Method for detecting protein impurities in protein samples

Summary:
This paper focuses on the importance of protein purity, including the detection methods of protein impurities, including electrophoresis, chromatography, sedimentation rate determination, mass spectrometry, and light scattering. No method can directly quantify the purity of a protein sample. Whether the ultimate goal is to interpret the analytical data, verify the quality of the process, or to ensure the safety of the biologic product, the determination of sample purity is a critical step.

With the development of proteinaceous biological products, protein purity has become a major problem in drug management. In the quality guidelines for biological products, it is pointed out that in addition to evaluating the purity of APIs and pharmaceutical products, it may be composed of expected products and a variety of product-related substances, and the possible impurity components should be evaluated. These impurities may be produced during the production process or related to the product, and their structure may be known, qualitative, or unknown.
Before evaluating the purity of a sample, first identify the type of impurity to be tested, such as nucleic acids, carbohydrates, lipids, unrelated proteins, isozymes, inactivated proteins, and determine the ability to distinguish between assumed impurities and targets in the solution to be tested. The physical and chemical properties (chemical analysis or physical characteristics) of proteins. The concentration of an impurity may have been reduced below the detection limit during the purification process, but there is still residue in the chromatographic peak. There is no doubt that the apparent purity depends on the chosen method of measurement and its sensitivity. Most separation methods can effectively remove non-protein impurities. The following is a brief introduction to the detection of protein impurities in protein samples.
1. Electrophoresis electrophoresis is the simplest and lowest cost, and is the most sensitive in determining the number of protein components in a sample, so it is most common. It is commonly used for the first screening of protein purity identification, and even for the identification of early high heterogeneity samples. If the expected impurity is different from the molecular weight of the target protein, SDS gel electrophoresis can distinguish impurities. For molecules with similar molecular weights but different amino acid compositions, SDS gel electrophoresis is generally inseparable, but in non-denaturing gel electrophoresis, it can be identified according to its different electrophoretic mobility.
However, there are some potential issues to be aware of when using this method. For denaturing gels, false negatives and false positives may occur. False negatives can occur if impurities co-migrate or impurities cannot enter the gel. Therefore, the whole piece of glue, including the concentrated glue and the separation glue, should be dyed, and it is necessary to check whether the protein fuel is suitable. The occurrence of false positives may be due to covalent modification during sample preparation, or uneven glue or oxidant residues. Similar problems can occur in non-denaturing gels as well. If the electrostatic charge of the impurity is zero or opposite to the target protein, the impurity will not appear on the gel and can be resolved by widening the pH range of non-denaturing gel electrophoresis.
2, chromatography
2.1 Gel Filtration Chromatography Gel filtration chromatography is one of the simplest methods for detecting impurities with different molecular weights of the target protein. This method is non-destructive and very fast. Because this method is a sample discrimination method, the sample is diluted as it passes through the gel column, so the sample to be tested needs to have a certain initial concentration. The specific amount of detection depends on the sensitivity when detecting impurities.
The sensitivity of gel filtration chromatography for detecting molecular size is lower than that of electrophoresis. The amount of material required for the experiment is greater than that of electrophoresis. Since gel filtration chromatography is usually carried out in a natural state, the results reflect the heterogeneity of the sample. However, if the protein is present as a stable oligomer (such as a fatty acid enzyme), it will exhibit heterogeneity in gel filtration chromatography. At the same time, fast, reversible self-linked proteins also exhibit an abnormal concentration of elution profile. In such cases, gel filtration chromatography can be repeated by selecting different portions from the asymmetric or broadened peaks.
2.2 Reversed-Phase High Performance Liquid Chromatography Reversed-phase high-performance liquid chromatography (reversed phase HPLC) uses a non-polar matrix such as a modified silicon medium as a stationary phase to reduce protein and stationary phase by organic solvents in the mobile phase. The affinity is eluted with a gradient of decreasing polarity to elute the protein.
Since the method is simple and rapid, and has ready-made instruments and equipment, which can be flexibly applied to protein separation of many different characteristics, this method has been generally used for the purity determination of protein samples. If necessary, the purity of the primary target can also be determined by changing the mobile phase to separate the sample under different gradients and conditions.
3, sedimentation rate determination method of sedimentation velocity, can be simple and rapid non-destructive to determine the purity of the protein, while the ratio of molecular mass and molecular size is very sensitive. The advantage of this method is that the measurement range is very wide; however, the limitation is that the sensitivity of the sample having a small difference in molecular mass is lower than that of the electrophoresis technique.
4. Mass spectrometry mass spectrometry can directly determine the distribution of covalent mass in a sample. This method can easily and easily determine impurities. Mass spectrometry can not only detect the presence of impurities, but also describe the quality characteristics of impurities, so mass spectrometry is often used to identify the source of impurities. Covalently modified modification features and sites can be identified by tandem mass spectrometry (MS/MS). Since impurities may have a similar mass-to-charge ratio to the main target, mass spectrometry combined with other methods (such as gel separation chromatography) can increase the accuracy of sample purity determination and achieve the best results.
5. Light Scattering Methods Some instruments currently measure individual samples by static or dynamic light scattering, or monitor high performance liquid chromatography and field-level separation processes. The ability to identify eluted proteins is enhanced by continuous determination of molecular size and apparent molecular mass to distinguish between analytes, multimers or impurities of the analyte. The light scattering method is very simple and non-destructive, while providing a molecular mass map of the elution time function. If the UV detection peak is asymmetrical, by means of multiple angle light scattering (MALS) analysis, it is possible that the apparent molecular weight of the main part of the peak is mol/L, and the edge is 2 mol/L, indicating that there may be a dimer. . However, the appearance of multiple molecular mass does not necessarily prove that there must be self-binding, and different methods are needed to verify the result.
No method can directly quantify the purity of a protein sample. Typically, protein purity determination involves assessing the amount of impurities to be determined or merely verifying the presence of impurities in the protein sample. Whether the ultimate goal is to interpret the analytical data, verify the quality of the process, or to ensure the safety of the biologic product, the determination of sample purity is a critical step.