Treatment of Hemophilia A with a Highly Purified Factor VIII Concentrate Prepared by Anti-FVIIIc Immunoaffinity Chromatography

1992 ◽  
Vol 67 (01) ◽  
pp. 019-027 ◽  
Author(s):  
Joseph E Addiego ◽  
Edward Gomperts ◽  
Liu Shu-Len ◽  
Patricia Bailey ◽  
Suzanne G Courter ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Virus Transmission ◽  
Human Albumin ◽  
Immunoaffinity Chromatography ◽  
Exchange Chromatography ◽  
Pharmacokinetic Studies ◽  
Enveloped Viruses ◽  
Clinically Significant ◽  
Detergent Mixture

SummaryTo reduce the risk of pathogenic virus transmission associated with the therapeutic administration of plasma-derived antihemophilic factor (FVIIIc), a process utilizing anti-FVIIIc immunoaffinity chromatography to isolate FVIIIc has been developed. In addition, the starting cryoprecipitate solution has been treated with an organic solvent/detergent mixture to inactivate lipid-enveloped viruses. A final ion exchange chromatography step is used to further remove contaminants, e.g., anti-FVIIIc antibody, potentially leached with FVIIIc during the immunoaffinity step. The purified FVTII is stabilized for lyophili-zation and storage by the addition of human albumin. The monoclonal anti-FVIIIc antibody used in the immunoaffinity step of the process is not detectable in the final preparation. Viral reduction studies performed at specific steps of the process demonstrate that 11 logs of human immunodeficiency virus (HIV) and greater than 4-5 logs of other lipid-enveloped viruses are inactivated within the first 30 s of exposure to the solvent/ detergent mixture and 4-5 logs of various model viruses, e. g. Endomyocarditis virus (EMC), are physically removed during washing of the immunoaffinity column. The lyophilized product is reconstituted using sterile water in a matter of seconds.The pharmacokinetics of Hemofil® M were compared to those obtained using a standard heat-treated concentrate (Hemofil® CT) in five severe factor VIII deficient hemophiliacs in a randomized, cross-over study. No statistically significant differences were observed in mean half life (p >0.6) or median recovery (p = 0.4) between the two preparations. No clinically significant adverse effects were observed in patients receiving either FVIII preparation.In addition, 43 patients at 18 different centers underwent pharmacokinetic studies, with a nominal dose of 50 u/kg FVIIIc Hemofil® M. The mean recovery was 103.6%, and the t 1/2 was 14.6 h. The recovery of FVIII in this group was as expected, providing an increase of assayed FVIII of approximately 2% per unit of FVTII/kg infused.Clinical trials using Hemofil® M have been initiated in 124 hemophilia A patients. The safety and efficacy of Hemofil® M has been established. To date, 0 of 60 patients tested have seroconverted to HIV. None of the previously untreated patients show clinical or laboratory evidence of Non-A, Non-B hepatitis (NANB), with 21 patients remaining negative as far as presence of antibodies to the Hepatitis C virus (a-HCV negative) at least 6 months after the initial infusion. There is no evidence of neoantigenicity, evidenced by seroconversion to murine antibody. An 8.7% (2 of 23) prevalence of anti-FVIIIc inhibitor development has been observed in previously untreated patients with FVIIIc⩽3%, receiving only the monoclonally purified solvent/ detergent treated FVIII concentrate while on study and on poststudy surveillance. All patients demonstrated clinical hemostasis following product use for either on demand bleeding or surgical prophylaxis.

PREPARATION OF HIGH SPECIFIC ACTIVITY PLASMA AHF BY ANTI-FVIIIc IMMUNOAFFINITY CHROMATOGRAPHY

1987 ◽  
Author(s):  
M GRIFFITH ◽  
S A LIU ◽  
G NESLUND ◽  
I TSANG ◽  
D LETTELIER ◽  
...  
Keyword(s):  
Organic Solvent ◽  
Specific Activity ◽  
Virus Titer ◽  
High Specific Activity ◽  
Immunoaffinity Chromatography ◽  
Exchange Chromatography ◽  
Experimental Conditions ◽  
Assay Sensitivity ◽  
Enveloped Viruses ◽  
Detergent Mixture

To reduce the risk of pathogenic virus transmission associated with the therapeutic administration of plasma derived AHF (FVIIIc) we have developed a process wherein anti-FVIIIc immunoaffinity chromatography is used to isolate FVIIIc from a cryoprecipitate solution which has been treated with an organic solvent/detergent mixture to inactivate 1-ipid-enveloped viruses. A final ion exchange chromatography step is used to further remove contaminants (eg. anti-FVIIIc antibody) eluted with FVIIIc during the immunoaffinity step. FVIIIc obtained in the process has a specific activity of 1500 to 2000 AHF units (one stage clotting assay) per mg of protein, representing a ≥120,000-fold purification from plasma. The purified FVIIIc is stabilized for lyophilization and storage by the addition of human albumin. Polyclonal anti-FVIIIc immunoblots reveal polypeptides with apparent mol wts between 90,000 and 230,000 (heavy chain) and 70,000 to 76,000 (light chain). The major လcontaminant” in the preparation is von Willebrand Factor, one mol (Mr = 250,000 subunit) per mol of FVIIIc. Fibrinogen and fibronectin are barely detectable in the final preparation at levels of 1.0 and 0.3 ng per AHF unit respectively, representing a 3 million-fold purification of FVIIIc relative to these proteins. Anti-FVIIIc antibody, used in the immunoaffinity step of the process, is not detectable in the preparation at levels of 2.0 ng/mL (i.e. ≤0.01 ng per AHF unit). The extent to which virus reduction is associated with the process was also evaluated. The addition of an organic solvent/detergent mixture to cryoprecipitate solution resulted in HIV (and other lipid-enveloped viruses) inactivation to levels below the level of assay sensitivity in <5 min; representing a measurable 4 to 5 log reduction in virus titer. In addition, substantial (nonenveloped and lipid-enveloped) virus reduction was associated with the immunoaffinity step used in the process (3,400 to 50,000-fold depending on the virus/experimental conditions studie). The overall process results in a high specific activity AHF preparation which also appears to be substantially improved over previous AHF preparations with respect to viral contamination.

The Prevalence of Anti-Factor VIII Antibodies in the Hemophilia A Population of the Quebec Province of Canada: Results of Testing with the Bethesda/Nijmegen Assay and with Three ELISA Assays.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1308-1308
Author(s):  
Anne-Marie Vincent ◽  
David Lillicrap ◽  
Angie Tuttle ◽  
Laurence Dedeken ◽  
Christine Demers ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Pharmacokinetic Studies ◽  
Severe Hemophilia ◽  
Elisa Plate ◽  
Blood Specimens ◽  
Highly Correlated ◽  
Recombinant Fviii ◽  
Negative Controls

Abstract Abstract 1308 Poster Board I-330 Introduction The development of anti-Factor VIII antibodies (FVIII Abs) is the most serious complication of the treatment of hemophilia A. The prevalence and incidence of FVIII Abs is usually assessed by the functional Bethesda/Nijmegen assay. We hypothesized that some FVIII Abs might not be detected by the Bethesda/Nijmegen assay but would be apparent in an ELISA assay. The FVIII Ab ELISA may be more sensitive or it may be detecting Abs against both functional and non-functional epitopes of FVIII. Subjects and Methods In the Province of Quebec (population 7.8 × 106), all subjects with hemophilia must be registered with one of the four designated hemophilia centers to be allowed to be treated with FVIII concentrates. Over the last year, blood specimens were obtained from all registered severe hemophilia A subjects to be tested for FVIII Abs. Citrated plasma specimens obtained more than 48 hours after the last FVIII treatment were tested with the Bethesda/Nijmegen assay and three described ELISAs (Haemophilia 2009;15:374-6) using as the coating antigen two different full-length recombinant FVIII (FLRFVIII) concentrates, Helixate® FS and Advate® respectively, and a B domain-deleted recombinant FVIII (BDDRFVIII) concentrate, Xyntha®. Six normal plasmas were used as negative controls on each ELISA plate. Mean and standard deviation (SD) of absorbance were calculated for the total of all the plates used for each of the three coating antigens. Results were considered positive with Bethesda unit (BU) ≥ 0.4 /mL and ELISA absorbance ≥ 3 standard deviations (SD) of the mean of the normal plasmas. Results At time of writing this abstract ≥ 80% of the target subjects have been tested and the remaining are being tested. Twelve out of 114 (10.5 %) are positive with the Bethesda/Nijmegen assay. Eleven out of these 12 are ELISA positive. Ten out of the 102 Bethesda negative subjects (9.8 %) are positive for the FLRFVIII ELISAs, all of them being negative for the BDDRFVIII ELISA. The titre of FVIII Abs measured by the Bethesda assay was highly correlated (R=0.93) with the titre measured with the three ELISAs. Conclusion Our observed prevalence (10.5%) of Bethesda positive subjects is comparable with values reported in similar unselected severe hemophilia A populations. There is no published literature with which to compare our observed prevalence of 9.8% of ELISA positive amongst Bethesda negative subjects. Bethesda negative plasmas that are positive for the FLRFVIII ELISAs and negative for the BDDRFVIII ELISA are presumed to have FVIII Abs directed against the B domain of the FLRFVIII concentrates. The clinical significance of this observation is presently unknown but is being investigated with pharmacokinetic studies. Disclosures No relevant conflicts of interest to declare.

Purification and characterization of factor VIII 1,689-Cys: a nonfunctional cofactor occurring in a patient with severe hemophilia A

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2117-2122
Author(s):  
DP O'Brien ◽  
EG Tuddenham
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Heavy Chains ◽  
Thrombin Cleavage ◽  
Before And After ◽  
Functional Defect ◽  
Thrombin Activation

We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after thrombin activation by Western blotting with monoclonal antibodies (MoAbs). Normal or patient citrated plasma was ultracentrifuged, cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void volume fractions were reduced and subjected to ion exchange chromatography yielding material of specific activity approximately 1,000 U/mg protein (VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is fully activatable by thrombin with proteolytic fragmentation as previously described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII 1,689-Cys has the normal distribution of factor VIII light and heavy chains prior to thrombin activation. After exposure to thrombin the heavy chain polypeptides were fully proteolysed but the light chain was totally resistant to cleavage. This is consistent with the demonstration in the patient's leucocyte DNA of a C to T transition in codon 1,689 converting Arg to Cys at the light chain thrombin cleavage site as previously described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light chain of Factor VIII 1,689-Cys is not released from von Willebrand factor (vWF) by thrombin, but this is not the sole cause of the functional defect since the protein purified free of vWF has no coagulant activity. We conclude that the functional defect in factor VIII 1,689-Cys is a consequence of failure to release the acidic peptide from the light chain upon thrombin activation.

scholarly journals МЕТОДОЛОГІЧНІ ПІДХОДИ ОДЕРЖАННЯ ФАКТОРА VIII ЗСІДАННЯ КРОВІ

2020 ◽  
Vol 79 (1-2) ◽  
pp. 102-108
Author(s):  
N. O. Shurko ◽  
V. L. Novak
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Coagulation Factor ◽  
Coagulation Factors ◽  
Modern Technology ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Protein Therapy ◽  
Biotechnological Approach

The article deals with basic methods used by modern technology to obtain coagulation factor VIII (FVIII). The blood plasma fractionation remains the only biotechnological approach to make life-saving protein therapy to treat human diseases. The biological medicines from human plasma play a vital role in the treatment of patients with different diseases. These products include a range of coagulation factors (FVIII, FIX, the prothrombin complex, Von Willebrand factor, fibrinogen etc.), immunoglobulins, protease inhibitors, anticoagulants and albumin. Four plasma proteins are commercially important for production: albumin, IgG, factor VIII, and factor IX. VIII is a coagulation factor in the blood, which is missing or defective in patients with Hemophilia A. Replacement therapy with FVIII concentrates constitutes the basis for hemophilia care. Cryoprecipitate was described in the mid 60's of the XX century as a first concentrate of antihemophilic FVIII.The main indications for the clinical use of cryoprecipitate were hypofibrinogenemia or disfibrinogenemia. Previously, cryoprecipitate was used for treatment of hemophilia A and von Willebrand’s disease. Traditional FVIII production methods included deposition steps, which were aimed at elimination of protein impurities such as fibrinogen, fibronectin and immunoglobulins. These technologies could use the combination of methods at low temperatures or the addition of protein precipitating substances (PEG, polyvinylpyrrolidone, dextran, ficol, percol etc.). Using chromatographic methods in FVIII production technology allowed receiving high purity and specific activity concentrate of FVIII. Ion exchange chromatography techniques are often used in order to isolate coagulation FVIII. These techniques include methods of affinity chromatography as well as the use of monoclonal antibodies to bind of FVIII. Nowadays, production of plasma concentrate of FVIII is used in combination with different chromatographic techniques.

scholarly journals Pharmacokinetic Studies of Factor VIII in Chinese Boys with Severe Hemophilia A

2018 ◽  
Vol 131 (15) ◽  
pp. 1780-1785 ◽  
Author(s):  
Zhen-Ping Chen ◽  
Pei-Jing Li ◽  
Gang Li ◽  
Ling Tang ◽  
Ying-Zi Zhen ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Pharmacokinetic Studies ◽  
Severe Hemophilia

Rational design of a fully active, long-acting PEGylated factor VIII for hemophilia A treatment

Blood ◽  
2010 ◽  
Vol 116 (2) ◽  
pp. 270-279 ◽  
Author(s):  
Baisong Mei ◽  
Clark Pan ◽  
Haiyan Jiang ◽  
Hendri Tjandra ◽  
Jonathan Strauss ◽  
...  
Keyword(s):  
Factor Viii ◽  
Rational Design ◽  
Hemophilia A ◽  
Treatment Options ◽  
Pharmacokinetic Studies ◽  
Functional Assay ◽  
Coagulation Activity ◽  
Long Acting

Abstract A long-acting factor VIII (FVIII) as a replacement therapy for hemophilia A would significantly improve treatment options for patients with hemophilia A. To develop a FVIII with an extended circulating half-life, but without a reduction in activity, we have engineered 23 FVIII variants with introduced surface-exposed cysteines to which a polyethylene glycol (PEG) polymer was specifically conjugated. Screening of variant expression level, PEGylation yield, and functional assay identified several conjugates retaining full in vitro coagulation activity and von Willebrand factor (VWF) binding.PEGylated FVIII variants exhibited improved pharmacokinetics in hemophilic mice and rabbits. In addition, pharmacokinetic studies in VWF knockout mice indicated that larger molecular weight PEG may substitute for VWF in protecting PEGylated FVIII from clearance in vivo. In bleeding models of hemophilic mice, PEGylated FVIII not only exhibited prolonged efficacy that is consistent with the improved pharmacokinetics but also showed efficacy in stopping acute bleeds comparable with that of unmodified rFVIII. In summary site-specifically PEGylated FVIII has the potential to be a long-acting prophylactic treatment while being fully efficacious for on-demand treatment for patients with hemophilia A.

Sensitization of CD4+ T Cells to Coagulation Factor VIII: Response in Congenital and Acquired Hemophilia Patients and in Healthy Subjects

2000 ◽  
Vol 84 (10) ◽  
pp. 643-652 ◽  
Author(s):  
Mark Reding ◽  
Huiyun Wu ◽  
Mark Krampf ◽  
David Okita ◽  
Brenda Diethelm-Okita ◽  
...  
Keyword(s):  
T Cells ◽  
Factor Viii ◽  
Cd4 T Cells ◽  
Healthy Subjects ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
Cd4 Cells ◽  
Acquired Hemophilia ◽  
Clinically Significant ◽  
Inhibit Factor

SummaryAntibodies (Ab) that inhibit factor VIII (fVIII) may develop in patients with hemophilia A and rarely in individuals without congenital fVIII deficiency (acquired hemophilia). Synthesis of fVIII inhibitors requires CD4+ T cells. We investigated the proliferative response of blood CD4+ cells from 11 patients with congenital or acquired hemophilia and 12 healthy subjects, to recombinant human fVIII, and to pools of overlapping synthetic peptides spanning the sequences of individual fVIII domains. All patients had CD4+ cells that responded to fVIII. The intensity of the responses fluctuated over time: several patients had brief periods when they did not respond to fVIII. All healthy subjects had transient CD4+ responses to fVIII, that were significantly lower than those of hemophilia patients. Also, healthy subjects responded to fVIII less frequently and for shorter periods than hemophilia patients. All patients and healthy subjects recognized several fVIII domains: the A3 domain was recognized most strongly and frequently. The transient sensitization of CD4+ cells to fVIII in healthy subjects suggests that inadequate tolerization of CD4+ cells to fVIII, due to lack of endogenous fVIII, is an important factor in the development of clinically significant anti-fVIII antibodies in hemophilia A.

Novel and Clinically Significant Factors Influencing the Pharmacokinetic Variability of Recombinant Factor VIII (Kogenate-FS) in Children.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3991-3991 ◽  
Author(s):  
Chris Barnes ◽  
David Lillicrap ◽  
Victor Blanchette ◽  
Ann Marie Stain ◽  
Janneth Pazmino-Canizares ◽  
...  
Keyword(s):  
Factor Viii ◽  
Half Life ◽  
Clotting Factor ◽  
Pharmacokinetic Studies ◽  
Pharmacokinetic Data ◽  
Limited Information ◽  
Pharmacokinetic Variability ◽  
Number Of Patients ◽  
The Mean ◽  
Clinically Significant

Abstract Understanding the pharmacokinetics of factor VIII (FVIII) is critical to maximise the clinical benefit of clotting factor replacement therapy. Little is known about the pharmacokinetics of recombinant FVIII (rFVIII) in children and current dosing regimes, largely based on adult pharmacokinetic data, may be inappropriate. Further, there is limited information about factors determing the pharmacokinetic variability of FVIII between children. We performed a pharmacokinetic study in 20 children with severe haemophilia A (mean age 12.8 years) following a bolus of 50 U/kg of rFVIII (Kogenate-FS, Bayer Health Care). FVIII:C levels were assessed at 10 time points to determine the effect of patient size, age and pre infusion vWF:Ag level on FVIII pharmacokinetics. The patients had no history of inhibitors to FVIII (>0.5 BU). The mean incremental FVIII recovery was 1.87 (U/ml)/(U/kg) (range 1.25– 2.76) which is significantly lower than adult pharmacokinetic studies. (Table 1) FVIII recovery showed a greater correlation with body surface area (BSA, Spearman correlation p = 0.037) than to weight (p = 0.048) suggesting dosing based on BSA may lead to more predictable FVIII recovery in children. The mean half-life of FVIII was 10.7 hours (range 7.8 to 15.3) and is reduced in comparison to adult studies. FVIII half-life showed a positive correlation with levels of vWF:Ag (p = 0.0001) (Figure 1) and half life was reduced in six patients with FVIII inhibitor titres between 0.05–0.15 Nijmegen modified BU (p = 0.06). The correlation of rFVIII half-life with vWF:Ag levels may provide a basis for pharmacological augmentation of vWF:Ag levels to prolong rFVIII half life and may be of major benefit in countries with a limited supply rFVIII. The finding that FVIII half-life is reduced in patients with inhibitor titres that have previously been regarded as clinically insignificant suggests that standards and methodologies for the measurement of inhibitors should be re-evaluated. This study shows that the pharmacokinetics of rFVIII in children are unique and that BSA, pre infusion levels of vWF and the presence of low, previously considered non-clinically significant inhibitors may affect the pharmacokinetics of Kogenate-FS in children. These relationships, and the potential to improve dosing schedules for FVIII in children, need further study. Comparison of results from our centre with other published pharmacokinetic studies of rFVIII. Reference Number of patients Mean age (range) (Years) Mean incremental recovery (U/ml)/(U/kg) Mean half life (hours) Mean clearance (ml/hr/kg) Mean volume of distribution steady state (ml/kg) ¥ Results represent pooled data of one rFVIII product prepared at two different sites assessed by one stage assay. Only week 1 of serial pharmacokinetic studies is presented. Barnes (2004) 20 12.8 (4.4 – 18.1) 1.9 10.7 4.1 59.2 Lee (1999)¥ 30 N/A 2.5 12.7 2.0 62.6 Fijnvandraat (1997) 12 34.0 (17 – 64) 2.4 11.3 3.2 44.8 Harrison (1991) 14 34.1 (23 – 72) 2.8 16.5 2.4 51.2 Schwartz (1990) 17 N/A 2.7 15.8 2.5 50.5 Figure Figure

scholarly journals Purification and characterization of factor VIII 1,689-Cys: a nonfunctional cofactor occurring in a patient with severe hemophilia A

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2117-2122 ◽  
Author(s):  
DP O'Brien ◽  
EG Tuddenham
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Heavy Chains ◽  
Thrombin Cleavage ◽  
Before And After ◽  
Functional Defect ◽  
Thrombin Activation

Abstract We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after thrombin activation by Western blotting with monoclonal antibodies (MoAbs). Normal or patient citrated plasma was ultracentrifuged, cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void volume fractions were reduced and subjected to ion exchange chromatography yielding material of specific activity approximately 1,000 U/mg protein (VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is fully activatable by thrombin with proteolytic fragmentation as previously described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII 1,689-Cys has the normal distribution of factor VIII light and heavy chains prior to thrombin activation. After exposure to thrombin the heavy chain polypeptides were fully proteolysed but the light chain was totally resistant to cleavage. This is consistent with the demonstration in the patient's leucocyte DNA of a C to T transition in codon 1,689 converting Arg to Cys at the light chain thrombin cleavage site as previously described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light chain of Factor VIII 1,689-Cys is not released from von Willebrand factor (vWF) by thrombin, but this is not the sole cause of the functional defect since the protein purified free of vWF has no coagulant activity. We conclude that the functional defect in factor VIII 1,689-Cys is a consequence of failure to release the acidic peptide from the light chain upon thrombin activation.

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