Protein analysis

[CSV]

Hello!
I'm about to develop an assay and stability indicating method targeting a hydrophobic protein (20kD). The protein can split into two chains (14 and 6 kD) and the ratio is to be monitored during stability studies.

I have tried RPC (Jupiter C18 150x4.6 mm 5 um 300 Ã…) with AcN gradients but nothing seems to elute (UV 215nm). Not even high concentrations of 2-PrOH works. I suppose a C18 packing may be too hydrophobic or maybe the protein precipitate on the column.
I've also tried AcN gradients on CN-packing but still nothing elutes!

Is there anyone with protein experience out there that can help me?! What other options do I have? Ion-exchange? HIC?
The customer has a rudimentary size-exclusion method with poor separation, but I doubt it will due as a quantitative method.

Thanks!

[HW Mueller]

Did you try pH adjustments, with maybe a max of ~30% organic? What mobile phase do they use for the SEC, what column material?

[CSV]

In the RPC (C18,CN) I used 0.1% formic acid in water (A) and AcN (B).

The size-exclusion column is a TosoHaas TSK-gel G2000SW 600x7.5 mm and the mobile phase comprises of AcN:Water(0.9% NaCl) 40:60 V/V.

[tom jupille]

If the protein(s) are very hydrophobic, then you may indeed be getting irreversible retention on C18 (by the time you get enough ACN in the mobile phase to elute the protein, the solubility is too low).

A shorter-chain column (C3 or C4) might help, but your experience with the CN column (which is effectively around a C3 or C4) suggests continuing problems.

HIC would certainly seem to be an option since you would be working with the native rather than denatured proteins (denatured are often more hydrophobic).

[HW Mueller]

If you want to stick with RP It might indeed help to reduce the organic, but play with the pH (as mentioned) and try salting in (chaotropic salts), rather than salting out (lyotropic salts). Or you may want to try other chaotropes like urea, maybe even without organic solvent. Detergents might be a last resort. (A book on protein purification might be very helpful)

If these are highly purified proteins than SEC with the Tosoh SuperSW columns might be the way to go. There should be no problem in separating those two proteins, you have to keep them solvated, though. You might have seen that I have an ongoing problem with a SW3000 (mw 150 000 proteins), because of irreversible changes after using TFA in mobile phases, alcohols also did strange things (mostly reversible though). I couldn´t find out what the G stands for in the SEC column you mentioned, but it might even be worthwhile to try that one also without org. modifyer, keeping the proteins solvated via pH and/or chaotropes (maybe just PBS, phosphate buffered saline, at pH 7?).
Results could be informative to many people here.

[CSV]

Thanks for the replies!

I'm going to order that SEC column and see what I can do with it.

CSV

[oldtimer]

An article that may help guide your RP HPLC dev is J. Chromatogr. A 1053(2004)299-305. That article details some of the dev parameters for up to 150KD proteins(antibodies) by RP HPLC. We routinely do RP HPLC using 0.1% TFA with ACN gradients for 60KD hydrophobic proteins on C8 and C4 columns; and we've found in many cases that increasing column temperature up to 60C can dramatically improve chromatography from smearing peaks and apparent non-elution to reasonably eluted and profiled peaks. The impact of changing column temp from 40 to 60C had a dramatic impact.

[CSV]

Is it enough to keep the column in 60 C or do I need to preheat the mobile phase?

[oldtimer]

Using an Agilent 1100 HPLC, plumbing allows preheating the mobile phase. We've used Agilents plumbed with and without preheating option, and I've not observed much impact on preheating the eluents. It's probably a case-by-case evaluation depending on such things as column size, critical pairs resolution, etc. Systematically evaluate impact on your application.