CE Infinite Mass Spectrometry Direct Coupled Full Column Imaging Isoelectric Focusing iCIEF

CE Infinite Mass Spectrometry Direct Coupled Full Column Imaging Isoelectric Focusing iCIEF

CE Infinite mass spectrometry direct coupled full column imaging with isoelectric focusing iCIEF

CEInfinite iCIEF-MS Online coulping platform

In the development and production of protein bioproducts, post-translational modifications are necessaryPTMs and degradation can generate charges on the target proteinheterogeneousBody. biopharmaceuticalcompanyRelying on capillary isoelectric focusingQuantify charge isomers using CIEF and imaging CIEF (iCIEF). Characterization of bioelectric charge isomers is useful for many life cycle stages, including cell line selection, stability studies, formulation, andprocessControl, but this characterization requires separating the protein into individual charges with sufficient purityDistillates of isomersUsed for downstream analysis.

In addition, the direct coupling of iCIEF with the mass spectrometer (MS) used for characterization will significantly simplify the characterization process.

AES has developed the CENimite prepared iCIEF system and a proprietary full column imaging detection (WCID) separation column for the separation of iCIEF WCID and online coupling with MS, allowing real-time observation of the separation focusing (iCIEF) dynamics and subsequent migration of charge isomer peaks. During the pressure transfer process, the electric field keeps the sample in the separation capillary focused. The inner diameter of the separation capillary is larger, while the inner diameter of the transport capillary is smaller. Therefore, this difference in inner diameter greatly reduces the diffusion and remixing of separated protein isomers upon leaving the column.

Details

Highlights of CENiteiCIEF-MS

• Directly combine iCIEF-MS with traditional low flow or high-sensitivity nanoflow ESI from Thermo Fisher Scientific, Waters, and Bruker.

• Any buffer or solution can be used as a supplement. It does not involve chemical migration. Protein charge variants are introduced into MS sequentially based on their pI.
  

• CE-MS can be directly converted from any existing iCIEF or CIEF method. Good separation can be achieved at both low and high sample concentrations.

ICIEF MS analysis of NIST mAb samples

ICIEF UV (red) and iCIEF MS (blue) clearly observe the profiles of charge isomers and obtain excellent MS spectra of all variant peaks, which allows us to deconvolve most complete NIST mAb charge variants.

The results indicate that the alkaline peaks B1 and B2 are mainly caused by the isomerization of C-terminal lysine, while the molecular weight difference between the acidic peak A1 and the main peak is small, which may be due to modifications such as deamidation. Further research and confirmation are needed through the preparation of iCIEF.

Customer feedback:

“ICIEF plays a crucial role in monitoring charge changes during the development of biopharmaceuticals. CE Infinite allows us to obtain quality readings of the product variants detected by iCIEF, which is of great help in understanding the chemical properties of charge variants. When CEInfinite is combined with the high-resolution and precise mass spectrometry provided by the Orbitrap MS system, a detailed understanding of product variants and product stability can be obtained.

In short, the use of iCIEF MS with CEInite is a very powerful characterization tool in biopharmaceutical development. "

-Dr. Dan Bach Kristensen

Symphogen Chief Scientist