Creative Biolabs is one of the world’s most trusted experts to provide independent antibody analysis. Based on our vast experience gained from analyzing hundreds of antibodies, we offer high quality molecular weight/mass measurement for Bispecific Antibody (BsAbs) to meet customers’ requirements. To measure the molecular weight/mass of BsAbs, we developed two major approaches: SEC multi-angle light scattering (SEC-MALS) analysis and Electrospray Ionization (ESI) based analysis.
Size-exclusion chromatography multi-angle light scattering (SEC-MALS) is a powerful technique to measure the mass of a macromolecule. SEC separates molecules by their sizes, and in some cases by molecular weights. However, it relies on similarity to a set of reference standards for accurate mass determination, and fails for special proteins. MALS takes advantage of the intensity and the angular dependence of the scattered light to determine absolute molar mass and size of the molecule in solution. SEC-MALS combines SEC with MALS, and therefore can measure the absolute molecular weight and root-mean-square (RMS) radius (refers to radius of gyration, Rg) simultaneously. Compared with SEC or MALS alone, SEC-MALS gives more accurate mass measurements. Besides, it is a useful approach to characterize proteins size and shape in native solution conditions.
Figure 1. (A) The equipment used for the SEC-MALS analysis is shown. (B) The SEC-MALS analyses for the core S-antibody complexes are shown (Louis, J. M., 2014).
Mass spectrometry (MS) is a powerful technique that offers both structure and molecular mass or concentration information of molecules after their conversion to ions. In the past few years, ESI-MS has arisen as a crucial technique in clinical laboratories. ESI-MS uses a “soft” ionization technique which is widely used in biological mass spectrometry. This technique allows the ionization of large biological molecules (such as proteins) without them being fragmented, and thus they can be measured in native forms. In addition, ESI-MS serves a sensitive and reliable tool for measuring biomolecules that are unable to be determined by other conventional techniques.
Figure 2. The schematic diagram of electrospray ionization mass spectrometry (Liebisch, G. 2016).
Electrospray ionization time-of-flight (ESI-TOF) is a type of MS, in which the molecule to be measured is ionized by forcing the sample solution via a small heated capillary into an electric field to generate a very fine mist of charged droplets. Afterwards, sample ions with different masses are accelerated to the same kinetic energy and the time taken by each ion to reach a detector at a known distance is detected. This time is based on the mass-to-charge ratio of the ion, and the mass of the molecular ions in the sample is confirmed.
With our well-established BsAbs analysis platform, the experienced scientists at Creative Biolabs are dedicated to helping you develop BsAbs. Our high quality molecular weight/mass measurement services will greatly contribute to the successes of your projects. Creative Biolabs also provides various other services related to BsAbs development. Please feel free to contact us for more information and a detailed quote.
References
1. Louis, J. M.; et al. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations. PloS one. 2013, 9(8): e104683.
2. Liebisch, G.; et al. High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Biochim Biophys Acta. 2006, 1761(1): 121-128.
3. Yu, Y.; et al. SEC-MALS method for the determination of long-chain branching and long-chain branching distribution in polyethylene.” Polymer. 2005, 46(14): 5165-5182.
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