Analysis on the application of SEC-MALS detection of molecular weight and molecular weight distribution

1. SEC-MALS instrument introduction and detection of molecular weight and molecular weight distribution principle

SEC-MALS, full name 18 Angle laser light scattering gel chromatography system, is a combination of dimensional exclusion chromatography and multi-angle light scattering technology. After separation by Size Exclusion Chromatography (SEC), each component enters the multi-angle light scattering detector (MALS). Light scattering occurs when laser irradiation is applied to the analyte, and the intensity of the scattered light is measured simultaneously by MALS from multiple angles. The scattered light intensity is directly proportional to the square of the molar mass, concentration and refractive index increment (see Figure 1), thus directly calculating the absolute molecular weight and molecular weight distribution information of the analyte. Compared with other molecular weight detection equipment (such as GPC, LC-MS, etc.), It has the characteristics of high accuracy, no need to use standard protein, independent elution retention time and direct accurate determination of absolute molecular weight of protein, which can meet the detection needs of the entire drug research and development, process and process development, product quality control and other links.

FIG. 1 Principle of SEC-MALS detection

2. SEC-MALS related applications and case sharing

SEC-MALS instrument can be used to characterize the molecular weight and molecular weight distribution of a variety of drugs or excipients, polymers, including peptides, antibodies, fusion proteins, AAV, hydroxyethyl starch, polyethylene glycol, polysaccharides and protein iron succinate, etc. The parameters that SEC-MALS can provide include, but are not limited to, the weight average molecular weight Mw, the number average molecular weight Mn, the Z average molecular weight Mz, the peak molecular weight Mp, and the molecular weight distribution (Mw/Mn) of the polymer.

Case 1 Herceptinomab SEC-MALS assay with weight average molecular weight and polymer analysis

The anti-tumor agent Trastuzumab (Herceptin) is a monoclonal antibody targeting the Her2 protein (encoded by ERBB2) and has been approved by the FDA as a first-line treatment for HER2-positive gastric cancer in combination with other chemotherapy agents. As a protein drug, the phenomenon of protein aggregation is widespread in the process of product fermentation, purification, preparation, storage, and even administration, and the formed polymers may lead to protein inactivity and increase immunogenicity, seriously affecting the quality index and production and application of the protein. Therefore, the study of protein drug polymers is a key quality attribute of the product.

Figure 1. SEC-MALS detection spectrum of Herceptin monoclonal antibody

Figure 2. Superimposed comparison diagram of molecular weight distribution of three batches of subsamples

Case 2: Weight average molecular weight and polymer analysis of S protein SEC-MALS

S protein is the most important target in the study of novel coronavirus. It exists in the form of homologous trimers on the surface of the virus, and its conformation form is very unstable and spontaneously changes. The weight average molecular weight and aggregation state of S protein can be detected by SEC-MALS.

Figure 3. SEC-MALS detection spectrum of S protein

Case study on molecular weight and molecular weight distribution of plasma matrix samples of 3 hydroxyethyl starch

Hyroxyethyl Starch (HES) is a non-ionic starch modification product. At present, it is considered to be the best plasma substitute, and is often used in the medical field as the treatment of hemorrhagic shock and blood thinner to maintain blood colloid osmotic pressure. Because of its unique characteristics and functions, hydroxyethyl starch has become the focus of attention again in recent years. The average weight molecular weight and molecular weight distribution of hydroxyethyl starch plasma matrix samples with different concentrations were detected by Hongren biological products analysis platform. The results showed that the molecular weight and molecular weight distribution of hydroxyethyl starch plasma matrix samples with different concentrations were basically the same.

Figure 4. SEC-MALS spectra of hydroxyethyl starch plasma samples

Case 4 Detection of molecular weight and molecular weight distribution of polysaccharide samples

The main active component of ericium ericium is polysaccharide, which can regulate the immune system by participating in the cellular immune reaction of the body, improve the immune activity of the body, and play an anti-tumor role. The polysaccharide is composed of glucose, galactose and mannose, most of which are glucans composed of main chain connected by β (1→3) bond and branch chain connected by β (1→6) bond. After polysaccharide extraction from hericium hericium extract, the extract polysaccharide components were analyzed and detected. Due to the relatively large weight average molecular weight of hericium hericium extract polysaccharide, there was no suitable polysaccharide standard for molecular weight correction when conventional GPC was used for detection. SEC-MALS was recommended to detect the weight average molecular weight and molecular weight distribution of polysaccharide samples.

Figure 5. SEC-MALS spectra of polysaccharides from hericium ericium extract