Evaluation of the iCE280 Analyzer as a potential high-throughput tool for formulation development
Ning Li1, Kendall Kessler, Laura Bass
1Department of Pharmaceutical Research and Development, Pfizer Global Biologics, St. Louis Laboratory, Pfizer Inc., St. Louis, MO 63017, USA. ning.x.li@pfizer.com
Journal of Pharmaceutical and Biomedical Analysis
|October 19, 2006
Summary
The iCE280 Analyzer effectively separates protein charge variants, offering high-throughput analysis for glycosylated and non-glycosylated therapeutics. While pegylated proteins remain challenging, the system demonstrates excellent linearity and precision for IgG charge variant determination.
Area of Science:
- Biopharmaceutical analysis
- Protein characterization
- Analytical chemistry
Background:
- Traditional capillary isoelectric focusing (cIEF) methods can be time-consuming.
- High-throughput analysis is crucial for efficient biopharmaceutical development.
- Accurate separation of protein charge variants is essential for quality control.
Purpose of the Study:
- To evaluate the iCE280 Analyzer as a high-throughput tool for determining isoelectric point (pI) and separating charge variants.
- To assess the performance of the iCE280 Analyzer using glycosylated, non-glycosylated, and pegylated protein therapeutics.
- To identify the sources of charge variants in protein therapeutics.
Main Methods:
- Isoelectric focusing (IEF) using the iCE280 Analyzer.
- Model proteins included glycosylated, non-glycosylated, and pegylated therapeutic proteins.
- Assay performance was evaluated for linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, reproducibility, and robustness.
Main Results:
- The iCE280 Analyzer achieved resolution for glycosylated and non-glycosylated proteins, but challenged with pegylated proteins.
- Charge variants were attributed to C-terminal lysine residues, sialic acid content, and deamidation.
- Excellent linearity (R² > 0.984) was observed for IgG species across a concentration range of 2-333 µg/ml.
- LOD and LOQ were determined to be 3 and 11 µg/ml, respectively.
- High precision and reproducibility were demonstrated with R.S.D. ≤ 0.2% for pI values and ≤ 1.4% for main peak area distribution.
- Robustness testing showed minimal impact from ±50% pharmalyte concentration deviation.
- The iCE280 Analyzer eliminated the mobilization step, increasing analytical throughput at least 2-fold.
Conclusions:
- The iCE280 Analyzer is a promising high-throughput tool for pI determination and charge variant separation of certain protein therapeutics.
- The method demonstrates robust performance, linearity, and precision suitable for biopharmaceutical quality control.
- Further optimization may be needed to fully resolve pegylated protein species.
