A method for determining the residual amount of trifluoroacetic acid in recombinant teriparatide

Abstract

The present invention discloses a method for determining the residual amount of trifluoroacetic acid in recombinant teriparatide. This method uses high performance liquid chromatography for detection, selects a C18 chromatographic column, and uses UV as the detector. The method of the present invention is accurate, rapid and convenient, and is applicable to the determination of trifluoroacetic acid in the stock solution of recombinant teriparatide.

Description

Technical Field

[0001] The present invention relates to a quality control method for recombinant teriparatide, and particularly to a method for determining the residual amount of trifluoroacetic acid in recombinant teriparatide by high performance liquid chromatography. Background Art

[0002] Recombinant teriparatide (rhPTH), a recombinant human parathyroid hormone analogue (PTH 1-34), is a therapeutic drug for postmenopausal women with osteoporosis developed by genetic engineering recombinant technology. It has the same amino acid sequence as the first 1-34 amino acids at the N-terminus of natural human parathyroid hormone (1-84) in the human body, and both have the same biological activity and physiological and pharmacological characteristics. Recombinant teriparatide is expressed from Escherichia coli, and after steps such as seed amplification, fermentation culture, centrifugal collection, high-pressure homogenization, inclusion body washing, inclusion body lysis, renaturation, Q Sepharose FF purification, ultrafiltration, enzymatic digestion, etc., the recombinant teriparatide stock solution is obtained after sterile filtration and aliquoting. The stock solution is formulated into finished products of different specifications and dosage forms, which are suitable for the treatment of postmenopausal women with osteoporosis at high risk of fractures.

[0003] Trifluoroacetic acid, abbreviated as TFA, is an organic compound with the chemical formula C2HF3O2. It is a perfluorinated derivative of acetic acid and also the simplest perfluorocarboxylic acid compound. TFA is miscible with water, fluorohydrocarbons, methanol, ethanol, ether, acetone, benzene, carbon tetrachloride, hexane, and can partially dissolve carbon disulfide and alkanes with more than six carbons. It is an excellent solvent for proteins and polyesters. TFA is commonly used in the reverse phase high performance liquid chromatography purification for eluting crude peptides from solid phase resins. TFA belongs to acidic organic solvents and is a class IV solvent in the pharmacopoeia. It is toxic in clinical use, so it cannot exist in excessive amounts in drugs. Moreover, inhalation, oral administration or percutaneous absorption of trifluoroacetic acid is harmful to the body and has a strong irritating effect on eyes, mucous membranes, respiratory tract and skin. Therefore, the residual amount of TFA in drugs must be strictly controlled.

[0004] TFA is used to prepare the buffer solution for RPC reverse chromatography in the purification process of recombinant teriparatide, such as KR102364696B1, CN103451219A, etc. There is no clear regulation for class IV solvents in the pharmacopoeia, and usually it is limited by the limit of class E solvents at 0.5%. Currently, the prior art discloses that the determination of TFA in some chemical drugs can be carried out by gas chromatography-mass spectrometry, but the sample treatment is troublesome, the equipment cost is high, and a quality control method for trifluoroacetic acid in recombinant teriparatide has not been established yet. Therefore, finding a specific and relatively economical detection method for recombinant teriparatide to accurately determine the residual amount of trifluoroacetic acid in recombinant teriparatide samples, so as to control the quality of recombinant teriparatide products, is a technical problem not solved by the prior art.

[0005] The present invention establishes a method for determining the residual amount of TFA in recombinant teriparatide by high performance liquid chromatography (HPLC). This method is simple, easy to operate, accurate in results, highly sensitive, and low in equipment cost. Summary of the Invention

[0006] The object of the present invention is to provide a method for analyzing and determining the residual amount of trifluoroacetic acid in recombinant teriparatide by high performance liquid chromatography, which can be used for the quality control of recombinant teriparatide products.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A method for determining the residual amount of trifluoroacetic acid in recombinant teriparatide, the determination method using high performance liquid chromatography, comprising the following steps:

[0009] (1) Prepare a sample of recombinant teriparatide and a reference solution of trifluoroacetic acid;

[0010] (2) Analyze the sample and the reference solution by high performance liquid chromatography;

[0011] (3) Calculate the residual amount of trifluoroacetic acid in the sample;

[0012] Among them, the chromatographic conditions of high performance liquid chromatography are as follows:

[0013] Chromatographic column: C18 chromatographic column;

[0014] Mobile phase: aqueous phosphoric acid solution: acetonitrile;

[0015] Isocratic elution.

[0016] In the determination method of the present invention, the pH of the aqueous phosphoric acid solution of the mobile phase is adjusted with NaOH, and the pH of the aqueous phosphoric acid solution is 3.0 ± 0.2. The content of phosphoric acid in the aqueous phosphoric acid solution is 0.08% - 0.15% (v / v%), preferably 0.1% - 0.15% (v / v%), and more preferably 0.1% (v / v%). In some preferred embodiments, the content of phosphoric acid in the aqueous phosphoric acid solution is 0.1% (v / v%), and the pH of the aqueous phosphoric acid solution is 3.0. In some embodiments, the proportion of the aqueous phosphoric acid solution in the mobile phase is 75% - 85%, preferably 75% - 80%, and more preferably 80%.

[0017] In the determination method of the present invention, the organic phase of the mobile phase is acetonitrile. For the high performance liquid analysis of recombinant teriparatide products, methanol or acetonitrile can be used as the organic phase, but the present invention finds that when methanol is used for detection, the column efficiency is significantly lower than that when acetonitrile is used for detection under the same peak area of the chromatographic peak. In some embodiments, the proportion of acetonitrile in the mobile phase is 15% - 25%, preferably 20% - 25%, and more preferably 20%.

[0018] In the determination method of the present invention, the ratio of the phosphoric acid aqueous solution to acetonitrile is 75-85:25-15, preferably 75-80:25-20, and more preferably 80:20.

[0019] In the determination method of the present invention, the flow rate of the mobile phase is 1.0-1.2 mL / min, preferably 1.0 mL / min.

[0020] In the determination method of the present invention, the column temperature of the C18 chromatographic column is 30-40 °C, preferably 35 °C.

[0021] In the determination method of the present invention, the detection wavelength of the UV detector is 200-220 nm, preferably 210 nm.

[0022] In the determination method of the present invention, the elution time is not less than 5 min, not less than 6 min, not less than 7 min, not less than 8 min, not less than 9 min, or not less than 10 min. In a preferred embodiment, the elution time is 10 min.

[0023] In the determination method of the present invention, the injection volume is 20 μL.

[0024] In the determination method of the present invention, the concentration of the trifluoroacetic acid reference substance is 0.5 μg / mL.

[0025] In some preferred embodiments, the determination method of the present invention comprises the following steps:

[0026] (1) Prepare a recombinant teriparatide sample and a trifluoroacetic acid reference substance solution;

[0027] (2) Analyze the sample and the reference substance solution by high performance liquid chromatography;

[0028] (3) Calculate the residual amount of trifluoroacetic acid in the sample;

[0029] Among them, the chromatographic conditions of the high performance liquid chromatography are as follows:

[0030] Chromatographic column: C18 chromatographic column;

[0031] Mobile phase: 0.1% phosphoric acid aqueous solution: acetonitrile = 80:20, and the pH of the phosphoric acid aqueous solution is 3.0;

[0032] Detection wavelength: 210 nm; isocratic elution, elution time: 10 min; flow rate: 1.0 mL / min; column temperature: 35 °C; injection volume: 20 μL.

[0033] In one embodiment, the recombinant teriparatide sample comprises a chromatographically collected solution of recombinant teriparatide. The chromatographically collected solution of recombinant teriparatide contains a chromatographic buffer substance, and the chromatographic buffer substance comprises disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride.

[0034] On the other hand, the present invention provides the application of the method for determining the residual amount of trifluoroacetic acid in the aforementioned recombinant teriparatide in the quality control of recombinant teriparatide.

[0035] In one embodiment, the quality control includes monitoring the residual amount of trifluoroacetic acid in the chromatographically collected solution of recombinant teriparatide. The chromatographically collected solution of recombinant teriparatide contains a chromatographic buffer substance, and the chromatographic buffer substance comprises disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride.

[0036] The determination method of the present invention can effectively separate trifluoroacetic acid without being interfered by the chromatographic buffer substances, which is beneficial to the monitoring of the content of trifluoroacetic acid during the production process of recombinant teriparatide. In addition, methodological experiments show that the linear range of trifluoroacetic acid in the determination method of the present invention is 0.02 μg / mL to 1 μg / mL, the detection limit is 0.05 ng, the quantification limit is 0.2 ng, and the recovery rate is 101.4%. Each index is significantly better than the existing literature reports, and the accurate detection of the residual amount of trifluoroacetic acid in recombinant teriparatide can be achieved, especially suitable for the detection of low-content trifluoroacetic acid.

[0037] The present invention overcomes the defects of the prior art such as complex operation, troublesome sample treatment and high equipment cost, and provides a method for detecting the residual amount of trifluoroacetic acid in recombinant teriparatide. This method is simple and fast to operate, accurate in result and high in sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Shows the linear regression curve of trifluoroacetic acid.

[0039] Figure 2 Shows the chromatograms of the blank solution (water), the control solution (SP buffer solution) and the trifluoroacetic acid reference solution.

[0040] Figure 3 Shows the chromatograms of the trifluoroacetic acid reference solution and the chromatographically collected solution of recombinant teriparatide by RPC.

[0041] Figure 4 Shows the chromatograms of the blank solution (water) and the chromatographically collected solution of recombinant teriparatide by SP. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] The present invention will be further described in detail below in conjunction with examples and drawings, but the embodiments of the invention are not limited thereto.

[0043] Analytical instrument: Waters e2695 high performance liquid chromatography; ultraviolet detector (UV detector).

[0044] Reagents and samples: ultrapure water; acetonitrile (HPLC); phosphoric acid (AR); trifluoroacetic acid (HPLC); recombinant teriparatide reference standard (NIFDC, 410019-201801); SP chromatography buffer: weigh sodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride, dissolve in purified water, adjust the pH value to 7.10 with 2 mol / L NaOH. The final concentration of sodium hydrogen phosphate in the buffer is 1.73 g / L, the final concentration of sodium dihydrogen phosphate is 1.08 g / L, and the final concentration of sodium chloride is 58.44 g / L.

[0045] Establishment of the method in Example 1

[0046] This example provides a method for determining the residual amount of trifluoroacetic acid in recombinant teriparatide. The specific steps of the determination method are as follows:

[0047] (1) Prepare recombinant teriparatide sample and trifluoroacetic acid reference solution;

[0048] (2) Analyze the sample and reference solution by high performance liquid chromatography;

[0049] (3) Calculate the residual amount of trifluoroacetic acid in the sample;

[0050] The chromatographic conditions of high performance liquid chromatography are as follows:

[0051] Chromatographic column: C18 chromatographic column (Shim-pack / VP-ODS 250*4.6 mm);

[0052] Mobile phase: 0.1% phosphoric acid aqueous solution: acetonitrile = 80:20, and the pH of the phosphoric acid aqueous solution is 3.0;

[0053] Detection wavelength of UV detector: 210 nm; isocratic elution, elution time: 10 min; flow rate: 1.0 mL / min; column temperature: 35 °C; injection volume: 20 μL.

[0054] The calculation method of the residual amount of trifluoroacetic acid in the sample is as follows:

[0055] Formula: M X = M R (A X / A R )

[0056] In the formula: M X : TFA residue amount of the sample, μg; M R : TFA sample loading amount of the reference, μg;

[0057] A X : The peak area of the TFA peak of the sample, mAU*s;

[0058] A R : The average peak area of the TFA of the reference substance, mAU*s;

[0059] Divide M obtained from the above formula X by the sample injection volume (0.02 ml) to obtain the residual amount of TFA in the sample (μg / ml). If there is no TFA chromatographic peak in the sample or the peak area is less than the peak area of the TFA peak at the detection limit, the residual amount of TFA in the sample is less than the detection limit.

[0060] Example 2 Influence of Chromatographic Conditions on the Detection Effect of Trifluoroacetic Acid

[0061] Verify the influence of the chromatographic conditions of the determination method described in Example 1 on the detection effect of trifluoroacetic acid through Comparative Examples 1-5.

[0062] 1) Comparative Example 1

[0063] This experimental example provides the influence of the selection of the organic phase in the mobile phase on the detection effect of trifluoroacetic acid. Methanol or acetonitrile is selected as the organic phase, and the organic phase ratio is designed to be 5%, 20%, and 25% for verification. The verification sample is a 0.5 μg / mL trifluoroacetic acid reference substance solution, and other chromatographic conditions and experimental steps refer to the determination method of Example 1.

[0064] The judgment indexes are the tailing factor and the number of theoretical plates (0.8 < tailing factor < 1.5, number of theoretical plates > 10000), where the European Pharmacopoeia and the British Pharmacopoeia stipulate that except as otherwise specified, the symmetry factor of the chromatographic peak of the reference substance solution for quantification in the chromatogram should be 0.8 - 1.5 when performing the determination of related substances or content. The results are shown in the following table:

[0065]

[0066] Through comparative analysis, it can be seen that when acetonitrile is used as the organic phase for detection at different organic phase ratios, the peak shape of trifluoroacetic acid and the detection column efficiency obtained are better than those of methanol.

[0067] 2) Comparative Example 2

[0068] This experimental example provides the influence of the acetonitrile ratio on the detection effect of trifluoroacetic acid. The ratio of acetonitrile in the mobile phase is set at 5%, 10%, 15%, 20%, and 25% for verification. The verification sample is a 0.5 μg / mL trifluoroacetic acid reference substance solution, and other chromatographic conditions and experimental steps refer to the determination method of Example 1. The judgment indexes are the tailing factor and the number of theoretical plates (0.8 < tailing factor < 1.5, number of theoretical plates > 10000). The results are shown in the following table:

[0069] Proportion of acetonitrile (%) 5％ 10％ 15％ 20％ 25％ Tailing factor 2.6 2.0 1.7 1.2 1.0 Number of theoretical plates 2573 2502 12155 19543 6262

[0070] From the data in the above table, it can be seen that the tailing factor of the trifluoroacetic acid peak decreases as the proportion of acetonitrile increases, reaching the optimal value of 1.0 at 25% acetonitrile, while the number of theoretical plates increases as the proportion of acetonitrile increases, reaching the highest at 20% acetonitrile and then rapidly decreasing. To ensure that both the peak shape and column efficiency are in a good state, considering the two judgment indexes comprehensively, the proportion of acetonitrile can be controlled at 15% - 25%, preferably 20%.

[0071] 3) Comparative Example 3

[0072] This experimental example provides the influence of the phosphoric acid content in the phosphoric acid aqueous solution on the detection effect of trifluoroacetic acid. Phosphoric acid aqueous solutions with four phosphoric acid contents of 0.05%, 0.08%, 0.1%, and 0.15% are selected for verification. The verification sample is a 0.5 μg / mL trifluoroacetic acid reference solution. Other chromatographic conditions and experimental procedures refer to the determination method of Example 1. The judgment indexes are the tailing factor and the number of theoretical plates (0.8 < tailing factor < 1.5, number of theoretical plates > 10000). The results are shown in the following table:

[0073]

[0074] From the above data, it can be seen that the higher the phosphoric acid content, the better the peak shape of trifluoroacetic acid, but the number of theoretical plates reaches the highest value at 0.08% and then decreases slightly as the phosphoric acid content increases. Therefore, through the comprehensive comparison of the two judgment indexes, the phosphoric acid content in the phosphoric acid aqueous solution can be controlled at 0.08% - 0.15%, preferably 0.1%.

[0075] 4) Comparative Example 4

[0076] This experimental example provides the influence of the flow rate of the mobile phase on the detection effect of trifluoroacetic acid. Three flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min are selected for verification. The verification sample is a 0.5 μg / mL trifluoroacetic acid reference solution. Other chromatographic conditions and experimental procedures refer to the determination method of Example 1. The judgment indexes are the tailing factor and the number of theoretical plates (0.8 < tailing factor < 1.5, number of theoretical plates > 10000). The results are shown in the following table:

[0077]

[0078] Comparing the above data, it can be seen that both the tailing factor and the number of theoretical plates of the trifluoroacetic acid peak decrease as the flow rate increases. Therefore, considering the peak shape and column efficiency comprehensively, the flow rate can be controlled at 1.0 - 1.2 mL / min, preferably 1.0 mL / min.

[0079] 5) Comparative Example 5

[0080] This experimental example provides the influence of the concentration of trifluoroacetic acid reference solution on the detection effect of trifluoroacetic acid. The trifluoroacetic acid standard was diluted with ultrapure water into three concentrations of 0.05 μg / mL, 0.5 μg / mL, and 1.0 μg / mL for verification. Each sample was injected twice. The chromatographic conditions and experimental procedures refer to the determination method of Example 1. The judgment indexes are the RSD of peak area, tailing factor, and theoretical plate number. The results are shown in the following table:

[0081]

[0082]

[0083] Note: NA represents that the RSD calculation is not performed for this item.

[0084] From the data in the above table, it can be concluded that when the concentration is less than 0.05 μg / mL, the chromatographic peak of trifluoroacetic acid is too small, which affects the accuracy of detection as a reference substance. When the concentration is greater than 1.0 μg / mL, the chromatographic peak of trifluoroacetic acid shows an obvious front extension, which is also not conducive to accurate detection. Therefore, the concentration of the trifluoroacetic acid reference solution is finally selected as 0.5 μg / mL.

[0085] Methodology verification of Example 3

[0086] The determination method provided in Example 1 was subjected to methodology verification. The specific chromatographic conditions are as follows:

[0087] Chromatographic column: Shim-pack / VP-ODS 250*4.6 mm;

[0088] Mobile phase: 0.1% phosphoric acid solution (PH = 3.0): acetonitrile = 80:20;

[0089] Detection wavelength: 210 nm; isocratic elution, elution time: 10 min; flow rate: 1.0 mL / min; column temperature: 35 °C; injection volume: 20 μL.

[0090] 1) System suitability

[0091] Two trifluoroacetic acid reference solutions with a concentration of 0.5 μg / mL were prepared separately, denoted as reference substance 1 and reference substance 2. Determination was carried out according to the above chromatographic conditions. Reference substance 1 was injected repeatedly 5 times, and reference substance 2 was injected repeatedly 2 times. Calculated from the results: the RSD of the retention time of trifluoroacetic acid in reference substance 1 was 0.7%, and the RSD of the peak area was 0.4%; the recovery rate of reference substance 2 relative to reference substance 1 was 99.8%. Among them, the detection results of reference substance 1 injected repeatedly 5 times are shown in the following table:

[0092]

[0093] It can be seen from the above data that the chromatographic conditions are very stable, with small differences in each parameter, and are applicable to the detection of trifluoroacetic acid residue.

[0094] 2) Linearity and range

[0095] Dilute the trifluoroacetic acid reference substance with ultrapure water to seven concentrations of 0.02 μg / mL, 0.04 μg / mL, 0.08 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and 1 μg / mL, and perform the determination according to the above chromatographic conditions. Statistically analyze the peak areas of each sample, and perform linear regression with the standard substance concentration (mg / mL) as the abscissa and the standard substance peak area (mAU*s) as the ordinate to obtain the Figure 1 linear curve shown. The results show that the determination method has a good linear relationship within the trifluoroacetic acid concentration range of 0.02 - 1 μg / mL, and the R 2 of the linear regression is 0.99997.

[0096] 3) Spiked recovery rate

[0097] Take 180 μL of the recombinant teriparatide standard solution and 20 μL of the 1.0 mg / mL trifluoroacetic acid reference solution, mix them evenly, and use it as the spiked recovery sample, and dilute two portions in parallel. The results show that the average recovery rate of trifluoroacetic acid is 101.4%.

[0098] 4) Repeatability

[0099] Take the trifluoroacetic acid standard substance, and use the SP chromatography buffer to prepare 6 portions of the test solution with a concentration of 0.5 μg / mL in parallel, inject 1 injection for each, and the RSD of the trifluoroacetic acid content calculated from the 6 portions of the test solution is 0.3%.

[0100] 5) Intermediate precision

[0101] Under the conditions of different times and different instruments, use the same chromatographic column for detection. Take the trifluoroacetic acid standard substance, and use SP chromatography buffers of different batches to prepare 6 portions of the test solution with a concentration of 0.5 μg / mL in parallel, inject 1 injection for each, and detect by different experimental personnel. The results show that under the conditions of different times and different instruments, the RSDs of the detection results of 6 portions of the test solution prepared in parallel by two experimental personnel are 0.3% and 0.3% respectively, and the RSD of the detection results of a total of 12 portions of the test solution is 3.2%.

[0102] 6) Detection limit and quantitation limit

[0103] Take the diluted TFA reference substance (0.1 μg / mL), add it into the sample vial, and inject 5 μL, 4 μL, 3 μL, 2 μL, 1 μL, 0.5 μL, 0.4 μL, and 0.2 μL respectively. Statistically calculate the detection results of each sample. Calculate the detection limit as 0.05 ng based on the peak closest to 3 with a signal-to-noise ratio between 3 and 5, and calculate the quantification limit as 0.2 ng based on the peak closest to 10 with a signal-to-noise ratio between 10 and 20.

[0104] Based on the above results of the methodological verification, it can be seen that the chromatographic analysis method established in the present invention has good linearity, high accuracy, good repeatability, low detection limit and quantification limit, and is applicable to the detection of the residual amount of trifluoroacetic acid in recombinant teriparatide.

[0105] Example 4 Determination of the Residual Amount of Trifluoroacetic Acid in Recombinant Teriparatide

[0106] Refer to the description of Example 1 in CN102399285B to construct a recombinant teriparatide Escherichia coli engineering bacterium, and then obtain the recombinant teriparatide RPC chromatography collected solution and SP chromatography collected solution according to the same fermentation and purification steps in Example 1 of this patent. CN102399285B is incorporated into this application by reference.

[0107] Detect according to the method for determining the residual amount of trifluoroacetic acid in recombinant teriparatide provided in Example 1.

[0108] Precisely measure the trifluoroacetic acid standard substance and dilute it with water to prepare a trifluoroacetic acid reference solution of 0.5 μg / mL.

[0109] Dilute the recombinant teriparatide RPC chromatography collected solution 10 times with ultrapure water as the sample solution to be measured.

[0110] Inject the recombinant teriparatide SP chromatography collected solution directly as the sample solution to be measured.

[0111] Measure the reference solution and the sample solution to be measured respectively. The chromatogram of the recombinant teriparatide RPC chromatography collected solution is shown in Figure 3 , and the calculation results show that the content of trifluoroacetic acid in the sample solution to be measured is 2.15 μg / mL; the chromatogram of the recombinant teriparatide SP chromatography collected solution is shown in Figure 4 , and the calculation results show that the content of trifluoroacetic acid in the sample solution to be measured is 0.013 μg / mL.

[0112] The peak emergence time, peak shape, etc. of the trifluoroacetic acid sample chromatograms in all examples are basically as shown in Figure 2 .

[0113] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for determining the residual amount of trifluoroacetic acid in recombinant teriparatide. The determination method uses high performance liquid chromatography and comprises the following steps: (1) Prepare a recombinant teriparatide sample and a trifluoroacetic acid reference solution; (2) Analyze the sample and the reference solution by high performance liquid chromatography; (3) Calculate the residual amount of trifluoroacetic acid in the sample; Among them, The chromatographic conditions for high performance liquid chromatography are as follows: Chromatographic column: C18 chromatographic column, column temperature is 35 °C; Mobile phase: 0.1% phosphoric acid aqueous solution: acetonitrile = 80:20, and the pH of the phosphoric acid aqueous solution is 3.0; Detection wavelength of the UV detector: 210 nm; isocratic elution, elution time: 10 min; flow rate: 1.0 mL / min; injection volume: 20 μL; The model of the C18 chromatographic column is Shim-pack / VP-ODS 250*4.6 mm; The recombinant teriparatide sample includes a recombinant teriparatide chromatography collection solution, and the recombinant teriparatide chromatography collection solution contains a chromatography buffer substance, and the chromatography buffer substance includes disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride.

2. The method for determining the residual amount of trifluoroacetic acid in recombinant teriparatide according to claim 1, wherein The concentration of the trifluoroacetic acid reference is 0.5 μg / mL.

3. The method for determining the residual amount of trifluoroacetic acid in recombinant teriparatide according to any one of claims 1-2, characterized in that, The calculation method for the residual amount of trifluoroacetic acid in the sample is as follows: M X = M R (A X / A R ); where M X : Residual amount of TFA in the sample, μg; M R : Loading amount of TFA in the reference substance, μg; A X : Peak area of TFA in the sample, mAU*s; A R : Average peak area of TFA in the reference substance, mAU*s.

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