Method for purifying novel coronary antigen peptide M

Abstract

The invention relates to the technical field of polypeptide vaccines, in particular to a purification method of novel coronal antigen peptide M. The novel coronal antigen peptide M23 is synthesized through a classical polypeptide solid-phase synthesis technology, and then the novel coronal antigen peptide M23 hydrochloride raw material medicine with the purity larger than or equal to 99.0%, good stability, stable acid radical content and low residual solvent content is finally obtained by reasonably controlling the process conditions of reverse-phase silica gel purification and salt conversion, rotary evaporation concentration and freeze-drying. And the purification yield is at a relatively high level.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide vaccines, and in particular to a method for purifying a novel coronavirus antigen peptide M. Background Art

[0002] The novel coronavirus (SARS-CoV-2) belongs to the β-genus coronavirus of the Coronaviridae family. Since its discovery and confirmation in late 2019, it has spread rapidly worldwide, posing a significant challenge to global public health systems and prompting countries to strengthen disease surveillance, testing capabilities, and medical resource allocation. Prevention and control measures for the novel coronavirus primarily include non-drug interventions, vaccination, and antiviral drugs. The development of safe and effective vaccines is also a key approach to controlling the infection and spread of the novel coronavirus. Vaccines can stimulate the human immune system to produce antibodies, reducing infection rates and the incidence of severe illness. Currently, the main novel coronavirus vaccines include mRNA vaccines, inactivated vaccines, protein subunit vaccines, and adenovirus vector vaccines. Due to the high variability of the novel coronavirus and its propensity to produce different variants, this property poses a challenge to immune escape for traditional vaccines (such as inactivated and mRNA vaccines), resulting in reduced efficacy. Therefore, the development of broad-spectrum, highly effective vaccines that can address viral mutations has become a research hotspot.

[0003] Peptide vaccines utilize the amino acid sequence of a known pathogen's epitope, artificially synthesized through chemical synthesis into short peptides as antigens. These peptides are designed to elicit an immune response, activating the immune system and producing a specific immune response. Peptide vaccines offer the following advantages: They target specific pathogen epitopes for immunization, providing specific protection; antigen design by screening conserved regions of pathogens effectively addresses immune escape caused by viral mutations; and synthetic peptides lack genetic material or pathogen structure, avoiding potential biosafety risks associated with traditional vaccines. The Chinese Center for Disease Control and Prevention's Institute of Viral Disease Prevention and Control patents, "SARS-CoV-2 Specific Peptides and Their Applications" and "Identification and Application of T Cell Dominant Epitopes of Novel Coronavirus in the Chinese Population," disclose multiple SARS-CoV-2-specific peptides covering the S, M, and N proteins of SARS-CoV-2. Among them, the novel coronavirus antigen peptide M23 (peptide sequence H-Pro-Lys-Glu-Ile-Thr-Val-Ala-Thr-Ser-Arg-The-Leu-Ser-Tyr-Tyr-Lys-Leu-OH, molecular weight approximately 1970 Da) relies primarily on cellular immunity to induce the production of specific killer T cells against the novel coronavirus, preventing immune escape by novel coronavirus variants. It is highly versatile and can be used as a sequential booster vaccine. Currently, no specific purification processes have been found for the novel coronavirus antigen peptides M and M23. Traditional peptide purification processes cannot meet the stringent purity and yield requirements for high-quality vaccine development. Therefore, it is urgent to develop a purification method and process suitable for the novel coronavirus antigen peptide M23 to confirm its clinical application value and lay the foundation for the subsequent large-scale production of novel coronavirus antigen peptides and the development of novel coronavirus polypeptide vaccine preparations. Summary of the Invention

[0004] In light of this, the present invention provides a method for purifying a novel coronavirus antigen peptide. This method optimizes and combines process parameters such as reverse silica gel purification, salt transfer purification, rotary evaporation, and lyophilization to ultimately obtain the novel coronavirus antigen peptide M23 hydrochloride with high purity, good stability, stable acid radical content, low residual solvent content, and high purification yield.

[0005] A method for purifying novel coronavirus antigen peptide M, comprising: dissolving a crude novel coronavirus antigen peptide M product and then sequentially performing reverse silica gel purification, salt conversion purification, rotary evaporation concentration, and freeze-drying to obtain a refined novel coronavirus antigen peptide M product;

[0006] The mobile phase of the reverse silica gel purification includes an aqueous phase and an organic phase; the aqueous phase is a 0.5-2.0 vol% phosphoric acid aqueous solution, and the organic phase is acetonitrile;

[0007] The mobile phase for the salt conversion purification comprises an aqueous phase and an organic phase, the aqueous phase comprises a 20-100 Mm ammonium chloride solution and 0.05-0.3% hydrochloric acid, and the organic phase is acetonitrile;

[0008] The rotary evaporation concentration is as follows: 30-50 vol% at 30-50°C;

[0009] The freeze-drying includes pre-freezing, sublimation drying and desorption drying;

[0010] The temperature of the desorption drying is 25-35°C.

[0011] The present invention optimizes and combines process parameters such as reverse silica gel purification, salt transfer purification, rotary evaporation, and lyophilization. Experimental results show that using the process parameters of the present invention to purify crude products yields refined novel coronavirus antigen peptide M with a purity of ≥99.0%, excellent stability, stable acid radical content, and low residual solvent (e.g., acetonitrile) content. Furthermore, both purification and yield remain high. Changing any of these process parameters significantly reduces the effectiveness.

[0012] In the purification method described herein, the novel coronavirus antigen peptide M includes M23. In specific embodiments, crude novel coronavirus antigen peptide M23 and other antigenic peptides (such as S protein antigenic peptide and N protein antigenic peptide) were purified using the purification method described herein. It was found that this method is suitable for purifying M23, but is not suitable for antigenic peptides with structures significantly different from M23.

[0013] In some embodiments, the aqueous phase of the reverse silica gel purification is a 0.5 vol% aqueous phosphoric acid solution, a 1 vol% aqueous phosphoric acid solution, a 1.5 vol% aqueous phosphoric acid solution, or a 2 vol% aqueous phosphoric acid solution.

[0014] In some embodiments, the aqueous phase for salt conversion purification is selected from any one of the following combinations:

[0015] 100Mm ammonium chloride solution and 0.1vol% hydrochloric acid, 20Mm ammonium chloride solution and 0.05vol% hydrochloric acid, 40Mm ammonium chloride solution and 0.2vol% hydrochloric acid, 70Mm ammonium chloride solution and 0.3vol% hydrochloric acid.

[0016] In some embodiments, the rotary evaporation concentration is 40% at 35°C, or 50% at 30°C, or 30% at 40°C, or 40% at 35°C, or 30% at 50°C.

[0017] In the present invention, the pre-freezing temperature is -50 to -30°C. In some embodiments, the freezing temperature is -50°C, -50°C, or -30°C.

[0018] In the present invention, the temperature of the sublimation drying is -10 to 0°C. In some embodiments, the temperature of the sublimation drying is -10°C, -5°C, 0°C

[0019] In some embodiments, the solvent used for dissolving is a 5-15 vol% acetonitrile aqueous solution, wherein the concentration of the acetonitrile aqueous solution is preferably 5 vol%, 10 vol%, or 15 vol%.

[0020] In some embodiments, the filler material for the reverse silica gel purification comprises C18 or C8.

[0021] In some embodiments, the filler material for the salt conversion purification comprises C18 or C8.

[0022] The present invention has found that the fillers used in reverse silica gel purification and salt transfer purification also have a certain impact on the yield and purity of purification. The yield and purity of purification using C4 fillers are significantly inferior to those of C18 and C8.

[0023] The present invention also provides a novel coronavirus antigen peptide M product obtained by any of the above purification methods.

[0024] The present invention also provides a novel coronavirus vaccine, which comprises the novel coronavirus antigen peptide M fine product as described above and an adjuvant.

[0025] The present invention first synthesizes the novel coronavirus antigen peptide M23 through classic polypeptide solid-phase synthesis technology. Then, by rationally controlling the process conditions of reverse-phase silica gel purification and salt conversion, rotary evaporation concentration, and lyophilization, the novel coronavirus antigen peptide M23 hydrochloride raw material is finally obtained with a purity of ≥99.0%, good stability, stable acid group content, and low residual solvent content, and the purification yield is at a high level. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the chromatogram of the lyophilized powder sample of antigen peptide M23 in experimental group 3;

[0027] Figure 2 This is the mass spectrum of the lyophilized powder sample of antigen peptide M23 in experimental group 3. DETAILED DESCRIPTION

[0028] The present invention provides a method for purifying novel coronavirus antigen peptides. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art can modify or appropriately alter and combine the methods and applications herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0029] The present invention uses classic Boc or Fmoc peptide solid-phase synthesis technology to synthesize a crude product of the novel coronavirus antigen peptide M23. This is followed by dissolution of the crude peptide, purification and salt conversion on reverse-phase silica gel, rotary evaporation, and lyophilization to obtain the novel coronavirus antigen peptide M23 hydrochloride API with a purity of ≥99.0%, excellent stability, stable acid group content, and low residual solvent content. The purification yield is high.

[0030] In a specific embodiment of the present invention, the crude novel coronavirus antigen peptide M23 was synthesized using Fmoc polypeptide solid phase synthesis technology. The specific method includes:

[0031] In some specific embodiments, the purification method of the present invention comprises the following steps:

[0032] 1) Dissolve the crude product: Dissolve the crude product in purified water: acetonitrile = 95-85%: 5-15%. Ultrasonication or stirring can be used to assist dissolution. After the solution is clear, filter it with a 0.45μm or 0.22μm microporous filter membrane and use it as the sample to be purified.

[0033] 2) First Purification: Purification was performed on a reverse-phase silica gel column chromatography medium (C18 or C8) using a 0.5-2.0% (v / v) phosphoric acid / water solution as the aqueous phase and acetonitrile as the organic phase. The detection wavelength was 220 nm. Purified fractions were collected according to the preparative peak fractions and analyzed by analytical HPLC. Qualified fractions (purity ≥ 97.5%) were transferred to the next step. Detection method: Mobile phase A was 0.1% (v / v) sulfuric acid / water solution, mobile phase B was acetonitrile, and the analytical column was a Waters Acquity UPLC CHS 1.7 μm 2.1*150 mm column or equivalent. The flow rate was 0.2 mL / min, the column temperature was 20°C, the detection wavelength was 220 nm, and the detection gradient was 19% to 21% of phase B in 14 min.

[0034] 3) Salt Transfer: Salt transfer was performed on a reverse-phase silica gel column chromatography using a 20-100 mM ammonium chloride and 0.05-0.3% (v / v) hydrochloric acid / water solution as the aqueous phase and acetonitrile as the organic phase. The monitoring wavelength was 220 nm. The salt transfer fractions were collected according to the preparative peak and analyzed by analytical HPLC. Qualified fractions (purity ≥99.0%) were transferred to the next step. The detection method was the same as that for the first purification step.

[0035] 4) Rotary evaporation: Rotary evaporation is performed at a temperature of 30-40° C., and the volume of the liquid removed is controlled to be 30-50% of the original liquid volume. After the rotary evaporation is completed, the sample solution proceeds to the next step.

[0036] 5) Freeze-drying: Pre-freeze at -50 to -30°C, perform sublimation drying at -10 to 0°C, and perform desorption drying at 25 to 35°C. The finished product, novel coronavirus antigen peptide M23 hydrochloride drug substance, is obtained.

[0037] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0038] The present invention will be further described below in conjunction with the embodiments:

[0039] Example

[0040] In this example, an experimental group and a control group were set up, wherein the experimental group was purified by the purification method of the present invention, and the control group was purified by the comparative example.

[0041] (1) Purification method of the present invention (experimental groups 1 to 4)

[0042] Taking experimental group 1 as an example, the specific purification method includes the following steps:

[0043] (1) Dissolution of crude product: Dissolve the crude product in purified water: acetonitrile = 95%:5% (i.e., 5% acetonitrile / water). Ultrasonication or stirring can be used to assist dissolution. After the solution is clear, filter it with a 0.45 μm or 0.22 μm microporous filter membrane and use it as the sample to be purified.

[0044] (2) First step purification: Purification was performed on a reversed-phase silica gel column chromatography medium (C18) using a 0.5% (v / v) phosphoric acid / water solution as the aqueous phase and acetonitrile as the organic phase. The detection wavelength was 220 nm. Purified fractions were collected according to the preparative peak fractions and tested by analytical HPLC. Qualified fractions (purity ≥ 97.5%) were transferred to the next step. Detection method: Mobile phase A was a 0.1% (v / v) sulfuric acid / water solution, mobile phase B was acetonitrile, and the analytical column was a Waters Acquity UPLC CHS 1.7 μm 2.1*150 mm or equivalent column. The flow rate was 0.2 mL / min, the column temperature was 20°C, the detection wavelength was 220 nm, and the detection gradient was 19→21% of phase B in 14 min.

[0045] (3) Salt conversion: Salt conversion was performed on a reverse-phase silica gel column chromatography medium (C18) using a 100 mM ammonium chloride and 0.1% (v / v) hydrochloric acid / water solution as the aqueous phase and acetonitrile as the organic phase. The monitoring wavelength was 220 nm. The salt conversion fractions were collected according to the preparative peak and analyzed by analytical HPLC. Qualified fractions (purity ≥99.0%) were transferred to the next step. The detection method was the same as that for the first purification step.

[0046] (4) Rotary evaporation: Rotary evaporation was performed at 35°C, and the volume of the liquid removed was controlled to be 40% of the original volume. After the rotary evaporation was completed, the sample solution entered the next step.

[0047] (5) Freeze drying: pre-freeze at -40°C, perform sublimation drying at -5°C, and perform desorption drying at 30°C. Finally, a freeze-dried powder sample - the novel coronavirus antigen peptide M23·hydrochloride raw material drug substance - is obtained.

[0048] The specific parameters involved in the purification methods of experimental groups 2 to 4, including crude product dissolution, first-step purification mobile phase and filler, salt conversion mobile phase and filler, rotary evaporation concentration, and lyophilization, are shown in Table 1. Other conditions were the same as those of experimental group 1.

[0049] Lyophilized powder samples were prepared according to the methods of experimental groups 1 to 4, and the purification yield, finished product purity, acid radical content, and acetonitrile content were tested as follows:

[0050] 1. Purity detection method:

[0051] Chromatographic system: ultra-high performance liquid chromatography;

[0052] Detector: UV detector;

[0053] Chromatographic column: Waters CSH C18 1.7 μm 3.0*150 mm;

[0054] Mobile phase A: 0.1% sulfuric acid;

[0055] Mobile phase B: acetonitrile;

[0056] Column temperature: 20°C;

[0057] Flow rate: 0.2 mL / min.

[0058] 2. Acid radical detection:

[0059] Chromatographic system: Thermo Fisher ion chromatograph;

[0060] Detector: conductivity detector;

[0061] Chromatographic column: Dionex IonPac™ AS19 RFIC™, 4 mm × 250 mm;

[0062] Mobile phase: potassium hydroxide;

[0063] Column temperature: 30°C;

[0064] Flow rate: 1.0 mL / min;

[0065] 3. Acetonitrile detection:

[0066] Chromatographic system: Agilent gas chromatograph;

[0067] Detector: hydrogen flame ionization detector;

[0068] Chromatographic column: DB-624, 30m×0.53mm, 3μm;

[0069] Carrier gas: helium;

[0070] Flow rate: 1.0 mL / min.

[0071] The test results are shown in Table 1. The chromatographic and mass spectrometric test results of the freeze-dried powder samples prepared in Experimental Group 3 are shown in Figures 1-2 .

[0072] Table 1

[0073]

[0074]

[0075] Purification is carried out within the parameter range of the present invention, with a high purification yield (between 67% and 73% overall), a finished product purity of ≥99.0%, good control of residual acid radicals (trifluoroacetate and phosphate) and very low content, a normal chloride ion content (5.0% to 7.0%), and a pharmacopoeia-compliant acetonitrile content (≤0.041%).

[0076] (2) Long-term stability test, accelerated stability test and test results

[0077] Long-term and accelerated stability studies were conducted on a lyophilized powder of the novel coronavirus antigen peptide M23 hydrochloride, prepared on a large scale using the purification method described in Experimental Group 3. The results are shown in Table 2.

[0078] Table 2 Stability results of experimental group 3

[0079]

[0080] The results showed that the freeze-dried powder sample prepared according to experimental group 3 of the present invention had the best stability at -20±5°C and 2-8°C, and was slightly less stable at 25±5°C and 60%±5% RH.

[0081] (III) Comparative Example Purification Method

[0082] The specific parameters for the purification methods of Comparative Examples 1-7, including crude product dissolution, the first purification mobile phase and filler, the salt conversion mobile phase and filler, rotary evaporation concentration, and lyophilization, are shown in Table 4. Other conditions were the same as those of Experimental Group 1. The purification yield, finished product purity, acid radical content, and acetonitrile content of the lyophilized powder samples of Comparative Examples 1-7 were measured, and the results are shown in Table 4.

[0083] Table 4 Purification parameters of Comparative Examples 1 to 7

[0084]

[0085] Compared with experimental group 1: After the comparative example 1 was replaced with reversed-phase silica gel C4 filler, the purification yield and finished product purity both decreased, indicating that the reversed-phase silica gel C4 filler was not ideal for the purification of the novel coronavirus antigen peptide M23.

[0086] Compared with Experimental Group 2: Comparative Examples 2 and 3 replaced the one-step purification mobile phase with a lower or higher concentration of phosphoric acid, and replaced the salt transfer purification mobile phase with a lower or higher concentration of hydrochloric acid. The purification yield and finished product purity were significantly reduced, indicating that the acid concentration was not conducive to the purification of the novel coronavirus antigen peptide M23 when it exceeded a certain range.

[0087] Compared with Experimental Group 3: the concentration of ammonium chloride used for salt conversion in Comparative Example 4 was relatively low, resulting in incomplete phosphate replacement, high phosphate content, and low chloride ion content; the rotary evaporation temperature in Comparative Example 5 was relatively high, resulting in a certain degree of degradation of the sample, and the purity was less than 99.0%.

[0088] Compared with experimental group 4: the volume of the spun-out solution in comparative example 6 was too low, resulting in the residual acetonitrile exceeding the 0.041% specified in the pharmacopoeia; the analytical drying temperature in comparative example 7 was too high, resulting in a certain degree of degradation of the sample during the freeze-drying process, and the purity was less than 99.0%.

[0089] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for purifying novel coronavirus antigen peptide M, characterized in that: The crude novel coronavirus antigen peptide M was dissolved and then subjected to reverse silica gel purification, salt conversion purification, rotary evaporation concentration, and freeze-drying to obtain the refined novel coronavirus antigen peptide M. The mobile phase of the reverse silica gel purification includes an aqueous phase and an organic phase; the aqueous phase is a 0.5-2.0 vol% phosphoric acid aqueous solution, and the organic phase is acetonitrile; The mobile phase for salt conversion purification comprises an aqueous phase and an organic phase, wherein the aqueous phase comprises a 20-100 Mm ammonium chloride solution and 0.05-0.3% hydrochloric acid, and the organic phase is acetonitrile.

2. The purification method according to claim 1, wherein The aqueous phase for the reverse silica gel purification is 0.5 vol% phosphoric acid aqueous solution, 1 vol% phosphoric acid aqueous solution, 1.5 vol% phosphoric acid aqueous solution or 2 vol% phosphoric acid aqueous solution.

3. The purification method according to claim 1, wherein The aqueous phase of the salt conversion purification is selected from any one of the following combinations: 100Mm ammonium chloride solution and 0.1vol% hydrochloric acid, 20Mm ammonium chloride solution and 0.05vol% hydrochloric acid, 40Mm ammonium chloride solution and 0.2vol% hydrochloric acid, 70Mm ammonium chloride solution and 0.3vol% hydrochloric acid.

4. The purification method according to claim 1, wherein The rotary evaporation concentration is as follows: 30-50 vol% at 30-50°C.

5. The purification method according to claim 4, wherein The rotary evaporation concentration is 40% at 35°C, 50% at 30°C, 30% at 40°C, 40% at 35°C, or 30% at 50°C.

6. The purification method according to claim 1, wherein The freeze-drying includes pre-freezing, sublimation drying and desorption drying; The drying temperature is 25°C to 35°C. The pre-freezing temperature is -50°C to -30°C; The temperature of the sublimation drying is -10°C to 0°C.

7. The purification method according to any one of claims 1 to 6, characterized in that The solvent used for the dissolution is a 5-15 vol% acetonitrile aqueous solution.

8. The preparation method according to any one of claims 1 to 7, characterized in that The filler material for reverse silica gel purification includes C18 or C8; and / or The filler for the salt conversion purification includes C18 or C8.

9. The novel coronavirus antigen peptide M product prepared by the preparation method according to any one of claims 1 to 8.

10. A novel coronavirus vaccine, characterized in that: It comprises the novel coronavirus antigen peptide M fine product as described in claim 9 and an adjuvant.

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