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

[Arthur Rowe]

Hi Borries

I think I agree with everything you write. It is indeed the higher
aggregates which concern the pharma people (or, in the last analysis, the
regulatory agencies) since patients do not usually develop anaphylactic
shock by exposure to a monomer-dimer equilibrating protein system. And
pre-dilution of samples prior to analysis does make things easier.

So why do they sometimes (but not of course always) demand that analysis be
done at formulation level of concentration? There may be rational reasons
for this. Proteins are more stable at high concentration than at low -
essentially because a 100 mg/ml protein solution is a self-protective
osmolyte, to put it into contemporary jargon. Hence dilution introduces an
extra (and potentially harmful) step, which can never in itself be
beneficial to their case for licensing. What happens after it is injected
into a patient is of course fascinating, but all the stability tests we do
are related to matters prior to that event.

And, at the end of the day, if you are doing analyses for a client, then the
client calls the shots as to what they require.

As regards reversible aggregation, I personally find that biopharma people
may (and often do) become interested when I find it happening in their
samples, but very seldom indeed do I get approached with an enquiry re a Kd
value. I wonder if that is your experience too (Borries, John, anyone else)?

Regards to all

Arthur


> 
> 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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