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Method for the crystallization of human serum albumin

a technology crystallization method, which is applied in the field of human serum albumin crystallization method, can solve the problems of inability to perform crystallization of albumin in a sufficiently controlled or reliable way, prevent the development of extensive knowledge of crystallization conditions necessary in the design of large-scale crystallization, and low ionic strength. , the effect of high ionic strength

Inactive Publication Date: 2006-08-10
GTC BIOTHERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides improved methods for producing crystalline human albumin from various sources such as human plasma, recombinant sources, and bodily fluids of transgenic mammals, plants, avians, bacteria, yeast, or insect cells. The method has a high purification power and can effectively separate crystalline albumin from other proteins, bacteria, fatty acids, or molecular species present in the starting material. The purity of albumin can be improved through a re-crystallization procedure. The methods also provide precise manipulation of process parameters such as pH, temperature, phosphate salt concentration, and caprylic acid or caprylate salt concentration to optimize the production of crystalline human albumin. The process of the invention involves changing phosphate salt concentration, controlling pH, and applying heating and cooling procedures in a planned manner. Overall, the invention provides a reliable and efficient method for producing high-purity crystalline human albumin."

Problems solved by technology

However, the crystallization conditions and procedures were very sparingly described.
On the basis of the data available from this citation it is not possible to perform crystallization of albumin in a sufficiently controlled or reliable way.
A review of the prior art literature indicates that while there are several methods of crystallization proposed the relevant citations do not teach a process that is efficient at an industrial or commercial scale, teach a method that is unavailable for use in the production of a therapeutic product or excipient, or provide a process only useful in the production of single crystals useful only in x-ray diffraction studies.
Thus, the limitations of the prior art prevent the development of the extensive knowledge of crystallizing conditions necessary in the design of a large-scale crystallization processes for hSA in a therapeutic, pharmaceutical excipient, or medical adjuvant role.
Moreover, the prior art does not provide teachings that provide for the use from sources other than human plasma.

Method used

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  • Method for the crystallization of human serum albumin
  • Method for the crystallization of human serum albumin
  • Method for the crystallization of human serum albumin

Examples

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Effect test

example 1

A Preferred Crystallization Process

[0064] This process description is made for the crystallization of a purified albumin solution and for that purposes describes the use of only a phosphate solution only. However, according to the current invention this method can be used on impure or only partially purified starting material, as may be found from transgenic or cell culture feedstreams, with the addition of additional steps provided herein. Variations of the inventive method, for example utilizing starting solutions with significant impure material, are presented below.

Step 1. Precipitation

[0065] Phosphate stock solution, containing 2.8 moles of NaH2PO4 and 1.2 moles of K2HPO4 dissolved in water and filled to 1.0 liter, was used in crystallization. This 4 M phosphate solution had pH 6.2 when measured after dilution to 0.5 M. Thereafter, 200 ml of a purified albumin solution was precipitated by adding 371 ml of 4.0M phosphate stock solution at a pH of 6.2. On the basis of the add...

example 2

Crystallization of Albumin with the Microdiffusion Method

[0071] Crystallization examples from 48 samples were prepared according to a hanging drop microdiffusion method known in the art. The sample solution contained purified 198 mg / ml of albumin and 3.2 mg / ml sodium caprylate. The liquid solution of albumin was prepared by mixing 3 μl of albumin solution with 3 μl of 1.8-2.3 M phosphate. The drops were allowed to equilibrate in the closed microdiffusion wells and refrigerated at 5° C. Crystals were produced in less than 24 hours according to this embodiment of the the current invention. According to the current invention, feedstreams from other source material, especially transgenic and cell culture sources, can also be utilized in conjunction with a microdiffusion hanging drop method.

[0072] The crystals were observed with microscope and photographed with digital camera. Information regarding the development of the crystals are provided on tables 19 and 20. These examples show th...

example 3

Crystallization of Albumin in Impure Starting Material

[0074] The following process description is originally made from a recombinant hSA starting material which contains much of impurities which interfere the crystallization. The first process steps are needed for removal of impurities. If more pure albumin is used, the number of steps is correspondingly reduced.

Step 1. Concentration

[0075] The starting material should be concentrated by ultrafiltration filtered as much as possible. High protein concentration will reduce the usage of phosphate and increase the yield of crystallizable rhSA. The material should also be filtered with water in the end of concentration procedure in order to reduce the salts originating from the previous process steps. Guideline for protein concentration: A280nm=150-200.

Step 2. Initial Precipitation with Phosphate

[0076] Add 2.5 volumes of 4 M phosphate (pH 6.2) into 1.5 volumes of rhSA concentrate. The final phosphate concentration at this step is 2...

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Abstract

The present invention relates to the purification and production of human albumin from various sources through crystallization and repeated crystallization. Basic features of the invented process include providing specific reaction conditions and precipitating reagents to maximize albumin crystallization. Solubility diagrams are utilized as the basis for process control of the invented method. The current invention specifically controls phosphate concentration, pH and temperature to precisely guide crystallization kinetics and crystal yield.

Description

FIELD OF THE INVENTION [0001] The present invention relates to methods for provide a highly reliable method and commercially viable method of crystallizing human albumin. More specifically, the current invention provides a method to produce crystalline human albumin purified from various albumin sources, specifically including from transgenic animals or other recombinant sources. BACKGROUND OF THE INVENTION [0002] The present invention is directed to an improved method of crystallizing human serum albumin (“hSA”)(herein, hSA will be used interchangeably with the term human serum albumin). This process is preferably done to enhance purification procedures for recombinant hSA that can then be utilized in therapeutic applications or as an excipient in pharmaceutical preparations. With regard to pharmaceutical preparations human albumin as purified herein can be used as a therapeutic agent or as an excipient. In either case suitable formulations can be found in REMINGTON'S PHARMACEUTICA...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/38C07K14/765
CPCC07K14/765C07K2299/00Y10S530/829Y10S530/83A61P1/18A61P11/00A61P11/16A61P13/12A61P17/02A61P7/00A61P7/04A61P7/08A61P7/10A61P9/10
Inventor VISURI, KALEVIUOTILA, SINIKKAFULTON, SCOTT P.COUTO, DANIEL E.
Owner GTC BIOTHERAPEUTICS INC
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