Onivyde® (irinotecan liposomal injection) was approved by the U.S. Food and Drug Administration (FDA) in 2015 for the treatment of metastatic pancreatic cancer (Zhang, 2016). Its liposomal formulation enhances drug stability, prolongs circulation time in the body, and promotes tumor-specific accumulation via the enhanced permeation and retention effect (Milano et al., 2022). Onivyde®, when combined with fluorouracil and leucovorin, is prescribed to patients who have progressed after gemcitabine-based therapy (Wang-Gillam, 2016). Clinical evidences have demonstrated that Onivyde® provides superior efficacy compared to free irinotecan, with significant improvements in overall survival (de Man, 2018). In 2023, it was also approved as a first-line treatment for metastatic pancreatic ductal adenocarcinoma, further solidifying its role in cancer therapeutics (Cui, 2024).

Given its growing clinical significance, strong market presence, and the impending patent expiration in 2025, generic manufacturers are keen to develop a bioequivalent version of Onivyde®. Regulatory approval for generics requires demonstrating Q1/Q2 sameness and bioequivalence to the reference product (Stern, 2021). This process requires comparative studies to ensure that the generic product matches the reference in both composition and performance (Ren, 2023). However, developing a generic version of Onivyde® is particularly challenging due to its complex structure and intricate production process. Onivyde® features a unique unilamellar lipid bilayer, with a size distribution around 110 nm (Milano et al., 2022). These liposomes, typically spherical or elliptical in shape, encapsulate irinotecan as needle-like crystals within their cavity, achieved through active remote loading process (Bernardi, 2023, Liu, 2019). This method creates a concentration gradient of sucrose octasulfate ester salts across the lipid bilayer, allowing irinotecan to cross the membrane and precipitate with the sucrose salts inside the liposome (Drummond, 2006). This advanced drug loading technology effectively stabilizing irinotecan within the liposomes but undoubtedly increases the complexity of manufacturing liposomal irinotecan. The multi-step manufacturing process poses additional challenges for maintaining sterility and quality control. As the results, characterization methods are essential to accurately identify and assess the product quality throughout the development process.

A robust in vitro release test (IVRT) is critical for assessing the quality and consistency of both the innovative and generic Onivyde® formulations. Such tests are essential for formulation development, quality control during production, and establishing bioequivalence with the reference products (Larsen, 2009). Moreover, a well-established IVRT can serve as a predictor of in vivo performance, supporting the establishment of an in vitro-in vivo correlation (IVIVC) (Shen and Burgess, 2015). A successful IVIVC, particularly a level A correlation, could serve as a surrogate for in vivo bioequivalence studies and streamline regulatory approval by reducing the need for extensive clinical studies. (Somayaji et al., 2016). However, standardized IVRT procedures or regulations for liposomal irinotecan products are still lacking. Considering the significance of IVRT for quality control and predicting in vivo behavior of liposomal irinotecan, it is essential to develop suitable IVRT methods for generics development and formulation comparison.

In this study, we have established a robust, reproducible, and discriminative in vitro accelerated release method for Onivyde® using the Agilent NanoDis® system, achieving a 24-hour release profile. This system integrates conventional dissolution equipment with tangential flow filtration, allowing for automated separation of the released drug from the intact liposomes (Mead, 2023, Taylor-Vine, 2023) (Fig. S1). Tangential Flow Filtration (TFF) in the Agilent NanoDis® system offers significant advantages over traditional dialysis and other sample separation methods for nanoparticle release testing. Unlike traditional dialysis, which often suffers from slow permeation rates and membrane saturation, TFF actively filters the medium and quickly separates the released drugs from nanoparticles (Lombardo, 2021). This dynamic filtration process minimizes clogging and avoids concentration polarization effects that can affect accuracy in dialysis. Additionally, TFF’s continuous flow prevents particle aggregation and reduces mechanical stress on the particles, making it overcome the severe drawbacks of other sample separation methods such like centrifugation, filtration, or centrifugal ultrafiltration (Patel, 2021). With its rapid, efficient separation capabilities, the NanoDis® system thus provides more accurate and reliable results on nanoparticle release, aligning with automated operation process. We optimized key release conditions, including materials and molecular weight cut-offs (MWCOs) of the filters, composition and pH of the release medium, and paddle speed. Our optimized IVRT successfully differentiates formulations of different qualities, evaluates batch-to-batch consistency, discriminates between mixtures with various percentages of liposomal irinotecan and free drug. Discriminative in vitro release methods can serve as important quality assessment tools throughout the entire lifecycle of a product. Overall, our IVRT method holds potential as a quality control and assurance characterization, to ensure consistent therapeutic performance across products. Moreover, it can also offer assessment of bioequivalence for generic Onivyde® formulations, facilitating regulatory agencies in the approval process of the related products.