(4iq) Deep Eutectic Solvent Excipients for Concentrated Protein Therapeutic Formulations

Drug delivery, biomaterials, ionic liquids, polymers, wound healing, medical devices

Teaching Interests

Biomaterials, polymer physics, drug delivery

Abstract

Concentrated protein therapeutic formulations have garnered significant attention in both commercial and research efforts due to patient-favored, facile administration via subcutaneous tissue. However, a challenge faced in formulation is the propensity of intermolecular electrostatic and hydrophobic interactions driving aggregation of therapeutic leading to an exponential increase in viscosity and thus a limit to their injectability (< 25cP). Such material phenomena leads to issues in both bioavailability and clinical translation, where resultant injection site swelling imparts both a transport barrier to the therapeutic, and severe pain onto the patient. Hence, improvement of injectability necessitates addition of excipients to limit therapeutic self-aggregation via a shielding-like effect.

Current excipient chemistries include a complex mixture of polysorbate surfactants, amino acids, disaccharides, salts, and hyaluronidase enzymes capable of local extracellular matrix (ECM) degradation for improved transport. However, further innovation upon clinically approved products is a prevalent need with only a 60-85% bioavailability for common subcutaneous therapies. Such formulations also retain a narrow colloidal stability window due to changes in molecular chemistry upon physical and thermal stress, thereby necessitating cold chain (5°C) storage conditions and short time (10-14 days) scales for maximum clinical efficacy. We seek to simplify and stabilize concentrated protein therapeutic formulations through use of deep eutectic solvents (DESs), a liquid salt (Tm < 100°C) chemistry with expansive charge and amphiphilicity profiles fostered via precise cation-to-anion ratio control. In this study, we investigate the extent of electrostatic and hydrophobic interactions between protein and ionic species of DESs, their relation to viscosity, and end-user bioavailability.

With an infinite library of cation and anion chemistries to choose from, we employed cholinium derived cations and organic carboxylic acid anions in synthesis of our chosen DESs due to their natural expression in various biological systems. Herein, we synthesized a library of 30 biocompatible cholinium and carboxylic acid based DESs in either 2:1, 1:1, or 1:2 cation-to-anion ratios, and formulated them with a polyclonal mixture of IgG antibody (100 – 300 mg/mL). Antibody was our therapeutic of choice for this initial study due to their increasing use in subcutaneous therapy and their lower bioavailability as compared to other protein biologics (ex. peptides). Initial in vitro screening strategies constituted rheological characterization of all 30 DESs formulated with antibody (100 mg/mL) and assays assessing thermal stability through techniques such as circular dichroism, differential scanning calorimetry, and dynamic light scattering, and functional capability using enzyme-linked immunosorbent assay (ELISA) at various incubation temperatures (5°C, room temperature, 37°C).

Ruling out formulations tending to higher viscosity values (>25cP) and displaying thermal denaturation, we arrived at 11 candidates to probe for in vivo experimentation (Figure 1A). Subcutaneous bioavailability of 11 different DESs formulated with IgG antibody (100 mg/mL) was compared to naked antibody formulated in saline within a mouse model. Results from an initial 6 day study indicated both comparable and far improved pharmacokinetics of DES-antibody in comparison to saline. Such a range can be attributed to a combination of different viscosity profiles and interactions between free DES ionic species and surrounding ECM encouraging tissue. From these 11 tested candidates, 5 DES-antibody formulations were probed for an extended study over 21 days. Relative bioavailability values for DES-antibody candidates and a saline control injected subcutaneously were compared to an intravenous injection of saline control. Select DES-antibody candidates displayed an increase by 2-5 fold (85-250%) in bioavailability as compared to saline control (47%) when injected into subcutaneous tissue (Figure 1B).

The high potential of our DES-antibody formulations for subcutaneous antibody therapy at a clinically relevant concentration (100 mg/mL) has been highlighted in our in vitro and in vivo studies thus far. This presents an exciting opportunity for incorporation of DESs as excipients in concentrated protein therapeutic formulations. We seek to further elucidate the relationship between DES and antibody aggregation through fundamental lenses of electrostatic and hydrophobic parameters as to probe higher the concentration barrier to an even greater extent (300 mg/mL). Currently, through optimization of implicative variables such as conductivity and surface tension for a range of neat DESs, we have extrapolated a mathematical model for prediction of viscosity in final formulation containing antibody (Figure 2). Ongoing studies demonstrate the potential of using facile techniques to predict injectability of concentrated protein therapeutic formulations in the absence of protein itself, leading to an effective cost saving measure in subcutaneous therapy development.

Figure Captions

Figure 1: A) Shear sweep rheology for synthesized DESs formulated at 10% (v/v) with 100 mg/mL of IgG antibody. B) 21 day serum concentration and bioavailability for candidate DES formulated at 10% (v/v) with 100 mg/mL IgG antibody in a subcutaneous mouse model.

Figure 2: Experimental viscosity (red), conductivity (black), and model predicted viscosity (green) of DES formulated with 300 mg/mL IgG antibody as a function of DES concentration. Vertical lines represent locations of minimum and maximum values for associated colored curve. Horizontal line (blue) represents viscosity threshold for injectable subcutaneous therapeutic formulations (25cP).