Innovative Methodology Enhances Bioequivalence Studies and Advances Anemia Treatment

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). FCM is a well-tolerated form of iron available in both tablet and injectable forms, making it particularly beneficial for patients with serious conditions such as Crohn's disease or ulcerative colitis. Thus, given the widespread prevalence and impact of anemia, having effective, reliable and affordable treatments available is crucial.

To ensure that patients receive the best possible care, rigorous clinical 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, a Conscio Group member, had extensive experience in analyzing complex analytes in various biological matrices, including determination of trace elements using ICP/MS. However, the determination of TBI in the presence of FCM posed a specific challenge. But why?

Project manager and bioanalytical expert Štěpán Eichler explains: “FCM, a high-dose iron complex, quickly saturates the body's transferrin protein, while FCM levels in the bloodstream remain extremely high (up to two orders higher within a few hours after dosing). This makes it difficult to accurately measure TBI, as the high concentration of FCM interferes with the analysis.” To overcome this, analysts needed to separate the transferrin protein from the much larger and more abundant FCM molecules, while ensuring high precision despite naturally occurring iron in the body.

Innovative Solutions for Precise Iron Speciation and Bioequivalence Studies

The development of a specialized on-line solution was the decisive factor for the project’s success. Using a fully bioinert HPLC system with size exclusion chromatography coupled to ICP/MS, the Quinta team achieved high separation efficiency for all relevant iron species, ensuring excellent selectivity and sensitivity for TBI measurement, allowing precise measurements even at low concentrations.

Moreover, during the ICP/MS measurement, an additional challenge arose. It was the absence of a suitable internal standard on the market to stabilize the ICP/MS signals. We addressed this by introducing a post-column addition technique, which further enhanced the method’s accuracy.

Finally, this approach was validated according to the ICH M10 bioanalytical guidelines, effectively handling concentration ratios of TBI to FCM up to 1:400.

Subsequently, the developed methodology was applied in a bioequivalence study involving anemia patients. The in-study quality control results demonstrated that the method was highly accurate, with total iron measurements ranging from 99.2% to 102.0% and transferrin-bound iron measurements from 99.2% to 100.4%. Precision was also confirmed, with coefficients of variation below 2.6% for TI and below 5.4% for TBI across the validated concentration range.

The incurred sample reanalysis (ISR) test showed that 100% of ISR results for TI and 97.5% for TBI met the acceptance criteria. These findings confirm that method is robust and reliable for bioequivalence studies of FCM products.

Ensuring Accurate Iron Measurement for Improved Patient Care and Product Development

In conclusion, accurate determination of iron levels by this method is crucial for patients suffering not only from anemia, but other iron deficiency diseases. Reliable measurement can lead to better monitoring of treatment efficacy, allowing healthcare providers to adjust therapies as necessary, ultimately improving patient outcomes and quality of life and reducing the complications associated with disease.

Moreover, for manufacturers of iron supplements and related products, our developed method can be modified to serve as a valuable tool for testing new formulations in the future. By demonstrating robust bioequivalence, this approach enhances confidence in the reliability and efficacy of their products, helping manufacturers better meet regulatory requirements, streamline product approvals, and bring effective treatments to market more quickly and at lower costs. ‘At Quinta-Analytica, our mission is to deliver precise and trustworthy services that guarantee the quality of medicines, ultimately benefiting patients and healthcare providers’, says Eichler.

The project was performed in collaboration with our partner İDE CSO, which provides broad range of services including clinical documentation and regulatory operations. For more information, visit www.quinta.cz and www.consciogroup.com.