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Purification of human serum albumin

A technology of human serum albumin and serum albumin, which is applied in the field of purification of human serum albumin and can solve problems such as feasibility limitations

Inactive Publication Date: 2004-09-01
TAURUS HSA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, although the recombinant production of hSA in transgenic animals has the potential to generate large quantities of pure hSA and artificial blood, its feasibility is limited by economic factors

Method used

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  • Purification of human serum albumin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1: Purification of hSA from transgenic bovine milk

[0071] Starting from lipid-free (skim) transgenic milk containing recombinant hSA product, the product stream was acidified with acetic acid (10-15%) to pH 4.2 to 4.8 to precipitate casein. Precipitated casein was removed by tangential flow microfiltration using a standard TFF system with filter cartridges comprising Ceramem 0.2 [mu]m ceramic blocks or A / G Technologies 750K cut-off hollow fibers.

[0072] The product was purified by affinity chromatography using Prometic Biosciences Blue SA column. The loading / washing buffer conditions contained NaCl at pH 8-9 at 50-250 mM ionic strength and a low concentration of Polysorbate 20 (Tween 20). The product was eluted with 20-50 mM phosphate buffer containing 10-30 mM octanoate pH 5.5-6.5.

[0073] Product crystallization was carried out in a batch reaction box, and 4M phosphate (4M NaH 2 PO 4 and 4M K 2 HPO 4 70:30 mixture by volume) and 1 to 3 mM caprylat...

Embodiment 2

[0081] Example 2: Purification of hSA using two different affinity columns

[0082] Starting from lipid-free (skim) transgenic milk containing recombinant hSA product, samples were clarified by acid precipitation of casein as described in Example 1. Dye ligand affinity chromatography was then performed as previously described.

[0083] Finally, ABD protein ligand chromatography was performed on the eluate from the dye ligand affinity purification. The ABD column equilibration and wash buffer used contained 10-50 mM acetic acid at pH 5.0-6.0 and 100-150 mM NaCl. The pH of the hSA samples was adjusted to 5.0-6.0 by adding enoic acid before loading onto the ABD column. hSA was eluted using 25-100 mM glycine buffer pH 2.5. The purification results are shown in Table 2.

[0084] Form 2

[0085] Process Representative Step hSA Yield ppm bSA ppm IgG

[0086]Clarified sample stream 99% 2×10 4 1×10 4

[0087] Dye ligand chromatography 95% 300 30

[0088] ABD protein lig...

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Abstract

The invention features methods of purifying human serum albumin (hSA) from endogenous serum albumin of the host cell producing the hSA. The methods include providing a sample comprising hSA and serum albumin of the host cell, applying the sample to an affinity column that binds hSA at a higher affinity than the serum albumin of the host cell, eluting bound hSA from the affinity column, and crystallizing the eluted has. The invention also features compositions comprising hSA produced by the methods of the invention.

Description

[0001] related application [0002] This application claims priority to USSN 60 / 297,884, filed June 13,2001. This patent is incorporated herein by reference. Background of the invention [0003] In the blood, serum albumin is one of the most abundant proteins. In the blood they act as carriers for hydrophobic molecules such as fatty acids and act as clarifiers by binding to organic molecules to sequester them until they can be removed. Due to their abundance in blood, serum albumins are a major determinant of the properties of blood, especially serum. [0004] During World War II, it was realized that hSA (human serum albumin) could be formulated into a physiologically applicable solution to make a substitute for human blood (artificial blood) to compensate for blood loss caused by trauma or surgery. See Cohn et al. (1946), J. Amer. Chem. Soc. 68:459-75. In fact, this artificial blood is an ideal blood substitute in most cases because the body can quickly replenish lost bl...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K35/14A61K38/00A61P7/08C07K1/22C07K1/30C07K14/765C12P21/02
CPCC07K14/765A61K38/00A61P7/08
Inventor S·富尔顿
Owner TAURUS HSA