VALIDATION OF AN HPLC METHOD FOR THE ANALYSIS OF THE CHARGE HETEROGENEITY OF THE RECOMBINANT MONOCLONAL-ANTIBODY IDEC-C2B8 AFTER PAPAIN DIGESTION

An HPLC procedure was validated for determining the purity with respec t to the charge variant distribution of the recombinant monoclonal ant ibody (MAb) IDEC-C2B8 by high-performance ion-exchange chromatography. Papain was used to fragment the molecule into Fab and Fc fragments pr ior to chromatographic analysis. Fragmentation allowed the resolution of the variants arising from the cyclization of glutamine to pyrogluta mate Bt the amino-terminus of the light and heavy chains (Fab-pE/Q var iants) from the variants resulting from the processing of the carboxy- terminal lysine residues of the heavy chains (Fc-Lys variants). The as say demonstrated good linearity, yielding correlation coefficients of >0.99 for total protein, Fc-Lys variants and Fab-pE/Q variants. Recove ry of total protein from the column was 95.7%. Sample recovery studies demonstrated a mean accuracy of 102% for a Fab fragment over the rang e 2-10% of the total protein. The limit of detection was 0.2 mu g and 0.1 mu g for Fc and Fab variants, respectively, The repeatability of t he assay and intermediate precision had relative standard deviation (R SD) values of <1%. Parameters of the papain digest (time, digest stabi lity, reagent stability, pH and papain vendor) and of the chromatograp hy (mobile phase pH, stability, buffer concentration, and column lot a nd aging) were evaluated for robustness and determined to be acceptabl e. Data are presented demonstrating the suitability of the assay for d etermining the product purity of a recombinant MAb. Published by Elsev ier Science B.V.