CN110568112A - Method for directly detecting exenatide fusion protein - Google Patents

Abstract

The invention relates to a method for measuring exenatide fusion protein, which directly measures exenatide fusion protein (molecular weight is more than 10000Da) in a sample by adopting a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) technology. The method of the invention preferably uses GL Sciences InertSustainBio C18 liquid chromatography column, electrospray ionization (ESI) tandem mass spectrometry detection. The invention successfully develops a simple and efficient pretreatment method of the biological sample, and the biological sample can be directly subjected to liquid quality detection after simple solid phase extraction. The method of the invention uses the exenatide fusion protein mutant as an internal standard protein, and can accurately quantify the exenatide fusion protein. The method of the invention realizes the direct quantitative analysis of the complete biological macromolecules (molecular weight is more than 10000Da) in the biological sample for the first time, and has high sensitivity. The present inventors successfully applied this method to preclinical pharmacokinetic studies of exenatide fusion proteins, and believe that this method as an analytical tool would drive the development of similar biopharmaceuticals.

Description

Method for directly detecting exenatide fusion protein

Technical Field

The invention belongs to the field of drug analysis, and relates to a method for directly measuring exenatide fusion protein by adopting a liquid chromatography-mass spectrometry combined technology, wherein the molecular weight of the exenatide fusion protein is more than 10000 Da.

Background

In the past decade, the development of innovative chemical drugs has been subject to difficulties and challenges, and has progressed slowly. On the contrary, since biopharmaceuticals are more and more focused by pharmaceutical companies due to their high specificity and low toxicity, and the patent protection of marketed biopharmaceuticals is about to expire, this will greatly promote the development of the biopharmaceutical market, and in the coming years, biopharmaceuticals will enter the high-speed development stage as new pharmaceutical entities. With the increasing interest and investment of enterprises and the increasing demand for biopharmaceuticals in the medical field, it is expected that by 2020, biopharmaceuticals will have a market share exceeding 1/3. The rapid development of biological drugs inevitably leads to an urgent need for quantitative analysis methods, and therefore, the development of simple and efficient quantitative methods becomes a difficult point and a hotspot in the research and development of biological drugs.

Polypeptide and protein drugs are generally large in molecular weight, poor in ionization efficiency, low in ion detection efficiency and large in ion variability, and the application of mass spectrometry in quantitative analysis of the polypeptide and protein drugs is severely limited by the factors. Therefore, the Ligand Binding Assay (LBA) has once become the only quantitative analysis method for polypeptide and protein drugs, and is widely used. However, as mass spectrometry technology improves and develops, the disadvantages of the LBA method are also gradually revealed. These disadvantages include: the LBA method is time consuming and costly to develop; some key reagents (e.g., antibodies, radiolabelling agents, etc.) are highly variable; the same LBA method is difficult to be simultaneously suitable for sample determination of different matrixes; LBA methods do not enable simultaneous quantification of multiple compounds; cross reaction is easy to occur, and the quantitative accuracy is poor.

In recent years, with the improvement of the cognitive level of researchers and the advent of various novel mass spectrometers, some researchers find that polypeptide and protein drugs can be hydrolyzed into a plurality of small peptides with relatively small molecular weights, specific peptide fragments are selected from the small peptides, and the in vivo conditions of the polypeptide and protein drugs are indirectly reflected by the quantification of the specific peptide fragments, so that the method is called an indirect LC-MS/MS method. The research strategy has been successfully applied to the quantitative analysis of polypeptide and protein drugs and is widely accepted. In Wang's research, KPC protein is first hydrolyzed into several small peptides, 3 specific peptide segments are selected from them, and the indirect LC-MS/MS method is applied to successfully realize the quantitative research of KPC protein [1 ]. Using the same study strategy, researchers successfully achieved quantitation of interleukin 21 (IL-21) in human and cynomolgus monkey serum and tissues [2 ]. In the work by fungen, the authors outlined the latest advances in enzymatic and immunocapture technologies for quantification of polypeptides, proteinaceous drugs [3 ].

however, the indirect LC-MS/MS method is a double-edged sword for researchers, and although the researchers obtain more accurate quantitative results, the sample pretreatment becomes complicated. Before quantitative analysis of an object to be detected, not only needs to optimize proteolysis conditions, but also needs to confirm specific peptide fragments, and needs to develop a simple and efficient peptide fragment extraction and purification method. Therefore, indirect LC-MS/MS is still available for academic research, but high-throughput applications are difficult to achieve. How to develop a simple, accurate, high-throughput method for quantifying polypeptides and proteins has become a significant challenge for researchers.

Because the quantitative research of polypeptide and protein medicines is still in the initial stage and corresponding research reports are less, the corresponding guidance principle is not provided in each country. The only guidance available at present is a white paper published in the journal of AAPS (american association of pharmaceutical scientists) in 2015, and the authors therein summarize the research results obtained in the application of LC-MS/MS method for quantifying polypeptide protein drugs in recent years. The authors indicate that the choice of quantitative analysis method depends mainly on the molecular weight of the test substance, and that when the molecular weight of the test substance is less than 10000Da, the complete test substance can be directly quantified by LC-MS/MS; when the molecular weight of the analyte is more than 10000Da, an indirect LC-MS/MS method is adopted to quantify the analyte [4 ].

The detection object of the invention is exenatide fusion protein-exenatide fusion protein with human fibronectin type III domain 10 or a fusion protein of a mutant thereof, and early pharmacodynamic studies find that the drug not only has the hypoglycemic effect, but also has a certain curative effect on Alzheimer's disease. The molecular weight of the compound is 15000-20000 Da which is far more than 10000Da, and the compound should be quantitatively researched by adopting a ligand binding test (LBA) or an indirect LC-MS/MS method theoretically, but the two methods are more complex and have longer development period, so that the detection requirement of a research object is difficult to meet.

Reference documents:

[1]Wang, H.; Drake, S. K.;Youn, J. H.; Rosenberg, A. Z.; Chen, Y.;Gucek, M.;Suffredini, A. F.; Dekker, J. P. Sci. Rep. 2017, 7,1-10.

[2] Palandra, J.; Finelli, A.; Zhu, M.; Masferrer, J.; Neubert, H. Anal Chem. 2013, 85, 5522-5529.

[3]Fung, E. N.; Bryan, P.;Kozhich, A. Bioanalysis.2016, 8,847-856.

[4]Jenkins, R.; Duggan, J. X.;Aubry, A. F.; Zeng, J.; Lee, J. W.;Cojocaru, L.;Dufield, D.;Garofolo, F.;et al.Aaps J. 2015, 17, 1-16.

Disclosure of Invention

Aiming at the defects in the prior art, the inventor tries to directly detect the exenatide fusion protein in a sample to be detected by adopting a liquid chromatography-mass spectrometry method. Through intensive research, a detection method which is simple in operation, accurate in result, good in specificity and high in sensitivity is successfully developed, and can be used for detecting exenatide fusion protein with molecular weight more than 10000 Da.

The technical scheme adopted by the invention is as follows:

A method for directly detecting exenatide fusion protein by using a liquid chromatography-mass spectrometry method, comprising the following steps: separating a sample to be detected suspected to contain target protein by using a High Performance Liquid Chromatography (HPLC) to obtain a part enriched with the target protein; subjecting the enriched target protein to an electrospray ionization source under conditions suitable to produce chromatographically detectable one or more target protein ions; determining the amount of one or more target protein ions by tandem mass spectrometry, wherein the determined ions comprise precursor ions and fragment ions.

When the method is used for quantitative determination of the exenatide fusion protein, the method further comprises the following steps: preparing a standard substance and a quality control substance, and respectively pretreating the standard substance, the quality control substance and a sample to be detected by using a solid-phase extraction column. The preparation method of the standard substance and the quality control substance comprises the following steps: preparing standard substance solutions with exenatide fusion protein concentrations of 5, 10, 20, 50, 100, 250 and 500ng/ml by using a blank matrix; and preparing quality control solution with exenatide fusion protein concentrations of 5, 15, 80 and 400ng/ml by using the blank matrix. The solvent of the standard may be water, glucose solution, mixed solution of acetonitrile and water, mixed solution of methanol and water, mixed solution of acetonitrile or methanol and ammonium salt aqueous solution, wherein the volume percentage of acetonitrile or methanol in each mixed solution is not more than 80%.

The sample to be detected can be a biological sample, can be from monkey, human, dog, rat, mouse or rabbit, and can be plasma, serum, bile, urine or cerebrospinal fluid.

The Solid Phase Extraction (SPE) pretreatment comprises the following specific operations: adding acetonitrile into the solid phase extraction column, and centrifuging to discard effluent; adding a balance solution into the solid phase extraction column, and centrifuging to discard an effluent; adding a sample to be detected into the solid phase extraction column, and centrifuging to discard an effluent; adding the leaching solution 1 and the leaching solution 2 into the solid-phase extraction column in sequence, and discarding effluent; adding an elution solution into the solid phase extraction column, and collecting an effluent liquid.

In a preferred embodiment of the invention, the solid phase extraction pretreatment is performed using MonoSpin^TMC18 centrifugal solid phase extraction column. In addition to the conventional mesopores (pore size 10 nm), a large number of through-holes (pore size 5 μm) were present in the SPE column. The existence of the through hole not only ensures the smooth passing of the biological matrix, avoids the phenomenon of cylinder blockage, but also ensures the effective retention of the macromolecular to-be-detected object.

The equilibrium solution and the leacheate 1 after the solid-phase extraction pretreatment can be formic acid aqueous solution, acetic acid aqueous solution or trifluoroacetic acid aqueous solution; the leacheate 2 can be a mixed solution of acetonitrile and water or a mixed solution of acetonitrile and an ammonium salt aqueous solution, wherein the volume percentage concentration of the acetonitrile is 1% -10%; the eluent can be a mixed solution of acetonitrile and water or a mixed solution of acetonitrile and an ammonium salt aqueous solution, wherein the volume percentage concentration of the acetonitrile is 20-80%. The aqueous ammonium salt solution may be an ammonium formate, ammonium acetate or ammonium sulfate solution, wherein the concentration of ammonium salt is 0.5-50 mM. A preferred eluent is acetonitrile/5 mM ammonium acetate (30/70, v/v).

Further, the liquid chromatographic conditions of the step (1) are that a liquid chromatographic column with a filler of octadecyl bonded phase silica gel is adopted, the pore diameter is 200 Å (20nm), a mobile phase A is an acetonitrile solution containing formic acid or acetic acid, a mobile phase B is an aqueous solution containing formic acid or acetic acid, and gradient elution is carried out.

In a preferred embodiment of the invention, HPLC tandem mass spectrometry can be performed using Shimadzu GL Sciences InertSustain Bio C18 LC Columns (100X 2.1mm, particle size 1.9 μm, pore size 20nm) or Columns of similar nature, at a column temperature of 40 ℃ and a sample size of 10 μ l.

The response strength of the target protein can be obviously improved by adding acid into the water phase of the liquid chromatography mobile phase, and formic acid or acetic acid can be added; the volume percentage concentration of the acid is 0.05-0.5%, preferably 0.05-0.2%, and more preferably 0.1%; the acid is preferably formic acid; the flow rate was 0.3ml/min and the elution was carried out in a gradient. The volume percentage concentration is the volume (ml) of acid contained in 100ml of water. The gradient elution procedure is shown in table 1.

TABLE 1 gradient elution conditions for liquid chromatography

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	30	70
0.5	30	70
			5	45	55
5.1	98	2
			6.5	98	2
6.6	30	70
			8	30	70

The electrospray ionization conditions of the step (2) are as follows: interface temperature: 300 ℃, atomizing gas flow: 3L/min, heating air flow: 10L/min, heating block temperature: 400 ℃, desolventizing gas temperature: 250 ℃, dry air flow: 10L/min.

The mass spectrum conditions of the step (3) are as follows: triple quadrupole mass spectrometer adopts positive ion mode detection, collision energy: 37eV, and the scanning mode is multi-reaction monitoring. In a preferred embodiment of the invention, the mass spectrometer uses an shimadzu LC-MS8060 triple quadrupole mass spectrometer, Q1 pre-biased: -16V, Q3 pre-biased: -34V, atomizing gas flow rate: 3L/min, heating air flow: 10L/min, interface temperature: 300 ℃, desolventizing gas temperature: 250 ℃, heating block temperature: 400 ℃, dry air flow: 10L/min, the ion reaction for the quantitative analysis of the exenatide fusion protein is m/z 1630.6 → 1573 respectively.

Specifically, the exenatide fusion protein is a fusion protein of exenatide and a 10 th domain of human fibronectin type III or a mutant thereof. Furthermore, the sequence of the fusion protein is SEQ ID NO. 1, and the code is FIM.

In a preferred embodiment of the invention, an internal standard, preferably an exenatide fusion protein mutant, is included in the hplc tandem mass spectrometry. The research result shows that the amino acid sequence is SEQ ID NO:2, the exenatide fusion protein mutant FIM-GA keeps high consistency with the exenatide fusion protein in the aspects of extraction efficiency and chromatographic behavior, and the mutant serving as an internal standard has no interference on the exenatide fusion protein.

The method of the invention determines that the exenatide fusion protein has good linearity (r) within the range of 5-500ng/ml²>0.99). The high performance liquid chromatography tandem mass spectrometry established by the invention meets the analysis requirements of CFDA on biological samples in the aspects of accuracy, precision, specificity, stability, matrix effect and the like. The method of the invention has been successfully applied to the detection of the blood concentration of the hypodermically injected exenatide fusion protein cynomolgus monkey.

The invention has the following beneficial effects:

1. the method of the invention realizes the direct quantitative analysis of the complete biological macromolecules (molecular weight is more than 10000Da) in the biological sample for the first time.

2. The sensitivity is very high, with a lower limit of quantitation of 5ng/ml (equivalent to 0.3 pmol/ml), which is very uncommon for the direct quantitation of intact proteins.

3. A simple and efficient pretreatment method of a biological sample is successfully developed, and the biological sample can be directly subjected to liquid quality detection after simple solid phase extraction. Compared with the traditional affinity capture method or enzymolysis method, the SPE method is time-saving, efficient, low in cost and easy to realize high-throughput application.

4. The method uses the designed and prepared exenatide fusion protein mutant as an internal standard protein, can accurately quantify the exenatide fusion protein, and ensures the reliability of results.

5. The present inventors successfully applied this method to preclinical pharmacokinetic studies of exenatide fusion proteins, and believe that this method as an analytical tool would drive the development of similar biopharmaceuticals.

the sequences provided in the invention are:

SEQ ID NO:1

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGTGGSGGGSVSSVPTNLEVVAATPTSLLISWDAPYAYSAAAVDYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAEVRSFCTDWPAEKSCKPLRGKPISINYRT

SEQ ID NO:2

HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGTGGSGGGSVSGAPTNLEVVAATPTSLLISWDAPYAYSAAAVDYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAEVRSFCTDWPAEKSCKPLRGKPISINYRT

Drawings

FIG. 1: a through hole structure of integral silicon dioxide and a pretreatment flow chart of a biological sample.

FIG. 2: influence of organic solvent ratio in SPE pretreatment eluent on recovery. (A) The eluent was acetonitrile/5 mM ammonium acetate (10/90, v/v); (B) the eluent was acetonitrile/5 mM ammonium acetate (30: 70, v/v); (C) the eluent was acetonitrile/5 mM ammonium acetate (70: 30, v/v)

FIG. 3: the effect of acid or base addition to SPE pretreatment eluents on recovery. (A) The eluent was acetonitrile/5 mM ammonium acetate (30/70, v/v); (B) eluent acetonitrile/5 mM ammonium acetate (30/70, v/v), 2% formic acid; (C) the eluent was acetonitrile/5 mM ammonium acetate (30/70, v/v), 2% aqueous ammonia was added.

FIG. 4: separation effect of different chromatographic columns on FIM protein. (A) Diamonsil C18 column (100X 4.6mm, particle size 5 μm, pore size 10nm, Dima); (B) ZORBAX Eclipse Plus C8 chromatographic column (100X 4.6mm, particle size 5 μm, pore size 10nm, Agilent); (C) GL Sciences InertSustain Bio C18 chromatographic column (100X 2.1mm, particle size 1.9 μm, pore size 20nm, Shimadzu).

FIG. 5: effect of different mobile phase systems on the chromatographic separation of FIM proteins. (A) Acetonitrile-water as mobile phase; (B) acetonitrile-5 mM ammonium acetate aqueous solution as mobile phase; (C) containing 0.05% formic acid acetonitrile-0.05% formic acid water solution as mobile phase; (D) containing 0.1% formic acid acetonitrile-0.1% formic acid water solution as mobile phase; (E) containing 0.2% formic acid acetonitrile-0.2% formic acid aqueous solution as mobile phase.

FIG. 6: human insulin and liraglutide as representative chromatograms of internal standards. (A) Chromatogram of 10ng/ml human insulin standard solution; (B) chromatogram of blank plasma with 10ng/ml human insulin added; (C) chromatograms of FIM protein and liraglutide standard solutions under optimal chromatographic conditions for FIM protein.

FIG. 7: electrophoretic detection map of FIM-GA internal standard protein

FIG. 8: representative chromatograms of FIM protein (4.52 min) and FIM-GA internal standard protein (4.41 min). (A) Blank monkey plasma samples; (B) adding a blank plasma sample of 5ng/ml FIM protein and 1 mu g/ml FIM-GA internal standard protein; (C) plasma samples collected 1h after subcutaneous administration of 1mg/kg FIM protein, 1. mu.g/ml FIM-GA internal standard protein was added.

FIG. 9: calibration graph of FIM

FIG. 10: mean plasma drug concentration-time curve (n = 2) after subcutaneous injection in monkeys of 1mg/kg FIM protein.

Detailed Description

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. Modifications and equivalents will occur to those skilled in the art and are intended to be included within the scope of the present invention.

Example 1: establishment and optimization for detecting exenatide fusion protein by LC-MS/MS method

1.1 instruments, materials and reagents

The instrument comprises the following steps: API LCMS-8060 LC MS, Shimadzu corporation, Japan; labsolutions chromatography workstations, Shimadzu Japan; model 17R high speed refrigerated centrifuge, thermo fisher technologies; VX-02 model multi-tube vortex oscillator; BM-40 pure water purification system, Beijing Zhongsheng Yang Yuan science and technology development Co.

Sample and reagent: 6 blank monkey plasma; the exenatide fusion protein FIM and FIM-GA internal standard protein are prepared by Beijing Huajinriqing biological medicine technology company Limited; chromatographic grade methanol and acetonitrile, purchased from Tianjin Cordcord science and technology Limited; chromatographic grade formic acid, purchased from Tianjin Guangfu Fine chemical research institute; ammonium acetate, available from tianjin shin & science and technology development ltd; purified water is made; other reagents were analytically pure.

1.2 selection of solid phase extraction column

100ng/ml of drug-containing plasma was pretreated using a ProElut C18 (particle size 50 μm, pore size 10nm, Dima Tech Co.) solid phase extraction column. Firstly, adding 3ml of methanol into a solid phase extraction column, and discarding effluent; adding 3ml of water, and discarding the effluent; 1ml of sample was loaded and the effluent collected. As a result, clogging of the column during the loading process and the drug-containing plasma could not be flowed down by pressurization or evacuation with the ear washing bulb.

100ng/ml of drug-containing plasma was pretreated using Shimadzu GL WondaSep MAX (particle size 70 μm, pore size 9 nm) and WondaSep MCX (particle size 70 μm, pore size 9 nm) solid phase extraction columns, respectively. Firstly, adding 3ml of methanol into a solid phase extraction column, and discarding effluent; adding 3ml of water, and discarding the effluent; 1ml of sample is loaded, and effluent is discarded; adding 3ml of water and 3ml of methanol/water (volume ratio of 5: 95) in sequence for leaching, and removing impurities; adding 1ml of methanol containing 2% formic acid to the WondApp MAX column for elution, adding 1ml of methanol containing 30% ammonia water to the WondApp MCX column for elution, and collecting the effluent. Blow-drying the effluent by nitrogen, redissolving by 100 mul of redissolving solvent (methanol/water containing 2% formic acid (volume ratio of 1: 1)), and taking 10 mul of redissolved matter for liquid chromatography-mass spectrometry detection. No analyte was detected in any of the results. And (3) repeatedly carrying out solid-phase extraction on the plasma containing the medicine, diluting the eluent by 10 times with a redissolving solvent, and directly carrying out liquid chromatography-mass spectrometry detection, wherein the result still does not detect the substance to be detected.

1.3 solid phase extraction pretreatment (SPE) method optimization

The drug-containing plasma samples were pretreated using a MonoSpin C18 centrifugal solid phase extraction column (mesoporous 10nm, through-hole 5 μm, Shimadzu corporation). Firstly, adding 300 mul of acetonitrile into a solid phase extraction column, centrifuging for 1min at the temperature of 4 ℃ and the centrifugal force of 2000g, and discarding effluent; adding 300 mul of aqueous solution containing 0.01% trifluoroacetic acid into the solid phase extraction column, centrifuging for 1min, and discarding effluent; sampling 100 mul, centrifuging for 1min, and discarding effluent; adding 300 mul of aqueous solution containing 0.01% trifluoroacetic acid and 300 mul of acetonitrile/5 mM ammonium acetate (volume ratio is 5: 95) in sequence for leaching, and removing impurities; and finally, adding 100 mul of eluent into the solid phase extraction column for elution, and collecting the effluent liquid. The through-hole structure of the monolithic silica and the pretreatment procedure of the biological sample are shown in FIG. 1.

3 kinds of eluents containing organic solvents in different proportions are designed, the same plasma sample containing the medicine is subjected to solid phase extraction pretreatment according to the steps and the parameters, and then liquid chromatography-mass spectrometry detection is carried out. The chromatogram is shown in fig. 2, and the result shows that the response value of the drug is reduced when the organic solvent ratio in the eluent is higher or lower, and the optimal ratio is acetonitrile/5 mM ammonium acetate =30:70 (volume ratio).

And (3) designing an eluent which does not contain acid and alkali and contains 2% formic acid and 2% ammonia water, respectively carrying out solid-phase extraction pretreatment on the same plasma sample containing the medicine according to the steps and the parameters, and then carrying out liquid chromatography-mass spectrometry detection. The chromatogram is shown in FIG. 3, and the results show that neither acid nor base addition to the eluate did improve recovery, so the most preferred SPE eluate was acetonitrile/5 mM ammonium acetate (30: 70 by volume).

1.4 selection of chromatography columns

The chromatographic column A is Diamonsil C18 (100X 4.6mm, particle size 5 μm, pore size 10nm, Dimakejiu Co.); the chromatographic column B is ZORBAX Eclipse Plus C8 (100X 4.6mm, particle size 5 mu m, pore size 10nm, Agilent company); the chromatographic column C was GLSciences InertSustainBio C18 (100X 2.1mm, particle size 1.9 μm, pore size 20nm, Shimadzu corporation).

Drug-containing plasma samples subjected to SPE pretreatment in the optimal manner of 1.3 were separated by the 3 types of chromatography columns described above, respectively, and subjected to mass spectrometry.

Mass spectrum conditions: adopting an electrospray ionization source (ESI), and selecting a scanning mode of multi-reaction monitoring (MRM) to carry out measurement in a positive ionization mode, wherein the ion reactions for FIM and internal standard FIM-GA quantitative analysis are m/z 1630.6 → 1573 and m/z 1625.1 → 1573.1 respectively; other mass spectral parameters are shown in table 2.

TABLE 2 Mass Spectrometry Condition parameters of Exenatide fusion proteins and internal reference proteins

The results are shown in FIG. 4. When the chromatographic column A is used for determination, the peak shape of the substance to be determined is poor and the substance cannot be separated from endogenous interfering components (4A); the chromatographic column B has an obvious peak shape tailing phenomenon, and the quantification of the object to be detected is seriously influenced (4B); the chromatographic column C has the best effect, the peak shape of the object to be detected is sharp and symmetrical, and the object to be detected and the endogenous interference component (4C) can be effectively separated. Therefore, finally we chose a GL Sciences inertsustatin Bio C18 chromatography column for isolation and analysis of exenatide fusion proteins.

1.5 optimization of the mobile phase of liquid chromatography

We first selected the commonly used organic phases methanol and acetonitrile for comparative experiments. FIM solution with concentration of 100ng/ml, organic phase with methanol and acetonitrile as mobile phase, GL Sciences InertSustain Bio C18 chromatographic column for multiple injection, and liquid chromatography-mass spectrometry detection, the data results are shown in Table 3. The results show that: when methanol is used as an organic phase for repeated detection, the area of the FIM peak is gradually reduced, which indicates that the object to be detected is unstable, and when acetonitrile is used as the organic phase, the FIM response value is stable. Therefore, acetonitrile was chosen as the organic phase of the mobile phase.

TABLE 3 chromatographic determination of FIM with methanol and acetonitrile as the mobile phase organic solvents, respectively

Under the condition of taking acetonitrile as an organic phase solvent, the optimization of the mobile phase formula is tried. 5 different mobile phase systems in the table 4 are respectively tested, the drug-containing plasma sample with the same concentration is used for carrying out liquid chromatography-mass spectrometry detection, the conditions of mass spectrometry are the same as 1.4, and the obtained chromatogram is shown in the figure 5. The result shows that the response intensity of the substance to be detected is obviously improved by adding a proper amount of formic acid (0.1%) into the mobile phase, and the response intensity of the exenatide fusion protein is reduced by adding ammonia salt (ammonium formate, ammonium acetate and the like), so that the final mobile phase system is 0.1% formic acid aqueous solution (A) -0.1% formic acid acetonitrile (B).

TABLE 4.5 different mobile phase systems

Therefore, the optimal chromatographic conditions are as follows: GL Sciences InertSustainBio C18 chromatographic column (100X 2.1mm, particle size 1.9 μm, pore size 20nm, Shimadzu corporation); mobile phase: 0.1% aqueous formic acid (a) -acetonitrile (B) containing 0.1% formic acid; sample introduction amount: 10 mu l; flow rate: 0.3 ml/min; column temperature: 40 ℃; the gradient elution conditions are shown in table 1.

1.6 selection of internal standards

1.6.1 internal standard of human insulin

We first chose human insulin as an internal standard. An internal standard solution containing 10ng/ml human insulin was prepared using a sample dilution (acetonitrile/5 mM ammonium acetate =30: 70) and assayed by LC-MS as in 1.5, and the results are shown in FIG. 6A. Meanwhile, a plasma sample containing 10ng/ml human insulin is prepared, and liquid chromatography-mass spectrometry detection is carried out after SPE pretreatment, and the result is shown in FIG. 6B. The results show that recovery of SPE pretreatment was low and the chromatographic peak profile of human insulin in monkey plasma samples was poor.

1.6.2 Liraglutide internal standards

we also tried liraglutide as an internal standard. Plasma samples of liraglutide were prepared and subjected to SPE pretreatment followed by liquid chromatography-mass spectrometry detection, with the results shown in fig. 6C. The result shows that the peak pattern of the liraglutide is poor, and the response value of the liraglutide is gradually reduced after continuous sample injection, so that the liraglutide is not suitable for serving as an internal standard. The specific data are shown in Table 5.

TABLE 5 chromatographic data for serial injection of liraglutide plasma samples

Time to peak (min)	Peak area
		5.175	146,512
5.175	87,873
		5.175	57,242
5.175	62,726
		5.175	49,117
5.175	49,729
		5.175	35,187
5.175	42,083
		5.175	35,411

1.6.3 FIM-GA internal Standard

In view of the fact that no suitable internal standard is available on the market, we designed a mutant FIM-GA with 2 amino acids different from the FIM protein, and the FIM-GA protein with the purity of more than 95% is obtained by performing ion exchange chromatography and hydrophobic chromatography purification after being expressed by using escherichia coli, and an electrophoretogram is shown in FIG. 7.

FIM-GA was tested for its suitability as an internal standard. Adding 1 mug/ml FIM-GA protein into the FIM plasma sample to prepare internal standard plasma, performing liquid chromatography-mass spectrometry detection after SPE pretreatment, and obtaining a chromatographic result shown in figure 8. The result shows that the chromatographic peak type of the FIM-GA internal standard protein is good, the elution time is closer to that of the exenatide fusion protein, the chromatographic behavior consistency is higher, and the method is ideal internal standard selection.

example 2 methodological validation of the assay of Exenatide fusion proteins by LC-MS/MS method

2.1 sensitivity and specificity

We selected blank monkey plasma from 6 different sources and performed SPE pretreatment and LC-MS detection in the optimal manner as in example 1, and the chromatographic results are shown in fig. 8A. Fig. 8A shows that no exenatide fusion protein was detected in the blank monkey plasma samples, demonstrating good specificity of the method.

Meanwhile, the signal-to-noise ratio of the lower limit of quantification (LLOQ) is analyzed, the detection result of the lower limit of quantification sample is shown in figure 8B, and the calculation result shows that the signal-to-noise ratio is far greater than 10, so that the method meets the quantification requirement, and the method is proved to have good sensitivity.

2.2 calibration Curve construction

Taking FIM stock solution (20.45 mg/ml), diluting with sample diluent to obtain calibration curve working solution with concentration of 50, 100, 200, 500, 1000, 2500, 5000 ng/ml. And diluting the FIM-GA internal standard stock solution (2.46 mg/ml) by using the same diluent to finally obtain the internal standard working solution with the concentration of 1 mug/ml. And respectively taking 20 mul of FIM calibration curve working solution, adding 180 mul of monkey blank plasma, and uniformly mixing to obtain drug-containing plasma samples with the concentrations of 5, 10, 20, 50, 100, 250 and 500ng/ml of calibration curve. And adding 20 mul of internal standard working solution and mixing uniformly again. The plasma sample containing the medicine is prepared at present, the substance to be detected and the internal standard stock solution are stored in a refrigerator at the temperature of 20 ℃ below zero, and the standard working solution is stored in a refrigerator at the temperature of 4 ℃.

Plasma samples were subjected to SPE pretreatment and quantitative analysis of the liquid quality as in example 1, and the calibration curve is shown in FIG. 9. The results show good linearity in the range of 5-500ng/ml, with a representative regression equation of y =0.0104051x +0.00146893 (r)²=0.9981)。

2.3 accuracy and precision

Taking FIM stock solution (20.45 mg/ml), diluting with sample diluent to obtain final working solution with concentration of 50, 150, 800, 4000 ng/ml. And diluting the FIM-GA internal standard stock solution (2.46 mg/ml) by using the same diluent to finally obtain the internal standard working solution with the concentration of 1 mug/ml. And respectively taking 20 mul of FIM quality control working solution, adding 180 mul of monkey blank plasma, and uniformly mixing to obtain drug-containing plasma sample concentrations of 5, 15, 80 and 400ng/ml for quality control. And adding 20 mul of internal standard working solution and mixing uniformly again. 6 samples were prepared in parallel for each quality control concentration, and the pretreatment by solid phase extraction and quantitative analysis of liquid quality were carried out in the optimum manner in example 1 for three days with the statistical results shown in Table 6. Accuracy and precision are expressed in relative error (RE%) and relative standard deviation (RSD%), respectively. The result meets the verification requirement of the biological sample analysis method (LLOQ is in the range of +/-20 percent, and the low, medium and high concentration quality control substances are in the range of +/-15 percent).

TABLE 6 detection of Intra-and diurnal precision and accuracy of exenatide fusion proteins in monkey plasma (n =6)

2.4 substrate Effect and recovery

2 quality control concentrations 15 and 400ng/ml were examined, each concentration being paralleled by 6 samples. Firstly, taking 20 mul of quality control working solution, adding 180 mul of diluent, adding 20 mul of internal standard protein, uniformly mixing to obtain a mixed standard solution, carrying out liquid chromatography-mass spectrometry analysis, and recording the measured peak area as a; preparing a drug-containing plasma quality control sample according to 2.3, carrying out SPE pretreatment and sample injection analysis according to the optimal mode of the example 1, and recording the measured peak area as b; the blank plasma was SPE pretreated as optimal for example 1, the collected eluent was blown dry with nitrogen, redissolved with the above "mixed standard" solution, analyzed by injection and the measured peak area was designated as c.

c/a x 100% is the matrix effect, and the determination result is 90-101%, which indicates that the FIM determined by the method is not interfered by endogenous substances in monkey plasma. b/c × 100% was the recovery rate, and the measurement result was 40%.

2.5 stability

The 2 concentrations of drug-containing plasma quality control samples were examined at 15, 400ng/ml, each concentration being paralleled by 6 samples. 3 stability experiments were performed separately: 1. standing at room temperature for 2.5 h; 2. the sample after SPE treatment according to the embodiment 1 is placed in an autosampler for 24 h; 3. freeze-thawing at-70 deg.C and 20 deg.C for three times.

The stability results are expressed in relative error (RE%) and relative standard deviation (RSD%). The results are shown in Table 7. The results show that FIM protein stability is good under all conditions examined.

Table 7 stability of FIM plasma samples under different storage conditions (n =6)

Example 3: application of LC-MS/MS method in determination of pharmacokinetics of exenatide fusion protein in monkey plasma

The experimental animal is a cynomolgus monkey purchased from Suzhou Xishan Zhongke experimental animals Co. The animal study protocol was conducted according to the guidelines of the animal protection and application committee of the Tianjin institute of medicine, approved by the science and technology committee of Tianjin City. Two cynomolgus monkeys were randomly selected, one each for male and female, and the experimental animals were fed with normal diet and water ad libitum during the experiment. The neck and back were given 1mg/kg FIM protein by subcutaneous injection, and the forelimb was bled for 1ml before and after administration at 0.083, 0.167, 0.333, 0.5, 1, 2, 4, 6, 8 and 12h, respectively. The collected blood sample was centrifuged at 4000g for 5min, and the upper plasma was collected, separately packed and frozen in a refrigerator at-70 ℃. And taking out the frozen sample when the sample is to be analyzed, and melting the frozen sample at room temperature.

The chromatogram of the plasma samples collected 1h after administration is shown in FIG. 8C, the mean plasma drug concentration-time curve is shown in FIG. 10, and the corresponding calculation results of the pharmacokinetic parameters are shown in Table 8. The results show that: the FIM protein is absorbed quickly in vivo after being administered subcutaneously, and the blood concentration in vivo reaches the maximum value 1h after administration; then rapidly eliminated, and the blood concentration is 1/20 below the maximum concentration 12h after administration; the elimination half-life is 2 h.

TABLE 8 pharmacokinetic parameters of FIM protein in monkeys after subcutaneous administration (n = 2)

sequence listing

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Tianjin research institute for New drug evaluation Co Ltd

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Claims (23)

1. A method for directly determining exenatide fusion protein in a sample, wherein the molecular weight of the exenatide fusion protein is more than 10000Da, comprising the following steps:

The method comprises the steps of separating a sample to be detected suspected to contain target protein by a High Performance Liquid Chromatography (HPLC) to obtain a part enriched with the target protein.

Subjecting the enriched target protein to an electrospray ionization source under conditions suitable to produce chromatographically detectable one or more target protein ions.

Determining the amount of one or more target protein ions by tandem mass spectrometry, wherein the determined ions comprise precursor ions and fragment ions.

2. The quantitative determination method according to claim 1, characterized by comprising the steps of:

The method comprises the steps of preparing a standard product and a quality control product.

And utilizing a solid phase extraction column to respectively pretreat the standard product, the quality control product and the sample to be detected.

And thirdly, respectively separating the standard substance, the quality control substance and the sample to be detected suspected to contain the target protein by High Performance Liquid Chromatography (HPLC) so as to obtain the enriched part of the target protein.

the enriched target protein is subjected to an electrospray ionization source under conditions suitable to generate chromatographically detectable target protein ion or ions.

Fifthly, determining the amount of one or more target protein ions by tandem mass spectrometry, wherein the determined ions comprise precursor ions and fragment ions.

3. The method of claim 1 or 2, wherein the sample comprises a biological sample.

4. The method of claim 3, wherein the biological sample is from a monkey, a human, a dog, a rat, a mouse, or a rabbit.

5. The method of claim 3, wherein the biological sample comprises plasma, serum, bile, urine, and cerebrospinal fluid.

6. A process according to claim 1 or 2, wherein the solid phase extraction comprises the steps of:

Adding acetonitrile into the solid phase extraction column, and centrifuging to discard effluent;

Adding a balance solution into the solid phase extraction column, and centrifuging to discard an effluent;

Adding a sample to be detected into the solid phase extraction column, and centrifuging to discard an effluent;

Adding the leaching solution 1 and the leaching solution 2 into the solid-phase extraction column in sequence, and discarding effluent;

Adding an elution solution into the solid phase extraction column, and collecting an effluent liquid.

Wherein the HPLC chromatographic conditions are as follows: adopts a liquid chromatographic column with octadecyl bonded phase silica gel as a filler and has a pore diameter(20nm), the mobile phase A is acetonitrile solution containing formic acid or acetic acid, the mobile phase B is aqueous solution containing formic acid or acetic acid, and gradient elution is carried out.

Wherein the electrospray ionization conditions are as follows: interface temperature: 300 ℃, atomizing gas flow: 3L/min, heating air flow: 10L/min, heating block temperature: 400 ℃, desolventizing gas temperature: 250 ℃, dry air flow: 10L/min.

Wherein the tandem mass spectrometry conditions are: triple quadrupole mass spectrometer adopts positive ion mode detection, collision energy: 37eV, and the scanning mode is multi-reaction monitoring.

7. The method of claim 6, wherein the equilibration solution may be an aqueous solution comprising formic acid, an aqueous solution comprising acetic acid, or an aqueous solution comprising trifluoroacetic acid; wherein the leaching solution 1 can be aqueous solution containing formic acid, aqueous solution containing acetic acid and aqueous solution containing trifluoroacetic acid; wherein the leaching solution 2 can be a mixed solution of acetonitrile and water or a mixed solution of acetonitrile and an ammonium salt water solution, wherein the volume percentage concentration of the acetonitrile is 1-10%; the elution solution can be a mixed solution of acetonitrile and water or a mixed solution of acetonitrile and an ammonium salt water solution, wherein the volume percentage concentration of the acetonitrile is 20-80%.

8. Elution solution 2 and according to claim 7, characterized in that the aqueous ammonium salt solution may be ammonium formate, ammonium acetate or ammonium sulphate solution, wherein the concentration of ammonium salt is 0.5-50 mM.

9. The method of claim 6, wherein the solid phase extraction column is GL Sciences C18。

10. The method of claim 6, wherein the HPLC gradient elution conditions are 0-0.5min, 70% B; 0.5-5min, 70% -55% B; 5-5.1min, 55% -2% B; 5.1-6.5min, 2% B; 6.5-6.6min, 2% -70% B; 6.6-8min, 70% B; the volume percentage concentration of the acid in the mobile phase A and the mobile phase B is 0.05 to 0.5 percent.

11. The method according to claim 10, wherein the concentration of the acid in mobile phases a and B is between 0.05% and 0.2% by volume.

12. the method of claim 11, wherein the acid is present in mobile phases a and B at a concentration of 0.1% by volume.

13. The method of any one of claims 7, 10-12, wherein the acid is formic acid.

14. The method of claim 6, wherein the liquid chromatography column is GL Sciences InertSustainBio C18 LC Columns.

15. The method of claim 2, wherein the standard and quality control are prepared by the following method:

and preparing standard solutions with the exenatide fusion protein concentrations of 5, 10, 20, 50, 100, 250 and 500ng/mL by using the blank matrix.

And preparing quality control solution with exenatide fusion protein concentrations of 5, 15, 80 and 400ng/mL by using the blank matrix.

16. The method according to claim 2, wherein the solvent of the standard may be water, glucose solution, mixed solution of acetonitrile and water, mixed solution of methanol and water, acetonitrile or mixed solution of methanol and ammonium salt aqueous solution, wherein the volume percentage of acetonitrile or methanol in each mixed solution is not more than 80%.

17. The method of claim 1 or 2, wherein said exenatide fusion protein is a fusion protein of exenatide with human fibronectin type iii domain 10 or a mutant thereof.

18. The method of claim 17, wherein the exenatide fusion protein has the sequence of seq id NO 1.

19. The method of claim 2, wherein the standard and quality control further comprise an internal standard protein.

20. The method of claim 19, wherein the internal standard protein is a mutant of exenatide fusion protein.

21. The method of claim 20, wherein the sequence of the internal standard protein is SEQ ID No. 2.

22. The method of claim 18, wherein the measured target protein precursor and fragment ions are 1630.6 and 1510.9, respectively.

23. The method of claim 21, wherein the measured internal standard protein precursor and fragment ions are 1625.1 and 1573.1, respectively.