Tween 20 peak in SEC

[Mattias]

I am analysing a protein by SEC, and the sample contains Tween 20 (polysorbate). The polysorbate gives a disturbing peak just where my protein elutes. I assume that it is the micells that I observe in this high MW region?

Any ideas of how to break these micells (without detroying the protein!)? Or other ideas of how to get rid of the interference? I have tried to use fluorecence detection instead (280/340 nm) but the response was not good enough.

Method uses Tris-buffer pH 7.4, polymer based column, MW protein about 50 kDa.

[Bryan Evans]

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).

[Mattias]

Hi Bryan,

This is an excellent idea! I have the column in-house already so I will give it a go ASAP.

Thanks /Mattias

[Mattias]

As maybe could be expected - the high MW peak from the polysorbate is just passing through the RAM column. So this nice idea did not work.

I am thinking of some kind of labelling of the protein instead. A small molecule should not alter the MW of the protein significantly and make it possible to detect by fluorescence.

The most obvious idea would be to add polysorbate to the mobile phase - but the column costs $3000 so I am a bit reluctant.

[Bryan Evans]

Hi Mattias -

Did you try this on Cadenza HS-C18?

[Mattias]

Yes, I connected a Cadenza HS-C18 (20*3 mm) just before the SEC column. I hoped that it would collect the polysorbate, which it probably does. But the high MW peak I get from injecting pure polysorbate solution (50 µg/ml) was still there at same size. I assume that this is the micells of polysorbate that I see - and they are also big enough to pass through Cadenza without any retention.

[Bryan Evans]

Some thoughts:

If the surfactant is aggregating into large miscelles:
- increasing column temperature / buffer concentration
could help to disperse the surfactant.

Not sure what effect this has on the activity of the protein

If it's overload:

- the data shows a 50x3 column (5uL injection) with ~0.5ug Tween 20
on column. Maybe a 2uL inj. is required for 20x3 column.

[HW Mueller]

It could be that memory is playing tricks, but aren´t there some commercial solutions to get rid of all kinds of detergents? Did I see this in Pierce? Calbiochem?

[danko]

I must admit: I’d rather optimize various conditions than add a second column in addition to the analytical one (the SEC).
Mattias, are there any aromatic amino acids in your protein? It’s almost difficult to imagine this large protein without aromatic AAs. For instance, I have a protein containing a single tryptophan and its fluorescence is huge. But one has to determine the excitation and the emission spectra accurately and adjust the detector in accordance to their maxima. I’m sure the excitation wavelength for many proteins is 280 nm, but did you confirm that by actual scan? Even more important is the emission wavelength it varies tremendously with pH, temperature and other conditions in the analyte’s environment.
You don’t describe your mobile phase, so it’s difficult to point at a potential optimization possibility but anyway here are some suggestions you might like to examine closer:

1. Determine the working wavelengths by scanning the spectra and make sure the solute (protein) is dissolved in mobile phase.
2. Degas the mobile phase by sparkling with He, because oxygen quenches fluorescence.
3. If your mobile phase is purely aqueous, try and add ca. 10% isopropanol. It should facilitate the fluorescence and maybe it could disperse the micelles – if that’s what you’re dealing with.
4. Finally, make sure the temperature isn’t too high. High temperature is another known florescence quenching factor.

I’m assuming you’ve chosen the right SEC column for the protein you’re dealing with (i.e. it’s not excluded together with these potential micelles etc.) If it shows that your protein is excluded, then you’ll need to find a column with larger pores and then you wouldn’t even need the fluorescence detector.

Good luck with your troubleshooting/optimization.

Best Regards

[vickig]

Mattias,

Did you solve your Tween/protein problem? We are have same problem w/ Tween 80. Any suggestions?

[ScottHorn]

Initially I'd try disrupting the micelles, if that is indeed what is causing the problem. You could raise the critical micelle concentration of the liquid by increasing salt concentration in your buffer. Also, diluting the sample before injection would lower the tween concentration and disrupt the micelles. If that fails, maybe micelles aren't the problem. I would then try a different column, that could better distinguish between the protein and whatever else is in there.

[vickig]

Thanks ScottHorn, however everything we have tried to disrupt the micelles has not worked. I believe the interference is due to micelles given the elution position. Adding Tween to the Mobile phase works somewhat, but prodcues a negative peak in the area of the aggregates we are trying to detect. I don't think an alternate column will help...remember that we are talking SEC here, so the options are limited, and the micelle peak is approx the same size as the aggregate.

[Uwe Neue]

How about retaining the protein on a suitable ion exchanger, where the Tween 80 will wash through, followed by elution with salt and subsequent SEC?

Alternatively, selective detection would work. Danko has proposed fluorescence. Also, a post-column derivatization that is selective for the protein will do. The classical ninhydrin reaction will do just fine.

[hplcuser]

Sepax SEC Columns: http://www.sepax-tech.com/SEC.php

The tween doesn't greatly affect the column life and there is data that can send you proving that.

[Ting Wu]

What is the concentration of the tween 20 in your sample?

Check this http://www.sepax-tech.com/application_n ... ween20.pdf