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Activity measurements of dalcinonacog alfa

Abstract Introduction Many recombinant and modified FIX products have been, and continue to be, developed with the aim of improving treatment for patients with haemophilia B. One such new product is dalcinonacog alfa, a recombinant FIX with modifications to provide improved features such as subcutaneous administration. Aim In view of previously observed assay discrepancies with modified FIX therapeutics, the aim of this study was to assess potential discrepancies in potency measurement of dalcinonacog alfa between and within different assay methods. Methods Potency of dalcinonacog alfa was measured against the 5th International Standard (IS) for FIX Concentrate and the 4th IS for FIX Plasma by One‐Stage Clotting Assay, using 9 different APTT reagents and 2 commercially available FIX chromogenic kits. Plasma‐derived concentrate and recombinant FIX samples were also included for comparison in every assay. Results Substantial discrepancies were observed when assaying dalcinonacog alfa using the one‐stage clotting assay against both standards. No statistically valid results were obtained when testing dalcinonacog alfa using either chromogenic kit. Increasing the incubation time with the activation reagent in both chromogenic kits resulted in valid assays and increased the potency to become more in line with potencies by one‐stage clotting assays. Increasing the incubation time in the chromogenic kits had no effect on the potencies of the plasma‐derived or recombinant samples. However, incubation time influenced in the one‐stage clotting assay using Dapttin. Conclusions Within and between assay method discrepancy was found when assaying dalcinonacog alfa. Methods for potency labelling and clinical monitoring should be given careful consideration.

There are two methods for measuring FIX activity: the onestage clotting assay (OSCA) and the functional chromogenic assay (ChA). Within these methods, there are multiple different reagents/kits. All licensed FIX therapeutics are potency labelled using the OSCA as described in the European Pharmacopeia (EP) 1 against the current WHO International Standard (IS) for FIX, Concentrate. For clinical monitoring of postinfusion samples, OSCA against a plasma standard traceable to the current WHO IS for FIX, Plasma is routinely used. 2 However, discrepancies between methods and reagents when assaying the extended half-life and full-length recombinant therapeutics have been reported and the compositional differences of the reagents/kits are believed to play a part in the observed discrepancies. [3][4][5][6][7][8][9] It is also clear that these modified products have different characteristics than the IS, which are either plasma-derived concentrate or normal plasma. Therefore, these potency evaluations deviate from the basic concept of biological standardization: 'like versus like'. 10,11 Nevertheless, statistically valid assays and their estimates can be obtained when these modified products are assayed against the IS, which justify potency labelling in International Units (IU). [3][4][5][6]8,[12][13][14] Catalyst Biosciences has developed a new rFIX therapeutic, dalcinonacog alfa (DA) (previously known as CB2679d/ISU304), which has been given orphan drug designation in Europe and the United States and has currently completed a Phase 1/2 proof of concept clinical trial. 15 DA is a full-length recombinant FIX molecule with amino acid substitutions in the protease domain that confer the product with increased potency, enhanced FVIIIa binding and reduced inhibition by antithrombin. These enhanced features allow for effective prophylaxis of patients by subcutaneous administration. 16,17 In view of the previously observed assay discrepancies with modified FIX therapeutics, the aim of this study was to assess any potential discrepancies in potency measurement of DA between and within different assay methods. To investigate this, one-stage clotting and chromogenic assays were carried out using a range of reagents and kits. Plasma-derived concentrate FIX (PD) and recombinant FIX (RB) samples were also included for comparison in every assay, and potencies were calculated against the 5th IS for FIX Concentrate (S1) and the 4th IS for FIX Plasma (S2).

| One-stage clotting assays
All test preparations, except S2, were prediluted to 1 IU/mL (for the initial assay, DA and RB were diluted based on assigned potency and subsequent assays based on the estimated potency from the first assay to ensure that the responses were in the same range as the standard) in FIX immune depleted plasma (Technoclone). All test preparations were further prediluted to approximately 0.1 IU/mL in Glyoxaline/HSA buffer. Assays were performed on an ACL TOP 550 automated coagulometer (Werfen)

Reagent
Phospholipid Activator

| Statistical analysis
Raw data were analysed using bioassay software Combistats Overall potency estimates for each reagent were calculated as geometric means (GM) and variability was estimated using the geometric coefficient of variation (%GCV). One-way ANOVA followed by Tukey's multiple comparisons test or an unpaired t test with the level of significance set at 5% (GraphPad Prism version 6.00 for Windows, GraphPad Software) were used to determine if there was a statistically significant difference between potency estimates from reagents or kits.
It is important to note that this study was not aimed at determining the accuracy of the labelled potency of DA and RB but focused on the agreement of values obtained by the different methods.

| RE SULTS
With the exception of DA vs S2 using the reagent APTT-SP, one or more statistically valid assays were achieved for all samples with all APTT reagents (see Figure 1A-F). Intra-assay variability for OSCAs when assayed against S1 were low for all samples and all reagents, with the majority of GCVs < 10%. This indicated that assays were precise with good reproducibility ( Table 2). Similar intra-assay variability was observed when samples were assayed against S2 (data not shown). Table 3 shows the overall inter-reagent variability for all preparations. Good agreement of potencies was obtained for PD with a GCV of ~3% against S1 and ~6% against S2. There was no discrepancy between OSCA reagents (P > .05). Higher variability was observed for RB with a GCV of ~15% against S1 and ~16% against S2, indicating that there was some disagreement in potency obtained with the different OSCA reagents. The highest OSCA potency estimate for RB was using Cephascreen (~11 IU/mL) and lowest using SynthaFax (~7 IU/mL) when assaying against S1 and S2 ( Figure 1C and D). The variability for DA was at least 2-fold higher than those obtained for RB with a GCV of 38% against S1 and 46% against S2.
TA B L E 2 Geometric mean (GM) and intra-assay variability expressed as geometric coefficient of variation (%GCV) for all reagents/ kits when testing plasma-derived FIX (PD), recombinant FIX (RB) and dalcinonacog alfa (DA) against the plasma-derived concentrate international standard: S1, using different one-stage clotting assays (OSCA) reagents and chromogenic assay (ChA) kits. No %GCV is given for APTT-SP as only 1 valid assay was obtained using this reagent and Synthafax giving the lowest value (~5 IU/mL vs S1) ( Table 2).
When testing was carried out with ChAs, statistically valid results were obtained for both PD and RB with low intra-assay variability ( Figure 1C-F and Table 3). No OSCA/ChA discrepancy was observed for PD while for RB there was a ~30% discrepancy between the OSCAs and ChAs with an overall GM for OSCAs of 9.2 IU/mL and ChAs of 7.1 IU/mL against S1 (Table 3). For DA, when ChAs were carried out according to the manufacturer's instructions, results for all assays against both standards were found to be statistically invalid due to non-parallelism. Median slope ratios for DA against both S1 and S2, using both kits, were ~1.3 meaning that the slope of the dose-response curve of the standards was steeper than the dose-response curve of DA. Median slope ratios for PD and RB against both standards were within the 0.9-1.11 limits set for non-parallelism (all 1.0, except for RB against S2 using the Rossix kit, which had a slope ratio of 0.9). However, when the chromogenic assay kits were deconstructed and the incubation time of DA with the FXIa containing 'activation reagent' was extended, dose-response curves with DA were parallel to both standards and statistically valid results were obtained (slope ratios close to 1.0). Figure 2 shows the potency obtained for DA and PD using the  Figure 4A against S1 and 4B against S2. There was a reduction in potency for DA and RB against both standards and a reduction in potency of PD when assayed against S2. However, this decrease in potency was only significantly different against S2 (t test DA, P < .001; RB, P < .001; PD, P < .05).

| D ISCUSS I ON
It is widely known that assay discrepancies exist when measuring the potency of both full-length and modified recombinant FIX products against plasma and plasma-derived Concentrate FIX standards using different methods and reagents. [4][5][6][7][8][9][12][13][14][18][19][20][21][22] In this study, the degree of assay discrepancies of a novel modified rFIX therapeutic, dalcinonacog alfa (DA), was investigated and compared to those observed when assaying a plasma-derived TA B L E 3 Overall geometric mean (GM) and method GM (one-stage clotting assays (OSCA) and chromogenic assays (ChA)) as well as inter-assay variability (% geometric coefficient of variation (GCV) or range where only 2 kits tested) for plasma-derived FIX (PD), recombinant FIX (RB) and dalcinonacog alfa (DA) against the plasma-derived concentrate international standard: S1 and the plasma international standard: S2  7,8,23,24 For DA in the OSCAs, there was a roughly 3-fold difference between the highest potency (CK Prest) and lowest potency (SynthFax) using both S1 and S2 standards. RB also showed lower potency when assayed using SynthaFax than the other APTT reagents, underestimation of Benefix with Synthfax has been previously reported. 18 With the exception of Dapttin, CK Prest gave significantly higher potency for DA than all the other APTT reagents (one-way ANOVA, P < .0001). However, when the assay was repeated using an incubation time of 300 seconds instead of 120 seconds, a lower potency was obtained, which was significantly different from the potency obtained by CK Prest (S1 which the required thrombin is generated during the reaction. Also, Rossix contains factor V, whereas Hyphen does not, and the kits also have different FXa substrates. Differences in potency estimation by the 2 ChA kits have been reported elsewhere when testing other extended half-life FIX therapeutics. 9 One study showed that in a small data set of patients treated with FC fusion FIX (Alprolix), the Rossix kit recovered close to expected activity but the Hyphen kit was ~30% lower, while in patients treated with albumin fusion FIX (Idelvion) both kits recovered higher than expected median potencies. 9 It is unclear how the differences in amino acid sequences between DA and wild-type FIX could account for the differences in their activation by FXIa in the ChAs.
However, DA binds FVIIIa ~8-to 10-fold better than wild-type FIX and is more resistant to inhibition by antithrombin, 16,17 which may play a different role in each of the assay types.

| CON CLUS ION
In conclusion, as seen for other modified FIX products, discrepancies were observed when assaying DA using different methods/reagents.
Despite the OSCA discrepancies, with the exception of APTT-SP, DA could be validly assayed against both the Concentrate FIX IS and the Plasma FIX IS using the APTT reagents investigated in this study.
The OSCA is the EP method and thus the expected potency labelling method in Europe. Due to the assay discrepancies within the OSCA method, the reagent used for potency labelling and clinical monitoring needs to be carefully considered. Further optimization of the chromogenic assays would be required before DA could be reliably measured using these methods.