Automated High-Throughput Matrix Assisted Laser Desorption Ionization Mass Spectrometry Methodology for Formulation Assessment of Polyethylene-Glycol-Conjugated Cytokine Proteins.

Biological therapeutics are major contributors to the pharmaceutical pipeline and continue to grow in sales and scope. Additionally, the field's understanding of cancer biology has advanced such that biopharmaceuticals can harness the power of the immune system for oncology treatments. Several of these novel therapeutics are engineered versions of naturally occurring proteins designed to improve therapeutic properties including potency, target engagement and half-life extension. Cytokines, such as interferons and interleukins, are a broad class of signaling proteins which modulate the body's immune response; engineered cytokines have entered the clinic as promising new immuno-oncology therapies. While these therapies hold great promise, their additional structural complexity introduces analytical challenges, and traditional analytical platforms may be ill-suited to effectively assess product development risks. Further, the pharmaceutical industry relies on streamlining approaches for high-throughput experimentation to achieve speed and efficiency for the discovery and development of new modalities. These demands necessitate the use of state-of-the-art techniques to rapidly characterize these new modalities and guide process development and optimization. Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) is a rapid, sensitive and automatable technique amenable for high-throughput analysis of proteins. In this work, we have developed an automated MALDI-MS platform to prepare, acquire and analyze molecular degradation in engineered PEGylated cytokines formulation samples. This orthogonal technique integrated seamlessly with current developability risk assessment workflows, ultimately enabling selection of a final formulation strategy for clinical development.

High-throughput infrared spectroscopy for quantification of peptides in drug discovery.

Peptides have gained an increasing importance in drug discovery as potential therapeutics. Discovery efforts toward finding new, efficacious peptide-based therapeutics have increased the throughput of peptide development, allowing the rapid generation of unique and pure peptide samples. However, high-throughput analysis of peptides may be still challenging and can encumber a high-throughput drug discovery campaign. We report herein a fit-for-purpose method to quantify peptide concentrations using high-throughput infrared spectroscopy (HT-IR). Through the development of this method, multiple critical method parameters were optimized including solvent composition, droplet deposition size, plate drying procedures, sample concentration, and internal standard. The relative absorbance of the amide region (1600-1750 cm-1) to the internal standard, K3Fe(CN)6 (2140 cm-1), was determined to be most effective at providing lowest interference for measuring peptide concentration. The best sample deposition was achieved by dissolving samples in a 50:50 v/v allyl alcohol/water mixture. The developed method was used on 96-well plates and analyzed at a rate of 22 min per plate. Calibration curves to measure sample concentration versus response relationship displayed sufficient linearity (R2 > 0.95). The repeatability and scope of detection was demonstrated with eighteen peptide samples that were measured with most values below 20% relative standard deviation. The linear dynamic range of the method was determined to be between 1 and 5 mg/mL. This developed HT-IR methodology could be a useful tool in peptide drug candidate lead identification and optimization processes.

Solid-to-Liposome Conformational Transition of Phosphatidylcholine and Phosphatidylserine Probed by Atomic Force Microscopy, Infrared Spectroscopy, and Density Functional Theory Calculations.

Liposomes are emerging therapeutic formulations for site-specific delivery of chemotherapeutic drugs. The efficiency and selectivity of drug delivery by these carriers largely rely on their surface properties, shape, and size. There is a growing demand for analytical approaches that can be used for structural and morphological characterization of liposomes at the single-vesicle level. AFM-IR is a modern optical nanoscopic technique that combines the advantages of scanning probe microscopy and infrared spectroscopy. Our findings show that AFM-IR can be used to probe conformational changes in phospholipids that take place upon their assembly into liposomes. Such conclusions can be made based on the corresponding changes in intensities of the lipid vibrational bands as the molecules transition from a solid state into large unilamellar vesicles (LUVs). This spectroscopic analysis of LUV formation together with density functional theory calculations also reveals the extent to which the molecular conformation and local environment of the functional groups alter the AFM-IR spectra of phospholipids. Using melittin as a test protein, we also examined the extent to which LUVs can be used for protein internalization. We found that melittin enters LUVs nearly instantaneously, which protects it from possible structural modifications that are caused by a changing environment. This foundational work empowers AFM-IR analysis of liposomes and opens new avenues for determination of the molecular mechanisms of liposome-drug interactions.

Rapid label-free cell-based Approach Membrane Permeability Assay using MALDI-hydrogen-deuterium exchange mass spectrometry for peptides.

Peptide therapeutics are a growing modality in the pharmaceutical industry and expanding these therapeutics to hit intracellular targets would require establishing cell permeability. Rapid measurement target-agnostic cell permeability of peptides is still analytically challenging. In this study, we demonstrate the development of a rapid high-throughput label-free methodology based on a MALDI-hydrogen-deuterium exchange mass spectrometry (MALDI-HDX-MS) approach to rank-order peptide cell membrane permeability using live THP-1 and AsPc-1 cells. Peptides were incubated in the presence of live cells and their permeability into the cells over time was measured by MALDI-HDX-MS. A differential hydrogen-deuterium exchange approach was used to distinguish the peptides outside of the cells from those inside. The peptides on the outside of the cells were labeled using sufficiently short exposure to deuterium oxide, while the peptides inside of the cells were protected from labeling as a result of permeation into the cells. The deuterium labeled and peak area ratios of unlabeled peptides were compared and plotted over time. The developed methodology, referred to as Cell-based Approach Membrane Permeability Assay (CAMPA), was applied to study an array of 24 diverse peptides including cell-penetrating peptides, stapled and macrocyclic peptides. The cell membrane permeability results observed by CAMPA were corroborated by previously reported in literature data. The CAMPA MALDI-MS analysis was fully automated including MS data processing using internally developed Python scripts. Moreover, CAMPA was demonstrated to be useful for differentiating passive and active cell transportation by using an endocytosis inhibitor in cell incubation media for selected peptides.

A chemoenzymatic strategy for site-selective functionalization of native peptides and proteins.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

Rapid antibody conformational screening by matrix-assisted laser desorption/ionization hydrogen-deuterium exchange mass spectrometry.

Recent advances in the field of cancer biology have accelerated the discovery and development of novel biopharmaceuticals. At the forefront of these drug development efforts are high-throughput screening, compressed timelines, and limited sample quantities, all characteristic of the discovery space. To meet program targets, large numbers of protein variants must be produced, screened, and characterized, presenting a daunting analytical challenge. Additionally, the higher-order structure is paramount for protein function and must be monitored as a critical quality attribute. Matrix-assisted laser desorption/ionization mass spectrometry has been utilized as an ultra-fast, automatable, sample-sparing analytical tool for biomolecules. Our group has published applications integrating hydrogen-deuterium exchange mass spectrometry with matrix-assisted laser desorption/ionization mass spectrometry for the rapid conformational characterization of small proteins, the current work expands this application to monoclonal and bi-specific antibodies. This study demonstrates the ability of the methodology, matrix-assisted laser desorption/ionization hydrogen-deuterium exchange mass spectrometry, to detect conformational differences between bi-specific antibodies from different expression hosts. These conformational differences were validated by orthogonal techniques including circular dichroism, nuclear magnetic resonance, and size-exclusion chromatography hydrogen-deuterium exchange mass spectrometry. This work demonstrates the utility of applying the developed methodology as a rapid conformational screening tool to triage samples for further analytical characterization.

Pd Reaction Intermediates in Suzuki-Miyaura Cross-Coupling Characterized by Mass Spectrometry.

Palladium-catalyzed Suzuki-Miyaura (SM) coupling is widely utilized in the construction of carbon-carbon bonds. In this study, nanoelectrospray ionization mass spectrometry (nanoESI-MS) is applied to simultaneously monitor precatalysts, catalytic intermediates, reagents, and products of the SM cross-coupling reaction of 3-Br-5-Ph-pyridine and phenylboronic acid. A set of Pd cluster ions related to the monoligated Pd (0) active catalyst is detected, and its deconvoluted isotopic distribution reveals contributions from two neutral molecules. One is assigned to the generally accepted Pd(0) active catalyst, seen in MS as the protonated molecule, while the other is tentatively assigned to an oxidized catalyst which was found to increase as the reaction proceeds. Oxidative stress testing of a synthetic model catalyst 1,5-cyclooctadiene Pd XPhos (COD-Pd-XPhos) performed using FeCl3 supported this assignment. The formation and conversion of the oxidative addition intermediate during the catalytic cycle was monitored to provide information on the progress of the transmetalation step.

Trapping-Enrichment Multi-dimensional Liquid Chromatography with On-Line Deuterated Solvent Exchange for Streamlined Structure Elucidation at the Microgram Scale.

At the forefront of chemistry and biology research, development timelines are fast-paced and large quantities of pure targets are rarely available. Herein, we introduce a new framework, which is built upon an automated, online trapping-enrichment multi-dimensional liquid chromatography platform (TE-Dt-mDLC) that enables: 1) highly efficient separation of complex mixtures in a first dimension (1 D-UV); 2) automated peak trapping-enrichment and buffer removal achieved through a sequence of H2 O and D2 O washes using an independent pump setup; and 3) a second dimension separation (2 D-UV-MS) with fully deuterated mobile phases and fraction collection to minimize protic residues for immediate NMR analysis while bypassing tedious drying processes and minimizing analyte degradation. Diverse examples of target isolation and characterization from organic synthesis and natural product chemistry laboratories are illustrated, demonstrating recoveries above 90 % using as little as a few micrograms of material.

Enantioselective UHPLC Screening Combined with In Silico Modeling for Streamlined Development of Ultrafast Enantiopurity Assays.

Enantioselective chromatography has been the preferred technique for the determination of enantiomeric excess across academia and industry. Although sequential multicolumn enantioselective supercritical fluid chromatography screenings are widespread, access to automated ultra-high-performance liquid chromatography (UHPLC) platforms using state-of-the-art small particle size chiral stationary phases (CSPs) is an underdeveloped area. Herein, we introduce a multicolumn UHPLC screening workflow capable of combining 14 columns (packed with sub-2 µm fully porous and sub-3 µm superficially porous particles) with nine mobile phase eluent choices. This automated setup operates under a vast selection of reversed-phase liquid chromatography, hydrophilic interaction liquid chromatography, polar-organic mode, and polar-ionic mode conditions with minimal manual intervention and high success rate. Examples of highly efficient enantioseparations are illustrated from the integration of chiral screening conditions and computer-assisted modeling. Furthermore, we describe the nuances of in silico method development for chiral separations via second-degree polynomial regression fit using LC simulator (ACD/Labs) software. The retention models were found to be very accurate for chiral resolution of single and multicomponent mixtures of enantiomeric species across different types of CSPs, with differences between experimental and simulated retention times of less than 0.5%. Finally, we illustrate how this approach lays the foundation for a streamlined development of ultrafast enantioseparations applied to high-throughput enantiopurity analysis and its use in the second dimension of two-dimensional liquid chromatography experiments.

Development of ProTx-II Analogues as Highly Selective Peptide Blockers of Nav1.7 for the Treatment of Pain.

Inhibitor cystine knot peptides, derived from venom, have evolved to block ion channel function but are often toxic when dosed at pharmacologically relevant levels in vivo. The article describes the design of analogues of ProTx-II that safely display systemic in vivo blocking of Nav1.7, resulting in a latency of response to thermal stimuli in rodents. The new designs achieve a better in vivo profile by improving ion channel selectivity and limiting the ability of the peptides to cause mast cell degranulation. The design rationale, structural modeling, in vitro profiles, and rat tail flick outcomes are disclosed and discussed.

Interrogation of solution conformation of complex macrocyclic peptides utilizing a combined SEC-HDX-MS, circular dichroism, and NMR workflow.

Recent technological and synthetic advances have led to a resurgence in the exploration of peptides as potential therapeutics. Understanding peptide conformation in both free and protein-bound states remains one of the most critical areas for successful development of peptide drugs. In this study it was demonstrated that the combination of Size-Exclusion Chromatography with Hydrogen-Deuterium Exchange Mass Spectrometry (SEC-HDX-MS) and Circular Dichroism Spectroscopy (CD) can be used to guide the selection of peptides for further NMR analysis. Moreover, the insights from this workflow guide the choice of the best biologically relevant conditions for NMR conformational studies of peptide ligands in a free state in solution. Combined information about solution conformation character and stability across temperatures and co-solvent compositions greatly expedites selection of optimal conditions for NMR analysis. In total, the combination of SEC-HDX-MS, CD, and NMR into a single complementary workflow greatly accelerates conformational analysis of peptides in the drug discovery lead optimization process.

Natural Trienoic Acids as Anticancer Agents: First Stereoselective Synthesis, Cell Cycle Analysis, Induction of Apoptosis, Cell Signaling and Mitochondrial Targeting Studies.

The first Z-stereoselective method was developed for the synthesis of unsaturated acids containing a 1Z,5Z,9Z-triene moiety in 61-64% yields using the new Ti-catalyzed cross-coupling of oxygen-containing and aliphatic 1,2-dienes as the key synthetic step. It was shown for the first time that trienoic acids with non-methylene-interrupted Z-double bonds show moderate cytotoxic activities against tumor cell lines (Jurkat, K562, U937, HL60, HeLa), human embryonic kidney cells (Hek293), normal fibroblasts and human topoisomerase I (hTop1) inhibitory activity in vitro. The synthesized acids efficiently initiate apoptosis of Jurkat tumor cells, with the cell death mechanism being activated by the mitochondrial pathway. A probable mechanism of topoisomerase I inhibition was also hypothesized on the basis of in silico studies resorting to docking. The activation and inhibition of the most versatile intracellular signaling pathways (CREB, JNK, NFkB, p38, ERK1/2, Akt, p70S6K, STAT3 and STAT5 tyrosine kinases) responsible for cell proliferation and for initiation of apoptosis were studied by multiplex assay technology (Luminex xMAP).

In silico method development for the reversed-phase liquid chromatography separation of proteins using chaotropic mobile phase modifiers.

Recent advances in biomedical and pharmaceutical processes has enabled a notable increase of protein- and peptide-based drug therapies and vaccines that often contain a higher-order structure critical to their efficacy. Hyphenation of chromatographic and spectrometric techniques is at the center of all facets of biopharmaceutical analysis, purification and chemical characterization. Although computer-assisted chromatographic modeling of small molecules has reached a mature stage across the pharmaceutical industry, software-based method optimization approaches for large molecules has yet to see the same revitalization. Conformational changes of biomolecules under chromatographic conditions have been identified as the major culprit in terms of sub-optimal modeling outcomes. In order to circumvent these challenges, we herein investigate the outcomes generated via computer-assisted modeling from using different chaotropic and denaturing mobile phases (trifluoroacetic acid, sodium perchlorate and guanidine hydrochloride in acetonitrile/water-based eluents). Linear and polynomial regression retention models using ACD/Labs software were built as a function of gradient slope, column temperature and mobile phase buffer for eight different model proteins ranging from 12 to 670 kDa (holo-transferrin, cytochrome C, apomyoglobin, ribonuclease A, ribonuclease A type I-A, albumin, y-globulin and thyroglobulin bovine). Correlation between experimental and modeled outputs was substantially improved by using strong chaotropic and denaturing modifiers in the mobile phase, even when using linear regression modeling as typically observed for small molecules. On the contrary, the use of conventional TFA buffer concentrations at low column temperatures required the used of polynomial regression modeling indicating potential conformational structure changes of proteins upon chromatographic conditions. In addition, we illustrate the power of modern computer-assisted chromatography modeling combined with chaotropic agents in the developing of new RPLC assays for protein-based therapeutics and vaccines.

Ultra-high-throughput SPE-MALDI workflow: Blueprint for efficient purification and screening of peptide libraries.

At the forefront of synthetic endeavors in the pharmaceutical industry, including drug discovery and high-throughput screening, timelines are tight and large quantities of pure chemical targets are rarely available. In this regard, the development of novel and increasingly challenging chemistries requires a commensurate level of innovation to develop reliable analytical assays and purification workflows with rapid turnaround that enables accelerated pharmacological evaluation. A small-scale automation platform enabling high-throughput analysis and purification to streamline the selection of candidate leads would be a transformative advance. Herein, we introduce an automation-friendly solid-phase extraction-matrix-assisted laser desorption/ionization (SPE-MALDI) platform applied to the high-throughput purification and analysis of peptide libraries. This advance enabled us to purify peptides from microgram levels in less than a day with results comparable to traditional high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS).

New synthetic analogues of natural 5Z,9Z-dienoic acids: Stereoselective synthesis and study of the anticancer activity.

A stereoselective method was developed for the synthesis of synthetic analogues of natural 5Z,9Z-dienoic acids by esterification of aliphatic and aromatic alcohols and carboxylic acids with (5Z,9Z)-1,14-tetradeca-5,9-dienedioic acid and (5Z,9Z)-1,14-tetradeca-5,9-dienediol, synthesized by Ti-catalyzed homo-cyclomagnesiation of the tetrahydropyran ether of hepta-5,6-dien-1-ol with Grignard reagents. In order to establish the effect of molecular structure on the antitumor activity, the obtained 5Z,9Z-dienoic acids were tested for the inhibitory activity against human topoisomerase I, the cytotoxic activity in vitro against several cancer and normal cell lines (Jurkat, HL-60, K562, U937, fibroblasts), the effect on the cell cycle, and apoptosis-inducing ability using flow cytofluorometry. In addition, the effect of the synthesized acids on the cancer cell production of some phosphorylated and unphosphorylated proteins responsible for proliferation and apoptosis was studied by a new multiplex assay technology, MAGPIX.

Total Synthesis of Natural Lembehyne C and Investigation of Its Cytotoxic Properties.

The first Z-stereoselective method for the synthesis of the natural marine alkynol lembehyne C, containing a 1Z,5Z,9Z-triene moiety, in 41% yield was developed using the new Ti-catalyzed cross-coupling of oxygenated and aliphatic 1,2-dienes as the key step. It was found for the first time that lembehyne C exhibits moderate cytotoxicity against Jurkat, K562, U937, and HL60 cancer cells and also efficiently induces apoptosis in Jurkat cells, with the cell death mechanism being activated by the mitochondrial pathway. The lembehyne C inhibition of the cell cycle follows the mitotic catastrophe mechanism.

Use of Raman spectroscopy and size-exclusion chromatography coupled with HDX-MS spectroscopy for studying conformational changes of small proteins in solution.

Protein-based drugs are a relatively new paradigm in modern therapeutics. These large molecule drugs often have much higher specificity and selectivity compared to small molecules that have been used in therapeutics for centuries. However, there are many analytical challenges associated with drug discovery and development of these new modalities. One of these analytical challenges concerns fast and robust assessment of peptides or small protein conformational structures in solution. In this study, we report a novel analytical approach that is based on Raman spectroscopy (RS) and size exclusion chromatography-hydrogen-deuterium exchange-mass spectrometry (SEC-HDX-MS) for probing conformational structures of proteins in solution. Specifically, we demonstrate that RS and SEC-HDX-MS can be used to probe temperature-induced changes in ubiquitin and insulin. We also show that a combination of these techniques provides a more comprehensive analysis and comparison of peptide or small protein conformational structures than by any one technique. Our results demonstrate that RS and SEC-HDX-MS allow for elucidation of sequential transformations in α-helix and ß-sheet content of these proteins. These findings suggest that the proposed approach can be used for a fast investigation of changes in protein or peptide secondary structures under different solution conditions.

Comprehensive online multicolumn two-dimensional liquid chromatography-diode array detection-mass spectrometry workflow as a framework for chromatographic screening and analysis of new drug substances.

The analysis of complex mixtures of closely related species is quickly becoming a bottleneck in the development of new drug substances, reflecting the ever-increasing complexity of both fundamental biology and the therapeutics used to treat disease. Two-dimensional liquid chromatography (2D-LC) is emerging as a powerful tool to achieve substantial improvements in peak capacity and selectivity. However, 2D-LC suffers from several limitations, including the lack of automated multicolumn setups capable of combining multiple columns in both dimensions. Herein, we report an investigation into the development and implementation of a customized online comprehensive multicolumn 2D-LC-DAD-MS setup for screening and method development purposes, as well as analysis of multicomponent biopharmaceutical mixtures. In this study, excellent chromatographic performance in terms of selectivity, peak shape, and reproducibility were achieved by combining reversed-phase (RP), strong cation exchange (SCX), strong anion exchange (SAX), and size exclusion chromatography (SEC) using sub-2-µm columns in the first dimension in conjunction with several 3.0 mm × 50 mm RP columns packed with sub-3-µm fully porous particles in the second dimension. Multiple combinations of separation modes coupled to UV and MS detection are applied to the LC × LC analysis of a protein standard mixture, intended to be representative of protein drug substances. The results reported in this study demonstrate that our automated online multicolumn 2D-LC-DAD-MS workflow can be a powerful tool for comprehensive chromatographic column screening that enables the semi-automated development of 2D-LC methods, offering the ability to streamline full visualization of sample composition for an unknown complex mixture while maximizing chromatographic orthogonality. Graphical Abstract.

New 1,3-Diynoic Derivatives of Natural Lembehyne B: Stereoselective Synthesis, Anticancer, and Neuritogenic Activity.

An original method has been developed for the synthesis of 1,3-dyine derivatives of natural lembehyne B in high yields (50-67%) and with high selectivity (>98%). The key stage of the synthesis is new Ti-catalyzed cross-cyclomagnesiation of oxygenated and aliphatic 1,2-dienes induced by Grignard reagents. For studying the effect of the structure on the antitumor and neuritogenic activities, a series of lembehyne B analogues with different distances between the terminal hydroxy group and the 1,3-diyne moiety was prepared and tested for neuritogenic activity on mouse neuroblastoma Neuro 2A cells and for cytotoxicity, induction of apoptosis, and effects on the cell cycle using Jurkat, U937, K562, HeLa, and Hek293 tumor cell lines.

Combination of HDX-MS and in silico modeling to study enzymatic reactivity and stereo-selectivity at different solvent conditions.

The higher-order structure of a protein defines its function, and protein structural dynamics are often essential for protein binding and enzyme catalysis. Methods for protein characterization in solution are continuously being developed to understand and explore protein conformational changes with regards to function and activity. The goal of this study was to survey the use of combining HDX-MS global conformational screening with in silico modeling and continuous labeling peptide-level HDX-MS as an approach to highlight regions of interest within an enzyme required for biocatalytic processes. We surveyed in silico modeling correlated with peptide level HDX-MS experiments to characterize and localize transaminase enzyme structural dynamics at different conditions. This approach was orthogonally correlated with a global Size-Exclusion-HDX (SEC-HDX) screen for global conformational comparison and global alpha-helical content measurements by circular dichroism. Enzymatic activity and stereo-selectivity of transaminases were compared at different reaction-solution conditions that forced protein conformational changes by increasing acetonitrile concentration. The experimental peptide-level HDX-MS results demonstrated similar trends to the modeling data showing that certain regions remained folded in transaminases ATA-036 and ATA-303 with increasing acetonitrile concentration, which is also associated with shifting stereoselectivity. HDX modeling, SEC-HDX and CD experimental data showed that transaminase ATA-234 had the highest level of global unfolding with increasing acetonitrile concentration compared to the other two enzymes, which correlated with drastically reduced product conversion in transamination reaction. The combined HDX modeling/experimental workflow, based on enzymatic reactions studied at different conditions to induce changes in enzyme conformation, could be used as a tool to guide directed evolution efforts by identifying and focusing on the regions of an enzyme required for reaction product conversion and stereoselectivity.