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Method of preparing a high-purity, virus safe, biologically active transferrin preparation
| Details |
Inventors: Bethke, Ulf; Kothe, Norbert; Rudnick, Dieter; Moller, Wolfgang; Kloft, Michael;
Assignee: Biotest Pharma GmbH (Dreieich, DE)
Primary Examiner: Rosen; Sam
Assistant Examiner:
Attorney, Agent or Firm: Sprung Horn Kramer & Woods
To provide a simple method of preparing biologically active transferrin that can be employed on an industrial scale and that will result in extremely pure and natural transferrin containing no viruses and appropriate for both in-vitro and in-vivo applications, the .gamma.-globulins are precipitated from the fraction containing the transferrin, the precipitant is removed from the residual liquid by ultrafiltration or gel filtration, the liquid is adjusted to a prescribed ionic concentration and protein concentration and (a) treated with .beta.-propiolactone and the solution is subjected to ultraviolet radiation or (b) treated with specific detergents and subjected to ion-exchange chromatography, and the transferrin is concentrated and filtered sterile. |
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DETAILED DESCRIPTION We claim: 1.
A method of preparing a high-purity, virus-safe biologically active, transferrin preparation from plasma fractions that contain transferrin, comprising a) removing the .
gamma.
-globulins from the fraction that contains the transferrin by precipitation, b) removing the precipitant from the residual liquid by ultrafiltration or gel filtration, c) adjusting the liquid to an ionic concentration of 0.
005 to 0.
5 M and to a protein concentration of 10 to 60 g/l and d) treating the liquid with (i) 0.
01 to 0.
5% .
beta.
-propiolactone at a pH of 6.
0 to 9.
0, adjusting the protein concentration to 2 to 25 g/l and then subjecting the solution to ultraviolet radiation at 254 nm, or (ii) 0.
05 to 5% tri-n-butyl phosphate and 0.
03 to 0.
4% sodium cholate or 0.
1 to 5% polyoxyethylenesorbitanmonooleate, e) subjecting the solution to ion-exchange chromatography, with an ionic strength that ensures that all the proteins except the transferrin are adsorbed, and f) concentrating and sterile filtering the transferrin.
2.
The method according to claim 1, wherein the precipitation is effected with ammonium sulfate or ethanol, thereby to remove the .
alpha.
-globulins.
3.
The method according to claim 1, wherein step (e) is anion-exchange chromatography.
4.
The method according to claim 1, wherein the chromatography is effected with an ionic concentration of 0.
02 to 0.
12 M at a pH of 6.
0 to 8.
0 that ensures that all the proteins except transferrin are adsorbed.
5.
The method according to claim 4, wherein the precipitation is effected with ammonium sulfate or ethanol, thereby to remove the .
alpha.
-globulins.
6.
The method according to claim 1, wherein between steps e) and f) a complexing agent is added to bind bound iron, followed by ultrafiltration or gel filtration to
Description:
The invention relates to a method of preparing a high-purity, virus-safe, biologically active, transferrin preparation from plasma fractions that contain transferrin.
Transferrin is the protein that transports iron in human and animal plasma, in which its concentration is approximately 2
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