Abstract
Recombinant factor VIIa (rFVIIa; NovoSeven, Novo Nordisk, Copenhagen, Denmark) is used to control bleeding in patients with hemophilia. A generic version of FVIIa was developed by AryoGen (Tehran, Iran). This study compared the composition and functional activities of AryoSeven and NovoSeven. Each product was compared at equigravimetric (1 mg/mL) stock solution for protein content. The proteomic profile was obtained using surface-enhanced laser desorption ionization mass spectrometry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was carried out to determine the protein profile and Western blotting was performed using a polyclonal rabbit antihuman FVIIa antibody. The FVIIa-related antigen was also measured using a commercially available enzyme-linked immunosorbent assay method. Functional assay included the prothrombin time correction in FVII-deficient plasma. The protein content was comparable in 2 products and the mass spectra analysis showed a single peak at 50 kDa in all products. The SDS-PAGE and immunoblotting studies were comparable. Both products exhibited similar coagulant properties in different assays.
Introduction
Commercially available recombinant coagulation factors are widely used in the management of bleeding disorders. A commercially available recombinant factor VIIa (FVIIa), namely, NovoSeven (Novo Nordisk; Copenhagen, Denmark) is approved for the management of bleeding in patients with hemophilia A having inhibitors. The FVIIa, when complexed with tissue factor, can activate FX to FXa and FIX to FIXa. Following complexation with other factors, FXa converts prothrombin to thrombin, which eventually converts fibrinogen to fibrin. The effect of FVIIa in patients with or without hemophilia has been evaluated in different in vitro experimental models. In 1 such model, supplementation of FVIIa at a concentration as low as 10 nmol/L with tissue factor increased both the rate and the level of thrombin generation in blood drawn from healthy volunteers and in patients with hemophilia A. 1 In another model, escalating dosage of FVIIa (NovoSeven) demonstrated a dose-dependent increase in thrombin generation in plasma from patients with hemophilia A. 1
NovoSeven is used for the treatment of bleeding episodes in patients with hemophilia A or hemophilia B who develop inhibitors to FVIII or FIX and also in patients with acquired hemophilia. 1,2 Furthermore, FVIIa is used for the prophylaxis of bleeding in those patients undergoing surgical interventions or invasive procedures. It is also used for the prophylaxis and treatment of intra- or perioperative bleeding episodes in patients with congenital FVII deficiency. 1,3
Mariani et al reported on the half-life, efficacy, and side effects of rFVIIa in patients with congenital FVII deficiency. 4 Wong et al reported on the successful administration of rFVIIa in newborns with severe congenital FVII deficiency associated with intracranial hemorrhage. 5 Mayer et al reported that rFVIIa did not improve survival or functional outcome after intracerebral hemorrhage despite the reduction in hematoma. 6 Ingerslev evaluated the efficacy and safety of rFVIIa in the prophylaxis of bleeding in various surgical procedures in patients with hemophilia having FVIII and FIX inhibitors. 7
Although, many studies regarding the efficacy and safety of rFVIIa (NovoSeven) have been reported, studies on a direct comparison with other rFVIIa products have not been possible until recently. 1
AryoSeven (AryoGen; Tehran, Iran) is a biosimilar product comparable to NovoSeven and is reported to have a similar efficacy and safety profile. 8,9 This product is clinically being used in Iran for the management of bleeding in patients with hemophilia. 10 Although the clinical studies have reportedly shown similar safety and efficacy profile of AryoSeven to NovoSeven, a comparison of the 2 products in terms of composition and functional properties is not reported.
The current study was designed to compare the biochemical and functional profiles of these 2 recombinant VIIa preparations in order to demonstrate their biochemical and procoagulant profiles. These comparative studies included protein content, molecular mass profile, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and immunoblotting studies utilizing a polyclonal (rabbit) antihuman factor VIIa antibody. Factor VIIa antigen levels were also measured. The functional studies included clot-based assays to measure the procoagulant properties of the 2 products in various systems.
Materials and Methods
Materials
Two batches of NovoSeven were purchased from Novo Nordisk. Six batches of AryoSeven were obtained from AryoGen. Per the product insert, all AryoSeven products contained 1.2 mg of rFVIIa. One batch of NovoSeven contained 2.4 mg and other 1.0 mg of rFVIIa, respectively. All batches were initially diluted in sterile water to obtain concentrations of 1 mg/mL solution. Further dilutions were made in saline to obtain working concentrations of 100 µg/mL.
Protein Quantitation
Protein content was determined in each of the rFVIIa products using the modified Lowry method. 11 The stock solution of 1 mg/mL was used for total protein quantitation. Purified bovine albumin standard (Thermo Scientific, Rockford, Illinois) was used for cross-referencing purpose.
Molecular Profiling Using Surface-Enhanced Laser Desorption Ionization Technique
Mass spectrometric analysis was carried out to compare molecular mass profiling of each rVIIa using surface-enhanced lasers desorption ionization (SELDI) method. The analysis was carried out utilizing ProChip mass spectrometer (PCS 4000) from Bio-Rad (Hercules, California). The protein composition of rVIIa was investigated using SELDI gold chip. Stepwise washing of the gold chip was carried out for 6 minutes in acetone; followed by 6 minutes in 0.01 N HCl; 6 minutes in a 50% methanol and 0.005 N HCl mixtures; and finally 6 minutes in 100% methanol. The chip was allowed to air dry for at least 10 minutes before the application of samples.
Each of the individual rFVIIa samples (100 µg/mL) was diluted (1:10) with Tris-HCl (pH 7.5) buffer. An energy-absorbing matrix (EAM) solution was prepared by dissolving sinapic acid in equal volumes of 100% acetonitrile and 0.1% trifluroacetic acid. The sinapic acid used allows for a saturated solution that cocrystallizes with the proteins in the sample; this cocrystallization allows for the ionization of the proteins in the sample. The diluted sample in the amount of 5 µL was added to 10 µL of EAM and mixed well. The resulting mixture of 5 µL was spotted on the gold chip. The gold chip was allowed to air dry for 1 hour before analysis on the SELDI. The chip was processed at 2 laser power settings,1500 nJ and 3500 nJ. At the 1500 nJ setting, data regarding the molecular weight (MW) range between 0 and 20 kDa were acquired and at the 3500 nJ setting, data from the MW range between 20 and 150 kDa were acquired.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Analysis
Laemmli sample buffer (Boston BioProducts, Ashland, Massachusetts) of 5 µL was added to 20 µL of rFVIIa at 1 mg/mL stock solution. The samples were then denatured and subjected to electrophoresis under reducing conditions through 4% to 20% gradient Tris-N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid–SDS polyacrylamide mini gels (Pierce Biotechnology, Rockford, Illinois). Gel was then stained with Coomassie blue (Bio-Rad Laboratories, Hercules, California) for band visualization.
Immunoblotting Studies Using Rabbit Antihuman FVIIa Antibody
In order to determine the immune reactive component in the NovoSeven and AryoSeven preparations, immunoblotting studies were carried out using a commercially available rabbit antihuman FVIIa antibody (Abcam; Cambridge, Massachusetts). Various batches of the 2 types of rFVIIa samples (same as those processed for SDS-PAGE analysis) were electrophoresed through 4% to 20% gradient gel and then were electrotransferred onto nitrocellulose membrane overnight at 4°C. Precision Dual Color Plus protein standards (Bio-Rad Laboratories) were included in each gel. After electrotransfer, the nitrocellulose membranes were blocked with 5% milk/Tris-buffer saline, pH 7.6, 0.1% Tween-20 (TBST) for 1 hour with gentle shaking, then briefly washed in TBST, and incubated for 1 hour at room temperature with rabbit antihuman FVIIa immunoglobulin G (IgG; 1.87 µg/mL in 5% milk/TBST). The blot was then washed and incubated for 30 minutes at room temperature with 1:100 000 dilutions in 5% milk/TBST of horseradish peroxidase-conjugated donkey antirabbit IgG (H + L; Thermo Scientific). The blots were then extensively washed and immunoreactive bands were detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology) followed by film exposure for about 15 seconds.
Factor VII Antigen Level Using Enzyme-Linked Immunosorbent Assay Method
The FVII antigen content (VII:AG) was measured in each rFVIIa preparation using a commercially available Zymutest FVII:AG enzyme-linked immunosorbent assay (ELISA) kit (Aniara, Westchester, Ohio). All samples at a working concentration of 1 µg/mL were diluted (1:20) in sample diluents prior to placement on to micro ELISA plate per assay specifications. Calibration standard was reconstituted with 2 mL of sample diluents. Further serial dilutions (1:2; 1:4; 1:10; 1:20) were made in sample buffer. The sample diluent was utilized as a blank. Each conjugate rabbit anti (human)-FVII horseradish peroxidase immunoconjugate was reconstituted with 7.5 mL of conjugate buffer and stored in original vial until use.
Diluted samples or standards in the amount of 50 µL were pipetted into wells in duplicates. The samples were incubated for 15 minutes at room temperature and then 200 µL of immunoconjugate was added to each well. The sample-loaded microplate was allowed to incubate for 1 hour at room temperature. After incubation, the plate was washed 5 times with wash buffer. Once the plate was washed and excess buffer was removed from the wells, 200 µL of tetramethylbenzidine/hydrogen peroxide substrate was added to each well and then the plate was incubated for 5 minutes at room temperature. Immediately following the incubation, 50 µL of 0.45 mol/L sulfuric acid (H2SO4) was added to stop color development. Color was allowed to stabilize for 10 minutes prior to reading at 450 nm utilizing the SpectraMax Plus 384 (Molecular Devices, LLC, Sunnyvale, California).
Functional FVII Activity Assay Using Clot-Based Method
Each of the 10 µg/mL stock solutions of the 2 lots of NovoSeven and 6 lots of AryoSeven were diluted to 1 µg/mL in saline. Factor VII-deficient plasma was supplemented with 1 µg/mL solutions of 8 agents to yield 100 ng/mL concentration. Individual samples at a concentration of 100 ng/mL were assayed for FVII levels using the functional method outlined subsequently.
Individual samples were diluted (1:10) in Owren Koller buffer (Aniara). Diluted sample of 0.1 mL was mixed with 0.1 mL of FVII-deficient plasma and transferred to an STA 4 instrument cuvette (Diagnostica Stago, Mount Olive, New Jersey). The samples were incubated for 3 minutes at 37°C. Prewarmed thromboplastin–calcium reagent (Innovin; Dade Behring, Germany) of 0.2 mL was then added and time to clot formation was recorded. A standard curve was constructed using a 1:10, 1:100, and 1:200 dilution of normal human plasma in Owren Koller buffer. Assuming the 1:10 dilution as 100% concentration, the graph representing the resulting clotting time versus percentage concentration was used to extrapolate percentage concentration of rFVIIa in the samples.
Procoagulant Activity as Measured by Clot-Based Method
Prothrombin time (PT) and activated partial thromboplastin time (aPTT) assays were performed in rFVIIa supplemented with whole blood and retrieved plasma samples at 1, 0.5, and 0.25 µg/mL concentration. The retrieved plasma was obtained from freshly drawn citrated human whole blood supplemented with both types of FVIIa preparations after centrifugation at 2500g for 15 minutes. The procoagulant effects of these preparations were also measured using the PT in FVII-deficient plasma supplemented with both types of FVIIa at a logarithmitic level of 1 to 0.001 µg/mL. The whole-blood studies were carried out on STA4 instrument. The clotting studies on the plasma samples were carried out on the ACL 300 plus instrument (Molecular Devices). For PT measurement, 2 different thromboplastin reagents, namely, RecombiPlasTin 2G (Instrumentation Laboratory Company, Bedford, Massachusetts) and Innovin (Dade Behring) reagents were used. Actin FSL reagent (Siemens Healthcare Diagnostics, Deerfield, Illinois) was utilized for aPTT measurement.
Results
This study was designed to compare various batches of NovoSeven and AryoSeven in various compositional and functional assays. Several batches of AryoSeven and NovoSeven were compared.
Protein Content
The mean protein content in 6 batches of AryoSeven was 2.37 ± 0.2 mg/mL which was comparable to 2 batches of NovoSeven 2.20 ± 0.7 mg/mL (Table 1).The determined values of protein were higher than the stated values in the package insert for both the products.
Protein concentration determination in various samples of AryoSeven and NovoSeven as Measured by Lowry Method.
Surface-Enhanced Laser Desorption Ionization Analysis
The composite data on the SELDI analysis of 6 batches of AryoSeven and 2 batches of NovoSeven are given in Figure 1 to determine MW. In the composite spectra of all rFVIIa, a prominent peak was found to be at 50 kDa. The 50-kDa peak represents FVII. No other peaks were detectable. The mean intensity of peak (0.8 unit) or level of expression was similar for both NovoSeven and AryoSeven.
Molecular profiling using surface-enhanced laser desorption/ionization (SELDI) analysis. The SELDI-time-of-flight (TOF) spectra assessing the molecular weight (MW) range of 30 to 60 kDa of recombinant factor VIIa (rFVIIa) compounds. The peak at 50 kDa is representative of FVIIa. There is no significant difference in intensity. Average intensity for AryoSeven was 0.815 ± 0.118 units and for NovoSeven was 0.859 ± 0.047 units.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Analysis
The SDS-PAGE studies were performed to evaluate the protein composition in both rFVIIa preparations. Figure 2 shows SDS-PAGE analysis of AryoSeven #1018 (lane 2), AryoSeven #1019 (lane 3), AryoSeven #1020 (lane 4), AryoSeven #1021 (lane 5), AryoSeven #1022 (lane 6), AryoSeven #1023 (lane 7), NovoSeven # BU60485 (lane 8), NovoSeven # JV60316 (lane 9), and MW marker (lane1). All rFVIIa preparations contain bands predominantly at 50 kDa. However, the band at 29 kDa was due to partial reduction in FVIIa upon addition of 2-mercaptoethanol, a component of Laemmli sample buffer.
Gel electrophoretic profile factor VIIa (FVIIa).
Immunoblotting Studies
As shown in Figure 3, all rFVIIa preparations have similar cross reactivity with rabbit antihuman rFVIIa antibody. All rFVIIa have 2 bands at 50 and 29 kDa. The MW of human FVII was reported to be around 50 kDa. 12 Under reducing conditions and upon addition of 2-mercaptoethanol, a component of Laemmli buffer generated a comparable band at 29 kDa in all rFVIIa preparations.
Immunoblot analysis of various recombinant factor VIIa (rFVIIa) preparations.
Factor VII Antigen Level
Factor VII-related antigen levels of all batches of rVIIa were measured using an ELISA method. The mean level of AryoSeven and NovoSeven was 83.5% ± 13.7% and 75% ± 15.1%, respectively, which was not statistically significant (P value = .47) as shown in Figure 4.
Factor VII (FVII)-related antigen. The mean of FVII antigen level of 2 batches of NovoSeven and 6 batches of AryoSeven is measured by enzyme-linked immunosorbent assay (ELISA) method.
Functional FVII Assay
Figure 5 shows percentage concentration of FVII/FVIIa in both NovoSeven and AryoSeven preparations supplemented in FVII-deficient plasma. Student unpaired, 2-tailed t test and GraphPad Prism 4 software for Windows Version 4.00 were used to calculate mean and standard deviations of the NovoSeven (n = 2) and AryoSeven lots (n = 6) showed no statistical significant difference (P = .30). The FVII/VIIa levels of AryoSeven and NovoSeven were 75.18% ± 15.26% and 60.55% ± 19.95%, respectively, at a concentration of 100 ng/mL.
Percentage concentration of factor VII (FVII)/factor VIIa (FVIIa) in 2 NovoSeven and 6 AryoSeven batches at 100 ng/mL. The mean percentage concentration of FVII/FVIIa of 2 batches of NovoSeven and 6 batches of AryoSeven is measured in FVII-deficient plasma.
Clot-Based Studies
Six batches of AryoSeven were supplemented in whole blood at final concentrations of 1, 0.5, and 0.25 µg/mL to measure clot-based assays such as PT and aPTT (Table 2). The corresponding mean PT levels were 6.6 ± 0.1, 6.8 ± 0.14, and 7 ± 0.11 seconds, respectively, and the corresponding mean aPTT readings were 41.8 ± 1.98, 44.5 ± 1.17, and 47 ± 2.68 seconds, respectively. The control PT and aPTT values in whole blood were 9.6 and 46.9 seconds, respectively.
Clotting Study on Factor VIIa Supplemented in Whole Blood.
Abbreviations: aPPT, activated partial thromboplastin time; Conc, concentration; PT, prothrombin time.
Similarly, PT and aPTT were also measured in retrieved plasma that was obtained from freshly drawn citrated human whole blood after centrifugation at 2500g for 15 minutes (Table 3). Based on the sensitivity and design of the instrument to measure hypocoagulant response rather than a procoagulant response, the calculated mean PT was 7 seconds at all concentrations. Similarly, the average aPTT was 27.3 ± 3.69, 28.6 ± 2.99, and 29.2 ± 2.94 seconds at concentrations of 1, 0.5, and 0.25 µg/mL, respectively. The control PT and aPTT values in retrieved plasma were 7.95 and 31.4 seconds, respectively.
Clotting Study on Factor VIIa Supplemented in Retrieved Plasma.
Abbreviations: aPPT, activated partial thromboplastin time; Conc, concentration; PT, prothrombin time.
Two batches of NovoSeven were also supplemented in both whole blood (Table 2) and retrieved plasma (Table 3) at the same concentration. In whole blood, at concentrations of 1, 0.5, and 0.25 µg/mL, the calculated mean PT was 6.8 ± 0.21, 7 ± 0.28, 7 ± 0.21 seconds and the mean aPTT was 43.1 ± 3.60, 43 ± 3.46, and 45.9 ± 1.69 seconds, respectively. These PT and aPTT results were comparable to AryoSeven. Similarly, in retrieved plasma, the PT and aPTT results were comparable with AryoSeven. As mentioned earlier, based on the sensitivity and design of the instrument to measure hypocoagulant response rather than a procoagulant response, the calculated mean PT was 7 seconds at all concentrations. Similarly, the mean aPTT was 24.3 ± 1.34, 24.4 ± 0.77, and 25.6 ± 1.41 seconds at concentrations of 1, 0.5, and 0.25 µg/mL, respectively.
The PT was also measured in FVII-deficient plasma utilizing different reagents such as RecombiPlasTin 2G (Figure 6) and Innovin(Figure 7). There were no statistically significant differences between NovoSeven and AryoSeven at different concentrations studied utilizing different prothrombin reagents.
NovoSeven and AryoSeven factor VIIa (FVIIa) supplemented in factor VII (FVII)-deficient plasma (prothrombin time [PT]: Innovin). Both the AryoSeven and NovoSeven preparations were diluted to obtain 0.001 to 1.0 µg/mL which supplemented in FVII-deficient plasma. Innovin was used for PT measurement.
NovoSeven and AryoSeven factor VIIa (FVIIa) supplemented in factor VII (FVII)-deficient plasma (prothrombin time [PT]: RecombiPlasTin). Both the AryoSeven and NovoSeven preparations were diluted to obtain 0.001 to 1.0 µg/mL which supplemented in FVII-deficient plasma. RecombiPlasTin 2G was used for PT measurement.
Discussion
The comparative studies carried out on the 6 batches of AryoSeven clearly indicate that the individual batches of AryoSeven are comparable. The studies carried out on the 6 batches of AryoSeven and 2 batches of NovoSeven to determine the compositional and functional characteristics also revealed comparable results.
The protein content of 6 batches of AryoSeven ranged from 1.97 to 2.58 mg/mL in contrast to NovoSeven batches which ranged from 1.69 to 2.75 mg/mL. The protein content as measured by Lowry method was higher than the absolute amount of FVIIa in these preparations. The reported amount of FVIIa in the finished product was 0.6 mg/mL. Since both products contain glycylglycine as excipient, the higher value may be due to the presence of this agent. The glycylglycine contains a peptide bond that may interfere in the protein determination.
The molecular mass profile as measured by SELDI analysis also revealed a distinct single peak for all batches of NovoSeven and AryoSeven with average MW of 50 kDa. The MW range was 30 to 60 kDa for all preparations. The average intensity at 50 kDa was 0.815 ± 0.118 unit for AryoSeven and 0.859 ± 0.047 unit for NovoSeven. No significant differences between 2 preparations in molecular mass profile and peak intensity were noted suggesting comparability of these 2 products.
A comparison of the MW profile of the different batches of AryoSeven and NovoSeven in the SDS-PAGE analysis under reduced conditions revealed 2 distinct bands in all batches of AryoSeven and NovoSeven. No other bands were visible in the MW range of 10 to 250 kDa. The 2 distinct bands represented a dense band at 50 kDa and a weaker band at 29 kDa. The relative intensity of these bands was also comparable. These results further validate the compositional biosimilarity of AryoSeven and NovoSeven.
This study was carried out to demonstrate the relative purity of the 2 groups of rFVIIa preparations. In order to further investigate the composition and relative purity of the AryoSeven and NovoSeven preparations, immunoblotting studies were carried out utilizing rabbit antihuman FVIIa/FVII antibody. This study revealed 2 distinct bands that were comparable in all products. The immunoblotting study revealed a major band at 50 kDa and a second band at 29 kDa. This study confirms the purity of products and their biosimilarity.
To further compare the 2 groups of rFVIIa preparations, the FVII-related antigen was measured utilizing a commercially available ELISA method at identical concentration of 1 µg/mL. The FVII-related antigen was in the range of 70% to 80% for these products. No significant difference was noted in the FVII antigen level between the 2 groups (P value = .47).
In the clot-based functional assays performed in the citrated whole blood and retrieved plasma, the 2 batches of FVIIa produced comparable effect in the PT and aPTT assays. In the 0.25 to 1.0 µg/mL range in comparison to the control with saline supplementation, the aPTT did not exhibit any significant reduction whereas there was a shortening of PT values in the whole blood which was identical in both the AryoSeven and the NovoSeven preparations. In the retrieved plasma, both products produced comparable effect on the PT and aPTT assays. There was a shortening of the aPTT which was similar in both groups of rFVIIa preparations. These studies showed that the 2 products have comparable effects on the clotting profile of whole blood and retrieved plasma upon supplementation of these agents.
The functional concentration of FVII/FVIIa in the 2 groups of rFVIIa preparations was also comparable. When these agents were supplemented at 100 ng/mL, it resulted in 60% to 75% rFVIIa levels in the FVII-deficient plasma. Although the AryoSeven showed a slightly higher value, the difference was not statistically significant (P = .30). These results also suggest that the functional activities for the 2 FVIIa preparations are comparable.
To further investigate the functionality of the AryoSeven and NovoSeven in human FVIIa-deficient plasma, 2 different prothrombin reagents, namely, RecombiPlasTin 2G and Innovin were used. In a logarithmic concentration range of 0.001 to 0.1 µg/mL, a concentration-dependent correction of the PT values was noted for both reagents and there was no difference between AryoSeven and NovoSeven. These studies further confirm the functionality of the FVIIa in the 2 products which was comparable in both products.
The result reported in this study utilized various methods to compare the composition and functional properties of AryoSeven and NovoSeven. In all of the studies carried out, the 2 products exhibited comparable profile. Thus, the biosimilar version of NovoSeven named the AryoSeven appears chemically and biologically equivalent to NovoSeven.
Although the clinical data on the comparison of NovoSeven and AryoSeven are somewhat limited, 2 recent studies have reported on the safety and efficacy of the 2 products in parallel investigations. 8,9 In these studies, the investigators have reported similar safety and efficacy of the 2 products in patients with hemophilia having inhibitors and in patients with congenital FVII deficiency. Taken together, the reported biochemical and functional comparison further validates the biosimilarity of the 2 rFVIIa products. Our studies warrant further investigations and postmarketing surveillance data to collect additional information on the similarity of the 2 products.
Footnotes
Acknowledgments
The authors are thankful to Dr Khoein for providing the AryoSeven preparations. We are also thankful to Dr Richard Kennedy, vice provost for research at the health science division of Loyola University, for his advice and support.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
This study was supported by internal research funds of the Hemostasis and Thrombosis Research Laboratories at the Loyola University Medical Center.