Protein A chromatography

Bruce Coryell brucec at brucec.mv.com
Wed Dec 13 03:33:13 EST 2000


Thanks for this eminently helpful post!

TPitarresi wrote:

> Hi Bruce,
>
> Try 2-4 M urea at neutral pH (filter with a .2 um filter, you don't want your
> cleaning solution to contain particulates) .  You can try 2 M Guanidine HCl as
> well.  If possible reverse the flow on the cartridge. 5 bed volumes usually
> does the job. Urea targets ppt proteins, it should have little effect on the
> binding activity of Protein A.  Watch your flow rates and pressure.  Urea will
> generate more pressure than most aqueous buffers.
>
> If the perseptive cartidge can handle alcohol, you can also try 20%-70%
> ethanol.  Don't jump straight to 70%, you run the risk of forming bubles in the
> cartridge due to outgasing.  Ethanol should target lipids and other hydrophobic
> contaminants.  Again 5 bed volumes is usually enough.  Like the urea ethanol is
> more viscous than most buffers, watch your flow rates and pressure.  Protein A
> should be stable with an ethanol wash.
>
> You can also try a warm detergent wash. 1% detergent solution in water warmed
> to 37%.  You will need extensive washing (15-20 bed volumes) to remove all the
> detergent. -- watch SDS it can reduce the binding capactity of Protein A over
> time.
>
> Try a very low pH wash-- Protein A is stable for short periods of time at pH 2.
>  A You could use glycine HCL.  Be careful-- Protein A will lose activity if
> left for an extend period.  Also--watch out for the base matrix.  You need to
> ask perseptive if it is stable to acid.
>
> regards,
> Tina
> Amersham Pharmacia Biotech






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