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

[Tom Laue]

Hi All-
I am following this discussion with great interest. I think it is time 
to revisit the J-O effect, but for a somewhat different reason. With the 
fluorescence optics, we are able to monitor sedimentation in serum 
(total protein concentrations of 70 - 100 mg/ml). There are several good 
biomedical reasons for being concerned about aggregation in serum, let 
alone in formulations. E.g., injection of monoclonal antibodies from 
formulation conditions into serum may or may not be benign with respect 
to aggregation- both homo- and hetero- aggregate formation could affect 
the immunogenicity, toxicity and pharmaco-kinetics. It will be important 
to disentangle aggregate formation from hydrodynamic nonideality.
You certainly can observe the J-O effect in serum. Sometimes the effect 
is spectacular- peaks in concentration where the slower material runs up 
against the boundary of the faster moving material. There are some 
papers that model the effect from some years ago (David Yphantis did 
some work on this, as did John Cann) and, of course, Arthur has done a 
great deal of work on it. Most of this work has focused on simple 
solutions. I'd be very interested in thoughts on these effects in 
biological fluids. Keep going with the discussion!
Thanks-
Tom

Arthur Rowe wrote:
>
>     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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-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
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Phone: 603-862-2459
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E-mail: Tom.Laue at unh.edu
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