CN111072766B

CN111072766B - Deamidation impurity of rhPTH (1-34), preparation method and application thereof

Info

Publication number: CN111072766B
Application number: CN201911400036.8A
Authority: CN
Inventors: 林桃; 李静; 刘月峰; 樊欣迎; 郭静雅; 闻亚磊
Original assignee: Beijing Bokangjian Gene Technology Co ltd
Current assignee: Beijing Bokangjian Gene Technology Co ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2022-02-22
Anticipated expiration: 2039-12-30
Also published as: CN111072766A

Abstract

The invention provides a deamidation impurity of rhPTH (1-34), a preparation method and application thereof. The preparation method comprises the following steps: carrying out heat treatment on the rhPTH (1-34) stock solution to carry out deamidation reaction on the rhPTH (1-34) stock solution to obtain a deamidated impurity crude product; purifying the deamidated impurity crude product by adopting a cation chromatography, and performing pH gradient elution to obtain the deamidated impurity of rhPTH (1-34). The method is characterized in that the stock solution of the rhPTH (1-34) generates deamidation impurities by adopting a heat treatment mode, and then the deamidation impurities are separated and purified by a cation exchange chromatography and eluted by pH gradient to obtain the deamidation impurities of the rhPTH (1-34) with higher purity, thereby providing a foundation for further research on quality control and clinical safety of the rhPTH (1-34) medicament.

Description

Deamidation impurity of rhPTH (1-34), preparation method and application thereof

Technical Field

The invention relates to the field of pharmaceutical impurities, in particular to a deamidation impurity of rhPTH (1-34), a preparation method and application thereof.

Background

In the case of drugs, especially biological drugs, the retention of the molecular configuration and biological activity of the active component protein or polypeptide depends on various covalent and non-covalent forces, and the maintenance of these forces is closely related to the microenvironment in which they are located, such as temperature, light, ion concentration, and mechanical shear force. Some conditions may change to cause impurities to be generated.

Drug impurities are substances present in drugs that have no therapeutic effect or affect the stability, efficacy, or even harm to the health of the human body. The relationship between the impurities of a drug and the safety of a drug is a complex relationship influenced by many factors. Most of impurities in some medicines have potential biological activity, and some impurities even interact with the medicines to influence the efficacy and safety of the medicines, so that toxic and side effects can be seriously generated. For example, degradation products in tetracycline cause fanconi syndrome, and byproducts of methotrexate produce exothermic reactions, etc. And the influence on the safety of the medicine, such as optical isomers of the medicine, R-isomer of the lissaxad and two metabolites in the body of the R-isomer of the lissaxad have strong embryotoxicity and teratogenicity.

Therefore, the research on the impurities of the medicine is very important for the safety, stability and quality control of the medicine or the establishment of quality standards. Protein deamidation is a common degradation reaction during protein storage. Non-enzymatic deamidation of many proteins during production and storage is one of the main causes of loss of protein activity, often resulting in changes in local hydrophilicity/hydrophobicity of the protein, which largely affects the spatial structure of the protein, reduces biological activity and even changes biological function. In addition, deamidation may also affect the metabolic kinetics of the protein.

For the protein drug rhPTH (1-34), deamidation reaction exists in the polypeptide stock solution of rhPTH (1-34), but for the drug, the structure and property of impurities generated after deamidation reaction, influence on the safety of the drug and clinical toxic and side effects are not reported in the prior art. The study and analysis of these problems depends on the ability to isolate the impurities from rhPTH (1-34). Therefore, how to separate the deamidated impurities from rhPTH (1-34) becomes a problem to be solved for studying the influence of the impurities on the quality and toxicity of the drug.

Disclosure of Invention

The invention mainly aims to provide a deamidation impurity of rhPTH (1-34), a preparation method and application thereof, aiming at solving the problem that the impurity cannot be fully understood and researched to influence the safety of the quality of the rhPTH (1-34) medicament due to the absence of the impurity in the prior art.

In order to accomplish the above objects, according to one aspect of the present invention, there is provided a method for preparing a deamidated impurity of rhPTH (1-34), the method comprising: carrying out heat treatment on the rhPTH (1-34) stock solution to carry out deamidation reaction on the rhPTH (1-34) stock solution to obtain a deamidated impurity crude product; purifying the deamidated impurity crude product by adopting a cation chromatography, and performing pH gradient elution to obtain the deamidated impurity of rhPTH (1-34).

Further, before the heat treatment, the preparation method also comprises the step of adjusting the pH value of the rhPTH (1-34) stock solution, preferably adjusting the pH value of the rhPTH (1-34) stock solution to be neutral, and more preferably adjusting the pH value to be 6.5-7.5.

Further, the heat treatment comprises: heating the rhPTH (1-34) stock solution after the pH value is adjusted at 60-90 ℃ to obtain deamidation impurities of the rhPTH (1-34); preferably, the heating time is 24-48 h; preferably, after heating and before obtaining crude product of deamidated impurities of rhPTH (1-34), the method further comprises the step of adjusting the pH value of the heated product, preferably, the pH value is adjusted to 5.5-6.5.

Further, the purification of the crude deamidated impurity product by cation exchange chromatography comprises: purifying the deamidated impurity crude product for the first time by adopting a first cation chromatographic column to obtain a first purified product; purifying the first purified product for the second time by adopting a second cation chromatographic column to obtain deamidation impurities of the rhPTH (1-34); preferably, the first cation chromatography column is selected from the group consisting of cation chromatography columns with packing of SP sepharose 25, SPFF, SP sepharose 50, SE52 or SE 92; preferably, the resolution of the second cation chromatography column is greater than the resolution of the first cation chromatography column; more preferably, the second cationic chromatography column is selected from the group consisting of cationic chromatography columns packed with Souece15S, Souece30S, SynChropak S300 or Ultrasil CX.

Further, a first purification of the crude deamidated impurity product using a first cationic chromatography column to obtain a first purified product comprising: sequentially carrying out balancing, sample loading and leaching on the first cation chromatographic column to obtain a first leaching column; performing pH gradient elution on the first elution column by using a first eluent and a second eluent to obtain a first purified product; wherein the first eluent is phosphate buffer solution with the concentration of 10mmol-30mmol/L, pH-5.5-6.5, and the second eluent is phosphate buffer solution with the concentration of 10mmol-30mmol/L, pH-7.5-8.5.

Further, the first elution column is eluted by using a pH gradient of the first elution solution and a second elution solution, wherein the pH gradient is as follows: 6.0-6.5, 6.5-7.0, 7.5-8.0 and 8.0-8.5, preferably, the pH gradient elution comprises: firstly, respectively adopting the following volume ratios of 50%: the first elution column is eluted for the first time by the first mixed solution of 50% of the first eluent and the second eluent until the pH value of an eluted product is unchanged; then sequentially adopting the following components in volume ratio of 75%: 25% and 90%: 10% and 100%: and (3) carrying out second elution on a second mixed solution of the first eluent and the second eluent of 0 until the pH of an elution product of the second elution is not changed, so as to obtain a first purified product.

Further, the second purifying the first purified product with a second cation chromatography column comprises: after the second cation chromatographic column is balanced, loaded and washed in sequence, a second washing column is obtained; performing pH gradient elution on the second elution column by using a third eluent and a fourth eluent to obtain deamidated impurities of rhPTH (1-34); wherein the third eluent is phosphate buffer solution with the concentration of 10mmol-30mmol/L, pH-5.5-6.5, and the fourth eluent is phosphate buffer solution with the concentration of 10mmol-30mmol/L, pH-7.5-8.5.

Further, performing pH gradient elution on the second elution column by using a third elution solution and a fourth elution solution, wherein the pH gradient is as follows: 6.0-6.5, 6.5-7.0, 7.5-8.0 and 8.0-8.5, preferably, the pH gradient elution comprises: firstly, respectively adopting the following volume ratios of 50%: eluting the second elution column for the first time by using a third mixed solution of 50% of a third eluent and a fourth eluent until the pH value of an eluted product is unchanged; then sequentially adopting the following components in volume ratio of 75%: 25% and 90%: 10% and 100%: and (3) carrying out secondary elution on a fourth mixed solution of the third eluent and the fourth eluent of 0 until the pH of an elution product of the secondary elution is not changed, so as to obtain the deamidation impurity of the rhPTH (1-34).

In order to achieve the above objects, according to a second aspect of the present invention, there is provided deamidation impurities of rhPTH (1-34) obtained by any of the above-mentioned preparation methods.

According to a third aspect of the present invention, there is provided the use of the above-described deamidated impurity of rhPTH (1-34) for the quality control of a drug of rhPTH (1-34).

By applying the technical scheme of the invention, the rhPTH (1-34) stock solution generates deamidation impurities by adopting a heat treatment mode, and then the deamidation impurities of the rhPTH (1-34) with higher purity are obtained by separation and purification by a cation exchange chromatography and pH gradient elution, thereby providing a foundation for further research on quality control and clinical safety of the rhPTH (1-34) medicament.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows a liquid phase diagram of the deamidated impurity as initially prepared in example 1 of the present invention;

FIG. 2 shows a liquid phase diagram of the deamidated impurity after the first purification step in example 1 of the present invention;

FIG. 3 shows a liquid phase detection pattern of an absorption peak after the first purification step in example 1 of the present invention;

FIG. 4 shows a liquid phase diagram of the deamidated impurities after the second purification step in example 1 of the present invention;

FIG. 5 shows a liquid phase detection pattern of an absorption peak after the second purification in example 1 of the present invention;

fig. 6 is a graph showing the comparison of the purified RRT ═ 0.89 deamidation impurity and system suitability for use in example 1 of the present invention;

FIG. 7 shows a liquid phase diagram of the deamidated impurity after the first purification step in example 2 of the present invention;

FIG. 8 shows a liquid phase detection pattern of an absorption peak after the first purification in example 2 of the present invention;

FIG. 9 shows a liquid phase diagram of the deamidated impurity after the second purification step in example 2 of the present invention;

FIG. 10 is a liquid phase detection pattern showing an absorption peak after the second purification in example 2 of the present invention;

FIG. 11 shows a liquid phase diagram of the deamidated impurities after purification by reverse phase chromatography in comparative example 1 of the present invention;

FIG. 12 is a liquid phase detection pattern showing an absorption peak after purification by reverse phase chromatography in comparative example 1 of the present invention;

FIG. 13 shows a liquid phase diagram of deamidated impurities after purification by gel filtration chromatography in comparative example 2 of the present invention;

FIG. 14 is a liquid phase detection pattern showing an absorption peak after purification by gel filtration chromatography in comparative example 2 of the present invention;

FIG. 15 shows a liquid phase diagram of a deamidated impurity after hydrophobic chromatography purification in comparative example 3 of the present invention;

FIG. 16 is a liquid phase detection pattern showing an absorption peak after purification by hydrophobic chromatography in comparative example 3 of the present invention;

FIG. 17 shows a liquid phase diagram of the deamidated impurity after anion chromatography purification in comparative example 3 of the present invention;

FIG. 18 is a liquid phase detection pattern showing an absorption peak after anion chromatography purification in comparative example 3 of the present invention;

FIG. 19 shows a liquid phase diagram of deamidated impurities after elution with a cation chromatographic purification salt gradient in comparative example 4 of the present invention;

FIG. 20 shows a liquid phase detection pattern of an absorption peak after gradient elution with an anion chromatography purified salt in comparative example 4 of the present invention.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

Interpretation of terms:

rh-PTH (1-34) stock: the application refers to a recombinant human parathyroid hormone (1-34) stock solution, which refers to a protein solution obtained by separating and purifying rhPTH (1-34) protein expressed by recombinant human parathyroid hormone (1-34) engineering bacteria according to the existing column chromatography method, wherein the concentration and the purity of the protein solution meet the quality standard (the quality standard of the original medicine is taken as reference).

CV: column Volume.

The prior art discloses that protein drugs are easy to generate deamidation reaction so as to contain deamidation impurities, and for the protein polypeptide drug rhPTH (1-34), the prior art controls the content of the deamidation impurities in the drugs from the aspect of monitoring the content of the deamidation impurities generated in the preparation process. And is unknown about the interaction between such impurities and the drug, or the clinical safety of the impurities themselves. In order to understand these problems more clearly, the present application aims to provide a method for preparing and purifying deamidated impurities of rhPTH (1-34), thereby providing a reference for establishing limit standards of impurities in raw liquid and preparation of rhPTH (1-34), and performing qualitative analysis and clinical safety analysis on the impurities.

In order to provide a method for preparing the deamidating impurity of rhPTH (1-34), the inventors have conducted intensive studies on the production process of the impurity, and found that in vitro, non-enzymatic deamidation occurs in many proteins during production and storage, resulting in loss of protein activity. The site of deamidation is more prevalent with Asn, usually occurring between Asn-Gly or Asn-Ser, and about 70% of deamidation results in the production of Asp. Further analyzing the possible position of deamidation reaction of rhPTH (1-34), the amino acid sequence of the rhPTH (1-34) stock solution polypeptide is as follows (SEQ ID NO: 1):

NH₂-Ser-Val-Ser-Glu-Ile-Gln-Leu- ⁸Met-His-Asn¹⁰-Leu-Gly-Lys-His-Leu-Asn¹⁶-Ser- ¹⁸Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn³³-Phe-COOH。

it is understood that the 10-, 16-and 33-positions are asparagine. In addition, in the stability test of the rhPTH (1-34) stock solution and the preparation, it was found that deamidation of asparagine is likely to occur.

In order to obtain deamidated impurities of rhPTH (1-34), in one exemplary embodiment of the present application, there is provided a process for preparing deamidated impurities of rhPTH (1-34), the process comprising: carrying out heat treatment on the rhPTH (1-34) stock solution to carry out deamidation reaction on the rhPTH (1-34) stock solution to obtain a deamidated impurity crude product; separating and purifying the deamidated impurity crude product by adopting a cation chromatography and carrying out pH gradient elution to obtain the deamidated impurity of rhPTH (1-34).

According to the preparation method of the deamidation impurity of the rhPTH (1-34), the heat treatment mode is adopted to enable the original rhPTH (1-34) solution to generate the deamidation impurity, and then the deamidation impurity of the rhPTH (1-34) with higher purity is obtained through separation and purification by a cation chromatography and elution by pH gradient. The deamidation impurity of the rhPTH (1-34) is separated and purified, which provides a basis for further research on the quality control and clinical safety of the rhPTH (1-34) medicament.

The temperature of the heat treatment is selected according to the conditions of impurity generation in the process of stability investigation of the rhPTH (1-34) stock solution, and the heat treatment is equivalent to change the direction to increase the rate of impurity generation in the stability investigation. Compared with deamidation impurities generated by other methods, the heat treatment has the advantages of high purity of the prepared deamidation impurities, short impurity preparation period, high impurity yield and the like.

Thus, all heat treatment conditions that produce deamidation impurities are suitable for use in this application. For example, the temperature and time of the heat treatment are not particularly limited. In a preferred embodiment, the heat treatment comprises: heating the rhPTH (1-34) stock solution after the pH value is adjusted at 60-90 ℃ to obtain deamidation impurities of the rhPTH (1-34); preferably, the heating time is 24-48 h. Heating for 24-48 h at 60-90 ℃ to obtain deamidating impurities with higher purity.

In the process of investigating the stability of rhPTH (1-34) stock solution, the stability of protein is damaged at high temperature, deamidation impurities are increased gradually along with the increase of time, and the temperature and the time have great influence on the deamidation impurities of the stock solution, and then the method of high temperature and prolonging the deamidation time is adopted when the deamidation impurities are prepared, and the optimal conditions for preparing the deamidation impurities are determined to be 70 ℃ and 30 hours through gradient investigation. With specific reference to the following table:

table 1:

table 2:

table 3:

in order to obtain deamidated impurities more easily, in a preferred embodiment, the preparation method further comprises the step of adjusting the pH of the rhPTH (1-34) stock solution before the heat treatment, preferably adjusting the pH of the rhPTH (1-34) stock solution to be neutral, more preferably 6.5-7.5. The purpose of this pH adjustment step is to produce deamidated impurities of higher purity under neutral conditions.

In a preferred embodiment, after heating and before obtaining the crude product of the deamidated impurities of rhPTH (1-34), the method further comprises the step of adjusting the pH of the heated product, preferably to 5.5-6.5. The purpose of the pH adjustment of the heated product was to maintain the pH of the crude rhPTH (1-34) deamidation impurity consistent with the pH of the loading buffer. Whereas adjustment to a pH of 5.5-6.5 enables the prepared deamidated impurities to bind better to the chromatography column during loading.

The step of separating and purifying the deamidated impurities can be obtained by improving the existing impurity separation method. To further increase the purity of the isolated deamidated impurities, in a preferred embodiment, the purification of the crude deamidated impurities by cationic chromatography comprises: purifying the deamidated impurity crude product for the first time by adopting a first cation chromatographic column to obtain a first purified product; and (3) purifying the first purified product for the second time by adopting a second cation chromatographic column to obtain the deamidation impurity of the rhPTH (1-34).

In the preferred embodiment, primary purification is firstly carried out by two times of cation chromatography purification, so as to obtain deamidation impurities with higher purity. And in the second step, the deamidated impurities with higher purity obtained in the first step are purified, so that the impurities with higher purity are obtained. According to the difference of the cationic chromatographic column packing used in the second purification step, the purity of the impurities obtained by separation is different, and when a cationic chromatographic column with higher resolution is adopted, the deamidation impurities with the purity of more than 90 percent can be obtained.

It should be noted that the selection of suitable ion exchange packing is made in accordance with the objective of the purification step and the conditions of the starting materials. Proper sample preparation is necessary to obtain optimal separation and avoid degradation of the performance of the chromatography column. The sample must be clear and free of particulate matter. To remove particulate matter, it is usually necessary to filter (filter pore size in buffer) or centrifuge (10000 g; 15min) the sample before loading. HiTrap was used as a desalted sample^TM5ml of desaling (volume up to 1.5ml) or HiPrep^TM26/10 desaling (volume up to 15ml) was switched to the buffer of choice. Very small volumes of sample without major contamination at high salt concentrations can be diluted with an initiation buffer to reduce the salt concentration to a level that does not interfere with filler binding.

According to the liquid phase map analysis, the relative retention time of the deacylated impurities and rhPTH (1-34) is obtained, according to the possible properties of the deacylated impurities, purification methods such as anion chromatography (including strong anion chromatography and weak anion chromatography), reversed phase chromatography, gel filtration chromatography, hydrophobic chromatography and the like are tried to purify the deamidated crude product, and liquid phase detection is carried out on the impurities prepared by different chromatographic fillers, so that the purification effect on the target deamidated impurities is not good, and the deamidated impurities with the purity of 45% are obtained at most.

In order to obtain target impurities with better purity, through multiple purification experiments, the impurities prepared by different chromatographic fillers are subjected to liquid phase detection, and finally, the cationic chromatographic fillers are determined to be used. The cation chromatographic packing is purified by SPFF packing initially, the deamidation impurity purity in the crude product of the amide impurity can be improved to 70 percent, and in order to not achieve more ideal effect, the secondary purification is carried out by SPFF packing later, and the purity is not improved more obviously.

In order to further obtain purer target impurities, two or more successive purifications with other strongly acidic ion chromatography columns were attempted, but no significant improvement was observed. Then, two times of purification is changed, different chromatographic columns and chromatographic fillers are adopted, and the purity can reach more than 90%. On the basis that the purity of the deamidation impurity of the target protein reaches 74 percent through the first-step purification, a second-step purification is carried out by using a pure strong acid cation chromatographic column, and the deamidation impurity with the purity of 95 percent, even 100 percent, meeting the requirement can be obtained.

A large amount of screening tests show that the purification effect is higher when the first cation chromatographic column is a strong-acid cation chromatographic column. Strongly acidic cation chromatography columns include, but are not limited to, chromatography columns with packing material SP sepharose 25, SPFF, SP sepharose 50, SE52 or SE 92. The second purification is fine purification, and the resolution of the purification is greater than that of the first purification. A chromatography column suitable for fine purification would be suitable for use in this application. To further improve the purification efficiency and purity, in another preferred embodiment the second cationic chromatography column is selected from the group consisting of columns packed with Souece15S, Souece30S, SynChropak S300 or Ultrasil CX.

In the present application, the first cation chromatographic column and the second cation chromatographic column of the above-mentioned kind are used to separate and purify deamidated impurities, and the target impurities with higher purity can be obtained, compared with other chromatographic columns.

In connection with the preparation of each of the above-described columns, the preservative solution and preservative need to be washed off before any packing is used. To obtain the best performance separation and reproducible results, it is recommended to use a pre-packed chromatography column. The required packed bed volume is determined by the amount of sample to be purified and the binding capacity of the packing. The column will have a binding capacity that exceeds the required binding capacity by a factor of about 5.

SP-Sepharose FF (SP-Sepharose FF) is a chromatographic separation medium with strong acid cationic groups formed by binding carboxymethyl groups to agarose microspheres. The product retains good hydrophilicity and large net frame structure of agarose, has good compatibility with bioactive macromolecules, and has the advantages of high ion exchange capacity, less non-specific adsorption, and high flow rate. There are mainly six specifications of 1ml, 5ml, 10ml, 20ml, 100ml and 500ml, the first three can be pre-packed columns. The recommended flow rate is 100-700 cm/h. The storage buffer was: 20% ethanol.

The specific use method is as follows:

1) balancing: equilibration was performed with 2-5 column volumes of the initial buffer solution, suggesting a flow rate of 100 cm/h. And observing the change of the detector until the parameters such as conductivity, pH value and the like are unchanged.

2) Loading: pretreatment of the sample: the sample was filtered through a 0.45 μm filter and loaded to avoid clogging the column. The loading amount is selected according to the nature of the sample and the amount of chromatography medium, and the optimal loading amount can also be found by linear experiments.

3) Leaching: and (4) leaching by using 2-3 column volumes of equilibrium buffer solution, and observing the change of the detector until parameters such as conductivity, pH value and the like are unchanged.

4) And (3) elution: constant elution, gradient elution, or step elution may be used. It is generally recommended to use gradient elution that increases salinity.

Tricorn

^TM10/100 chromatography column (SOURCE 15S packing), SOURCE15S packing is ideal for rapid, high resolution purification and easy scale-up. Robust, monodisperse spherical particles with controlled size, provide excellent flowability, can be loaded up to 25mg protein/ml, and are stable in organic solvents and at extreme pH values.

Tricorn

^TM10/100 is a high performance chromatography column for optimization studies of high resolution purification or amplification purification. The SOURCE15S chromatographic pre-packed column was used for rapid separation, the separation time was less than 3min with 1mL SOURCE chromatographic column when the flow rate was 9.6mL/min, and about 20min when the flow rate was 1mL/min using a peristaltic pump.

In the first purification step, specific conditions for purification may be appropriately set depending on the selected column. In a preferred embodiment, the first purification of the crude deamidated impurity using a first cationic chromatography column to obtain a first purified product comprises: sequentially carrying out balancing, sample loading and leaching on the first cation chromatographic column to obtain a first leaching column; performing pH gradient elution on the first elution column by using a first eluent and a second eluent to obtain a first purified product; wherein the first eluent is 10mmol-30mmol/L, pH ═ 5.5-6.5 phosphate buffer solution, and the second eluent is 10mmol-30mmol/L, pH ═ 7.5-8.5 phosphate buffer solution.

The adoption of pH gradient elution is helpful for separating the target deamidation impurities from the stock solution and other irrelevant impurities, and the gradient of pH is controlled to be between 5.5 and 8.5, so that the target protein, the stock solution and other impurity proteins can be separated due to different charge quantities, and the deamidation impurities with higher purity can be obtained.

In the case where the types and pH values of the first eluent and the second eluent are determined, the specific pH gradient can be optimally selected. In a preferred embodiment, subjecting the first elution column to pH gradient elution with a first eluent and a second eluent to obtain a first purified product comprises: firstly, respectively adopting the following volume ratios of 50%: the first elution column is eluted for the first time by the first mixed solution of 50% of the first eluent and the second eluent until the pH value of an eluted product is unchanged; then sequentially adopting the following components in volume ratio of 75%: 25% and 90%: 10% and 100%: and (3) carrying out second elution on a second mixed solution of the first eluent and the second eluent of 0 until the pH of an elution product of the second elution is not changed, so as to obtain a first purified product.

In the preferred embodiment, the elution is performed according to the pH gradient, and a large part of the raw solution in which the deamidation reaction does not occur can be separated, so that the purity of the first purified product obtained is relatively high.

In the second purification step, the specific operation of the second purification may vary depending on the chromatography column used. In a preferred embodiment, the second purification of the first purified product using a second cationic chromatography column comprises: after the second cation chromatographic column is balanced, loaded and washed in sequence, a second washing column is obtained; performing pH gradient elution on the second elution column by using a third eluent and a fourth eluent to obtain deamidated impurities of rhPTH (1-34); wherein the third eluent is 10mmol-30mmol/L, pH ═ 5.5-6.5, and the fourth eluent is 10mmol-30mmol/L, pH ═ 7.5-8.5 phosphate buffer.

In a preferred embodiment, subjecting the second elution column to a pH gradient elution with a third elution solvent and a fourth elution solvent to obtain the deamidated impurities of rhPTH (1-34) comprises: firstly, respectively adopting the following volume ratios of 50%: eluting the second elution column for the first time by using a third mixed solution of 50% of a third eluent and a fourth eluent until the pH value of an eluted product is unchanged; then sequentially adopting the following components in volume ratio of 75%: 25% and 90%: 10% and 100%: and (3) carrying out secondary elution on a fourth mixed solution of the third eluent and the fourth eluent of 0 until the pH of an elution product of the secondary elution is not changed, so as to obtain the deamidation impurity of the rhPTH (1-34).

The two elution purification processes are cation elution, so the used eluent is the same. It should be noted that, since the conventional elution is performed by using a salt gradient elution, the above-mentioned step of elution is also performed by screening of various elution modes (including a salt gradient elution) to obtain the above-mentioned preferable pH gradient elution.

In a second exemplary embodiment of the present application, there is provided a deamidation impurity of rhPTH (1-34) prepared by any of the above-mentioned methods, wherein the purity of the deamidation impurity of rhPTH (1-34) is 90-100%.

In a third exemplary embodiment of the present application, there is provided the use of the above-described deamidated impurity of rhPTH (1-34) for the quality control of a drug of rhPTH (1-34).

The following examples are presented to further illustrate the benefits of the present application.

Example 1

1. Experimental reagent

Na₂HPO₄-12H₂O、NaH₂PO₄-H ₂0. NaOH, absolute ethyl alcohol, NaCl, HCl, rhPTH (1-34) stock solution.

2. Instrument and instrument thereof

pH detector, peristaltic pump, air blast sterilizer, AKTA Exploer100 protein purifier, analytical balance, chromatographic column XK16(SPFF packing), and Tricorn ^TM10/100 chromatography column (SOURCE 15S packing).

3. Solution preparation:

and (3) buffer solution A: 20mmol/L, pH ═ 5.8 phosphate buffer; and (3) buffer solution B: 0.2mol/L NaOH solution;

and (3) buffer C: 20mmol/L, pH ═ 8 phosphate buffer; and (3) buffer solution D: 20% absolute ethyl alcohol.

4. The experimental steps are as follows:

4.1 preparation of deamidating impurities

The stock solution of rhPTH (1-34) was adjusted to pH 7.0. The mixture was placed in an oven (70 ℃, 30h), and then the pH was adjusted to 5.8 and placed in a refrigerator for further use.

Figure 1 shows the liquid phase diagram of the initial preparation of 0.89 deamidated impurity. In the liquid phase diagram, the substance with RT-22.465 is deamidation impurity to be prepared, the substance with RT-25.159 is rhPTH (1-34) stock solution, and RRT-0.89.

From the above figure, it can be seen that: the deamidation impurity purity of preliminary preparation is lower, needs design experiment, purifies this deamidation, obtains the higher RRT of purity 0.89 deamidation impurity to follow-up analysis and research to impurity.

4.2 purification of deamidated impurities

4.2.1 purification concept:

because the prepared deamidation impurities have low purity, the first step needs to use a chromatographic column XK16(SPFF filler) to carry out primary purification on the deamidation impurities to obtain the deamidation impurities with high purity. And in the second step, the deamidated impurity with high purity obtained in the first step is purified to obtain the deamidated impurity with the purity of 0.89, wherein the purity of the deamidated impurity meets the requirement.

4.2.2 purification step:

4.2.2.1 first step: purification using column XK16(SPFF Filler)

1) The column was rinsed with 2CV of ultra pure water to remove ethanol at a flow rate of 1cm/min (elution requires attention to the pressure of the column and its chromatographic system, too high a flow rate would result in too high a pressure, too low a flow rate, and longer purification time. The flow rate at elution at the time of the experiment was set directly to 1 cm/min).

2) The column was cleaned with buffer B in a 1CV clean volume; the rinsing was carried out at a linear speed of 1 cm/min.

3) Washing the column with ultrapure water to neutrality, at a volume of about 5 CV; the rinsing was carried out at a linear speed of 1 cm/min.

4) The column was equilibrated at a linear velocity of 1cm/min for about 5CV using buffer A.

5) And (4) zero setting of an ultraviolet baseline, monitoring an ultraviolet absorption peak by selecting a UV280 wavelength, and loading the prepared deamidated impurities at the linear speed of 1 ml/min.

6) After the loading is finished, the sample is rinsed by using the buffer solution A, the rinsing volume is 2CV, and the linear velocity is 1 cm/min.

7) Performing pH gradient elution by using a buffer solution A and a buffer solution C, setting the gradient of the buffer solution C to be 50%, increasing the gradient of the buffer solution C to be 75%, 90% and 100% after the pH value of the buffer solution C is unchanged, and using the buffer solution D to be 0-30% after the pH value of the buffer solution C is unchanged; and (3) carrying out gradient elution by 20CV, and observing UV220, UV280 and UV214 during loading, rinsing and gradient elution, observing a spectrum, ensuring that each absorption peak is collected, and marking each absorption peak.

Fig. 2 shows the first-step purification pattern, and the liquid phase detection pattern of each absorption peak of the purification pattern is shown as fig. 3, wherein the ultraviolet absorption peak 4 is 0.89 deamidated impurity with purity of 74.75% and the liquid phase detection pattern is shown as RRT.

4.2.2.2 second step: purification was performed using a Tricorn 10/100 chromatography column (SOURCE 15S packing)

1) The column was rinsed with 2CV of ultra pure water to remove ethanol at a linear velocity of 1 cm/min.

7) The pH gradient elution was performed using buffer A and buffer C, and the UV280 was observed during the purification process to ensure that each absorption peak was collected.

FIG. 4 shows the second step of purification, which is to examine each absorption peak collected, and find that the peak is the elution peak of deamidation impurity, and the purity of deamidation impurity is more than 90%. The purity of the product can reach 100% after the product is diluted to the working concentration. The liquid phase detection pattern is shown in FIG. 5.

4.3 conclusion:

comparing the final RRT ═ 0.89rhPTH deamidation impurity with the system applicability, as shown in FIG. 6, the deamidation impurity prepared is consistent with the impurity generated in the process of stock solution stability investigation, and the purity of the deamidation impurity meets the requirement of impurity research.

Example 2

Two-pass cation chromatography

The chromatographic column and the chromatographic packing of the two purifications are the same, and the chromatographic packing is as follows: spf, column types were all: XK 16/20.

(1) First purification of deamidated impurities (packing: SPFF, column type: XK16/20)

The column was washed with 2CV of ultrapure water to remove ethanol at a flow rate of 1cm/min (elution requires attention to the pressure of the column and its chromatographic system, too high a flow rate would result in too high a pressure, too low a flow rate, and longer purification time).

2) The column was cleaned with buffer B in a 1CV clean volume and rinsed at a line speed of 1 cm/min.

The first purification pattern is shown in FIG. 7. The purity of the first purified impurities obtained by performing liquid phase detection on the obtained purified crude liquid is 73.. 06%, and a liquid phase detection map is shown in fig. 8. It can be seen that the effect is better after the first purification, but the purity is still not high enough. Thus, a second purification was performed (using the same column and packing).

(2) The secondary purification step is the same as the primary purification.

The secondary purification pattern is shown in fig. 9, because the chromatographic packing is unchanged, the used chromatographic purification method is the same, only one protein elution peak is provided, and the target deamidation impurity cannot be separated from other impurities. The liquid phase detection spectrum is shown in fig. 10, and it is found from the liquid phase detection that the purity is improved by the secondary purification using the same filler, but the improvement is not significant enough (the purity is 74.73%).

Comparative example 1

Reverse phase chromatography (packing Source15 RPC; column Tricorn ^TM10/100 column)

1 reversed phase chromatography step:

1.1 pretreatment of a chromatographic column: 3CV of water; 0.5mol/LNaOH 2 CV; buffer E (10mmol/L PB, 500mmol/L NaCl)2CV and buffer F (10mM PB, 10% ethanol) 5-6CV, followed by washing.

1.2 chromatographic column pretreatment: phase A: buffer F (10mM PB, 10% ethanol); phase B: buffer G (80% ethanol), gradient 0-100% phase B; 2CV is added, and a gradient 100-0B phase is set; 2CV punching; then 7CV was equilibrated with buffer F. And (5) ultraviolet zero setting.

1.3, loading and leaching: after the loading was completed, the sample was rinsed with 40% buffer G (80% ethanol).

1.4 elution: and (3) preparing the buffer solution F and the buffer solution G into a buffer solution G with the volume concentration of 40-60% for elution, recording the map of the buffer solution G, and collecting the ultraviolet absorption peak.

1.5 impurity washing: 100% buffer G (80% ethanol) washes to baseline plateau.

1.6 post-treatment of chromatographic column: 50% of each AB, 3CV of water, 5CV of 0.5mol/L NaOH, 5CV of water, and 4.5CV of 20% ethanol, and the mixture was washed successively.

The pattern of the reverse phase chromatography is shown in FIG. 11. And then respectively carrying out liquid phase detection on different ultraviolet absorption peaks, wherein the purity of deamidated impurities of 2-mesh ultraviolet absorption peaks is the highest and is 42%, and a liquid phase detection map is shown in figure 12.

Comparative example 2

Gel filtration chromatography (Filler Sephadex)^TMG-25; chromatographic column XK50/30)

1. A chromatography step:

1.1 chromatographic column treatment: 3CV of water; 0.5mol/L NaOH 2 CV; buffer C (PB, 10mmol/L, pH 7.0)2CV, followed by rinsing.

1.2 the prepared crude deamidated liquid is loaded, and the loading volume does not exceed 30% of the column volume.

1.3 after loading, the target protein peak was collected in buffer C (PB, 10mmol/L, pH 7.0).

The chromatogram is shown in FIG. 13. And (3) carrying out molecular sieve chromatography on the deamidated impurity crude pure solution, collecting gel filtration chromatography solution, and carrying out liquid phase detection. As shown in FIG. 14, the liquid phase detection spectrum revealed that the deamidated impurities separated by gel filtration chromatography had a purity of only 33%.

Comparative example 3

Hydrophobic chromatography (Filler type: Phenyl Sepharose6 Fast Flow, column: XK16/40)

1. A chromatography step:

1.1 chromatographic column treatment: 3CV of water; 0.5mol/L NaOH 2 CV; buffer D (10mmol/L PB; 500mmol/L (NH)₄)₂SO₄pH 7.0)2CV, and washing was performed sequentially.

1.2 the prepared crude deamidation solution is diluted to 0.5mmol/L ammonium sulfate by using 3mol/L ammonium sulfate mother liquor, and then the sample is loaded by hydrophobic chromatography.

1.3 after loading, buffer D (10mmol/L PB; 500mmol/L (NH)₄)₂SO₄ Balance 2 CV.

1.4 elution was performed using buffer E (10mmol/L PB; pH 7.0), and the elution peak was collected and subjected to liquid phase detection.

The chromatography purification pattern is shown in FIG. 15, the purified chromatography liquid is collected and subjected to liquid phase detection, and the liquid phase pattern is shown in FIG. 16, wherein the impurity purity is up to 41%.

Comparative example 4

Anion chromatography (packing type: QSepharose TMhigh Performance, column model: XK16/20)

1. A chromatography step:

1.1 the column was rinsed with 2CV of ultra pure water to remove ethanol at a linear velocity of 1 cm/min.

1.2 the column was cleaned with NaOH in a volume of 1CV and rinsed at a line speed of 1 cm/min.

1.3 the column was rinsed to neutrality with ultra pure water at a volume of about 5CV and the rinsing was carried out at a line speed of 1 cm/min.

1.4 Using buffer E (8mmol/L acetate buffer pH4.0) at a linear velocity of 1cm/min to equilibrate the column for about 5 CV.

And (5) adjusting the ultraviolet baseline to zero, adjusting the pH value of the deamidated impurity crude liquid to 4.0, and loading.

1.6 after the completion of the sample application, the mixture was equilibrated with buffer E (8mmol/L acetate buffer pH4.0) at an equilibrium volume of 2CV and a linear velocity of 1 cm/min.

1.7 elution: phase A buffer solution E (8mmol/L acetate buffer solution pH4.0), phase B buffer solution F (8mmol/L acetate buffer solution pH4.0; 500mmol/LNaCl), gradient 0-100% B; washing with 15CV, performing gradient elution, collecting elution peak, and performing liquid phase detection.

The anion chromatogram is shown in FIG. 17. Collecting purified chromatographic solution, and performing liquid phase detection on the purified chromatographic solution, wherein the liquid phase chromatogram is shown in FIG. 18, and the impurity purity is up to 45%.

Comparative example 5

Cation chromatography (chromatography packing SPFF, chromatography column: XK16/20)

1. The experimental steps are as follows:

1.4 the column was equilibrated with buffer C (10mmol/L PB; pH 7.0) at a linear velocity of 1cm/min for about 5 CV.

And 1.5, adjusting the ultraviolet baseline to zero, and carrying out sample loading.

1.6 after the end of the sample application, the mixture was equilibrated with buffer C (10mmol/L PB; pH 7.0) at an equilibrium volume of 2CV and a linear velocity of 1 cm/min.

1.7 elution: phase a buffer C (10mmol/L PB; pH 7.0), phase B buffer G (10mmol/L PB; 500mmol/L nacl, pH 7.0), set up gradient 0-100% B; washing with 15CV, performing salt gradient elution, collecting elution peak, and performing liquid phase detection.

As shown in fig. 19, the purification pattern shows that deamidated impurities cannot be effectively separated from other hetero-proteins by using salt gradient elution, and then the liquid phase detection is performed on the purified sample by using salt gradient elution. The liquid phase spectrum is shown in figure 20, and the impurity purity is up to 43.7%. The purity of the desired deamidated impurity can be greatly improved using the same column and packing and using a pH gradient elution (see example 1).

From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the method is characterized in that the stock solution of the rhPTH (1-34) generates deamidation impurities by adopting a heat treatment mode, and then the deamidation impurities are separated and purified by a cation exchange chromatography and eluted by pH gradient to obtain the deamidation impurities of the rhPTH (1-34) with higher purity, thereby providing a foundation for further research on quality control and clinical safety of the rhPTH (1-34) medicament.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Beijing Bokangjian Gene science and technology Co., Ltd

<120> deamidation impurity of rhPTH (1-34), preparation method and application thereof

<130> PN124703BJBKJ

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 34

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 1

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Thr Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe

Claims

1. A process for the preparation of deamidated impurities of rhPTH (1-34), said process comprising:

carrying out heat treatment on the rhPTH (1-34) stock solution to carry out deamidation reaction on the rhPTH (1-34) stock solution to obtain a deamidated impurity crude product;

adjusting the pH value of the rhPTH (1-34) stock solution to 6.5-7.5 before the heat treatment;

heating the rhPTH (1-34) stock solution after the pH value is adjusted at 70-90 ℃ to obtain deamidated impurities of the rhPTH (1-34);

the heating time is 25-48 h;

purifying the deamidated impurity crude product by adopting a cation chromatography, and performing pH gradient elution to obtain the deamidated impurity of the rhPTH (1-34);

the purification of the crude deamidated impurity product by cation exchange chromatography comprises:

purifying the deamidated impurity crude product for the first time by adopting a first cation chromatographic column to obtain a first purified product;

purifying the first purified product for the second time by adopting a second cation chromatographic column to obtain the deamidated impurity of the rhPTH (1-34);

the rhPTH (1-34) stock solution refers to recombinant human parathyroid hormone (1-34) stock solution;

the recombinant human parathyroid hormone (1-34) stock solution separates and purifies rhPTH (1-34) protein expressed by the recombinant human parathyroid hormone (1-34) engineering bacteria, and the obtained protein solution with the concentration and the purity meeting the quality standard of the original medicine.

2. The process of claim 1, wherein after said heating and before obtaining said crude product of said deamidated impurities of rhPTH (1-34), said process further comprises the step of adjusting the pH of said heated product.

3. The method according to claim 2, wherein the pH is adjusted to 5.5 to 6.5.

4. The method of claim 1, wherein the first cation column is selected from the group consisting of cation columns having a packing material of SPSepharose 25, SPFF, SP Sepharose 50, SE52, and SE 92.

5. The method of claim 4, wherein the second cation chromatography column has a resolution greater than the first cation chromatography column.

6. The method of claim 5, wherein the second cationic chromatography column is selected from the group consisting of a cationic chromatography column packed with source15S, source 30S, Synchropak S300, and Ultrasil CX.

7. The method of claim 6, wherein the first purifying the crude deamidated impurity with a first cationic chromatography column to obtain a first purified product comprises:

sequentially carrying out balancing, loading and leaching on the first cation chromatographic column to obtain a first leaching column;

performing pH gradient elution on the first elution column by using a first eluent and a second eluent to obtain a first purified product;

wherein the first eluent is phosphate buffer solution with the concentration of 10mmol-30mmol/L, pH-5.5-6.5, and the second eluent is phosphate buffer solution with the concentration of 10mmol-30mmol/L, pH-7.5-8.5.

8. The method of claim 7, wherein the first elution column is eluted with a pH gradient using the first eluent and the second eluent, wherein the pH gradient is as follows: 6.0-6.5, 6.5-7.0, 7.5-8.0 and 8.0-8.5.

9. The method of claim 8, wherein the pH gradient elution comprises:

firstly, respectively adopting the following volume ratios of 50%: carrying out first elution on the first elution column by using 50% of first mixed solution of the first eluent and the second eluent until the pH value of an eluted product is unchanged;

then sequentially adopting the following components in volume ratio of 75%: 25% and 90%: 10% and 100%: and (3) performing second elution on a second mixed solution of the first eluent and the second eluent, wherein the second mixed solution is 0, until the pH of an elution product of the second elution is not changed, so as to obtain the first purified product.

10. The method of claim 1, wherein the second purifying the first purified product using a second cationic chromatography column comprises:

after the second cation chromatographic column is balanced, loaded and washed in sequence, a second washing column is obtained;

performing pH gradient elution on the second elution column by using a third eluent and a fourth eluent to obtain the deamidated impurities of the rhPTH (1-34);

wherein the third eluent is phosphate buffer solution with the concentration of 10mmol-30mmol/L, pH-5.5-6.5, and the fourth eluent is phosphate buffer solution with the concentration of 10mmol-30mmol/L, pH-7.5-8.5.

11. The method of claim 10, wherein the second elution column is eluted with a pH gradient using a third eluent and a fourth eluent, wherein the pH gradient is as follows: 6.0-6.5, 6.5-7.0, 7.5-8.0 and 8.0-8.5.

12. The method of claim 11, wherein the pH gradient elution comprises:

firstly, respectively adopting the following volume ratios of 50%: eluting the second elution column for the first time by using a third mixed solution of 50% of the third eluent and the fourth eluent until the pH value of an eluted product is unchanged;

then sequentially adopting the following components in volume ratio of 75%: 25% and 90%: 10% and 100%: and (3) carrying out secondary elution on a fourth mixed solution of the third eluent and the fourth eluent of 0 until the pH of an elution product of the secondary elution is not changed, so as to obtain the deamidation impurity of the rhPTH (1-34).

13. Deamidating impurities of rhPTH (1-34) produced by the process of any one of claims 1-12.

14. Use of the deamidated impurity of rhPTH (1-34) of claim 13 in the quality control of a drug of rhPTH (1-34).