Amino Acid-based Ionic Liquid Delivery Systems for Subcutaneous Administration of High Concentration Biologics

Introduction: Subcutaneous administration of high-concentration antibody solutions is a promising strategy for enhanced therapeutic outcomes, but challenges persist in achieving efficient delivery due to viscosity and stability issues. For instance, high-concentration antibody solutions of concentrations >100mg/ml are often highly viscous due to accelerated protein aggregation and unfolding aided by undesirable protein-protein interactions. Injection of highly unstable viscous solutions can cause low absorption rates in the subcutaneous space, leading to delayed onset of action. High-viscosity solutions may cause injection site reactions or difficulties with administration and cause localized reactions such as pain, erythema, or swelling at the injection site. High-concentration antibody delivery is patient-compliant as it often requires fewer and less frequent doses, making treatment more manageable and less disruptive to daily life. This approach can also improve health disparities by enhancing access to effective treatments in underserved communities, where healthcare resources and consistent medical care are often limited. In this study, we explore a novel class of Amino acid ionic liquids as potential drug delivery systems that enable the delivery of high-concentration monoclonal antibodies through the subcutaneous injection route. In vitro experiments demonstrate enhanced stability and reduced viscosities, while, in vivo experiments demonstrate increased bioavailability and tissue penetration. Our IL-based system, proven safe in vitro and in vivo, shows potential for efficient subcutaneous delivery while improving therapeutic efficacy and patient compliance.

Materials and

Methods: Ionic liquid synthesis and formulation preparation: Solid forms of acids deployed as anions and bases as cations were separately dissolved in vials with a minimal amount of ultrapure water. 60°C with a molar ratio of either 1:1, cation:anion. Excess water was removed through rotary evaporation set to 60°C and 20 mbar for 2 hours, followed by placement in a vacuum oven set to 60°C for 2 days.

Thermal stability of antibody-containing ionic liquid formulations: To assess the thermal stability of IgG-containing solutions with ionic liquid, solutions were made in vials, sealed with parafilm, and incubated at either 4˚C, room temperature, and 37˚C for different time points. Samples were then sampled out of vials and used for a range of stability and function assays, ( ELISA, SDS page, and Circular Dichroism )

Viscosity measurements
600uL of ionic liquid with antibody solution was loaded onto a rheometer (TA Instruments HR 20 Discovery Hybrid) with a 40mm 2° conical plate adapter set to 25°C. Shear sweeps were performed on samples (n = 3-5) at rates between 1 to 1000 s−1 with five points per decade. Viscosity values at 1 Hz were reported and used as a screen of formulations for later stability and in vivo experimentation.

In Vivo Pharmacokinetics and Bioavailability experiments
Female C57BL/6 mice, 5 weeks old. Mice were subcutaneously injected on the right flank with 100ul of Ionic Liquid -mIgG formulation or Control. Subsequently, 20ul of blood was also collected from the tail vein at the above-mentioned time intervals.

Results, Conclusions, and Discussions: This study explores the use of ionic liquids (ILs), salts with melting points below 100°C, to address these issues. ILs, particularly those synthesized from amino acid precursors, exhibit charge shielding properties that neutralize interactions between proteins, thus maintaining stability and bioactivity. Our focus was on amino acid-based ionic liquids (AAILs), which are biodegradable, non-toxic, and can be customized for specific needs. These ILs offer environmental benefits and high thermal stability, making them ideal for biomedical applications. We synthesized a novel series of AAIL formulations compatible with high-concentration IgG antibodies and assessed their physicochemical properties, including viscosity, conductivity, and stability. Rheological analysis helped determine the optimal IL concentration to reduce viscosity while preserving stability.

In vitro assessments showed that our IL formulations stabilize high concentration antibodies(100mg/ml) under high stress conditions, where IgG formulated with the IL retained effective binding after extended exposure to high temperatures of 37oC and 50oC (Figure 1), reducing the need for cold chain logistics, particularly beneficial in low-resource settings. We also evaluated the cytotoxicity of these ILs to ensure their safety for subcutaneous use.

In vivo experiments in animal models evaluated the pharmacokinetics and tissue distribution of high-concentration IgG (100mg/ml) delivered subcutaneously using the IL formulation compared to conventional delivery of IgG using saline. Our results indicated enhanced bioavailability and prolonged circulation time of antibodies delivered via the IL-based system, with improved tissue penetration and reduced systemic toxicity. Interestingly, we observed a total bioavailability of 83% when using an IL-based subcutaneous delivery system, an almost 2 fold increase when compared to traditional subcutaneous delivery systems, which was shown to have a bioavailability of 47% (Figure 2)

Overall, our findings highlight the potential of ILs as a versatile platform for the efficient subcutaneous delivery of high-concentration antibody solutions, offering opportunities for enhanced therapeutic efficacy and patient compliance in the treatment of various diseases. Further optimization and translational studies are warranted to advance the clinical application of this promising delivery strategy.

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