Enabling accurate measurement of activated factor XI (FXIa) in therapeutic immunoglobulin products.

BACKGROUND AND OBJECTIVES: In 2010, an intravenous immunoglobulin (IVIG) product was removed from the market due to an association with serious thromboembolic events. Investigations revealed that factor XIa (FXIa) was present as a process-related impurity. This study investigated the ability of two commercial FXIa assays to measure FXIa in immunoglobulin preparations and conducted a survey of FXIa activity in marketed immunoglobulin products. MATERIALS AND METHODS: Factor XIa assays were modified to include spiking of samples with FXIa before testing. An immunoglobulin product and its excipient were used to assess the ability of the assays to recover the spiked FXIa levels. RESULTS: The Biophen FXIa assay required a high pre-dilution of the sample to obtain statistically valid results and complete FXIa recovery. The ROX FXIa assay was more sensitive, giving statistically valid results at a lower sample pre-dilution and FXIa spike level. This modified ROX FXIa assay was used to assay 17 lots of immunoglobulin products for FXIa. Two product lots had measurable FXIa levels without the need for spiking. A further 3 lots produced detectable but not statistically valid FXIa results when left unspiked. Spiking produced statistically valid assays and recoveries above 100%, demonstrating inherent FXIa. CONCLUSION: This study shows marketed immunoglobulin products can contain detectable levels of FXIa. Spiking brings the FXIa levels into the quantifiable range of the assay, allowing measurement of inherent FXIa. Accurate measurement is important to inform on 'safe' levels of FXIa in these products and allow future safety guidelines to be set.

The traceability of commercial plasma calibrators to the plasma International Standards for factor VIII and factor IX.

Accurate measurement of coagulation factors is essential, especially for diagnosis of deficiency. Clinical laboratories use commercially available plasma calibrators, which should be traceable to the relevant plasma International Standard (IS). This study assessed the relationship between the plasma IS for factors IX (FIX) and VIII (FVIII) and some commonly used commercial calibrators. Calibrators from seven manufacturers were assayed for FIX and FVIII activity by one-stage clotting assay (OSCA) using different activated partial thromboplastin time (APTT) reagents and deficient plasmas, or chromogenic assay (CA). Results were calculated relative to the 4th IS Factors II,VII,IX,X, Plasma or the 6th IS Factor VIII/VWF, Plasma. Results for each calibrator were similar across the APTT reagents and deficient plasmas used. All calibrators showed a recovery of 90%-111% of the manufacturers' values, except calibrator C, which had recovery of around 85%. CA gave similar results, with good recovery for all but calibrator C. Similar low recoveries for OSCA and CA were found for a different lot of calibrator C and for a different calibrator product from manufacturer C. When all calibrators from manufacturer C were assayed by OSCA using the manufacturer's own deficient plasmas and APTT reagents, the mean recovery was still below 90%. Overall, there was good traceability of the international unit between the IS and commercial calibrator plasmas. Calibrators from one manufacturer consistently yielded lower than expected values for FIX and FVIII. This could lead to an over-estimation of the coagulation factor content in patient samples and demonstrates the importance of careful choice of calibrator.

Potency estimates for recombinant factor IX in the one-stage clotting assay are influenced by more than just the choice of activated partial thromboplastin time reagent.

The activated partial thromboplastin time (APTT) one-stage clotting (OSC) assay is used to potency label factor IX (FIX) therapeutics and to monitor the treatment of deficient patients. Studies have highlighted differences in potency estimates for FIX therapeutics depending on what activator type the APTT reagent contains. During the study to replace the 4th International Standard (IS) for FIX Concentrate, it was noted that for the recombinant FIX (rFIX) candidates, a potency difference of 20% was obtained by some between laboratories using the reagent DAPTTIN. This study was designed to investigate this discrepancy. Two plasma-derived (pdFIX) and 2 rFIX materials were assayed against the 4th IS for FIX concentrate (07/182) using the OSC assay. Three different APTT reagents were used (DAPTTIN, SynthAFax and SynthASil), plus 4 different activation times. The pdFIX samples were not affected by activation time or APTT reagent, with expected potencies recovered in all assays and at all activation times. For rFIX, expected potencies were recovered using SynthASil, but SynthAFax generated potencies around 20% lower. This was consistent across the activation times. For DAPTTIN, potencies for rFIX dropped progressively and by up to 30% as activation time increased from 120 to 300 seconds. This study demonstrates that activation time as well as choice of APTT reagent can result in discrepancies in the potency estimation of rFIX. These factors should be taken into account when assaying rFIX.

Establishment of the World Health Organization First International Standard for Factor XII, Plasma, Human.

Until recently, the role of factor XII (FXII) in hemostasis was not considered to be important since patients with FXII deficiency do not present with bleeding. The activation of FXII by agents including mast cells and platelet polyphosphates suggests that it may have a role in thrombogenesis. The inhibition of FXII therefore presents an option for antithrombotic therapy, and antibodies and inhibitors are already in development. Assays for FXII will be required to support these technologies, and an international standard (IS) for FXII would be useful for the development of these methods and for the clinical monitoring of patients. The purpose of this study was to develop an IS for FXII, with values for functional activity (FXII:C) and antigen (FXII:Ag). Double-spun normal plasma was pooled, filled into siliconized glass ampoules, and freeze-dried to prepare the candidate material. Data from 20 laboratories using the one-stage clotting assay were used to assign the functional activity value in units (u). The antigen value was calculated using data from eight laboratories that carried out antigen assays. Each laboratory was requested to collect two local normal plasma pools. Units of activity and antigen were calculated relative to these pools, as is usual for new coagulation factor analytes. The amount of activity or antigen in 1 ml of normal plasma from each pool was taken to be 1 unit. A total of 566 donors were used across the pools for the FXII:C study and 216 donors for the FXII:Ag study. The overall geometric mean per ampoule for FXII:C was 0.86 u and for FXII:Ag was 0.80 u. The inter-laboratory variation was 10 and 11%, respectively (expressed as the geometric coefficient of variation). Based on these data, the candidate was deemed suitable for use as an IS for FXII. In 2017, the candidate was established by the World Health Organization (WHO) Expert Committee on Biological Standardization as the WHO first IS for blood coagulation FXII, Plasma (National Institute for Biological Standards and Control code 15/180). The values assigned were 0.86 international units (IU) of functional activity (FXII:C) per ampoule and 0.80 IU/ampoule of antigen (FXII:Ag).