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Dynamic light scattering (DLS) has emerged as a suitable assay for evaluating the particle size and distribution of intravenously administered iron-carbohydrate complexes. However, the protocols lack standardization and need to be modified for each iron-carbohydrate complex analyzed. The present protocol describes the application and special considerations for the analysis of iron sucrose.
Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally heterogeneous nanoparticle complexes, which exhibit varying sensitivity to the conditions required for the methodologies available to physicochemically characterize these agents. Currently, the critical quality attributes of iron-carbohydrate complexes have not been fully established. Dynamic light scattering (DLS) has emerged as a fundamental method to determine intact particle size and distribution. However, challenges still remain regarding the standardization of methodologies across laboratories, specific modifications required for individual iron-carbohydrate products, and how the size distribution can be best described. Importantly, the diluent and serial dilutions used must be standardized. The wide variance in approaches for sample preparation and data reporting limit the use of DLS for the comparison of iron-carbohydrate agents. Herein, we detail a robust and easily reproducible protocol to measure the size and size distribution of the iron-carbohydrate complex, iron sucrose, using the Z-average and polydispersity index.
Iron sucrose (IS) is a colloidal solution comprised of nanoparticles consisting of a complex of a polynuclear iron-oxyhydroxide core and sucrose. IS is widely employed to treat iron deficiency among patients with a wide variety of underlying disease states who do not tolerate oral iron supplementation or for whom oral iron is not effective1. IS belongs to the drug class of complex drugs as defined by the Food and Drug Administration (FDA), which is a class of drugs with complex chemistry commensurate with biologicals2. The regulatory evaluation of complex drug products may require additional orthogonal physicochemical methods and/or preclinical or clinical studies to accurately compare follow-on complex drugs3,4. This is important because several studies have reported that the use of IS versus a follow-on IS product does not produce the same clinical outcomes. This underscores the criticality of the use of novel and orthogonal characterization techniques that are suitable for detecting differences in the physicochemical properties between IS products5,6.
The accurate elucidation of the size and size distribution of IS is of clinical importance, as particle size is a major influential factor in the rate and extent of opsonization-the first critical step in the biodistribution of these complex drugs7,8. Even slight variations in the particle size and particle size distribution have been related to changes in the pharmacokinetic profile of iron-oxide nanoparticle complexes9,10. A recent study by Brandis et al. showed that particle size measured by DLS was significantly different (14.9 nm ± 0.1 nm vs. 10.1 nm ± 0.1 nm, p < 0.001) when comparing a reference listed drug and a generic sodium ferric gluconate product, respectively11. The consistent batch-to-batch quality, safety, and efficacy of iron-carbohydrate products are entirely dependent on the manufacturing process scale-up, and potential manufacturing drift must be carefully considered9. The manufacturing process may result in residual sucrose, and this will vary based on the manufacturer12. Any modifications in the manufacturing process variables can lead to significant changes in the final complex drug product with regard to the structure, complex stability, and in vivo disposition9.
To assess drug consistency and predict the drug's in vivo behavior, contemporary orthogonal analytical methodologies are required to determine the physicochemical properties of complex nanomedicines. However, there is a lack of standardization of methodologies, which can result in a high degree of interlaboratory variation in result reporting13. Despite the recognition of these challenges by global regulatory authorities and the scientific community14, most of the physicochemical characteristics of IS remain poorly defined, and the full complement of critical quality attributes in the context of available regulatory guidance documents have not been defined15. The draft product-specific guidance documents issued by the FDA for iron-carbohydrate complexes suggest DLS as a procedure to evaluate the size and size distribution of follow-on products16,17.
Several publications have detailed established DLS protocols to determine IS nanoparticle dimensions13,18. However, because the sample preparation, procedure conditions, instrumentation, and instrumentation setting parameters are different among the published methods, the DLS results cannot be directly compared in the absence of a standardized method to interpret the results13,18. The diversity in methodologies and data-reporting approaches limit the appropriate evaluation of these characteristics for comparative purposes19. Importantly, many of the DLS protocols previously published to evaluate IS do not account for the effect of the diffusion of sucrose in the suspension due to the presence of free sucrose, which has been shown to spuriously elevate the Z-average-calculated hydrodynamic radii of the nanoparticles in colloidal solutions13,18. The present protocol aims to standardize the methodology for the measurement of the particle size and distribution of IS. The method has been developed and validated for this purpose.
1. Operating the machine
The method described was validated according to ICH Q2(R1)20, which involved the measurement of test solutions under varying conditions. The precision was only 0.5% RSD for the Z-average size, while a maximum of 3.5% RSD was calculated for the PDI. The mean results from different analysts and days only differed by 0.4% for the Z-average size and 1.5% for the PDI. Statistics were calculated from 12 measurements performed by two analysts on varying days. Neither changes in the test concentration in ...
DLS has become a fundamental assay for the determination of the size and size distribution of nanoparticles for applications in drug development and regulatory evaluation. Despite advances in DLS techniques, methodologic challenges still exist regarding the diluent selection and sample preparation, which are especially relevant for iron-carbohydrate complexes in colloidal solutions. Importantly, DLS methods for iron-carbohydrate nanomedicines have not yet been studied extensively in the biological milieu (e.g., the plasm...
M.B., E.P., M.W., and A.B. are employees of Vifor Pharma. G.B. is a consultant for Vifor Pharma.
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Name | Company | Catalog Number | Comments |
Equipment | |||
Zetasizer Nano ZS | Malvern | NA | equipped with Zetasizer software 7.12, Helium Neon laser (633 nm, max. 4 mW) and 173° backscattering geometry |
Materials | |||
Disposable plastic cuvettes | |||
LLG-Disposable plastic cells | LLG labware | LLG-Küvetten, Makro, PS; Order number 9.406011 | |
low-particle water | (The use of freshly deionized and filtered (pore size 0.2 μm) water is recommended). | ||
Microlitre pipette | |||
Venofer 100 mg/5 mL | Vifor Pharma | ||
Volumetric flask 25 mL | |||
Nanosphere | Thermo | 3020A | Particle Standard |
Software | |||
Origin Pro v.8.5 | Origin Lab Corporation |
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