CN108918694A - Derivatization detection method before a kind of remaining HPLC column of MSX - Google Patents
Derivatization detection method before a kind of remaining HPLC column of MSX Download PDFInfo
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Abstract
The invention discloses derivatization detection methods before a kind of remaining HPLC column of MSX.Standard solution is drawn including precision or solution to be measured is added in centrifuge tube, triethylamine solution and phenyl isothiocyanate solution is added, is reacted at room temperature after mixing, adds n-hexane mixing, it stands, obtains derivatization standard items or derivatization sample to be tested after removing phase membrane filtration；Derivatization standard items or derivatization sample to be tested are loaded in HPLC column and are detected respectively；Sample introduction is analyzed for the chromatographic condition detected according to above-mentioned HPLC, draws standard curve with various concentration gradient concentration x, the peak area y of standard solution；The peak area of the MSX of solution to be measured is substituted into resulting standard curve again, to calculate the content of MSX in solution to be measured.It is quick and convenient, accuracy is high, reproducible, at low cost, applicability is good, be able to satisfy the detection needs of different MSX residual quantity levels, medicament research and development and the quality of production control in have great application value.
Description
Technical field
The present invention relates to derivatizations before the detection method of biochemistry more particularly to a kind of remaining HPLC column of MSX to detect
Method.
Background technique
Chinese hamster ovary cell (Chinese Hamster Ovary, CHO) is most popular foreign protein eukaryon
One of expression system.Wherein CHO-GS cell system is by the target gene of foreign protein and glutamine synthelase (GS) gene mark
The expression vector transfection of note is in Chinese hamster ovary celI, by the mortifier L-Methionine sulphoxide imine (L- that glutamine synthelase is added
Methionine Sulphoximine, MSX), the destination protein gene that can make GS gene and be attached thereto expands together, reaches
The purpose for improving destination protein gene expression dose, to improve destination protein yield.
But the chemistry of MSX, physics and toxicity properties are not yet completely studied, in recombinant protein product
Impurity residual may have influence to drug safety.According to Chinese Pharmacopoeia 2015 editions《The guidance of 9102 drug impurity analysis of general rule
Principle》Requirement, MSX belongs to technique related impurities, and drug standard answers strict regulations toxic impurities content.But MSX is in purple
There is no absorption peak outside, lack corresponding coloration method yet, causes current MSX method for detecting residue research not perfect, lack logical
With the detection method of property.It is reported that (HPLC-MS), which is used in conjunction, using high performance liquid chromatography and mass spectrum can detecte MSX content,
But equipment needed for this method is expensive, and cost is difficult to vast pharmaceutical manufacturer and research institution is born.Therefore, MSX residual inspection
It surveys and is difficult to be promoted in conventional medicament research and development and enterprise practical production.
Summary of the invention
The purpose of the present invention is to provide derivatization detection method before a kind of remaining HPLC column of measurement MSX, this method letters
It is single reliable, reproducible, convenient for being used in medicament research and development and actual production.
To achieve the above object, the present invention provides derivatization detection method before a kind of remaining HPLC column of MSX, and feature exists
In,
Sample pre-treatments：Precision is drawn standard solution or solution to be measured and is added in centrifuge tube, and triethylamine solution and different is added
Thiocyanic acid phenyl ester solution, reacts at room temperature after mixing, adds n-hexane and is uniformly mixed, stands, remove phase membrane filtration
After obtain derivatization standard items or derivatization sample to be tested；
HPLC detection：Derivatization standard items or derivatization sample to be tested are loaded in HPLC column and are detected respectively；
Obtain result：Sample introduction is analyzed for the chromatographic condition detected according to above-mentioned HPLC, with the various concentration gradient of standard solution
Concentration is x, peak area is that y draws standard curve；Again by the peak area of the MSX of solution to be measured, resulting standard curve is substituted into, from
And calculate the content of MSX in solution to be measured.
Further, in the sample pre-treatments step, standard solution or solution to be measured：Triethylamine solution：Isothiocyanic acid benzene
Ester solution：The volumetric usage ratio (1~3) of n-hexane：(0.5~2)：(0.5~2)：(2~5)；Preferably, volumetric usage ratio is
2：1：1：4.
Further, in the sample pre-treatments step, the reaction time at room temperature is 0.5-1.5h；Preferably, room temperature
The lower reaction time will be 1h.
Further, in the sample pre-treatments step, n-hexane is added and is uniformly mixed as n-hexane vortex oscillation is added；It is excellent
Choosing, the time of vortex oscillation is 5-15min；It is furthermore preferred that the time of vortex oscillation is 10min.
Further, in the sample pre-treatments step, the time of standing is 20-40min；Preferably, the time of standing is
30min。
Further, in the sample pre-treatments step, filter membrane is using 0.45 μm of filter membrane.
Further, the condition of the HPLC is that mobile phase uses mobile phase A and Mobile phase B；Wherein mobile phase A is
The mixed solution of pH6.5,0.1mol/L sodium acetate and acetonitrile, Mobile phase B are 80 volume % acetonitrile solutions；Preferably, it flows
Phase A is the pH6.5 of 93 parts by volume, the mixed solution of the acetonitrile of 0.1mol/L sodium acetate and 7 parts by volume；
Further, the condition of the HPLC is that elution starts the mobile phase A using 100% volume；Start to be changed within 15 minutes
+ 15 volume % Mobile phase B of 85 volume % mobile phase A；Start within 18 minutes to be changed to+24 volume % mobile phase of 76 volume % mobile phase A
B；Start within 25 minutes to be changed to+40 volume % Mobile phase B of 60 volume % mobile phase A；Start within 30 minutes to be changed to 60 volume % mobile phase As
+ 40 volume % Mobile phase Bs；Start within 30.01 minutes to be changed to 100 volume % Mobile phase Bs；Start within 40.01 minutes to be changed to 100 volume %
Mobile phase A.
Further, the condition of the HPLC is, using 3000 high performance liquid chromatograph of DIONEX UltiMate；
Preferably, the chromatographic column used is reverse phase C18 chromatographic column；It is furthermore preferred that be Sepax AAA, specification be 4.6mm ×
250mm×5μm；
Optional, chromatogram column temperature is 36 DEG C.
Further, the condition of the HPLC is flow rate of mobile phase 1.0mL/min；
Optional, sample volume is 100 μ L；
Optional, UV Detection wavelength is 254nm.
For MSX, disclosed documents and materials there is no to show that derivatization is suitable for MSX or has suitable derivatization
Reagent.The present invention is studied by the chemical structure to MSX, it is believed that its chemical composition includes amino acid similar structures and sulfoxide
Two parts of imine structure, wherein amino acid similar structures have the potentiality as derivatization target spot.And phenyl isothiocyanate is
A kind of wide spectrum derivatization reagent that can be reacted with amino acid and imino acid, phenyl isothiocyanate and free amino acid reaction life
At thiocyanic acid phenyl ester-amino acid derivativges can be detected at ultraviolet 254nm.Therefore, the present invention creatively uses different
Thiocyanic acid phenyl ester performs the derivatization MSX, is examined using Universal efficient liquid chromatogram (HPLC) collocation UV detector to MSX
It surveys, and matches the detection common reverse phase C18 chromatographic column of small molecule compound, it is easy to operate, separating degree is good, to make
MSX remains to obtain fast and accurately quantitative detection, achieves unexpected technical effect.
The invention has the advantages that：
(1) detection method provided by the invention is easy to operate, quick and convenient, accuracy is high, reproducible, can treat test sample
Product MSX residual quantity is quantitatively judged, and has great application value in medicament research and development and the control of the quality of production.
(2) sample to be tested pre-column derivatization and detection time are shorter, can quickly detect to batch samples, detection
Speed is fast, at low cost, and applicability is good.
(3) accuracy in detection is high, and MSX has good linear relationship, energy within the scope of 1.5625 μm of ol/L~50 μm ol/L
Meet the detection needs of different MSX residual quantity levels.
(4) versatility of method provided by the invention is high, required instrument, derivatization reagent etc. be vast pharmaceutical manufacturer and
Research institution routinely configures, easy to operate, low in cost, is adapted for promoting and using on a large scale.
Detailed description of the invention
Fig. 1 is 1.5625 μm of ol/L standard solution HPLC test maps.1 is the peak MSX.
Fig. 2 is 3.1250 μm of ol/L standard solution HPLC test maps.1 is the peak MSX.
Fig. 3 is 6.2500 μm of ol/L standard solution HPLC test maps.1 is the peak MSX.
Fig. 4 is 12.5000 μm of ol/L standard solution HPLC test maps.1 is the peak MSX.
Fig. 5 is 25.0000 μm of ol/L standard solution HPLC test maps.1 is the peak MSX.
Fig. 6 is 37.5000 μm of ol/L standard solution HPLC test maps.1 is the peak MSX.
Fig. 7 is 50.0000 μm of ol/L standard solution HPLC test maps.1 is the peak MSX.
Fig. 8 is MSX canonical plotting.X-axis is MSX concentration (μm ol/L), and y-axis is peak area (mAUmin).
Fig. 9 is test map after water derivatization.
Figure 10 is containing 12.5 μm of ol/L MSX and 12.5 μm of ol/L MET mixing sample HPLC test maps.1 be the peak MSX, 2
For the peak MET.
Figure 11 is sample to be tested HPLC test map.
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, prepared by standard solution
50 μm of ol/L MSX standard reserving solutions are prepared：It weighs 0.9012g MSX and is dissolved in 0.1mol/L sodium acetate (pH
6.5)：Acetonitrile=93:In 7 (V/V) solution, it is settled to 1L.
25 μm of ol/L MSX standard solution are prepared：Above-mentioned 50 μm of ol/L MSX standard reserving solution is added isometric
0.1mol/L sodium acetate (pH 6.5)：Acetonitrile=93:7 (V/V) solution.
Various concentration gradient standard solution is prepared：Draw 1000,750,500,250,125,62.5,31.25 μ L's respectively
50 μm of ol/L MSX standard reserving solutions, are separately added into the 0.1mol/L acetic acid of 0,250,500,750,875,937.5,968.75 μ L
Sodium (pH6.5)：Acetonitrile=93:7 (V/V) solution are settled to 1mL, obtain concentration difference 50,37.5,25,12.5,6.25,
3.125, the concentration gradient standard solution of 1.5625 μm of ol/L.Concentration of standard solution gradient preparation method such as table 1.
1 various concentration gradient standard solution of table is with tabulation
2, derivatization reagent is prepared
Triethylamine solution：14mL triethylamine and 86mL acetonitrile are mixed evenly gained.
Phenyl isothiocyanate solution：It is made into the acetonitrile solution of the phenyl isothiocyanate of 12.5 μ L/mL.
3, Derivatization Method
Precision draws 200 μ L standard solution or solution to be measured (Chinese hamster ovary celI recombinant human nerve growth factor stoste, lot number
L01, Wei Ming biological medicine Co., Ltd) it is added in 1.5mL centrifuge tube, the above-mentioned triethylamine solution of 100 μ L is added and 100 μ L are above-mentioned
Phenyl isothiocyanate solution, mixing, is placed in and reacts 1h at room temperature, add 400 μ L n-hexanes, vortex oscillation 10min.It stands
30min is removed mutually with obtaining derivatization sample to be tested after 0.45 μm of membrane filtration.
The preparation method of the Chinese hamster ovary celI recombinant human nerve growth factor stoste is：Use the weight containing mature 118 amino acid
Group growth factor of human nerve DNA sequences encoding (mature peptide, 118 amino acid, GenBank No.V01511) and glutamine close
It transfects at the expression vector of enzyme (GS) genetic marker in Chinese hamster ovary cell (Chinese hamster ovary celI), Chinese hamster ovary celI seed cell is in CD
FortiCHO culture medium (article No.：A1148301, GIBCO company of the U.S.) in culture 10d after take recombinant human nerve growth factor CHO
Cell fermentation liquid, 4000rpm are centrifuged 5min, take supernatant to be purified, purification process is shown in Chinese patent application file
CN106478801A obtains Chinese hamster ovary celI recombinant human nerve growth factor stoste using the method purifying of embodiment 1.
4, HPLC-UV testing conditions
Testing conditions are：
High performance liquid chromatograph：3000 high performance liquid chromatograph of DIONEX UltiMate (including online degasser, quaternary
Pump, automatic injector, column oven configure 7 work station of DAD detector C hromeleon).
Chromatographic column：Sepax AAA, specification are 4.6mm × 250mm × 5 μm, i.e. internal diameter 4.6mm, column length 250mm, filler grain
5 μm of diameter；
Chromatogram column temperature：36℃；
Mobile phase：Mobile phase A is 0.1mol/L sodium acetate (pH6.5)-acetonitrile=93:7 (V/V), Mobile phase B 80%
(V/V) acetonitrile solution；
Elution program：Mobile phase A and Mobile phase B gradient elution ratio are shown in Table 2；
2 mobile phase A of table and Mobile phase B gradient elution volume ratio table
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 15 | 85 | 15 |
| 18 | 76 | 24 |
| 25 | 60 | 40 |
| 30 | 60 | 40 |
| 30.01 | 0 | 100 |
| 40 | 0 | 100 |
| 40.01 | 100 | 0 |
| 45 | 100 | 0 |
Flow rate of mobile phase：1.0mL/min；
Sample volume：100μL；
UV Detection wavelength：254nm；
5, prepared by standard curve
After various concentration gradient standard solution is performed the derivatization by above-mentioned derivatising condition, according to above-mentioned chromatographic condition sample introduction
Analysis, with concentration be x, peak area is that y draws standard curve, the results are shown in Table 4.Various concentration gradient standard solution HPLC detection is folded
Map is added to see that Fig. 1-Fig. 7, standard curve are shown in Fig. 8.In Fig. 1-7,1 be the peak MSX, peak height from high to low be respectively 50,37.5,25,
12.5, the MSX standard solution of 6.25,3.125,1.5625 μm of ol/L.From table 3 and Fig. 1-2 can be seen that MSX 1.5625~
It is in good linear relationship, regression equation y=0.3339x-0.4705, R within the scope of 50 μm of ol/L2=0.9986.
3 standard curve testing result table of table
| Standard solution number | Concentration (μm ol/L) | Peak area (mAUmin) |
| 7 | 1.5625 | 0.2300 |
| 6 | 3.1250 | 0.5837 |
| 5 | 6.2500 | 1.6480 |
| 4 | 12.5000 | 3.6126 |
| 3 | 25.0000 | 7.4376 |
| 2 | 37.5000 | 12.3519 |
| 1 | 50.0000 | 16.2277 |
6, specificity is tested
Above-mentioned 25 be prepared μm ol/L MSX standard solution, 100 μ L is taken, the L-Methionine (MET) of 25 μm of ol/L is added
100 μ L of aqueous solution, preparation contain the mixed solution of 12.5 μm of ol/L MSX and 12.5 μm of ol/L L-Methionines (MET).It takes respectively
Water, each 200 μ L of above-mentioned gained mixed solution after respectively performing the derivatization by above-mentioned derivatising condition, are measured by above-mentioned testing conditions.
Test map is shown in Fig. 9, Figure 10.1 is the peak MSX in Figure 10, and 2 be the peak MET.
It can be seen that water derivatization sample in the position MSX without absorption peak from Fig. 9 and Figure 10；The retention time of MSX is
The retention time of 13.747min, MET are 24.803min, and separating degree between the two is that 13.24, MET does not influence MSX residual quantity
Detection, illustrate that this method specificity reaches requirement.
7, accuracy is tested
50 μm of ol/L MSX standard reserving solutions of 80,100,120 μ L are added in 50 μ L testing sample solutions respectively, then mend
Add 0.1mol/L sodium acetate (pH6.5)：Acetonitrile=93:7 (V/V) solution to 200 μ L are prepared into containing 20,25,30 μm of ol/L MSX
The testing sample solution of standard items, it is to be measured by each 3 parts of above-mentioned testing conditions measurement after being performed the derivatization by above-mentioned derivatising condition
Sample as a result, calculate the rate of recovery and relative standard deviation RSD, the results are shown in Table 5.As can be seen from Table 5, three concentration levels
RSD is respectively less than 2%, illustrates that this method accuracy is high.
4 accuracy experimental result table of table
8, Intermediate precision is tested
25 μm of ol/L MSX standard solution are taken, after derivatization, 0 is placed respectively, is surveyed respectively by above-mentioned testing conditions afterwards for 24 hours
Determine as a result, record peak area, calculates relative standard deviation RSD, the results are shown in Table 5.As can be seen from Table 5, different time detects
RSD is 0.720%, less than 2%, illustrates that this method Intermediate precision is high.
5 Intermediate precision experimental result table of table
9, repeated experiment
50 μm of ol/L MSX standard items stock solutions of 80,100,120 μ L are taken respectively, then add 0.1mol/L sodium acetate
(pH6.5)：Acetonitrile=93:7 (V/V) solution to 200 μ L prepare 20,25,30 μm of ol/L MSX standard solution, after derivatization,
By above-mentioned testing conditions respectively measure 3 parts of samples as a result, calculating average peak area, standard deviation and relative standard deviation, as a result
It is shown in Table 6.The RSD of three concentration levels is respectively less than 2%, illustrates that this method is reproducible.
6 repeated experiment result table of table
10, the remaining detection of MSX in sample to be tested
Analyte sample fluid after step 3 of learning from else's experience derivatization is detected by above-mentioned same detection condition, calculates sample to be tested
The peak area of middle MSX substitutes into resulting standard curve, calculates the content of MSX in sample to be tested.The result is shown in Figure 11.From Figure 11
As can be seen that sample to be tested HPLC test map shows that Chinese hamster ovary celI recombinant human nerve growth factor is former not in the position MSX appearance
MSX residual is guaranteed lower than detection limit, safety in liquid.
Protection scope of the present invention is not limited to remaining to MSX in Chinese hamster ovary celI recombinant human nerve growth factor stoste
Detection, for other kinds of sample (such as Chinese hamster ovary celI recombinant protein, the stoste of antibody and preparation), those skilled in the art
It has the ability that corresponding pretreatment mode is selected to obtain sample to be tested, the chromatographic condition and standard curve in the present embodiment are to it
It is equally applicable.
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, preferred embodiment is not considered as limiting the invention, and those skilled in the art are not departing from this hair
Above-described embodiment can be changed, modified, replaced and become within the scope of the invention in the case where bright principle and objective
Type.
Claims (10)
1. derivatization detection method before a kind of remaining HPLC column of MSX, which is characterized in that
Sample pre-treatments：Precision draws standard solution or solution to be measured is added in centrifuge tube, and triethylamine solution and different sulphur cyanogen is added
Acid phenenyl ester solution, reacts at room temperature after mixing, adds n-hexane and is uniformly mixed, stands, and obtains after removing phase membrane filtration
To derivatization standard items or derivatization sample to be tested；
HPLC detection：Derivatization standard items or derivatization sample to be tested are loaded in HPLC column and are detected respectively；
Obtain result：Sample introduction is analyzed for the chromatographic condition detected according to above-mentioned HPLC, with the various concentration gradient concentration of standard solution
It is that y draws standard curve for x, peak area；Again by the peak area of the MSX of solution to be measured, resulting standard curve is substituted into, to count
Calculate the content of MSX in solution to be measured.
2. derivatization detection method before the remaining HPLC column of MSX as described in claim 1, which is characterized in that before the sample
It manages in step, standard solution or solution to be measured：Triethylamine solution：Phenyl isothiocyanate solution：The volumetric usage ratio (1 of n-hexane
~3)：(0.5~2)：(0.5~2)：(2~5)；Preferably, volumetric usage ratio is 2：1：1：4.
3. derivatization detection method before the remaining HPLC column of MSX as described in claim 1, which is characterized in that before the sample
It manages in step, the reaction time at room temperature is 0.5-1.5h；Preferably, the reaction time is 1h at room temperature.
4. derivatization detection method before the remaining HPLC column of MSX as described in claim 1, which is characterized in that before the sample
It manages in step, n-hexane is added and is uniformly mixed as n-hexane vortex oscillation is added；Preferably, the time of vortex oscillation is 5-
15min；It is furthermore preferred that the time of vortex oscillation is 10min.
5. derivatization detection method before the remaining HPLC column of MSX as described in claim 1, which is characterized in that before the sample
It manages in step, the time of standing is 20-40min；Preferably, the time of standing is 30min.
6. derivatization detection method before the remaining HPLC column of MSX as described in claim 1, which is characterized in that before the sample
It manages in step, filter membrane is using 0.45 μm of filter membrane.
7. derivatization detection method before the remaining HPLC column of MSX as described in claim 1, which is characterized in that the item of the HPLC
Part is that mobile phase uses mobile phase A and Mobile phase B；Wherein mobile phase A is pH6.5, the mixing of 0.1mol/L sodium acetate and acetonitrile
Solution, Mobile phase B are 80 volume % acetonitrile solutions；Preferably, mobile phase A is the pH6.5 of 93 parts by volume, 0.1mol/L acetic acid
The mixed solution of sodium and the acetonitrile of 7 parts by volume.
8. derivatization detection method before the remaining HPLC column of MSX as described in claim 1, which is characterized in that the item of the HPLC
Part is that elution starts the mobile phase A using 100% volume；Start within 15 minutes to be changed to+15 volume % of 85 volume % mobile phase A stream
Dynamic phase B；Start within 18 minutes to be changed to+24 volume % Mobile phase B of 76 volume % mobile phase A；Start within 25 minutes to be changed to 60 volume % stream
Dynamic phase A+40 volume % Mobile phase B；Start within 30 minutes to be changed to+40 volume % Mobile phase B of 60 volume % mobile phase A；30.01 minutes
Start to be changed to 100 volume % Mobile phase Bs；Start within 40.01 minutes to be changed to 100 volume % mobile phase As.
9. derivatization detection method before the remaining HPLC column of MSX as described in claim 1, which is characterized in that the item of the HPLC
Part is, using 3000 high performance liquid chromatograph of DIONEX UltiMate；
Preferably, the chromatographic column used is reverse phase C18 chromatographic column；It is furthermore preferred that be Sepax AAA, specification be 4.6mm ×
250mm×5μm；
Optional, chromatogram column temperature is 36 DEG C.
10. derivatization detection method before the remaining HPLC column of MSX as described in claim 1, which is characterized in that the item of the HPLC
Part is flow rate of mobile phase 1.0mL/min；
Optional, sample volume is 100 μ L；
Optional, UV Detection wavelength is 254nm.
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| CN114166964A (en) * | 2021-11-23 | 2022-03-11 | 上海臻格生物技术有限公司 | Rapid detection method for methionine sulfoxide imide |
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| SOO HEAN GARY KHOO等: "Metabolic characterization of a hyper-productive state in an antibody producing NS0 myeloma cell line", 《METABOLIC ENGINEERING》 * |
| 张莹 等: "异硫氰酸苯酯柱前衍生法测定复方氨基酸注射液(3AA)中氨基酸含量", 《天津药学》 * |
| 曾文珊 等: "重组人甲状旁腺激素1-34的氨基酸组成分析", 《药物生物技术》 * |
| 池小彬 等: "基于过甲酸氧化法HPLC-PITC柱前衍生对胱氨酸、蛋氨酸的测定", 《南昌航空大学学报(自然科学版)》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114166964A (en) * | 2021-11-23 | 2022-03-11 | 上海臻格生物技术有限公司 | Rapid detection method for methionine sulfoxide imide |
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