Page 29 - Pharmaceutical- Guide to Biopharmaceutical

Characterization Quality Control

Glycan Analysis RF-20Axs

Analysis of Glycans by HPLC Detection benefits

of Fluorescence-Marked Glycans Cell Line Optimization
click here • The low noise and excellent S/N ratio ensure ample sensitivity for glycan analysis.
• Cell temperature control functionality enables highly reproducible data acquisition.
Operating Principle and Features Measurement Method and Conditions
• Standard, semi-micro, inert, and other cells can be selected based on the given analysis.
Glycans can affect the safety and efficacy of biopharmaceuticals. One Glycans in an antibody drug were analyzed by HPLC with detection by
technique used to analyze glycans is to mark them with fluorescence the high-sensitivity RF-20Axs fluorescence detector. An Aeris PEPTIDE
and then analyze them by HPLC using a fluorescence detector. XB-C18 core-shell analytical column was used. The column packing Culture
Shimadzu RF-20Axs fluorescence detectors offer low noise and good S/ material penetration was optimized for analyzing peptides and other
N levels (compared to previous models, as shown in Fig. 1) to provide macromolecules, which makes it effective for separating glycans and
excellent sensitivity and linearity for glycan analysis. Glycan fluorescent contaminants in antibody drugs.
labeling methods include those using pyridylamino (PA)-glycan and Two types of antibody drugs were treated with trypsin and
2-aminobenzamide (2-AB)-labeled glycan. Either type of fluorescent- Glycopeptidase F was used to cleave glycans. Then the glycans were
labeled glycans can be analyzed in the same manner. fluorescently derivatized by PA and used for analysis (Table 1).
Results
RF-20AXS
S/N:84 Peak differences noticed between the chromatograms for antibody Purification
drugs A and B after about 50 minutes of elution (*) clearly indicated
the drugs contained different glycan levels. In addition, many peaks
with different response levels were observed (Fig. 2 and 3).
mV
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min 40
mV Antibody Drug A
RF-10AXL 40 *
(Previous model) S/N:3 Antibody Drug A
* Characterization

0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
Fig. 1 Chromatograms of 10 fmol PA-Glycan
0 0.0 10 20 30 40 50 60 min
mV Fig. 2 Chromatogram of PA-Glycans from Antibody Drug A
Table 1 Analytical Conditions 0.0 10 20 30 40 50 60 min
40
Column: Aeris PEPTIDE XB-C18 mV Antibody Drug B
(150 mm × 2.1 mm I.D., 1.7 μm) 40 Specifications Quality Control
Mobile phase A: 20 mmol/L Ammonium Formate Antibody Drug B
(pH 4.5) 0.0095 % (v/v) Formic acid-water
Instrument RF-20Axs fluorescence detector
Mobile phase B: 20 mmol/L Ammonium Formate
0.0095 % (v/v) Formic acid-Methanol Light source Xenon lamp, low-pressure mercury lamp (To check wavelength accuracy)
Time Program (B. Conc.): 0 % (0 min) → 5 % (60 min) →
→ 10 % (70 min) → * Wavelength range Excitation wavelength from 200 to 900 nm, Fluorescence wavelength from 200 to 900 nm
→ 100 % (70.01 min- 80 min) →
→ 0 % (95.01 – 110 min) * Cell temperature control range (Room temperature - 10 °C) to 40 °C
Flowrate: 0.4 mL/min Cell Standard conventional cell Volume: 12 μL Pressure capacity: 2 MPa
Column Temp.: 40 °C 0
Injection Volume: 3 µL Optional semi-micro cell Volume: 3 μL Pressure capacity: 2 MPa
0 0.0 10 20 30 40 50 60 min
Detection: RF-20Axs Pharmacokinetics
(Ex: 320 nm, Em: 400 nm) Optional inert cell Volume: 12 μL Pressure capacity: 2 MPa
0.0 10 20 30 40 50 60 min
Sampling rate Max. 100 Hz (1 wavelength mode)
Fig. 3 Chromatogram of PA-Glycans from Antibody Drug B
Conclusion Function Four-wavelength detection, wavelength scanning
Application Examples (Shimadzu Application News No.) Operating environment 4 to 35 °C
Glycans in antibody drugs can be analyzed using HPLC by fluorescent Dimensions W 260 mm × D 500 mm × H 210 mm
labeling the glycans after trypsin digestion. RF-20Axs detectors offer • Analysis of 2-AB glycans (L483)
high sensitivity and low noise. They can also be connected to an LC- • Quantitative analysis of favipiravir spiked in plasma (L570) Weight 18 kg Others
2060 series integrated HPLC system (refer to p. 24). Power requirement 100 to 240 V AC, 400 VA, 50/60 Hz
This analysis of glycans in antibody drugs was achieved with help from professor Kenichiro Tadoroki of the Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University
of Shizuoka.

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