CN103163234A - Deletion peptide, non-peptide impurities and detection method for peptide phase 11 of solid-phase synthesis thymulin alpha1 - Google Patents
Deletion peptide, non-peptide impurities and detection method for peptide phase 11 of solid-phase synthesis thymulin alpha1 Download PDFInfo
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Abstract
The invention discloses a deletion peptide, non-peptide impurities and a detection method for peptide phase 11 of solid-phase synthesis thymulin alpha1. The detection method comprises the following steps: eluting a sample of the peptide phase 11 of the solid-phase synthesis thymulin alpha1in a gradient mode by means of a liquid chromatography (LC)/mass spectrometer (MS), and obtaining an ion flow spectrum; if obtained ion flow spectrum has a peak with a peak area of 9.46% at the time of 4.44 min and has a peak with the peak area of 8.19% at the time of 14.86min, it shows that the deletion peptide is contained in the peptide phase 11 of the solid-phase synthesis thymulin alpha1; and if the obtained ion flow spectrum has a peak with a peak area of 0.91 at the time of 22.64min, it shows that the non-peptide impurities are contained in the peptide phase 11 of the solid-phase synthesis thymulin alpha1. According to the detection method, the fact that whether the deletion peptide or non-peptide impurities are contained in the peptide phase 11 of solid-phase synthesis thymulin alpha1 or not can be accurately and sensitively identified, and therefore the quality or purity of the thymulin alpha1 can be effectively guaranteed and controlled.
Description
Technical field
The present invention relates to disappearance peptide, non-peptide impurity and detection method in the synthetic Thymosin alpha 1 of solid phase, relate in particular to disappearance peptide, non-peptide impurity and the detection method in 11 peptide stages of the synthetic Thymosin alpha 1 of solid phase, belong to the synthetic Thymosin alpha 1 of solid phase field.
Background technology
Thymosin alpha 1 is isolated a kind of active peptides from thymosin fraction 5 (TF-5), 28 amino acid residues, consists of, and molecular weight is 3108.37.In TF-5, the content of Thymosin alpha 1 is 0.6%, is the important activity component of human thymocyte hormone.For example Thymosin alpha 1 can be regulated the growth of T lymphocyte, differentiation and ripe.In addition, Thymosin alpha 1 can be repaired impaired T lymphocyte.Although the Thymosin alpha 1 separated from TF-5 is without obvious toxicity, by artificial solid phase, synthetic commercially available Thymosin alpha 1 can cause bad reaction because of impure.
At present, the working specification of the synthetic Thymosin alpha 1 of artificial solid phase is stereotyped, does not almost optimize space.Commercially available protected amino acid and the purity of related reagent also are enough to support artificial solid phase synthesis of high purity Thymosin alpha 1.Under the not amiss prerequisite of working specification, whether the synthetic Thymosin alpha 1 of artificial solid phase is pure, depends on the situation of the disappearance peptide that it contains.
Different from small-molecule drug, the biologically active of polypeptide is very strong.In artificial solid phase in synthetic Thymosin alpha 1, even exist micro-disappearance peptide also can cause serious toxic and side effect.The key of controlling the quality of the synthetic Thymosin alpha 1 of artificial solid phase is to control the disappearance peptide.At present both do not disclose the disappearance peptide situation in the synthetic Thymosin alpha 1 of artificial solid phase both at home and abroad, openly do not measured the method for the disappearance peptide in the synthetic Thymosin alpha 1 of artificial solid phase yet.This state is not suitable with the role in clinical of Thymosin alpha 1.In order to improve this situation, ensure the clinical drug safety of Thymosin alpha 1, formed the present invention.
Summary of the invention
Whether the 11 peptide stages that one of purpose of the present invention is to provide a kind of synthetic Thymosin alpha 1 of detection solid phase that can be accurate, sensitive contain the method for disappearance peptide or non-peptide impurity;
Another object of the present invention is to provide the disappearance peptide spectrum in 11 peptide stages of the synthetic Thymosin alpha 1 of solid phase;
Three of purpose of the present invention is that the disappearance peptide spectrum in 11 peptide stages of the synthetic Thymosin alpha 1 of solid phase is applied to the quality control of Thymosin alpha 1 solid phase in synthetic.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
Whether a kind of 11 peptide stages of detecting the synthetic Thymosin alpha 1 of solid phase contain the method for disappearance peptide or non-peptide impurity, comprise the following steps: adopt the LC/MS combined instrument to carry out gradient elution in 11 peptide stage samples of the synthetic Thymosin alpha 1 of solid phase and obtain the ion flow pattern; If the ion flow pattern obtained the peak that peak area is 9.46% occurs or occurs that when 14.86min peak area is respectively 8.19% peak, illustrates that 11 peptides of the synthetic Thymosin alpha 1 of solid phase contain the disappearance peptide in the stage when 4.44min; If the peak that peak area is 0.91% appears in the ion flow pattern obtained when 22.64min min, illustrate that 11 peptides of the synthetic Thymosin alpha 1 of solid phase contain non-peptide impurity in the stage.
The amino acid sequence of 11 peptides of Thymosin alpha 1 of the present invention is shown in SEQ ID No.1.
Described gradient elution is preferably with 0.1% formic acid/acetonitrile gradient wash-out, and flow velocity is 0.4ml/min, and gradient is: 0-5min, 0.1% formic acid/acetonitrile=97/3; 5-30min, 0.1% formic acid/acetonitrile=75/25.
LC in the inventive method in the LC/MS combined instrument adopts Agilent 1200 auto injections, and the specification of Waters Xterra RP18 analytical column is 5 μ m, 3.0 * 150mm; MS in the LC/MS combined instrument adopts Bruker Solatix FT-ICR-MS, ion current detecting device.
The present invention further provides the disappearance peptide spectrum in 11 peptide stages of the synthetic Thymosin alpha 1 of solid phase, its amino acid sequence is shown in SEQ ID No.2 or SEQ ID No.3;
Quality control substance during disappearance peptide spectrum provided by the present invention or non-peptide impurity are synthetic as the Thymosin alpha 1 solid phase, be applied to improve quality or the purity that solid phase is synthesized Thymosin alpha 1.
The inventive method can be accurate, sensitive the 11 peptide stages that identify the synthetic Thymosin alpha 1 of solid phase whether contain disappearance peptide or non-peptide impurity, can effectively ensure and control the quality of Thymosin alpha 1, in the synthetic Thymosin alpha 1 of preparation high-purity solid phase, significant application value be arranged.
The accompanying drawing explanation
The ion current of Fig. 1 Thymosin alpha 1 11 fragments of peptides Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn.
The mass spectrum of Fig. 2 Thymosin alpha 1 11 fragments of peptides Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn.
The mass spectrum of Fig. 3 Thymosin alpha 1 11 fragments of peptides disappearance peptide Glu-Lys-Lys-Val-Val-Glu-Glu-Ala-Glu-Asn.
The mass spectrum of 11 fragments of peptides disappearance peptide Glu-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn of Fig. 4 Thymosin alpha 1.
The mass spectrum of the non-peptide impurity that 11 fragments of peptides of Fig. 5 and Thymosin alpha 1 coexist.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can modify or replace details and the form of technical solution of the present invention, but these modifications and replacement all fall within the scope of protection of the present invention.
1. prepare Fmoc-Asn (Trt)-Wang Resin
Take 10g (3.1mmol) Wang Resin and be placed in the synthetic post of solid phase reaction.Add 60ml DCM swelling resin 10min, take DCM away.Wash resin three times with DMF, each 50ml, blow two minutes, takes solvent away.Take 5.5g (9.3mmol) Fmoc-Asn (Trt) and 1.38g (10.23mmol) HOBt in the ground triangular flask, add 50ml DMF to dissolve.Add 2.15ml (13.95mmol) DIC activation 5min under cooling in ice-water bath.Amino acid after activation is added in the resin washed with DMF, then add 0.23g (1.86mmol) DMAP, blow and stirred with nitrogen, room temperature reaction 2-4h.Reaction is washed resin three times with DMF after finishing, and each 50ml, blow two minutes, drains solvent.The resin that takes a morsel, shrink three times with methyl alcohol, and vacuum drying, to constant weight, is surveyed substitution value, as substitution value reaches expection, resin is sealed to unreacted hydroxyl with acetic anhydride, after capping 2-4h, with DMF, washes resin three times, and each 50ml, blow two minutes, takes solvent away.With methyl alcohol shrinkage resin three times, each 50ml, air blowing 5min, take solvent away, and vacuum drying, to constant weight, is surveyed substitution value.As substitution value reaches expection.
2.Fmoc-Asn the swelling of (Trt)-Wang Resin and de-Fmoc
Fmoc-Asn obtained above (Trt)-Wang Resin also is placed in the synthetic post of solid phase reaction, adds 60mlDCM swelling 10min, then takes DCM away.Use the DMF washing resin, use 50ml DMF at every turn, wash 2min at every turn, wash altogether three times.The DMF solution of the piperidine that is 20% by concentration takes off Fmoc, takes off twice altogether, and each 50ml once takes off 3min, another de-8min.Then use the DMF washing resin, wash 2min with 50mlDMF at every turn, wash altogether four times.Finally with DCM, wash Asn (Trt)-Wang Resin, wash 2min at every turn, wash altogether twice.Take solvent away.Resin is to the triketohydrindene hydrate displaing yellow.
3. prepare Glu (OtBu)-Asn (Trt)-Wang Resin
Take 2.12g (4.98mmol) Fmoc-Glu (OtBu) and 0.74g (5.5mmol) HOBt in dry ground triangular flask, dissolve with 50ml DMF, it is cooling that solution is put ice bath.Add 1.15ml (7.47mmol) DIC activation 5min.Then the amino acid after activation is added in Asn obtained above (Trt)-Wang Resin and reacts 2h.Take reactant liquor away.Use the DMF washing resin, use 50ml DMF at every turn, wash 2min at every turn, wash altogether three times.Resin does not develop the color to triketohydrindene hydrate.The DMF solution of the piperidine that is 20% by concentration takes off Fmoc, takes off twice altogether, each 50ml, de-3min, de-8min for the second time for the first time.Then use the DMF washing resin, wash 2min with 50ml DMF at every turn, wash altogether four times.Finally use the DCM washing resin, wash 2min at every turn, wash altogether twice.Take solvent away.Resin is to the triketohydrindene hydrate displaing yellow.
4. preparation
Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-SertBu)-Ser(tBu)-Glu-(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-Asn(Trt)-Wang
Resin
According to the method for preparing Glu (OtBu)-Asn (Trt)-Wang Resin successively by 1.55g (4.98mmol) Fmoc-Ala, 2.12g (4.98mmol) Fmoc-Glu (OtBu), 2.12g (4.98mmol) Fmoc-Glu (OtBu), 2.53g (7.47mmol) Fmoc
-Val, 2.53g (7.47mmol)fmoc
-Val, 3.17g (7.47mmol)fmoc-Glu (OtBu), 3.50g (7.47mmol) Fmoc-Lys (Boc), 4.67g (9.96mmol) Fmoc-Lys (Boc), 4.23g (9.96mmol) Fmoc-Glu (OtBu), 4.67g (9.96mmol) Fmoc-Lys (Boc), 3.52g (9.96mmol) Fmoc-Leu, 4.09g (9.96mmol) Fmoc-Asp (OtBu), 4.67g (9.96mmol) Fmoc-Lys (Boc), 3.96g (9.96mmol) Fmoc-Thr (tBu), 3.96g (9.96mmol) Fmoc-Thr (tBu), 3.52g (9.96mmol) Fmoc-Ile, 3.17g (7.47mmol) Fmoc-Glu (OtBu), 2.86g (7.47mmol) Fmoc-SertBu), 2.86g (7.47mmol) Fmoc-SertBu), 2.97g (7.47mmol) Fmoc-Thr (tBu), 3.07g (7.47mmol) Fmoc-Asp (OtBu), 2.53g (7.47mmol) Fmoc-Val, 2.32g (7.47mmol) Fmoc-Ala, 3.10g (9.96mmol) Fmoc-Ala, 4.09g (9.96mmol) Fmoc-Asp (OtBu) and 3.8l g (9.96mmol) Fmoc-Ser (tBu) are coupled on Glu (OtBu)-Asn (Trt)-Wang Resin and de-Fmoc.
5. preparation
Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-Ser(tBu)-Glu-(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-Asn(Trt)-WangResin
Under 0 ℃, 1.18ml (12.45mmol) acetic anhydride is mixed with 50ml DMF in dry ground triangular flask, then add 0.44ml (2.49mmol) DIPEA activation 5min.By acetic anhydride and Ser (tBu)-Asp (OtBu)-Ala-Ala-Val-Asp (OtBu)-Thr (tBu)-Ser (tBu)-Ser (tBu)-Glu (OtBu)-Ile-Thr (tBu)-Thr (tBu)-Lys-(Boc)-Asp (OtBu)-Leu-Lys (Boc)-Glu (OtBu)-Lys (Boc)-Lys (the Boc)-Glu (OtBu) after activation
-Val-Val-glu-(OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asn (Trt)-Wang Resin reacts 2h.Take reactant liquor away.DMF washing 4 times for resin, each 50ml DMF washes 2min at every turn.50ml DCM is used in DCM washing 2 times for resin at every turn, washes 3min at every turn.Take solvent away.Resin does not develop the color to triketohydrindene hydrate.Resin shrinks 3 times with MeOH, uses 50ml MeOH at every turn, shrinks 5min at every turn, drains.Obtain 18.5g dry peptide resin.Low temperature is preserved.
From
Ac-Ser (tBu)-Asp (OtBu)-Ala-Ala-Val-Asp (OtBu)-Thr (tBu)-Ser (tBu)-Ser (tBu)-Glu-(OtBu)-Ile-Thr (tBu)-Thr (tBu)-Lys (Boc)-Asp (OtBu)-Leu-Lys (Boc)-Glu (OtBu)-Lys (Boc)-Lys (Boc)-Glu (OtBu)-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asn (Trt)-WangResin cuts Thymosin alpha 1
Under 0 ℃ by the peptide resin of 18.5g drying and 180ml lysate (TFA/TIS/H
2o) reaction 0.5h, room temperature reaction 2.5h.After reaction finishes, with sand core funnel isolated by filtration resin.A small amount of TFA washing three times for resin.The filtrate merged goes out the Thymosin alpha 1 crude product with the ether sedimentation of the 1800ml of 0 ℃.Centrifuging goes out the Thymosin alpha 1 crude product.The Thymosin alpha 1 crude product washs thick peptide three times with the ether of 0 ℃, uses N
2blow away remaining ether, be placed in vacuum dryer and be dried to constant weight, obtain 6.27g Thymosin alpha 1 crude product, yield is 81%, and content is 68%.
Test example
The evaluation of the disappearance peptide spectrum of Fmoc-Glu (OtBu)-Lys (Boc)-Lys (Boc)-Glu (OtBu)-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu (OtBu)-Asn (Trt)-Wang Resin
Take a morsel
Fmoc-Glu (OtBu)-Lys (Boc)-Lys (Boc)-Glu (OtBu)-Val-Val-Glu (OtBu)-Glu (OtBu)-Ala-Glu-(OtBu)-Asn (Trt)-Wang Resin is not purified, according to standard method cutting deprotection.11 fragments of peptides Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn of the Thymosin alpha 1 obtained are at LC (Agilent 1200 auto injections, Waters Xterra RP18 analytical column, 5 μ m, 3.0 analyzed on * 150mm)/MS (Bruker Solatix FT-ICR-MS, ion current detecting device) combined instrument.With 0.1% formic acid/acetonitrile gradient wash-out, gradient is in Table 1.The ion flow pattern is shown in Fig. 1.Fig. 1 consists of 4 peaks, and they appear at respectively 4.44min, 5.85min, and 14.86min and 22.64min, their peak area is respectively 9.46%, 81.43%, and 8.19% and 0.91%.
The mass spectrogram of correspondence that appears at the peak of 5.85min is shown in Fig. 2, and the quality of this ion is 1303.69768, is that the Theoretical Mass several 1302.63033 of 11 fragments of peptides Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn of Thymosin alpha 1 adds H.
The mass spectrogram of correspondence that appears at the peak of 4.44min is shown in Fig. 3, the quality of this ion is 587.81044 * 2, is that Thymosin alpha 1 11 fragments of peptides Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn disappearance Glu Theoretical Mass several 1173.58774 add 2H.
The mass spectrogram of correspondence that appears at the peak of 14.86min is shown in Fig. 4, the quality of this ion is 1175.58020, is that the Theoretical Mass several 1174.53537 of 11 fragments of peptides Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn disappearance Lys of Thymosin alpha 1 adds H.
The mass spectrogram of correspondence that appears at the peak of 22.64min is shown in Fig. 5, and the quality of this ion is 1088.54119, and the disappearance peptide corresponding with 11 fragments of peptides Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn of Thymosin alpha 1, be not non-peptide impurity.
Claims (7)
1. whether the 11 peptide stages of detecting the synthetic Thymosin alpha 1 of solid phase contain the method that lacks peptide or non-peptide impurity, comprise the following steps: adopt the LC/MS combined instrument to carry out gradient elution in 11 peptide stages samples of the synthetic Thymosin alpha 1 of solid phase and obtain the ion flow pattern; If the ion flow pattern obtained the ion current peak that peak area is 9.46% occurs or occurs that when 14.86min peak area is respectively 8.19% ion current peak, illustrates that 11 peptides of the synthetic Thymosin alpha 1 of solid phase contain the disappearance peptide in the stage when 4.44min; If the ion current peak that peak area is 0.91% appears in the ion flow pattern obtained when 22.64min, illustrate that 11 peptides of the synthetic Thymosin alpha 1 of solid phase contain non-peptide impurity in the stage.
2. it is characterized in that in accordance with the method for claim 1: the amino acid sequence of 11 peptides of described Thymosin alpha 1 is shown in SEQ ID No.1.
3. in accordance with the method for claim 1, it is characterized in that: described gradient elution is that flow velocity is 0.4ml/min with 0.1% formic acid/acetonitrile gradient wash-out, and gradient is: 0-5min, 0.1% formic acid/acetonitrile=97/3; 5-30min, 0.1% formic acid/acetonitrile=75/25.
4. in accordance with the method for claim 1, it is characterized in that: the LC in described LC/MS combined instrument adopts Agilent 1200 auto injections, and the specification of Waters Xterra RP18 analytical column is 5 μ m, 3.0 * 150mm; MS in the LC/MS combined instrument adopts Bruker Solatix FT-ICR-MS, ion current detecting device.
5. it is characterized in that in accordance with the method for claim 1: the amino acid sequence of described disappearance peptide is shown in SEQ ID No.2 or SEQ ID No.3.
6. the disappearance peptide of 11 peptides in the stage of the synthetic Thymosin alpha 1 of solid phase composed, and it is characterized in that: its amino acid sequence is shown in SEQ ID No.2 or SEQ ID No.3.
7. disappearance peptide spectrum claimed in claim 6 improves the quality of the synthetic Thymosin alpha 1 of solid phase or the application in purity as quality control substance.
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| CN113092646A (en) * | 2021-04-07 | 2021-07-09 | 海南中和药业股份有限公司 | Gas phase analysis method for determining content of N, N-diisopropylcarbodiimide in polypeptide |
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Effective date of registration: 20201222 Address after: No. 584, xinqiong village, Dapu Town, Yongchun County, Quanzhou City, Fujian Province, 362600 Patentee after: Ye Shenghai Address before: 570311 No.4 Road, phase II, Yaogu Industrial Park, Haikou national high tech Zone, Hainan Province Patentee before: HAINAN HERUI PHARMACEUTICAL Co.,Ltd. |
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Effective date of registration: 20210112 Address after: Liu'an Development Zone, Yongchun County, Quanzhou City, Fujian Province (east side of Taoxi bridge) Patentee after: Yongchun County Product Quality Inspection Institute Fujian fragrance product quality inspection center, national incense burning product quality supervision and Inspection Center (Fujian) Address before: No. 584, xinqiong village, Dapu Town, Yongchun County, Quanzhou City, Fujian Province, 362600 Patentee before: Ye Shenghai |
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