(T1030-01-02) Demonstrating the Excipient Effect In Vitro for Pre-clinical Formulation Analysis

Purpose: Across historical literature and the current scientific community, there is a large gap in knowledge surrounding subcutaneous drug injection, as the mechanisms of uptake of the drug to the blood and lymphatics, the events that occur in vivo upon injection, and the immunological responses and how they impact the overall action of the injected drug is largely unknown. Due to this end, pharmaceutical companies use thousands of animals every year to evaluate candidate formulations intended for use in a human environment. Often, these studies give little feedback on how each excipient within a formulation impacts the post-injection phenomena that will dictate bioavailability. When developing novel formulations, scientists go through a trial-and-error development process, altering excipient and buffer concentrations and testing these formulation candidates in vast numbers of animal studies before finalizing pharmaceutical composition which may still require optimization later. Understanding how each excipient aids or hinders the active pharmaceutical ingredient’s (API’s) release in a biomimetic environment prior to pre-clinical testing is paramount in guiding decisions on formulation composition, ensuring drug candidates with only the highest likelihood of success are tested at later stages. To this end, we looked to employ an in vitro subcutaneous injection site simulator (‘SCISSOR’) to determine each excipient’s (buffers, surfactants, cryoprotectants) effect on formulation behavior post-injection. Herein, we describe the pharmacokinetic behavior of a commercially available monoclonal antibody formulation following modification of its excipients before injection.

Methods: Formulation preparation: A commercial formulation of denosumab was obtained from a local pharmacy. To remove each excipient, 1 mL of the solution was placed in a 10kDa cutoff dialysis cassette (Slide-A-Lyzer™ G3 Dialysis Cassettes, 10K MWCO, Thermo A52971) and dialyzed against a citrate buffer @pH=6.0 or phosphate buffer @ pH=7.4 with (or without) each excipient of interest (polysorbate 20, sorbitol, buffer). SCISSOR Assays: The subcutaneous injection site simulator “SCISSOR” was obtained from Pion Inc., USA. The system comprises an artificial extracellular matrix (ECM) (simulating the subcutaneous space) filled into a cartridge suspended in a bicarbonate-based buffer at pH ~ 7.4, maintained by introducing CO2(g) of top of the solution that provided “infinite sink” conditions. The system was equilibrated to 34 °C for 30 min prior to injection inside the cartridge. For all injectates, a 200 mL aliquot was introduced via Eppendorf Repeater® E3 pipette using modified 1 mL Eppendorf Combitip advanced tips provided by Pion Inc., USA (Part number 10900-0012) fitted to a 25GX 5/8"Med Vet International Terumo needle at 15 µL/s. The %release of each compound was monitored via in situ fiber-optic UV-Vis probes from a Rainbow spectrophotometer unit (Pion Inc., USA).

Results: pH / Buffer Effect: Citrate and Acetate buffer were exchanged over a period of 18 hours. The unadulterated injectable released 102 ± 1 % over 67 hours and the injectable dialyzed against citrate buffer resulted in 91 ± 6 % over the same time span. When the citrate buffer was exchanged for phosphate buffer, the release dropped to 76 ± 3% over 50 hours. Surfactant and Cryoprotectant Effect: The concentrations of polysorbate 20 and sorbitol were altered to determine how the presence of a surfactant will affect post-injection events and release behavior. The unadulterated sample released 102 ± 1 % over 67 hours, while the sample with only surfactant as an excipient resulted in immediate release, with 58 ± 25% releasing immediately post-injection. The removal of polysorbate 20 and sorbitol resulted in a maximum release of 56 ± 18% at 43 hours.

Conclusion: Multiple variations of the same commercially available subcutaneously administered formulation of denosumab were assayed within the SCISSOR platform to show how each excipient effects the post-injection phenomena that will dictate the pharmacokinetic behavior of the injectate. Citrate (pH=6.0) and phosphate (pH=7.4) were exchanged as the solvent for the injectable solution. Not only did this result in different release profiles, but the pH that resided closer to the commercially available formulation produced a faster and complete release of the antibody. The surfactants effect on the release profile can be seen by the “early release” kinetics observed when only a surfactant is included without another thickening agent. The formulation could not achieve full release with the polysorbate removed from the system, most likely due to the tendency of biomolecules to aggregate over time in pristine aqueous solutions. Cryoprotectants were shown to negate the effects that a surfactant has on aqueous solutions. The SCISSOR platform showed reflux along the needle pathway will result if surfactants are the only excipients. The addition of sorbitol allowed controlled and complete release of the API over the expected timespan. In conclusion, the SCISSOR platform demonstrated how each excipient can affect a monoclonal antibody-based formulation, taking a small step in developing an in vitro tool for pre-clinical formulation analysis.