Method for analyzing related impurities in virus-like particle medicine

Abstract

The invention discloses a method for analyzing related impurities in a virus-like particle drug, which adopts a tandem chromatography system consisting of a large-aperture size exclusion chromatographic column A and a small-aperture composite chromatography chromatographic column B which are directly connected, the column B has a separation mechanism of volume exclusion and hydrophobic interaction, chromatographic signals are detected, and the related impurities in the virus-like particle drug are analyzed. Synchronous separation, identification and quantification of virus-like particle related components, capsid protein related components and free oligonucleotide related components are realized by analyzing the peak appearance sequence of each chromatographic peak and the absorption signal intensity ratio under different wavelengths. The method effectively solves the problem of co-elution of different types of impurities, especially capsid proteins and free oligonucleotides in a traditional chromatographic method, has the advantages of high separation degree, accurate quantification and good repeatability, and is suitable for process development and quality control of virus-like particle drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the analysis of biological drug impurities, in particular to a method for analyzing related impurities in virus-like particle drugs. BACKGROUND

[0002] Virus-like particles (VLPs) are a class of hollow particles self-assembled by viral structural proteins, which do not contain viral genetic material and are therefore non-infectious, but retain the immunogenicity of viruses. They are important carriers for current vaccines and gene drugs (such as HPV vaccines and mRNA vaccines for COVID-19), and the VLPs encapsulating immunoadjuvants or therapeutic oligonucleotides are representatives of the new generation of biological drugs. Quality control of VLP drugs is the key to their clinical application, and impurity analysis is one of the core links. The impurities in VLP drugs mainly include: capsid protein-related components (such as capsid proteins that have not been polymerized during VLP assembly), free oligonucleotide-related components (such as mRNA fragments that have not been encapsulated in mRNA vaccines), and super-large impurities formed by the polymerization of VLPs themselves. These impurities not only reduce the effectiveness of the drug (such as free oligonucleotides that cannot be delivered by VLP carriers), but also can cause adverse reactions (such as empty protein monomers that can induce non-specific immune reactions), so their content needs to be strictly controlled.

[0003] Currently, the analysis methods for VLP drugs and their impurities still face great challenges. Conventional analysis methods, such as single size exclusion chromatography (SEC) or ion exchange chromatography (IEC), often fail to achieve efficient separation of all key impurities, especially small molecular weight impurities. Size exclusion chromatography is based on the difference in hydrodynamic volume, but its resolution is limited, and it is not effective for separating capsid protein monomers and free oligonucleotides with smaller molecular weights and similar sizes. These components often overlap due to their close elution times, forming a broad co-elution peak, which makes it impossible to achieve accurate quantification. Ion exchange chromatography can separate based on charge differences, but for complex systems such as VLPs, where large molecular assemblies coexist with free small molecules and their charge properties may be similar, its selectivity is also insufficient, and it is difficult to distinguish VLPs, capsid proteins, and free oligonucleotides in one analysis. Therefore, existing analysis methods cannot simultaneously solve the separation problem of different molecular weight impurities in VLP drugs, especially the separation efficiency of small molecular impurities (capsid protein and free oligonucleotide-related components) is extremely low, which becomes a bottleneck for the quality control of VLP drugs. SUMMARY

[0004] The purpose of the present application is to provide a method for analyzing related impurities in virus-like particle drugs, which solves the problem that existing technology cannot simultaneously effectively separate multiple types of impurities (especially small molecular impurities) in VLP drugs.

[0005] Technical solution: The method for analyzing related impurities in virus-like particle drugs provided by the application adopts a tandem chromatography system, wherein the tandem chromatography system comprises a macroporous size exclusion chromatography column A and a small-pore composite chromatography column B which are directly connected; the chromatography column B has separation mechanisms of specific volume exclusion and hydrophobic interaction; chromatography signals are detected, and absorption signal intensities and peak order of each chromatography peak are compared to analyze impurities.

[0006] Preferably, an ultraviolet detector is used to simultaneously detect signals at 260 nm and 280 nm wavelengths; and absorption signal intensities and peak order of each chromatography peak at 260 nm and 280 nm are compared to identify and quantify virus-like particle related components, capsid protein related components and free oligonucleotide related components.

[0007] Preferably, the separation protein range of the chromatography column A is 50 kDa to 10,000 kDa, and further preferably 50 kDa to 7,500 kDa.

[0008] Preferably, the separation protein range of the chromatography column B is 100 Da to 1,250 kDa, and further preferably 5 kDa to 1,250 kDa; and the filler surface is bonded with an alkyl hydrophobic group, and further preferably a butyl hydrophobic group.

[0009] Preferably, the filler pore size of the chromatography column A is 1,000-2,000 Å, and further preferably 1,000 Å.

[0010] Preferably, the filler pore size of the chromatography column B is 100-300 Å, and further preferably 300 Å.

[0011] Preferably, the filler particle size of the chromatography column A is 5-10 μm, and further preferably 5 μm.

[0012] Preferably, the filler particle size of the chromatography column B is 1.8-3 μm, and further preferably 3 μm.

[0013] Preferably, the column A is an SRT SEC-1000 chromatography column, and the column B is a BioMix SEC-300 chromatography column.

[0014] Preferably, the mobile phase used is 0.8-1.2 times phosphate buffer + (0.4-0.6) M sodium chloride, pH 6.5-7.5.

[0015] Further preferably, the mobile phase used is 1 times phosphate buffer + 0.5 M sodium chloride.

[0016] Preferably, the flow rate of chromatographic analysis is 0.5-1.0 mL / min, and the column temperature is 20-30℃.

[0017] Preferably, the ultraviolet detector is a diode array detector.

[0018] Preferably, the identification is based on the fact that the 260 nm signal of the virus-like particle related components is significantly stronger than the 280 nm signal; the 260 nm signal of the capsid protein related components is relatively strong; and the 260 nm absorption of the free oligonucleotide related components is much stronger than the 280 nm. Preferably, the identification is performed by calculating the ratio of the 260 nm and 280 nm absorption signals, and the ratio between 1.5 and 2.0 is for the capsid protein related components, and the ratio greater than 2.0 is for the oligonucleotide related components.

[0019] Further preferably, the method for analyzing the related impurities in the virus-like particle drug of the present application comprises: (1) Selection of chromatographic column and series connection: Column A (large pore size SEC column): a large pore size SEC chromatographic column with a separation range of 50 kDa to 7500 kDa (such as SRT SEC-1000, 1000 Å) is selected, which is used to separate large molecular weight VLP related components (such as complete VLP, VLP related polymer impurities, etc.).

[0020] Column B (small pore size composite chromatographic column): a small pore size composite chromatographic column with a separation range of 1 kDa to 1250 kDa (such as BioMix SEC-300, 300 Å, which is spherical silica with butyl hydrophobic groups bonded to the surface) is selected, which has volume exclusion composite hydrophobic interaction and is used to finely separate small molecular weight capsid protein related components (such as unaggregated capsid protein monomers or oligomers) and free oligonucleotide related components (such as unencapsulated mRNA fragments, degraded oligonucleotides, etc.).

[0021] Series connection mode: the outlet of column A is directly connected to the inlet of column B to form a series connection chromatographic chromatography system.

[0022] (2) Selection of mobile phase The preferred is 1 times PBS buffer with a pH value of 6.5-7.5. More preferably, a certain proportion of sodium chloride is added to the 1 times PBS buffer to enhance the hydrophobic interaction between the sample and the chromatographic column. Preferably, the concentration of sodium chloride is 0.5 M. The phosphate buffer can maintain the pH stability of the system, provide a certain ionic strength, inhibit the electrostatic interaction, and improve the separation effect.

[0023] If the mobile phase without sodium chloride to enhance the hydrophobic interaction is selected, the capsid protein related components and the free oligonucleotide related components with close molecular weights are easily wrapped in a wide peak, and the separation degree is poor, which cannot be integrated and quantitatively calculated.

[0024] (3) Sample treatment and injection Dilute the sample to be tested to 0.5-2 mg / mL (by VLP) with mobile phase.

[0025] (4) Chromatographic condition setting Flow rate: 0.5-1.0 mL / min (too low flow rate will prolong the analysis time, too high flow rate will reduce the resolution); Column temperature: 20-30 ℃ (maintain the stability of column packing, reduce sample denaturation); Detection wavelength: 260 nm (nucleic acid characteristic wavelength) and 280 nm (protein characteristic wavelength) are detected simultaneously (using a diode array detector). Compare the absorption characteristics of the two wavelengths, and identify the peak attribution by the order of peak elution: VLP-related components: first peak, signals at both 260 nm and 280 nm, and the 260 nm signal is significantly stronger; Capsid protein-related components: peak elution order is later than VLP-related components, signals at both 260 nm and 280 nm, and the 260 nm signal is stronger.

[0026] Free oligonucleotide-related components: peak elution order is later than capsid protein-related components, 260 nm absorption is much stronger than 280 nm, and the 280 nm signal is weak due to almost no protein.

[0027] (5) Elution and detection The sample flows through column A and column B in turn: VLP-related components of large molecular weight (such as intact VLP) are first separated by column A, and then eluted into column B; Capsid protein and free oligonucleotide-related components of small molecular weight are further separated on column B by volume exclusion (separating molecules of different sizes) and hydrophobic interaction (separating molecules with different hydrophobicity), and finally detected by the detector.

[0028] Invention principle: Because the pore size of the first chromatographic column is larger, the related components can all enter the pores, and it is suitable for the separation of VLP and its super large impurities, so the VLP super large polymer impurities flow out first, followed by the VLP main component. And the remaining small molecular weight components are fully permeable on the first large-pore chromatographic column, and they flow out at the same time, which achieves the complete separation of super large components, large components and small components. The pore size of the second chromatographic column is smaller than the VLP related components, so the VLP and its super large molecular weight impurities do not enter the pores, and the super large polymer impurities still flow out first, and the VLP main component flows out later. The order of the components out of the peak does not change. For small molecular weight components, the small pore size of the second chromatographic column and the combined hydrophobic effect can separate this part in detail. The larger the molecular weight, the earlier the peak, and the smaller the molecular weight, the later the peak. Weak hydrophobicity, strong hydrophobicity. Because the shell protein contains a large amount of conjugated cyclic structure of aromatic amino acids, this structure can make it have a higher absorption value at 280 nm. Oligonucleotides contain almost no such structure, so their absorption value at 280 nm is very weak, but their bases (purine and pyrimidine) have conjugated double bond structures and have strong absorption of ultraviolet light near 260 nm. Therefore, by comparing the peak area ratio of 260 nm:280 nm, the ratio between 1.5~2.0 should be the shell protein and VLP related components, and the ratio greater than 2.0 should be the oligonucleotide related components.

[0029] Beneficial effects: Compared with the prior art, the present application has the following obvious advantages: 1. The present application has excellent impurity separation ability through the "specific pore size combination of series column" and "combined separation mechanism (volume exclusion + hydrophobicity)", which realizes the separation of different molecular weight components in VLP drugs, especially in the separation of shell protein and free oligonucleotide related components. It has a good advantage, solves the problem of poor separation degree or co-elution of traditional methods, and realizes the baseline separation of different molecular weight components; 2. Precise quantification: the percentage content of different molecular weight components in VLP drugs can be accurately calculated respectively; 3. High repeatability and practicability: the method is stable and fast, and is very suitable for process development and quality control of VLP drugs. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The sample spectrum of the SRT SEC-1000 series BioMix SEC-300 SEC method of the present application for detecting HBcAg VLP packaged CpG oligonucleotide.

[0031] Figure 2 The sample integral spectrum of 260 nm HBcAg VLP packaged CpG oligonucleotide.

[0032] Figure 3Figure 1 shows the chromatogram of HBcAg VLPs encapsulating CpG oligonucleotides detected by SRT SEC-1000 in series with Zenix-C SEC-300 SEC method. DETAILED DESCRIPTION

[0033] The technical solutions of the present application are further described below in combination with the drawings, examples and comparative examples.

[0034] Example 1

[0035] The sample analyzed was: HBcAg VLPs encapsulating CpG oligonucleotides, 1 mg / mL, solvent system: 10 mM Tris-HCl, pH 8.0, E. coli expression system, preparation method according to patent CN200480015952.5.

[0036] Instrument and reagents: Agilent 1260 Infinity II HPLC system equipped with a diode array detector.

[0037] Chromatographic column: one SRT SEC-1000 (5 μm, 1000 Å, 7.8 mm x 30 cm) as column A, and one BioMix SEC-300 (3 μm, 300 Å, 7.8 mm x 30 cm) as column B were connected in series.

[0038] Mobile phase: 1x PBS buffer + 0.5 M NaCl, pH 7.0, filtered through a 0.45 μm filter and degassed before use.

[0039] Chromatographic conditions: Flow rate: 0.8 mL / min; Column temperature: 25°C; Detection wavelength: 260 nm & 280 nm; Injection volume: 10 μL; Sample concentration: ~1 mg / mL (calculated as VLPs); Analysis process: The sample was injected and analyzed, and the chromatograms at 260 nm and 280 nm were recorded, and the analysis spectrum is shown in Figure 2 .

[0040] The sample showed one peak and a small peak before it in region 1, and multiple peaks in regions 2 and 3.

[0041] Region 1 is VLP-related component. There are signals at both 260 nm and 280 nm, and the 260 nm signal is stronger, which confirms that this component is VLP carrying nucleic acid. Peak 1 is identified as the main peak of VLP (retention time is 17.637 min), and the small peak Peak 2 in front of it is a large impurity of VLP-related polymer (retention time is 16.364 min). According to the peak area of the 260 nm chromatogram, the VLP-related component accounts for 43.37% as calculated in Table 1.

[0042] Region 2 is capsid protein-related component. Since its retention time is later than that of the main peak of VLP, the molecular weight of this component is much smaller than that of the main peak of VLP. There are absorptions at both 260 nm and 280 nm, so this component contains both protein and oligonucleotide components, and therefore this part is a complex of capsid protein (such as dimer, trimer, hexamer) and oligonucleotide. According to the peak area of the 260 nm chromatogram, the capsid protein-related component accounts for 38.77% as calculated in Table 1.

[0043] Region 3 is free oligonucleotide-related component. Its retention time is much later than that of the main peak of VLP, and the 260 nm ultraviolet response is significantly higher than that of 280 nm, which has the typical spectral characteristics of nucleic acid, which is free oligonucleotide and its degradation fragments. According to the peak area of the 260 nm chromatogram, the free oligonucleotide-related component accounts for 17.86% as calculated in Table 1.

[0044] Table 1: Retention time of each peak at 260 and 280 nm and peak area and peak area ratio calculation table

[0045] Example 2

[0046] The difference from Example 1 is that the column A: SRT SEC-1000 (5 μm, 1000 Å, 7.8 mm x 30 cm) is replaced by SRT SEC-2000 (5 μm, 2000 Å, 7.8 mm x 30 cm).

[0047] Under the conditions, the chromatograms at 260 nm and 280 nm were recorded. The results show that the VLP-related component, the capsid protein-related component and the free oligonucleotide-related component can still achieve obvious baseline separation. The peak order of each chromatographic peak and the absorption signal intensity ratio characteristics (such as 260 nm / 280 nm ratio) are consistent with those of Example 1, which conforms to the component identification rule. By calculating the peak area at 260 nm, the three types of components can be successfully quantitatively analyzed.

[0048] Comparative Example 1 The difference from Example 1 is that the chromatographic column B is replaced by Zenix-C SEC-300 (3 μm, 300 Å, 7.8 mm x 30 cm), and the others remain unchanged.

[0049] The chromatographic column only has volume exclusion effect without hydrophobic effect. The closely related components of capsid protein and free oligonucleotide components are poorly separated, and cannot be effectively separated and quantitatively calculated. Details are shown in Table 2. Figure 3 .

[0050] Comparative Example 2 The difference from Example 1 is that the two chromatographic columns in series are replaced by a single chromatographic column SRT SEC-500 (5 μm, 500 Å, 7.8 mm x 30 cm), and the others remain unchanged.

[0051] The VLP and its polymer impurities are all excluded on the chromatographic column, and cannot be effectively separated; the closely related components of capsid protein and free oligonucleotide components are poorly separated, and cannot be quantitatively calculated.

Claims

1. A method of analyzing related impurities in a virus-like particle drug, characterized by, The serial chromatographic system comprises a macropore size exclusion chromatographic column A and a small pore composite chromatographic column B connected directly, the column B has specific volume exclusion and hydrophobic interaction separation mechanism; the chromatographic signal is detected, the absorption signal intensity and peak sequence of each chromatographic peak are compared, and the impurities are analyzed.

2. The method of claim 1, wherein, The ultraviolet detector is used to detect the signals at 260 nm and 280 nm simultaneously; the virus-like particle related components, capsid protein related components and free oligonucleotide related components are identified and quantified by comparing the absorption signal intensity and peak sequence of each chromatographic peak at 260 nm and 280 nm.

3. The method of claim 1, wherein, The filler pore size of the column A is 1000-2000 Å.

4. The method of claim 1, wherein, The filler pore size of the column B is 100-300 Å.

5. The method of claim 1, wherein, The column A is an SRT SEC-1000 chromatographic column, and the column B is a BioMix SEC-300 chromatographic column.

6. The method of claim 1, wherein, The mobile phase used is 0.8-1.2 times phosphate buffer + (0.4-0.6) M sodium chloride, pH 6.5-7.

5.

7. The method of claim 1, wherein, The flow rate of the chromatographic analysis is 0.5-1.0 mL / min, and the column temperature is 20-30℃.

8. The method of claim 1, wherein, The ultraviolet detector is a diode array detector.

9. The method of claim 1, wherein, The filler particle size of the column A is 5-10 μm, and the filler particle size of the column B is 1.8-3 μm.

10. The method of claim 1, wherein, The identification is based on the calculation of the ratio of the absorption signals at 260 nm and 280 nm; the ratio between 1.5 and 2.0 is the capsid protein related component, and the ratio greater than 2.0 is the oligonucleotide related component.

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