PB0226 - Assay of the factor VIIIa mimetic, Mim8, using one-stage clotting assay requires a product-specific reference material

Background: The human bispecific antibody, Mim8, is in clinical trials for treatment of haemophilia A. Mim8 binds factor (F) X and FIXa, enabling activation of FX and bypassing the need for FVIII.

Aims: This study investigates the suitability of FVIII one-stage clotting (OSC) assays for measuring Mim8.

Methods: Mim8 reference material (RM) and drug product (DP) (both 200 µg/ml) were diluted to 20 µg/ml in imidazole buffer, followed by 1/10 in FVIII-deficient plasma and 1/3 in buffer. For comparison, the FVIII plasma International Standard (IS), diluted 1/3 in buffer, and the calibrator for emicizumab (a licensed FVIII mimetic), diluted 1/10 in deficient plasma and 1/3 in buffer (~3 µg/ml) were also included. All materials were assayed (n≥3) using a range of activated partial thromboplastin time (APTT) reagents and data were analysed by parallel line bioassay. Linear portions of the responses were used for calculations. Parallelism was assessed using the ratio of the standard and test slopes (acceptability 0.95-1.07).

Results: A representative result is shown in Figure 1, using APTT reagent Actin FS. The Mim8 DP was not parallel to the FVIII IS or the emicizumab calibrator (ratio of slopes for DP 1.15 and 0.84, respectively). However, the Mim8 DP was parallel to the RM (ratio of slopes 1.02), indicating a valid comparison could be made. The same trend for non-parallelism of Mim8 to the IS or emicizumab calibrator was observed with other APTT reagents. Using RM as the assay standard, 100% recovery of DP was achieved across the 6 APTT reagents studied (Figure 2), with no assay discrepancy observed.

Conclusion(s): In conclusion, Mim8 should not be assayed against the FVIII IS nor the emicizumab calibrator. For accurate measurement of Mim8, a specific reference material must be used. With this, no APTT reagent discrepancy was observed, therefore inter-laboratory discrepancy would be reduced.