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Hi All,
Did anyone here have some experience of plasma protein-binding of
macromoleculars? I have a fusion protein(~180kd), and I want evaluate
the plasma protein-binding of it. Ultracentrifugation seemed like not
applicable, since the largest cutoff MW of Millipore mcrocon is 100kd,
equillibrum dialysis neither.
Can somebody give me a suggestion? SPR? column? or others?
Thanks
Ning
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The following message was posted to: PharmPK
What is the target? Molecules of that size and structure would be
susceptible-obviously- to binding to targets and possibly, after
repeated
dosing, to anti drug antibodies formed in response. One possible
approach
is to use size exclusion chromatography or PAGE (not SDS). Incubate
your
drug in plasma and compare with incubates in buffer alone. Water's new
Synapse MS may also provide some insight. Additional approaches to
look at
binding include Biacore and Microcal.
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Dear Ed,
Thanks for your response. As you mentioned, the anti-drug antibody for
the molecular with such MW is always the problem.
From PD view, we test the plasma / serum protein binding of the small
molecular drug just because only the unbound drug can achieve the
pharmacological effect. However, if it' s the same to the
macromolecular? if the conventional binding pattern can give us some
real information? I'm afraid this is the case by case issue. Maybe
some no, but for the drug we testing now, it's supposed to act with
the receptors in the bonemarrow, if the the bound drug can have
effecacy as the same as unbound? I'm a little bit confused.
Since regulatory guys wants this parameter, I'm seeking if the PAGE
can help me. Biacore is a potential method, but the reproductbility is
the problem.
Thanks!
Ning
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The following message was posted to: PharmPK
At the very least, size matters. The movement of your molecule,
including
rotation, affects kinetics of your drug. If you have a something
binding
to your drug the kinetics will be impacted, by simply changing size
and the
movement. If the agent binding to the drug also directly or indirectly
impacts binding to the target (substituting for the target or steric
hindrance) your drug's activity will be minimized or, at worst,
neutralized.
Anti drug antibodies are sometimes a problem, you may also have less
specific antibodies present- for example if your molecule was raised in
mice, pre-existing anti mouse antibodies could bind to your
molecule. The
target you are looking for may also exist in the plasma in a free form
(for
examples soluble TNF receptors) or cells in whole blood may include your
target on their surface.
The more info,the better the resolution
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Do you see any difference in recovery of your material after
incubation with
buffer vs whole blood or plasma? If you did not see a difference...???
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Hi Ed,
I'm not sure if you mean the serum stability of my drug, it seemed
like stable in serum. I'm still seeking methods, focus on the affinity
chromatographics, such as HSA column.
Thanks
Ning
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The following message was posted to: PharmPK
Indirectly. Your drug could be bound to targets on intact cells, on
cell
fragments in whole blood or to soluble targets in plasma or whole
blood.
Comparing recovery from whole blood serum or plasma to that of buffer
should
indicate the presence of a binding issue. You may also want to use
material
from your target disease population since they may have greater
amounts of
target.
If the recovery is the same-you do not have a binding issue here-
you may
have one in vivo due to induction of antibody, etc. Do this type of
experiment first then expand to more exotic approaches if you need to
later.
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part of recovery is related to stability part is related to strict
recovery. Compare the two.
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