(W0930-03-18) In Vitro Release Method Development for Onivyde^® Using Agilent NanoDis^® System

Purpose: Onivyde^® is a liposomal injection formulated with the active pharmaceutical ingredient irinotecan, first approved by the U.S. Food and Drug Administration (FDA) in 2015. In combination with fluorouracil (5-FU) and leucovorin, Onivyde^® is prescribed for patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy. Additionally, it received supplemental approved as the first-line treatment for metastatic pancreatic ductal adenocarcinoma in 2023. Despite its robust sales and effective treatment for pancreatic cancer, development of generic version of Onivyde^® remains challenging due to its complex manufacturing process and distinctive characteristics. The FDA has issued draft guidance for characterizing pegylated liposomal products, stressing the importance of in vitro release tests (IVRT). A dependable IVRT holds several key benefits for development of generic Onivyde^®. It can aid in selecting lead formulations during development process and ensure consistent quality control for both innovative and generic products in the later stages. Additionally, a robust IVRT has the potential to predict Onivyde^®'s in vivo performance and behavior, enabling the establishment of an in vitro-in vivo correlation (IVIVC). Such a correlation, particularly if classified as level A, could serve as a surrogate for in vivo bioequivalence studies, thereby reducing clinical burdens. However, standardized IVRT procedures or regulations for assessing the quality and safety of generic irinotecan liposomal products are absent. Thus, this research aims to establish a reliable and reproducible in vitro release methodology for Onivyde^® using the Agilent NanoDis^® system.

Methods: The IVRT of Onivyde^® was conducted using an Agilent NanoDis^® system. Initially, 300 µL of Onivyde^® was introduced into 900 mL of release medium. The paddle speed and filter settings were configured accordingly, and temperature was set at 37°C. Release samples of 1 mL were collected at specific time intervals of 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hours, and replaced with an equal volume of fresh release medium. Meanwhile, an equal amount of free irinotecan solution was placed in the release medium as a positive control to represent the complete release. To assess the cumulative release (%) of irinotecan from the liposome formulations, the following equation was used: Cumulative Release _{irrinotecan ^(%)} = Flu_released / Flu_control  × 100%. Where Flu_released represents the fluorescence intensity of irinotecan released from liposome formulations, and Flu_control represents the fluorescence intensity of free irinotecan. Multiple factors were evaluated to optimize the IVRT of Onivyde^®. These factors included the concentration of ammonium bicarbonate in 4.05 mg/ml HEPES release medium (0, 0.25, 0.5, 1, 2, 3, 5 mM), the pH of the release medium (4.5, 5.5, 6.5, 7.4, 8.5), paddle speed (25, 50, 100 rpm) and the molecular weight cut-off (MWCO) of filters (30, 100, 300, 500 KD).

Results: The effects of ammonium bicarbonate on release profiles of Onivyde^® were depicted in Figure 1A. A higher concentration of ammonium bicarbonate in the release medium corresponded to a faster cumulative release rate. Without the addition of ammonium bicarbonate, the release percentage remained around 20% after 24 hours. However, at concentrations of 2, 3, and 5mM ammonium bicarbonate, irinotecan was rapidly released from liposome, reaching 90% release within 0.5h. When ammonium bicarbonate concentrations were 0.25, 0.5 and 1mM, Onyvide^® achieved 80% release within 24h. We selected 0.25 mM ammonium bicarbonate for subsequent experiments. The influence of pH of release medium on Onivyde^® was illustrated in Figure 1B. As the pH increased, the drug release rate gradually accelerated. We chose physiological pH 7.4 to continue the experiment. We also investigated the impact of paddle speed on the Onivyde^® release, as shown in the Figure 2. The released curve was slightly slower at 25rpm compared to those at 50 rpm and 100 rpm. Finally, we explored the influence of the filter MWCO on the release of Onivyde^®, as displayed in the Figure 3. With filters of 100, 300, and 500 KD, the drug release curves were nearly identical. However, when using a 30 KD filter, irinotecan had a much slower release rate. To ensure a fully penetration of free drug through the filter, we chose a 500 KD filter in our IVRT.

Conclusion: Building upon these experimental findings, the IVRT for Onivyde^® based on Agilent NanoDis^® system was successfully established. The optimized release medium consisted of 0.25mM ammonium bicarbonate in HEPES solution with a pH of 7.4. The method employed filters with 500 KD MWCO and maintained a paddle speed of 50rpm.


Figure 1. (A) Effect of ammonium bicarbonate concentrations on the release profiles of Onivyde®. The release experiments were performed in HEPES solution at various ammonium bicarbonate concentrations (pH 7.4) at 37°C. (B) Effect of pH values of release media containing 0.25mM ammonium bicarbonate in HEPES solution on the release profiles of Onivyde® at 37°C. A 50rpm paddle speed and 500 KD filters were applied (n = 3, mean ± SD).


Figure 2. Effect of paddle speed on the release profiles of Onivyde®. The release medium was 0.25 mM ammonium bicarbonate in HEPES (pH 7.4) at 37°C and experiments were performed with 500 KD filters (n = 3, mean ± SD).


Figure 3. The release profile of Onivyde® using filters with different molecular weight cut-off. The release media was 0.25 mM ammonium bicarbonate in HEPES (pH 7.4) at 37°C and paddle speed was 50 rpm (n = 3, mean ± SD).