CN101798335B

CN101798335B - Purification method of thymosin extrasin alpha 1

Info

Publication number: CN101798335B
Application number: CN 201010138995
Authority: CN
Inventors: 戴柱; 覃亮政; 马亚平; 袁建成
Original assignee: Hybio Pharmaceutical Co Ltd
Current assignee: Hybio Pharmaceutical Co Ltd
Priority date: 2010-03-30
Filing date: 2010-03-30
Publication date: 2013-02-13
Anticipated expiration: 2030-03-30
Also published as: CN101798335A

Abstract

The invention provides a technical method applicable to the industrialized purification of thymosin extrasin alpha 1, which comprises the following steps: using octadecylsilane chemically bonded silica as a fixed phase; using a trifluoroacetic acid water solution as a phase A, using acetonitrile as a phase B; controlling the gradient (B%) between 10 and 60 percent; carrying out gradient elution on a crude peptide solution; and carrying out the step of salt conversion, wherein the thymosin extrasin with trifluoroacetic acid salt is converted into salt-free thymosin extrasin by a reverse-phase efficient liquid phase chromatography method. The invention adopts the one-step reverse-phase efficient liquid phase chromatography method for purification, and uses the reverse-phase efficient liquid phase chromatography method for converting the salt, so the purified thymosin extrasin alpha 1 has high purification and high yield, and the industrialization requirement can be met.

Description

A kind of purification process of thymosin α1

Technical field

The present invention relates to the purification process of a peptide species, relate in particular to the purification process of thymosin α1.

Background technology

Thymosin α1 (Thymosin α 1) is to obtain a kind of small molecule bioactive polypeptide by separation and purification in the thymosin fraction 5 (TF-5), formed by the arrangement of 28 seed amino acids, molecular weight 3108.37, its content accounts for 0.6% of TF-5, has higher immune-enhancing activity.The Thymosin alpha 1 mechanism of action mainly is to act on the differentiation of T lymphocyte, growth and ripe stages, thereby regulates cellular immune function, and enhancing body diseases prevention and resistance against diseases are the good polypeptide drugs of a kind of market application foreground.

Summary of the invention

The present invention proposes a processing method that is suitable for industrialization purifying thymosin α1, use reversed-phased high performace liquid chromatographic purifying thymosin α1, purity height and yield are good, reach industrialized requirement.

For achieving the above object, the technical scheme taked of the present invention may further comprise the steps into:

1) take octadecylsilane chemically bonded silica as stationary phase, take the aqueous solution of trifluoroacetic acid as the A phase, acetonitrile is the B phase, and gradient B%:10%-60% carries out gradient elution with thick peptide solution;

The concentration of described " aqueous solution of trifluoroacetic acid " is 0.01%-1%, is preferably 0.05%-0.3%.Described " thick peptide " refers to adopt liquid phase synthesizing method or solid-phase synthesis to obtain, and not yet passes through the thymosin α1 that the thick peptide of thymosin α1 of refinement treatment or purity can not fulfilling medicinals." thick peptide solution " refers to that thick peptide is dissolved in the resulting solution of pure water, and pure water meets the water for injection standard, preferred ultrapure water.The preferred preferred chromatographically pure of the purity grade of " acetonitrile ".Step 1) hereinafter also is expressed as purifying.

2) turn salt: adopt reversed-phased high performace liquid chromatographic trifluoroacetate to be changed into salt-free.

The stationary phase of described " RPLC " is eight alkyl silane bonded silica gels.Described " turning salt " refers to 50% acetonitrile wash-out the thymosin α1 trifluoroacetate be changed into salt-free thymosin α1 again with the water flushing that contains 5% acetonitrile.

The purifying scale comprises following specification chromatographic column: 5cm * 25cm (pillar diameter * length), 15cm * 25cm, 30cm * 25cm.

Embodiment

Embodiment one:

1. sample preparation: thick peptide dissolves with ultrapure water, makes sample dissolve the rear membrane filtration of use fully, the collection filtrate for later use.

2. purifying:

Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm * 25cm.Moving phase: A phase: 0.05% trifluoroacetic acid aqueous solution, B phase: acetonitrile, flow velocity: 50-80ml/min.Detect wavelength: 230nm.Gradient: B%:10%-60% (elution time 50min).Sample size is 1.5-2g.

Purge process: rinse chromatographic column well the back balance loading with the acetonitrile more than 50%, applied sample amount is 1.5-2g.Linear gradient elution 50min collects the purpose peak, and the thymosin α1 solution of collecting is for subsequent use after water temperature is no more than 35 ℃ of lower vacuum rotary steams to be concentrated into about 10-20mg/mL.

3, turn salt: chromatographic column: the chromatographic column take eight alkyl silane bonded silica gels as stationary phase, pillar diameter and length are: 5cm * 25cm.Rinse chromatographic column well rear loading with the acetonitrile solution more than 50%, applied sample amount 1.5-2g, with the water flushing 15-30min that contains 5% acetonitrile, use at last 50% acetonitrile wash-out, collect the purpose peak, the thymosin α1 solution of collecting is no more than in water temperature goes to suitable large small vessels after 35 ℃ of lower vacuum rotary steams are concentrated into about 150-200mg/mL.Can obtain purity after the lyophilize greater than 98% thymosin α1.

Embodiment two:

2. purifying:

Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15cm * 25cm.Moving phase: A phase: 0.3% trifluoroacetic acid aqueous solution; B phase: acetonitrile, flow velocity: 450-550ml/min.Detect wavelength: 230nm.Gradient: B%:10%-60% (elution time 50min).Sample size is 10-15g.

Purge process: rinse chromatographic column well the back balance loading with the acetonitrile more than 50%, applied sample amount is 10-15g.Linear gradient elution 50min collects the purpose peak, and the thymosin α1 solution of collecting is for subsequent use after water temperature is no more than 35 ℃ of lower vacuum rotary steams to be concentrated into about 10-20mg/mL.

3, turn salt: chromatographic column: the chromatographic column take eight alkyl silane bonded silica gels as stationary phase, pillar diameter and length are: 15cm * 25cm.Rinse chromatographic column well rear loading with the acetonitrile solution more than 50%, applied sample amount 10-15g, with the water flushing 15-30min that contains 5% acetonitrile, use at last 50% acetonitrile wash-out, collect the purpose peak, the thymosin α1 solution of collecting is no more than in water temperature goes to suitable large small vessels after 35 ℃ of lower vacuum rotary steams are concentrated into about 150-200mg/mL.Can obtain purity after the lyophilize greater than 98% thymosin α1.

Embodiment three:

2. purifying:

Purification condition: chromatographic column: the chromatographic column take octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm * 25cm.Moving phase: A phase: 0.1% trifluoroacetic acid aqueous solution; B phase: acetonitrile, flow velocity: 1900-2200ml/min.Detect wavelength: 230nm.Gradient: B%:10%-60% (elution time 50min).Sample size is 55-75g.

Purge process: rinse chromatographic column well the back balance loading with the acetonitrile more than 50%, applied sample amount is 55-75g.Linear gradient elution 50min collects the purpose peak, and the thymosin α1 solution of collecting is for subsequent use after water temperature is no more than 35 ℃ of lower vacuum rotary steams to be concentrated into about 10-20mg/mL.

3, turn salt: chromatographic column: the chromatographic column take eight alkyl silane bonded silica gels as stationary phase, pillar diameter and length are: 30cm * 25cm.Rinse chromatographic column well rear loading with the acetonitrile solution more than 50%, applied sample amount 55-75g, with the water flushing 15-30mi n that contains 5% acetonitrile, use at last 50% acetonitrile wash-out, collect the purpose peak, the thymosin α1 solution of collecting is no more than in water temperature goes to suitable large small vessels after 35 ℃ of lower vacuum rotary steams are concentrated into about 150-200mg/mL.Can obtain the thymosin α1 that purity requires greater than 98% conformance with standard after the lyophilize.

In sum, operation is simple and feasible, elaboration purity is higher than 98%, ideal yield coefficient, purifying cost are lower in order to upper method purifying thymosin α1, reaches industrialized requirement.

Claims

1. the purification process of a thymosin α1 may further comprise the steps:

1) take octadecylsilane chemically bonded silica as stationary phase, take the aqueous solution of the trifluoroacetic acid of 0.05%-0.3% as the A phase, acetonitrile is the B phase, and gradient B%:10%-60% carries out gradient elution with thick peptide solution;

2) turn salt: take eight alkyl silane bonded silica gels as stationary phase, the water flushing with containing 5% acetonitrile changes into trifluoroacetate salt-free with 50% acetonitrile wash-out again.