Qualification of a select one‐stage activated partial thromboplastin time‐based clotting assay and two chromogenic assays for the post‐administration monitoring of nonacog beta pegol

Abstract

Background Nonacog beta pegol (N9-GP) is an extended half-life, glycoPEGylated recombinant human factor IX that is under development for the prophylaxis and treatment of bleeding episodes in hemophilia B patients. Considerable reagent-dependent variability has been observed when one-stage clotting assays are used to measure the recovery of recombinant FIX products, including N9-GP. Objective To qualify select one-stage clotting and chromogenic FIX activity assays for measuring N9-GP recovery. Methods The accuracy and precision of the one-stage clotting assay (with the STA-Cephascreen activated partial thromboplastin [APTT] reagent) and the ROX Factor IX and BIOPHEN Factor IX chromogenic assays for measuring N9-GP recovery were assessed in N9-GP-spiked hemophilia B plasma samples in a systematic manner at three independent sites, with manufacturer-recommended protocols and/or site-specific assay setups, including different instruments. Results For each of the three FIX activity assays qualified on five different reagent-instrument systems, acceptable intra-assay and interassay accuracy and precision, dilution integrity, reagent robustness and freeze-thaw and short-term sample stabilities were demonstrated. The STA-Cephascreen assay showed a limited reportable range at one of the three qualification sites, and the BIOPHEN Factor IX assay showed suspect low-end sensitivity at one of the three qualification sites. An individual laboratory would account for these limitations by adjusting the assay's reportable range; thus, these findings are not considered to impact the respective assay qualifications. Conclusion The one-stage clotting assay with the STA-Cephascreen APTT reagent, the ROX Factor IX chromogenic assay and the BIOPHEN Factor IX chromogenic assay are considered to be qualified for the measurement of N9-GP in 3.2% (0.109 m) citrated human plasma.

FIXing postinfusion monitoring: Assay experiences with N9‐GP (nonacog beta pegol; Refixia®; Rebinyn®)

“…These results demonstrate that chromogenic coagulation assays produce accurate and consistent measurements that are independent of the PEGylation status of the FIX molecule and that the PEG moiety attached to N9‐GP does not interfere with the reagents used in the available chromogenic assays (Table ). FIX chromogenic assays can also reliably quantify the total FIX activity in samples containing both N9‐GP and unmodified FIX, providing additional advantage…”

Measurement of extended half‐life recombinant factor IX products in clinical practice

“…Currently, there is no international consensus of the degree of variation between reagents or assay methods measuring the same parameter which could be expected to alter the clinical management of patients. About 20%‐30% difference has been considered acceptable in several studies . At FIX:C below 10 IU/dL, the calculation of percentages can magnify small differences in activity which may not be clinically significant.…”

Evaluation of chromogenic factor IX assays by automated protocols

“…The over‐recovery of N9‐GP by most silica‐based APTT reagents has recently been explained by Rosén et al, who found that N9‐GP was prematurely converted to activated FIX (FIXa) during the contact activation phase of the clotting assay. Another recent three‐site study measuring recovery of N9‐GP at four spike levels using Rossix and Hyphen CSA concluded these assays were able to measure N9‐GP using a ± 30% of target as acceptable . The suitability of CSA for measuring the third current EHL product, rFIX‐Fp, remains unclear .…”