Tween 20 peak in SEC

[amirtamboli]

Hi Mattias

Below is an application for Tween 20 in IgG aqueous solution
using Cadenza HS-C18:

http://www.silvertonesciences.com/files/TI384E.pdf

In the chromatogram - Tween 20 detergent is retained while the
IgG antibody is excluded.

Perhaps it is possible to follow similar experimental condtions
and collect the 50kDa protein at the void volume (free of Tween 20).

Dear Mr Evans
Can you send me the above application, mentioned in link as it is not opening and i am in need of tween 20 application

[Vlad Orlovsky]

Brian is no longer with the company as far as I know.

[amirtamboli]

can you please solve my problem

[prepcolumn]

can you please solve my problem

amirtamboli

here is the application

http://www.imtaktusa.com/site_media/fil ... TI384E.pdf

[amirtamboli]

can you please solve my problem

amirtamboli

here is the application

http://www.imtaktusa.com/site_media/fil ... TI384E.pdf

suggested one already tried with different column and shown good result but it has tailing

[biochemist]

Hi Amir,

You could try incorporating 25% IPA in your mobile phase, then using fluorescence detection.

Development of a sensitive size exclusion HPLC method with fluorescence detection for the quantitation of recombinant human erythropoietin (r-HuEPO) aggregates
http://www.sciencedirect.com/science/ar ... 8506004092

The method is for Polysorbate 80, but do let us know if it works for Polysorbate 20 as well.

[Mattias]

Being the thread starter, maybe I should tell how it went...

We gave up and introduced a placebo solution in the method. So now we subtact the peak area of the tween peak in the placebo sample from the aggregates peak area in the sample.

Not the best solution, but we ran out of time. It is never too late to give up.

[biochemist]

Mattias,

Congratulations on solving your problem! Did you face any problems during the validation?

I'm curious - does the Tween 20 elute in a consistent way (size, shape and retention time)? We tended to get wildly different results for a placebo, depending on the Tween 20 concentration - sometimes it would be well-behaved, and sometimes not. What column are you using?

[Mattias]

The Tween 20 peak is fairly consistent in placebo samples, but the precision is pretty bad anyhow. The Tween peak is about 50% of the aggregate peak area in stability samples and close to 100% of the peak area in newly produced batches.

The validation was more focused on using the method as a limit test for aggregates, rather than calculating the precision at different concentrations. It was acceptable, but not fantastic. The column is a TSK column.