CN108373499B - A kind of purifying and ionic control method of Teriparatide acetate - Google Patents
A kind of purifying and ionic control method of Teriparatide acetate Download PDFInfo
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- CN108373499B CN108373499B CN201810119038.9A CN201810119038A CN108373499B CN 108373499 B CN108373499 B CN 108373499B CN 201810119038 A CN201810119038 A CN 201810119038A CN 108373499 B CN108373499 B CN 108373499B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
Abstract
The present invention provides the purifying and ionic control method of a kind of Teriparatide acetate, comprising the following steps: 1): thick peptide solution is carried out first time purifying using the acetonitrile system of ammonium bicarbonate aqueous solution；2): the eluent of above-mentioned collection being carried out second using sodium sulphate/sulfuric acid acetonitrile system and is purified；3): first using ammonium acetate aqueous solution acetonitrile system to elute the eluent of collection, then carry out efficient liquid phase with the elution of aqueous acetic acid acetonitrile system and turn salt；4): will turn that hydrochloric acid is added after the eluent after salt is concentrated under reduced pressure.The invention has the following advantages that being combined by addition hydrochloric acid progress acetate plasma content control after repeatedly purifying and turning salt; use different chromatographic systems; the Teriparatide acetic acid purity salt that prepare with scale obtains is greater than 99%; methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10%; acetate ion content≤5%; ≤ 0.1%, total recovery is greater than 60% for chloride ion content≤4%, trifluoroacetic acid root and sulfate radical content.
Description
Technical field
The present invention relates to a kind of purification process of polypeptide, more particularly to meet the Teriparatide acetic acid of 40 quality requirement of USP
The purifying and ionic control method of salt.
Background technique
Teriparatide (PTH (1-34)), English name Teriparatide are a kind of polypeptide drugs of long-chain, peptide sequence are as follows:
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-Val-His-Asn-Phe-OH
Teriparatide is the 1-34 amino acid fragment of human parathyroid hormone (PTH), by Eli Lilly company, the U.S. through gene
Recombinant technique research and development are made, and drug is made in the form of acetate, was approved by the FDA in the United States listing in 2002 years.It is first
It is approved for the bone formation-promoter for the treatment of severe osteoporosis, is had a vast market foreground.
Document and the patent for being related to Teriparatide synthesis at present are more, are related to isolating and purifying less, especially this hair
Bright purifies for the first time using the progress of high pressure reverse-phase chromatography and purifies for the second time and hydrochloric acid progress acetate etc. is added after turning salt
Ion concentration control combines prepare with scale (a batch can obtain 200 grams of finished product or more) purity and ion concentration meets USP 40
Middle Teriparatide acetic acid salt bulk drug quality requirement be even more without reference to.
For the purification process of this product, existing patented technology carries out disclosure.Chinese patent CN106167522A disclose by
Crude product is dissolved in 30% acetonitrile solution, is purified with ammonium acetate buffer, and control pH is 4.0~7.0, acetic acid/acetonitrile C18 column
Turn salt, the method that teriparatide acetate is made, although the teriparatide acetate acetate that purity reaches 99% or more can be obtained,
Single miscellaneous content is not pointed out, while acetic acid radical content is not described in final teriparatide acetate obtained.
Chinese patent CN102993293A disclose by sulfuric acid and acetic acid mixed aqueous solution ammonium hydroxide tune pH value to 5.0~
6.0 buffer is purified, and the sample for collecting 95%~99% adjust pH value to cool down to putting after 5.5~7.0 to 2~8 DEG C
Product is precipitated, is collected by centrifugation after precipitating with after 60% phosphate aqueous solution, then be diluted with water one times, turns salt after upper reversed-phase column, finally
Teriparatide acetate is made.Its yield does not embody in the patent.Sample is dissolved with 60% phosphoric acid before turning salt, and it is unstable that there are products
It degrades, introduce the unstable risk of new impurity, yield.Acetic acid radical content is not retouched in final teriparatide acetate obtained
It states, the content of other anion used in technique is not also studied.
Patent CN102731643A discloses a kind of purification process of Teriparatide, dissolves thick peptide using water, then uses
0.2%TFA/ acetonitrile is mobile phase, C18 column separating purification, and 0.2% acetic acid/acetonitrile C18 column turns salt, total recovery 20.5~
32.2%, do not study the concrete content of anion in largest single impurity and teriparatide acetate equally yet.
To solve impurity of the existing technology and the indefinite problem of ion concentration, acquisition meets 40 quality requirement of USP
Teriparatide bulk pharmaceutical chemicals, also need to conduct further research to purifying and turn salt method.
Summary of the invention
The purpose of the present invention is to provide the works of a kind of large-scale separation purifying of Teriparatide acetate and ionic control
Process, with high purity and yield is good, content≤5% of acetate after freeze-drying, obtains the Te Lipa for meeting 40 quality requirement of USP
Peptide bulk pharmaceutical chemicals reach the requirement of industrialized production, solve the indefinite difficulty of impurity and ion concentration existing in the prior art
Topic
To achieve the goals above, the present invention provides the following technical solution: a kind of purifying of Teriparatide acetate and
Ionic control method, comprising the following steps:
Step 1): thick peptide solution is subjected to the purifying of first time gradient elution using chromatographic column, collects eluent；The chromatography
The mobile phase that column uses includes mobile phase A and Mobile phase B, and the mobile phase A is ammonium bicarbonate aqueous solution, with acetic acid or ammonium hydroxide tune
PH, the Mobile phase B are acetonitrile；
Step 2): the eluent of above-mentioned collection is subjected to second of gradient elution purifying using chromatographic column, collects eluent；
The mobile phase that the chromatographic column uses includes mobile phase A and Mobile phase B, and the mobile phase A is aqueous sodium persulfate solution, with sulfuric acid tune
PH, the Mobile phase B are acetonitrile；
Step 3): the eluent collected in step 2) is subjected to efficient liquid phase using chromatographic column and turns salt, wherein first using acetic acid
The elution of aqueous ammonium acetonitrile system, then eluted with aqueous acetic acid acetonitrile system；
Step 4): hydrochloric acid is added after the eluent after step 3) transfer salt is concentrated under reduced pressure, makes the residual quantity control of acetate
System is below 5%.
Preferably, the pH of mobile phase A is 7.0~9.5 in the step 1), the concentration of ammonium hydrogen carbonate is 0.01~
0.2mol/L。
As further preferred, the pH of mobile phase A is 8.0 in the step 1), and the concentration of ammonium hydrogen carbonate is 0.1mol/
L。
Preferably, the pH of mobile phase A is 2.0~4.0 in the step 2), the concentration of sodium sulphate is 0.05~
0.2mol/L。
As further preferred, the pH of mobile phase A is 3.0 in the step 2), and the concentration of sodium sulphate is 0.1mol/L.
Preferably, the amount for adjusting hydrochloric acid used in acetic acid radical content in the step 4) is 1~4N.
As further preferred, the amount that hydrochloric acid used in acetic acid radical content is adjusted in the step 4) is 3.3N.
As further preferred, the target peptide solution that 1~4 equivalent 1N hydrochloric acid is added is transferred to size in the step 4)
It is freeze-dried in suitable stainless steel pallet, obtained Teriparatide purity is greater than 99%, methionine sulfoxide Teriparatide
Impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% teriparatide acetate, acetate ion content≤5%, chlorine from
≤ 0.1%, purifying yield is 60% for sub- content≤4%, trifluoroacetic acid root and sulfate radical content.
Preferably, the stationary phase that the step 1) to chromatographic column described in step 3) uses is octadecylsilane bonded silica
Glue or eight alkyl silane bonded silica gels.
Preferably, further including steps of in above-mentioned steps 1) following sample treatment is carried out before, by thick peptide
It is dissolved using the acetonitrile solution of volume ratio 5~10% according to 5~20g/L of concentration, then uses 0.22~0.45 μm of filter membrane
It is stand-by to collect filtrate for filtering.
As it is further preferably, a kind of large-scale separation purifying of Teriparatide acetate and the side of ionic control
Method, specifically includes the following steps:
Step 1): dissolving thick peptide according to 5~20g/L of concentration with the acetonitrile solution of volume ratio 5~10%, using 0.22~
0.45 μm of membrane filtration.
Step 2): first separation purifying is carried out to filtered crude product using high performance liquid chromatograph device: passing through DAC-
CXTH200 prepares dynamic axial pressured column, by thick peptide solution with octadecylsilane chemically bonded silica or eight alkyl silane bonded silicas
Glue is that the chromatographic column progress of stationary phase is gradient-purified, by 0.01~0.2mol/L ammonium bicarbonate aqueous solution ammonium hydroxide tune pH value 7.0
Solution after~9.5 is A phase, and acetonitrile is B phase, and gradient: B%:10%~30% is eluted；Flow velocity is 1000~1400ml/min,
Detection wavelength is 220nm, Fractional Collections.
Step 3): dynamic axial pressured column is prepared for acetonitrile in the qualified solution collected in step 2 by DAC-CXTH200
Ratio be down to 10% and carry out second directly below and purify, with octadecylsilane chemically bonded silica or eight alkyl silane bonded silicas
Glue is that the chromatographic column of stationary phase carries out gradient elution purifying, is used with the aqueous sodium persulfate solution that molar concentration is 0.05~0.2mol/L
Solution after sulfuric acid tune pH value 2.0~4.0 is A phase, and acetonitrile is B phase, and gradient: B%:10~30% is eluted；Flow velocity be 1000~
1400ml/min, Detection wavelength 220nm, Fractional Collections.
Step 4): carrying out efficient liquid phase with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels and turn salt, will
Step 3) acquired solution rinses 20~30min with 3~5% acetonitrile ammonium acetate solution, and the concentration of ammonium acetate is 10~100mM,
Then it is eluted with aqueous acetic acid acetonitrile system, collects teriparatide solutions, the volume ratio of acetic acid is in the aqueous acetic acid
0.1~0.2%, acetonitrile ratio is 35% or more.
Step 5): will turn 35 DEG C of eluent or less after salt reduced pressures, and the hydrochloric acid that 1~4N is added adjusts product after freeze-drying
Middle acetate ion content controls the residual of acetate 5% hereinafter, sample is placed in freeze drier freeze-drying.
The purifying and ionic control method of a kind of Teriparatide acetate provided by the invention, compared with the prior art have with
Lower advantage: by carrying out primary purifying and secondarily purified control impurity using high pressure reverse-phase chromatography, and hydrochloric acid is added after turning salt
It carries out the control of acetate plasma content to combine, repeatedly purify and turns salt process using different chromatographic systems, scale system
Standby (a batch can obtain 200 grams of finished product or more) purity and ion concentration meet the Teriparatide acetate of quality requirement in USP 40
Bulk pharmaceutical chemicals, obtained Teriparatide purity are greater than 99%, methionine sulfoxide Teriparatide impurity summation≤0.50% and it is maximum not
Know it is single it is miscellaneous≤0.10%, acetate ion content≤5%, chloride ion content≤4%, trifluoroacetic acid root and sulfate radical content≤
0.1%, purifying total recovery is greater than 60%.
Detailed description of the invention
It is that the chromatography spectrogram and HPLC spectrogram of Teriparatide acetate are obtained using embodiment three below.
Fig. 1 is that the 1st purifying chromatographs spectrogram in embodiment one.
Fig. 2 is the target peak HPLC spectrogram collected after the 1st chromatography in embodiment one.
Fig. 3 is that the 2nd purifying chromatographs spectrogram in embodiment one.
Fig. 4 is the 2nd target peak HPLC spectrogram collected after purification in embodiment one.
Fig. 5 is the chromatography of ions figure that sample is directly lyophilized after two transfer salt of embodiment.
Fig. 6 is the chromatography of ions figure of addition hydrochloric acid freeze-drying sample after two transfer salt of embodiment.
Specific embodiment
Embodiment one:
1. sample treatment: the thick peptide of every gram of Teriparatide is dissolved in 50ml volume ratio: acetonitrile: water=5:95 acetonitrile solution
In, for ultrasound to being completely dissolved, it is stand-by to collect filtrate for 0.45 μm of membrane filtration.
2. the 1st purifying:
Purification condition: chromatographic column: using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured column of stationary phase,
Pillar diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the ammonium bicarbonate aqueous solution of 0.1mol/L, ammonium hydroxide
Adjusting pH is 8.0；B phase: acetonitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Gradient: B%:10~30%, 45min, sample introduction
Amount is 80g.
Purification process: loading after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solution.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is greater than
The target peptide solution of largest single impurity≤0.10% is diluted with water one times at 95% and relative retention time RRT 0.9~1.1, to
With.Target peptide components recoveries 82%.1st purifying chromatography spectrogram is as shown in Figure 1, the target peak HPLC spectrogram collected after chromatography
As shown in Figure 2.
3. the 2nd purifying:
Purification condition: chromatographic column: using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured column of stationary phase,
Pillar diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the aqueous sodium persulfate solution of 0.1mol/L, sulfuric acid tune
PH is 3.0；B phase: acetonitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Gradient: B%:10%~30% elutes 45min,
Sample volume is 80g.
Purification process: by chromatographic column loading after mobile phase A balance 1CV, applied sample amount 4L sample solution.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is greater than
99.5%, methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% target peptide solution adds
Water dilutes one times, for use.Target peptide components recoveries 84%.What the 2nd purifying was collected after chromatographing spectrogram as shown in figure 3, chromatographing
Target peak HPLC spectrogram is as shown in Figure 4.
4. turn salt and ionic control
Turn salt condition: chromatographic column: using eight alkyl silane bonded silica gels as the DAC-20 dynamic axial pressured column of stationary phase, column
Sub- diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the ammonium acetate aqueous solution of 0.02mol/L；B phase: second
Nitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Sample volume is 80g.
Turn salt process: loading after 1CV after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solution.Gradient is set
The gradient isocratic elution 15min of 5%B phase；It then is 50% acetonitrile solution elution samples of 0.1% acetic acid with mass concentration,
It is concentrated under reduced pressure into 10~20mg/ml at 35 DEG C of target peptide solution of collection, 3 equivalent 1N hydrochloric acid are added, for use.Target peptide components return
Yield 97%.
5. freeze-drying
Target peptide solution obtained by upper step is transferred in sizeable stainless steel pallet, purity is obtained after freeze-drying
99.5%, the teriparatide acetate of methionine sulfoxide Teriparatide impurity summation 0.38% and maximum unknown list miscellaneous 0.06%,
Acetate ion content 3.9%, chloride ion content 3.6%, trifluoroacetic acid radical content 0.09% and sulfate radical content 0.08% are pure
Changing total recovery is about 66.8%.
Embodiment two:
1. sample treatment: the thick peptide of every gram of Teriparatide is dissolved in 50ml volume ratio: acetonitrile: water=5:95 acetonitrile solution
In, for ultrasound to being completely dissolved, it is stand-by to collect filtrate for 0.45 μm of membrane filtration.
2. the 1st purifying:
Purification condition: chromatographic column: using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured column of stationary phase,
Pillar diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the ammonium bicarbonate aqueous solution of 0.01mol/L, acetic acid
Adjusting pH is 7.0；B phase: acetonitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Gradient: B%:10%~30%, 45min, into
Sample amount is 80g.
Purification process: loading after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solution.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is greater than
The target peptide solution of largest single impurity≤0.10% is diluted with water one times at 95% and relative retention time RRT 0.9~1.1, to
With.Target peptide components recoveries 80%.
3. the 2nd purifying:
Purification condition: chromatographic column: using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured column of stationary phase,
Pillar diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the aqueous sodium persulfate solution of 0.05mol/L, sulfuric acid tune
PH is 2.0；B phase: acetonitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Gradient: B%:10%~30% elutes 45min,
Sample volume is 80g.
Purification process: by chromatographic column loading after mobile phase A balance 1CV, applied sample amount 4L sample solution.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is greater than
99%, methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% target peptide solution adds water
One times of dilution, for use.Target peptide components recoveries 80%.
4. turn salt and ionic control
Turn salt condition: chromatographic column: using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured column of stationary phase,
Pillar diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the ammonium acetate aqueous solution of 0.02mol/L；B phase: second
Nitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Sample volume is 80g.
Turn salt process: loading after 1CV after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solution.Gradient is set
The gradient isocratic elution 15min of 5%B phase；It then is 50% acetonitrile solution elution samples of 0.1% acetic acid with mass concentration,
It is concentrated under reduced pressure into 10~20mg/ml at 35 DEG C of target peptide solution of collection, takes half that 1.0 equivalent 1N hydrochloric acid are added, for use.Target
The peptide components rate of recovery 95%.
5. freeze-drying
The target peptide that 1.0 equivalent 1N hydrochloric acid of 1.0 equivalent 1N hydrochloric acid target peptide solutions and addition are not added obtained by upper step is molten
Liquid is transferred to respectively in sizeable stainless steel pallet, and purity is obtained after the two freeze-drying and is all larger than 99%, methionine is sub-
Sulfone Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% teriparatide acetate, chloride ion content≤
4% ,≤0.1%, purifying total recovery is about 60.8% for trifluoroacetic acid root and sulfate radical content.Difference be the former acetate from
Sub- content 8%, the latter's acetate ion content 4%, the effect that hydrochloric acid is added herein are to control the content of acetate ion.Turn salt
The chromatography of ions figure of sample is directly lyophilized afterwards as shown in figure 5, the peak of serial number 1 is acetate ion in figure, the peak of serial number 2 is
Chloride ion, the peak of serial number 5 are sulfate ion；Turn to add after salt the chromatography of ions figure that sample is lyophilized after hydrochloric acid as shown in fig. 6,
The peak of serial number 2 is acetate ion in figure, and the peak of serial number 4 is chloride ion, and the peak of serial number 7 is sulfate ion.
Embodiment three:
1. sample treatment: the thick peptide of every gram of Teriparatide is dissolved in 50ml volume ratio: acetonitrile: water=5:95 acetonitrile solution
In, for ultrasound to being completely dissolved, it is stand-by to collect filtrate for 0.45 μm of membrane filtration.
2. the 1st purifying:
Purification condition: chromatographic column: using eight alkyl silane bonded silica gels as the DAC-20 dynamic axial pressured column of stationary phase, column
Sub- diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the ammonium bicarbonate aqueous solution of 0.01mol/L, ammonium hydroxide tune
PH is 7.5；B phase: acetonitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Gradient: B%:10%~30%, 45min, sample introduction
Amount is 80g.
Purification process: loading after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solution.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is greater than
The target peptide solution of largest single impurity≤0.10% is diluted with water one times at 95% and relative retention time RRT 0.9~1.1, to
With.Target peptide components recoveries 81%.
3. the 2nd purifying:
Purification condition: chromatographic column: using eight alkyl silane bonded silica gels as the DAC-20 dynamic axial pressured column of stationary phase, column
Sub- diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the aqueous sodium persulfate solution of 0.05mol/L, sulfuric acid tune pH
It is 2.0；B phase: acetonitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Gradient: B%:10~30% elutes 45min, sample introduction
Amount is 80g.
Purification process: by chromatographic column loading after mobile phase A balance 1CV, applied sample amount 4L sample solution.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is greater than
99%, methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% target peptide solution adds water
One times of dilution, for use.Target peptide components recoveries 82%.
4. turn salt and ionic control
Turn salt condition: chromatographic column: using eight alkyl silane bonded silica gels as the DAC-20 dynamic axial pressured column of stationary phase, column
Sub- diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the ammonium acetate aqueous solution of 0.02mol/L；B phase: second
Nitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Sample volume is 80g.
Turn salt process: loading after 1CV after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solution.Gradient is set
The gradient isocratic elution 15min of 5%B phase；It then is 50% acetonitrile solution elution samples of 0.1% acetic acid with mass concentration,
It is concentrated under reduced pressure into 10~20mg/ml at 35 DEG C of target peptide solution of collection, 2.0 equivalent 1N hydrochloric acid are added, for use.Target peptide components
The rate of recovery 94%.
5. freeze-drying
Target peptide solution obtained by upper step is transferred in sizeable stainless steel pallet, purity is obtained after freeze-drying
99.2%, the teriparatide acetate of methionine sulfoxide Teriparatide impurity summation 0.48% and maximum unknown list miscellaneous 0.08%,
Acetate ion content 4.8%, chloride ion content 3.5%, trifluoroacetic acid radical content 0.06%, sulfate radical content 0.08%%,
Purifying total recovery is about 62.4%.
Example IV:
1. sample treatment: the thick peptide of every gram of Teriparatide is dissolved in 50ml volume ratio: acetonitrile: water=5:95 acetonitrile solution
In, for ultrasound to being completely dissolved, it is stand-by to collect filtrate for 0.45 μm of membrane filtration.
2. the 1st purifying:
Purification condition: chromatographic column: using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured column of stationary phase,
Pillar diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the ammonium bicarbonate aqueous solution of 0.2mol/L, ammonium hydroxide
Adjusting pH is 9.0；B phase: acetonitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Gradient: B%:10%~30%, 45min, into
Sample amount is 80g.
Purification process: loading after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solution.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is greater than
The target peptide solution of largest single impurity≤0.10% is diluted with water one times at 95% and relative retention time RRT 0.9~1.1, to
With.Target peptide components recoveries 83%.
3. the 2nd purifying:
Purification condition: chromatographic column: using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured column of stationary phase,
Pillar diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the aqueous sodium persulfate solution of 0.2mol/L, sulfuric acid tune
PH is 3.0；B phase: acetonitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Gradient: B%:10%~30% elutes 45min,
Sample volume is 80g.
Purification process: by chromatographic column loading after mobile phase A balance 1CV, applied sample amount 4L sample solution.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is greater than
99%, methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% target peptide solution adds water
One times of dilution, for use.Target peptide components recoveries 80%.
4. turn salt and ionic control
Turn salt condition: chromatographic column: using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured column of stationary phase,
Pillar diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the ammonium acetate aqueous solution of 0.02mol/L；B phase: second
Nitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Sample volume is 80g.
Turn salt process: loading after 1CV after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solution.Gradient is set
The gradient isocratic elution 15min of 5%B phase；It then is 50% acetonitrile solution elution samples of 0.1% acetic acid with mass concentration,
It is concentrated under reduced pressure into 10~20mg/ml at 35 DEG C of target peptide solution of collection, 4.0 equivalent 1N hydrochloric acid are added, for use.Target peptide components
The rate of recovery 91%.
5. freeze-drying
Target peptide solution obtained by upper step is transferred in sizeable stainless steel pallet, purity is obtained after freeze-drying
99.1%, the teriparatide acetate of methionine sulfoxide Teriparatide impurity summation 0.44% and maximum unknown list miscellaneous 0.09%,
Acetate ion content 4.2%, chloride ion content 3%, trifluoroacetic acid radical content 0.07% and sulfate radical content equal 0.05% are pure
Changing total recovery is about 60.4%.
Embodiment five:
1. sample treatment: the thick peptide of every gram of Teriparatide is dissolved in 50ml volume ratio: acetonitrile: water=5:95 acetonitrile solution
In, for ultrasound to being completely dissolved, it is stand-by to collect filtrate for 0.45 μm of membrane filtration.
2. the 1st purifying:
Purification condition: chromatographic column: using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured column of stationary phase,
Pillar diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the ammonium bicarbonate aqueous solution of 0.01mol/L, ammonium hydroxide
Adjusting pH is 9.5；B phase: acetonitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Gradient: B%:10%~30%, 45min, into
Sample amount is 80g.
Purification process: loading after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solution.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is greater than
The target peptide solution of largest single impurity≤0.10% is diluted with water one times at 95% and relative retention time RRT 0.9~1.1, to
With.Target peptide components recoveries 80%.
3. the 2nd purifying:
Purification condition: chromatographic column: using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured column of stationary phase,
Pillar diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the aqueous sodium persulfate solution of 0.05mol/L, sulfuric acid tune
PH is 4.0；B phase: acetonitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Gradient: B%:10~30% elutes 45min, into
Sample amount is 80g.
Purification process: by chromatographic column loading after mobile phase A balance 1CV, applied sample amount 4L sample solution.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is greater than
99%, methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% target peptide solution adds water
One times of dilution, for use.Target peptide components recoveries 81%.
4. turn salt and ionic control
Turn salt condition: chromatographic column: using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured column of stationary phase,
Pillar diameter and filling length are as follows: 20*25cm.Mobile phase A: molar concentration is the ammonium acetate aqueous solution of 0.02mol/L；B phase: second
Nitrile.Flow velocity: 1200ml/min.Check wavelength 220nm.Sample volume is 80g.
Turn salt process: loading after 1CV after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solution.Gradient is set
The gradient isocratic elution 15min of 5%B phase；It then is 50% acetonitrile solution elution samples of 0.1% acetic acid with mass concentration,
It is concentrated under reduced pressure into 10~20mg/ml at 35 DEG C of target peptide solution of collection, 3.5 equivalent 1N hydrochloric acid are added, for use.Target peptide components
The rate of recovery 94%.
5. freeze-drying
Target peptide solution obtained by upper step is transferred in sizeable stainless steel pallet, purity is obtained after freeze-drying
99.3%, the teriparatide acetate of methionine sulfoxide Teriparatide impurity summation 0.5% and maximum unknown list miscellaneous 0.10%, vinegar
Acid ion content 5%, chloride ion content 4%, trifluoroacetic acid root and sulfate radical content equal 0.1%, purifying total recovery are about
60.9%.
The related substance of resulting teriparatide acetate and ion concentration are produced using the technique of embodiment one to embodiment five
Meet the requirement of USP 40, other indices also meet USP 40, while also being compliant with the requirement of EP 9.0, can be used as acetic acid spy
Vertical pa peptide bulk pharmaceutical chemicals export US and European, while can also meet Chinese market requirements of customs declaration.
Comparative example one:
The mobile phase A used is purified by the 1st time in embodiment three and replaces with ammonium acetate buffer solution, with acetic acid or ammonium hydroxide tune
PH finally turns after salt also with HCl treatment, remaining technique and each parameter are the same as embodiment three by secondarily purified.
Obtain purity 90.0% after final freeze-drying, methionine sulfoxide Teriparatide impurity summation 0.62% and it is maximum not
Know single miscellaneous 0.15% teriparatide acetate, acetate ion content 3.9%, chloride ion content 4.8%, trifluoroacetic acid root contains
Amount 0.18% and sulfate radical content 0.20%, purifying total recovery is about 60.2%.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, several simple deductions and replacement can also be made, all shall be regarded as belonging to of the invention
Protection scope.
Claims (7)
1. the purifying and ionic control method of a kind of Teriparatide acetate, it is characterised in that the following steps are included:
Step 1): thick peptide solution is subjected to the purifying of first time gradient elution using chromatographic column, collects eluent；The chromatographic column makes
Mobile phase includes mobile phase A and Mobile phase B, and the mobile phase A is ammonium bicarbonate aqueous solution, with acetic acid or ammonium hydroxide tune pH,
The Mobile phase B is acetonitrile；
Step 2): the eluent of above-mentioned collection is subjected to second of gradient elution purifying using chromatographic column, collects eluent；It is described
The mobile phase that chromatographic column uses includes mobile phase A and Mobile phase B, and the mobile phase A is aqueous sodium persulfate solution, with sulfuric acid tune pH,
The Mobile phase B is acetonitrile；
Step 3): carrying out efficient liquid phase using chromatographic column for the eluent collected in step 2) and turn salt, wherein first using ammonium acetate water
The elution of dissolved in acetonitrile system, then eluted with aqueous acetic acid acetonitrile system；
Step 4): being added hydrochloric acid after the eluent after step 3) transfer salt is concentrated under reduced pressure, and controls the residual quantity of acetate
5% or less；
The pH of mobile phase A is 7.0~9.5 in the step 1), and the concentration of ammonium hydrogen carbonate is 0.01~0.2mol/L；The step
It is rapid 2) in the pH of mobile phase A be 2.0~4.0, the concentration of sodium sulphate is 0.05~0.2mol/L；Vinegar is adjusted in the step 4)
The amount of hydrochloric acid used in acid group content is 1~4N.
2. according to the method described in claim 1, it is characterized by: in the step 1) mobile phase A pH be 8.0, carbonic acid
The concentration of hydrogen ammonium is 0.1mol/L.
3. according to the method described in claim 1, it is characterized by: in the step 2) mobile phase A pH be 3.0, sulfuric acid
The concentration of sodium is 0.1mol/L.
4. according to the method described in claim 1, it is characterized by: adjusting hydrochloric acid used in acetic acid radical content in the step 4)
Amount is 3.3N.
5. according to the method described in claim 1, it is characterized by: the mesh of 1~4 equivalent 1N hydrochloric acid will be added in the step 4)
Mark peptide solution, which is transferred in sizeable stainless steel pallet, to be freeze-dried, and obtained Teriparatide purity is greater than 99%,
Methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% teriparatide acetate, acetate
≤ 0.1%, purifying yield is 60% for ion concentration≤5%, chloride ion content≤4%, trifluoroacetic acid root and sulfate radical content.
6. described in any item methods according to claim 1~5, it is characterised in that: step 1) to chromatographic column described in step 3) makes
Stationary phase is octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels.
7. described in any item methods according to claim 1~5, it is characterised in that: further include steps of in above-mentioned step
It is rapid 1) to carry out following sample treatment before, by thick peptide using the acetonitrile solution of volume ratio 5~10% according to 5~20g/ of concentration
Then L dissolution uses 0.22~0.45 μm of membrane filtration, it is stand-by to collect filtrate.
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| CN109897099A (en) * | 2019-03-27 | 2019-06-18 | 哈尔滨吉象隆生物技术有限公司 | A kind of preparation method of Teriparatide |
| CN112940102A (en) * | 2020-12-30 | 2021-06-11 | 苏州天马医药集团天吉生物制药有限公司 | Purification method of Somalutide |
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Effective date of registration: 20200609 Address after: Building C5, Jiangsu life science and Technology Innovation Park, No.9, Weidi Road, Qixia District, Nanjing, Jiangsu Province, 210046 Co-patentee after: Meiyaoxing (Nanjing) Pharmaceutical Co.,Ltd. Patentee after: NANJING HANXIN PHARMACEUTICAL TECHNOLOGY Co.,Ltd. Address before: 210038 Jiangsu city of Nanjing province and the economic and Technological Development Zone Road No. 5 Patentee before: Meiyaoxing (Nanjing) Pharmaceutical Co.,Ltd. |