Optimization of a DEAE ion-exchange workflow for a porcine brain-derived vimentin-containing preparation

Proteins of similar molecular mass can co-extract, co-elute, and co-migrate during SDS-PAGE, complicating assessment of tissue-derived vimentin preparations. We optimized a self-packed XK16/20 diethylaminoethyl fast-flow (DEAE-FF) workflow for obtaining a porcine brain-derived preparation containing vimentin and added orthogonal identity, composition, and quality-control analyses. Five process variables were evaluated by single-factor experiments and an L16(4^5) design using an equal-weight composite of SDS-PAGE target-band proportion and apparent target-band recovery. Column packing was assessed with an acetone UV254 tracer. The preparation was characterized by bicinchoninic acid assay, SDS-PAGE, anti-vimentin immunoblotting, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and a gel-clot Limulus amebocyte lysate limit test. Trial 9 (pH 7.0, 1:5 solid-to-liquid ratio, 30 min ultrasonication at 430 W, and 1.0 M NaCl in the full-strength elution buffer) produced the highest composite score. The acetone tracer yielded a peak at 12.4 min with an asymmetry factor of 0.944, and the 100% elution step had the highest protein concentration among the evaluated eluates. Three verification batches showed an SDS-PAGE target-band proportion of 89.76 ± 1.82%. LC-MS/MS of the approximately 53-57 kDa gel region identified porcine vimentin (P02543; 33% sequence coverage, 15 peptides, 10 unique peptides, and 18 peptide-spectrum matches) together with multiple co-purifying cytoskeletal protein records. Endotoxin was below assay-derived upper bounds of 0.03 and 0.125 EU/mg. The workflow produced a compositionally heterogeneous, vimentin-containing preparation rather than homogeneous vimentin.