[RASMB] concentration range for non-ideal behavior

[John Philo]

John, first I am not very clear about what you mean by "failed to observe
any evidence of non-ideal behavior". Based on excluded volume alone one
would expect the sedimentation coefficient of any globular protein to be at
least ~4.5% less at 5 mg/mL than at infinite dilution (and more so for an
asymmetric molecule like an antibody). So surely you must be seeing at least
that effect, correct?  If not, I would guess the decrease from non-ideality
is being masked by weak rapid self-association, exactly as Jack said.

 

If what you mean by "failed to observe any evidence of non-ideal behavior"
is just that you can get a fairly good fit to something like a c(s) model
without considering non-ideality, then yes at 5 mg/mL the fits might not
look too bad. If all you are worried about is properties of major species,
doing this may be okay, but as you go up in concentration at some point the
non-ideality means the fitting model is fundamentally wrong and you will
likely get false minor peaks. So a lot depends on exactly what is the
purpose of the study.

 

John

 

From: RASMB [mailto:rasmb-bounces at list.rasmb.org] On Behalf Of John J.
Correia
Sent: Wednesday, January 21, 2015 3:29 PM
To: John Sumida; rasmb at rasmb.org
Subject: Re: [RASMB] concentration range for non-ideal behavior

 

John, The nonideality above 1 mg/ml rule depends upon the numerical
contribution from a (1 + Ksc) term where c is on a weight scale. Ks is
phenomenological and not easy to guess at. If Ks = 0.01 for example at 1
mg/ml the term starts to be significant. Thus if Ks is smaller than higher
concentrations are required to nonideality effects. Since nonideality is
either charge and/or excluded volume dependent, small molecules will not
contribute much and uncharged molecules will not contribute. Thus working
with small macromolecules at their pI will give ideal behavior over a wide
range of concentrations. In addition mAb's often self-associate albeit
weakly thus potentially masking nonideality. 

 

From: John Sumida <jpsumida at u.washington.edu
<mailto:jpsumida at u.washington.edu> >
Date: Tuesday, January 20, 2015 at 8:00 PM
To: "rasmb at rasmb.org <mailto:rasmb at rasmb.org> " <rasmb at rasmb.org
<mailto:rasmb at rasmb.org> >
Subject: [RASMB] concentration range for non-ideal behavior

 

Dear RASMB,

 

My recollection is that concentrations in excess of 1.0 mg/ml should be
avoided due to the possibility of non-ideal interactions that would
complicate the analysis of SV and SE measurements.  However in two recent
cases (in one case a mAb was being studied, and in another a globular viral
capsid protein), we exceeded this value by a factor of five and failed to
observe any evidence of non-ideal behavior. In both cases, it was possible
to fit both SV and SE data without assuming any non-ideal interactions when
the experiment was performed in PBS or HEPES.

 

I would be curious to hear if it is the experience of the community that
non-ideality is commonly to be expected at concentrations >1.0mg/ml or
whether the concentration cutoff for non-ideality is less of an actual
cutoff being more a function of the particular properties of the molecule
being studied.

 

Best regards

John Sumida

University of Washington

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