Comparative in Vitro and in Vivo Study of Various Factor VIII Preparations

1979 ◽  
Author(s):  
J.P. Allain ◽  
F. Verroust ◽  
J.P. Soulier
Keyword(s):  
Factor Viii ◽  
High Purity ◽  
Half Life ◽  
Hemophilia A ◽  
Post Injection ◽  
High Doses ◽  
Antibody Levels ◽  
Similar Risk

A comparison of nine commercial and non-commercial Factor VIII preparations was made. They consisted of llyophilized cryoprecipitate, 4 intermediate and 4 high purity concentrates. Protein, Fibrinogen, Factor VIII complex, IgG, anti-A and anti-B antibody levels were measured. Factor VIII:C content varied from 4-7 u/ml in cryoprecipitate, 12-31 u/ml in intermediate and 21-40 u/ml in high purity concentrates. These three categories of Factor VIII preparations can be better defined by 2 ratios: u FVIII/mg proteins and u F VIII/mg fibrinogen. They were respectively < 0.5 and < 1 in cryo, 0.5-1 and 1-3 in intermediate purity concentrates, > 1 and > 3 in high purity concentrates. The F VIII :C/F VIII : AG ratio ranged from 0.3 to 0.6 in any preparation. The F VIII:C/F VIII :VWF ratio was always lower than 1.Each preparation was injected to several classic hemophilia A patients for treatment of minor hemorrhages. The peak of activity was always found 1 hour post-injection and the recovery ranged from 80 to 105%. The Factor VIII half-life ranged from 10 to 12.5 hours. No significant differences in half-life or recovery was found, and the clinical efficacy was similar. With the exception of fibrinogen load, all products carry similar risk for hepatitis, anti-IgG immunization and hemolysis. The differences lie in the ease of injection, the price and the yield of Factor VIII from starting plasma. Nevertheless, high purity concentrates should be used when high doses are reauired for surgery or treatment of patients with inhibitor.

scholarly journals Recombinant VWF Fragments Improve Bioavailability of Subcutaneous Factor VIII in Hemophilia A Mice

Blood ◽  
2020 ◽  
Author(s):  
Nadine Vollack-Hesse ◽  
Olga Oleshko ◽  
Sonja Werwitzke ◽  
Barbara Solecka-Witulska ◽  
Christoph Kannicht ◽  
...  
Keyword(s):  
Factor Viii ◽  
Half Life ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Dependent Manner ◽  
High Concentrations ◽  
Von Willebrand ◽  
Willebrand Factor

Conventional treatment of hemophilia A (HA) requires repetitive intravenous (IV) injection of coagulation factor VIII (FVIII). Subcutaneous (SC) administration of FVIII is inefficient because of binding to the extravascular matrix, in particular to phospholipids (PL), and subsequent proteolysis. To overcome this, recombinant dimeric fragments of von Willebrand factor (VWF) containing the FVIII stabilizing D3 domain were engineered. Two fragments, called VWF-12 and VWF-13, demonstrated high binding affinity to recombinant human FVIII (rhFVIII) and suppressed PL-binding in a dose-dependent manner. High concentrations of VWF fragments did not interfere with the functional properties of full-length VWF in vitro. The HA mouse model was used to study the effects of VWF-12 or VWF-13 on the in vivo pharmacokinetics of rhFVIII, demonstrating (i) no significant impact on rhFVIII recovery or half-life after a single IV administration; (ii) enhanced bioavailability (up to 18.5 %) of rhFVIII after SC administration; (iii) slow absorption (cmax 6h) and prolonged half-life (up to 2.5-fold) of rhFVIII after SC administration. Formation of anti-FVIII antibodies was not increased after administration of rhFVIII/VWF-12 SC compared to rhFVIII IV. A single SC dose of rhFVIII/VWF-12 provided protection in the HA tail bleeding model for up to 24h. In conclusion, recombinant VWF fragments support FVIII delivery through the SC space into vascular circulation without interfering with VWF or FVIII function. Slow resorption and excretion of FVIII after SC administration highlight the potential application of VWF fragments for SC FVIII prophylaxis in HA.

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

In Vitro and In Vivo Characterization of Full-Length rFVIII Modified with PEG Via Coupling to Primary Amino Groups.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3147-3147 ◽  
Author(s):  
Peter L. Turecek ◽  
Jürgen Siekmann ◽  
Herbert Gritsch ◽  
Katalin Váradi ◽  
Rafi-Uddin Ahmad ◽  
...  
Keyword(s):  
Half Life ◽  
Hemophilia A ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Full Length ◽  
Thrombin Inhibitor ◽  
Knock Out ◽  
Knock Out Mice

Abstract Chemical modification of recombinant therapeutic proteins with PEG has been shown to enhance the biological half-life. Here we assess the effect of PEGylation on FVIII. Full-length rFVIII bulk drug substance from protein-free fermentation (Advate process, Baxter) was conditioned into a buffer suitable for coupling to polyethylene glycol succinimidyl succinate (linear PEG, 5 kDa PEG chain length). PEG was covalently bound by amine coupling preferentially to lysine residues of FVIII at neutral pH. PEG was removed by ion-exchange chromatography and the PEG-FVIII derivative was concentrated by ultra-diafiltration. The conjugates thus obtained retained about 30–40% of the activity of non-modified rFVIII. The specific activity decreased with the amount of PEG linked to the FVIII molecule. In SDS-PAGE and immunoblot studies PEGylated rFVIII showed a band pattern similar to unmodified FVIII with full-length, heavy chain fragments of 180 kDa and 120 kDa and the light chain fragment of 80 kDa. PEGylation also occurred to a high extent in the B domain of FVIII. All bands appeared broadened due to the attachment of polymeric PEG. The maintenance of functionality of FVIII was demonstrated by its potential to be activated and inactivated by thrombin. In the assay PEGylated and unmodified FVIII were incubated with 1 nM thrombin. Sub-samples were drawn at intervals up to 40 minutes and added to a mixture of FIXa, FX, phospholipid vesicles and Ca2+ containing a thrombin inhibitor. After 3 minutes incubation at 37°C the amount of activated FX (FXa) was measured using a FXa-specific chromogenic substrate. Unmodified rFVIII showed a typical picture of an immediate increase in FXa activity and a subsequent decline with no further FXa generation after 15 minutes. PEGylated rFVIII was activated to the same extent as unmodified FVIII but the decay in FXa generation was slower and did not reach the zero level, even 40 minutes after incubation. The formation of the typical thrombin cleavage fragments, with unmodified as well as PEGylated rFVIII, was demonstrated in a Western blot analysis. The slower inactivation by thrombin was also seen there. The pharmacokinetic properties of PEGylated rFVIII compared with rFVIII were investigated in hemophilia A knock-out mice. Both preparations were applied at a dose of 200 IU rFVIII/kg and groups of mice (n=5) were exsanguinated at several time points up to 24 hours. Terminal half-life for PEGylated rFVIII was calculated at 4.9 hours compared with 1.9 hours for unmodified rFVIII in hemophilia A knock-out mice. AUC was approximately doubled. These results indicate that rFVIII can be biochemically modified with PEG whilst at least partly retaining its major functions, but at the same time prolonging its survival in the circulation of hemophilic mice.

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.

Expression of therapeutic levels of factor VIII in hemophilia A mice using a novel adeno/adeno-associated hybrid virus

2004 ◽  
Vol 92 (08) ◽  
pp. 317-327 ◽  
Author(s):  
Dmitri Gnatenko ◽  
Yong Wu ◽  
Jolyon Jesty ◽  
Andrea Damon ◽  
Patrick Hearing ◽  
...  
Keyword(s):  
Factor Viii ◽  
Quantitative Pcr ◽  
Hemophilia A ◽  
High Titer ◽  
Small Nuclear Rna ◽  
Genomic Integration ◽  
Novel Approach ◽  
Fviii Activity

SummaryWe have generated an E1a/E1b/E3-deleted adeno/adeno-associated (Ad/AAV) hybrid virus driven by a small nuclear RNA (pHU1-1) promoter for expression of a B domain-deleted (Thr761-Asn1639) factor VIII transgene (FVIIIΔ761-1639). Productive replication of Ad/AAV/FVIIIΔ761-1639 in AAV repexpressing cells resulted in generation of monomeric and dimeric mini-adenoviral (mAd) replicative forms that retained the AAV integration elements (mAd/FVIIIΔ761-1639). In vitro studies using Ad/AAV/FVIIIΔ761-1639 generated ∼2-logs greater FVIII activity than mAd/FVIIIΔ761-1639. To determine its capacity for in vivo excision and/or genomic integration, Ad/AAV/FVIIIΔ761-1639 was injected by tail vein into three groups of hemophilia A mice (2 X 1011 vp [n = 3]; 4 X 1011 vp [n = 3]; 8 X 1011 vp [n = 3]), with clear concentration-dependent increase in FVIII activity (range 160-510 mU/ml; plasma activity 16% – 51% of normal). Peak activity was seen by Day (D) 5, with slow return to baseline by D28 (0.1 – 0.9% activity); in only 3/9 mice was loss of FVIII activity associated with development of anti-FVIII antibodies. Quantitative-PCR using genomic DNA isolated from D28 liver, spleen, heart, lungs, and kidney demonstrated the highest concentration in liver (∼10 genomes/ cell), with little to no organ toxicity at early (D5 or 6) or late (D28) post-infusion time points. There was no evidence for spontaneous transgene excision or genomic integration in vivo as evaluated by quantitative PCR and genomic blotting. These data establish (i) the feasibility and applicability of developing high-titer Ad/AAV hybrid viruses for FVIII delivery using a small cellular promoter, (ii) the potential utility of this virus for generation of “gutted” monomeric and dimeric mAD/FVIII retaining AAV integration elements, and (iii) that the development of strategies for regulated Rep68/78 co-expression may provide a novel approach for excision, integration, and long-term FVIII transgene expression.

Evaluation of an Adenoviral Vector Encoding Full-Length Human Factor VIII in Hemophiliac Mice

1999 ◽  
Vol 81 (02) ◽  
pp. 234-239 ◽  
Author(s):  
Sheila Connelly ◽  
Julie Andrews ◽  
Angela Gallo-Penn ◽  
Luigina Tagliavacca ◽  
Randal Kaufman ◽  
...  
Keyword(s):  
Factor Viii ◽  
Adenoviral Vector ◽  
Human Factor ◽  
Hemophilia A ◽  
Adenoviral Vectors ◽  
Full Length ◽  
Human Factor Viii ◽  
Immunohistochemical Analyses

SummaryAdenoviral vectors provide a promising gene therapy system for the treatment of hemophilia A. Potent vectors encoding a human factor VIII (FVIII) cDNA were developed that mediated sustained FVIII expression in normal and hemophiliac mice and complete phenotypic correction of the bleeding disorder in hemophiliac mice and dogs (Connelly and Kaleko, Haemophilia 1998; 4: 380-8). However, these studies utilized vectors encoding a truncated version of the human FVIII cDNA lacking the B-domain (BDD FVIII). In this work, an adenoviral vector encoding the human full-length (FL) FVIII cDNA was generated and characterized. While functional FL FVIII was secreted in vitro, expression of the FL protein was not detected in the plasma of vector-treated hemophiliac mice. Unexpectedly, the FL FVIII vector-treated animals demonstrated phenotypic correction of the bleeding defect as measured by a tail-clip survival study. FL FVIII protein was visualized in the mouse livers using human FVIII-specific immunohistochemical analyses. These data demonstrate that adenoviral vector-mediated in vivo expression of BDD FVIII is more efficient than that of the FL protein and that phenotypic correction can occur in the absence of detectable levels of FVIII.

Issues with the Assay of Factor VIII Activity in Plasma and Factor VIII Concentrates

2000 ◽  
Vol 84 (12) ◽  
pp. 942-948 ◽  
Author(s):  
Henry Kingdon ◽  
Kenneth Mann ◽  
Gilbert White ◽  
Roger Lundblad
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factors ◽  
Chromogenic Substrate ◽  
In Vitro Measurements ◽  
Factor Viii Concentrates ◽  
The One

SummaryA review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VIII concentrates such as Hemofil ® M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements, it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor Villa during the assay process.

Bioengineering of coagulation factor VIII for improved secretion

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3412-3419 ◽  
Author(s):  
Hongzhi Z. Miao ◽  
Nongnuch Sirachainan ◽  
Lisa Palmer ◽  
Phillip Kucab ◽  
Michael A. Cunningham ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Mrna Levels ◽  
Golgi Transport ◽  
Recombinant Fviii ◽  
Therapy Strategies

Abstract Factor VIII (FVIII) functions as a cofactor within the intrinsic pathway of blood coagulation. Quantitative or qualitative deficiencies of FVIII result in the inherited bleeding disorder hemophilia A. Expression of FVIII (domain structure A1-A2-B-A3-C1-C2) in heterologous mammalian systems is 2 to 3 orders of magnitude less efficient compared with other proteins of similar size compromising recombinant FVIII production and gene therapy strategies. FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose-binding lectin LMAN1. Bioengineering strategies can overcome each of these limitations. B-domain-deleted (BDD)-FVIII yields higher mRNA levels, and targeted point mutations within the A1 domain reduce interaction with the ER chaperone immunoglobulin-binding protein. In order to increase ER to Golgi transport we engineered several asparagine-linked oligosaccharides within a short B-domain spacer within BDD-FVIII. A bioengineered FVIII incorporating all of these elements was secreted 15- to 25-fold more efficiently than full-length FVIII both in vitro and in vivo. FVIII bioengineered for improved secretion will significantly increase potential for success in gene therapy strategies for hemophilia A as well as improve recombinant FVIII production in cell culture manufacturing or transgenic animals. (Blood. 2004;103: 3412-3419)

scholarly journals Development of a Pegylated Dimeric Recombinant Factor VIII-Fc Fusion Protein for the Treatment for Hemophilia a Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3783-3783
Author(s):  
Bin Liu ◽  
Xiaoshan Wang ◽  
Xueqin Li ◽  
Haixia Yan ◽  
Shuya Wang ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Factor Viii ◽  
Treatment Option ◽  
Half Life ◽  
Hemophilia A ◽  
Binding Activity ◽  
Recombinant Factor Viii ◽  
Fc Fusion Protein ◽  
In Vivo Tests

Abstract Hemophilia A is a hereditary bleeding disorder resulting from reduced factor FVIII activity. It occurred in 1/5000 male. Currently, the treatment option is with the factor FVIII replacement therapy. A long-acting recombinant monomeric FVIII-Fc fusion protein product (Eloctate®) has been approved in 2014 by the US FDA, it requires to infuse the drug for every 3 days or twice a week. There is a clinical need to develop longer half-life product to extend the treatment option to once a week infuse for hemophilia A patients. Recently, we have developed a dimeric recombinant factor VIII-Fc (drFVIII-Fc) fusion protein therapeutic candidate, which is entering the clinical development in China. To generate a longer half-life recombinant FVIII product, we have developed a PEGylation method to PEGylated this drFVIII-Fc fusion protein to PEGdrFVIII-Fc. We have analyzed and characterized the fusion protein by various analytic methods, as well as in vivo animal tests. It was shown that PEGdrFVIII-Fc fusion protein has been modified with about five Y type of 40kd PEG; the remaining activity is around 700 IU/mg, and the in vivo tests in cynomolgus monkey demonstrated that the fusion protein has a half-life of about 37 hours. The data also showed that there was no detectable affinity binding activity of vWF to a PEGdrFVIII-Fc fusion protein, as compared with the binding activity of 5.16X10-4M for the molecule of a drFVIII-Fc fusion protein. In conclusion, we are able to generate a PEGylated form of a drFVIII-Fc molecule with the relevant specific activity and has been shown the molecule with the prolonger half-life in the in vivo tests. The further biochemical analysis demonstrated PEGdrFVIII-Fc fusion protein with no detectable vWF binding activity, which might explain why its half-life is longer than vWF's ~15hours half-life in vivo. This molecule is likely to be used as a once-weekly treatment option for hemophilia A patients. Currently, we are in the development stage of an IND filing in China. Disclosures No relevant conflicts of interest to declare.

A Novel Deletion in the Fviii B-Domain That Reduces Transgene Size While Preserving FVIII Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3182-3182
Author(s):  
Yi-Lin Liu ◽  
Hua Zhu ◽  
Alexander Schlachterman ◽  
Heesoon Chang ◽  
Rodney M. Camire ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Dose Group ◽  
Hemophilia A ◽  
Specific Activity ◽  
Post Injection ◽  
Vector Genome ◽  
Fviii Activity

Abstract Hemophilia A is an inherited X-linked bleeding disorder caused by a deficiency in Factor VIII (FVIII). Clinically significant improvement of hemophilia phenotype can be achieved with low circulating factors, thus makes it a good target disease for gene therapy. Adeno-associated virus (AAV) vectors have proven successful for the delivery of the factor IX gene in humans with hemophilia B. For the treatment of hemophilia A, a problem in the packaging of the rFVIII cDNA or various B-domainless derivatives (i.e. rFVIII-SQ) in AAV vectors is the large size of the insert, which combined with required elements, can exceed the packaging capacity of AAV (~5 kb). This difficulty limits the choice of both promoter and regulatory elements when designing an expression cassette for AAV vectors. Here we developed strategies to overcome these limitations by (1) development of a novel FVIII B-domain deleted molecule (2) construction of a short liver-specific promoter. We further tested these vectors in a series of in vitro and in vivo experiments. Factor VIII-SQ is a well-characterized derivative of FVIII and has been used by several groups in a gene therapy setting; the recombinant protein is used clinically to treat hemophilia A. We have constructed a shorter version of FVIII-SQ, by deleting the entire B-domain. In addition, we have engineered this FVIII to be intracellularly processed using a PACE-furin recognition site such that the protein is secreted from cells as two chains (FVIII-RKR; fully processed heavy and light chains). This FVIII-RKR along with FVIII-SQ was transiently expressed in COS-1 cells and conditioned media was collected at 24, 48 and 72 hrs post transfection. Using a combination of ELISA and functional assays we were able to demonstrate that FVIII-RKR was efficiently secreted from these cells. The data also revealed that FVIII-RKR has a 4–8-fold increase in specific activity compared to FVIII-SQ. We further tested whether FVIII-RKR could function in an in vivo setting. Plasmid DNA (50μg) containing FVIII-RKR or FVIII-SQ with liver-specific mouse transthyretin (mTTR) promoter were introduced into hemophilia A (HA) mice hydrodynamically via tail vein. Two out of four mice in the SQ group and three out of four mice in the RKR group had significant shortening of the clotting time at days 1 and 3 post injection, indicating that this shortened version of FVIII is functional in vivo. To address FVIII long-term expression we synthesized AAV vectors and delivered to immuno-deficient HA mice through hepatic portal vein. AAV vectors containing an expression cassette of mTTR promoter and FVIII-SQ have been administered. Expression of physiological FVIII levels was observed in high dose group (4.0E+12 vector genome per animal, n=4). FVIII activity averages 1.88 U/ml by Coamatic assay or 0.81 U/ml by aPTT assay at 12 weeks post injection. In low dose group (1.0E+12 vector genome per animal, n=5) therapeutic level of FVIII is achieved, 0.59 U/ml by Coamatic assay or 0.23 U/ml by aPTT assay at 12 weeks post injection. Finally, AAV vectors with FVIII-RKR have been produced and shown to have similar packaging efficiency to AAV-FVIII-SQ. Studies are currently underway with AAV-FVIII-RKR to evaluate the ability of this vector to drive long-term expression of functional protein. In summary, we developed a novel FVIII molecule that has high specific activity and is suitable for efficiently packaging in the AAV vectors.

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