Use of Plasma with High Levels of lonised Calcium in the Production of Model Scale Goagulation Factor Concentrates

1990 ◽  
Vol 64 (03) ◽  
pp. 374-378 ◽  
Author(s):  
A Farrugia ◽  
S Douglas ◽  
J James ◽  
G Whyte ◽  
R Herrington
Keyword(s):  
Factor Viii ◽  
Large Scale ◽  
Coagulation Factor ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Blood Collection ◽  
Half Strength ◽  
Factor Viii Concentrates ◽  
Coagulation Factor Concentrates ◽  
Thrombogenic Potential

SummaryWe have attempted to exploit the Ca2+ -dependent stability of factor VIII in producing factor VIII concentrates of higher yield. Plasma levels of ionised calcium were increased in two ways: (a) whole blood collection into half-strength citrate CPD anticoagulant, leading to free Ca2+ levels of ca 120 µM and (b) apheresis collection of plasma which was then recalcified to free Ca2+ levels of ca 300 µM under heparin cover. Coagulation factor concentrates were prepared using model versions of our industrial scale manufacturing methods. Factor VIII yield was increased through low citrate collection. This did not compromise factor IX yield or thrombogenic potential. Use of recalcified heparinised plasma did not lead to any improvement in factor VIII yield and resulted in a marked drop in factor IX recovery, possibly from interference by heparin of factor IX binding in ion-exchange chromatography. The benefits accruable through the use of half-strength citrate CPD anticoagulant support the continued evaluation of this preservative in large scale blood collection and fractionation. The deleterious effects of heparin in charge-mediated plasma fractionations may pose serious difficulties in harvesting vitamin K dependent factors.

F VIII Inhibitors in Haemophilia A Patients after Continuous Infusion of Factor VIII Concentrates.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3100-3100
Author(s):  
Ch. von Auer ◽  
J. Oldenburg ◽  
M. von Depka ◽  
C. Escuriola-Ettinghausen ◽  
W. Kreuz ◽  
...  
Keyword(s):  
Factor Viii ◽  
Continuous Infusion ◽  
Orthopaedic Surgery ◽  
Coagulation Factor ◽  
Haemophilia A ◽  
Missense Mutations ◽  
Factor Viii Concentrates ◽  
Coagulation Factor Concentrates ◽  
Major Orthopaedic Surgery ◽  
Mild Haemophilia

Abstract Continuous infusion (CI) of coagulation factor concentrates has been used since the early 1990s. Recent reports of the occurrence of an inhibitor (inh) after CI have raised concerns about this method of treatment. We conduct a retrospective study to investigate the development of inh after CI of FVIII concentrates in Germany. 99 haemophilia treating physicians in Germany were contacted and asked to answer a questionnaire. So far data of 13 departments have been reported and analyzed. Three of these 13 centers never conducted a CI, in 5 no inh were detected and in 5 haemophilia centers 10 patients with inh development after CI were registered. 5 of these patients were suffering from severe, 1 from moderate and 4 from mild haemophilia (age between 7 months and 57 years). Indications for treatment were major bleeds and surgical procedures. Plasma derived (6) and recombinant (4) factor concentrates were given in various infusion sets. Data concerning infused amount (4300 to >100000 IE), exposure days (1 to >100) and inh characteristic (alloantibodies, 3 LR, 7 HR) were collected. Regarding the genotype, we found 4 missense-mutations, 2 intron-22-inversions, 1 small deletion, 3 were unknown. In our own center we found no inh in 81 patients with major orthopaedic surgery and bolus infusion of factor VIII concentrate compared to 2 inh in 8 patients with major orthopaedic surgery and CI of FVIII. In conclusion we found only in 2 patients the typical gene mutation for inh development. Strikingly the inh developed very often in patients with mild haemophilia. These findings agree with published results. There might be an uncommon inh-pathomechanism due to CI or patients with mild haemophilia might exhibit a much higher prevalence of inhibitor development when treated with an “intensive FVIII-treatment” such as CI.

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.

Factor VIII Inhibitors in Haemophilia A Patients after Continuous Infusion of Factor VIII Concentrates.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3198-3198
Author(s):  
Charis von Auer ◽  
Johannes Oldenburg ◽  
Manuela Krause ◽  
Wolfgang Miesbach ◽  
Inge Scharrer ◽  
...  
Keyword(s):  
Factor Viii ◽  
Continuous Infusion ◽  
Coagulation Factor ◽  
Splice Site Mutation ◽  
Missense Mutations ◽  
Site Mutation ◽  
Study Results ◽  
Factor Viii Concentrates ◽  
Coagulation Factor Concentrates ◽  
Mild Haemophilia

Abstract Continuous infusion (CI) of coagulation factor concentrates has been used in haemophilia treatment since the early 1990s. Recent reports of the occurrence of an inhibitor (inh.) after CI have raised concerns about this method of treatment. We conduct a retrospective study to investigate the development of inh. After CI of factor VIII (FVIII) concentrates in Germany and report on further study results. A questionnaire was sent to 100 haemophilia treating colleagues in Germany. So far 42 colleagues have answered, in 19 haemophilia treatment centers CI was used as treatment modality, 5 of them still use it today. Altogether 200 CI were conducted in 128 patients (pat.). In 14 pat. Inh. Development was detected. 5 of these pat. Were suffering from severe, 1 from moderate and 8 from mild haemophilia, their median age was 27 years. Indications for treatment before inh. Detection were major bleeds and surgical procedures. Plasmaderived (9) and recombinant (4) factor concentrates were given in various infusion sets. Data concerning gene mutation, exposure days, inhibitor characteristic, former change of product, concomitant medication, vaccination and blood transfusion were collected. Regarding the genotype, we found 4 missense mutations, 2 intron-22-inversions, 1 small deletion, 1 splice site mutation, 6 were unknown. In our study strikingly the inh. Developed very often in pat. With mild or moderate haemophilia and genotypes that exhibit an inh. Prevalence of < 10 %. Our further data confirm the assumption that pat. With mild haemophilia might exhibit a much higher prevalence of inh. Development when treated with an “intensive FVIII - treatment” such as CI. On the other hand there might be an uncommon inh. pathomechanism in connection with CI, resulting in a peculiar group of pat. developing the inh. after this way of application. A prospective multicenter study to investigate the inhibitor development after CI should be conducted.

A Survey of the Effectiveness of Prothrombin Complex Concentrates in Controlling Hemorrhage in Patients with Hemophilia and Anti-Factor VIII Antibodies

1980 ◽  
Vol 44 (01) ◽  
pp. 039-042 ◽  
Author(s):  
Philip M Blatt ◽  
Doris Ménaché ◽  
Harold R Roberts
Keyword(s):  
Factor Viii ◽  
Ad Hoc ◽  
Hemophilia A ◽  
Working Party ◽  
International Committee ◽  
Factor Ix ◽  
Prothrombin Complex Concentrates ◽  
Factor Viii Concentrates

SummaryThe treatment of patients with hemophilia A and anti-Factor VIII antibodies is difficult. Between July 1977 and June 1978, a survey was carried out by an ad hoc working party of the subcommittee on Factor IX concentrates of the International Committee on Thrombosis and Hemostasis to assess the effectiveness of Prothrombin Complex Concentrates in controlling hemorrhage in these patients. The results are presented in this paper and, although subjective, support the view that these concentrates are not as effective in patients with inhibitors as Factor VIII concentrates are in patients without inhibitors.

scholarly journals Gene Therapy in Hemophilia: Recent Advances

2021 ◽  
Vol 22 (14) ◽  
pp. 7647
Author(s):  
E. Carlos Rodríguez-Merchán ◽  
Juan Andres De Pablo-Moreno ◽  
Antonio Liras
Keyword(s):  
Gene Therapy ◽  
Adverse Events ◽  
Factor Viii ◽  
Coagulation Factor ◽  
Cost Savings ◽  
Factor Ix ◽  
Clotting Factors ◽  
Advanced Therapies ◽  
Potential Benefits

Hemophilia is a monogenic mutational disease affecting coagulation factor VIII or factor IX genes. The palliative treatment of choice is based on the use of safe and effective recombinant clotting factors. Advanced therapies will be curative, ensuring stable and durable concentrations of the defective circulating factor. Results have so far been encouraging in terms of levels and times of expression using mainly adeno-associated vectors. However, these therapies are associated with immunogenicity and hepatotoxicity. Optimizing the vector serotypes and the transgene (variants) will boost clotting efficacy, thus increasing the viability of these protocols. It is essential that both physicians and patients be informed about the potential benefits and risks of the new therapies, and a register of gene therapy patients be kept with information of the efficacy and long-term adverse events associated with the treatments administered. In the context of hemophilia, gene therapy may result in (particularly indirect) cost savings and in a more equitable allocation of treatments. In the case of hemophilia A, further research is needed into how to effectively package the large factor VIII gene into the vector; and in the case of hemophilia B, the priority should be to optimize both the vector serotype, reducing its immunogenicity and hepatotoxicity, and the transgene, boosting its clotting efficacy so as to minimize the amount of vector administered and decrease the incidence of adverse events without compromising the efficacy of the protein expressed.

TT virus contaminates first-generation recombinant factor VIII concentrates

Blood ◽  
2001 ◽  
Vol 98 (8) ◽  
pp. 2571-2573 ◽  
Author(s):  
Alberta Azzi ◽  
Riccardo De Santis ◽  
Massimo Morfini ◽  
Krystyna Zakrzewska ◽  
Roberto Musso ◽  
...  
Keyword(s):  
Factor Viii ◽  
First Generation ◽  
Second Generation ◽  
Human Albumin ◽  
Factor Ix ◽  
Recombinant Factor Viii ◽  
Tt Virus ◽  
Recombinant Product ◽  
Before And After ◽  
Factor Viii Concentrates

Abstract Recombinant factor VIII and factor IX concentrates, human-plasma–derived albumin, and samples from previously untreated patients with hemophilia were examined for the presence of TT virus (TTV) by using polymerase chain reaction testing. Blood samples from the patients were obtained prospectively before and every 3 to 6 months after therapy was begun. TTV was detected in 23.5% of the recombinant-product lots and 55.5% of the albumin lots tested. Only first-generation factor VIII recombinant concentrates stabilized with human albumin were positive for TTV, whereas all second-generation (human protein–free) concentrates were negative for the virus. In 59% of patients treated with either first- or second-generation recombinant factor concentrates, TTV infection developed at some point after the initial infusion. Infection with TTV in these patients before and after treatment did not appear to be clinically important. Thus, first-generation recombinant factor VIII concentrates may contain TTV and the source of the viral contamination may be human albumin.

scholarly journals Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 942-952 ◽  
Author(s):  
L Zhang ◽  
A Jhingan ◽  
FJ Castellino
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Coagulation Factor ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Human Protein ◽  
Complete Disappearance ◽  
Carboxyglutamic Acid

Abstract To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.

IN VIVO STUDIES ON RED CELLS AND PLATELETS STORED IN HALFSTRENGTH CITRATE

1987 ◽  
Author(s):  
C Prewse ◽  
K Bell ◽  
B Griffin
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Red Cells ◽  
In Vivo Studies ◽  
Dramatic Improvement ◽  
Half Strength ◽  
Survival Studies ◽  
Cellular Components

We have previously shown that donation of blood into anticoagulants containing half the normal amount of citrate results in a dramatic improvement in the stability of coagulation factor VIII and has no adverse effect on the in vitro qualities of red cells or platelets during storage. To confirm the viability of stored cellular components we are now performing autologous survival studies in healthy volunteers using radiolabelled cells from red cells and platelets stored for 35 and 5 days respectively. Results to date indicate a 24 hour survival of 80% for red cells stored at a haematocrit of 0.70 for 35 days. Infusion of Ill-In oxine labelled platelets after storage for 5 days in full or half-strength citrate gave recoveries of 40% and survivals of 7 days. These encouraging results suggest use of halfstrength citrate may be a route to increasing factor VIII supply without any additional donor recruitment. Further in vitro studies have also been performed on cellular components and reveal adequate in vitro quality for half-strength citrate blood held at room temperature for 20 hours prior to component preparation.

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