Charge variant analysis of protein-based biopharmaceuticals using two-dimensional liquid chromatography hyphenated to mass spectrometry

Abstract The profile of charge variants represents an important critical quality attribute of protein-based biopharmaceuticals, in particular monoclonal antibodies and must therefore be closely controlled. In this work, 2D-LC methods for charge variant analysis were developed using a strong cation-exchange chromatography (SCX) as first dimension (1D) separation. Non-porous SCX (3 µm) particle columns and different mobile phases were evaluated using a test mixture of some standard proteins of different size and pI (comprising myoglobin, bovine serum albumin, cytochrome c, lysozyme and β-lactoglobulin) and two monoclonal IgG1 antibodies (NIST mAb and Secukinumab). The most promising 1D eluent for SCX was a salt-mediated pH-gradient system using a ternary mobile phase system with 2-(N-morpholino)ethanesulfonic acid, 1,3-diamino-2-propanol and sodium chloride. For the second dimension (2D), a desalting reversed-phase liquid chromatography (RP-LC) was chosen to enable the hyphenation of the charge variant separation with mass spectrometric (MS) detection. While for intact mAbs the 2D just served for desalting without additional selectivity, the 2D contributed some orthogonal selectivity for the mAb fragment separation. Various core-shell and monolithic columns were tested and variables such as gradient time and flow rate systematically optimized. Unexpectedly, a C4 400 A column (3.4 µm diameter with 0.2 µm porous shell) provided higher peak capacities compared to the same 1000 A column (2.7 µm diameter with 0.5 µm porous shell). A thinner shell appeared to be more advantageous than wider pores under high flow regime. An ultra-fast RP-LC method with a run time of one minute was developed first using trifluoroacetic acid and later with formic acid as additive for better MS compatibility. The successful hyphenation of the two orthogonal separation modes SCX and RP-LC could be demonstrated in the multiple heart-cutting and the full comprehensive mode. MS analysis using a high-resolution quadrupole time-of-flight instrument enabled to identify different glycoforms and some major charge variants of the antibody at the intact protein level as well as on the subunit level (Fc/2, Lc, Fd’) in a middle-up approach by 2D-LC-ESI-MS analysis.