Problem: Purity and charge isoform analysis of therapeutic proteins by slab gel is labor intensive, time consuming and only semi quantitative. The complexity of biologics requires robust analyses in biopharmaceutical development and quality control laboratories. Many laboratories continue to apply manual technologies to address questions regarding protein purity, heterogeneity and identity. These techniques, like SDS-PAGE and isoelectric focusing, often lack the reproducibility, quantitation and precision necessary to obtain accurate answers. As more therapeutic proteins enter the market, an even larger number accumulate in biopharmaceutical drug pipelines. In order to improve assay quality and manage the growing workload, automated systems are needed to provide qualitative and quantitative information.

cIEF separation of three different therapeutic MAb molecules using advanced cIEF technology

Solution: Over the last six years, capillary electrophoresis (CE) technology has been replacing SDS-PAGE for purity analysis. CE-savvy analysts have proven the value of this technology. As CE adoption increases, there is a growing need for systems that general analysts can routinely operate for extended periods, with minimal training. Recently, in collaboration with a number of biopharmaceutical companies, Beckman Coulter launched a new CE platform: the PA 800 plus Pharmaceutical Analysis System. This robust and easy-to-use characterization platform performs quantitative, qualitative and automated methods for protein purity by SDS-gel analysis, charge isoform distribution by capillary isoelectric focusing (cIEF) and glycan analysis by CZE.

The analyst simply selects the method and types in the number of samples at hand. The software instantly calculates the sequence table and the amount of reagent vials needed and provides a visual diagram of their location in the vial trays. After placing these in the PA 800 plus system, the instrument is up and running with a single mouse-click. The instrument status is visible from across the lab, making it easy for the analyst to run multiple systems or perform other tasks. There is a noticeable change of color on the instrument’s large screen signals when the assays are completed, and advanced reports are generated.

Of particular importance is the optimization scientists have achieved with cIEF over the last two years. Charge heterogeneity characterization by cIEF now utilizes high-concentration phosphoric acid and sodium hydroxide as anolyte and catholyte respectively, as well as acetic acid as chemical mobilizer. This combination increases the resolution and eliminates the cathodic drift of the pH gradient, which is a common problem in IEF. The use of iminodiacetic acid and arginine as anodic and cathodic stabilizers protects gradient linearity and stability from isotachophoretic decay, and yields extremely reproducible separations. New proprietary synthetic peptide pI markers do not have tertiary or quaternary structures or PTMs, adding precision and accuracy to pI estimation. As an example, an intermediate precision study in which three therapeutic monoclonal antibodies were separated by cIEF achieved RSD for pI of less than 0.1% (n=25) and RSD of isoform group area composition lower than 3%.

In all, this new platform significantly increases operational efficiency for therapeutic protein characterization assays in development and quality control. The robust methods minimize user-related variations facilitating portability in today’s multi-user, multiinstrument and multi-facility biopharmaceutical enterprise.

For more information, go to www.beckman.com