Protein A chromatography

Anthea Scothern anthea.scothern at umist.ac.uk
Wed Dec 13 04:37:21 EST 2000


"Bruce Coryell" <brucec at brucec.mv.com> wrote in message
news:3A353592.85E9D9EC at brucec.mv.com...
> Does anyone have experience with cleaning and regenerating a Perseptive
> Biosystems Protein A cartridge.  I  have a Protein A cartridge that is
> badly fouled and generating high back pressures, but no money in budget
> for another one.   Any  hope?  Obviously any cleaning method would need
> to not denature or cleave the Protein A ligands...

Hi Bruce,
I assume that you are using Poros resin.  I don't use Protein A resin, but
the cleaning & regeneration guidelines for the cation, anion, heparin and
metal chelate resins are all fairly similar.  So from my Poros HS operating
instructions:

Chemical Resistance
pH 1-14 (up to 0.5M NaOH, 1M HCl)
Ionic strength: 0-5M all common salts
Buffer additives:  all common agents including 8M urea, 6M guanidine/HCl,
ethylene glycol, and detergents.  Cationic detergents not recommended as
they can bind.
Do not expose to strong oxidisers such as hypochlorite/nitric acid or strong
reducing agents such as sulphite.
Solvents: water, 0-100% alchohols, acetonitrile, other common organic
solvents.

Cleaning & Regenerating
Simple: 1-5 column volumes of 1-2M salt
Complex:
1-5 column volumes of 1M NaCl/1M NaOH followed by
1-5 column volumes of 1M acetic or hydrochloric acid followed by
water then starting buffer to equilibrate
To remove lipids & lipoproteins:
50% methanol, isopropanol or acetonitrile with the acid or base

It recommends that you inject viscous or aggressive solvents directly,
making the volume as large as possible and using a low flow rate.
Some of these may be too aggressive for Protein A - go carefully!  I use
self-pack columns. An engineer told me that if normal cleaning didn't work,
then I could unpack the column into a 50ml falcon tube and leave overnight
on a roller with the cleaning solution, then spin down, resuspend in packing
solvent and repack the column.  I've done this for several of my columns and
it works OK.  Check if you have a packing device before you unpack your
column though!
You can also get high pressure if the inlet frits inside the column are
blocked - try replacing those if the cleaning doesn't help.
My experience with these resins is that they block very easily - we filter
all of our buffers, water, acids, ethanol before use and we NEVER put cell
extracts over the column!  We precipitate the protein with ammonium sulphate
and then re-dissolve the pellet and de-particulate that by centrifugation.

Hope some of this helps,

Anthea Scothern

Posttranscriptional Control Group,
Department of Biomolecular Science,
UMIST,
Manchester,
UK







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