Page 9 - Pharmaceutical M5 Biopharma

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Sponsored Feature  9

Glycans Addressing both fast screening and full profiling

Full Characterization and Monitoring

At later stages of biotherapeutics development, full characterization of glycan profile is
required in order to define the reference glycan profile of a product for later QA/QC.
High-resolution chromatography is needed for in-depth characterization, in order to
separate and identify structural isomers that pose a health risk, e.g. alpha-1,3 galactose.
Retention time gives reliable identification, and reproducibility of chromatography is
the key driver for method selection.

Purified Enzymatically Label HPLC
Protein Release Glycan and Purify LC/MS

Glycan standard R.T. %RSD Area %RSD
2-AB Man5 0.273 0.743
2-AB G2 0.245 0.684
2-AB G2FS1 0.196 0.589
Reproducibility of HILIC mode glycan separation, tested with 2-AB labeled
glycan standard (40 fmol each)

Application News: Fluorescence HPLC
High-Sensitivity Analysis of chromatogram of labelled
2-AB Glycans by RF-20Axs glycan mixture derived from
Florescence Detector monoclonal antibody
sample.

LAAN-A-LC-E260
Application High Performance Liquid Chromatography
News High-Sensitivity Analysis of 2-AB Glycans by RF-20Axs
Florescence Detector
No.L483
Glycans, present in antibody-drug products have an Table 1 Analytical Conditions
effect on their safety and efficacy, therefore requiring System Column : Prominence : TSKgel Amide-80 (150 mm L. × 2.0 mm I.D., 3 µm)
that the types and quantities of the glycans present be
investigated. Due to the culture conditions, the diversity Mobile Phase : A: 50 mmol/L Ammonium formate pH 4.4
B: Acetonitrile
and heterogeneity of the glycan structures cannot be Time Program : B.Conc. 73 % (0 min) → 60 % (48 min) → 0 % (49 - 53 min)
avoided so their management must be implemented Flowrate : 0.4 mL/min (0 - 48 min, 58.01 - 80 min)
→ 73 % (54 - 80 min)
during the production process.
In Application News No. L452, the analysis of a 0.2 mL/min (48.01 - 58 min)
Column Temp. : 40 °C
pyridylamino (PA)-glycan using a fluorescence detector Injection Vol. : 2 µL
was intr oduced. Her e, the analysis of a Detection : Ex 330 nm, Em 420 nm
2-aminobenzamide-labelled glycan (2-AB glycan) is Flow Cell : Conventional cell
introduced. As in Application News No. L452, the *Preparation of Mobile Phase A
world's highest sensitivity fluorescence detector, the RF- water, about 340 µL formic acid was added to obtain a pH of 4.4.
After dissolving 3.15 g (50 mmol) ammonium formate in 1 L distilled
20Axs, was used for detection.
n Analysis of Low Concentration Standard Solution
In this study, the fluorescent-labeled glycans that were mV (×0.1)
used include 2-AB Man-5, 2-AB G2, and 2-AB G2FS1 1.00
(Prozyme). Their structures are shown in Fig. 1. ■ Peaks
The analytical conditions that were used are shown in 1. 2-AB Man5, 2. 2-AB G2, 3. 2-AB G2FS1
Table 1. The glycans were separated using hydrophilic 0.75
interaction chromatography (HILIC). Fig. 2 shows the
results of analysis of a 0.5 nmol/L standard solution 0.50
using a 2 µL (1 fmol) injection. As can be confirmed
from the obtained data, sufficient sensitivity is achieved
even using an ultralow amount injection. The limits of 0.25 1 2
detection (S/N=10) and quantitation (S/N=3.3), 3
respectively, are shown in Table 2.
0.00
-0.25
2-AB Man5:
2-AB oligomannose 5 2-AB 0 10 20 30 40 min
2-AB labeled asialo-, galactosylated 2-AB Fig. 2 Chromatogram of 1 fmol Each of 2-AB-Labeled Glycans
2-AB G2 (NA2):
(0.5 nmol/L each, 2 µL injection)
biantennary
2-AB mono-sialylated-, galactosylated 2-AB Table 2 Limits of Detection and Quantitation
2-AB G2FS1 (A1F):
biantennary, core-substituted with fucose
Glycan standard LOD (fmol) LOQ (fmol)
: Galactose : Mannose : Fucose 2-AB Man5 0.44 1.33
: N-Acetylglucosamine : N-Acetylneuraminic acid 2-AB G2 0.45 1.36
2-AB: 2-Aminobenzamide 2-AB G2FS1 0.50 1.48
Fig. 1 Structures of 2-AB Glycans Used in This Study
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