In recent years, the use and number of

bio-therapeutics has increased

significantly.
For these largely protein-based
therapies, the quantitation of aggregates
is of particular concern given their
potential effect on efficacy and
immunogenicity.
This need has renewed interest in size-
exclusion chromatography (SEC).
Size Exclusion Chromatography (SEC) is
the separation technique based on the
molecular size of the components.
Separation is achieved by the differential
exclusion from the pores of the packing
material, of the sample molecules as they
pass through a bed of porous particles.
The principle feature of SEC is its gentle
non-adsorptive interaction with the sample,
enabling high retention of bio-molecular
activity.
Definition
Size-exclusion
chromatography (SEC)
is a chromatographic
method in which
molecules in solution
are separated by their
size, not by molecular
weight. It is usually
applied to large
molecules or
macromolecular
complexes such as
proteins and industrial
polymers.
Mechanism
Molecules larger than the
pore size can not enter the
pores and elute together as
the first peak in the
chromatogram
Molecules that can enter
the pores will have an
average residence time in
the particles that depends
on the molecules size and
shape . Different molecules
therefore have different
total transit times through
the column

Proteins are prone to interact with surface charged
sites of chromatographic stationary phases.
chromatographic stationary phases and mobile
phases have been used to mitigate nonideal
interactions.
Factors Can Be Used To Manipulate SEC
Separations
flow rate,
column length,
mass load,
volume load
Salt Concentration
Mobile Phase pH
Column Dimensions
Particle Size
Figure 5 SEC chromatograms of
antibody-A analyzed using TSKgel
G3000SWxl column;
(a) at various flow-rates of 0.5mL=min,
0.4mL=min, 0.3mL=min, 0.2mL=min, and
0.1mL=min as labeled in the figure;
(b) using mobile phase consisting of 0%,
10%, 20%, 30%, 40%, 50%, or 60%
acetonitrile as labeled in the figure with
0.1% TFA, and 0.1% formic acid in Milli-
Q water;
(c) using mobile phase consisting of 20%
acetonitrile with 0.1% formic acid and with
0%, 0.02%, 0.05%, or 0.1% TFA in Milli-Q
water as labeled
in the figure.
A 4-component protein mixture
was separated on a Zorbax GF- 250 column
(2509.4mm) using a mobile phase of 200mM
sodium phosphate, pH 7.0. The injection
volume was varied from 2 to 200 mL] and
ambient temperature was used. Detection,
represented on the y-axis, was carried out at
230 nm. The flow rate was 2ml=min.
Resolution (Rs) between BSA and ovalbumin
are shown. Peak Identities: 1BSA-dimer;
2BSA; 3ovalbumin; 4lysozyme and
5sodium azide.
Figure 6 Effect of injection volume on
separation efficiency in SEC.

(B) For comparability, elution
volumes were normalized to
column void volumes.
Figure 7 Effect of particle size (dp).
(A) Overlay of single injection
chromatograms of a mAb
sample
(1.0 g=L) analyzed on
AQCUITY BEH200(1.7 mm).
Zenix SEC-250 (3 mm) and
TSKgel 3000 SWxl
(5 mm).