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3
APPLICATION NOTE
Proteins were prepared as previously
1
; specifically in
duplicate at 2.5 to 15 mg/ml in different formulation
buffers. Overall scattering intensity was measured and
plotted vs. concentration, and a linear regression line
was fit. A high-concentration measurement of the same
molecule and formulation was then measured at 40
mg/ml. Deviation of the 40 mg/ml measurement from
the linear regression line was used as a parameter for
ranking candidates. Deviation above the regression
line (positive SLS deviation values) shows an increased
propensity for aggregation; deviation below the linear
regression line (negative SLS deviation values) indicates
a lower propensity for aggregation, and therefore a
good result for future development.
Experiments were run in duplicates on the Prometheus
Panta, Wyatt DynaProII, and Avacta Analytical Optim
1
(Avacta Group plc, UK, denoted as "microcuvette")
device to compare the deviation rankings for each
candidate. The results were evaluated by the values
obtained and the deviation between the duplicate
measurements.
Results
DLS measurements to determine k
D
Candidates with positive k
D
values indicate repulsive
forces between molecules, and were considered a
better formulation compared to those with negative k
D
values in Figure 1.
k
D
of five mAbs in different buffer systems
20
15
10
5
0
-5
-10
-15
-20
-25
-30
Citrate pH 4.5 Citrate pH 5.5 Histidine pH 5.5 Histidine pH 6.0 Histidine pH 6.5
Mol 1
Phosphate pH 7.0 Phosphate pH 7.5
Mol 2 Mol 3 Mol 4 Mol 5
k
D
(ml/g)
Figure 1: k
D
values for five different candidates in seven different buffers. Candidates with positive k
D
values are considered optimal for further development due to their
repulsive forces, which reduce the chance for aggregation at higher concentrations.
