Hello!

Is your Protein tagged? If not, you could introduce a His- or Strep-Tag at one End of your chain, where it doesn't interfere with function or folding. You could also introduce a cleavage site to enzymatically remove the Tag, for example using TEV-Protease. Using immobilized Protease is of course the easiest option, otherwise you'd have to remove it afterwards.

If you cannot introduce a tag for whatever reason, one option is to optimize your separating conditions (like increasing the salt concentration in the buffer to reduce non-specific interactions). Additionally, you could follow up your SEC with an additional chromatography step, e.g. Anion- or Cation Exchange. If your protein has any sugar residues, an additional affinity column is also an option.

I am assuming your protein will not survive a reverse-phase chromatography, otherwise this is a good option as well.

In conclusion: Introduce a Tag, if possible. Either way, introducing a third separation step will hopefully increase your purity, although you will likely need to screen different methods and/or optimize the ones available to you.

Good luck!