Cation exchange-HPLC and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain.

A unique, late-eluting "basic peak" (relative to the "main peak") was observed by weak cation exchange-HPLC (WCX) for a recombinant monoclonal antibody (mAb) sample. Peak fractions were collected, desalted, and analyzed by high-resolution MS using a top-down characterization approach that provided accurate masses of intact mAb charge isoforms and a comprehensive profile of the structural heterogeneity. The individual light (L) and heavy (H) chain subunits from the main and basic peaks were analyzed by reversed-phase (RP) HPLC/MS after disulfide bond reduction and cysteine alkylation. Three mAb isoforms were detected, and their modifications were localized to H chain. Bottom-up characterization using RP-HPLC/MS peptide mapping and accurate mass measurements identified three distinct H chain C-terminal peptides ending in glycine, lysine, or alpha-amidated proline. The combined analyses showed that the main WCX peak mAb isoform contained two unmodified L chains and two H chains terminating in glycine. Each mAb isoform that coeluted in the basic peak consisted of two unmodified L chain subunits and a single H chain ending in glycine, but the second H chain terminated in lysine for one isoform and alpha-amidated proline for another isoform. The WCX elution positions of the isoforms were consistent with their respective net charge. To the best of our knowledge, the occurrence of C-terminal alpha-amidation in mAbs has not been reported previously.

"Top Down" characterization is a complementary technique to peptide sequencing for identifying protein species in complex mixtures.

At present, mass spectrometry provides a rapid and sensitive means for making conclusive protein identifications from complex mixtures. Sequencing tryptic peptides derived from proteolyzed protein samples, also known as the "Bottom Up" approach, is the mass spectrometric gold standard for identifying unknowns. An alternative technology, "Top Down" characterization, is emerging as a viable option for protein identifications, which involves analyzing the intact unknowns for accurate mass and amino acid sequence tags. In this paper, both characterization methods were employed to more comprehensively differentiate two early-eluting peaks in a process-scale size-exclusion chromatography (SEC) step for a recombinant, immunoglobulin gamma-1 (IgG-1) fusion protein. The contents of each SEC peak were enzymatically digested, and the resulting peptides were mapped using reversed-phase (RP) HPLC-ion trap MS. Many low-level UV signals were observed among the fusion protein-related peptide peaks. These unknowns were collected, concentrated, and analyzed using nanoelectrospray (nanoES) collision-induced dissociation (CID) tandem (MS/MS) mass spectrometry for identification. The peptide sequencing experiments resulted in the identification of twenty host cell-related proteins. Following peptide mapping, the contents of the two SEC peaks were protein mass profiled using on-line RP HPLC coupled to a high-resolution, quadrupole time-of-flight (Qq/TOF) MS. Unknown proteins were also collected, concentrated, and dissociated using nanoES CID MS/MS. Intact protein CID experiments and accurate molecular weight information allowed for the identification of three full length host cell-derived proteins and numerous clips from these and additional proteins. The accurate molecular weight values allowed for the assignment of N- and C-terminal processing, which is difficult to conclusively access from peptide mapping data. The peptide-mapping experiments proved to be far more effective for making protein identifications from complex mixtures, whereas the protein mass profiling was useful for assessing modifications and distinguishing protein clips from full length species.