Implementing reversed-phase and hydrophilic interaction liquid chromatography into the characterization of DTPA-ramucirumab conjugate before radiolabeling.

Radioimmunoconjugates represent a promising class of therapeutics and diagnostics. The characterization of intermediate chelator-antibody products, i.e., without the radionuclide, is frequently omitted, bringing significant uncertainty in the radioimmunoconjugate preparation. In the present study, we explored the utility of reversed-phase (RPLC) and hydrophilic interaction (HILIC) liquid chromatography with UV detection to characterize ramucirumab stochastically conjugated with p-SCN-Bn-CHX-A"-DTPA chelator (shortly DTPA). The conjugation was well reflected in RPLC chromatograms, while chromatograms from HILIC were significantly less informative. RPLC analyses at the intact level confirmed that the conjugation resulted in a heterogeneous mixture of modified ramucirumab. Moreover, the RPLC of DTPA-ramucirumab confirmed heterogeneous conjugation of all subunits. The peptide mapping did not reveal substantial changes after the conjugation, indicating that most parts of ramucirumab molecules remained unmodified and that the DTPA chelator was bound to various sites. Eventually, the RPLC method for analysis of intact ramucirumab was successfully applied to online monitoring of conjugation reaction in 1 h intervals for a total of 24 h synthesis, which readily reflected the structural changes of ramucirumab in the form of retention time shift by 0.21 min and increase in peak width by 0.22 min. The results were obtained in real-time, practically under 10 min per monitoring cycle. To the best of our knowledge, our study represents the first evaluation of RPLC and HILIC to assess the quality of intermediates during the on-site preparation of radioimmunoconjugates prior to radiolabeling.

Easy, Robust, and Repeatable Online Acid Cleavage of Proteins in Mobile Phase for Fast Quantitative LC-MS Bottom-Up Protein Analysis─Application for Ricin Detection.

Sample preparation involving the cleavage of proteins into peptides is the first critical step for successful bottom-up proteomics and protein analyses. Time- and labor-intensiveness are among the bottlenecks of the commonly used methods for protein sample preparation. Here, we report a fast online method for postinjection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics. The chemical cleavage is achieved in 0.1% formic acid within 35 s in a capillary heated to 195 °C installed upstream of the analytical column, enabling the generated peptides to be separated. The peptides generated by the optimized method covered the entire sequence except for one amino acid of trastuzumab used for the method development. The qualitative results are extraordinarily stable, even over a long period of time. Moreover, the method is also suitable for accurate and repeatable quantification. The procedure requires only one manual step, significantly decreasing sample transfer losses. To demonstrate its practical utility, we tested the method for the fast detection of ricin. Ricin can be unambiguously identified from an injection of 10 ng, and the results can be obtained within 7-8 min after receiving a suspicious sample. Because no sophisticated accessories and no additional reagents are needed, the method can be seamlessly transferred to any laboratory for high-throughput proteomic workflows.

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial.

The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

Evaluation of strategies for overcoming trifluoroacetic acid ionization suppression resulted in single-column intact level, middle-up, and bottom-up reversed-phase LC-MS analyses of antibody biopharmaceuticals.

A wide range of strategies for efficient chromatography and high MS sensitivity in reversed-phase LC-MS analysis of antibody biopharmaceuticals and their large derivates has been evaluated. They included replacing trifluoroacetic acid with alternative acidifiers, relevancy of elevated column temperature, use of dedicated stationary phases, and counteraction of the suppression effect of trifluoroacetic acid in electrospray ionization. At the column temperature of 60 °C, which significantly reduces in-column protein degradation, the BioResolve RP mAb Polyphenyl, BioShell IgG C4 columns performed best using mobile phases with full or partial replacement of trifluoroacetic acid with difluoroacetic acid in the analysis of intact antibodies. Similarly, 0.03% trifluoroacetic acid in combination with 0.07% formic acid is a good alternative in analyzing antibody chains at 60 °C. Collectively, the addition of 3% 1-butanol to the mobile phase acidified with 0.1% formic acid was the most efficient approach to simultaneously achieving good chromatographic separation and MS sensitivity for intact and reduced antibody biopharmaceuticals. Moreover, this mobile phase combined with the BioResolve RP mAb Polyphenyl column was subsequently demonstrated to provide excellent results for peptide mapping of antibody biopharmaceuticals fully comparable with those obtained using a state-of-the-art column for peptide separation, thus opening an avenue for a single-column multilevel analysis of these biotherapeutics.