(T1430-07-46) Advanced Characterization of Injectafer^®: Carboxymaltose Structure, Iron Core Structure, and Interaction of Carboxymaltose with Iron Core

Purpose: Nearly 5 million Americans and 20 percent African-American and Mexican-American women are suffering from anemia, which is mainly caused by iron deficiency [1]. Oral iron formulations such as ferrous salt are not suitable for chronic iron deficiency anemia treatment due to hypersensitivity reactions, poor absorption, and frequent dosing. IV iron formulations are a method to increase iron availability, such as high molecular weight iron dextran, iron sucrose, or ferric gluconate. However, they also have significant severe adverse reactions. CSL Vifor patented Injectafer^® which is a high molecular weight intravenous iron complex that allows the administration of a maximum of 1000 mg iron in a single IV dose without causing transferrin oversaturation. The carbohydrate ligands, also known as carboxymaltose, strongly interact with the polynuclear iron-oxyhydroxide/oxide core in Injectafer^® [2]. The in vivo stability of Injectafer^® is partially influenced by the properties of the carboxymaltose ligands, including their molecular weight, molecular weight distribution, molecular structure, and composition. One of the prerequisites in creating a generic version of Injectafer^® is developing a complete understanding of the structure of the iron core, the maltodextrin used to synthesize carboxymaltose, and how the carboxymaltose interacts with the iron core. Therefore, our focus is to characterize the carboxymaltose ligand, the iron core structure, and the interaction of the carboxymaltose ligand with the iron core in Injectafer^®.

Methods: To isolate the carboxymaltose, Injectafer^® was acidified to dissolve the ferric oxide/hydroxide core. The pH of the solution was adjusted to 1.0 and purified by removing free iron using ion exchange chromatography. The excess amount of salts in the isolated carboxymaltose was removed by dialysis overnight. Preparative-scale size-exclusion chromatography (SEC) was used to understand the distribution of different molecular weight species of carboxymaltose in Injectafer^®. Mössbauer spectra of iron core were obtained at different blocking temperatures from 240 to 5K. Injectafer^® was diluted in deuterated water at 100x, 200x, 400x, 800x, and 1600x to understand the interaction of carboxymaltose with the iron core. ¹H NMR spectra (Bruker Avance-III-800) were obtained for both the isolated carboxymaltose from Injectafer^® and diluted Injectafer^®.

Results: Carboxymaltose structure: The ¹H NMR results support the elemental analysis of dried Injectafer^® that approximately 95-100 mg of carboxymaltose are present in 1 mL of Injectafer^®. Preparative SEC was used to separate the fractions based upon the retention time of the column. Figure 1 shows the ¹H NMR spectra of the various fractions collected from the preparative SEC separation. Based on the ¹H NMR spectra, the amount of isolated carboxymaltose present in the SEC fractions# 4, 5, 6, 7, and 8 are 19, 45, 25, 8, and 0.67 percent respectively. The peak intensity of a-1,6 branching (~ 4.8 ppm) is the highest in fraction# 4 in Figure 1, suggesting a highly branched carboxymaltose polymer with the highest molecular weight of ~ 17 kDa. In addition, fractions # 5 and 6 have relatively low-branched polymers, with an average molecular weight of 7 and 1.4 kDa respectively compared to fraction# 4. The low molecular weight maltodextrin elutes in the later SEC fractions. The last two fractions contain mostly linear polymers with an average molecular weight of 474 and 294 Da. Iron core structure: Mössbauer spectra of Injectafer^® were acquired at different blocking temperatures from 240 to 5K at different temperature increments. For higher temperatures, such as from 240 to 60K, the paramagnetic doublet was broadened, and there were no distinct sextet spectra observed (data not shown). However, as the temperature decreased, the sextet feature was prominent as shown in Figure 2. Carboxymaltose-iron core interaction: At 400x dilution of Injectafer^®, the peaks at ~ 4.1 ppm, corresponding to the protons closest to the carboxy group, began to appear in ¹H NMR spectra in Figure 3, which is an indication of the presence of the carboxy group in carboxymaltose. Moreover, the peak intensity of the terminal group at ~3.35 ppm increased relative to the peaks at 3.5 to 4 ppm in diluted Injectafer^®, which was indicative that either the low molecular weight or both the low and high molecular weight carboxymaltose was being observed in all the diluted Injectafer^®.

Conclusion: Carboxymaltose in Injectafer^® contains both high molecular weight highly branched and low molecular weight linear polymers. The lower blocking temperature indicates a smaller particle size of approximately 4-5 nm and low crystallinity of iron cores in Injectafer^®, consistent with the reported literature. It also suggested that the iron core is akageneite (ß-FeOOH)-like structure and Injectafer^® does not contain any iron (II). At 0.125 mg/ml iron concentration (400x dilution), the carboxymaltose started to become observable in the ¹H NMR spectrum. Further studies are required to quantify the degree of interaction of carboxymaltose with the iron core.

References: 1. Miller, Jeffery L et al. "Iron deficiency anemia: a common and curable disease." Cold Spring Harbor perspectives in medicine 3.7 (2013): a011866.
2. Zou, Peng, et al. "Physicochemical characterization of iron carbohydrate colloid drug products." The AAPS journal 19 (2017): 1359-1376.

Acknowledgements: This research was funded by the U.S Food and Drug Administration (75F40121C00189).


Figure 1: 1H NMR spectra of SEC fractions of isolated carboxymaltose from Injectafer®. Each 1H NMR spectrum was integrated to quantify with respect to the internal standard, d4-TSP [3-(Trimethylsilyl) propanoic acid sodium salt], at 2 mg TSP/ml of D2O concentration. Each spectrum was normalized based on the TSP peak followed by 3x, 1.3x, 4x, and 54x factors of multiplication of fractions #4, 6, 7, and 8 respectively.


Figure 2: Mössbauer spectra of Injectafer® at 5 K temperature.


Figure 3: 1H NMR spectra of diluted Injectafer® in D2O at 100x, 200x, 400x, 800x, and 1600x concentration (from top to bottom).