RP of large proteins

[dorunrun]

A little help would be appreciated. I am trying to develop a separation for an acidic protein that is 60-70 kD. It is produced in a cell line and during fermentation the product is sometimes pyruvulated. This is what I'm trying to separate. I have a decent method but the pyruvalated form elutes as a backside peak and consequently I could be over or under estimating the impurity. The current method specifics are as follows
C4 column
A: 0.1% TFA in water
B: 0.08%TFA in ACN
gradient 30-45% in 20 Minutes
90% flush and then re-equilibration.

I have tried numerous other modifiers (HFBA, TEA, TBA, PO4,), different pH's (higher pH loses all peak shape), different columns (C8, CN, phenyl), different gradient conditions, longer, shorter etc and different MP.

I'm at the point where I'm ready to give up and try IEX.

Any ideas form anyone who has used RP with proteins.
Thanks

[SIELC_Tech]

If you give me your email I can send your a brochure for separation of proteins by Promix mixed mode columns. With Promix columns you have at least two mechanisms to separate your proteins : reverse phase and ion-exchange. Check application for insulins:

http://hplcmethods.com/compound_212.php

We believe that this is first separation of humalog and humulin (without ion-pairing reagent).

We can do a free screening for your sample on Promix columns.

Contact us if you need more information.

Regards,

Vlad

[Koen Hollebekkers]

I would always advise to try both modes, RP and IEX. If one of the modes is not sufficient enough, try 2-D LC.
I have no experience with mixed mode analytical columns but in my opinion the peak capacity is a big issue when separating cell lysates.
I have no experience with your method and have no suggestions for your method.

Good luck

[HW Mueller]

How about methanol or the propanols with different phosphate buffers?