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Characterisation


way of manufacturing could result in different products. These findings leave their mark on the reflection papers of the European Medicines Agency (EMA) for intravenous liposomal and iron-based nano-colloidal generic products.11–13 This article provides an overview of different characterisation methods, which have been used successfully to characterise and compare NBCDs. A special focus is put on the


characterisation of iron carbohydrate drugs with regard to requirements set out in the EMA reflection paper.


Physicochemical characterisation Although NBCDs can be widely characterised by physicochemical methods, this cannot assure sufficient biosimilarity between the products by itself.11


However, analytical tools are essential for quality characterisation (Chemistry, Manufacturing and Controls; CMC) and reproducibility of manufacturing of each batch. They provide information about size, purity, molecular weight and structure of the complexes. Furthermore, data on stability can be regarded as surrogates for the in vivo behaviour of the complexes. Some parameters, for example the size or polymorphism, require additional methods to verify results.12–14


An example toolbox for a


physicochemical characterisation of iron carbohydrate complexes is provided in Table 1.


Size, morphology and molecular weight With regards to the administration route of NBCDs, the size of the complexes should be determined, because it has a direct influence on the tissue distribution and elimination of the drugs. The importance of these measurements is underlined by the fact that the EMA reflection paper on liposomes, as well as on iron-based nano-colloidal products, recommend the determination of the size.13, 14


The two most common


techniques for the determination of size of NBCDs are either dynamic light scattering (DLS) or microscopic methods. These methods, however, are based on different physical principles and provide different information. It is therefore advisable to combine them. Whereas DLS measures the


hydrodynamic diameter of the particles, microscopic methods such as transmission electron microscopy (TEM), scanning electron microscopy


Stability/ purity


Table 1: Quality characterisation for intravenous iron-based nano-colloidal products14 Parameter


Size Size of particle Size of iron core Molecular weight Structure Morphology of the core Polymorphic form of iron core Charge


Structure and composition of carbohydrate matrix


Possible methods DLS


AFM TEM


XRPD GPC


TEM (+EDX) SEM


Mößbauer spectroscopy XRPD


Zeta potential NMR MS IR


UV-VIS


Degradation kinetics Amount of labile iron


Ratio divalent/trivalent iron


Acid degradation of iron (III) Ferrozine® Cerimetry


assay Mößbauer spectroscopy


(SEM) or atomic force microscopy (AFM) are helpful to obtain information about the geometric diameter of the whole complex or its single components. In the case of iron carbohydrates, TEM images enable the size determination of the iron core itself.6


Moreover, the high


number of counted particles with DLS enables the determination of the size distribution of the complexes and their polydispersity index (PDI); most microscopic methods are hampered by the large amount of observations needed to determine the size distribution of the complexes. A clear advantage of the microscopic methods is that they are perfectly suited for the determination of shape and morphology of the complexes. The vesicles in liposomal preparations for example can easily be grouped in uni- or multi-lamellar vesicles by images from cryo-electron microscopy (cryo- EM). Due to the wide information gained, microscopic methods should be part of every NBCD characterisation. Besides size, the molecular weight is also of interest. Gel permeation chromatography (GPC), which is a type of size exclusion chromatography, is a standard method for molecular weight determination. Separation is based on the hydrodynamic volume of samples and their distribution in the porous stationary phase. GPC determines the


References Jahn et al, 20116


Kudasheva et al, 200416 Fütterer et al, 20137


,


Kudasheva et al, 200416 Fütterer et al, 20137


Balakrishnan et al, 200924 Jahn et al, 20116


Balakrishnan et al, 200924 Kudasheva et al, 200416 Jahn et al, 20116


Fütterer et al, 20137 Jahn et al, 20116 Jahn et al, 20116 Jahn et al, 20116 Jahn et al, 20116


Kudasheva et al, 200416 Jahn et al, 20116 Jahn et al, 20116 Neiser et al, 20158 Jahn et al, 20116


,


, Neiser et. al8


viscosity molecular weight (Mv). Other


parameters such as the PDI and other molecular units can be calculated from the obtained data.


Structure


The ideal methods for structure characterisation are dependent on the type of NBCD. Some methods are suitable for different complex structures, whereas other methods cover more specific features of the corresponding formulation. Spectroscopic methods as nuclear magnetic resonance (NMR), infrared (IR), UV-Visible (UV-Vis) and inductively coupled plasma mass spectrometry (ICP-MS) are suitable for studying the composition of several NBCDs. Besides composition determination in terms of elemental analysis, some methods can provide additional information about the complex structure: NMR, for example, has been used to determine the liposomal encapsulation efficiency, 15


an


important characteristic for liposomes,12 whereas UV-Vis spectrometry was utilised for the investigation of the complex geometry. 16


The zeta potential


provides information about the stability of colloidal formulations. It is recommended for liposomes and for iron-based nano-colloidal products. 13, 14 Iron oxide hydroxides exist in several


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