Polysorbate 80 (PS80) is a non-ionic surfactant commonly used in biopharmaceutical products to improve protein stability and reduce aggregation. Quantification of PS80 in immunoglobulin G (IgG) formulations is analytically challenging due to the combination of low surfactant concentration and high protein content. At Octapharma AB, PS80 in subcutaneous immunoglobulin (SCIg) is currently quantified using a colorimetric method that involves several time-consuming preparation steps and the use of hazardous chemicals. Therefore, the aim of this study was to develop and evaluate an alternative spectrophotometric method for quantification of PS80 in SCIg. 

To reduce the protein interference, a sample preparation workflow consisting of ethanol precipitation, sodium chloride addition and ultrafiltration using 30kDa centrifugal filters was developed. PS80 was quantified spectrophotometrically at 234 nm using several calculation approaches, including external calibration, one-point calibration, absorbance coefficient, standard addition and matrix-matched calibration. The sample preparation workflow enabled more efficient protein removal where protein concentration decreased by 99.99%. External calibration showed high linearity within the investigated concentration range, although recovery experiments indicated remaining matrix effects. Among the evaluated approaches, matrix-matched calibration showed the best recovery. 

Compared to the existing colorimetric method, the developed spectrophotometric workflow reduced analysis time and avoided the use of hazardous solvents. Although additional optimization and validation would still be required before possible routine implementation, the results indicate that spectrophotometric quantification of PS80 in SCIg may be possible when combined with sufficient protein removal and appropriate calibration strategy.