Development, validation, and implementation of a robust and quality control-friendly focused peptide mapping method for monitoring oxidation of co-formulated monoclonal antibodies.

Monoclonal antibody (mAb) coformulation containing two therapeutic proteins provides benefits of improved therapeutic efficacy and better patient compliance. Monitoring of the individual mAb stability in the coformulation is critical to ensure its quality and safety. Among post-translational modifications (PTMs), oxidation is often considered as one of the critical quality attributes (CQAs) as it potentially affects the structure and potency. Although hydrophobic interaction chromatography (HIC) and reversed phase liquid chromatography (RPLC) have been used to monitor overall protein oxidation, mass spectrometry of peptide digests resolved by LC methods can afford superior selectivity and sensitivity for specific PTMs. With the advent of the Quadrupole Dalton (QDa) mass spectrometer as an affordable add-on detector, implementation of targeted oxidation assays in development and quality control (QC) laboratories is now feasible. In this study, as the first effort to implement MS-based methods for antibody coformulation in QC laboratories, we developed and validated a high-throughput and robust focused peptide mapping method using QDa for simultaneous site-specific monitoring of oxidation of methionine and tryptophan residues in heavy-chain (HC) complementary determining regions (CDRs) of two co-formulated mAbs. The method was validated in terms of accuracy, precision, linearity, range, quantitation limit (QL), specificity, and solution stability per recommendations in ICH Q2. The method robustness was systematically assessed involving multiple sample preparation and instrument method parameters. The method met the validation criteria in GMP laboratories with excellent robustness and was implemented in both GMP and development environments.

Site-Selective Synthesis of Insulin Azides and Bioconjugates.

A synthetic method to access novel azido-insulin analogs directly from recombinant human insulin (RHI) was developed via diazo-transfer chemistry using imidazole-1-sulfonyl azide. Systematic optimization of reaction conditions led to site-selective azidation of amino acids B1-phenylalanine and B29-lysine present in RHI. Subsequently, the azido-insulin analogs were used in azide-alkyne [3 + 2] cycloaddition reactions to synthesize a diverse array of triazole-based RHI bioconjugates that were found to be potent human insulin receptor binders. The utility of this method was further demonstrated by the concise and controlled synthesis of a heterotrisubstituted insulin conjugate.

Nickel-Catalyzed Asymmetric Alkene Hydrogenation of α,ß-Unsaturated Esters: High-Throughput Experimentation-Enabled Reaction Discovery, Optimization, and Mechanistic Elucidation.

A highly active and enantioselective phosphine-nickel catalyst for the asymmetric hydrogenation of α,ß-unsaturated esters has been discovered. The coordination chemistry and catalytic behavior of nickel halide, acetate, and mixed halide-acetate with chiral bidentate phosphines have been explored and deuterium labeling studies, the method of continuous variation, nonlinear studies, and kinetic measurements have provided mechanistic understanding. Activation of molecular hydrogen by a trimeric (Me-DuPhos)3Ni3(OAc)5I complex was established as turnover limiting followed by rapid conjugate addition of a nickel hydride and nonselective protonation to release the substrate. In addition to reaction discovery and optimization, the previously unreported utility high-throughput experimentation for mechanistic elucidation is also described.

Ion mobility analysis of molecular dynamics.

The combination of mass spectrometry and ion mobility spectrometry (IMS) employing a temperature-variable drift cell or a drift tube divided into sections to make IMS-IMS experiments possible allows information to be obtained about the molecular dynamics of polyatomic ions in the absence of a solvent. The experiments allow the investigation of structural changes of both activated and native ion populations on a timescale of 1-100 ms. Five different systems representing small and large, polar and nonpolar molecules, as well as noncovalent assemblies, are discussed in detail: a dinucleotide, a sodiated polyethylene glycol chain, the peptide bradykinin, the protein ubiquitin, and two types of peptide oligomers. Barriers to conformational interconversion can be obtained in favorable cases. In other cases, solution-like native structures can be observed, but care must be taken in the experimental protocols. The power of theoretical modeling is demonstrated.

An IMS-IMS threshold method for semi-quantitative determination of activation barriers: Interconversion of proline cis↔trans forms in triply protonated bradykinin.

Collisional activation of selected conformations by multidimensional ion mobility spectrometry (IMS-IMS), combined with mass spectrometry (MS), is described as a method to determine semi-quantitative activation energies for interconversion of different structures of the nonapeptide bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). This analysis is based on a calibration involving collision-induced dissociation measurements of ions with known dissociation energies (i.e., "thermometer" ions) such as leucine enkephalin, BK, and amino acid-metal cation systems. The energetic barriers between six conformations of [BK+3H]3+ range from 0.23 ±0.01 to 0.55 ±0.03 eV. Prior results indicate that the major peaks in the IMS distributions correspond to specific combinations of cis and trans configurations of the three proline residues in the peptide sequence. The analysis allows us to directly assess pathways for specific transitions. The combination of structural assignments, experimentally determined barrier heights, onset of the quasi-equilibrium region, and dissociation threshold are used to derive a semi-quantitative potential energy surface for main features of [BK+3H]3+.

Cis-trans isomerizations of proline residues are key to bradykinin conformations.

A recent ion mobility-mass spectrometry (IM-MS) study of the nonapeptide bradykinin (BK, amino acid sequence Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)) found evidence for 10 populations of conformations that depend upon the solution composition [J. Am. Chem. Soc. 2011, 133, 13810]. Here, the role of the three proline residues (Pro(2), Pro(3), and Pro(7)) in establishing these conformations is investigated using a series of seven analogue peptides in which combinations of alanine residues are substituted for prolines. IM-MS distributions of the analogue peptides, when compared to the distribution for BK, indicate the multiple structures are associated with different combinations of cis and trans forms of the three proline residues. These data are used to assign the structures to different peptide populations that are observed under various solution conditions. The assignments also show the connectivity between structures when collisional activation is used to convert one state into another.

From solution to the gas phase: stepwise dehydration and kinetic trapping of substance P reveals the origin of peptide conformations.

Past experimental results and molecular dynamics simulations provide evidence that, under some conditions, electrospray ionization (ESI) of biomolecules produces ions that retain elements of solution phase structures. However, there is a dearth of information regarding the question raised by Breuker and McLafferty, "for how long, under what conditions, and to what extent, can solution structure be retained without solvent?" (Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 18145). Here, we use cryogenic ion mobility-mass spectrometry to experimentally probe the structural evolution of the undecapeptide substance P (SP) during the final stages of ESI. The results reveal that anhydrous SP conformers originate from evaporation of cluster ions, specifically, [SP + 2H](2+) (H2O)n (n = 0 to ∼50) and [SP + 3H](3+) (H2O)n (n = 0 to ∼30), and that major structural changes do not occur during the evaporative process. In the case of [SP + 3H](3+), the results demonstrate that a compact dehydrated conformer population can be kinetically trapped on the time scale of several milliseconds, even when an extended gas phase conformation is energetically favorable.

Analytical Performance Evaluation of Identity, Quality-Attribute Monitoring and new Peak Detection in a Platform Multi-Attribute Method Using Lys-C Digestion for Characterization and Quality Control of Therapeutic Monoclonal Antibodies.

The use of multi-attribute method (MAM) for identity and purity testing of biopharmaceuticals offers the ability to complement and replace multiple conventional analytical technologies with a single mass spectrometry (MS) method. Phase-appropriate method validation is one major consideration for the implementation of MAM in a current Good Manufacturing Practice (cGMP) environment. We developed a MAM workflow for therapeutic monoclonal antibodies (mAbs) with optimized sample preparation using lysyl endopeptidase (Lys-C) digestion. In this study, we evaluated the assay performances of this platform MAM workflow for identity, product quality attributes (PQAs) monitoring and new peak detection (NPD) for single and coformulated mAbs. An IgG4 mAb-1 and its coformulations were used as model molecules in this study. The assay performance evaluation demonstrated the full potential of the platform MAM approach for its intended use for characterization and quality control of single mAb-1 and mAb-1 in its coformulations. To the best of our knowledge, this is the first performance evaluation of MAM for mAb identity, PQA monitoring, and new peak detection (NPD) in a single assay, featuring 1) the first performance evaluation of MAM for PQA monitoring using Lys-C digestion with a high-resolution MS, 2) a new approach for mAb identity testing capable of distinguishing single mAb from coformulations using MAM, and 3) the performance evaluation of NPD for MAM with Lys-C digestion. The developed platform MAM workflow and the MAM performance evaluation paved the way for its GMP qualification and enabled clinical release of mAb-1 in GMP environment with MAM.

Number of solution states of bradykinin from ion mobility and mass spectrometry measurements.

Ion mobility and mass spectrometry measurements have been used to examine the populations of different solution structures of the nonapeptide bradykinin. Over the range of solution compositions studied, from 0:100 to 100:0 methanol:water and 0:100 to 90:10 dioxane:water, evidence for 10 independent populations of bradykinin structures in solution is found. In some solutions as many as eight structures may coexist. The solution populations are substantially different than the gas-phase equilibrium distribution of ions, which exhibits only three distinct states. Such a large number of coexisting structures explains the inability of traditional methods of characterization such as nuclear magnetic resonance spectroscopy and crystallography to determine detailed structural features for some regions of this peptide.

Identification of ortho-Substituted Benzoic Acid/Ester Derivatives via the Gas-Phase Neighboring Group Participation Effect in (+)-ESI High Resolution Mass Spectrometry.

Benzoic acid/ester/amide derivatives are common moieties in pharmaceutical compounds and present a challenge in positional isomer identification by traditional tandem mass spectrometric analysis. A method is presented for exploiting the gas-phase neighboring group participation (NGP) effect to differentiate ortho-substituted benzoic acid/ester derivatives with high resolution mass spectrometry (HRMS1). Significant water/alcohol loss (>30% abundance in MS1 spectra) was observed for ortho-substituted nucleophilic groups; these fragment peaks are not observable for the corresponding para and meta-substituted analogs. Experiments were also extended to the analysis of two intermediates in the synthesis of suvorexant (Belsomra) with additional analysis conducted with nuclear magnetic resonance (NMR), density functional theory (DFT), and ion mobility spectrometry-mass spectrometry (IMS-MS) studies. Significant water/alcohol loss was also observed for 1-substituted 1, 2, 3-triazoles but not for the isomeric 2-substituted 1, 2, 3-triazole analogs. IMS-MS, NMR, and DFT studies were conducted to show that the preferred orientation of the 2-substituted triazole rotamer was away from the electrophilic center of the reaction, whereas the 1-subtituted triazole was oriented in close proximity to the center. Abundance of NGP product was determined to be a product of three factors: (1) proton affinity of the nucleophilic group; (2) steric impact of the nucleophile; and (3) proximity of the nucleophile to carboxylic acid/ester functional groups. Graphical Abstract ᅟ.

Semi-automated screen for global protein conformational changes in solution by ion mobility spectrometry-massspectrometry combined with size-exclusion chromatography and differential hydrogen-deuterium exchange.

Development of methodologies for studying protein higher-order structure in solution helps to establish a better understanding of the intrinsic link between protein conformational structure and biological function and activity. The goal of this study was to demonstrate a simultaneous screening approach for global protein conformational changes in solution through the combination of ion mobility spectrometry-mass spectrometry (IMS-MS) with differential hydrogen-deuterium exchange (ΔHDX) on the size-exclusion chromatography (SEC) platform in a single on-line workflow. A semi-automated experimental setup based on the use of SEC on-column conditions allowed for tracking of protein conformational changes in solution as a function of acetonitrile concentration. In this setup, the SEC protein elution data was complemented by the ΔHDX profile which showed global protein conformational changes as a difference in the number of deuterons exchanged to protons. The ΔHDX data, in turn, was complemented by the changes in the drift time by IMS-MS. All three orthogonal techniques were applied for studying global higher-order structure of the proteins ubiquitin, cytochrome c and myoglobin, in solution simultaneously. The described approach allows for the use of a crude sample (or mixture of proteins) and could be suitable for rapid comparison of protein batch-to-batch higher-order structure or for optimizing conditions for enzymatic reactions.

Cis→Trans Isomerization of Pro(7) in Oxytocin Regulates Zn(2+) Binding.

Ion mobility/mass spectrometry techniques are employed to investigate the binding of Zn(2+) to the nine-residue peptide hormone oxytocin (OT, Cys(1)-Tyr(2)-Ile(3)-Gln(4)-Asn(5)-Cys(6)-Pro(7)-Leu(8)-Gly(9)-NH2, having a disulfide bond between Cys(1) and Cys(6) residues). Zn(2+) binding to OT is known to increase the affinity of OT for its receptor [Pearlmutter, A. F., Soloff, M. S.: Characterization of the metal ion requirement for oxytocin-receptor interaction in rat mammary gland membranes. J. Biol. Chem. 254, 3899-3906 (1979)]. In the absence of Zn(2+), we find evidence for two primary OT conformations, which arise because the Cys(6)-Pro(7) peptide bond exists in both the trans- and cis-configurations. Upon addition of Zn(2+), we determine binding constants in water of KA = 1.43 ± 0.24 and 0.42 ± 0.12 µM(-1), for the trans- and cis-configured populations, respectively. The Zn(2+) bound form of OT, having a cross section of Ω = 235 Å(2), has Pro(7) in the trans-configuration, which agrees with a prior report [Wyttenbach, T., Liu, D., Bowers, M. T.: Interactions of the hormone oxytocin with divalent metal ions. J. Am. Chem. Soc. 130, 5993-6000 (2008)], in which it was proposed that Zn(2+) binds to the peptide ring and is further coordinated by interaction of the C-terminal, Pro(7)-Leu(8)-Gly(9)-NH2, tail. The present work shows that the cis-configuration of OT isomerizes to the trans-configuration upon binding Zn(2+). In this way, the proline residue regulates Zn(2+) binding to OT and, hence, is important in receptor binding. Graphical Abstract ᅟ.

From solution to the gas phase: factors that influence kinetic trapping of substance P in the gas phase.

Substance P (RPKPQQFFGLM-NH2) [M + 3H](3+) ions have been shown to exist as two conformers: one that is kinetically trapped and one that is thermodynamically more stable and therefore energetically preferred. Molecular dynamics (MD) simulations suggested that the kinetically trapped population is stabilized by interactions between the charge sites and the polar side chains of glutamine (Q) located at positions 5 and 6 and phenylalanine (F) located at positions 7 and 8. Here, the individual contributions of these specific intramolecular interactions are systematically probed through site-directed alanine mutations of the native amino acid sequence. Ion mobility spectrometry data for the mutant peptide ions confirm that interactions between the charge sites and glutamine/phenylalanine (Q/F) side chains afford stabilization of the kinetically trapped ion population. In addition, experimental data for proline-to-alanine mutations at positions 2 and 4 clearly show that interactions involving the charge sites and the Q/F side chains are altered by the cis/trans orientations of the proline residues and that mutation of glycine to proline at position 9 supports results from MD simulations suggesting that the C-terminus also provides stabilization of the kinetically trapped conformation.

Conformation types of ubiquitin [M+8H]8+ Ions from water:methanol solutions: evidence for the N and A States in aqueous solution.

Ion mobility and mass spectrometry measurements are used to examine the gas-phase populations of [M+8H](8+) ubiquitin ions formed upon electrospraying 20 different solutions from 100:0 to 5:95 water:methanol that are maintained at pH = 2.0. Over this range of solution conditions, mobility distributions for the +8 charge state show substantial variations. Here we develop a model that treats the combined measurements as one data set. By varying the relative abundances of a discrete set of conformation types, it is possible to represent distributions obtained from any solution. For solutions that favor the well-known A-state ubiquitin, it is possible to represent the gas-phase distributions with seven conformation types. Aqueous conditions that favor the native structure require four more structural types to represent the distribution. This analysis provides the first direct evidence for trace amounts of the A state under native conditions. The method of analysis presented here should help illuminate how solution populations evolve into new gas-phase structures as solvent is removed. Evidence for trace quantities of previously unknown states under native solution conditions may provide insight about the relationship of dynamics to protein function as well as misfolding and aggregation phenomena.

Differentiation of compact and extended conformations of di-ubiquitin conjugates with lysine-specific isopeptide linkages by ion mobility-mass spectrometry.

Modification of ubiquitin, a key cellular regulatory polypeptide of 76 amino acids, to polyubiquitin conjugates by lysine-specific isopeptide linkage at one of its seven lysine residues has been recognized as a central pathway determining its biochemical properties and cellular functions. Structural details and differences of distinct lysine-isopeptidyl ubiquitin conjugates that reflect their different functions and reactivities, however, are only partially understood. Ion mobility spectrometry (IMS) combined with mass spectrometry (MS) has recently emerged as a powerful tool for probing conformations and topology involved in protein interactions by an electric field-driven separation of polypeptide ions through a drift gas. Here we report the conformational characterization and differentiation of Lys63- and Lys48-linked ubiquitin conjugates by IMS-MS. Lys63- and Lys48-linked di-ubiquitin conjugates were prepared by recombinant bacterial expression and by chemical synthesis using a specific chemical ligation strategy, and characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry, circular dichroism spectroscopy, and molecular modeling. IMS-MS was found to be an effective tool for the identification of structural differences of ubiquitin complexes in the gas phase. The comparison of collision cross-sections of Lys63- and Lys48-linked di-ubiquitin conjugates showed a more elongated conformation of Lys63-linked di-ubiquitin. In contrast, the Lys48-linked di-ubiquitin conjugate showed a more compact conformation. The IMS-MS results are consistent with published structural data and a comparative molecular modeling study of the Lys63- and Lys48-linked conjugates. The results presented here suggest IMS techniques can provide information that complements MS measurements in differentiating higher-order polyubiquitins and other isomeric protein linkages.

Evidence for a quasi-equilibrium distribution of states for bradykinin [M + 3H]3+ ions in the gas phase.

Multidimensional ion mobility spectrometry coupled with mass spectrometry (IMS-IMS-MS) techniques are used to select and activate six different gas-phase conformations of bradykinin [M + 3H](3+) ions. Drift time distributions as a function of activation voltage show that at low voltages selected structures undergo conformational transitions in what appears to be a pathway dependent fashion. Over a relatively wide range of intermediate activation voltages a distribution of states that is independent of the initial conformation selected for activation (as well as the activation voltage in this intermediate region) is established. This distribution appears to represent an equilibrium distribution of gas-phase structures that is reached prior to the energy required for dissociation. Establishment of a quasi-equilibrium prior to dissociation results in identical dissociation patterns for different selected conformations. A discussion of the transition from solution-like to gas-phase structures is provided.