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By analyzing proteins as interacting elements instead of in
isolation, the ProteomeLab™ XL-A/XL-I more closely
approximates true physiological conditions by considering the
protein's shape (folded or unfolded), composition (assembled or
unassembled), stoichiometry (associative state) and heterogeneity
(aggregation) to accelerate lead optimization.
Protein Heterogeneity
The ability to characterize sample heterogeneity is
an important part of process development, formulation and QA/QC. The
accompanying graphs are distribution plots with heterogeneity
visually indicated by peaks. The position of each peak indicates the
sedimentation coefficient, while the area gives the amounts of that
species. The graph (left) uses the g(s)-distribution method to
resolve three non-interacting proteins. Levels of <10% by weight
can be quantified. For more in-depth analysis, third party software
is available for enhanced application versatility and sensitivity.
The graph (right) is a distribution plot of a highly stressed
monoclonal antibody sample, with heterogeneity visually indicated by
higher resolution peaks. Use of the more sophisticated c(s)
distribution method provides enhanced resolution by removing the
effects of diffusion. Levels of aggregates or other variants can be
quantified at levels well below <1% by weight.
Interacting Systems
The ProteomeLab XL-A/XL-I is adept at helping
determine yes/no binding of a small molecule to a target protein. In
the top-most example, a small-molecule inhibitor (the green square)
of a hormone-receptor interaction could bind to either the receptor
(A) or the hormone (B). The inhibitor uniquely absorbs at 321 nm and
has a low molecular weight and therefore does not sediment. If it
binds to a protein however, the bound molecule will sediment with
the apparent molecular weight of the protein and the complex
monitored at 321 nm. The left-hand graph represents sedimentation
equilibrium of an inhibitor + receptor mixture, with the absence of
curvature for the inhibitor (red) indicating that the compound does
not bind to the receptor. The right-hand graph shows a pronounced
curve, indicating that the inhibitor binds to the hormone.
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Molecular Conformation
The
characterization of
molecular conformation is a powerful quantitative method for
proving to regulatory agencies that changes in manufacturing
processes produce products with identical solution
conformations. It's also used in preclinical studies to verify
that recombinant proteins are properly folded, and as an aid
in selecting mutants or engineered forms of a protein. This
example demonstrates molecular conformation under varying salt
conditions. In TE buffer (top) an oligonucleosome structure
was shown to have a sedimentation coefficient of between 27-30
S (visually indicated by a vertical distribution plot). In 1.8
mM MgCI2, the same oligonucleosome structure was
shown to have a sedimentation coefficient of between 33-52 S
(visually indicated by a non-vertical distribution plot). The
solution's molecular mass of the oligonucleosome in these two
salts' concentrations remained the same (demonstrated by
sedimentation equilibrium), indicating that the
oligonucleosome folded into a more compact conformation in the
presence of salt.
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Stoichiometry
Data describing a protein's stoichiometry can
be useful early in drug development, and in providing
characterization and comparability data to regulatory
agencies. The data below is for a sequence homolog of a tumor
necrosis factor, part of a family of proteins that are all
trimers. Size exclusion chromatography indicated that this
protein is a monomer, suggesting either that it is not a true
trimer or that it was not properly folded after expression in
E. coli. In this form of plot, a single species gives a
straight line with a slope proportional to the solution mass.
The blue line shows the slope predicted for a monomer, the red
for a trimer. Clearly, this protein is indeed a trimer, as
expected.
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