Tween 80 (also known as Polysorbate 80) is used in the manufacture of protein solution formulations to help solubilize and stabilize the protein. It is one of a series of materials (including Tween 20, 40 and 60) which are fatty acid esters of sorbitan polyethoxylates. The various Tweens differ in the type of fatty acid present; Tween 80 is an oleate.

Tween 80 is added into the formulation at relatively high concentrations (~0.1%), and then removed later in the manufacturing process. WCAS has developed an LC-MS method to determine the residual Tween 80 concentration down to approximately one part per million.

Testing for Tween 80 

The LC separation yields a series of overlapping peaks, since Tween 80 is a mixture of isomers and congeners. Ionization is accomplished using electrospray, which gives multiply charged ions. By selecting a group of ions which correspond to a narrow band of congeners, a single chromatographic peak is obtained from the complicated Total Ion Current chromatogram (see chromatogram below).

For samples with high levels of Tween 80 (~0.05% or higher) or low salt concentrations, the samples are diluted into water and analyzed directly. For lower concentration samples, solid-phase extraction is used to separate the Tween 80 from the protein and buffer salts, which can adversely affect electrospray ionization. In either case, standards are prepared in the same manner as the samples, which 

accounts for any potential losses due to the preparation. This enables determination of Tween 80 from samples which originally contained high concentrations (>100 mM) of buffer salts.

We have seen some evidence that Tween 80 from different sources can yield somewhat varying results. For that reason, it is recommended that a sample of the Tween actually used in the manufacturing process be supplied with any samples submitted for analysis. The method currently in place is applicable only to Tween 80 in the absence of other Tweens; we are investigating approaches for samples which may contain more than one.

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Formerly West Coast Analytical Service (WCAS) and Bodycote Testing Group