TECHNICAL NOTE
2
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The result is that a positive slope for k
D
is considered an ideal formulation, as it is less
likely to result in increased viscosity or aggregation at high protein concentrations.
Negative k
D
values indicate conditions where the protein favors self-
association while positive values indicate repulsive interactions between
protein molecules. The latter is a more desirable scenario for developing a
therapeutic molecule.
The diffusion coefficient D is specifically related to the r
H
by the Stokes-Einstein equation
D = k
B
T/6πɳr
H
. (k
B
= Boltzmann constant; T = temp in Kelvin; ɳ = dynamic viscosity). This
shows that D is inversely related to the measured particle size; hence as the diffusion
coefficient gets larger, the measured particle size is smaller.
The k
D
is dependent on both the sequence of the candidate and its buffer environment.
Changes to a candidate's primary sequence can affect its self-interaction propensity,
for example by introducing hydrophobic patches to the solvent-exposed region of
the protein. Alternatively, the same candidate can behave differently depending on
the concentration of salt or the pH of the buffer it is formulated in. Increasing salt
concentration is generally assumed to shield the surface charges of the protein and
reduce electrostatic interactions between molecules.
Here we show that DLS experiments done in the Prometheus Panta give exemplary k
D
data for evaluating candidates' self-association propensity. First, we demonstrate how
changes in the diffusion coefficient as a result of concentration changes result in different
measured hydrodynamic radii of NIST mAb. Secondly, we demonstrate how changes to
the buffer formulation can have an impact on the self-association of a representative
mAb.
