No Touching! Abrasion of adsorbed protein Is the root cause of subvisible particle formation during stirring

This study addressed the effect of contact sliding during stirring of a monoclonal antibody solution onprotein aggregation, in particular, in the nanometer and micrometer size range. An overhead stirring setupwas designed in which the presence and magnitude of the contact between the stir bar and thecontainer could be manipulated. A solution of 0.1 mg/mL of a monoclonal antibody (IgG) in phosphatebuffered saline was stirred at 300 rpm at room temperature. At different time points, samples were takenand analyzed by nanoparticle tracking analysis, flow imaging microscopy, and size-exclusion chromatography.In contrast to nonecontact-stirred and unstirred samples, the contact-stirred sample containedseveral-fold more particles and showed a significant loss of monomer. No increase in oligomer contentwas detected. The number of particles formed was proportional to the contact area and the magnitude ofthe normal pressure between the stir bar and the glass container. Extrinsic 9-(2,2-dicyanovinyl) julolidinefluorescence indicated a conformational change for contact-stirred protein samples. Presence ofpolysorbate 20 inhibited the formation of micron-sized aggregates. We suggest a model in whichabrasion of the potentially destabilized, adsorbed protein leads to aggregation and renewal of the surfacefor adsorption of a fresh protein layer.Drug Delivery Technolog