[RASMB] analysis of biotech samples [was: non-ideality in SV]

[Carlos Alfonso Botello]

Te mando la puntilla de Allen.

Carlos
>  (3) The regulatory agencies would indeed like 
> to know the exact distribution of oligomers, 
> both reversible and irreversible, in the 
> product vial, even for products at 100+ mg/mL. 
> Unfortunately they don't always understand that 
> this may be technically impossible with current 
> technology and current theoretical 
> understanding of the strong non-ideality effects that may be present.

We've developed some approximate methods for 
interpretation of static light scattering, 
sedimentation equilibrium, and osmotic pressure 
of highly concentrated (> 100 mg/ml) protein 
solutions containing multiple macrosolute species 
(including both reversibly and irreversibly 
formed oligomers).  We know the conditions under 
which the approximations in the model ought to be 
valid, and the methods seem to work pretty well 
when applied to data obtained under those 
conditions.  Analyses of data obtained by 
different methods from the same protein yield 
internally consistent results.  If you are 
interested, check out the following publications:

Minton AP. 2008.  Effective hard particle model 
for the osmotic pressure of highly concentrated 
binary protein solutions.  Biophys. J 94: L57-59.

Jiménez M, Rivas G, Minton AP. 2007. Quantitative 
characterization of weak self-association in 
concentrated solutions of immunoglobulin G via 
measurement of sedimentation equilibrium and 
osmotic pressure.  Biochemistry 45: 13356-60.

Minton AP. 2007. Static light scattering from 
concentrated protein solutions.  I. General 
theory for protein mixtures and application to 
self-associating proteins.  Biophys J 93: 1321-8.

Minton AP. 2007. The effective hard particle 
model provides a simple, robust and broadly 
applicable description of nonideal behavior in 
concentrated solutions of bovine serum albumin 
and other non-associating proteins.  J Pharm Sci 96: 3466-69.

Zorrilla S, Jimenez M, Lillo P, Rivas G, Minton 
AP. 2004. Sedimentation equilibrium in a solution 
containing an arbitrary number of solute species 
at arbitrary concentrations: theory and 
application to concentrated solutions of ribonuclease. Biophys Chem 108:89-100.














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Dr. Carlos Alfonso
Servicio de Ultracentrifuga Analítica
Centro de Investigaciones Biológicas
C/ Ramiro de Maeztu, 9.
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E-mail: uanalitica at cib.csic.es
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