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High Sensitivity HPLC Profiling of 2-AB Glycans �Kenichiro Tanaka, William Hedgepeth　Shimadzu Scientific Instruments, Inc., Columbia MD

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Introduction

Glycans in antibody drugs may play roles in the antigenicity, pharmacokinetics and high-order structural stability of drugs, which could adversely affect drug safety and effectiveness. It is therefore necessary to investigate which glycans are present in antibody pharmaceuticals. Non-uniformity of glycans due to the instability of culture conditions used for antibody drugs is a concern that requires rigorous management of the production process.

Here, using an amide column TSKgel Amide-80 and a Prominence high-performance liquid chromatograph (HPLC), we introduce an example of the analysis of glycans. A high-sensitivity fluorescence detector was used for the analysis. A mixture of three glycans was injected to the system and LOD was found to be below 1 fmol.

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Materials

Reagents and Standards Reagents: l  Glyko 2-AB-(Human IgG N-Linked Glycan Library) 200 pmol, l  Glyko 2-AB-(Man-5) 100 pmol, Glyko 2-AB-(NA2) 100 pmol, and l  Glyko 2-AB-(A1F) 100 pmol were purchased from Prozyme.

l  Formic acid and ammonium formate were purchased from Sigma-Aldrich. l  Water was made in-house using a Millipore Milli-Q Advantage A10 Ultrapure Water Purification System. l  Acetonitrile was purchased from Honeywell. Standard solutions: l  Each glycan standard was dissolved in 50 uL of water. l  The stock solution of Glyko 2-AB-(Human IgG N-Linked Glycan Library) was diluted 50 times with water/

acetonitrile=1/1. l  The stock solutions of individual glycan standards were equally mixed and diluted with water/

acetonitrile=1/1 to 100, 50, 20, 10, 5, 2, 1, 0.5 ng/L each. l  They were transferred to a 1.5 mL vial for analysis.

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Materials

Fig. 1: Structures of the three glycans

2-AB-Man5

2-AB-G2 (NA2)

2-AB-G2FS1 (A1F)

: GlcNAc : Gal : Man : Fuc : NeuNAc

2-AB: 2-aminobenzamide

2-AB

2-AB

2-AB

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System

The standard solutions were injected to a Shimadzu Prominence system consisting of:

l  Two LC-20AD pumps

l  DGU-20A5R degassing unit

l  SIL-20AC autosampler

l  CTO-20AC column oven

l  RF-20Axs fluorescence detector equipped with a conventional flow cell

l  CBM-20A system controller

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Results

Sensitivity and linearity Sensitivity, linearity, and repeatability were checked using the three glycans as a standard solution. Table 1 shows the analytical conditions and Fig. 2 shows the chromatogram of 1 fmol each of standard solution. A standard as low as 1 fmol was able to be detected. Table 2 shows the limits of detection for respective glycans (signal to noise ratio: 3.3) and the limits of quantification (signal to noise ratio: 10). Fig. 3 shows the chromatogram of repeated analysis of 40 fmol (20ng/L) each of standard solution (n=6). As a result, less than 1 % relative standard deviation of retention times and peak areas were obtained. Table 3 shows the relative standard deviations. Seven different levels of glycan standard solutions ranging from 2 to 200 fmol (1-100 ng/L) were used for the linearity evaluation. The correlation coefficient (R2) of determination was higher than 0.999. The calibration curves are shown in Fig. 4.

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Results

Table 1: Analytical Conditions

System : Prominence HPLC Column : TSKgel Amide-80 (150 mm L. x 2.0 mm I.D., 3 µm) Mobile Phase : A: 50 mmol/L ammonium formate pH 4.4

B: Acetonitrile Time Program : B Conc. 73 % (0 min) - 60 % (48 min) - 0% (49-53 min) - 73% (54-80min) Flow Rate : 0.4 mL/min (0-48 min, 58.01-80min), 0.2 mL/min (48.01-58 min) Column Temperature : 40 ̊C Injection Volume : 2 µL Detection : Ex 330 nm, Em 420 nm Flow Cell : Conventional cell

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Results

Fig. 2: Chromatogram of 1 fmol (0.5 ng/L) each of glycan standards

0 10 20 30 40 min

-0.25

0.00

0.25

0.50

0.75

1.00mV(x0.1)

■Peaks 1. 2-AB-Man5, 2. 2-AB-G2, 3. 2-AB-G2FS1

1 2

3

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Results Fig. 3: Chromatogram of 40 fmol (20 ng/L) each of glycan standards

0 10 20 30 40 min

0.0

1.0

2.0

3.0

4.0

5.0

uV(x1,000)

■Peaks 1. 2-AB-Man5, 2. 2-AB-G2, 3. 2-AB-G2FS1

1 2 3

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Results

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    

Glycan  standard     LOD  (fmol)   LOQ  (fmol)  2-­‐AB-­‐Man5      0.44              1.33    2-­‐AB-­‐G2      0.45          1.36    

2-­‐AB-­‐G2FS1      0.50        1.48    

Table 3: Relative standard deviations of retention times and peak areas

Table 2: Limit of detection and quantitation

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Fig. 4: Calibration Curves

Results

2-AB-Man5 1-100 nmol/L (2-200 fmol) R2=0.9997

2-AB-G2 1-100 nmol/L (2-200 fmol) R2=0.9997

2-AB-G2FS1 1-100 nmol/L (2-200 fmol) R2=0.9997

0.0 25.0 50.0 75.0 Conc.0

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75000

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150000Area

0.0 25.0 50.0 75.0 Conc.0

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50000

75000

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Area

0.0 25.0 50.0 75.0 Conc.0

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Results

Separation of glycan mixture: The glycan mixture (2-AB-(Human IgG N-Linked Glycan Library)) was injected. Fig. 5 shows the chromatogram. Many glycans were successfully separated.

10 20 30 min

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0mV Fig. 5: Chromatogram of a 160 fmol (80 ng/L) of

glycan mixture

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Conclusions

1.  The Prominence system equipped with a RF-20Axs fluorescence detector enables very high-sensitivity analysis of glycans.

2.  An established method was successfully applied to glycan profiling with excellent sensitivity .

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