Developing a robust in vitro release method for a polymeric nanoparticle: Challenges and learnings

Nanomedicines have emerged as a promising approach for targeted therapeutic delivery and specifically as a beneficial alternative to conventional cancer therapies. They can deliver higher concentrations of chemotherapeutic agents at the tumour site compared to healthy tissue, hence provide improved drug efficacy and lower systemic toxicity (Bor et al., 2019, Ahlawat et al., 2018). Although nanoparticle research has started as early as in the 1990′s, currently there are only nine approved nanomedicines for the treatment of various types of cancers, liposomal formulations in their majority (Anselmo and Mitragotri, 2019).

AZD2811, is a potent and selective inhibitor of Aurora B kinase activity, which is related to cell progression regulation during mitosis. Its water-soluble dihydrogen phosphate prodrug, AZD1152, has been tested in clinical trials in various tumours, including acute myeloid leukaemia (AML) and has demonstrated efficacy. However, toxicity related to immunosuppression was also reported. The long infusion times of the prodrug in combination with its toxicity profile, posed challenges for the successful administration to patients. For this reason, the active pharmaceutical ingredient (API) AZD2811 was formulated as a polymeric nanoparticle formulation to enable controlled API release from the formulation in the blood, which would improve the toxicity profile while maintaining its efficacy (Floc’h et al., 2017, Floc’h et al., 2019).

The Accurins^TM nanoparticle technology which was selected to provide controlled drug release following IV infusion is based on a block copolymer formulation comprising poly-L,D-lactide (PLA) and poly(ethylene glycol) (PEG). An ion pairing approach was taken to increase AZD2811 encapsulation efficiency and decrease the API release rate. Following investigation of a range of counter ions, pamoic acid was identified as the optimal counter ion (Ashton, 2016).

Development of an in vitro release (IVR) method for nanomedicines is challenging because of the uniqueness and range of different formulation design approaches, as well as the complex nature of drug release mechanisms which may result in inherent variability. Regulatory guidance on the development of dissolution or release methods is limited as a one-size fits-all approach is not appropriate (Dadhaniya, 2015, Bao, 2022).

The FDA has published a guidance for industry on the topic of “Drug products, including biological products, that contain nanomaterials”, which provides recommendations rather than requirements around characterization methods and testing that demonstrate product quality (FDA, 2022). In this guidance, IVR is only highlighted as a potential critical quality attribute (CQA), depending on its impact on product performance, including quality, safety and efficacy.

The USP provides further recommendations rather than requirements in USP 〈1001〉 ‘In vitro release test methods for parenteral drug preparations’ (United States Pharmacopeia, 2023). This USP article emphasises the importance of drug release and provides selected information on some of the most commonly applied methods for the testing of nanosuspensions and liposomes. In addition, the USP stimuli articles ‘In-Vitro Product Performance of Parenteral Drug Products’ (D’Arcy, 2022) and ‘Testing the In-Vitro Product Performance of Nanomaterial based Drug Products’ (Wacker, 2022) provide some recommendations for the development of separation and isolation methods used to determine the released fraction of the API. They also present considerations for validation of the release assay and discuss the current practices and limitations in test apparatus, conditions, medium selection and separation techniques.

Currently, there is an increasing interest, both in the academic and the industrial space, in the development of nanosized long-acting injectable formulations, particularly for the treatment of chronic conditions, which release the drug over a few weeks or even months. Subsequently, the duration of the IVR testing of modified release formulations has been increased from a few days to significantly longer periods of time, with all the challenges that this brings (Bao, 2022). Examples of such parenteral formulations include PLGA/PLA-based microspheres (such as Bydureon®), bioresorbable solid implants (such as Zoladex®) and in situ forming depot technologies (Gonella, 2022).

The replacement of conventional oral therapies with parenteral therapies for the treatment of long-term conditions would reduce the frequency of dosing and subsequently improve patient compliance and quality of life. Due to the limited regulatory guidance available, the development of an IVR method can prolong the drug product development and delay delivery of life-changing medicines to patients (Dadhaniya, 2015).

The development of an IVR method is not only critical for the full characterisation of a formulation and a regulatory requirement, but also crucial in establishing an in vitro-in vivo correlation (IVIVC) or an in vitro-in vivo relationship (IVIVR) when an IVIVC is not feasible (Polli, 2000). When an IVIVR is available, then any side effects of the drug product could be predicted based on out-of-trend IVR performance (Bao, 2022).

A commercial IVR method is required to discriminate product and process differences that may have an impact on the quality and clinical performance of the drug product (United States Pharmacopeia, 2023), thus supporting the drug product throughout its life cycle. This can be achieved by comparing the performance of batches manufactured within a defined space of product and process parameters that result in a formulation that meets its specifications with that of variants produced outside this defined space and should be able to identify the batches that demonstrate bioequivalence to the pivotal clinical batch. Finally, it is recommended that the method should provide complete release profile, where the API has reached a plateau of at least 85% release (FDA, 2022).

The IVR method used to measure the release profile of the different ion pairs from the AZD2811 polymeric nanoparticles as well as for the release of clinical batches was fit-for-purpose at that early stage of drug product development; it provided clinically relevant release data and was discriminative between nanoparticle batches made with different processing parameters, expected to impact the IVR rate. Since an in vitro-in vivo correlation was not feasible (batches with different IVR profiles were not discriminated in vivo based on preclinical PK data), the principle of clinical relevance was employed instead. According to this, IVR data were clinically relevant when they fell within a pre–defined IVR safe space, within which drug product variants were anticipated to be bioequivalent to one another. The development of a biorelevant IVR method in a release medium representing the physiological environment was not the aim of this work, since it could introduce robustness challenges.

The ability of the fit-for-purpose IVR method to discriminate between batches with varying IVR rates was of great significance, since it could identify nanoparticles that were not suitable for use in clinical trials either due to slow API release, which would not result in the desired efficacy, or due to the fast API release, which could result in toxicity (Song, 2016). Despite its discriminative ability though, this method lacked inherent robustness. Issues such as day-to-day reproducibility, complex and laborious manual intervention needed for sampling and input material variability (release medium surfactant quality) resulted in variability in the IVR data.

To improve the robustness and minimise the variability seen in IVR data generated during product development for AZD2811 polymeric nanoparticles, an investigation was carried out to identify the root causes of the data variability. Extensive method development work followed around the selection of apparatus, isolation technique to separate the released API from the formulation and release medium composition.

Materials

Polysorbate 20 (product number P1379 and P7949), methyl-β-cyclodextrin (MeβCD, product number 779873), Triton™ X-100 (product number T8787), sodium dodecyl sulfate (SDS, product number L3771), Brij S100 (product number 466387), benzyl alcohol, ethyl acetate, dimethyl sulphoxide (DMSO), high pressure liquid chromatography (HPLC) grade trifluoroacetic acid (TFA), sodium chloride (NaCl) pellets, potassium chloride (KCl), di-sodium phosphate (Na₂HPO₄), sodium phosphate (NaH₂PO₄), potassium