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Analysis of Trypsin-Digested Rituximab (C18U 2D)

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Peptide mapping is a type of validation test required for biopharmaceutical drugs. For the peptide mapping, biopharmaceutical drugs are first digested by enzymes to fragmentize their peptide. Then they are generally separated by a reversed-phase chromatography and then used for primary structural analysis.  
The C18U series column is a silica-based reversed phase column especially designed for UHPLC use. The use of sub-2-micron organic silica hybrid particles provides superior separation efficiency and mechanical strength. In this application, trypsin-digest of an antibody drug was analyzed by the C18U 2D, using the advantage of its high separation efficiency. Peaks of various digested peptides were detected.  
In this application, trypsin digest of an antibody drug was prepared from a commercial rituximab formulation (anti-CD20 monoclonal antibody). Rituximab was enzyme digested using a protein digestion kit modified with trypsin.

Sample : 5 μL
Tryptic digest of Rituximab formulation
(Injection solution)
0.5 mg/mL


Column       : Shodex C18U 2D (2.0 mm I.D. x 150 mm)
Eluent       : (A); 0.1 % TFA in H2O (B); 0.1 % TFA in CH3CN
               Linear gradient:
               (B %) 10 to 40 % (0 to 25 min), 40 % (25 to 30 min), 90 % (30 to 35 min)
Flow rate    : 0.2 mL/min
Detector     : UV (220 nm) (small cell volume)
Column temp. : 40 °C

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