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Measuring Particle Size Distribution of Nanoparticle Enabled Medicinal Products, the Joint View of EUNCL and NCI-NCL. a Step By Journal of Controlled Release 299 (2019) 31–43 Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel Review article Measuring particle size distribution of nanoparticle enabled medicinal T products, the joint view of EUNCL and NCI-NCL. A step by step approach combining orthogonal measurements with increasing complexity ⁎ ⁎⁎ Caputo F.a, , Clogston J.b, Calzolai L.c, Rösslein M.d, Prina-Mello A.e,f, a Univ. Grenoble Alpes, CEA, LETI, F-38000 Grenoble, France b Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA c European Commission, Joint Research Centre, Via Enrico Fermi 2749, 21027 Ispra, VA, Italy d Swiss Federal Laboratories for Materials Research and Testing, Laboratory for Particles—Biology Interactions, EMPA, Lerchenfeldstrasse 5, St. Gallen CH-9014, Switzerland e Laboratory for Biological Characterisation of Advanced Materials (LBCAM), Department of Clinical Medicine, Trinity Translational Medicine Institute (TTMI), School of Medicine, Trinity College Dublin, Dublin 8, Ireland f AMBER Centre and CRANN Institute, Trinity College Dublin, Dublin 2, Ireland ARTICLE INFO ABSTRACT Keywords: The particle size distribution (PSD) and the stability of nanoparticles enabled medicinal products (NEP) in complex Nanomedicine biological environments are key attributes to assess their quality, safety and efficacy. Despite its low resolution, Nanoparticles enabled medicinal products dynamic light scattering (DLS) is the most common sizing technique since the onset of NEP in pharmaceutical Physico-chemical properties technologies. Considering the limitations of the existing sizing measurements and the challenges posed by complex Particle size distribution NEPs both scientists and regulators encourage the combination of multiple orthogonal high-resolution approaches to Morphology shed light in the NEP sizing space (e.g. dynamic light scattering, electron microscopy, field flow fractionation coupled Standard operating procedures (SOPs) to online sizing detectors, centrifugal techniques, particle tracking analysis and tunable resistive pulse sensing). The pharmaceutical and biotechnology developers are now challenged to find their own pragmatic characterisation ap- proaches, which should be fit for purpose and minimize costs at the same time, in a complicated landscape whereonly a few standards exist. In order to support the community, the European Nanomedicine Characterisation Laboratory (EUNCL) and the US National Cancer Institute Nanotechnology Characterization Laboratory (NCI-NCL) have jointly developed multiple standard operating procedures (SOPs) for NEP assessment, including the measurements of particle size distribution, and are offering wide access to their ‘state of the art’ characterisation platforms, in additionto making SOPs publicly available. This joint perspective article would like to present the NCI-NCL and EUNCL multi- step approach of incremental complexity to measure particle size distribution and size stability of NEPs, consisting of a quick preliminary step to assess sample integrity and stability by low resolution techniques (pre-screening), followed by the combination of complementary high resolution sizing measurements performed both in simple buffers and in complex biological media. Test cases are presented to demonstrate: i) the need for employing at least one high- resolution sizing technique, ii) the importance of selecting the correct sizing techniques for the purpose, and iii) the robustness of utilizing orthogonal sizing techniques to study the physical properties of complex NEP samples. Abbreviations: AF4, asymmetric flow field flow fractionation; AUC, analytical ultracentrifugation; DCS, Differential centrifugal sedimentation; DLS, Dynamiclight scattering; EMA, European medical agency; EUNCL, European Nanomedicine Characterisation Laboratory; FDA, US Food and Drug Administration; FFF, field flow fractionation; MALS, multi angle light scattering; MNs, multinationals; NCI-NCL, National Cancer Institute Nanotechnology Characterization Lab; NEP, nanoparticle enabled medicinal products; NTA, nanoparticle tracking analysis; PCS, photon correlation spectroscopy; PdI, polydispersity index; PSD, particle size distribution; PTA, Particle tracking analysis; SEC, size exclusion chromatography; SMEs, small and medium enterprises; SOP, standard operating procedure; TEM, Transmission electron microscopy; TRPS, Tunable Resistive Pulse Sensing; UV, ultra-violet. ⁎ Corresponding author. ⁎⁎ Corresponding author at: Laboratory for Biological Characterisation of Advanced Materials (LBCAM), Department of Clinical Medicine, Trinity Translational Medicine Institute (TTMI), School of Medicine, Trinity College Dublin, Dublin 8, Ireland; AMBER Centre and CRANN Institute, Trinity College Dublin, Dublin 2, Ireland. E-mail addresses: [email protected], [email protected] (F. Caputo), [email protected] (A. Prina-Mello). https://doi.org/10.1016/j.jconrel.2019.02.030 Received 20 December 2018; Received in revised form 20 February 2019; Accepted 20 February 2019 Available online 21 February 2019 0168-3659/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). F. Caputo, et al. Journal of Controlled Release 299 (2019) 31–43 1. Introduction addition, several articles are available where lessons learned and knowledge are shared with the scientific community [3–6,12,13]. The application of nanotechnology in healthcare has a tremendous From this close interaction, it emerged that average particle size, potential to address a variety of medical conditions by providing better particle size distribution (PSD) and aggregation behaviors in complex diagnostics and therapy. A broad range of nanoparticles enabled med- biological media are, among others, critical quality attributes for the icinal products (NEP) have been investigated for medical applications, preclinical characterisation of NEP [3,5,14–17]. In fact, they influence including liposomes, polymeric nanoparticles, lipid-based nano- the body absorption, biodistribution and excretion, during their phar- particles, micelles, nanocrystals, metal colloids, metal oxides, and many macological targeting and off targets effects [1,18,19]. Importantly, the others [1]. Unfortunately, when compared to the extensive research particle size and polydispersity can significantly change due to inter- activities in research laboratories and industries (e.g., SMEs and MNs), actions of plasma proteins with the NEP surface. In biological media the clinically approved nanomedicines are still very limited [2]. Among NEPs may aggregate increasing the average population size and even the extensive number of NEP published in literature, only about 50 sediment or, on the contrary, aggregation may be reduced, or they can candidates have successfully crossed the “valley of death”, and thus start to degrade, producing populations of smaller particles. translated from the research laboratories to scale up approved product Due to the key role in determining the efficacy and safety of NEP for use in clinical setups. Very often NEPs fail to reach late clinical [1], particle size, polydispersity and size stability should be controlled phases due to the lack of pre-clinical characterisation protocols, which in simple and in biological media from the early development stages, are needed to correlate their physico-chemical properties with their ideally from their design. At later stages, e.g. during routine manu- biological effects [1,3,4]. If compared to “classical” small molecule facturing, average size and polydispersity are key attributes to be drugs, the assessment of the quality, safety and efficacy profiles of NEPs controlled for quality purposes. According to the regulators' guidelines, demands the investigation of many additional physico-chemical prop- including EMA [8–10] and FDA [7,11], the NEP developers should not erties, including their chemical composition, average particle size and only characterise the particle size distribution of their products im- polydispersity, dispersion stability, particle shape, surface charge, drug mediately after their synthesis, but also study i) the reproducibility of loading and drug release, surface coating and hydrophobicity [1–3,5]. the manufacturing procedure (batch to batch consistency), ii) the long Moreover, the physico-chemical properties of NEP, including the size, term stability of the formulation during storage, iii) measure the na- physical and chemical stability, surface properties and drug release noparticle size in the final product administered to the patient andfi- profile, need to be investigated, not only in their original formulation, nally iv) analyze the changes of size and shape of the NEP after being in but also when dispersed in biological complex environments, since contact with physiological media (e.g., interactions with serum pro- different pH, high ionic strengths and the interactions with plasmacan teins) [3]. modify the physical properties of the particles, thus influencing their Many techniques are being used to measure the size distribution of safety and efficacy profiles [3,6,7]. To complicate matters even more, nanoparticle formulation in medicinal products, including electron the uniqueness of every NEP imposes a different methodological
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