No Touching! Abrasion of Adsorbed Protein Is the Root Cause of Subvisible Particle Formation During Stirring

Abstract

This study addressed the effect of contact sliding during stirring of a monoclonal antibody solution on protein aggregation, in particular, in the nanometer and micrometer size range. An overhead stirring set-up was designed in which the presence and magnitude of the contact between the stir bar and the container could be manipulated. A solution of 0.1 mg/mL of a monoclonal antibody (IgG) in phosphate buffered saline was stirred at 300 rpm at room temperature. At different time points, samples were taken and analyzed by nanoparticle tracking analysis, flow imaging microscopy, and size-exclusion chromatography. In contrast to non-contact-stirred and unstirred samples, the contact-stirred sample contained several-fold more particles and showed a significant loss of monomer. No increase in oligomer content was detected. The number of particles formed was proportional to the contact area and the magnitude of the normal pressure between the stir bar and the glass container. Extrinsic 9-(2,2-dicyanovinyl) julolidine fluorescence indicated a conformational change for contact-stirred protein samples. Presence of polysorbate 20 inhibited the formation of micron-sized aggregates. We suggest a model in which abrasion of the potentially destabilized, adsorbed protein leads to aggregation and renewal of the surface for adsorption of a fresh protein layer.

Effects of Tubing Type, Formulation, and Postpumping Agitation on Nanoparticle and Microparticle Formation in Intravenous Immunoglobulin Solutions Processed With a Peristaltic Filling Pump

“…resulting in protein particles in the bulk solution. [3][4][5] These stresses are prominent during the final solution handling step in protein drug product manufacturing: filling pump operation. The filling process is notorious for causing protein particles.…”

Synergistic effects of flow and interfaces on antibody aggregation

“…It has been proposed that accounting for both exposure to interfaces and hydrodynamic stresses may better explain protein aggregation than shear exposure alone (Bee, Stevenson et al, ; Kalonia et al, ; Sediq, van Duijvenvoorde, Jiskoot, & Nejadnik, ; Thomas & Geer, ). In fact, it is now established that air–liquid, liquid‐liquid, and solid–liquid interfaces can significantly promote and amplify protein aggregation (Bee et al, ; Bee, Stevenson et al, ; Jones, Kaufmann, & Middaugh, ; Kalonia et al, ; Maa & Hsu, ; Mahler et al, ; Mehta, Lewus, Bee, Randolph, & Carpenter, ; Perevozchikova, Nanda, Nesta, & Roberts, ; Sediq et al, ; Thirumangalathu et al, ). For instance, shaking or foaming of protein solutions potentially leads to considerable aggregation and precipitation phenomena (Bee et al, ; Maa & Hsu, ; Mahler et al, ).…”

Using extensional flow to reveal diverse aggregation landscapes for three IgG1 molecules

“…These developing in silico approaches complement established accelerated stress studies that are performed in vitro to predict the shelf‐life and stability of biologics (Jain et al, 2017; Yang et al, 2013). Various methods are employed to generate such data including heating (Cheng et al, 2012; Hamrang et al, 2015), stirring (Luo et al, 2011; Sediq, Van Duijvenvoorde, Jiskoot, & Nejadnik, 2016), shaking (Kiese, Papppenberger, Friess, & Mahler, 2008; Rudiuk, Cohen‐Tannoudji, Huille, & Tribet, 2012), and simulation of transportation (Fleischman, Chung, Paul, & Lewus, 2017). The extent of aggregation, however, can be heavily dependent on the type of accelerated stress employed (Fleischman et al, 2017; Joubert, Luo, Nashed‐Samuel, Wypych, & Narhi, 2011; Tamizi & Jouyban, 2016).…”