Genetic fusion to albumin improves the pharmacokinetic properties of factor IX

2009 ◽  
Vol 102 (10) ◽  
pp. 634-644 ◽  
Author(s):  
Thomas Weimer ◽  
Ulrich Kronthaler ◽  
Wiegand Lang ◽  
Stefan Schulte ◽  
Hubert J. Metzner
Keyword(s):  
Half Life ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Factor Ix ◽  
Recurrent Bleeding ◽  
Haemophilia B ◽  
Pharmacokinetic Properties ◽  
Genetic Fusion ◽  
In Vivo Recovery

SummaryHaemophilia B is a X-chromosome linked disease characterised by a deficiency of functionally active coagulation Factor IX (FIX). Patients with severe haemophilia B at risk of recurrent bleeding are treated approximately twice a week in a prophylactic setting by application of FIX concentrates.To increase convenience and compliance of the therapy it is desirable to reduce the dosing frequency by improving the pharmacokinetic properties of FIX. Here a concept of rFIX (recombinant factor IX) albumin fusion proteins (rIX-FPs) with cleavable linker peptides derived from the FIX activation sequence is presented. Constructs of the genetic fusion of FIX to albumin via cleavable linkers were expressed in mammalian cells and characterised after purification. In vitro activation studies with FXIa demonstrated that cleavage of the linker and the activation peptide proceeded comparably well. In a clotting assay the rIX-FPs with cleavable linker showed a 10- to 30-fold increase in the molar specific clotting activity compared to fusion proteins with non-cleavable linkers. Furthermore, in-vivo recovery, terminal half-life and the AUC of rIX-FPs in rats and rabbits as determined by FIX antigen measurements were significantly increased compared to rFIX (BeneFIX®). In FIX deficient (FIX−/−) mice the in-vivo recovery and the AUC were also significantly increased.The efficacy in reducing bleeding time was shown in FIX−/−mice by a tail tip bleeding model. The results suggest that rIX-FPs with a cleavable linker between FIX and albumin are a promising concept that may support the use of the albumin fusion technology to extend the half-life of FIX.

IN VIVO RECOVERY AND HALF-LIFE OF A STEAM-TREATED FACTOR IX (FIX) CONCENTRATE

1987 ◽  
Author(s):  
S Mörsdorf ◽  
E Seified ◽  
M Köhler ◽  
F Fasco ◽  
P Hellstern ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Body Weight ◽  
Half Life ◽  
Factor Ix ◽  
Risk Of Infection ◽  
Haemophilia B ◽  
Before And After ◽  
The Mean ◽  
In Vivo Recovery

The introduction of heat treatment of FVIII or FIX concentrates has reduced the risk of infection, however, has raised the question of a reduced haemo-statical effect. Therefore, the in vivo recovery and half-life of a steam-treated FIX concentrate (S-TIM4, Immuno) were investigated in 10 haemophilia B patients from two haemophilia centers. Patients mean age was 33 y (range 17-51 y) and the mean body weight (BW> was 67 kg (range 44-81 kg). Basal FIX levels ranged from 0.007 to 0.03 (median 0,007) U/ml. The patients had not received FIX concentrate at least 7 d prior to the study. Patients 1-4 received 4 different lots, patients 5-10 received one single lot. Blood was drawn before and after 15, 30 min, 1h, 4 h, 8 h, 10 h, 12 h, 24 h and additionally 48 h in patients 1-4. FIX levels were measured using FIX deficient plasma from Immuno (patients 1-10) in center 1, additionally in patients 5-10 using FIX deficient plasma from MerzDade. In vivo recovery and half-life were calculated according to Allain (1980, 1984) and given in % and h, respectively. Results: The table shows the dose and the calculated in vivo recovery and half-life, according to the FIX measurements in center 1 (C1) or center 2 (C2).Although the apparently longer half-life of patients 1-4 may in part be explained by the longer period of FIX measurements in center 1, the exclusive use of one single lot of FIX concentrate suggests an influence of the lot transfused in these patients. However, laboratory signs of DIC were not present.

Recombinant Human Coagulation Factor IX Expressed in HEK293 Cells: Influence of Serin Phosphorylation and Tyrosine Sulfation on Pharmacokinetic Properties in FIX-Knock-out Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4098-4098
Author(s):  
Ernst Boehm ◽  
Michael Dockal ◽  
Meinhard Hasslacher ◽  
Artur Mitterer ◽  
Eva M Muchitsch ◽  
...  
Keyword(s):  
Factor Ix ◽  
Hek293 Cells ◽  
High Yield ◽  
High Similarity ◽  
Tyrosine Sulfation ◽  
Knock Out ◽  
Pharmacokinetic Properties ◽  
Knock Out Mice ◽  
In Vivo Recovery

Abstract Recombinant factor IX (rFIX) expressed in Chinese hamster ovary (CHO) cells has been shown to be safe and effective in clinical studies, but differs in pharmacokinetics from plasma-derived FIX (pdFIX). In clinical studies, CHO-derived rFIX had a 30–50 % lower in-vivo recovery when compared to pdFIX, whereas mean residence time and terminal half-life did not differ between preparations. Although rFIX shows high similarity to pdFIX in structure and function, differences in glycosylation and gamma-carboxylation degree can be detected. Moreover, although experimental proof has yet to be published, the lower degree of phosphorylation of amino acid serine 155, and the lower degree of sulfation of tyrosine 158 have been hypothesized to be causative for the lower in-vivo recovery of rFIX. These two modifications occur at less than 20 % for the tyrosine-sulfation and at less than 1 % for the serine phosphorylation in rFIX, whereas pdFIX has both modifications to more than 90 % completed. We identified human HEK293 cells to perform rFIX phosphorylation and sulfation to a higher extent than CHO cells. A rFIX-producing cell line derived from HEK293 cells was generated by stable transfection, and was adapted to suspension culture conditions to allow lab-scale fermentation. rFIX was produced and purified from a single fermentation run using two different down-stream process schemes: the first was able to enrich high-phosphorylated and -sulfated rFIX; the second to purify total rFIX from the supernatant at high yield. For pharmacokinetic comparison, these HEK293 materials, CHO-derived rFIX, and a pdFIX preparation were formulated in the same buffer. Determination of phosphorylation and sulfation by mass spectrometry showed a phosphorylation and sulfation degree of 50 % plus a 20 % single modification (phosphorylation or sulfation) for the HEK293-material purified by the modification enrichment method versus 15 % for both modifications plus a 15 % single modification for the material purified by the high-yield protocol. The values for CHO-derived rFIX and pdFIX were similar to those in the literature. Oligosaccharide mapping revealed glycosylation differences among CHO-, HEK293-, and pdFIX preparations, but high similarity between both HEK293-derived materials. We compared the pharmacokinetics of the various FIX preparations in FIX-knock-out mice. In-vivo recovery and area under the curve were statistically significantly higher for the high phosphorylated and sulfated HEK293-material than for total rFIX derived from HEK293 cells. However, these two parameters were lower for both HEK293-derived rFIX preparations than for CHO-derived rFIX, and lower for CHO-derived rFIX than for pdFIX. This may be due to glycosylation differences between these FIX preparations. Mean residence times and terminal half-lives were similar for all preparations. In summary, these findings emphasize that the degree of rFIX-sulfation and -phosphorylation influences the pharmacokinetic properties of rFIX.

Pharmacokinetic Properties of Recombinant Factor VIII Compared with a Monoclonally Purified Concentrate (Hemofil® M)

1992 ◽  
Vol 68 (04) ◽  
pp. 433-435 ◽  
Author(s):  
M Morfini ◽  
G Longo ◽  
A Messori ◽  
M Lee ◽  
G White ◽  
...  
Keyword(s):  
Factor Viii ◽  
Half Life ◽  
Hemophilia A ◽  
Recombinant Dna ◽  
Volume Of Distribution ◽  
Pharmacokinetic Properties ◽  
Recombinant Fviii ◽  
Reported Data ◽  
In Vivo Recovery

SummaryA recombinant FVIII preparation, Recombinate™, was compared with a high-purity plasma-derived concentrate, Hemofil® M, in 47 hemophilia A patients in a cross-over evaluation of pharmacokinetic properties. The recombinant material showed a significantly lower clearance, volume of distribution, and higher in vivo recovery, but a similar half-life to the plasma-based product.In a comparison with reported data from other standard concentrates, the recombinant preparation exhibited potentially better pharmacokinetic properties in that its clearance was slower and its half-life was longer.We conclude that the recombinant DNA method of preparation does not adversely affect the biological and pharmacological characteristics of the factor VIII molecule.

scholarly journals Purified factor IX using monoclonal immunoaffinity technique: clinical trials in hemophilia B and comparison to prothrombin complex concentrates

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 568-575 ◽  
Author(s):  
HC Kim ◽  
CW McMillan ◽  
GC White ◽  
GE Bergman ◽  
MW Horton ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Vitamin K ◽  
Half Life ◽  
Viral Infections ◽  
Factor Ix ◽  
Hemophilia B ◽  
Terminal Phase ◽  
Factor Ix Deficiency ◽  
In Vivo Recovery

Abstract Replacement therapy for hemophilia B (factor IX deficiency) using prothrombin complex concentrate (PCC) has been associated with serious complications of thromboembolic events and transmission of viral infections. Monoclonal antibody-purified factor IX (Mononine) provides a highly purified factor IX concentrate, while eliminating other vitamin K-dependent factors (II, VII, and X). Mononine was evaluated for in vivo recovery, half-life, and for its safety and efficacy in 10 patients with hemophilia B. The in vivo recovery of factor IX with Mononine was a 0.67 +/- 0.14 U/dL (mean +/- SD) increase per 1U/kg of infused factor IX, and the biologic half-life (t1/2), determined using the terminal phase of elimination, was 22.6 +/- 8.1 hours. Comparison of in vivo recovery of other vitamin K-dependent factors following a single infusion of either Mononine or PCC showed that, whereas Mononine infusion caused no changes in other vitamin K-dependent factors or in prothrombin activation fragment (F1+2), PCC infusion was associated with significant increases of factors II (2.7 U/dL per 1 U/dL of IX increase) and X (2.2 U/dL for 1 U/dL for 1 U/dL of IX). Patients who used Mononine as their sole therapeutic material during the 12-month period showed an excellent response in hemostasis for their bleeding episodes. Their experience with long-term use of Mononine was at least equivalent to their previous experience with PCC in the frequency and amount of factor usage. No patients developed antibody against mouse IgG or an increase in IX inhibitor during the 12-month period. These results indicate that monoclonal antibody-purified factor IX concentrate provides hemostatically effective factor IX replacement while avoiding extraneous thrombogenic substances.

A Pharmacokinetic Study of the Plasma-Derived Factor IX AlphaNine® and Its Comparison with the Recombinant Factor IX BeneFIX®, in Previously Treated Patients with Severe Hereditary Hemophilia B.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3955-3955
Author(s):  
Vicente R. Cortina ◽  
T. Lissichkov ◽  
K. Zavilska ◽  
M. Matysiak ◽  
L. Gercheva ◽  
...  
Keyword(s):  
Half Life ◽  
Factor Ix ◽  
Hemophilia B ◽  
Severe Haemophilia ◽  
Open Label ◽  
Recombinant Factor Ix ◽  
Previously Treated Patients ◽  
Previously Treated ◽  
In Vivo Recovery

Abstract Objectives The objective of the present study was two fold: first, to determine the pharmacokinetic (PK) profile of the plasma-derived FIX concentrate AlphaNine® in patients with congenital severe haemophilia B (FIX:C 2%). To do this, two PK studies were carried out one six months after the first. The second objective was a comparison of the Alphanine® PK profile with the recombinant Factor IX, BeneFIX®. Patients and methods The first study was a prospective, five-center, open-label, comparative, PK study carried out in 25 severe hemophilia B patients who received 2 single doses of 65–75 IU/kg of AlphaNine® within 6 months (t=0 and t=6). The following parameters were assessed: in vivo recovery, half-life, AUC, mean residence time and clearance. As an extension of the study, a single dose of 65–75 IU/kg of BeneFIX® was administered in 9 out of 25 patients, after a wash-out period of 7–15 days. Results Table 1 summarizes the results obtained when comparing AlphaNine® within a period of time of 6 months (PK1 vs PK2) in 25 patients. Table 2 shows the results obtained when comparing the in vivo recovery of AlphaNine ® vs BeneFIX ® in the 9 patients studied. Conclusions These results confirm that AlphaNine® PK has similar profile as other plasma derived FIX products presently available to treat Hemophilia B patients. In addition, our results show that the recombinant FIX studied, BeneFIX® has a reduced in vivo recovery when is compared to AlphaNine®. Table 1 Parameter AlphaNine® (PK1) t=0 m AlphaNine® (PK2) t=6 m Results are expressed as Mean (SD) In vivo recovery (IU/dl:IU/kg) 1.0 (0.2) 1.2 (0.4) Half-life (h) 34.5 (6.2) 33.7 (5.4) Clearance (ml/min) 0.07 (0.01) 0.07 (0.01) AUC0-inf (IUxh/dl) 1602 (312) 1644 (360) MRT0-inf (h) 35.8 (5.4) 34.6 (5.2) Table 2 Parameter AlphaNine® (PK2) BeneFIX® Results are expressed as Mean (SD); * p<0.05 for the comparison of the in vivo recovery for the BeneFIX® group with the AlphaNine® PK2 In vivo recovery (IU/dl:IU/kg) 1.3 (0.5) 0.8 (0.2)*

IB1001, an Investigational Recombinant Factor IX, Pharmacokinetics in Pediatric Patients with Hemophilia B.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2225-2225
Author(s):  
Edward D. Gomperts ◽  
Shashikant Apte ◽  
Utpal Chaudhuri ◽  
Joseph M John ◽  
Vijay Ramanan ◽  
...  
Keyword(s):  
Half Life ◽  
Research Funding ◽  
Pediatric Patients ◽  
Robust Regression ◽  
Factor Ix ◽  
Hemophilia B ◽  
Maximum Plasma ◽  
Recombinant Factor Ix ◽  
In Vivo Recovery

Abstract Abstract 2225 Introduction IB1001 is a recombinant factor IX product being investigated for the treatment and prevention of bleeding in individuals with hemophilia B. Pharmacokinetics (PK) in adults (>12 years) demonstrated that IB1001 had results similar to the currently available recombinant FIX with respect to parameters such as terminal phase half-life and incremental recovery. We report the interim findings from a PK assessment in children <12 years, with severe hemophilia B (FIX <2%), >50 prior exposure days to FIX, and no history of or currently detectable inhibitor to FIX. Methods Non-randomized, open-label PK study with patients receiving 75±5 IU/kg of IB1001 following a washout period of ≥4 days from a previous FIX infusion. Factor IX levels were determined pre-infusion and at 15–30 minutes, 4–6, 24–26, and 68–72 hours post-infusion. Additional samples could be drawn at 1–3 and 10–14 hours. Calculated PK parameters were: half-life (β-phase t1/2, determined using a robust regression approach [Lee ML et al. XVIth ISTH Congress, Florence, Italy, 1997]) but generally assuming a single compartmental model because of the small number of points, maximum plasma concentration (Cmax), in vivo recovery (IVR) and AUC(0-∞) (determined by the trapezoidal rule). In addition, the AUC(0-t) and mean residence time (MRT) were calculated. Results When compared to the findings previously reported with IB1001 in adult (≥12 years of age) subjects (Martinowitz U et al. Haemophilia, 18, 2012), the results in pediatric patients demonstrate a more rapid metabolism of factor IX as is indicated by the shorter terminal half-life (mean±SD of 19.3±7.8 h versus 29.6±18.2 h in adults) and the smaller AUC0-∞ (mean±SD of 1059±264 versus 1668±598 in adults). In addition, the in vivo recovery was lower (mean±SD of 0.69±0.21) versus that seen in adults (mean±SD of 0.98±0.22). These results are similar to those reported by Berntorp et al (Haemophilia, 7, 2001) with nonacog alfa. Conclusions The pharmacokinetics of IB1001 has previously been shown to be non-inferior to nonacog alfa, another recombinant factor IX, in hemophilia B individuals >12 years of age. The current study is intended to provide information on children <12 and, particularly, <6 years of age. IB1001 is metabolized faster and has a lower recovery than the comparable findings in patients >12 years of age. Although the study is ongoing, these may represent important implications for the potential use of IB1001 in pediatric patients. Disclosures: Gomperts: Inspiration Biopharmaceuticals Inc: Consultancy. Apte:Inspiration Biopharmacauticals Inc: Research Funding. Chaudhuri:Inspiration Biopharmaceuticals Inc: Research Funding. John:Inspiration Biopharmaceuticals Inc: Research Funding. Ramanan:Inspiration Biopharmaceuticals Inc: Research Funding. Liesner:Inspiration Biopharmaceuticals Inc: Research Funding. Shapiro:Inspiration Biopharmaceuticals Inc: Honoraria, Research Funding. Mills:Inspiration Biopharmaceuticals Inc: Employment. Lee:Inspiration Biopharmaceuticals Inc: Employment.

In vivo recovery and half-life time of a steam-treated factor IX concentrate in hemophilia B patients

1988 ◽  
Vol 57 (6) ◽  
pp. 341-345 ◽  
Author(s):  
M. K�hler ◽  
E. Seifried ◽  
P. Hellstern ◽  
G. Pindur ◽  
C. Miyashita ◽  
...  
Keyword(s):  
Half Life ◽  
Life Time ◽  
Factor Ix ◽  
Hemophilia B ◽  
In Vivo Recovery

scholarly journals Purified factor IX using monoclonal immunoaffinity technique: clinical trials in hemophilia B and comparison to prothrombin complex concentrates

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 568-575
Author(s):  
HC Kim ◽  
CW McMillan ◽  
GC White ◽  
GE Bergman ◽  
MW Horton ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Vitamin K ◽  
Half Life ◽  
Viral Infections ◽  
Factor Ix ◽  
Hemophilia B ◽  
Terminal Phase ◽  
Factor Ix Deficiency ◽  
In Vivo Recovery

Replacement therapy for hemophilia B (factor IX deficiency) using prothrombin complex concentrate (PCC) has been associated with serious complications of thromboembolic events and transmission of viral infections. Monoclonal antibody-purified factor IX (Mononine) provides a highly purified factor IX concentrate, while eliminating other vitamin K-dependent factors (II, VII, and X). Mononine was evaluated for in vivo recovery, half-life, and for its safety and efficacy in 10 patients with hemophilia B. The in vivo recovery of factor IX with Mononine was a 0.67 +/- 0.14 U/dL (mean +/- SD) increase per 1U/kg of infused factor IX, and the biologic half-life (t1/2), determined using the terminal phase of elimination, was 22.6 +/- 8.1 hours. Comparison of in vivo recovery of other vitamin K-dependent factors following a single infusion of either Mononine or PCC showed that, whereas Mononine infusion caused no changes in other vitamin K-dependent factors or in prothrombin activation fragment (F1+2), PCC infusion was associated with significant increases of factors II (2.7 U/dL per 1 U/dL of IX increase) and X (2.2 U/dL for 1 U/dL for 1 U/dL of IX). Patients who used Mononine as their sole therapeutic material during the 12-month period showed an excellent response in hemostasis for their bleeding episodes. Their experience with long-term use of Mononine was at least equivalent to their previous experience with PCC in the frequency and amount of factor usage. No patients developed antibody against mouse IgG or an increase in IX inhibitor during the 12-month period. These results indicate that monoclonal antibody-purified factor IX concentrate provides hemostatically effective factor IX replacement while avoiding extraneous thrombogenic substances.

Fusion of Factor IX to Factor XIII-B Sub-Unit Improves the Pharmacokinetic Profile of Factor IX

2018 ◽  
Vol 118 (12) ◽  
pp. 2053-2063 ◽  
Author(s):  
Sandra Le Quellec ◽  
Nathalie Enjolras ◽  
Eloïse Perot ◽  
Jonathan Girard ◽  
Claude Negrier ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Half Life ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Pharmacokinetic Profile ◽  
Factor Ix ◽  
Severe Haemophilia ◽  
Optimal Care ◽  
Haemophilia B

AbstractProphylaxis is currently considered the optimal care for severe haemophilia. For patients and their families one of the major difficulties with prophylaxis is the need for frequent venipunctures. The half-life of standard factor IX (FIX) concentrates is approximately 18 hours, which requires 2 or 3 intravenous infusions per week to achieve bleeding prevention in patients with severe haemophilia B. Prolonging the half-life of FIX can therefore reduce the frequency of infusions. Recently, extended half-life recombinant FIX (rFIX) concentrates have been developed. We designed a new rFIX molecule fused to coagulation FXIII-B sub-unit. This sub-unit is responsible for the long half-life of the FXIII molecule (10–12 days). The rFIX-LXa-FXIIIB fusion protein contains a short linker sequence cleavable by activated FX (FXa), to separate rFIX from the carrier protein as soon as traces of FXa are generated, leaving rFIX free to perform its enzymatic role in the tenase complex. The rFIX-LXa-FXIIIB fusion protein was expressed in human hepatic Huh-7 cells and Chinese hamster ovary cells, and both wild-type rFIX (rFIX-WT) and rFIX-LXa-FXIIIB showed similar clotting activity and thrombin generation capacity in vivo after injection in haemophilia B mice compared with rFIX-WT. The half-life of the rFIX-LXa-FXIIIB molecule in WT mice and rats was 3.9- and 2.2-fold longer, respectively, compared with rFIX-WT. A potential advantage of this new molecule is its capacity to bind to fibrinogen via FXIII-B, which might accelerate fibrin clot formation and thus improve haemostatic capacity of the molecule.

scholarly journals In silico comparison of pharmacokinetic properties of three extended half-life factor IX concentrates

2021 ◽  
Author(s):  
Tim Preijers ◽  
Laura Bukkems ◽  
Max van Spengler ◽  
Frank Leebeek ◽  
Marjon Cnossen ◽  
...  
Keyword(s):  
Half Life ◽  
Real Life ◽  
Area Under The Curve ◽  
Human Albumin ◽  
Factor Ix ◽  
Target Values ◽  
Pharmacokinetic Properties ◽  
Dosing Regimens ◽  
Human Igg1

Abstract Purpose Pharmacokinetic (PK) differences between the extended half-life (EHL) factor IX (FIX) concentrates for hemophilia B exist, which may influence hemostatic efficacy of replacement therapy in patients. Therefore, we aimed to evaluate the PK properties of three EHL-FIX concentrates and compare them to a standard half-life (SHL) recombinant FIX (rFIX) concentrate. Methods Activity-time profiles of PEGylated FIX (N9-GP), FIX linked with human albumin (rIX-FP), FIX coupled to human IgG1 Fc-domain (rFIXFc), and SHL rFIX were simulated for 10,000 patients during steady-state dosing of 40 IU/kg once weekly (EHL-FIX) and biweekly (rFIX) using published concentrate specific population PK models. Results Half-lives were respectively 80, 104, and 82 h for N9-GP, rIX-FP, and rFIXFc versus 22 h for rFIX. Between the EHL concentrates, exposure was different with area under the curve (AUC) values of 78.5, 49.6, and 12.1 IU/h/mL and time above FIX target values of 0.10 IU/mL of 168, 168, and 36 h for N9-GP, rIX-FP, and rFIXFc, respectively. N9-GP produced the highest median in vivo recovery value (1.70 IU/dL per IU/kg) compared with 1.18, 1.00, and 1.05 IU/dL per IU/kg for rIX-FP, rFIXFc, and rFIX, respectively. Conclusions When comparing EHL products, not only half-life but also exposure must be considered. In addition, variation in extravascular distribution of the FIX concentrates must be taken into account. This study provides insight into the different PK properties of these concentrates and may aid in determination of dosing regimens of EHL-FIX concentrates in real-life.

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