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NTP/dNTP chromatographic separation method and system based on intelligent control

A technology of intelligent control and separation methods, applied in separation methods, chemical instruments and methods, solid adsorbent liquid separation, etc., can solve the problems of inconvenient sampling, analysis, separation and collection, keeping the chromatographic cabinet at low temperature, disadvantages, etc.

Active Publication Date: 2021-12-28
上海蔚之星生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the biological preparation of Document 1 and Document 2, it is necessary to separate and remove nucleosides, bases, or unreacted nucleotides produced by side reactions, or diphosphate compounds as intermediates, etc. by anion exchange resin chromatography; Because triphosphate compounds are very easy to degrade, they usually need to be carried out in a low-temperature chromatography cabinet; however, it is very inconvenient to frequently take samples for analysis, control instruments, separate and collect, etc., and too frequent is also very unfavorable for keeping the low temperature of the chromatography cabinet

Method used

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  • NTP/dNTP chromatographic separation method and system based on intelligent control
  • NTP/dNTP chromatographic separation method and system based on intelligent control
  • NTP/dNTP chromatographic separation method and system based on intelligent control

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Embodiment 1

[0058] The present embodiment is chromatographed 5'-ATP is used in the separation process figure 1 Intelligent chromatographic separation system shown, the specific process is as follows:

[0059]In 1L containing 40mmol / L adenosine, 200mmol / L phosphate buffer (pH = 6.5), 200mmol / L glucose and 5mmol / L magnesium phosphate reaction solution was added 70g of frozen S. cerevisiae, the reaction 32 ℃ 4 h, the molar conversion up to 90%, wherein the reaction mixture still contains unreacted adenosine, intermediate AMP, ADP and the like. The 5'-ATP reaction solution prepared above was filtered to remove yeast, a clear solution is obtained 5'-ATP reactions.

[0060] By chromatography on a smart system like, washing with water, gradient elution, wherein the cation exchange resin in an amount of 100ml (Na type), the washing process of deionized water in an amount of 1 ~ 2L, a flow rate of 100ml / h, an anion exchange resin in an amount of 200ml (CI type), the eluent is employed by wei...

Embodiment 2

[0062] The present embodiment is chromatographed 5'-dATP is used in the separation process figure 1 Intelligent chromatographic separation system shown, the specific process is as follows:

[0063] Cerevisiae in 1L containing 40mmol / L dAMP, 200mmol / L phosphate buffer (pH = 6.5), 200mmol / L glucose and 5mmol / L magnesium phosphate reaction solution was added 70g frozen, 32 deg.] C for 4 hours, the molar conversion rate up to 85%. The solution was filtered to remove yeast, to obtain a clear solution.

[0064] Comp chromatographed by smart systems, washing, elution, wherein the cation exchange resin in an amount of 100ml (Na type), the washing process of deionized water in an amount of 1 ~ 2L, a flow rate of 100ml / h, an anion exchange resin in an amount of 200ml ( Cl form), eluent using mass percentage concentration of 0 ~ 5% NaCl gradient elution, flow rate of 20 ~ 30ml / h. Press control system reports after chromatography, collecting an initial concentration of 5g / L, fin...

Embodiment 3

[0066] The present embodiment is chromatographed 5'-CTP is used in the separation process figure 1 Intelligent chromatographic separation system shown, the specific process is as follows:

[0067] Formulated 1L cerevisiae containing 40mmol / L CMP, 200mmol / L phosphate buffer (pH = 6.5), 200mmol / L glucose and 5mmol / L of magnesium phosphate, the reaction solution was added, the 70g frozen, the other conditions of Example 1 . The final available CTP 8g, content 98.1%.

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Abstract

The invention discloses an NTP / dNTP chromatographic separation method and system based on intelligent control, NTP / dNTP chromatographic separation is performed in a low-temperature chromatographic cabinet, and real-time online detection and intelligent control are performed according to the NTP / dNTP chromatographic separation process of a nucleic acid protein detector and an HPLC detector arranged outside the low-temperature chromatographic cabinet. Through real-time online detection of the nucleic acid protein detector, whether NTP / dNTP flows out in the sample loading and water washing process is monitored, so that whether the process runs normally or not is known; the gradient elution process is controlled and the NTP / dNTP is collected through timed online sampling and detection by an HPLC (High Performance Liquid Chromatography) detector. According to the method, the situation that nucleoside triphosphate is degraded due to the fact that a low-temperature chromatography cabinet is frequently opened for sampling or an instrument is regulated and controlled can be avoided, and meanwhile high-quality operation of the chromatography separation process is guaranteed.

Description

Technical field [0001] The present invention belongs to the field of biochemical separation techniques, in particular, to an intelligent control NTP / dNTP chromatographic separation based method and system. Background technique [0002] Nucleoside triphosphate (NTP) or deoxynucleoside triphosphates (dNTPs), may be a phosphoric acid as a raw material by Saccharomyces cerevisiae, or baker's yeast, biotransformation to form the corresponding triphosphate compounds can also be produced by a method such as acetate kinase; for example: using 5'-adenosine monophosphate (AMP) may generate 5'-triphosphate (of ATP); the use of 2'-deoxy-5'-adenylic (dAMP) may generate a 2'-deoxy-5'-adenosine triphosphate phosphate (of dATP); acid using 5'-cytidine (CMP) may be generated cytidine 5'-triphosphate (CTP) and the like. [0003] The conventional method of preparing a nucleoside triphosphate are as follows: [0004] Document 1: Chinese patent CN discloses an adenosine triphosphate (ATP) productio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/10
CPCB01D15/10
Inventor 邱蔚然欧伶王金涛陈修足王军周洁
Owner 上海蔚之星生物科技有限公司