A method for detecting NGF purity using SHS capillary gel electrophoresis
By using SDS and SHS as denaturants in NGF purity detection and capillary gel electrophoresis separation in SHS gel, the problems of weak affinity and incomplete protein denaturation of traditional CE-SDS methods were solved, and better separation effect and purity detection accuracy were achieved.
Patent Information
- Application Number
- CN202411774615.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-05
- Publication Date
- 2025-04-04
AI Technical Summary
The traditional CE-SDS method has weak affinity when detecting NGF purity and incomplete denaturation of proteins, resulting in poor separation effect.
Sodium dodecyl sulfate (SDS) and ammonium cetyl sulfate (SHS) were used as denaturants, and the protein conformation was fully opened by heating, and then capillary gel electrophoresis was performed in the SHS gel.
The separation effect of NGF is significantly improved, the accuracy of purity detection is improved, and the diffusion peaks and polymer impurity peaks present in traditional methods are eliminated.
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Figure CN119757505A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological experiments, and in particular to a method for detecting the purity of NGF by utilizing SHS capillary gel electrophoresis. Background Art
[0002] Purity determination is an important test index for protein drugs, which directly reflects the level of protein purification process and product quality. Therefore, purity determination is an indispensable test item in protein drug production process optimization, production process control and release analysis, and is also one of the important indicators for protein stability evaluation. The accurate characterization of protein purity mainly involves two aspects, one is the effective separation of protein molecules and impurities, and the other is the high-sensitivity detection and quantification of target proteins and impurities. In order to avoid the deviation of one detection method in protein purity detection, at least two separation methods are generally selected for detection to obtain relatively accurate purity information.
[0003] With the development of technology, analytical methods for protein purity continue to emerge, and the most common ones include chromatography, electrophoresis, mass spectrometry, immunochemistry, colorimetry, etc. Different analytical techniques are based on different principles, and the results obtained reflect the different characteristics of proteins in terms of size heterogeneity. Among them, size exclusion chromatography (SEC-HPLC) and capillary gel electrophoresis (CGE) are the two most common methods for analyzing the purity of protein drugs. A study by the China National Institute for Inspection and Quarantine showed that SEC can more objectively evaluate the percentage of aggregates in a sample, while CGE has a better separation effect on fragment impurities.
[0004] Nerve growth factor (NGF), as a neuronal growth regulatory factor with dual biological functions of neuronal nutrition and neurite growth promotion, plays an important regulatory role in the development, differentiation, growth, regeneration and expression of functional characteristics of central and peripheral neurons. At present, the methods reported in the literature to characterize the purity of NGF mainly include SEC-HPLC, reverse phase chromatography (RP-HPLC), hydrophobic interaction chromatography (HIC-HPLC) and polyacrylamide gel electrophoresis (SDS-PAGE). However, there are few reports on the study of CGE method to characterize the purity of NGF.
[0005] As one of the typical CGEs, capillary gel electrophoresis with sodium dodecyl sulfate (CE-SDS) is an important method for characterizing the purity / size heterogeneity of protein drugs (especially monoclonal antibodies) in the current biopharmaceutical field. Due to its strong hydrophobic properties, NGF has a weak affinity with SDS and shows poor separation effect in the CE-SDS system. Studies have shown that adding protein denaturants with longer alkyl chains, such as hexadecyl ammonium sulfate (SHS), to the gel can effectively increase the binding of the denaturant to the protein, thereby improving the separation effect of the protein. Summary of the invention
[0006] The purpose of the present invention is to overcome the limitations of weak affinity and incomplete protein denaturation of traditional CE-SDS, and to use SDS and SHS as denaturants, first completely open the protein conformation by heating, and then separate by SHS gel to obtain a better separation effect.
[0007] In order to achieve the above object, a method for detecting the purity of NGF using SHS capillary gel electrophoresis is designed, comprising the following steps: S1. Add sodium dodecyl sulfate (SDS) and hexadecyl ammonium sulfate (SHS) simultaneously to the sample buffer for NGF denaturation; S2. Separation in a gel system containing sodium dodecyl sulfate (SDS) and hexadecyl ammonium sulfate (SHS).
[0008] Preferably, the sample buffer comprises 0.1% hexadecyl ammonium sulfate (SHS) and 1.0% sodium dodecyl sulfate (SDS) to achieve the denaturation effect of the NGF sample.
[0009] Preferably, in step S1, during the sample processing, the conformation of the NGF protein is completely opened by heating under the conditions of the sample buffer system.
[0010] Preferably, the gel system comprises a gel buffer, and the concentration of the surfactant in the gel buffer is 0.2% SDS and 0.2%-0.5% SHS.
[0011] Preferably, step S2 is performed by capillary electrophoresis during the separation process, and the voltage used is 50-200 volts.
[0012] Preferably, the step also includes S3 method for detecting the purity of NGF, which evaluates the purity of NGF by observing the peak area and retention time of the target protein in the sample.
[0013] Compared with the prior art, the present invention has the following advantages: Aiming at the structural specificity of NGF protein, the present invention provides an application of SHS in the characterization of NGF purity. In this system, the limitations of weak affinity and incomplete protein denaturation of traditional CE-SDS are overcome, and a better separation effect is achieved. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 , is the non-reducing purity chromatogram of NGF in SDS gel buffer and SDS sample buffer system; Figure 2 , is the non-reducing purity chromatogram of NGF in SHS+SDS gel buffer and SDS sample buffer system; Figure 3 , are the non-reducing purity chromatograms of NGF in SHS+SDS gel buffer and SHS sample buffer systems; Figure 4 , are the non-reducing purity chromatograms of NGF in SHS+SDS gel buffer and SHS+SDS sample buffer systems. DETAILED DESCRIPTION
[0015] In order to make the purpose and principle of the present invention more clear, it is further described below in conjunction with the accompanying drawings and specific embodiments.
[0016] Embodiment 1 See also Figure 1 , Figure 2 This example provides a method for detecting the purity of NGF using SHS capillary gel electrophoresis. Recombinant human NGF is used as the research object, and the sample is denatured using SDS sample buffer and separated in a gel buffer containing SDS or SDS+SHS. The experimental results are shown in FIG. Figure 2 Shown: Compared to Figure 1 As shown in the SDS gel system separation, NGF protein can obtain better separation effect in the SDS+SHS gel system, but there is still a relatively obvious diffusion peak after the main component. It is speculated that the complex after the protein and SDS are combined is unstable, and SDS and the complex are separated to different degrees, resulting in different molecular weights of the complex and the appearance of diffusion peaks.
[0017] Embodiment 2 See also Figure 3 , This example is based on Example 1, but uses SHS as a denaturant to denature the sample and separate it in a gel system containing SDS and SHS. The experimental results are shown in Figure 3 As shown in the figure: compared with Example 1, the diffuse peak obviously disappeared, indicating that the component corresponding to the peak type is a pseudo component caused by the unstable binding of the protein surface with SDS, and is not a real impurity. Although the diffuse peak is significantly improved, an obvious high molecular impurity peak is found after the main peak. It may be a product-related impurity (polymer formed by covalent bonding), or it may be a pseudo high molecular impurity formed by the fact that 0.1% SHS in the sample buffer is not enough to completely bind to the protein.
[0018] Embodiment 3 See also Figure 4 In this example, based on Example 2, in order to eliminate the influence of protein conformation, SDS and SHS were added to the sample buffer at the same time. The two denaturants acted in combination and were separated in a gel system containing SDS and SHS. The experimental results are shown in Figure 4As shown: compared with Example 2, the high molecular impurity peak after the main peak disappears, confirming that the impurity component is not actually present in the sample, but is a conformational heterogeneity caused by the protein not being completely combined with the denaturant during the pretreatment process. The combined action of SDS and SHS can eliminate this effect.
[0019] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent substitutions or changes according to the technical solutions and novel concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A method for detecting the purity of NGF using SHS capillary gel electrophoresis, characterized in that: S1. Add sodium dodecyl sulfate (SDS) and hexadecyl ammonium sulfate (SHS) simultaneously to the sample buffer for NGF denaturation; S2. Separation in a gel system containing sodium dodecyl sulfate (SDS) and hexadecyl ammonium sulfate (SHS).
2. A method for detecting the purity of NGF by SHS capillary gel electrophoresis as claimed in claim 1, characterized in that: The sample buffer comprises 0.1% hexadecyl ammonium sulfate (SHS) and 1.0% sodium dodecyl sulfate (SDS), which is used to achieve the denaturation effect of the NGF sample.
3. A method for detecting the purity of NGF by SHS capillary gel electrophoresis as claimed in claim 1, characterized in that In the step S1, during the sample processing, the conformation of the NGF protein is completely opened by heating under the conditions of the sample buffer system.
4. A method for detecting the purity of NGF by SHS capillary gel electrophoresis as claimed in claim 1, characterized in that The gel system includes a gel buffer, and the concentration of the surfactant in the gel buffer is 0.2% SDS and 0.2%-0.5% SHS.
5. A method for detecting the purity of NGF by SHS capillary gel electrophoresis as claimed in claim 1, characterized in that The step S2 is performed by capillary electrophoresis during the separation process, and the voltage used is 50-200 volts.
6. A method for detecting the purity of NGF by SHS capillary gel electrophoresis as claimed in claim 1, characterized in that It also includes the S3 NGF purity detection method, which evaluates the purity of NGF by observing the peak area and retention time of the target protein in the sample.