TECHNICAL NOTE
3
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Results
Impact of concentration on r
H
determination
NISTmAb (RM 8671) is a standard mAb used as a
reference material for antibody formulation and
development. It is a well-characterized IgG-derived
antibody that is known to behave optimally in a
standard buffer. Typical calibration measurements
are made at 10 mg/ml in a 12.5 mM histidine buffer at
pH 6.0. With this concentration and buffer condition,
expected hydrodynamic diameter is 9.96 nm, i.e. an r
H
of
4.98 nm2.
Measured
rH
(nm)
Measured
rH
(nm)
[NISTmAb] mg/ml [Protein] mg/ml
NISTmAb
Lysozyme
1
3
5
Figure 1a compares the r
H
determination for lysozyme
and NISTmAb. Note that while there is no significant
difference in r
H
for lysozyme between 1 mg/ml and 10
mg /ml (r
H
= 1.7±0.1 to 1.9±0.1, respectively), NISTmAb
appears to become smaller as concentration increases.
The Prometheus Panta gives an r
H
value of 4.9 ± 0.1 nm
at 10 mg/ml, which is in good agreement with published
standards. This concentration-dependent change
in measured r
H
is due to self-association behavior in
the 12.5 mM histidine pH 6.0 buffer, which affects the
diffusion coefficient and therefore the measured r
H
.
Figure 1: Figure 1: A) NISTmAb prepared at varying concentrations in 12.5 mM histidine pH 6 buffer (teal); lysozyme prepared at varying concentrations in
25 mM histidine pH 5 buffer supplemented with 130 mM NaCl (purple). Results (n=2) for each concentration plotted against r
H
. B) Same experiment with focus on
NISTmAb to emphasise the relationship between concentration and measured size.
A B
