[RASMB] non-ideality in velocity [was: interference optics]

[Schoenfeld, Hans-J.]

 Hi Borries,
with your statements regarding the importance to distinguish reversible
oligomers from irreversible aggregates I completely agree.
This distinction is as important as it may be trivial, at least for
(physico)chemists. The two different types of association are often
mixed up by people with a non-chemistry (law of mass action) background.
I heard statements like: "everything that is not the monomer peak is
unwanted contamination per definition" or "we have no chance to get a
molecule accepted that shows two peaks in SEC". Consequently this would
mean that any active biological system that undergoes (slow)
self-oligomerization is unwanted...
Two further comments (sorry, if already given by others):
1.) On-line light scattering (one angle is sufficient) in SEC is not
only useful to measure accurate molecular mass, but in this context it
is also helpful to detect small traces of irreversible but soluble
medium-sized aggregates that appear in the exclusion volume (in this
context of course, it is more a qualitative than quantitative method).
2.) The AF4 separation technology (asymmetrical-flow
field-flow-fractionation) may be an alternative method that avoids
"filtering of aggregates" before quantitative analysis.
Best regards from Basel,
Hans-Joachim.

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org]
On Behalf Of Borries Demeler
Sent: Tuesday, April 08, 2008 17:21
To: Arthur Rowe
Cc: rasmb at server1.bbri.org
Subject: Re: [RASMB] non-ideality in velocity [was: interference optics]

> 
> John -
> 
> Yes, I take your point. I think we both know the 'biotech crowd', and 
> the desire which they can often have for results obtained under 
> 'formulation conditions'. But of course there may be a real problem 
> involved in correcting for Johnston-Ogston effects.

John and Arthur,

Aside from the points about J-O effect and other non-ideality effects, I
think that the premise of the 'biotech crowd' of evaluating formulations
exclusively at high concentration should be re-evaluated. Formulation
conditions often require high concentrations of the solute, and drug
companies are interested in the percentage of undesirable aggregate. 

I think it is not unreasonable to separate the "aggregates" into
2 kinds: reversible oligomerization (not necessarily undesirable) and
irreversible aggregation. The amount of reversible aggregate is clearly
related to concentration, the higher the concentration, the more you
have.  Once the formulation is injected, the solute is diluted in the
blood stream and presumably at a concentration resembling a low
concentration AUC experiment (or lower), so why worry about it? If one
really were interested in the Kd, and if it were high, an SE experiment
at higher concentration may be more appropriate to determine the amount
of reversible oligomerization, without having to worry about pitfalls of
SV at high concentration.

This leaves the irreversible aggregates, which really are undesirable.
Even upon dilution, the percent of irreversible aggregation will not
change, because, by definition, those aggregates are irreversible, and
therefore any method measuring the formulation at low concentration
should provide the correct percentage, with the added benefit of not
having to worry about J-O effects, boundary sharpening, and Weiner
skewing etc.

So my question is: Why are drug companies interested in the reversible
kind, or is there another reason to measure at high concentration?

Incidentally, it has been my experience that SEC-MALS is not so great at
detecting large irreversible aggregates, because they can get stuck in
the SEC column and you never see them in the MALS detector. However, the
reversible oligomerization may be detected fine by MALS .

Comments?

-Borries
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