I use manual columns, not FPLC.
On 8/9/21, 9:23 AM, "Antibody Discussion Forum on behalf of ABRF - Frances
Weis-Garcia" <email obscured> on behalf of <email obscured>>
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Thanks Laura.
Trying to get away from the FPLC ...
I'll ask some "old time" purification people who may be some "old school"
methods.
On 8/9/21, 9:43 AM, "Antibody Discussion Forum on behalf of Bover,Laura"
<email obscured> on behalf of <email obscured>> wrote:
MabselectSure (protein A based Column). We don’t see differences and in
fact protein A works better for all Ig (in spite what literature says). The tip
was giving to me by the company that made humanization of our Star antibody:
that use mabselect for all
Laura Bover, PhD
(working from home during COVID-19 Cell 713-703-1951<tel:713-703-1951>)
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Director Monoclonal Antibody Core Facility
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On Aug 9, 2021, at 7:50 AM, Weis-Garcia, Frances/Sloan Kettering
Institute <email obscured>> wrote:
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Yes Laura. This is exactly the stage to which I am referring.
How do you purify that 100 ml of media?
Over a Prot G resin using an FPLC?
Be well
Frances
Sent on the fly from my mobile device
On Aug 8, 2021, at 1:28 PM, David Blum <email obscured>> wrote:
Frances: I understand. This might not be the best option, but it
should
be good for processing multiple samples quickly without using the FPLC.
You could also batch bind with the resin you use now and spin down at a
speed that doesn't deform the beads. Good luck!
David
On Sat, Aug 7, 2021 at 1:30 PM Weis-Garcia, Frances/Sloan Kettering
Institute <email obscured>> wrote:
Thanks David. The binding capacity is pretty low relative to standard
resins and the beads are much more expensive.
0.6 mg rabbit IgG / ml Pierce™ Protein G Magnetic Beads
while
20.0 mg human IgG / ml Cytiva Protein G Sepharose™ 4 Fast Flow Media
(formerly GE LifeSciences / Formerly Pharmacia ... boy am I old)
I know I want to use the FastFlow resin (or something similar) ...
right
now I am more looking for a process that does not involve an FPLC if I
can
help it. I have one but the person who has used it is retireing and
this
is the only thing we will use it for, so do I need it is the question.
A lab (Ivo this was Pop Council) used to grow up a 100s of mls of each
hybridoma then line the bottles of conditioned media up on a shelf,
allowing the media to flow through a tube into a column below it and
pass
over the a prot G resin over night by gravity. Some how they set it up
so
the columns DID NOT DRY OUT. The next morning they would wash the
columns
by gravitty again in series and then elute them one at a time. Sounds
like
a great way to do a 5 to 20 at once, and I am sure I could figure this
out,
but before I spend time on this, was wondering if anyone else already
tackled this or had a better mouse trap.
Frances
ZRC 1563
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Mobile 516-967-0656
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On 8/6/21, 7:41 PM, "Antibody Discussion Forum on behalf of David Blum"
<
<email obscured> on behalf of <email obscured>> wrote:
Have you tried mag beads? We use them for histagged proteins at
small
scale but there are protein coupled beads too.
https://urldefense.com/v3/__https://www.thermofisher.com/order/catalog/product/88848__;!!PfbeBCCAmug!1wQ534Un_7nW4on1VYmclsK5q_UbNqiHri00ok_zzvKRc6MMxWrL3JWPqjdjH53Y$
On Fri, Aug 6, 2021 at 6:47 PM Weis-Garcia, Frances/Sloan Kettering
Institute <email obscured>> wrote:
Happy Friday.
Do any of you produce and purify small amounts of the new MAbs you
make
for further characterization prior to committing to a few or one
winner?
If so, how do you do this. Currently we make 350 ml exhausted media
(hybridoma adapted to 1% FBS prescreened for ultra-low levels of
bovine IgG
+ 1 ng/ml IL6) and then purify it on a Prot G column using and
FPLC. We
get 1 – 8 mg, but I would like to scale this back and simply it if
possible
because we usually do not need that much.
Thanks.
Frances
Frances Weis-Garcia, Ph.D.
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Immunology Program
Memorial Sloan Kettering Cancer Center
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