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.
Highlights
The advent of hybridoma (Kohler & Milstein, 1975) and phage display technologies (Winter, Griffiths, Hawkins, & Hoogenboom, 1994) has allowed monoclonal antibodies to revolutionize the biotechnology industry (Aggarwal, 2014)
The presence of aggregates during development can lead to a decreased yield and an increase in time to market, due to the need to optimize manufacturing conditions/formulation (Cromwell, Hilario, & Jacobson, 2006; Zurdo et al, 2015). monoclonal antibodies (mAbs)-based biologics are susceptible to aggregation throughout their lifetime, from over-expression in the cell (Kramarczyk, Kelley, & Coffman, 2008) and downstream processing (Skamris et al, 2016; Yu et al, 2016) to the final fill-finish step at high concentration (Cromwell et al, 2006; Rathore & Rajan, 2008)
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)
Summary
The advent of hybridoma (Kohler & Milstein, 1975) and phage display technologies (Winter, Griffiths, Hawkins, & Hoogenboom, 1994) has allowed monoclonal antibodies (mAbs) to revolutionize the biotechnology industry (Aggarwal, 2014). The ability to assess aggregation propensity is essential for any biopharmaceutical, including mAb-based products, to progress successfully from molecule to market (Jain et al, 2017; Tiller & Tessier, 2015; van der Kant et al, 2017) In silico analyses, such as TANGO (FernandezEscamilla, Rousseau, Schymkowitz, & Serrano, 2004), Zyggregator (Tartaglia & Vendruscolo, 2008), Waltz (Oliveberg, 2010), Aggrescan (Conchillo-Solé et al, 2007), and PASTA (Trovato, Seno, & Tosatto, 2007) can be used to predict the presence of aggregation-prone regions (APRs) within proteins using protein primary sequence information alone.
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