 20,000 s