Page 24 - Application Handbook - Liquid Chromatography

LAAN-A-LC-E265

Application High Performance Liquid Chromatography

News Peptide Mapping of Antibody Drugs by Nexera-i

No.L488

Peptide mapping by HPLC is one of the important quality Table 1 shows the analytical conditions. Here, the Aeris
assurance tests used for verifying the primary structure 1.7 µm PEPTIDE XB-C18 100 Å small-particle core-shell
of antibody drugs. Typically, following enzymatic column and the Nexera-i integrated UHPLC system was
digestion of the antibodies, separation is conducted used. Mobile phase A was 0.1 % trifluoroacetic acid
using a traditional reversed phase column. Due to the (TFA) in water and mobile phase B was 0.08 % TFA in
large number of peaks that require separation, the use acetonitrile. To ensure proper gradient performance
of small-particle columns and core shell columns for with TFA, an optional 300 µL mixer was used.
peptide analysis has spread in recent years. Fig. 2 shows the chromatogram of IgG tryptic digest, in
In order to compare elution profiles for identity and which an extremely large number of peaks are clearly
mutation confirmation, a highly repeatable system is separated.
required. The Nexera-i integrated UHPLC is the ideal
system for such an analysis. Here, the Nexera-i is used
in the analysis of IgG (human immunoglobulin G)
tryptic digest. Table 1 Analytical Conditions
Column : Aeris 1.7 µm PEPTIDE XB-C18 100 Å
(150 mm L. × 2.0 mm I.D., 1.7 µm)
Mobile Phase : A: 0.1 % trifluoroacetic acid in water
B: 0.08 % trifluoroacetic acid in acetonitrile
Time Program : B.Conc. 0 % (0 min) → 45 % (90 min)
→ 100 % (90.01 - 95 min) → 0 % (95.01 - 110 min)
n Analysis of IgG Tryptic Digest Flowrate : 0.2 mL/min
For this investigation, after reduction and alkylation of Column Temp. : 60 ˚C
: 10 µL
Injection Vol.
IgG, tryptic enzyme digestion was used as shown in Detection : LC-2040C 3D at 215 nm
Fig. 1 for sample preparation. Flow Cell : High-speed high-sensitivity cell

mAU (�10)
4.5
10 mg/mL human IgG in water 20 µL
6 mol/L guanidine hydrochloride in 4.0
0.25 mol/L Tris buffer (pH 7.5) 80 µL
3.5
0.5 mol/L dithiothreitol in water 2 µL
Incubate at 37 ˚C for 30 min 3.0
0.5 mol/L iodoacetamide in water 4.8 µL 2.5

Incubate at room temperature for 30 min in the dark

2.0
0.5 mol/L dithiothreitol in water 2 µL
1.5
0.25 mol/L Tris buffer (pH 7.5) 700 µL
1 mg/mL trypsin in 1 mmol/L HCl 4 µL 1.0
Incubate at 37 ˚C for 20 hours

0.5
Trifluoroacetic acid 1 µL
0.0
Inject to UHPLC
0.0 25.0 50.0 75.0 min
Fig. 1 Sample Preparation Fig. 2 Chromatogram of IgG Tryptic Digest