Protein cleanup with SAX

[Mattias]

Hi,

I have close to no experience with working with proteins, but I was asked to explore the possibilities of doing online cleanup of a product containing FSH and a preservative (I am not sure if I am allowed to tell which, but it is a small, relatively hydrophobic one). The cleanup would be performed by a column-switch, and the aim was to remove the preservative before a size-exclusion analysis.

My idea was to use a small SAX column and bind the protein at pH 7.4 (which is higher than the isoelectric point of FSH) and then elute the protein to the SEC column with the SEC mobile phase which has a very high salt concentration.

I have tried a Agilent BIO-SAX column, 10*4.6 mm and a 10 mM Trisbuffer at pH 7.4 for loading the protein (0.4 ml/min). But I have not been able to get any retention at all of the protein.

Is this approach possible or should I give up?

(we are using a RAM column for clean-up at the moment, but it gives a lot of band broadening. A combination of concentration and clean-up step would be preferable!)

Thanks!

[tom jupille]

Anion exchange is certainly used to purify FSH, but most of the work that I've seen uses weak exchangers and low-pressure columns (things like DEAE cellulose), which makes sense for prep.

Those prep materials generally have much higher ion exchange capacities than the silica-based HPLC packings, which makes them more retentive, so one possibility would be to look for a higher capacity column, possibly resin-based.

[Mattias]

Thanks Tom!

I will look into some other column material for this "on-off" separation.

However, I doubt that the Agilent column has too low capacity since I am only injecting about 60 IU per injection. Would it improve if I increased the pH further and lowered the ion-strength (from 10 mM)?

I also learned that the product contains a lot of salt, which I assume is like injecting a sample dissolved in acetonitrile on a RP-column...

[DaveM]

I've not had a lot of success with silica-based columns for anion exchange; perhaps it's just the proteins I've been looking at. The most success I've had so far is the Agilent BioMonolithQA, although I have found you need to be incredibly careful about cleaning it, ie. more often than the instructions say, and that's with pure protein, not lysates or anything. I'd like to try a polymeric column some time but have never got round to it.

What does 'a lot of salt' and 'very high salt' mean? Generally I find that anything above 150mM NaCl will cause problems with protein retention (although yes this will change with pH). But it's simple to get around, just dilute the sample before applying it to the column (if you can of course; I once had to give in to a protein that precipitated below 300mM salt). It will still elute in a small volume, even if it goes on in a big one.

As an aside, if very high salt is above 1M and the column says it can't handle it, I've done successful SEC at 1.5M NaCl without damaging the column, I just didn't let the flow stop ant any point and washed the system quickly and thoroughly at the end.

DM

[tom jupille]

Ion exchange capacity is the equivalent to surface area on a reversed-phase column. It affects how much you can load, but it also affects retention, because it determines the phase ratio.

Retention (k') is the product of the analyte's distribution coefficient (which is contolled by the system chemistry) multiplied by the phase ratio. For a given mobile phase ionic strength, therefore, a lower capacity column will give a lower k'.

All of that said, the high salt concentration in your diluent might well be causing the problem. It *is* exactly analogous to a high-organic diluent injected into a reverses-phase system as you suggest.

[Mattias]

Thank you for your replies,

DaveM> The sample is dissolved in a a 0.15 M buffer and the mobile phase that I will use to elute the protein contains 0.2M NaCl.

I have tried to load the protein using a weak buffer as mobile phase (2 mM at pH 7.4) and only injectíng a very small volume. But it doesn't matter how little I inject or the ionstrength of the SAX mobile phase - FSH has no retention on this column. I understand why you had little luck with the silica based columns for this purpose!

I will look into the Amersham range of ion exchange columns, which clearly are designed to work with very large molecules.