(T1130-11-64) Successful Validation of a Thrombin Generation Assay (TGA) for the Release of an Intravenous Immunoglobulin (IVIG) Drug

Purpose: Plasma-derived therapies are drug products derived from human plasma using fractionation processes where target proteins in plasma are purified to homogeneity. They are used to treat a wide variety of rare diseases including disorders of hemostasis (hemophilia and antithrombin III deficiency to primary (autoimmune lymphoproliferative syndrome) and secondary immunodeficiencies (AIDS). Even small amounts of contaminating coagulation factors remaining in these preparations pose a significant risk to the patient and can result in thrombotic/thrombolytic events, and in some cases death. Quantitative measurement of these trace contaminants can result in considerable challenges for analytical method development. Manufacturers of these drug products are required to investigate the ability of their production process to remove procoagulant activities making them safe for release into the clinic.  Here we explore a quantitative analytical method that measures factor XIa (FXIa)-like activity in an intravenous immunoglobulin (IVIG) drug as a function of thrombin generation.

Methods: The validated thrombin generation assay (TGA) was designed and performed on the Calibrated Automated Thrombogram® platform (CAT) from Diagnostica Stago, Inc. TGA is a universal test capable of assessing global hemostatic balance. The CAT method is both specific and sensitive, thus giving increased relevance to hemostatic testing. This assay measures thrombin generation utilizing the specific fluorogenic thrombin substrate Z-Gly-Gly-Arg 7-amido-4-methylcoumarin hydrochloride (ZGGR-AMC). We used a modified TGA to detect FXIa-like activity in IVIG samples. Reaction mixtures contained IVIG sample, FXI deficient plasma, relipidated tissue factor, and assay buffer.  Thrombin formation was initiated by the addition of ZGGR-AMC/CaCl₂.

Results: The assay was validated using two lots of IVIG containing varying levels of FXIa. Eight runs were performed over four days by three different specialists. Peak thrombin in IVIG test samples was compared to a standard curve prepared using purified human FXIa (0.031 – 2.000 mU/mL). For each run, the r² for the non-linear regression curve for response vs. concentration was ≥97%, and the %CV between replicates was ≤20%. The detection limit was 0.25 mU/mL FXIa.  For each lot of IVIG, the 90% CI for recovery at each level across runs was within 70-143% from the nominal level and 50-200% at the LLOQ. The %CV for the within-run and between-run precision for the measured results was ≤30% and ≤50% for the LLOQ. The r² for the linear regression for measured values vs. nominal values was ≥95%. The quantitation range, defined as the range that spans the lowest and highest levels that pass the criteria for accuracy, precision and linearity, was 0.85-7.43 mU/mL with a LLOQ of 0.85 mU/mL.

Conclusion: Validation of this assay provides a solution for assessing hemostatic risk in plasma-derived therapies. This method is a powerful release assay that ensures patient safety by quantitation of a process contaminant that poses a risk to normal hemostasis.

Acknowledgements: I have no disclosures or conflict of interest