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Chromatographic processes and purified compounds thereof

A chromatographic method and a mixture technology, which can be applied to ion pair reagents in the field of reversed-phase preparative chromatography, and can solve problems such as resolution reduction.

Inactive Publication Date: 2012-05-23
BIOCON LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] It has been observed in conventional chromatography that the resolution between impurities and the protein of interest decreases as the load on the column increases

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Crude insulin aspart material of ~75% purity was diluted 10-fold with purified water before adding IPA to a final concentration of 5%. in Kromasil TM ( -13μ-C8) column to purify the crude mixture. Mobile phase A was 1% hexanesulfonic acid (HSA) (w / v) in 250 mM sodium acetate, pH 4.0 and mobile phase B was isopropanol. In mobile phase A, a gradient elution of 18%-22% mobile phase B was performed over 20 column volumes. The addition of HSA effectively removes the desocta (desocta) insulin aspart and also observes that the des leader sequence desB-arginine insulin aspart precursor is reduced from a level of -2.77% to less than 0.27%, and the overall purity achieved is ~97.85%.

[0077] Control 2

[0078] Crude insulin aspart material of ~75% purity was diluted 10-fold with purified water, then ethanol was added to a final concentration of 10%. in Kromasil TM ( -13μ-C8) column to purify the crude mixture. Mobile phase A was 25 mM ammonium sulfate in 250 mM sodium...

Embodiment 2

[0080] Crude insulin aspart material of ~75% purity was diluted 10-fold with purified water, then ethanol was added to a final concentration of 10%. in Kromasil TM ( -13μ-C8) column to purify the crude mixture. Mobile phase A was 1% hexanesulfonic acid (HSA) (w / v) in 250 mM sodium acetate in 25 mM ammonium sulfate, pH 4.0 and mobile phase B was ethanol. A gradient elution of 25%-35% of mobile phase B in mobile phase A was performed over 20 column volumes. Addition of HSA reduced the des leader sequence desB-arginine insulin aspart precursor from a level of -3% to less than 0.3%. The desocta insulin aspart was completely removed and the level of monoglycosylated insulin aspart decreased from -2% to 0.3%. The overall purity achieved was -98%.

[0081] Control 3

[0082] Crude insulin aspart material of ~67% purity was diluted 10-fold with purified water, then methanol was added to a final concentration of 10%. in Kromasil TM ( -13μ-C8) column to purify the crude mix...

Embodiment 3

[0084] Crude insulin aspart material of ~67% purity was diluted 10-fold with purified water, then methanol was added to a final concentration of 10%. in Kromasil TM ( -13μ-C8) column to purify the crude mixture. Mobile phase A was 1% hexanesulfonic acid (HSA) (w / v) in 250 mM ammonium acetate in 25 mM ammonium sulfate, pH 4.0 and mobile phase B was methanol. A gradient elution of 55%-65% of mobile phase B was performed in mobile phase A over 20 column volumes. Addition of HSA purified the des leader sequence desB-arginine insulin aspart precursor from a level of -2% to less than 1%. The monoglycosylated insulin aspart impurity was reduced from 3% to less than 0.5%. Desocta (de-eight) insulin aspart decreased from -10% to less than -2%. The overall purity achieved was -92%.

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PUM

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Abstract

The present disclosure demonstrates the utility of ion pairing agents in the preparative scale of purification. More particularly, the disclosure relates to the usage of ion pairing agents in RP preparative linear chromatography enabling high purity of the desired end product. The disclosure shows that ion-pairing agents have dramatic effect on desired purity of polypeptides.

Description

technical field [0001] The present invention illustrates the use of ion pairing agents (ion pairing agents) in preparative scale purification. More specifically, the present invention relates to the use of ion-pairing reagents in reversed-phase preparative chromatography, enabling the desired high purity of the final product to be obtained. The present invention shows that ion pairing reagents have a significant effect on the desired purity of polypeptides. Background technique [0002] A number of different chromatographic procedures were applied to achieve the desired end result (regarding purity and yield). Reverse phase chromatography is one of the most efficient purification methods employed. Reverse-phase liquid chromatography ("RP-LC") and reverse-phase high-performance liquid chromatography ("RP-HPLC") are commonly used to purify molecules such as peptides and proteins produced by synthetic or recombinant methods. RP-LC and RP-HPLC methods can efficiently separate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/20C07K7/64C07K7/16C07K14/62
CPCC07K14/46C07K7/06C07K7/16C07K1/20C07K14/62B01D15/32
Inventor 尼特斯·戴夫克里萨纳·海坦亚·古拉苏恩达雷什·尚卡尔哈里什·耶尔
Owner BIOCON LTD
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