Page 11 - Application Handbook - Liquid Chromatography

Application No.L452A
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n Analysis of Glycans in Antibody Drugs Table 2 Analytical Conditions
According to the pretreatment procedure of Fig. 3, the Instrument : Nexera X2
glycans were extracted from 2 types of antibody drugs, Column : Aeris PEPTIDE XB-C18
TM
and following purification, were subjected to (150 mm L. × 2.1 mm I.D., 1.7 µm)
fluorescent derivatization by PA (pyridylamination). Mobile Phases : A) 20 mmol/L Ammonium Formate
Fig. 4 shows the chromatograms of PA-glycans from 0.0095 % (v/v) Formic Acid-Water (pH 4.5)
B) 20 mmol/L Ammonium Formate
antibody drugs, and Table 2 shows the analytical 0.0095 % (v/v) Formic Acid-Methanol
conditions used. Comparing the peaks in the vicinity of Time Program : B Conc. 0 % (0 min) → 5 % (60 min) → 10 % (70 min)
50 minutes for the drugs A and B, respectively, the → 100 % (70.01 min → 80 min)
quantity of glycans associated with that peak in → 0 % (80.01 min → 90 min)
antibody drug A is much greater than that in drug B. Flowrate : 0.4 mL/min
The peak response is quite different for many other Column Temp. : 40 ˚C
peaks, which illustrates the formulation differences Detection : RF-20AXS (Ex = 320 nm, Em = 400 nm)
Injection Vol. : 3 µL
between drug manufacturers.

mV
40
Sample (Antibody drugs) "OUJCPEZ ESVH "

Ultra ltration

Tryptic digestion

Extraction of glycans by Glycopeptidase F
0

Puri cation of glycans by Blot Glyco ˎ 0.0 10 20 30 40 50 60 min
mV
40
Pyridylamination "OUJCPEZ ESVH #

UHPLC
ˎ Blot Glyco : SUMITOMO BAKELITE CO., LTD.

Fig. 3 Sample Preparation

0
Analysis of the glycans in the antibody drugs was conducted with the
kind cooperation of Kenichiro Todoroki, Ph.D. of the Laboratory of 0.0 10 20 30 40 50 60 min
Analytical and Bio-Analytical Chemistry, School of Pharmaceutical
Sciences, University of Shizuoka. Fig. 4 Chromatograms of PA-Glycans from Antibody Drugs
TM
For further information regarding the Aeris column, please contact
TEL: +81-3-5835-0126, gsupport@glc.shimadzu.co.jp

First Edition: Jan. 2014

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