In the field of biopharmaceutical research, accurate determination of protein purity is essential to ensure the quality and safety of products. Protein purity is closely related not only to its biological activity and structural stability, but also to its efficacy and safety. Therefore, researchers need efficient and accurate analytical methods to assess the purity of protein samples. As a commonly used method, SEC assay technology plays an important role in protein purity analysis.
1. Principle: The SEC determination technique is based on the differences in molecular size and conformation of proteins in the stationary phase-packed column. Stationary phase-packed columns are typically constructed of porous materials, where larger molecules are unable to enter the pores and therefore pass through the bed relatively quickly, while smaller molecules enter the pores more, resulting in longer times for them to pass through the bed.
In the SEC assay, the protein molecules in the sample solution form a dynamic equilibrium in the column. As the sample passes through the SEC column, the larger protein molecules pass through the column bed more quickly, while the smaller impurity molecules are relatively trapped in the pores, resulting in a purity separation. Ultimately, the purity level of the protein can be determined by detecting changes in the absorbance or fluorescence signal of the sample on the column bed.
2. Advantages and disadvantages:
1.Advantages:11. High efficiency: SEC assay technology can quickly and effectively separate proteins and impurities, providing high-purity samples. Compared with other separation techniques, SEC has a higher resolution and separation efficiency.
1.2 No special conditions: Compared with other separation techniques, such as ion exchange chromatography, affinity chromatography, etc., SEC determination technology does not require special solution conditions or ligands, making it easier to operate and apply.
1.3Maintain biological activity: SEC assay technology does not require high temperature or high concentration of solution during separation, so it can better maintain the biological activity and structural integrity of the protein.
2.Disadvantages: 21 Inability to resolve isomers of the same molecular weight: SEC assay techniques cannot resolve proteins with the same molecular weight but different conformations, such as monomers and aggregates of the same molecular weight. In this case, further identification and analysis in combination with other analytical methods is required.
2.2 Column effects may occur: Differences in bed packing materials and column sizes may lead to variations in results, so it is necessary to select the appropriate bed and standards when using the SEC assay technique, and to strictly control the experimental conditions.
3. Application cases:
SEC assay technology is widely used in biopharmaceutical research and manufacturing. Here are some common use cases:
1.Determine the purity of proteins: SEC assay techniques can quickly separate proteins and other impurities such as subunits, polymers, oligomers, etc., to determine the purity level of protein samples.
For example, in a drug discovery process, researchers used SEC technology to analyze the purity of a recombinant protein sample. The results showed that the SEC spectrum showed obvious peak-like separation, and the absorbance signal corresponding to the protein peak was high, indicating the presence of higher purity protein in the sample.
2.Protein aggregate analysis: SEC assay techniques can be used to determine the polymeric state of a protein, such as the proportion of distribution between monomers and aggregates, as well as the relative size of aggregates.
In an antibody study, scientists assessed the aggregate content of antibody samples using SEC assay techniques. The SEC profile shows the presence of multiple peaks in the sample, each representing a different size of aggregate. From the relative area of the peaks and the location of the peaks, the researchers were able to infer the content and size distribution of the various aggregates in the sample.
3.Biological Activity Preservation Assessment: SEC assay technology can detect whether a protein retains its biological activity during isolation. The effect of the separation on the structure and function of the protein can be assessed by comparing the activity of the sample before and after.
For example, a recombinant enzyme sample was purified using the SEC assay. By comparing with the unpurified sample, they found that the purified sample did not show a significant decrease in activity, indicating that the SEC assay technology maintained the biological activity of the enzyme well.
As a powerful tool for efficient measurement of protein purity, SEC assay technology plays an important role in biopharmaceutical research. The principle is simple, easy to operate, and it is able to provide high-quality samples. However, it should be noted that SEC assay techniques cannot resolve proteins with the same molecular weight but different conformations, so other analytical methods need to be considered in combination in practical applications. Through the rational selection of column beds and standards, as well as the combination of other technical means, the advantages of SEC assay technology can be fully utilized to ensure the quality and safety of protein products.