Post-translational modifications of protein drugs commonly include glycosylation, N-terminal pyroglutamate, C-terminal lysine truncation, oxidation, deamidation, etc. These different combinations of post-translational modifications can result in a myriad of charge isomers in protein molecules. Moreover, monoclonal antibodies undergo degradation and breakage under specific conditions from low molecular weight fragments. These isomers have an important impact on the stability and biological functions of protein drugs in measuring the quality of biological preparations. Therefore, appropriate methods are required for analysis. Creative Proteomics has invented a comprehensive set of solutions to detect the purity of monoclonal antibodies and protein charge isomers by capillary electrophoresis (CE).
1. CE-SDS for monoclonal antibody purity and size variant detection
CE-SDS protein gel electrophoresis uses liquid gel as the sieving medium. It distinguishes samples of different molecular weights according to the different migration times in the gel so as to detect size variants.
For pretreatment, the monoclonal antibodies are mixed with β-mercaptoethanol or iodoacetamide and then analyzed for purity by reduction and non-reduction. Two analytical method modes are applied: High Resolution (HR) and High Speed (HS) modes. In the HR mode, the distance from the inlet to the detection window is 20.0 cm, while in the HS mode, the distance from the inlet to the detection window is 10.2 cm. Although the resolution of the HS mode is slightly reduced, it results in a faster analysis compared to the HR mode (about 15 min for HS and 30 min for HR).
By reduction treatment, light chain, non-glycosylated heavy chain, and heavy chain of monoclonal antibodies were obtained. In the non-reduction method, light chain, heavy chain, 2 heavy & 1 light chain, non-glycosylated IgG as well as IgG's main body were obtained.
In addition, laser-induced fluorescence (LIF) can be used to detect highly sensitive molecular size isomers of monoclonal antibodies, which is 10 times more sensitive than UV.
2. Detection of isoelectric point and charge isomers
Capillary Isoelectric Focusing (cIEF) and Capillary zone electrophoresis CZE (CZE) can be used to characterize protein charge isomers.
The amphoteric electrolyte filled in and forms a continuous pH gradient under the action of the electric field formed by the acid (anode) solution and the alkaline (cathode) solution so that the amphoteric compounds migrate toward their respective isoelectric points (pI), allowing the peaks of different charge isomers to be obtained. Using the same separation method, CE is able to perform using the cIEF analysis of multiple monoclonal antibodies over a wide pH range.
We use imaging capillary isoelectric focusing (icIEF) via an iCE3 system to provide highly accurate, reproducible charge profiles for proteins, glycoproteins, and peptides.