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

Abstract

Monoclonal antibodies (mAbs) currently dominate the biopharmaceutical sector due to their potency and efficacy against a range of disease targets. These proteinaceous therapeutics are, however, susceptible to unfolding, mis‐folding, and aggregation by environmental perturbations. Aggregation thus poses an enormous challenge to biopharmaceutical development, production, formulation, and storage. Hydrodynamic forces have also been linked to aggregation, but the ability of different flow fields (e.g., shear and extensional flow) to trigger aggregation has remained unclear. To address this question, we previously developed a device that allows the degree of extensional flow to be controlled. Using this device we demonstrated that mAbs are particularly sensitive to the force exerted as a result of this flow‐field. Here, to investigate the utility of this device to bio‐process/biopharmaceutical development, we quantify the effects of the flow field and protein concentration on the aggregation of three mAbs. We show that the response surface of mAbs is distinct from that of bovine serum albumin (BSA) and also that mAbs of similar sequence display diverse sensitivity to hydrodynamic flow. Finally, we show that flow‐induced aggregation of each mAb is ameliorated by different buffers, opening up the possibility of using the device as a formulation tool. Perturbation of the native state by extensional flow may thus allow identification of aggregation‐resistant mAb candidates, their bio‐process parameters and formulation to be optimized earlier in the drug‐discovery pipeline using sub‐milligram quantities of material.

Synergistic effects of flow and interfaces on antibody aggregation

“…Different types of stresses and flow fields have been suggested to be critical in promoting the aggregation of proteins (Bee, Stevenson et al, ; Bekard, Asimakis, Bertolini, & Dunstan, ; Elias & Joshi, ). In addition to shaking and wall shear rate, elongational strain and cavitation shocks have also been recently proposed to potentially trigger protein aggregation (Dobson et al, ; Duerkop, Berger, Dürauer, & Jungbauer, ; Willis et al, ). Despite the considerable research efforts of the past decades, there still is no clear consensus regarding the type and magnitude of hydrodynamic stress required to induce protein aggregation, and mechanistic explanations of the processes involved remain elusive (Bee, Stevenson et al, ; Bekard, Barnham, White, & Dunstan, ; Biddlecombe et al, ; Dobson et al, ; Thomas & Geer, ).…”

The uniqueness of flow in probing the aggregation behavior of clinically relevant antibodies

“…This group of antibodies, dubbed the (Clinical) 33 herein, showed varied responses in the assays employed previously (Supplementary Table I and Figure S1) . The magnitude of aggregation triggered by the EFD is strongly dependent on protein concentration, strain rate (which is controlled by changing the velocity of the syringe driver) pass number (the number of times the fluid is shuttled through the capillary) and solution conditions (eg, arginine succinate buffer suppressing flow‐induced aggregation), yielding a complex response surface to flow . Here, the large number of antibodies to be studied, together with sample limitations, precluded such a detailed study.…”

“…In addition, we showed that a candidate mAb with poor biophysical and pharmacokinetic properties (MEDI1912_WFL) was more sensitive to hydrodynamic flow (in terms of quantity of aggregate produced) than its derivative (MEDI1912_STT), rationally engineered to improve its biophysical characteristics . Finally, we used the EFD to identify which factors (protein concentration, sequence, buffer components, and flow conditions) are key to modulating the flow‐induced aggregation of three model IgG1 mAbs . These data show the utility of hydrodynamic force as a potential developability assay.…”

“…It is becoming clear, however, that many aggregation pathways initiate from activated, partially unfolded metastable states that may not be detected by these techniques, especially in accelerated stability assays . To address this issue, we previously developed a device that exerts hydrodynamic forces, similar to those applied during bioprocessing, onto proteins . In this method, fluid is passed between two syringes coupled via a narrow glass capillary (one fifteenth of the syringes' diameter) (Figure A).…”

“…These data show the utility of hydrodynamic force as a potential developability assay. As these studies used only three IgG1 mAbs, our understanding of the behavior of a broader range of therapeutic mAbs, or how the EFD relates to other methods of assessing aggregation, remained unknown.…”