Page 10 - Application Handbook - Liquid Chromatography

LAAN-A-LC-E230A

Application High Performance Liquid Chromatography

News High Sensitivity Profiling of Glycans in Antibody

Drugs Using RF-20AXS
No.L452A

Glycans, or sugar chains, in antibody drugs play roles in
the antigenicity, pharmacokinetics, and stability of
higher-order structure, which could adversely affect RF-20AXS
their safety and effectiveness. Further, there is also S/N:84
concern about the non-uniformity of these glycans due
to instability of antibody drug culture conditions, which
has heightened the necessity to manage their
production process. However, while there is currently
no glycan test method specified in the Japanese
Pharmacopoeia, there is wide demand for an
assessment method. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
Here, we introduce an example of analysis of glycans in
antibody drugs using the Nexera X2 ultra high RF-10AXL
S/N:3
performance liquid chromatograph with the RF-20AXS
high-sensitivity fluorescence detector. For the analysis,
the Phenomenex core-shell, high-speed analytical
Aeris PEPTIDE XB-C18 column was used. Since the
TM
permeability of the packing material is optimized for
analysis of high-molecular compounds such as
peptides, the column is useful for separation of glycans
and impurities in antibody drugs.
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min

n Sensitivity and Linearity of Detectors in PA-Glycan Analysis Fig. 1 Chromatograms of 10 fmol PA-Glycan
The sensitivity and linearity of the RF-20AXS fluorescence
detector was evaluated using a pyridylamino (PA)-
glycan (PA-Sugar Chain 009, Takara Bio Inc.). Table 1
shows the analytical conditions. Area
Fig. 1 shows a comparison of the sensitivity obtained in 100000 R = 0.999
analysis of a PA-glycan at 10 fmol (5 nmol/L, 2 µL 90000
injected) using the fluorescence detectors RF-20AXS and
the previous model RF-10AXL connected in series. 80000
Excellent results were obtained with the RF-20AXS, with 70000
a good S/N ratio and low noise. Fig. 2 shows the 60000
calibration curve results obtained with the RF-20AXS
fluorescence detector over a concentration range of 1 – 50000
100 fmol (0.5 – 50 nmol/L, 2 µL injected). 40000
There is significant improvement in performance 30000
compared to the previous model, and these results 20000
demonstrate that the RF-20AXS fluorescence detector is
suitable for verification not only of the main peak, but 10000
of the trace level impurities as well. 0
0 25 50 75 100 (fmol)
Loading Amount
Table 1 Analytical Conditions
Instrument : Nexera X2 Fig. 2 Calibration Curve (1 – 100 fmol Injected)
Column : Shim-pack XR-ODS Ⅲ(50 mm L. × 2.0 mm I.D., 1.6 µm)
Mobile Phase* : A) 20 mmol/L Ammonium Formate
0.0095% (v/v) Formic Acid-Water (pH 4.5)
B) 20 mmol/L Ammonium Formate
0.0095% (v/v) Formic Acid-Methanol
A/B=95/5 (v/v)
Flowrate : 0.5 mL/min
Column Temp. : 40 ˚C *Mobile Phase Preparation
Detection : RF-20Axs (Ex = 320 nm, Em = 400 nm) 1.26 g (20 mmol) ammonium formate (M.W.: 63.026) was dissolved in
Injection Vol. : 2 µL 1 L of distilled water or methanol, and 95 µL of formic acid was added.