Charge Variant Analysis of Cetuximab (2) (SP-FT 4A)
Antibody drugs are produced from animal cells, and thus modification of proteins after their translation may result in variation of product’s uniformity. Consistent uniformity is important for maintaining the quality of biopharmaceuticals. Since uniformity variation can influence the functionalities of produced drugs, various quality assessments are required. One of them is analysis of charge variant (charge isomer). Charge variants are often observed for 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 cetuximab drug. A pH gradient method 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 Cetuximab formulation 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) Thermo Scientific CX-1 pH gradient buffer A (pH5.6)
(B) Thermo Scientific CX-1 pH gradient buffer B (pH10.2)
*Each buffer was used after diluted 10-fold with H2O
Low pressure linear gradient ; (1) B % = 0 % to 35 % (20 min)
(2) B % = 0 % to 55 % (20 min)
Flow rate : 0.5 mL/min
Detector : UV (280 nm)
Column temp. : 40 °C
(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.