This application discusses a unique charge variant analysis method using an ion exchange column.
Antibody drugs are produced from animal cells, and modification of proteins after their translation may result in variation of product’s uniformity. Consistent uniformity is important for the quality of biopharmaceuticals. Since uniformity variation can influence the functionalities of produced drugs, various quality assessments are required.
One of them is charge variant analysis (charge isomer). Often, charge variants are observed in monoclonal antibodies, which are produced by deamidation of aspartyl residue or incomplete processing of H chain C-terminal lysine residue. Therefore, strict management of their profiles becomes important.
IEC SP-FT 4A, a strong cation exchange column packed with non-porous gel, was used for the analysis of commercial trastuzumab drug. A salt gradient method by increasing the NaCl concentration was used. SP-FT 4A provided higher separation efficiency in a shorter analysis time of charge variant than other company’s weak cation exchange column.
Sample : 5 μL
5 mg/mL (in H2O)
Column : (1) Shodex IEC SP-FT 4A (4.6 mm I.D. x 10 mm ; 2.7 μm) (2) Weak cation exchange column from other manufacturer (4.0 mm I.D. x 250 mm ; 10 μm) Eluent : (A) 20 mM *MES buffer (pH7.0) / (B) (A) + 0.5 M NaCl Low pressure linear gradient ; (1) B % = 0 % to 10 % (10 min) (2) B % = 0 % to 20 % (20 min) Flow rate : (1) 0.5 mL/min (2) 1.5 mL/min Detector : UV (280 nm) Column temp. : 40 °C *MES : 2-(N-Morpholino) ethanesulfonic acid
(Caution) Backpressure will differ depends on the LC system used and the analytical conditions such as eluent compositions and column temperature. Please set the system pressure (sum of column and system pressures) less than 20 MPa.