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Purification process for glycosylated hemoglobin

A technology of glycosylated hemoglobin and hemoglobin, applied in the field of medical immunity, can solve the problems of undiscovered separation of HbAlc

Active Publication Date: 2015-06-10
桂林英美特生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report about the use of CM-Sepharose Fast F1ow chromatography column to separate HbAlc

Method used

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  • Purification process for glycosylated hemoglobin
  • Purification process for glycosylated hemoglobin
  • Purification process for glycosylated hemoglobin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] 1. Instruments and columns used:

[0045] 1.1 Instruments: simple protein purification system (including constant flow pump, UV detector, recorder) (Ametsham Pharmacia Biotech Inc), GradiFrac TM Programming (Ametsham Pharmacia Biotech Inc), Waters600 high-phase liquid chromatography (US Warers Company), etc. .

[0046] 1.2 Column: CM-Sepharose Fast F1ow column (fast flow CM Sepharose, GE Healthcare), SOURCE15S4.6 / 100PE ion exchange resin (Amersham Pharmacia Biotech Inc).

[0047] 2. Preparation of hemoglobin solution:

[0048] Take the blood sample (anticoagulated whole blood) and centrifuge at 4°C at low speed (2000r / min, 10-15min), take out the plasma with a disposable pipette, add 1×PBS (pH 7.2 to 7.4), and gently mixed to obtain a mixed solution. Centrifuge the mixture at 2000 rpm for 20 minutes at 4°C, remove the supernatant, add 1×PBS (pH 7.2 to 7.4) to the pellet, mix gently, centrifuge at 2000 rpm for 20 minutes at 4°C, and absorb the supernatant. Then add 1×P...

Embodiment 2

[0080] Repeat Example 1, the difference is that the hemoglobin solution prepared in the second step is not subjected to the saccharification step, but is directly separated and purified on the CM-Sepharose Fast Flow column.

[0081] After testing, after purifying by the process of this embodiment, the HbAlc purity is 99.53% (substance fraction), and its specific data are as shown in Table 3, and its high-performance liquid chromatogram is as follows image 3 shown.

[0082] table 3:

[0083]

Embodiment 3

[0085] Repeat Example 1, the difference is that the preparation steps of 4.1 mobile phase and the gradient linear elution step of 4.4 HbAlc are changed, and the specific changes are as follows:

[0086] 4.1 Preparation of mobile phase:

[0087] Liquid A: 25mmol / L NaAc+0.01%NaN 3 (final concentration), pH=5.8; Preparation method reference example 1.

[0088] Solution B: 25mmol / L NaAc+280mmol / L NaCl+0.02%NaN 3 (final concentration), pH=5.8; refer to Example 1 for the preparation method.

[0089] 4.4 Gradient linear elution of HbAlc:

[0090] Control the elution flow rate at 3-7mL / min, carry out elution according to the following elution conditions, collect the elution peak, and replace it with a new tube for collection when another peak occurs:

[0091] 1) Within the eluent time of 0.5 times column volume: solution A: 100%→80%;

[0092] 2) Within the eluent time of 1 column volume: liquid A: 80%, liquid B: 20%;

[0093] 3) Within the eluent time of 1 times the column volum...

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Abstract

The invention discloses a purification process for glycosylated hemoglobin (HbAlc). According to the process, hemoglobin liquid is taken and introduced into a CM-Sepharose Fast Flow chromatographic column, and mobile phase gradient elution is carried out so as to obtain a high-purity HbAlc antigen, wherein a mobile phase is composed of a solution A and a solution B, the solution A is 15 to 25 mmol / L NaAc with a pH value of 5.6 to 5.8 or a mixture of 15 to 25 mmol / L NaAc and 0.01 to 0.05% NaN3 with a pH value of 5.6 to 5.8, and solution B is mixture of 15 to 25 mmol / L NaAc, 280 to 320 mmol / L NaCl and 0.01 to 0.05% NaN3 and has a pH value of 5.6 to 5.8. Unlike the prior art, the purification process provided by the invention uses CM-Sepharose Fast Flow column chromatography to obtain the high-purity HbAlc antigen.

Description

technical field [0001] The invention relates to the technical field of medical immunity, in particular to a purification process of glycosylated hemoglobin. Background technique [0002] Glycosylated hemoglobin (GHb) is a compound formed by non-enzymatic interaction between hemoglobin (Hh) and carbohydrates in red blood cells in blood. Human Hh is mainly HbA (95 ~ 97%), HbA2 (<3%), HbF (<1%). The part of HbA that is bound to sugar is HbAl, and the part that is not bound to sugar is HbAO. According to the different carbohydrates bound, GHb is divided into HbAlal, HbAla2, HbAlb, and HbAlc, of which HbAlc accounts for about 80% of HbAl, and is a stable addition compound formed by the amino terminus of one or two β chains of Hh and glucose carbonyl. The content of HbAlc is significantly positively correlated with the blood sugar concentration, which can directly reflect the average level of blood sugar control in patients with elevated blood sugar in the past 2 to 3 mont...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/805C07K1/16
Inventor 蔡豪斌李珏燕苏时萍
Owner 桂林英美特生物技术有限公司