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Peptide orientation affects selectivity in ion-exchange chromatography.

ABSTRACT: Here we demonstrate that separation of proteolytic peptides, having the same net charge and one basic residue, is affected by their specific orientation toward the stationary phase in ion-exchange chromatography. In electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with an anion-exchange material, the C-terminus of the peptides is, on average, oriented toward the stationary phase. In cation exchange, the average peptide orientation is the opposite. Data with synthetic peptides, serving as orientation probes, indicate that in tryptic/Lys-C peptides the C-terminal carboxyl group appears to be in a zwitterionic bond with the side chain of the C-terminal Lys/Arg residue. In effect, the side chain is then less basic than the N-terminus, accounting for the specific orientation of tryptic and Lys-C peptides. Analyses of larger sets of peptides, generated from lysates by either Lys-N, Lys-C, or trypsin, reveal that specific peptide orientation affects the ability of charged side chains, such as phosphate residues, to influence retention. Phosphorylated residues that are remote in the sequence from the binding site affect retention less than those that are closer. When a peptide contains multiple charged sites, then orientation is observed to be less rigid and retention tends to be governed by the peptide's net charge rather than its sequence. These general observations could be of value in confirming a peptide's identification and, in particular, phosphosite assignments in proteomics analyses. More generally, orientation accounts for the ability of chromatography to separate peptides of the same composition but different sequence.

SUBMITTER: Alpert AJ

PROVIDER: S-EPMC2884984 | biostudies-other | 2010 Jun

REPOSITORIES: biostudies-other

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Project description:Charge variants are important attributes of monoclonal antibodies, including antibody-drug conjugates (ADCs), because charge variants can potentially influence the stability and biological activity of these molecules. Ion exchange chromatography (IEX) is widely used for charge variants analysis of mAbs and offers the feasibility of fractionation for in-depth characterization. However, the conjugated linker-drug on ADCs could potentially affect the separation performance of IEX, considering IEX separation relies on surface charge distribution of analyte and involves the interaction between analyte surface and IEX stationary phase. Here, we investigated weak cation exchange chromatography (WCX) for its application in analyzing three ADCs (two broad distribution ADCs and an ADC with controlled conjugation sites) and the 2-drug/4-drug loaded species isolated from the two broad distribution ADCs using hydrophobic interaction chromatography. The major peaks in WCX profile were characterized via fraction collection followed by capillary electrophoresis-sodium dodecyl sulfate or peptide mapping. Results suggested that both the number of drug loads and conjugation sites could impact WCX separation of an ADC. The hypothesis was that the linker drugs could interfere with the ionic interaction between its surrounding amino acids on the mAb surface and column resin, which reduced the retention of ADCs on WCX column in this study. Our results further revealed that WCX brings good selectivity towards positional isomers, but limited resolution for different drug load, which causes the peak compositions of the two broad-distribution ADCs to be highly complex. We also compared results from WCX and imaged capillary isoelectric focusing (icIEF). Results showed that separation in icIEF was less influenced by conjugated linker drugs for the ADCs studied in this work, and better alignment was found between the two techniques for the ADC with controlled conjugate sites. Overall, this work provides insights into the complexity of WCX analysis of ADCs, which should be considered during method development and sample characterization.

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Peptide orientation affects selectivity in ion-exchange chromatography.

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