Inducing protein aggregation by extensional flow

Abstract

Relative to other extrinsic factors, the effects of hydrodynamic flow fields on protein stability and conformation remain poorly understood. Flow-induced protein remodeling and/or aggregation is observed both in Nature and during the large-scale industrial manufacture of proteins. Despite its ubiquity, the relationships between the type and magnitude of hydrodynamic flow, a protein's structure and stability, and the resultant aggregation propensity are unclear. Here, we assess the effects of a defined and quantified flow field dominated by extensional flow on the aggregation of BSA, β 2 -microglobulin (β 2 m), granulocyte colony stimulating factor (G-CSF), and three monoclonal antibodies (mAbs). We show that the device induces protein aggregation after exposure to an extensional flow field for 0.36-1.8 ms, at concentrations as low as 0.5 mg mL −1 . In addition, we reveal that the extent of aggregation depends on the applied strain rate and the concentration, structural scaffold, and sequence of the protein. Finally we demonstrate the in situ labeling of a buried cysteine residue in BSA during extensional stress. Together, these data indicate that an extensional flow readily unfolds thermodynamically and kinetically stable proteins, exposing previously sequestered sequences whose aggregation propensity determines the probability and extent of aggregation.extensional flow | aggregation | unfolding | bioprocessing | antibody P roteins are dynamic and metastable and consequently have conformations that are highly sensitive to the environment (1). Over the last 50 y the effect of changes in temperature, pH, and the concentration of kosmatropic/chaotropic agents on the conformational energy landscape of proteins has become well understood (1). This, in turn, has allowed a link to be established between the partial or full unfolding of proteins and their propensity to aggregate (2). The force applied onto a protein as a consequence of hydrodynamic flow has also been observed to trigger protein aggregation and has fundamental (3), medical (4), and industrial relevance, especially in the manufacture of biopharmaceuticals (5-8). Although a wealth of studies have been performed (7,(9)(10)(11)(12)(13), no consensus has emerged on the ability of hydrodynamic flow to induce protein aggregation (7,14,15). This is due to the wide variety of proteins used (ranging from lysozyme, BSA, and alcohol dehydrogenase to IgGs), differences in the type of flow field generated (e.g., shear, extensional, or mixtures of these), and to the presence or absence of an interface (16). A shearing flow field (Fig. 1A, Top) is caused by a gradient in velocity perpendicular to the direction of travel and is characterized by the shear rate (s −1 ). This results in a weak rotating motion of a protein alongside translation in the direction of the flow. An extensional flow field (Fig. 1A, Bottom) is generated by a gradient in velocity in the direction of travel and is characterized by the strain rate (s −1 ). A protein in this type of flow would experien...

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

“…There is thus a need to develop stress tests that more closely replicate the conformational ensemble generated during processing and transport. In light of this, we and others have shown that hydrodynamic extensional flow fields encountered during the nano‐filtration, pumping, and fill‐finish steps of bio‐processing can trigger protein aggregation (Charm & Wong, 1981; Dobson et al, 2017; Simon, Krause, Weber, & Peukert, 2011; Wolfrum, Weichsel, Siedler, Weber, & Peukert, 2017). Using a reciprocating extensional and shear flow device (EFD) (Figure 1a), we showed that extensional flow fields can induce the conformational unfolding/remodeling of bovine serum albumin (BSA, Figure 1b), leading to aggregation that was characterized and quantified by an array of biophysical techniques including DLS, NTA, and TEM (Dobson et al, 2017).…”

“…In light of this, we and others have shown that hydrodynamic extensional flow fields encountered during the nano‐filtration, pumping, and fill‐finish steps of bio‐processing can trigger protein aggregation (Charm & Wong, 1981; Dobson et al, 2017; Simon, Krause, Weber, & Peukert, 2011; Wolfrum, Weichsel, Siedler, Weber, & Peukert, 2017). Using a reciprocating extensional and shear flow device (EFD) (Figure 1a), we showed that extensional flow fields can induce the conformational unfolding/remodeling of bovine serum albumin (BSA, Figure 1b), leading to aggregation that was characterized and quantified by an array of biophysical techniques including DLS, NTA, and TEM (Dobson et al, 2017). By subjecting five other globular proteins that varied in sequence, size, and structure to such flow stresses, we demonstrated that the aggregation propensity of proteins differed based on their fold, sequence, and the fluid fields to which they are subjected (Dobson et al, 2017).…”

“…Using a reciprocating extensional and shear flow device (EFD) (Figure 1a), we showed that extensional flow fields can induce the conformational unfolding/remodeling of bovine serum albumin (BSA, Figure 1b), leading to aggregation that was characterized and quantified by an array of biophysical techniques including DLS, NTA, and TEM (Dobson et al, 2017). By subjecting five other globular proteins that varied in sequence, size, and structure to such flow stresses, we demonstrated that the aggregation propensity of proteins differed based on their fold, sequence, and the fluid fields to which they are subjected (Dobson et al, 2017). For mAb‐based biotherapeutic scaffolds we observed a wide‐range of sensitivity to flow‐induced aggregation under identical conditions (strain rate, pass number, protein concentration and buffer), dependent on the protein sequence (Dobson et al, 2017).…”

“…By subjecting five other globular proteins that varied in sequence, size, and structure to such flow stresses, we demonstrated that the aggregation propensity of proteins differed based on their fold, sequence, and the fluid fields to which they are subjected (Dobson et al, 2017). For mAb‐based biotherapeutic scaffolds we observed a wide‐range of sensitivity to flow‐induced aggregation under identical conditions (strain rate, pass number, protein concentration and buffer), dependent on the protein sequence (Dobson et al, 2017). Accordingly, the aggregation‐prone antibody (MEDI1912_WFL, WFL herein, Figure 1b) was found to be most sensitive to extensional flow, while its rationally engineered aggregation‐resistant derivative (MEDI1912_STT, STT herein, Figure 1b (Dobson et al, 2016) showed markedly decreased aggregation (∼85 and ∼5% aggregation, respectively).…”

“…Accordingly, the aggregation‐prone antibody (MEDI1912_WFL, WFL herein, Figure 1b) was found to be most sensitive to extensional flow, while its rationally engineered aggregation‐resistant derivative (MEDI1912_STT, STT herein, Figure 1b (Dobson et al, 2016) showed markedly decreased aggregation (∼85 and ∼5% aggregation, respectively). An unrelated mAb, mAb1, showed intermediate behavior (Dobson et al, 2017). For BSA, the extent of aggregation is dependent on the magnitude of the strain rate, the total exposure time to the extensional flow event (controlled by both the number of passes and plunger velocity) and the protein concentration, producing a complex aggregation landscape.…”