Ferric Carboxymaltose injection is an iron supplement product used to treat iron deficiency anemia via the intravenous route. For the bioequivalence study, the recently revised FDA OGD suggests measuring Ferric Carboxymaltose-Associated Iron from serum. Direct measurement of Ferric Carboxymaltose is more challenging than indirect measurement (i.e., Total Iron-Transferrin bound Iron). The Lambda team has developed an innovative and selective Bioanalysis method using Inductively Coupled Plasma Mass Spectrometry (ICP-MS).
Challenges:
kDA filter selection: The selection of an appropriate kDA mass cut-off filter plays a critical role in iron component removal and the selective measurement of Ferric Carboxymaltose-Associated Iron.
Selection of Buffer solution: Optimization and the right selection of a buffer solution for the complete removal of free and transferrin-bound iron from samples containing FCM and Transferrin protein-bound iron.
Assay Parameter Optimization: Optimization of centrifugation parameters and sample processing steps to make the method robust.
Solutions:
Extraction Method:
- Since the Ferric Carboxymaltose molecular weight is 150,000 Daltons, a simple ultrafiltration technique is used. An ultrafilter with a molecular weight cut-off of 30,000 Daltons is selected for the extraction of Ferric Carboxymaltose-Associated Iron.
- Serum was mixed with a buffer solution to release the iron bound with transferrin protein. The treated serum sample was added to an ultrafilter and subjected to centrifugal force to remove the free iron and iron released from the Transferrin protein.
- The filtrate (Elute-lower part) containing the free iron shall be discarded, and the upper part of the filter contains Ferric Carboxymaltose-Associated Iron. As a final step, the residual upper part of the filter was taken and treated with further processing and sample cleanup.
- Processed samples shall be analyzed on an ICP-MS instrument to measure Ferric Carboxymaltose-Associated Iron.
ICP-MS Methodology:
- Methods were developed on an ICP-MS instrument. The ICP-MS platform is more selective, sensitive, and compliant with 21 CFR part 11. Additionally, ICP-MS is the most sensitive tool for elemental analysis, helping us develop Ferric Carboxymaltose-Associated Iron in serum samples.
Matrix Selection for CC/QCs and Approach to correct Endogenous Iron:
- Authentic serum was chosen as the matrix for calibration standards and quality control samples, ensuring better resemblance to the study samples. To account for endogenous iron levels, a background subtraction approach was utilized to calculate the concentration of the analyte accurately.
- CCs and QCs were prepared using the Ferric Carboxymaltose-Associated Iron formulation. Additionally, Efficiency QCs were prepared using Ferric Carboxymaltose formulation + the expected maximum concentration of Transferrin-bound Iron using Holo transferrin standard (These Efficiency QCs are prepared to mimic the study samples).
Key Outcomes:
- Calibration curve found linear for 5 µg/mL to 600 µg/mL range.
- The method is enabled to remove free Iron and Transferrin-bound iron from the sample and measure only Ferric Carboxymaltose-Associated Iron. During method development, Low QCs of FCM-associated Iron and Efficiency QC (FCM + Transferrin Iron) were analyzed, demonstrating the complete removal of Transferrin-bound Iron.
- Efficiency LLOQ (FCM 5µg/mL + Transferrin Iron 5µg/mL) and LQC (FCM 15µg/mL + Transferrin Iron 5µg/mL) were prepared, and accurate quantification of FCM concentration reveals complete removal of Transferrin Iron 5µg/mL.
- High throughput method capable of completing around 100 samples per day. Method Validation will be planned in upcoming months.
Conclusion:
- The methods comply with regulatory requirements for directly measuring Ferric Carboxymaltose-Associated Iron in serum, ensuring the accuracy and reliability of the analytical data.
- The developed method is accurate, precise, and reproducible for the selective detection of Ferric Carboxymaltose-Associated Iron.
Extensive Experience:
Lambda has accumulated extensive ICP-MS expertise in method development, validation, sample preparation, and quantitative analysis of elemental therapeutic drugs.
| Formulation | Analyte | Assay Status | Study Experience |
|---|---|---|---|
| Iron Sucrose | Total Iron | Validated Assay | 3 Study Completed |
| Transferrin Bound Iron | Validated Assay | ||
| Ferumoxytol | Total Iron | Developed and Ready for Validation | 1 Study Completed |
| Transferrin Bound Iron | Developed and Ready for Validation | ||
| Ferumoxytol Associated Iron | Developed and Ready for Validation | – | |
| Ferric Carboxymaltose | Total Iron | Validated Assay | 1 Study Completed |
| Transferrin Bound Iron | Validated Assay | ||
| Ferric Carboxymaltose Associated Iron | Developed and Ready for Validation | – | |
| Potassium Chloride | Potassium | Developed and Ready for Validation | – |
| Potassium Citrate | Potassium | Developed and Ready for Validation | – |
| Lithium Carbonate | Lithium | Upcoming Development | – |
Lambda Therapeutic Research offers a wide range of bioanalytical capabilities specializing in cell-based assays, biomarkers, immunogenicity, and pharmacokinetics (PK). With state-of-the-art facilities and a team of experienced scientists, Lambda provides comprehensive solutions for the analysis of small molecules, biomarkers, biologics, and therapeutic trace elements.
