Significance Proteins are inherently sensitive to environmental factors that include hydrodynamic flow. Flow-induced protein remodeling is used in vivo and can also trigger the aggregation of therapeutic proteins during manufacture. Currently, the relative importance of shear and extensional hydrodynamic flow fields to aggregation remains unclear. Here we develop a flow device that subjects proteins to a defined and quantified flow field that is dominated by extensional flow. We show that extensional flow is crucial to induce the aggregation of globular proteins and that flow-induced aggregation is dependent on both protein structure and sequence. These observations rationalize the diverse effects of hydrodynamic flow on protein structure and aggregation propensity seen in both Nature and in protein manufacture.

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.

Proteins 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⇓⇓⇓–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 experience an extensional force between the front (faster flow) and the rear (slower flow), potentially leading to elongation of the molecule as directly observed for a single DNA molecule (17, 18). The majority of protein aggregation studies to date have considered shear flows within capillaries (3) or through using viscometric-type devices (10, 16). On the whole, these studies show that globular proteins are generally resistant to shear flow in the absence of an interface (3, 14, 19). By contrast, Simon et al. (11) showed increased aggregation of BSA with increasing extensional flow. Many operations within biopharmaceutical manufacture such as filtration, filling, and pumping (5, 20) also create extensional flow fields. This observation, together with the importance of extensional flow fields to thrombosis (21) and spider silk spinning (22), suggests a link between extensional flow and protein aggregation.

Fig. 1. Design of extensional flow apparatus and validation of the generated flow field using CFD. (A) The differences between shear- (Top) and extensional flow (Bottom). Solid black arrows indicate velocity; dashed lines show streamlines indicative of flow direction. The orange dot in both diagrams represents a protein as a sphere in the flow. The curved red arrow represents rotation due to shear. Straight red arrows indicate the relative velocity of the protein, which differs before and after the contraction in the extensional flow. (B) Schematic of the extensional flow apparatus showing two syringes connected by a single capillary. (C) Image of the extensional flow device. (D, i) A 3D schematic of the contraction geometry where the barrel of the syringe meets the capillary (dotted red line shows the location of the contraction). The 2D axisymmetric approximation used for CFD analysis is superimposed in blue. CFD results of the extensional flow region showing the flow-velocity (D, ii) and strain-rate (D, iii) profiles for a typical flow with a plunger velocity (average inlet velocity) of 8 mms−1 (centerline strain rate = 11,750 s−1). (D, iv) Velocity and strain rate along a streamline located on the axis of symmetry at a plunger velocity of 8 mms−1.

To assess the relative importance of extension and shear to flow-induced aggregation, we have developed a low-volume flow device, characterized using computational fluid dynamics (CFD), which uses a rapid constriction to generate an extensional flow field followed by flow within a capillary that generates a shearing flow. Using our device, it is possible to deconvolute the effects of shearing and extensional flow fields. We demonstrate that extensional flow can trigger the aggregation of BSA and that the extent of aggregation is dependent on the total exposure time, strain rate, and protein concentration. We also show that the aggregation of a range of globular, natively folded proteins (β 2 m, G-CSF, and three mAbs) under extensional flow is diverse and is particularly damaging to therapeutic proteins (G-CSF and mAbs) under conditions analogous to those encountered during their manufacture. Finally, we show directly that the device triggers the aggregation of BSA by inducing partial unfolding and that the extent of aggregation is strain-rate- and protein-concentration-dependent, suggesting that aggregation occurs by interaction of partially unfolded proteins whose population is induced by extensional flow.

Discussion Whereas prior work has demonstrated that hydrodynamic flow can induce the unfolding of supercoiled plasmid DNA (20, 27), polymers (26), von Willebrand factor (21, 46), and other proteins (11), the relative ability of shear and extensional flow to induce aggregation for these different systems has remained unclear. To address this question, we designed a device to generate an extensional flow field that would subject natively folded globular proteins to high and well-defined strain rates. Using this device, we demonstrated that extensional flow has the ability to induce the aggregation of BSA. This, together with previously published studies, suggests that while shear and extensional flow fields can both induce aggregation (11, 31), their ability to do so is protein dependent. For example, both spider silk and von Willebrand factor have been observed to undergo shear-induced remodeling [which nonetheless are exposed to mixed shear/extensional flows in vivo (22, 46)]. As both of these proteins are evolved to respond to low levels of hydrodynamic force, it may be that their response to shear is atypical for globular, stably folded proteins. The latter proteins are relatively insensitive to shear flow where the presence of an interface is often required to induce aggregation (19). Repeating our experiments on a variety of proteins demonstrated that the extent of aggregation caused by extensional flow depends on the structure, topology, concentration, and precise sequence of the protein. In addition to delineating these determinants, we have shown using in situ cysteine labeling that extensional flow can induce conformational remodeling. The theoretical considerations and data discussed above (Fig. 4), suggest that extensional flow can catalyze the partial/full unfolding of proteins. A critical rate of energy transfer must, however, be reached to allow the unfolding barrier to be traversed during exposure to the flow force. Superficially, hydrodynamic forced unfolding is similar to mechanical unfolding of single protein molecules using optical tweezers or the atomic force microscope. These single-molecule forced unfolding studies have shown that mechanical strength is related to the ability of regions local to the points of force application to resist extension by contrast with traditional measures of stability such as thermal or chemical denaturation (45, 47). If flow-induced aggregation occurs from a partially or fully unfolded state, then the threshold strain rate (i.e., that required to bring about exposure of an aggregation-prone region) will be protein dependent. As a consequence, natively folded globular proteins will be generally recalcitrant to shear flow, whereas inherently extensible unstructured proteins are not. After the initial partial unfolding step, the likelihood of two (or more) unfolded molecules interacting productively is dependent on the affinity of the exposed aggregation-prone regions, the protein concentration, and the rate at which the protein regains its native structure, rationalizing the diverse sensitivity observed for the highly homologous IgG pair (MEDI1912_WFL and STT, Fig. 3A). Furthermore, as both the unfolding and aggregation steps are likely to dependent on factors such as pH, temperature, and ionic strength, even the same protein may display different extensional flow behavior in different environments. In summary, we have shown the utility of characterizing the behavior and dispersity of protein solutions subjected to well-defined hydrodynamic flows to deconvolve the effects of shear, extensional flow, protein topology, and sequence on their unfolding and aggregation properties. The results have revealed the sensitivity of proteins to unfolding and consequent aggregation under extensional flow in a manner dependent on the protein sequence and structure. The approach adopted will aid the rational redesign of protein sequences that are more robust to bioprocessing and help to understand how flow has been used by nature in biological processes as diverse as silk spinning and blood clotting.

Methods Characterization of Flow Geometry Using CFD. CFD (using the general finite-element simulation package Comsol Multiphysics) was used to visualize and quantify the flow field generated by the extensional flow device. This allowed the velocity, strain rate, and exposure time, among other parameters, to be calculated. A description of the CFD model, along with details of how to obtain the strain rate, is given in SI Appendix. Extensional Flow Apparatus and Experiments. Two 1-mL gas-tight syringes with inner bore diameter of 4.61 mm (Hamilton Syringes model 1001 RN Valco SYR) were modified to take a glass capillary tube of inner diameter 0.3 mm with a compression fitting (Hamilton Syringes RN 1 mm) producing an abrupt contraction with diameter ratio ∼15:1 producing a 238-fold increase in velocity. Protein solutions were stressed for a defined number of passes at a given plunger velocity, then the rig stopped, dissembled, and the solution expelled slowly from the syringe. Control samples were incubated at ambient temperature for the duration of a given stress experiment (e.g., 10 passes at a plunger velocity of 8 mm s−1 takes 1 min to complete). See SI Appendix. All experiments were performed at least twice unless otherwise stated. Protein Preparation. BSA (Sigma-Aldrich) was purified by gel filtration chromatography using a Superdex 200 (26/60) gel filtration column (GE Healthcare) equilibrated with 25 mM ammonium acetate buffer, pH 5.1 and stored in aliquots at −20 °C. Before stressing experiments, the protein was concentrated using a centrifugal concentrator with a 30-kDa cutoff filter (Merck Millipore). After filtration through a 0.22-μm membrane (Merck Millipore), the concentration was determined by UV spectroscopy (SI Appendix, Table S5) and adjusted as necessary. G-CSF C3 (37) was overexpressed in BL21[DE3]pLysS cells transformed with a pET23a_G-CSF C3 vector and purified as described in SI Appendix. Extensional flow experiments with G-CSF C3 were performed in filtered (0.22 μm) and degassed 25 mM sodium phosphate, 25 mM sodium acetate buffer, pH 7.0. β 2 m was purified as described (48) and extensional flow experiments performed in filtered (0.22 μm) and degassed 25 mM sodium phosphate buffer, pH 7.2. Antibodies were provided by MedImmune Ltd. Antibodies were prepared by dialyzing into 0.22 μm filtered and degassed 150 mM ammonium acetate buffer, pH 6.0, diluting before stressing experiments as appropriate. Insoluble Protein Pelleting Assay. After stressing for the desired number of passes, the apparatus was dissembled and 200 μL of protein solution ultracentrifuged using a Beckmann Coulter Optima TLX Ultracentrifuge equipped with a TLA100 rotor at 30,000 rpm for 30 min at 4 °C. Then, 150 μL of supernatant was removed and diluted to 2 mL (BSA) or 250 μL (all other proteins) in 6 M guanidine hydrochloride (Gdn HCl) 25 mM TrisHCl buffer, pH 6. The pellet and remaining supernatant were diluted in the same buffer to 2 mL (BSA) or 250 μL (all other proteins) and incubated overnight. The amount of protein in the pellet was then calculated by measuring the protein concentration of this solution, the supernatant after ultracentrifugation, and the protein solution in the absence of extensional flow using UV-visible spectroscopy (see SI Appendix, Table S5 for extinction coefficients). This procedure was performed in duplicate. Biophysical Characterization of Polydispersity. Experimental procedures for DLS, NTA, TEM, and FCS are described in SI Appendix. IAEDANS (5-[2-(Iodoacetamido)Ethylamino]Naphthalene-1-Sulfonic Acid) Labeling of BSA. A 5 mg mL−1 BSA solution (25 mM ammonium acetate, pH 5.1) was mixed with 5 mM IAEDANS and stressed for 0–100 passes at a plunger velocity of 8 mm s−1 (strain rate = 11,750 s−1). TCEP at 0.5 mM was added to the tube before stressing as required. In another experiment 5 mg mL−1 BSA was stressed for 0–100 passes in the presence or absence of TCEP (this protein was left for the same length of time as the extensional flow experiment above). Subsequently, this protein was mixed with 5 mM IAEDANS and incubated for the same time as the protein was stressed for in the presence of IAEDANS above. The IAEDANS labeling was quenched with SDS/PAGE loading buffer containing 200 mM DTT. The diluted samples (∼100 µg) were then analyzed by SDS/PAGE [using a 12% wt/vol (37.5:1 acrylamide:bis-acrylamide) gel]. Fluorescent bands in the gel were excited by UV light provided by a UV-trans illuminator (Syngene Gel documentation). The intensities of the fluorescent bands were analyzed with the Gene Tool software supplied with the instrument. The gel was then stained with Coomassie Brilliant Blue.

Acknowledgments We thank Prof. Joanne Tipper and Dr. Saurabh Lal for their help with NTA, and Mr. David Sharples for access to the ultracentrifuge facility. We thank Prof. Peter Olmsted for many insightful discussions at the inception of this work. J.D. and A.K. are cofunded by MedImmune Ltd. and the University of Leeds. L.F.W. is funded by the Engineering and Physical Sciences Research Council Centre for Innovative Manufacturing in Emergent Macromolecular Therapies, UK (EP/I033270/1), and S.E.R. and D.J.B. acknowledge funding by the ERC (European Research Council) (FP7/2007-2013 Grant Agreement 32240). N.K. holds a Chair in Pharmaceutical Processing at the University of Leeds funded by GlaxoSmithKline plc and the Royal Academy of Engineering. The DLS instrument was funded by the Medical Research Council (G0900958), the TEM by the Wellcome Trust (108466/Z/15/Z), and the FCS apparatus partly funded by the ERC (as above).