Chromatography Forum

Hi every one in the forum, I have problems in quantifying polysorbate 80 in protein formulations. Can any one help me out by suggesting some methods that works fine in quantifying the excipient (polysorbate 80)

Different methods I tried are like:
Hydrolysis by KOH (1M to 0.1M) and differnt temperatures
Result: Good linearity, 0.99 Regression with standards, but high percent variation in protein samples (its 20 to 30%)
SEC (cmc concept): keeping polysorbate 80 in mobile phase, this also fine with standard curve but in protein samples polysorbate 80 peak is coeluting with aggregate peaks
Precipitation of protein by ACN and Cold Acetone and detection by spectrophotometer with bradfords reagent: here also fine with standards, but the samples give interference of proteins(unable to get 100% precipitation)
RP HPLC (mobile phase with 8(organic):2(phosphate buffer) with pH 2.8
Standard curve is fine, but protein samples gives high variation in results
Planning for ELSD and CAD to optimize. If any suggestions regarding the above said methods or any new points please let me know

Here are two methods for polysorbate, the bottom one is used by two pharma companies in US to quantify Polysorbate 80 in protein mixture (they also have two amino acids and sugar, but all of them elute before Tween 80:

http://www.sielc.com/compound_423.html


Vlad Orlovsky
SIELC Technologies

Hi Vlad Orlovsky, thanks for the link

If you have any more suggestions regarding the polysorbate 80 analysis in biopharmaceutical (protein, mAbs etc) by any analytical method. Please let me know

Below is an application for Tween 20 in IgG solution on Cadenza HS-C18:
http://www.imtaktusa.com/site_media/fil ... TI384E.pdf

Cadenza HS-C18 consists of hydrophillic / hydrophobic hybrid ODS technology.
Proteins elute via hydrophillic ligands.

An alternative to Cadenza HS-C18 would be Presto FF-C18.
But, we do not yet have data for this.