Mobile phase containing arginine provides more reliable SEC condition for aggregation analysis

The critical role of mobile phase composition in size exclusion chromatography of protein pharmaceuticals

“…Greater protein recovery with arginine was clearly shown in a study using new resin by Yumioka et al34 A new column was packed with TSK G3000SWXL resin from the same lot that was used for the data shown as squares in Figure 3 and then equilibrated with 0.1 M phosphate, pH 6.8 containing 0.2 M arginine (instead of 0.2 M NaCl). As shown by the black triangles in Figure 3, with arginine the first injection showed a significantly higher aggregate content of 3.5% rather than 2.7% for the first injection in 0.2 M NaCl.…”

“…Since the monomer peak area was relatively independent of the injection order or the cosolvent used (arginine vs. NaCl), it is evident that recovery of aggregates is greater in 0.2 M arginine than in 0.2 M NaCl. Other amino acids (glycine, lysine, histidine, and glutamic acid), Na 2 SO 4 and ethanol were not effective in enhancing the recovery 34…”

A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates

“…Numerous studies have evaluated the addition of of organic modifiers or other additives, such as arginine [11,105] to mitigate these secondary interactions. These additives are often used to aid in protein recovery.…”

“…The reduced recovery of aggregates in SEC chromatography is an area of wide concern. [14,105] One strategy often used is the addition of arginine to the mobile phase to reduce secondary interactions. [11,101] Arginine acts as a binder to the analyte in solution, thus preventing it from interacting with the stationary phase.…”

Strategies for the Assessment of Protein Aggregates in Pharmaceutical Biotech Product Development

“…For instance, low solution ionic strength (e.g., 50 mM or less) may encourage hydrophobic interactions of the eluting protein with the column matrix, thereby slowing elution, affecting resolution and peak shape. Addition of arginine to the eluent may inhibit interaction between solute and column matrix (24). Detergents in the sample (as opposed to the eluent), though nominally of small molecular weight, can behave as large molecules if they form micelles (above their critical micelle concentration), appearing in the chromatogram as UV-absorbing peaks (25) and potentially also giving rise to light scattering and fluorescence signals.…”