RP method development for pegylated protein

[Rocnex1]

Hi All,

I am trying an RP method for a pegylated protein, MW around 27 KDa, PI round 7.

I am using an Agilent Zorbax 300SB-C3 column (3.0x450mm, 3.5 um) with a cuard column (same packing material, 4.6x12.5 mm). MP is A) 0.1% TFA in water B) 0.1% TFA in ACN. Flow rate 0.8 ml/min, column temperature @60 degrees C.

First I tried a gradient from 5% B to 100% B in 20 minutes to see where my protein will come out, Picture 1 below. There was a pretty big tail. When I tried injecting pure water or the buffer in which the protein was dissolved, saw a bunch of junk peaks aroud the time the protein came out.

If I want to optimize the method, let's say, remove the tail, which way should I go, manipulating the gradient, using different ion-pairing agent, different solvent, different column?

Please advise. Thanks a lot!

Regards,

Rocnex1

Protein


Water
[/img]

Buffer

[tom jupille]

You have two separate problems here: the tail and the junk. Let's address the junk issue first.

The peak profiles from your buffer and water look qualitatively similar, so I would guess that the junk is actually coming from your A mobile phase. To check, run a series of dummy gradients (no injection at all) with different pre-gradient equilibration times and see if the size of the junk peaks correlates with the equilibration time. There was a related thread a few weeks ago that will show you what I mean:
http://www.sepsci.com/chromforum/viewtopic.php?t=3951
If the size of the peaks does track the equilibration time, you have to find out where the junk is coming from. Possibilities include the water, the TFA, the ACN, etc. etc.

Now to address the tailing problem. Don't lose sight of the fact that that the tail may be "real" (i.e. you have distribution of molecules with different degrees of PEGylation, and what you are seeing is the "envelope" encompassing that distribution).

Assuming that you in fact have a single "molecular species", the first thing to check is simple overload. Try injecting only 1/10 the amount of protein (this is why you have to get the junk peaks out of the way first!). If the tailing improves, and especially if the retention time increases, then you were simply above the linear range of your system.

If the tailing does not improve, then the next thing would be to try a different column (same general type; there are lots of them out there!).

[ccyting]

I don't have any experience on HPLC of PEG-protein. I can imagine with 27,000 D molecular weight, your peak shape is not too bad at all. I will not run PEG-protein with 60C column temperature. It may create PEG stability problem inside the column. YMC (now Waters) has a size exclusion column for protein separation and will give a very sharp peak with aqueous mobile phase. I really don't know the junk peaks. With todays reagents quality, we should not see those peaks at all. How about your injection volume? As Tom mentioned, try different volume injection to trouble shoot the problem. Good luck.