A small amount of surfactant may be added to antibody drugs for the purpose of preventing aggregation or adsorption of IgG and surfactant concentration control is important in quality control. Polysorbate 80 (Tween 80) in a commercial antibody drug was measured by Evaporative light sacttering (ELS) detection using the column for polymer reverse phase chromatography ODP2 HP-4D without pretreatment.
Under alkaline conditions, IgG is not retained on the column but eluted together in a hold-up volume, and polysorbate 80 is retained on the column in reverse phase mode, allowing polysorbate 80 to completely separate from IgG and can be analyzed without pretreatment (deproteinization). A calibration curve was prepared from peak area values ​​of 4.3 to 5.5 min (double-logarithmic plot) and showed good linearity. Detection of 10 μg/mL was possible and the recovery rate of 50 μg/mL was as good as 93%. In this method, it is possible to analyze polysorbate 80 quickly and efficiently by subjecting the antibody drug to HPLC with only dilution.

Sample: 50 µL
Commercial antibody-drug formulation
14-fold dilution with H₂O