WO2021114788A1 - Teriparatide impurity f - Google Patents

Abstract

The present invention relates to the technical field of impurity synthesis, and particularly to a teriparatide impurity F. It is discovered that aspartimide impurities will be produced during the production and storage process of teriparatide, named as impurity F, and the discovery and synthesis of the impurity is beneficial to the quality control of teriparatide active pharmaceutical ingredient; by using the synthesis method, the purity of a crude peptide can reach 42%, and the purity of a refined peptide can reach 92% after purification. Compared with the prior art, a product has a high purity, is easy to purify, and has a correspondingly increased yield.

Description

A Teriparatide Impurity F

This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on December 10, 2019, the application number is 201911259992.9, and the invention title is "a teriparatide impurity F", the entire content of which is incorporated into this application by reference in.

Technical field

The present invention relates to the technical field of impurity synthesis, in particular to a teriparatide impurity F.

Background technique

Teriparatide is a fragment 1-34 in human parathyroid hormone. This fragment has the same biological activity as human parathyroid hormone. It was developed by Eli Lilly Company of the United States for primary osteoporosis, Hypogonadal osteoporosis and osteoporosis in menopausal women. The peptide sequence is: H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu- Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp ³⁰ -Val ³¹ -His-Asn-Phe-OH.

Teriparatide produces a variety of impurities during its production and storage. The impurities currently reported include Trp23(C=O)-teriparatide oxidation impurities, Trp23(OH)-teriparatide oxidation impurities, and Met8 (O)-teriparatide oxidation impurity, Met18(O)-teriparatide oxidation impurity, Met8,18(O)-teriparatide oxidation impurity, etc. In order to ensure the safety and effectiveness of teriparatide drugs, the content of impurities in the product must be controlled. The discovery and synthesis of new impurities have important practical significance for the quality control of teriparatide bulk drugs.

Summary of the invention

In view of this, the present invention provides a teriparatide impurity F. The discovery and synthesis of this impurity is beneficial to the quality control of teriparatide raw materials; by adopting the synthesis method of the present invention, the purity of the crude peptide can reach 42%, and the purity of the refined peptide can reach 92% after purification.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a teriparatide impurity F, the structural formula of which is shown in formula I:

In the research and development process of this application, it was found that aspartimide impurities will be produced during the production and storage process, named as impurity F, that is, the affinity between ^{30 #} Asp and 31 ^{# Val in teriparatide residues} The nuclear substitution reaction forms a five-membered ring. The peptide sequence is:

H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu- Arg-Lys-Lys-Leu-Gln-Asu-Val-His-Asn-Phe-OH. Its amino acid sequence is shown in SEQ ID NO:1.

The invention also provides a method for synthesizing teriparatide impurity F, which includes the following steps:

Step A: Using solid phase synthesis method, using resin as carrier, sequentially coupling Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(ODmab)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc- Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser( tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu )-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH and Boc-Ser(tBu)-OH to obtain peptide resins with protecting groups; the PG protecting group in Asp(PG) is All or Dmab Protecting base

Step B: Remove the PG protecting group of Asp(PG) and close the ring to obtain a peptide resin;

Step C: The peptide resin is cleaved to obtain teriparatide impurity F.

The inventors used the methods reported in the literature (DBU catalysis or acid degradation), and the purity of the target product obtained was less than 1%. According to the conventional synthesis method (such as HOAt/PyAop/DIPEA ring closure), the purity of the target product obtained was only about 1%. , The products with higher purity cannot be obtained. The present invention provides a method for preparing teriparatide impurity F. The mechanism is that the ^{exposed carboxyl group of the side chain of 30 #} Asp and diphenyl azide phosphate form a more active acyl azide, which is combined with 31 ^# Val. The amino group undergoes nucleophilic substitution to obtain the target product. The product obtained by the method has high purity, is easy to purify, and the yield is correspondingly improved.

Preferably, in step A, the resin is Wang Resinn, and the degree of substitution is 0.1-3.0 mmol/g.

In the specific embodiment provided by the present invention, the substitution degree of Wang Resinn is 0.5 to 0.8 mmol/g.

Preferably, in step A, the coupling agent used for coupling is one of HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA or HOAt/PyAop/DIPEA Or several.

Preferably, in step A, the reagent used for coupling to remove the Fmoc protective group is a 10%-30% piperidine solution, and the solvent used to dissolve the amino acid and the piperidine solution is NMP, THF, DCM, DMF or DMSO. One or more.

Preferably, in step A, the reagent for removing the Fmoc protective group used for coupling is a 20% piperidine solution.

Preferably, in step B, the PG protecting group in Asp(PG) is an All protecting group, the catalyst used for removal is tetrakistriphenylphosphine palladium, and the scavenger is phenylsilane or morpholine.

Preferably, in step B, the PG protecting group in Asp(PG) is a Dmab protecting group, and the scavenger used for removal is a 1% to 5% hydrazine hydrate-DMF solution.

Preferably, in step B, the PG protecting group in Asp(PG) is a Dmab protecting group, and the scavenger used for removal is a 2% hydrazine hydrate-DMF solution.

Preferably, in step B, the ring is closed by diphenyl azide phosphate.

Preferably, in step C, the lysis solution used for lysis is a trifluoroacetic acid solution containing a capture agent, and the capture agent is _{one or more of PhSMe, PhOH, EDT, H 2} O, TIS, and PhOMe.

Preferably, the capture agent is TIS.

Preferably, the volume ratio of trifluoroacetic acid to TIS in the lysis solution is (90-99): (1-10).

Preferably, the volume ratio of trifluoroacetic acid to TIS in the lysis solution is 95:5.

The present invention provides a teriparatide impurity F. The structural formula of teriparatide impurity F is shown in formula I. The advantages of the present invention are as follows:

The present invention found that teriparatide will produce aspartimide impurities during its production and storage process, named as impurity F, that is, nucleophilicity occurs between ^{30 #} Asp and 31 ^{# Val in teriparatide residues.} The substitution reaction forms a five-membered ring. The discovery and synthesis of this impurity is conducive to the quality control of teriparatide API;

Using the method reported in the literature (DBU catalysis or acid degradation), the purity of the target product obtained is less than 1%. According to the conventional synthesis method (HOAt/PyAop/DIPEA ring closure), the purity of the target product obtained is only about 1%, and neither can be obtained. A product with higher purity; while using the method of the present invention, the purity of the crude peptide can reach 42%, and the purity of the refined peptide can reach 92% after purification. Compared with the prior art, the product obtained by the present invention has high purity, is easy to purify, and the yield is correspondingly improved.

Description of the drawings

Figure 1: Synthetic route of impurity F;

Figure 2: HPLC profile of impurity F crude peptide (Example 3);

Figure 3: HPLC profile of impurity F refined peptide (Example 8);

Figure 4: The first-order mass spectrum of impurity F;

Figure 5: The first-level mass spectrum of impurity F after digestion;

Figure 6: The secondary mass spectrum of the fragment with the parent ion M/Z of 1455 after impurity F digestion;

Figure 7: The secondary mass spectrum of the fragment with the parent ion M/Z of 886 after impurity F digestion;

Figure 8: The secondary mass spectrum of the fragment with the parent ion M/Z of 702 after impurity F digestion;

Figure 9: The secondary mass spectrum of the fragment with the parent ion M/Z of 872 after impurity F digestion;

Figure 10: The parent ion M/Z after impurity F digestion is the comparison of the theoretical secondary fragments of the 1455 segment of the measured secondary fragment peak and the target peptide sequence;

Figure 11: The parent ion M/Z is 886 after impurity F is digested with the measured secondary fragment peak and the theoretical secondary fragment comparison of the target peptide sequence;

Figure 12: Comparison of the measured secondary fragment peak of the fragment with the parent ion M/Z of 702 after impurity F digestion with the theoretical secondary fragment of the target peptide sequence;

Figure 13: Comparison of the measured secondary fragment peak of the fragment with the parent ion M/Z of 872 after impurity F digestion with the theoretical secondary fragment of the target peptide sequence.

Detailed ways

The present invention discloses a teriparatide impurity F. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant persons can make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The abbreviations and their English meanings are as follows:

The method for specifically preparing teriparatide impurity F provided by the present invention includes the following steps:

1. Using Wang Resin as the carrier, the substitution degree is 0.1～3.0mmol/g, according to the peptide solid phase synthesis method, according to the peptide sequence, the protected amino acids are coupled in sequence from the C-terminus to the N-terminus. Among them, 30 ^# Asp uses Fmoc-Asp( PG)-OH, PG is All or Dmab protecting group, 1 ^# Ser uses Boc-Ser(tBu)-OH, and other residues use conventional amino acids. One or more of the coupling agents HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA are used to couple Fmoc-Asn( Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(PG)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc) -OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH , Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc -His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Met -OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH and Boc- Ser(tBu)-OH. PG is the All or Dmab protecting group, the reagent for removing the Fmoc protecting group is 20% piperidine solution, and the solvent used to dissolve the amino acid and 20% piperidine solution is one of NMP, THF, DCM, DMF and DMSO Or several.

2. Remove the PG protecting group. If PG is All, use tetrakistriphenylphosphine palladium catalysis and phenylsilane or morpholine as scavenger to remove; if PG is Dmab, use 2% hydrazine hydrate-DMF solution to remove.

3. First, use diphenyl azide phosphate to close the ring, and then use a trifluoroacetic acid solution containing a trapping agent to crack to obtain impurity F. The trapping agent is one of PhSMe, PhOH, EDT, H ₂ O, TIS, and PhOMe Or several, more preferably, the lysate is a TFA/TIS composition, wherein the volume ratio of TFA to TIS is 95:5.

The specific route is shown in Figure 1.

The teriparatide impurity F provided by the present invention and the reagents or instruments used in the preparation method thereof can be purchased from the market.

The following examples further illustrate the present invention:

Example 1: Coupling of amino acids

Weigh 62.5g (50mmol) of Wang Resin with a degree of substitution of 0.8mmol/g, add it to the solid phase reaction column, wash twice with DMF, swell the resin with DMF for 30 minutes, and weigh 19.37g (50mmol) of Fmoc-Phe- OH, 8.1g (60mmol) HOBt and 6.1g (5mmol) DMAP were dissolved in DMF, added 8.2g (65mmol) DIPCDI under ice bath, added to solid phase reaction column, reacted at room temperature for 1 hour, and washed with DMF 6 times. Then add 79.1g (1000mmol) pyridine and 102.1g (1000mmol) acetic anhydride mixture to seal the resin for 6 hours, wash with DMF 6 times, shrink and drain with methanol to obtain 71.4g Fmoc-Phe-Wang Resin, and the detection substitution degree is 0.3mmol/ g.

20% piperidine solution removes the Fmoc protective group (reaction time 5+7 minutes), and DMF washes 6 times. According to the peptide sequence, one or more of the coupling agents HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA are used for coupling in sequence Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(OAll)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc- Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc -Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu -OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH , Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val- OH and Boc-Ser(tBu)-OH.

Example 2: Coupling of amino acids

Weigh 100.0 g (50 mmol) of Wang Resin with a substitution degree of 0.5 mmol/g, add it to the solid phase reaction column, wash twice with DMF, swell the resin with DMF for 30 minutes, and weigh 38.74 g (100 mmol) of Fmoc-Phe- OH, 16.2g (120mmol) HOBt and 52.1g (100mmol) PyBOP were dissolved in DMF, 25.9g (200mmol) DIPEA was added under ice bath, added to the solid phase reaction column, reacted at room temperature for 2 hours, and washed with DMF 6 times. Then add 79.1g (1000mmol) pyridine and 102.1g (1000mmol) acetic anhydride mixture to seal the resin for 6 hours, wash with DMF 6 times, shrink and drain with methanol to obtain 115.4g Fmoc-Phe-Wang Resin, and the detection substitution degree is 0.26mmol/ g.

20% piperidine solution removes the Fmoc protective group (reaction time 5+7 minutes), and DMF washes 6 times. According to the peptide sequence, one or more of the coupling agents HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/DIPCDI, HATU/HOAt/DIPEA and HOAt/PyAop/DIPEA are used for coupling in sequence Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(ODmab)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc- Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc -Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu -OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH , Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val- OH and Boc-Ser(tBu)-OH.

Example 3: Synthesis of impurity F

Add 12.4 g (0.5 eq) of tetratriphenylphosphine palladium, 23.2 g (10 eq) of phenylsilane and 500 mL of dichloromethane to the peptide resin reaction column of Example 1, and react at room temperature for 1 hour. Remove the reaction solution and dichloromethane. Washed six times, washed with tetrahydrofuran three times, transferred to a three-necked flask, added 43.4g (20eq) of triethylamine, 117.9g (20eq) of diphenyl azide phosphate and 500mL of tetrahydrofuran, heated to reflux and reacted for 8 hours, cooled to room temperature, Filter, wash with tetrahydrofuran six times, shrink three times with methyl tert-butyl ether, and dry under vacuum to obtain 140 g of peptide resin.

Add lysis buffer (TFA:TIS=95:5, volume ratio) 1.2L to lyse at room temperature for 2 hours, filter, pour the filtrate into 12L ether to settle, centrifuge, wash, vacuum dry to obtain impurity F crude peptide 90g, purity 42.96% (Figure 2 ,Table 1).

Table 1

Example 4: Synthesis of impurity F

Add 2% hydrazine hydrate-DMF solution to the peptide resin reaction column of Example 2 to remove the Dmab protecting group (reaction time 10+10 minutes), wash with DMF 6 times, wash with tetrahydrofuran three times, transfer to a three-necked flask, and add triethyl Amine 30.4g (10eq), 82.6g (10eq) of diphenyl azide phosphate and 1L of tetrahydrofuran, heated to reflux for 15 hours, cooled to room temperature, filtered, washed with tetrahydrofuran six times, methyl tert-butyl ether shrinked three times, vacuum 200 g of peptide resin was obtained by drying.

Add lysis solution (TFA:H ₂ O=95:5, volume ratio) 1.6L for room temperature lysis for 2 hours, filter, pour the filtrate into 16L ether to settle, centrifuge, wash, and vacuum dry to obtain 120g of impurity F crude peptide with a purity of 40.35%.

Example 5: Synthesis of impurity F

Weigh 1/10 of the peptide resin of Example 1, add 1.2 g (0.5 eq) of tetrakistriphenylphosphine palladium, 2.3 g (10 eq) of phenylsilane and 50 mL of dichloromethane, and react at room temperature for 1 hour. The reaction solution is removed. Wash with dichloromethane for six times, weigh 1.6g (12mmol) HOAt and 5.2g (10mmol) PyAOP in DMF, add 2.6g (20mmol) DIPEA under ice bath, add to solid phase reaction column, react at room temperature for 15 hours, The reaction solution was removed, washed with DMF six times, MTBE contracted three times, and dried in vacuum to obtain 15 g of peptide resin. 150 mL of lysate (TFA:TIS=95:5, volume ratio) was added for lysis at room temperature for 2 hours, filtered, and the filtrate was poured into 1.5L of ether to settle, centrifuged, washed, and vacuum dried to obtain 9g of crude impurity F peptide with a purity of about 1%.

Example 6: Synthesis of impurity F

Take 5 g of teriparatide peptide resin, add 50% DBU-DMF solution, react at room temperature for 7 days, extract the reaction solution, wash with DMF six times, shrink three times with methyl tert-butyl ether, add the lysate (TFA:TIS) after vacuum drying =95:5, volume ratio) 50mL was lysed at room temperature for 2 hours, filtered, and the filtrate was poured into 500mL of ether to settle, centrifuged, washed, and vacuum dried to obtain 2g of crude impurity F peptide with a purity of 0.4%.

Example 7: Synthesis of impurity F

Dissolve 0.1 g of teriparatide refined peptide in 5 mL purified water, adjust the pH to 3 with dilute hydrochloric acid, heat to 90° C. and react for 3 days. The purity of impurity F is 0.8% by HPLC.

Example 8: Purification of Impurity F

The impurity F obtained in Example 2 was purified by HPLC. The sample amount was 30g/time. The fractions were collected according to the chromatographic peak, starting from about 10% at the beginning of the main peak, and the fractions were collected according to the chromatographic peak. Stop collecting at about 10%. The fraction was concentrated by rotary evaporation and lyophilized to obtain 15.5 g of impurity F refined peptide with a purity of 92.13% (Figure 3, Table 2).

1. HPLC preparation and purification chromatographic conditions are as follows:

(1) Mobile phase A1: 0.1% TFA solution, mobile phase B1: pure acetonitrile;

(2) Novasep LC150 chromatographic system, 15cm column with Welch Ultimate H S-C18 8μm

Packing (the theoretical plate number of the column efficiency value is not less than 5000);

(3) Monitoring wavelength: 220nm;

(4) Flow rate: 500mL/min

(5) Purification gradient:

Time (min)	Mobile phase A1 (%)	Mobile phase B1 (%)
0	90	10
5	75	25
105	65	35
115	30	70

2. HPLC analysis and detection chromatographic conditions are as follows:

(1) Mobile phase: Phase A: Acetonitrile: ammonium sulfate buffer = 10: 90 (V: V), Phase B: Acetonitrile: ammonium sulfate buffer = 50: 50 (V: V);

(2) Chromatographic column: Waters BEHPeptide 300-C18-1.7μm 2.1*100mm;

(3) Flow rate: 0.4mL/min;

(4) Monitoring wavelength: 214nm;

(5) Column temperature: 60℃;

(6) Detection gradient:

Time (min)	Mobile phase A (%)	Mobile phase B (%)
0	100	0
5	65	35
29	61	39
30	0	100
35	0	100
35.1	100	0
40	100	0

Table 2

Example 9: Confirmation of structure of impurity F

In order to determine the structure of impurity F, it was subjected to primary mass spectrometry and treated with Trypsin enzyme to analyze the primary and secondary mass spectra after digestion.

1. Experimental conditions:

(1) Instrument model: MALDI-TOF-TOF, Autoflex Speed;

(2) Primary mass spectrometry

Laser wavelength and frequency: ND: YAG 355nm, 1000Hz;

Polarity: Positive;

Operation mode: RP_700-3500;

Detector voltage: 2000V;

Analysis software: FlexAnalysis;

Target model: MTP 384 ground steel;

Substrate: DHB is dissolved into 20mg/mL with 30% acetonitrile aqueous solution;

Matrix and sample: mix in a 1:1 volume ratio, dry naturally, and put into the mass spectrometer test chamber.

(3) Secondary mass spectrometry

Laser wavelength and frequency: ND: YAG 355nm, 1000Hz;

Polarity: Positive reflector;

Operation mode: LIFT;

Detector voltage: 2000V;

Analysis software: FlexAnalysis, BioTools, Sequence Editor;

Target model: MTP 384 ground steel;

Substrate: DHB is dissolved into 20mg/mL with 30% acetonitrile aqueous solution;

Matrix and sample: mix in a 1:1 volume ratio, dry naturally, and put into the mass spectrometer test chamber.

(4) Sample treatment before digestion: the impurity F refined peptide was dissolved with 0.1% TFA solution into an aqueous solution with a concentration of about 0.1 mg/mL, and the solution was analyzed by mass spectrometry.

(5) Treatment of digested samples: Dissolve the impurity F refined peptide with 50mM ammonium bicarbonate solution into an aqueous solution with a concentration of about 1mg/mL, digest it with Trypsin, and analyze the primary and secondary mass spectra after digestion.

(6) The results are as follows:

1) The first-stage mass spectrum result of impurity F is shown in Figure 4.

2) Figure 5 shows the mass spectrum result of impurity F after digestion.

3) The results of the secondary mass spectrometry after impurity F digestion are shown in Figure 6-9.

4). The results of MALDI-TOF-TOF secondary mass spectrometry processing with BioTools software are shown in Figure 10-13.

(7) Data analysis

Table 3: Comparison of measured mass spectra and theoretical values of impurity F

sample	Theoretical value of primary mass spectrometry	Measured value of primary mass spectrometer
Impurity F	4100.727	4100.824

Table 4: Comparison of actual mass spectra and theoretical values after impurity F digestion

Fragment peptide sequence	Theoretical value of primary mass spectrometry	Measured value of primary mass spectrometer
SVSEIQLMHNLGK	1,455.762	1,455.732
HLNSMER	886.420	886.461
VEWLR	702.393	702.387
LQDVHNF	872.426	872.406

A. It can be seen from Table 3 that the primary mass spectrum of impurity F is consistent with the theoretical value.

B. Trypsin can selectively hydrolyze the carboxyl terminal peptide bond of lysine or arginine in protein. From Table 4, it can be seen that the secondary fragment peak of the peptide fragment after impurity F digestion is consistent with the theory, the parent ion M/Z is 1455 The secondary fragment peaks of the peptides of 886, 702, and 872 are consistent with the theoretical fragment peaks of peptides SVSEIQLMHNLGK, HLNSMER, VEWLR, and LQDVHNF respectively. The LQDVHNF peptide is obtained by hydrolysis of LQ(Asu)VHNF during the digestion process.

Therefore, the peptide sequence of impurity F can be determined as: H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu -Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asu-Val-His-Asn-Phe-OH (SEQ ID NO:1).

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims (10)

1. A teriparatide impurity F is characterized in that its structural formula is as shown in formula I:

   
2. The teriparatide impurity F according to claim 1, wherein the synthesis method comprises the following steps:

   Step A: Using solid phase synthesis method, using resin as carrier, sequentially coupling Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Val-OH, Fmoc-Asp(ODmab)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc- Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser( tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(OtBu )-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH and Boc-Ser(tBu)-OH to obtain a peptide resin with protecting groups; the PG protecting group in Asp(PG) is All Or Dmab protecting group;

   Step B: Remove the PG protecting group of Asp(PG) and close the ring to obtain a peptide resin;

   Step C: The peptide resin is cleaved to obtain teriparatide impurity F.
3. The teriparatide impurity F according to claim 2, wherein in step A, the resin is Wang Resinn, and the degree of substitution is 0.1-3.0 mmol/g.
4. The teriparatide impurity F according to claim 2, characterized in that, in step A, the coupling agent used in the coupling is HOBt/DIPCDI, HOBt/PyBop/DIPEA, HBTU/HOBt/DIPEA, HOAt/ One or more of DIPCDI, HATU/HOAt/DIPEA or HOAt/PyAop/DIPEA.
5. The teriparatide impurity F according to claim 2, characterized in that, in step A, the reagent for removing the Fmoc protective group used in the coupling is a 10%-30% piperidine solution to dissolve amino acid and piperidine The solvent used in the pyridine solution is one or more of NMP, THF, DCM, DMF or DMSO.
6. The teriparatide impurity F according to claim 2, wherein in step B, the PG protecting group in the Asp(PG) is an All protecting group, and the catalyst used for the removal is tetratriphenyl Phosphine palladium, scavenger is phenylsilane or morpholine.
7. The teriparatide impurity F according to claim 2, wherein in step B, the PG protecting group in the Asp(PG) is a Dmab protecting group, and the scavenger used for the removal is 1% to 5% hydrazine hydrate-DMF solution.
8. The teriparatide impurity F according to claim 2, characterized in that, in step B, the ring closure adopts diphenyl azide phosphate ring closure.
9. The teriparatide impurity F according to any one of claims 2 to 8, wherein in step C, the lysate used in the lysis is a trifluoroacetic acid solution containing a capture agent, and the capture agent is One or more of PhSMe, PhOH, EDT, H ₂ O, TIS, and PhOMe.
10. The teriparatide impurity F according to claim 9, wherein the capture agent is TIS, and the volume ratio of trifluoroacetic acid to TIS in the lysate is (90-99): (1-10).

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