Anemia is a serious medical condition affecting millions of people worldwide, characterized by a lack of healthy red blood cells to carry adequate oxygen to the body’s tissues. This condition can lead to fatigue, weakness, and various health complications if left untreated. According to the World Health Organization, anemia affects approximately 1.62 billion people globally, with iron deficiency being the most common cause. Anemia can be treated with various medications, including iron supplements, vitamin B12 injections, and specific iron complexes like Ferric carboxymaltose (FCM). Given the widespread prevalence and impact of anemia, having effective and reliable treatments available is crucial.
To ensure that patients receive the best possible care, rigorous bioequivalence and bioanalytical studies are essential for developing and approving new medications. Due to this need, Quinta-Analytica received a request to provide bioanalytical services for the bioequivalence study of a product containing FCM. The FDA guidance for the bioequivalence of this product requires assessment based on the determination of total iron (TI) and transferrin-bound iron (TBI) levels in human serum.
The bioanalytical team at Quinta-Analytica had previous experience with the determination of trace elements in biological matrices using inductively coupled plasma mass spectrometry (ICP/MS), but the determination of TBI in the presence of FCM posed an analytical challenge. But why?
The bioanalytical specialist Štěpán Eichler explains: “FCM is a high molecular weight iron complex that is administered at high doses. After dosing, the glycoprotein transferrin becomes saturated with iron to maximum capacity, while the concentration of FCM continues to rise to values up to two orders higher within a few hours. This makes FCM a strong interferent for TBI determination.”
This necessitated the separation of the protein transferrin (85 kDa) at low concentration levels from the extremely high dosages of the high molecular weight complex FCM (150 kDa) and other endogenous proteins containing iron. Furthermore, since TBI is also an endogenous compound, the method must be extremely accurate and precise after subtracting relatively high endogenous levels compared to the low expected increase in TBI concentration after administration of the test item.
Initially, we tested off-line approaches based on membrane microcentrifugation or solid-phase extraction, but the equilibrium between each form with such different concentration levels and non-specific protein interactions did not allow for the determination of TBI with sufficient accuracy and precision. Therefore, we developed an on-line solution using a fully bioinert HPLC system with size exclusion chromatography coupled to ICP/MS. This provided high separation efficiency for all expected iron species, along with excellent selectivity and sensitivity for TBI. One of the key issues was also the absence of a suitable internal standard (a high-molecular-weight and non-serum-endogenous species containing a specific element) to suppress ICP/MS signal variability, so a solution for post-column addition was successfully developed. The novel method using the correction of endogenous levels of TBI was then validated according to the ICH M10 bioanalytical guideline (EMEA/FDA) in the concentration ratio TBI/FCM up to 1:400.
The developed methodology for total iron and transferrin-bound iron determination was then applied to a bioequivalence study with anemia patients. In-study QC results showed that the method was accurate (TI: 99.2-102.0% / TBI: 99.2-100.4%) and precise (TI: CV<2.6% / TBI: CV <5.4%) over the validated concentration range. The incurred sample reanalysis (ISR) test showed that 100% of the ISR results for TI, as well as 97.5% for TBI, met the acceptance criteria. Overall, these findings confirm that the method is robust and reliable for bioequivalence studies of FCM products for anemia treatment.
We are proud to have accepted this analytical challenge, which has enhanced our ability to measure total iron and transferrin-bound iron levels. This experience has deepened our expertise and confirmed our confidence to handle similar samples in the future. At Quinta-Analytica, a Conscio company, we are committed to delivering precise and reliable bioanalytical services. We look forward to leveraging our enhanced methodologies to support future bioequivalence studies and contribute to advancements in anemia treatment.
The project was performed in collaboration with our partner İDE CSO which was responsible for clinical part of the FCM study. İDE CSO, based in Ankara, Turkey provides broad range of services including clinical documentation, regulatory operations, and conducting bioequivalence studies.
CONSCIO GROUP is a network of quality science organizations committed to ensuring good health for all by improving the safety and reliability of food and pharmaceuticals.
Conscio Group members are Quinta-Analytica, Gen-Plus, LVA, EL Labs, HyServe, Ekolab and Reference Analytics.
Visit www.consciogroup.com and www.ide-cso.com for more information.
