Tosoh
customer magazine
mAb Aggregate Analysis by UHPLC
Antibody therapeutics are enjoying high growth rates. In 2013, six of the top ten best-selling global drug brands
were monoclonal antibodies (mAbs) and more than 400 monoclonals were in clinical trials. The characterization
of these complex biomolecules is a major challenge in process monitoring and quality control. The main product
characteristics to be monitored are aggregate and fragment content, glycosylation pattern and charged iso-
forms.
Figure 2 :
COMPARISON OF ANTIBODY ANALYSIS RESULTS
Mouse-human chimeric mAb;
1:
trimer;
2:
dimer;
3:
monomer ;
4:
fragment
Figure 1
CALIBRATION CURVES
Protein standardon 2 µmy TSKgel UP-SW3000 and 1.7 µmy competitor column
The standard method used in biopharmaceutical QC for mAb aggregate
and fragment analysis is size exclusion chromatography (SEC). A new se-
ries of 2 micron silica based UHPLC columns with 25 nm (250 Å) pore
size can be applied to either increase speed or improve resolution of the
separation of antibody fragments, monomers, and dimers.
Compared to a commercially available 1.7 micron UHPLC column the cali-
bration curves of the new TSKgel UP-SW3000 2 μm column (see page 3)
shows a slightly shallower slope in the region of the molecular weight of
-
globulin. These differences in the separation range and steepness of
the curves are related to a slight difference in pore size (25 nm for TSKgel
versus 20 nm for the 1.7 µm material).
The separation of an antibody sample on the new 2 μm packing compared
to the competitor UHPLC column shows that the small difference in pore
sizes results in a better separation in the molecular weight range of anti-
bodies, fragments, and aggregates. Due to the wider separation window
the resolution between monomer and dimer as well as dimer and trimer
is slightly higher with TSKgel UP-SW3000 although particle size is slightly
larger than in the competitor column. Moreover, also the fragment peak is
more clearly separated from the monomer peak.
TSKgel UP-SW3000 is ideally suited for the analysis of the aggregate and
fragment contents of antibody preparations. It features the same pore size
as the renowned TSKgel G3000SW
XL
and TSKgel Super mAb columns
while improving resolution through a smaller particle size. Based on the
optimized pore size and the high degree of porosity the resolution in the
molecular weight range of immunoglobulins is even superior to a competi-
tive UHPLC column with slightly smaller particle and pore size.
05
Application
Monoclonals II
