CN106226428B - A kind of method of recombinant human nerve growth factor content in quick detection Chinese hamster ovary celI fermentation liquid - Google Patents
A kind of method of recombinant human nerve growth factor content in quick detection Chinese hamster ovary celI fermentation liquid Download PDFInfo
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Abstract
The invention discloses a kind of methods of recombinant human nerve growth factor content in quickly detection Chinese hamster ovary celI fermentation liquid.The specific chromatographic column for using butylsilane bonded silica gel reverse phase C4 column；Mobile phase: A is 0.1 volume % trifluoroacetic acid aqueous solution, B is 0.1 volume % trifluoroacetic acid acetonitrile solution, 0.22 μm of membrane filtration, it is used after ultrasonic degassing 10min, become gradient elution, the concentration of polar organic solvent is incremented by for continuity: 0-30min is raised to 40 volume %B, 30-35min by 10 volume %B and is raised to 90 volume %B by 40 volume %B, 35-36min drops to 10 volume %B, 36-41min by 90 volume %B and maintains 10 volume %B 5min；Finally carry out content calculating.The detection method accuracy is high, precision is good, and system suitability, the rate of recovery, linear relationship, stability are good.
Description
Technical field
The present invention relates to recombined humans in field of biological technology detection, more particularly to a kind of quickly detection Chinese hamster ovary celI fermentation liquid
The method of nerve growth factor content.
Background technique
Nerve growth factor (nerve growth factor, NGF) is the neurotrophic factor being found earliest, is tool
There is neurotrophic and promote a kind of nerve growth regulatory factor of enation double biological function, to various kinds of cell
Especially the development, differentiation, growth, regeneration of maincenter and peripheral neurons and the expression of functional characteristic all have important regulation and make
With.Nerve growth factor has important clinical value, has been widely used for maincenter and peripheral nervous disease at present
In treatment, in terms of also having the function of antiviral, anti-inflammatory, adjusting human immune system, promoting the Non nervous systems such as wound reparation.Mind
It include tri- subunits of α, β, γ through growth factor, wherein β subunit has complete biological activity, at present the nerve of clinical use
Growth factor is mainly derived from the mouse nerve growth factor (m β NGF) of mouse submandibular gland extraction.Compared with mouse nerve growth factor,
Recombinant human nerve growth factor (rh β NGF) has easy acquisition, high yield, high activity, low in cost special with non-immunogenicity etc.
Point establishes efficient, stable growth factor of human nerve recombinant expression system with important clinical meaning and commercial value.
With the development of detection technique, 2015 editions Chinese Pharmacopoeias provide the detection method of injection mouse nerve growth factor
Mouse nerve growth factor stoste and the content detection of finished product in addition to it can use traditional Lowry method, can also use ELISA method and
Molecular sieve high-efficient liquid chromatography (HPLC) method.Wherein protein content detection method is ELISA method in conventional cell fermentation liquid,
Protein content in different fermentations liquid can be measured with high throughput, to preferably be screened to high yielding cell sarain.But mind
Through the raw antibody of growth factor relatively difficult labour, people's nerve that presently commercially available Anti-NGF Antibody cannot be recombinated with Chinese hamster ovary celI is raw
The long factor is combined, therefore can not achieve the ELISA detection of recombinant human nerve growth factor content in Chinese hamster ovary celI fermentation liquid.
Relative to ELISA method, HPLC method has many advantages, such as that analysis is easy quickly, the concentration range of linearity is big, quantitative accurate.
2015 editions Chinese Pharmacopoeias and other open source literatures mainly use molecular sieve HPLC, such as Zhang Jinlong, Ren Jun, Fu Ling, and is waited to utilize
SEC-HPLC measures purity [J] biotechnology communications of mouse nerve growth factor, 2013,24 (5): 691-694.；Aries,
Chapter is built forever, and Huang takes off with flying speed, and waits HPLC detection recombination human nerve growth factor β stoste purity and content [J] China new drug miscellaneous
Will, 2015,24 (4): 400-402.；The method that Zhou Zhiwen, Zhang Hongshan, Wang Hong defend measurement nerve growth factor content:
CN, CN1982891[P]. 2007.；Bai Haifen, nerve growth factor contains in a kind of color beautiful nerve growth factor preparation of of Feng
The measuring method of amount: CN, CN104931598A [P] 2015..But the separation principle of molecular sieve HPLC method is according to albumen
Molecular size range it is different, and for complex sample (such as recombinant human nerve growth factor Chinese hamster ovary celI fermentation liquid) middle-molecular-weihydroxyethyl phase
Close hypoproteinosis separating capacity.It is examined in other open source literatures about to natural and recombination nerve growth factor purity, content
Survey method also uses reverse phase C18 column, and such as Sha Yunfei, Huang Tao is quick, Yang Bei, and rp-hplc determination pig brain is waited to mention
Take content [J] Fudan Journal of nerve growth factor in object: medicine, 2005,32 (1): 108-109.；Jiang Jing, Yu Shu
Duckweed, Jiang Guixiang wait recombination human nerve growth factor β (β-rhNGF) [J] China of the column chromatography eluting CHO expression of two step of
Bioengineering magazine, 2008,28 (10): 84-89.But C18 column hydrophobicity is larger, preferable to the separating effect of small molecule,
But it is poor to the separating effect of polypeptide and albumen, especially in complicated protein sample detection.
Therefore, that there is an urgent need in the art to a kind of detection methods is simple, separating degree is good, the accurate Chinese hamster ovary celI fermentation liquid of result
The HPLC rapid detection method of middle recombinant human nerve growth factor content.
Summary of the invention
In view of the deficiencies in the prior art, as antibody is difficult the growth factor of human nerve knot recombinated with Chinese hamster ovary celI in ELISA method
It closes, molecular sieve, C18 column HPLC method lack to quick detectability of complex sample etc., and the purpose of the present invention is to provide one kind
The HPLC rapid detection method of recombinant human nerve growth factor content in easy, accurate Chinese hamster ovary celI fermentation liquid.
To achieve the above object, inventor passes through a large amount of experimental study, final to obtain quickly detection Chinese hamster ovary celI fermentation liquid
The method of middle recombinant human nerve growth factor content, which is characterized in that step are as follows:
1) sample treatment: taking recombinant human nerve growth factor Chinese hamster ovary celI fermentation liquid, and 4000rpm is centrifuged 5min, takes supernatant
Liquid carries out HPLC detection after crossing 0.22 μm of filter membrane；
2) HPLC is detected: chromatographic column is butylsilane bonded silica gel reverse phase C4 column；Detector is UV detector；Detect wave
A length of 280nm；Flow velocity is 1.0ml/min；Column temperature is 30 DEG C；Sampling volume is 20 μ L；Mobile phase: A is 0.1 volume % trifluoro second
Aqueous acid, B are 0.1 volume % trifluoroacetic acid acetonitrile solution, and 0.22 μm of membrane filtration uses after ultrasonic degassing 10min, become ladder
The concentration of degree elution, polar organic solvent is incremented by for continuity: 0-30min is raised to 40 volume %B, 30-35min by 10 volume %B
90 volume %B, 35-36min, which are raised to, by 40 volume %B drops to 10 10 volume %B of volume %B, 36-41min maintenance by 90 volume %B
5min；
3) content calculates: with recombinant human nerve growth factor main peak peak in Recombinant Chinese cobra NGF Chinese hamster ovary celI fermentation liquid
Area is calculated by the equation of linear regression of recombinant human nerve growth factor standard items standard curve.
Further, the recombinant human nerve growth factor standard items standard curve the preparation method comprises the following steps:
Accurately weighing recombinant human nerve growth factor standard items is configured to 1.0mg/ml standard items stock solution, then is diluted to not
With the standard solution of concentration, the standard solution of diluted concentration gradient, 20 μ L Injection volumes, with nerve growth factor main peak are taken
Peak area and concentration make linear regression, obtain equation of linear regression.
Because the growth factor of human nerve that commercially available Anti-NGF Antibody cannot be recombinated with Chinese hamster ovary celI is combined, therefore
It can not achieve the ELISA detection of recombinant human nerve growth factor content in Chinese hamster ovary celI fermentation liquid.Simultaneously 2015 editions Chinese Pharmacopoeias and
The molecular sieve in document HPLC method having disclosed cannot separate nerve growth factor and foreign protein, cause testing result empty
It is high；And reverse phase C18 column polarity is big, and it is poor to the separation sensitivity of albumen, it cannot be guaranteed that nerve growth factor appearance region is without dry
It disturbs.It is limited that experiment progress is caused above, therefore the prior art is caused not have recombinant human nerve growth factor in Chinese hamster ovary celI fermentation liquid
Effective detection method, applicant is creative to detect recombinant human nerve growth factor using C4 column, then terraced by becoming
Degree method widens the gradient of the nonpolar liquid phase (acetonitrile) near nerve growth factor appearance time, achieves fabulous application
Effect can directly detect recombinant human nerve growth factor in Chinese hamster ovary celI fermentation liquid and contain to be implemented without and carry out purifying
Amount.
Detection method of the invention is using C4 column to the small albumen of hydrophobic difference is effectively divided each other in complex sample
From ability, improve recombinant human nerve growth factor and other eggs in Chinese hamster ovary celI fermentation liquid by preferably becoming gradient elution method
White separating degree, realization are used for quickly detecting its content with recombinant human nerve growth factor main peak peak area.The present invention is opposite
For existing HPLC detection method of content, do not need directly examine Chinese hamster ovary celI fermentation liquid progress more purification
It surveys, there is simple and efficient, high sensitivity, it is thin to be advantageously applied to recombinant human nerve growth factor CHO for the good feature of accuracy
In the screening and industrialization of born of the same parents' strain.
In the embodiment of the present invention, recombinant human nerve growth factor standard items equation of linear regression related coefficient is 0.9999,
It is linear good within the scope of 0-1.0mg/mL.The detection method accuracy, high precision are good, system suitability, the rate of recovery, linear
The methods of relationship, stability test learn the quick inspection that index is able to satisfy recombinant human nerve growth factor in Chinese hamster ovary celI fermentation liquid
It surveys.
Detailed description of the invention
Fig. 1 is recombinant human nerve growth factor standard chromatogram.
Fig. 2 is recombinant human nerve growth factor canonical plotting.
Fig. 3 is recombinant human nerve growth factor Chinese hamster ovary celI fermentation liquid chromatogram.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.Embodiment
In particular technique or condition person is not specified, described technology or conditions or according to the description of product according to the literature in the art
Book carries out.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
Embodiment 1:
1, sample treatment
Use DNA sequences encoding containing growth factor of human nerve (mature peptide, 118 amino acid, GenBank No.
V01511 mammalian cell fermentation expression recombinant human nerve growth factor), preferred mammal cell are Chinese hamster ovum
Nest cell (Chinese hamster ovary celI).In order to enable recombinant human nerve growth factor correctly to fold, process and secrete into culture solution, encode
DNA sequence dna should contain signal peptide sequence and recombinant human nerve growth factor leader peptide sequences.Chinese hamster ovary celI is cultivated in CD FortiCHO
It cultivates in base (article No.: A1148301, GIBCO company of the U.S.) to density 2 × 106A/ml, 90% or more vigor, that is, be inoculated with
It ferments in CD FortiCHO culture medium, cultivates initial density 5 × 105.Daily the measurement density of cell, vigor and
Glucose content in culture solution, and Feed C culture medium (article No.: A1327504, GIBCO company of the U.S.) is supplemented, make in culture solution
The final concentration of 4g/L of glucose, fermentation ends when cell viability≤70%.Recombinant human nerve growth factor CHO is taken after fermentation 10d
Cell fermentation liquid, 4000rpm are centrifuged 5min, take supernatant, after crossing 0.22 μm of filter membrane, carry out real-time HPLC detection.
2, chromatographic condition
(1) chromatographic column: butylsilane bonded silica gel reverse phase C4 column；
(2) detector: UV detector；
(3) Detection wavelength: 280nm；
(4) mobile phase: A is 0.1 volume % trifluoroacetic acid aqueous solution, and B is 0.1 volume % trifluoroacetic acid acetonitrile solution, 0.22 μ
M membrane filtration uses after 10 min of ultrasonic degassing, becomes gradient elution, and the concentration of polar organic solvent is incremented by for continuity: 0-
30min is raised to 40 volume %B, 30-35min by 10 volume %B and is raised to 90 volume %B, 35-36min by 90 volume %B by 40 volume %B
It drops to 10 volume %B, 36-41min and maintains 10 volume %B 5min.C4 chromatographic column has in separation complex sample each other in the present invention
The ability of the small albumen of hydrophobic difference, then by widening the organic solvent (nonpolarity near nerve growth factor appearance time
Phase) gradient, the ratio of organic solvent is slowly increased in 0-30min, to further promote nerve growth factor and fermentation liquid
In other foreign proteins separating effect.After realizing separation target, change gradient, fast lifting organic solvent ratio, punching
Wash the remaining foreign protein of chromatographic column.Mobile phase is returned to initial proportion again, and continues 5min, balances chromatographic column, it can be quick
It is detected next time.
(5) flow velocity: 1.0ml/min；
(6) column temperature: 30 DEG C；
(7) sampling volume: 20 μ L.
(8) content calculates: being calculated with recombinant human nerve growth factor main peak peak area by standard curve.
3, prepared by standard solution
Accurately weigh recombinant human nerve growth factor standard items (article No.: 11050-HNAC-1, Divine Land Yi Qiao, Beijing biology skill
Art Co., Ltd) it is configured to 1.0mg/ml standard items stock solution, then it is diluted to various concentration (0.2,0.4,0.6,0.8mg/ml)
Standard solution.
4, standard curve
Take the standard solution of diluted concentration gradient, 20 μ L Injection volumes, with recombinant human nerve growth factor peak area and
Concentration makees linear regression, obtains equation of linear regression.
5, system suitability
With 20 μ L continuous sample introduction of 1.0mg/ml recombinant human nerve growth factor standard solution, 6 needle, chromatogram is recorded, is calculated
System suitability.
6, recovery test
It dilutes or is added a certain amount of standard items respectively in standard solution, the upper press proof of various various concentrations is made,
Respectively into 3 needles, each sample recombinant human nerve growth factor content is calculated, the rate of recovery is calculated as detectable concentration divided by practical dense
Degree.
7, detection limit, quantitative limit test
Certain density test solution is configured, 20 μ L sample introductions are taken, records chromatogram, detection is limited to s/n=3, quantitatively be limited to
s/n=10。
8, precision test
The recombinant human nerve growth factor standard solution for taking 1.0mg/ml, in ought in a few days continuous sample introduction 6 times, calculate peak face
Long-pending and retention time withinday precision；And it is primary in sample introduction each in 6 days, calculate the precision in the daytime of peak area and retention time
Degree.
9, sample measures
The recombinant human nerve growth factor Chinese hamster ovary celI fermentation liquid for taking fermentation 10d is carried out according to the sample treatment under 1
Processing, is measured according to the chromatographic condition under 2, and peak area is substituted into the standard curve prepared in 4, calculates weight in fermentation liquid
Group growth factor of human nerve content.
10, interpretation of result
It according to the standard solution preparation method under 3, is measured according to the chromatographic condition under 2, recombinant human nerve growth
Factor standard product chromatogram is shown in Fig. 1.1.0mg/ml recombinant human nerve growth factor standard items only have 1 color as can be seen from Figure 1
Spectral peak, appearance time 23.748min, theoretical cam curve 10654, tailing factor 1.15.Recombinant human nerve growth factor
Canonical plotting is shown in Fig. 2, according to the standard solution preparation method under 3, is measured according to the chromatographic condition under 2, under 4
Method linear regression is made with peak area-concentration, the standard curve obtained isy=833953x+ 6496, R2=0.9999(yFor peak
Area mAu min,xFor drug concentration mg/mL), it is seen that recombinant human nerve growth factor is linear good within the scope of 0-1.0mg/mL
It is good.
In system suitability, the retention time RSD of 6 sample introductions is 0.07%, and the RSD of peak area is 0.51%, theoretical
Number of plates mean value is 9697-10341, tailing factor 1.08-1.21, shows that this method system suitability indices reach
Testing requirements.In recovery test, the rate of recovery 100.28-101.67%, RSD 0.82% shows that this method authenticity is very high.Inspection
It surveys and limits, in quantitative limit test, minimum detectable concentration is 10 μ g/ml, and detection is limited to 0.2 μ g, and minimum quantitative concentrations are 30 μ g/ml,
It is quantitatively limited to 0.6 μ g, illustrates that this method sensitivity is high.In precision test, withinday precision retention time RSD=0.05%,
Peak area RSD=0.51%, day to day precision retention time RSD=0.10%, peak area RSD=0.74%, withinday precision in the daytime
Precision RSD is respectively less than 2%, illustrates that the precision of this method is high.
The recombinant human nerve growth factor Chinese hamster ovary celI fermentation liquid of fermentation 10d is taken to carry out HPLC detection, obtained recombined human mind
Fig. 3 is seen through growth factor C HO cell fermentation liquid chromatography figure.From figure 3, it can be seen that recombinant human nerve is raw in Chinese hamster ovary celI fermentation liquid
Long factor main peak appearance time be 23.751min, theoretical cam curve 9756 suitable with standard items appearance time, trail because
Son is 1.23, separating degree 4.73.Recombinant human nerve growth factor main peak is integrated, linear regression will be substituted into after peak area
Equation, measuring concentration is 0.254mg/mL.
Above experiment results proved this method accuracy is high, precision is good, the rate of recovery, linear relationship, system suitability etc.
Index is able to satisfy the quick detection of recombinant human nerve growth factor content in Chinese hamster ovary celI fermentation liquid.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective
In the case where can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.
Claims (2)
1. a kind of method of recombinant human nerve growth factor content in quickly detection Chinese hamster ovary celI fermentation liquid, which is characterized in that step
Are as follows:
1) sample treatment: taking recombinant human nerve growth factor Chinese hamster ovary celI fermentation liquid, and 4000rpm is centrifuged 5min, takes supernatant, mistake
HPLC detection is carried out after 0.22 μm of filter membrane；
2) HPLC is detected: chromatographic column is butylsilane bonded silica gel reverse phase C4 column；Detector is UV detector；Detection wavelength is
280nm；Flow velocity is 1.0ml/min；Column temperature is 30 DEG C；Sampling volume is 20 μ L；Mobile phase: A is 0.1 volume % trifluoroacetic acid water
Solution, B are 0.1 volume % trifluoroacetic acid acetonitrile solution, and 0.22 μm of membrane filtration uses after ultrasonic degassing 10min, become gradient and wash
De-, the concentration of polar organic solvent is incremented by for continuity: 0-30min is raised to 40 volume %B by 10 volume %B, and 30-35min is by 40
Volume %B is raised to 90 volume %B, 35-36min and drops to 10 10 volume %B 5min of volume %B, 36-41min maintenance by 90 volume %B；
3) content calculates: with recombinant human nerve growth factor main peak peak area in Recombinant Chinese cobra NGF Chinese hamster ovary celI fermentation liquid
It is calculated by the equation of linear regression of recombinant human nerve growth factor standard items standard curve.
2. the method for quickly detecting recombinant human nerve growth factor content in Chinese hamster ovary celI fermentation liquid described in claim 1, feature
Be, the recombinant human nerve growth factor standard items standard curve the preparation method comprises the following steps:
Accurately weighing recombinant human nerve growth factor standard items is configured to 1.0mg/ml standard items stock solution, then is diluted to different dense
The standard solution of degree takes the standard solution of diluted concentration gradient, 20 μ L Injection volumes, with nerve growth factor main peak peak face
Long-pending and concentration makees linear regression, obtains equation of linear regression.
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| CN108918694B (en) * | 2018-05-02 | 2021-05-25 | 未名生物医药有限公司 | HPLC pre-column derivatization detection method for MSX residues |
| CN114252519B (en) * | 2020-09-23 | 2023-11-24 | 舒泰神（北京）生物制药股份有限公司 | Method for determining purity of nerve growth factor |
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| CN104931598A (en) * | 2014-03-21 | 2015-09-23 | 舒泰神（北京）生物制药股份有限公司 | Method for determining content of nerve growth factor (NGF) in nerve growth factor preparation |
| CN105223283A (en) * | 2014-07-01 | 2016-01-06 | 舒泰神（北京）生物制药股份有限公司 | The assay method of nerve growth factor content |
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| WO2000061634A2 (en) * | 1999-04-12 | 2000-10-19 | The Regents Of The University Of California | Ecotin-variant modulators of serine proteases |
| CN1566941A (en) * | 2003-07-08 | 2005-01-19 | 北京三诺佳邑生物技术有限责任公司 | Method for measuring nerve growth factor content in preparation |
| CN104931598A (en) * | 2014-03-21 | 2015-09-23 | 舒泰神（北京）生物制药股份有限公司 | Method for determining content of nerve growth factor (NGF) in nerve growth factor preparation |
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