1. Operating the machine
<ol>
	<li>Starting up the machine and software 
	NOTE: Supplemental Figure S1A-D describes the steps for starting up the machine and software.
	<ol>
		<li>Switch on the instrument at least 30 min before starting the measurements, and then start the PC.</li>
		<li>Double-click on the instrument software icon to start the program.</li>
		<li>Enter the username and password in the login window. Ensure that each user has their own account.</li...

<ol>
	<li>Auerbach, M., Gafter-Gvili, A., Macdougall, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+iron:+A+framework+for+changing+the+management+of+iron+deficiency.">Intravenous iron: A framework for changing the management of iron deficiency.</a> Lancet Haematology. 7 (4), e342-e350 (2020).</li><li>Generic drugs: FDA should make public its plans to issue and revise guidance on nonbiological complex drugs. US Government Accountability Office Available from: <a target="_blank" href="https://www.gao.gov/products/gao-18-80">https://www.gao.gov/products/gao-18-80</a> (2017)</li><li>Klein, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EU+regulatory+landscape+of+non-biological+complex+drugs+(NBCDs)+follow-on+products:+Observations+and+recommendations.">The EU regulatory landscape of non-biological complex drugs (NBCDs) follow-on products: Observations and recommendations.</a> European Journal of Pharmaceutical Sciences. 133, 228-235 (2019).</li><li>Astier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+select+a+nanosimilar.">How to select a nanosimilar.</a> Annals of the New York Academy of Sciences. 1407 (1), 50-62 (2017).</li><li>Rottembourg, J., Kadri, A., Leonard, E., Dansaert, A., Lafuma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+two+intravenous+iron+sucrose+preparations+have+the+same+efficacy.">Do two intravenous iron sucrose preparations have the same efficacy.</a> Nephrology Dialysis Transplantation. 26 (10), 3262-3267 (2011).</li><li>Aguera, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+original+versus+generic+intravenous+iron+formulations+in+patients+on+haemodialysis.">Efficiency of original versus generic intravenous iron formulations in patients on haemodialysis.</a> PLoS One. 10 (8), e0135967 (2015).</li><li>Alphandery, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+oxide+nanoparticles+for+therapeutic+applications.">Iron oxide nanoparticles for therapeutic applications.</a> Drug Discovery Today. 25 (1), 141-149 (2020).</li><li>Arami, H., Khandhar, A., Liggitt, D., Krishnan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+delivery,+pharmacokinetics,+biodistribution+and+toxicity+of+iron+oxide+nanoparticles.">In vivo delivery, pharmacokinetics, biodistribution and toxicity of iron oxide nanoparticles.</a> Chemical Society Reviews. 44 (23), 8576-8607 (2015).</li><li>Nikravesh, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+safety+and+efficacy+of+intravenous+iron-carbohydrate+nanomedicines:+From+production+to+clinical+practice.">Factors influencing safety and efficacy of intravenous iron-carbohydrate nanomedicines: From production to clinical practice.</a> Nanomedicine. 26, 102178 (2020).</li><li>Pai, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+DFO-chelatable+labile+iron+release+profiles+among+commercially+available+intravenous+iron+nanoparticle+formulations.">In vitro and in vivo DFO-chelatable labile iron release profiles among commercially available intravenous iron nanoparticle formulations.</a> Regulatory Toxicology and Pharmacology. 97, 17-23 (2018).</li><li>Brandis, J. E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+physicochemical+properties+of+the+iron+nanoparticle+drug+products:+Brand+and+generic+sodium+ferric+gluconate.">Evaluation of the physicochemical properties of the iron nanoparticle drug products: Brand and generic sodium ferric gluconate.</a> Molecular Pharmaceutics. 18 (4), 1544-1557 (2021).</li><li>Di Francesco, T., Sublet, E., Borchard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomedicines+in+clinical+practice:+Are+colloidal+iron+sucrose+ready-to-use+intravenous+solutions+interchangeable.">Nanomedicines in clinical practice: Are colloidal iron sucrose ready-to-use intravenous solutions interchangeable.</a> European Journal of Pharmaceutical Sciences. 131, 69-74 (2019).</li><li>Di Francesco, T., Borchard, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+and+easily+reproducible+protocol+for+the+determination+of+size+and+size+distribution+of+iron+sucrose+using+dynamic+light+scattering.">A robust and easily reproducible protocol for the determination of size and size distribution of iron sucrose using dynamic light scattering.</a> Journal of Pharmaceutical and Biomedical Analysis. 152, 89-93 (2018).</li><li>The Center for Research on Complex Generics. US Food and Drug Administration Available from: <a target="_blank" href="https://www.fda.gov/drugs/guidance-compliance-regulatory-information/center-research-complex-generics">https://www.fda.gov/drugs/guidance-compliance-regulatory-information/center-research-complex-generics</a> (2021)</li><li>Drug products, including biological products, that contain nanomaterials - Guidance for industry. Center for Drug Evaluation and Research. FDA-2017-D-0759. US Food and Drug Administration Available from: <a target="_blank" href="https://www.fda.gov/regulatory-information/search-fda-guidance-documents/drug-products-including-biological-products-contain-nanomaterials-guidance-industry">https://www.fda.gov/regulatory-information/search-fda-guidance-documents/drug-products-including-biological-products-contain-nanomaterials-guidance-industry</a> (2022)</li><li>US Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Draft+guidance+on+ferric+oxyhydroxide.">Draft guidance on ferric oxyhydroxide.</a> US Food and Drug Administration. , (2021).</li><li>Zou, P., Tyner, K., Raw, A., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+characterization+of+iron+carbohydrate+colloid+drug+products.">Physicochemical characterization of iron carbohydrate colloid drug products.</a> The AAPS Journal. 19 (5), 1359-1376 (2017).</li><li>D'Mello, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+landscape+of+drug+products+containing+nanomaterials+in+the+United+States.">The evolving landscape of drug products containing nanomaterials in the United States.</a> Nature Nanotechnology. 12 (6), 523-529 (2017).</li><li>Q2 (R1) Validation of analytical procedures: Text and methodology guidance for industry. FDA-2017-D-6821. US Food and Drug Administration Available from: <a target="_blank" href="https://www.fda.gov/regulatory-information/search-fda-guidance-documents/q2r1-validation-analytical-procedures-text-and-methodology-guidance-industry">https://www.fda.gov/regulatory-information/search-fda-guidance-documents/q2r1-validation-analytical-procedures-text-and-methodology-guidance-industry</a> (2005)</li><li>Reflection paper on the data requirements for intravenous iron-based nano-colloidal products developed with reference to an innovator medicinal product. European Medicines Agency Available from: <a target="_blank" href="https://www.ema.europe.eu/en/documents/scientific-guideline/reflection-paper-data-requirements-intravenous-iron-based-nano-colloidal-products-developed_en.pdf">https://www.ema.europe.eu/en/documents/scientific-guideline/reflection-paper-data-requirements-intravenous-iron-based-nano-colloidal-products-developed_en.pdf</a> (2015)</li><li>Fischer, K., Schmidt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitfalls+and+novel+applications+of+particle+sizing+by+dynamic+light+scattering.">Pitfalls and novel applications of particle sizing by dynamic light scattering.</a> Biomaterials. 98, 79-91 (2016).</li><li>Caputo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric-flow+field-flow+fractionation+for+measuring+particle+size,+drug+loading+and+(in)stability+of+nanopharmaceuticals.+The+joint+view+of+European+Union+Nanomedicine+Characterization+Laboratory+and+National+Cancer+Institute+-+Nanotechnology+Characterization+Laboratory.">Asymmetric-flow field-flow fractionation for measuring particle size, drug loading and (in)stability of nanopharmaceuticals. The joint view of European Union Nanomedicine Characterization Laboratory and National Cancer Institute - Nanotechnology Characterization Laboratory.</a> Journal of Chromatography A. 1635, 461767 (2021).</li><li>Yusa, S., Narain, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+6+-+Polymer+characterization.">Chapter 6 - Polymer characterization.</a> Polymer Science and Nanotechnology. , 105-124 (2020).</li></ol>

M.B., E.P., M.W., and A.B. are employees of Vifor Pharma. G.B. is a consultant for Vifor Pharma.

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...

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 &plusmn; 0.1 nm vs. 10.1 nm &plusmn; 0.1 nm, p &lt; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer Nano ZS</td>
			<td>Malvern</td>
			<td>NA</td>
			<td>equipped with Zetasizer software 7.12, Helium Neon laser (633 nm, max. 4 mW) and 173&#176; backscattering geometry</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable plastic cuvettes&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LLG-Disposable plastic cells</td>
			<td>LLG labware</td>
			<td>LLG-K&#252;vetten, Makro, PS; Order number 9.406011</td>
			<td></td>
		</tr>
		<tr>
			<td>low-particle water</td>
			<td></td>
			<td></td>
			<td>&#160;(The use of freshly deionized and filtered (pore size 0.2 &#956;m) water is recommended).</td>
		</tr>
		<tr>
			<td>Microlitre pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Venofer 100 mg/5 mL</td>
			<td>Vifor Pharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Volumetric flask 25 mL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanosphere</td>
			<td>Thermo</td>
			<td>3020A</td>
			<td>Particle Standard</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Origin Pro v.8.5&#160;</td>
			<td>Origin Lab Corporation</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dynamic light scattering analysis for the determination of the particle size of iron-carbohydrate complexes

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.

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 ...

Watch this Scientific Journal Video about Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes at JoVE.com

Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes

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 ...

Dynamic light scattering, or DLS, is a fundamental method to evaluate intact particle size and distribution. However, analysis of iron carbohydrate nanoparticles does pose some challenges. An advantage of DLS include wide available instrumentation, easy to perform the analysis, and establish protocols for the analysis of nanomaterials.One of the goals of DLS is to evaluate the poly dispersity profile of the nanoparticles, which may ultimately affect the interaction with the biological milieu. For the demonstration of the procedure, will be Cintia Batista Marques, a PhD candidate from University of Geneva. After starting the machine in the instrument software, select the storage location in the opened window, name the measurement file, and confirm the details by clicking on save.Select the required standard operating procedure from the dropdown list in the instrument interface. If an older SOP is needed, select browse for SOP from the list and confirm the selection by clicking on the green arrow. To begin the measurement process, click on the green start button at the bottom of the instrument interface screen.Then, fill one milliliter of the undiluted particle standard into a polystyrene cuvette and close it with the lid. Once filled, ensure that there are no air bubbles. If any air bubbles are present, remove them by tapping lightly on the cuvette.Place the cuvette in the cell holder of the instrument with the arrow mark facing forward and close the measuring chamber cover. Load the unit parameter SOP and enter sample name SST 20 nanometer particle standard in the start window. Additionally, add a note that includes an identifier number and the expiry date of the standard.To measure iron sucrose solution, pipette 0.5 milliliters of an iron sucrose solution with 2%mass by volume iron content into a 25 milliliter volumetric flask and fill up to the mark with low particle water, resulting in a solution containing 0.4 milligrams iron per milliliter. Now place the plastic cuvette containing the measuring solution in the device with the arrow mark facing forward and close the lid. Load the parameter SOP again and enter sample name batch number in the start window.Start the measurement and when it finishes, indicated by an acoustic signal, close the measurement window. Calculate the mean value of six individual measurements. Mark the individual measurements in the records view of measurement file and right click on create average result.Add the name of the mean value under sample name. Then confirm by clicking on okay. Wait for the software to create a new record at the end of the list and look for a name entered as well as the average result in that record.The size distribution plots by intensity, volume, and number are shown. Size distribution by intensity impacted by a second peak is provided as an example of a bad result. Poor quality data showed an additional signal at 5, 000 nanometers.The size distribution by number differed by up to a factor of two from the proposed intensity based Z average. Only slightly lower values were calculated by the size distribution by volume. When using DLS to characterize iron carbohydrate nanoparticles, it is important to ensure correct SOP, sample dilution, and also cuvette filling.Asymmetrical flow field flow fractionation and size exclusion chromatography and small angle x-ray scattering can be performed as additional orthogonal physico-chemical methods. Validated protocols for DLS greatly improve the robustness of initial and comparative characterization of iron carbohydrate nanoparticles. However, the sample preparation and the bias of the technique toward large particle sizes needs to be considered in the context of orthogonal methodologies.

dynamic-light-scattering-analysis-for-determination-particle-size

Research

JoVE Journal

Medicine

1.9K ビュー.  Vifor Pharma Management Ltd. 動的光散乱(DLS)は、無傷の粒子サイズと分布を評価するための基本的な方法です。ただし、鉄炭水化物ナノ粒子の分析にはいくつかの課題があります。DLSの利点には、幅広い機器、分析の実行が容易、ナノ材料の分析のためのプロトコルを確立することが含まれます。DLSの目標の1つは、最終的に生物学的環境との相互作用に影響を与える可能性のあるナノ粒子のポ?...