Production of highly purified clotting factor IX by a combination of different chromatographic methods

Hoffer, Lutz; Schwinn, Horst; Josić, Djuro

doi:10.1016/s0021-9673(99)00348-9

Cited by 35 publications

(30 citation statements)

References 18 publications

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“…The molecular weight of this protein is between 55.000 and 65.000 Da and its concentration in human plasma is about 5µg/mL (0.1µmol) in healthy individuals (Hoffer et al, 1999). The absence or a significant deficiency of hFIX causes hemophilia B, an X-linked recessive disorder of blood coagulation, also known as Christmas disease.…”

Section: Introductionmentioning

confidence: 99%

Production of specific polyclonal antibodies anti-human factor IX

Chaves

Rodrigues

Ferreira

et al. 2006

Braz. arch. biol. technol.

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Section: Introductionmentioning

confidence: 99%

Production of specific polyclonal antibodies anti-human factor IX

Chaves

Rodrigues

Ferreira

et al. 2006

Braz. arch. biol. technol.

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“…Q-Sepharose XL resin resulted in poorer FVIII activity recovery in the eluate fraction and poorer purification factor. It has been described that Q-Sepharose XL may activate coagulation factors (Hoffer et al 1999), which could be contributing to the low FVIII activity recovered in our experiments.…”

Section: Resultsmentioning

confidence: 66%

Purification of coagulation factor VIII using chromatographic methods. Direct chromatography of plasma in anion exchange resins

et al. 2010

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Coagulation factor VIII (FVIII) concentrates are used in the treatment of patients with Hemophilia A. Human FVIII was purified directly from plasma using anion exchange chromatography followed by gel filtration. Three Q-Sepharose resins were tested, resulting in 40% recovery of FVIII activity using Q-Sepharose XL resin, about 80% using Q-Sepharose Fast Flow and 70% using the Q-Sepharose Big Beads. The vitamin K-dependent coagulation factors co-eluted with FVIII from the anion exchange columns. In the second step of purification, when Sepharose 6FF was used, 70% of FVIII activity was recovered free from vitamin K-dependent factors.

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“…A standard curve of the hFIX protein present in normal human plasma was utilised to interpret the clotting activity. When compared to normal human clotting activity (that of adults with a FIX concentration of 5 lg ml -1 of blood), which normally reach 100% when measured with the aPTT method (Hoffer et al 1999;Acar et al 2006), the coagulation rates of 1.0-1.4% observed in the soybean TSP extracts were low. However, in most cases, haemophilia B patients usually require FIX supplementations with 10% of clotting activity to provide proper coagulation rates, and a significant haemostatic effect can be achieved with the elevation of the FIX levels in the blood to only 0.8 IU dl -1 (Kay et al 2000;Goldenberg et al 2008).…”

Section: Discussionmentioning

confidence: 90%

“…A dilution series with lyophilised human plasma containing the reference concentration of FIX (1 IU ml -1 or 5 lg ml -1 ) was performed in order to interpret the clotting activity. The clotting time was associated with the relative hFIX activity to provide blood coagulation (Hoffer et al 1999;Arruda et al 2004;Zhang et al 2007) apoplast and oil bodies and an absence of detectable hFIX protein in the cell wall and starch grains (Fig. 3a).…”

Section: Discussionmentioning

confidence: 94%

Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds

et al. 2010

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The seed-based production of recombinant proteins is an efficient strategy to achieve the accumulation, correct folding, and increased stability of these recombinant proteins. Among potential plant molecular farming systems, soybean [Glycine max (L.) Merrill] is a viable option for the production of recombinant proteins due to its high protein content, known regulatory sequences, efficient gene transfer protocols, and a scalable production system under greenhouse conditions. We report here the expression and stable accumulation of human coagulation factor IX (hFIX) in transgenic soybean seeds. A biolistic process was utilised to co-introduce a plasmid carrying the hFIX gene under the transcriptional control of the α' subunit of a β-conglycinin seed-specific promoter and an α-Coixin signal peptide in soybean embryonic axes from mature seeds. The 56-kDa hFIX protein was expressed in the transgenic seeds at levels of up to 0.23% (0.8 g kg(-1) seed) of the total soluble seed protein as determined by an enzyme-linked immunosorbent assay (ELISA) and western blot. Ultrastructural immunocytochemistry assays indicated that the recombinant hFIX in seed cotyledonary cells was efficiently directed to protein storage vacuoles. Mass spectrometry characterisation confirmed the presence of the hFIX recombinant protein sequence. Protein extracts from transgenic seeds showed a blood-clotting activity of up to 1.4% of normal plasma. Our results demonstrate the correct processing and stable accumulation of functional hFIX in soybean seeds stored for 6 years under room temperature conditions (22 ± 2°C).

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Production of highly purified clotting factor IX by a combination of different chromatographic methods

Cited by 35 publications

References 18 publications

Production of specific polyclonal antibodies anti-human factor IX

Production of specific polyclonal antibodies anti-human factor IX

Purification of coagulation factor VIII using chromatographic methods. Direct chromatography of plasma in anion exchange resins

Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds

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