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Early-stage cell line screening is a vital step in developing biosimilars of therapeutic monoclonal antibodies (mAbs). While the quality of the manufactured antibodies is commonly assessed by charge-based separation methods employing UV absorbance detection, these methods lack the ability to identify resolved mAb variants. We evaluated the performance of microfluidic capillary electrophoresis coupled to mass spectrometry (MCE-MS) as a rapid tool for profiling mAb biosimilar candidates from clonal cell lines. A representative originator sample was used to develop the MCE-MS method. The addition of dimethylsulfoxide (DMSO) to the background electrolyte yielded up to 60-fold enhancement of the protein MS signal. The resulting electropherograms consistently provided resolution of mAb charge variants within 10 min. Deconvoluted mass spectra facilitated the identification of basic variants such as C-terminal lysine and proline amidation, while the acidic variants could be assigned to deamidated forms. The MCE-MS method also allowed the identification of 18 different glycoforms in biosimilar samples. To mimic early-stage cell line selection, samples from five clonal cell lines that all expressed the same biosimilar candidate mAb were compared to their originator mAb. Based on the similarity observed in charge variants and glycoform profiles acquired by MCE-MS, the most promising candidate could be selected. The MCE-MS method demonstrated good overall reproducibility, as confirmed by a transferability study involving two separate laboratories. This study highlights the efficacy of the MCE-MS method for rapid proteoform screening of clonal cell line samples, underscoring its potential significance as an analytical tool in biosimilar process development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Therapeutic vaccines that elicit cytotoxic T cell responses targeting tumor-specific neoantigens hold promise for providing long-term clinical benefit to patients with cancer. Here we evaluated safety and tolerability of a therapeutic vaccine encoding 20 shared neoantigens derived from selected common oncogenic driver mutations as primary endpoints in an ongoing phase 1/2 study in patients with advanced/metastatic solid tumors. Secondary endpoints included immunogenicity, overall response rate, progression-free survival and overall survival. Eligible patients were selected if their tumors expressed one of the human leukocyte antigen-matched tumor mutations included in the vaccine, with the majority of patients (18/19) harboring a mutation in KRAS. The vaccine regimen, consisting of a chimp adenovirus (ChAd68) and self-amplifying mRNA (samRNA) in combination with the immune checkpoint inhibitors ipilimumab and nivolumab, was shown to be well tolerated, with observed treatment-related adverse events consistent with acute inflammation expected with viral vector-based vaccines and immune checkpoint blockade, the majority grade 1/2. Two patients experienced grade 3/4 serious treatment-related adverse events that were also dose-limiting toxicities. The overall response rate was 0%, and median progression-free survival and overall survival were 1.9 months and 7.9 months, respectively. T cell responses were biased toward human leukocyte antigen-matched TP53 neoantigens encoded in the vaccine relative to KRAS neoantigens expressed by the patients' tumors, indicating a previously unknown hierarchy of neoantigen immunodominance that may impact the therapeutic efficacy of multiepitope shared neoantigen vaccines. These data led to the development of an optimized vaccine exclusively targeting KRAS-derived neoantigens that is being evaluated in a subset of patients in phase 2 of the clinical study. ClinicalTrials.gov registration: NCT03953235 ., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Native mass spectrometry is a rapidly emerging technique for fast and sensitive structural analysis of protein constructs, maintaining the protein higher order structure. The coupling with electromigration separation techniques under native conditions enables the characterization of proteoforms and highly complex protein mixtures. In this review, we present an overview of current native CE-MS technology. First, the status of native separation conditions is described for capillary zone electrophoresis (CZE), affinity capillary electrophoresis (ACE), and capillary isoelectric focusing (CIEF), as well as their chip-based formats, including essential parameters such as electrolyte composition and capillary coatings. Further, conditions required for native ESI-MS of (large) protein constructs, including instrumental parameters of QTOF and Orbitrap systems, as well as requirements for native CE-MS interfacing are presented. On this basis, methods and applications of the different modes of native CE-MS are summarized and discussed in the context of biological, medical, and biopharmaceutical questions. Finally, key achievements are highlighted and concluded, while remaining challenges are pointed out., (© 2023 The Authors. Proteomics published by Wiley-VCH GmbH.)

SARS-CoV-2 has resulted in high levels of morbidity and mortality world-wide, and severe complications can occur in older populations. Humoral immunity induced by authorized vaccines wanes within 6 months, and frequent boosts may only offer transient protection. GRT-R910 is an investigational self-amplifying mRNA (samRNA)-based SARS-CoV-2 vaccine delivering full-length Spike and selected conserved non-Spike T cell epitopes. This study reports interim analyses for a phase I open-label dose-escalation trial evaluating GRT-R910 in previously vaccinated healthy older adults (NCT05148962). Primary endpoints of safety and tolerability were assessed. Most solicited local and systemic adverse events (AEs) following GRT-R910 dosing were mild to moderate and transient, and no treatment-related serious AEs were observed. The secondary endpoint of immunogenicity was assessed via IgG binding assays, neutralization assays, interferon-gamma ELISpot, and intracellular cytokine staining. Neutralizing antibody titers against ancestral Spike and variants of concern were boosted or induced by GRT-R910 and, contrasting to authorized vaccines, persisted through at least 6 months after the booster dose. GRT-R910 increased and/or broadened functional Spike-specific T cell responses and primed functional T cell responses to conserved non-Spike epitopes. This study is limited due to small sample size, and additional data from ongoing studies will be required to corroborate these interim findings., (© 2023. The Author(s).)

Characterization of charge heterogeneity is an essential pillar for pharmaceutical development and quality control of therapeutic monoclonal antibodies (mAbs). The highly selective and commonly applied capillary zone electrophoresis (CZE) method containing high amounts of ε-aminocaproic acid (EACA) provides a detailed and robust charge heterogeneity profile of intact mAb variants. Nevertheless, the exact location of protein modifications within these charge profiles remains ambiguous. Electrospray ionization mass spectrometry (ESI-MS) is a promising tool for this purpose; however, EACA is incompatible with electrospray. In this context, we present a two-dimensional CZE-CZE-MS system to combine efficient charge variant separation of intact mAbs with subsequent peptide analysis after in-capillary digestion of selected charge variants. The first dimension is based on a generic CZE(EACA) method in a fused silica capillary. In the second dimension, a neutral-coated capillary is used for in-capillary reduction and digestion with Tris(2-carboxyethyl)phosphine (TCEP) and pepsin, followed by CZE separation and MS/MS-characterization of the resulting peptides. The setup is demonstrated using stressed and nonstressed mAbs where peaks of basic, main, and acidic variants were transferred in a heart-cut fashion, digested, and characterized on the peptide level. Sequence coverages of more than 90% were obtained for heavy chain (HC) and light chain (LC) for four different mAbs, including low-abundance variants (<2% of the main peak). Frequently observed modifications (deamidation, oxidation, etc.) could be detected and localized. This study demonstrates a proof-of-concept for identification and localization of protein modifications from CZE charge heterogeneity profiles and, in this way, is expected to support the development and quality control testing of protein pharmaceuticals.

The development of therapeutic cancer vaccines remains an active area, although previous approaches have yielded disappointing results. We have built on lessons from previous cancer vaccine approaches and immune checkpoint inhibitor research to develop VBIR, a vaccine-based immunotherapy regimen. Assessment of various technologies led to selection of a heterologous vaccine using chimpanzee adenovirus (AdC68) for priming followed by boosts with electroporation of DNA plasmid to deliver T cell antigens to the immune system. We found that priming with AdC68 rapidly activates and expands antigen-specific T cells and does not encounter pre-existing immunity as occurs with the use of a human adenovirus vaccine. The AdC68 vector does, however, induce new anti-virus immune responses, limiting its use for boosting. To circumvent this, boosting with DNA encoding the same antigens can be done repetitively to augment and maintain vaccine responses. Using mouse and monkey models, we found that the activation of both CD4 and CD8 T cells was amplified by combination with anti-CTLA-4 and anti-PD-1 antibodies. These antibodies were administered subcutaneously to target their distribution to vaccination sites and to reduce systemic exposure which may improve their safety. VBIR can break tolerance and activate T cells recognizing tumor-associated self-antigens. This activation lasts more than a year after completing treatment in monkeys, and inhibits tumor growth to a greater degree than is observed using the individual components in mouse cancer models. These results have encouraged the testing of this combination regimen in cancer patients with the aim of increasing responses beyond current therapies., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Charge detection mass spectrometry (CDMS) provides mass domain spectra of large and highly heterogeneous analytes. Over the past few years, we have multiplexed CDMS on Orbitrap instruments, an approach termed Individual Ion Mass Spectrometry (I 2 MS). Until now, I 2 MS required manual adjustment of injection times to collect spectra in the individual ion regime. To increase sample adaptability, enable online separations, and reduce the barrier for entry, we report an automated method for adjusting ion injection times in I 2 MS for image current detectors like the Orbitrap. Automatic Ion Control (AIC) utilizes the density of signals in the m / z domain to adjust an ensemble of ions down to the individual ion regime in real-time. The AIC technique was applied to both denatured and native proteins yielding high quality data without human intervention directly in the mass domain.

Checkpoint inhibitor (CPI) therapies provide limited benefit to patients with tumors of low immune reactivity. T cell-inducing vaccines hold promise to exert long-lasting disease control in combination with CPI therapy. Safety, tolerability and recommended phase 2 dose (RP2D) of an individualized, heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA (samRNA)-based neoantigen vaccine in combination with nivolumab and ipilimumab were assessed as primary endpoints in an ongoing phase 1/2 study in patients with advanced metastatic solid tumors (NCT03639714). The individualized vaccine regimen was safe and well tolerated, with no dose-limiting toxicities. Treatment-related adverse events (TRAEs) >10% included pyrexia, fatigue, musculoskeletal and injection site pain and diarrhea. Serious TRAEs included one count each of pyrexia, duodenitis, increased transaminases and hyperthyroidism. The RP2D was 10 12 viral particles (VP) ChAd68 and 30 µg samRNA. Secondary endpoints included immunogenicity, feasibility of manufacturing and overall survival (OS). Vaccine manufacturing was feasible, with vaccination inducing long-lasting neoantigen-specific CD8 T cell responses. Several patients with microsatellite-stable colorectal cancer (MSS-CRC) had improved OS. Exploratory biomarker analyses showed decreased circulating tumor DNA (ctDNA) in patients with prolonged OS. Although small study size limits statistical and translational analyses, the increased OS observed in MSS-CRC warrants further exploration in larger randomized studies., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Biology is driven by a vast set of molecular interactions that evolved over billions of years. Just as covalent modifications like acetylations and phosphorylations can change a protein's function, so too can noncovalent interactions with metals, small molecules, and other proteins. However, much of the language of protein-level biology is left either undiscovered or inferred, as traditional methods used in the field of proteomics use conditions that dissociate noncovalent interactions and denature proteins.Just in the past few years, mass spectrometry (MS) has evolved the capacity to preserve and subsequently characterize the complete composition of endogenous protein complexes. Using this "native" type of mass spectrometry, a complex can be activated to liberate some or all of its subunits, typically via collisions with neutral gas or solid surfaces and isolated before further characterization ("Native Top-Down MS," or nTDMS). The subunit mass, the parent ion mass, and the fragment ions of the activated subunits can be used to piece together the precise molecular composition of the parent complex. When performed en masse in discovery mode (i.e., "native proteomics"), the interactions of life─including protein modifications─will eventually be clarified and linked to dysfunction in human disease states.In this Account, we describe the current and future components of the native MS toolkit, covering the challenges the field faces to characterize ever larger bioassemblies. Each of the three pillars of native proteomics are addressed: (i) separations, (ii) top-down mass spectrometry, and (iii) integration with structural biology. Complexes such as endogenous nucleosomes can be targeted now using nTDMS, whereas virus particles, exosomes, and high-density lipoprotein particles will be tackled in the coming few years. The future work to adequately address the size and complexity of mega- to gigadalton complexes will include native separations, single ion mass spectrometry, and new data types. The use of nTDMS in discovery mode will incorporate native-compatible separation techniques to maximize the number of proteoforms in complexes identified. With a new wave of innovations, both targeted and discovery mode nTDMS will expand to include extremely scarce and exceedingly heterogeneous bioassemblies. Understanding the proteinaceous interactions of life and how they go wrong (e.g., misfolding, forming complexes in dysfunctional stoichiometries and configurations) will not only inform the development of life-restoring therapeutics but also deepen our understanding of life at the molecular level.

Preclinical studies show that locoregional CTLA-4 blockade is equally effective in inducing tumor eradication as systemic delivery, without the added risk of immune-related side effects. This efficacy is related to access of the CTLA-4 blocking antibodies to tumor-draining lymph nodes (TDLNs). Local delivery of anti-CTLA-4 after surgical removal of primary melanoma, before sentinel lymph node biopsy (SLNB), provides a unique setting to clinically assess the role of TDLN in the biological efficacy of locoregional CTLA-4 blockade. Here, we have evaluated the safety, tolerability, and immunomodulatory effects in the SLN and peripheral blood of a single dose of tremelimumab [a fully human immunoglobulin gamma-2 (IgG2) mAb directed against CTLA-4] in a dose range of 2 to 20 mg, injected intradermally at the tumor excision site 1 week before SLNB in 13 patients with early-stage melanoma (phase 1 trial; NCT04274816). Intradermal delivery was safe and well tolerated and induced activation of migratory dendritic cell (DC) subsets in the SLN. It also induced profound and durable decreases in regulatory T cell (T reg ) frequencies and activation of effector T cells in both SLN and peripheral blood. Moreover, systemic T cell responses against NY-ESO-1 or MART-1 were primed or boosted ( N = 7), in association with T cell activation and central memory T cell differentiation. These findings indicate that local administration of anti-CTLA-4 may offer a safe and promising adjuvant treatment strategy for patients with early-stage melanoma. Moreover, our data demonstrate a central role for TDLN in the biological efficacy of CTLA-4 blockade and support TDLN-targeted delivery methods.

The coronavirus disease 2019 (COVID-19) pandemic continues to spread globally, highlighting the urgent need for safe and effective vaccines that could be rapidly mobilized to immunize large populations. We report the preclinical development of a self-amplifying mRNA (SAM) vaccine encoding a prefusion stabilized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein and demonstrate strong cellular and humoral immune responses at low doses in mice and rhesus macaques. The homologous prime-boost vaccination regimen of SAM at 3, 10 and 30 μg induced potent neutralizing antibody (nAb) titers in rhesus macaques following two SAM vaccinations at all dose levels, with the 10 μg dose generating geometric mean titers (GMT) 48-fold greater than the GMT of a panel of SARS-CoV-2 convalescent human sera. Spike-specific T cell responses were observed with all tested vaccine regimens. SAM vaccination provided protective efficacy against SARS-CoV-2 challenge as both a homologous prime-boost and as a single boost following ChAd prime, demonstrating reduction of viral replication in both the upper and lower airways. The SAM vaccine is currently being evaluated in clinical trials as both a homologous prime-boost regimen at low doses and as a boost following heterologous prime., (© 2022. The Author(s).)

A functional understanding of the human body requires structure-function studies of proteins at scale. The chemical structure of proteins is controlled at the transcriptional, translational, and post-translational levels, creating a variety of products with modulated functions within the cell. The term "proteoform" encapsulates this complexity at the level of chemical composition. Comprehensive mapping of the proteoform landscape in human tissues necessitates analytical techniques with increased sensitivity and depth of coverage. Here, we took a top-down proteomics approach, combining data generated using capillary zone electrophoresis (CZE) and nanoflow reversed-phase liquid chromatography (RPLC) hyphenated to mass spectrometry to identify and characterize proteoforms from the human lungs, heart, spleen, small intestine, and kidneys. CZE and RPLC provided complementary post-translational modification and proteoform selectivity, thereby enhancing the overall proteome coverage when used in combination. Of the 11,466 proteoforms identified in this study, 7373 (64%) were not reported previously. Large differences in the protein and proteoform level were readily quantified, with initial inferences about proteoform biology operative in the analyzed organs. Differential proteoform regulation of defensins, glutathione transferases, and sarcomeric proteins across tissues generate hypotheses about how they function and are regulated in human health and disease.

The combined use of electrospray ionization run in so-called "native mode" with top-down mass spectrometry (nTDMS) is enhancing both structural biology and discovery proteomics by providing three levels of information in a single experiment: the intact mass of a protein or complex, the masses of its subunits and non-covalent cofactors, and fragment ion masses from direct dissociation of subunits that capture the primary sequence and combinations of diverse post-translational modifications (PTMs). While intact mass data are readily deconvoluted using well-known software options, the analysis of fragmentation data that result from a tandem MS experiment - essential for proteoform characterization - is not yet standardized. In this tutorial, we offer a decision-tree for the analysis of nTDMS experiments on protein complexes and diverse bioassemblies. We include an overview of strategies to navigate this type of analysis, provide example data sets, and highlight software for the hypothesis-driven interrogation of fragment ions for localization of PTMs, metals, and cofactors on native proteoforms. Throughout we have emphasized the key features (deconvolution, search mode, validation, other) that the reader can consider when deciding upon their specific experimental and data processing design using both open-access and commercial software.

Native mass spectrometry (nMS) is a rapidly growing method for the characterization of large proteins and protein complexes, preserving "native" non-covalent inter- and intramolecular interactions. Direct infusion of purified analytes into a mass spectrometer represents the standard approach for conducting nMS experiments. Alternatively, CZE can be performed under native conditions, providing high separation performance while consuming trace amounts of sample material. Here, we provide standard operating procedures for acquiring high-quality data using CZE in native mode coupled online to various Orbitrap mass spectrometers via a commercial sheathless interface, covering a wide range of analytes from 30-800 kDa. Using a standard protein mix, the influence of various CZE method parameters were evaluated, such as BGE/conductive liquid composition and separation voltage. Additionally, a universal approach for the optimization of fragmentation settings in the context of protein subunit and metalloenzyme characterization is discussed in detail for model analytes. A short section is dedicated to troubleshooting of the nCZE-MS setup. This study is aimed to help normalize nCZE-MS practices to enhance the CE community and provide a resource for the production of reproducible and high-quality data., (© 2021 Wiley-VCH GmbH.)

We report a novel platform [native capillary zone electrophoresis-top-down mass spectrometry (nCZE-TDMS)] for the separation and characterization of whole nucleosomes, their histone subunits, and post-translational modifications (PTMs). As the repeating unit of chromatin, mononucleosomes (Nucs) are an ∼200 kDa complex of DNA and histone proteins involved in the regulation of key cellular processes central to human health and disease. Unraveling the covalent modification landscape of histones and their defined stoichiometries within Nucs helps to explain epigenetic regulatory mechanisms. In nCZE-TDMS, online Nuc separation is followed by a three-tier tandem MS approach that measures the intact mass of Nucs, ejects and detects the constituent histones, and fragments to sequence the histone. The new platform was optimized with synthetic Nucs to significantly reduce both sample requirements and cost compared to direct infusion. Limits of detection were in the low-attomole range, with linearity of over ∼3 orders of magnitude. The nCZE-TDMS platform was applied to endogenous Nucs from two cell lines distinguished by overexpression or knockout of histone methyltransferase NSD2/MMSET, where analysis of constituent histones revealed changes in histone abundances over the course of the CZE separation. We are confident the nCZE-TDMS platform will help advance nucleosome-level research in the fields of chromatin and epigenetics.

Current proteomic approaches disassemble and digest nucleosome particles, blurring readouts of the 'histone code'. To preserve nucleosome-level information, we developed Nuc-MS, which displays the landscape of histone variants and their post-translational modifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and the co-occurrence of oncogenic H3.3K27M with euchromatic marks (for example, a >15-fold enrichment of dimethylated H3K79me2). Nuc-MS is highly concordant with chromatin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout of nucleosome-level biology.

Capillary zone electrophoresis (CZE) based on electrophoretic mobility in the liquid phase and ion mobility spectrometry (IMS) based on mobilities in the gas phase are both powerful techniques for the separation of complex samples. Protein glycosylation is one of the most common post-translational modifications associated with a wide range of biological functions and human diseases. Due to their high structural variability, the analysis of glycans still represents a challenging task. In this work, the first on-line coupling of CZE with drift tube ion mobility-mass spectrometry (DTIM-MS) has been perfomed to further improve separation capabilities for the analysis of native and 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled N-glycans. In this way, a complexity of glycan signals was revealed which could not be resolved by these techniques individually, shown for both native and APTS-labeled glycans. Each individual glycan signal separated in CZE exhibited an unexpectedly high number of peaks observed in the IMS dimension. This observation could potentially be explained by the presence of isomeric forms, including different linkages, and/or gas-phase conformers. In addition, the type of sialic acid attached to glycans has a significant impact on the obtained drift time profile. Furthermore, the application of α2-3 neuraminidase enabled the partial assignment of peaks in the arrival time distribution considering their sialic acid linkages (α2-3/α2-6). This work is a showcase for the high potential of CZE-DTIM-MS, which is expected to find various applications in the future. Graphical abstract ᅟ.

Background : There is an unmet demand for community-scale fecal sludge treatment units (FSTUs) that serve communities of between 1,000 and 50,000 people and are able to operate in non-sewered and off-grid environments. An emerging industry standard for FSTUs includes as a key criteria energy independence in steady-state. Theoretically, there is sufficient thermal energy available in fecal sludge to provide the electrical power needed to run the FSTU. However, such a system had never been implemented. Methods : Biomass Controls has previously demonstrated the thermal treatment of fecal sludge using the Biogenic Refinery, a thermal FSTU deployed in three sites in India. In this article we describe testing where a Biogenic Refinery was paired with a thermal fluid heat exchanger and organic Rankine cycle generator to generate electrical power. Results : This Biogenic Refinery combined heat and power system generated sufficient electrical power to offset electrical parasitic loads in steady-state operation and produce a surplus of 1.2 kWe. Conclusions : The results of the study demonstrate that there is an excess of energy available and reliable mechanisms to generate electrical energy using an FSTU. Additional steps are necessary to transition to a true off-grid FSTU., Competing Interests: No competing interests were disclosed.

Multidimensional separation techniques play an increasingly important role in separation science, especially for the analysis of complex samples such as proteins. The combination of reversed-phase liquid chromatography in the nanoscale and CZE is especially beneficial due to their nearly orthogonal separation mechanism and well-suited geometries/dimensions. Here, a heart-cut nano-LC-CZE-MS setup was developed utilizing for the first time a mechanical 4-port valve as LC-CE interface. A model protein mixture containing four different protein species was first separated by nano LC followed by a heart-cut transfer of individual LC peaks and subsequent CZE-MS analysis. In the CZE dimension, various glycoforms of one protein species were separated. Improved separation capabilities were achieved compared to the 1D methods, which was exemplarily shown for ribonuclease B and its different glycosylated forms. LODs in the lower μg/mL range were determined, which are considerably lower compared to traditional CZE-MS. In addition, this study represents the first application of an LC-CE-MS system for intact protein analysis. The nano-LC-CZE-MS system is expected to be applicable to various other analytical challenges., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Capillary electrophoresis (CE) offers fast and high-resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user-friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano-electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE-MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two-dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE-modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Neoantigens, which are expressed on tumor cells, are one of the main targets of an effective antitumor T-cell response. Cancer immunotherapies to target neoantigens are of growing interest and are in early human trials, but methods to identify neoantigens either require invasive or difficult-to-obtain clinical specimens, require the screening of hundreds to thousands of synthetic peptides or tandem minigenes, or are only relevant to specific human leukocyte antigen (HLA) alleles. We apply deep learning to a large (N = 74 patients) HLA peptide and genomic dataset from various human tumors to create a computational model of antigen presentation for neoantigen prediction. We show that our model, named EDGE, increases the positive predictive value of HLA antigen prediction by up to ninefold. We apply EDGE to enable identification of neoantigens and neoantigen-reactive T cells using routine clinical specimens and small numbers of synthetic peptides for most common HLA alleles. EDGE could enable an improved ability to develop neoantigen-targeted immunotherapies for cancer patients.

Electromigration separation techniques often demand certain compounds in the electrolyte to achieve the required selectivity and efficiency. These compounds, including the electrolyte itself, ampholytes, polymeric compounds for sieving, complexing agents, tensides, etc. are often non-volatile. Thus, interference with the electrospray ionization process is a common issue, impeding direct coupling of such electrolyte systems to mass spectrometry. Still, several options exist to obtain mass spectra after separation, including offline fractionation, alternative ionization, dilution, or the change to volatile constituents. In the first part of this article, these methods are discussed. However, all of these options are a compromise of separation performance and sensitivity of mass spectrometric detection. Two-dimensional capillary electrophoresis-mass spectrometry (CE-CE-MS) systems represent a promising alternative to the aforementioned challenges, as they allow the use of existing methods with best separation performance in combination with sensitive mass characterization. In this context, the second part of this article is dedicated to the advantages, limitations, and applications of this approach. Finally, an outlook towards future developments is given.

Antitumor rejection by the immune system is a complex process that is regulated by several factors. Among these factors are the quality and quantity of mutational events that occur in cancer cells. Perhaps one of the most important types of mutations that influence antitumor immunity is the neoantigen, that is, a non-self-antigen that arises as a result of somatic mutation. Recent work has demonstrated that neoantigens hold significant promise for developing new diagnostic and therapeutic modalities. Therapeutic targeting of neoantigens is important for achieving benefit following therapy with immune checkpoint blockade agents or for cancer vaccines targeting mutations. Here, we review our understanding of neoantigens and discuss new developments in the quest to use them in cancer immunotherapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Capillary zone electrophoresis (CZE) is a powerful tool that is progressively being applied for the separation of monoclonal antibody (mAb) charge variants. Mass spectrometry (MS) is the desired detection method concerning identification of mAb variants. In biopharmaceutical applications, there exist optimized and validated electrolyte systems for mAb variant quantification. However, these electrolytes interfere greatly with the electrospray ionization (ESI) process. Here, a heart-cut CZE-CZE-MS setup with an implemented mechanical four-port valve interface was developed that used a generic ε-aminocaproic acid based background electrolyte in the first dimension and acetic acid in the second dimension. Interference-free, highly precise mass data (deviation less than 1 Da) of charge variants of trastuzumab, acting as model mAb system, were achieved. The mass accuracy obtained (low parts per million range) is discussed regarding both measured and calculated masses. Deamidation was detected for the intact model antibody, and related mass differences were significantly confirmed on the deglycosylated level. The CZE-CZE-MS setup is expected to be applicable to a variety of antibodies and electrolyte systems. Thus, it has the potential to become a compelling tool for MS characterization of antibody variants separated in ESI-interfering electrolytes. Graphical Abstract Two-dimensional capillary zone electrophoresis mass spectrometry for the characterization of intact monoclonal antibody (mAb) charge variants. A generic, but highly electrospray-interfering electrolyte system was used as first dimension for mAb charge variant separation and coupled to a volatile electrolyte system as second dimension via a four-port nanoliter valve. In this way, interference-free and precise mass spectrometric data of separated mAb charge variants, including deamidation products, were obtained.

CIEF represents an elegant technique especially for the separation of structural similar analytes, whereas MS is a state-of-the-art instrumentation for the identification and characterization of biomolecules. The combination of both techniques can be realized by hyphenating CIEF with CZE-ESI-MS applying a mechanical valve. During the CZE step, the remaining ESI-interfering components of the CIEF electrolyte are separated from the analytes prior to MS detection. In this work, a multiple heart-cut approach is presented expanding our previous single heart-cut concept resulting in a dramatical reduction of analysis time. Moreover, different sample transfer loop volumes are systematically compared and discussed in regard to peak width and transfer efficiency. With this major enhancement, model proteins (1.63-9.75 mg/L), covering a wide pI range (5-10), and charge variants from a deglycosylated model antibody were analyzed on intact level. The promising CIEF-CZE-MS setup is expected to be applicable in different bioanalytical fields, e.g. for the fast and information rich characterization of therapeutic antibodies., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In order to ensure the stability of pharmaceutical products appropriate manufacturing and storage conditions are required. In general, the degradation of active pharmaceutical ingredients (APIs) and subsequent formation of degradation products affect the pharmaceutical quality. Thus, a fast and effective detection and characterization of these substances is mandatory. Here, the applicability of Raman spectroscopy and CZE for the characterization of the degradation of effervescent tablets containing acetylsalicylic acid (ASA) and ascorbic acid (AA) was evaluated. Therefore, a degradation study was performed analyzing tablets from two different manufacturers at varying conditions (relative humidity (RH) 33%, 52% and 75% at 30°C). Raman spectroscopy combined with principal component analysis could be successfully applied for the fast and easy discrimination of non-degraded and degraded effervescent tablets after a storage period of approximately 24h (RH 52%). Nevertheless, a clear identification or quantification of APIs and degradation products within the analyzed tablets was not possible, i.a. due to missing reference materials. CZE-UV enabled the quantification of the APIs (ASA, AA) and related degradation products (salicylic acid (SA); semi-quantitative also mono- and diacetylated AA) within the complex tablet mixtures. The higher the RH, the faster the degradation of ASA and AA as well as the formation of the degradation products. Mono- and diacetylated AA are major primary degradation products of AA for the applied effervescent tablets. A significant degradation of the APIs was detected earlier by CZE (6-12h, RH 52%) than by Raman spectroscopy. Summarized, Raman spectroscopy is well-suited as quick test to detect degradation of these tablets and CZE can be utilized for further detailed characterization and quantification of specific APIs and related degradation products., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Capillary electrophoresis is commonly applied for the analysis of pharmaceutical products due to its high separation efficiency and selectivity. For this purpose, electrospray-ionization-(ESI)-interfering additives or electrolytes are often required, which complicates the identification of impurities and degradation products by mass spectrometry (MS). Here, a capillary zone electrophoresis (CZE) method with ultraviolet (UV) absorption detection for the simultaneous determination and quantification of ascorbic acid and acetylsalicylic acid in effervescent tablets was developed. Related degradation products were identified via CZE-CZE-MS. Systematic optimization yielded 100 mM tricine (pH = 8.8) as appropriate background electrolyte, resulting in baseline separation of ascorbic acid, acetylsalicylic acid, and related anionic UV-active degradation products. The CZE-UV method was successfully validated regarding the guidelines of the Food and Drug Administration. The validated method was applied to trace the degradation rate of the active pharmaceutical ingredients at defined ambient conditions. A heart-cut CZE-CZE-MS approach, including a 4-port-nL-valve, was performed for the identification of the observed degradation products. This 2D setup enables a precise cutting of accurate sample volumes (20 nL) and the independent operation of two physically separated CZE dimensions, which is especially beneficial regarding MS detection. Hence, the ESI-interfering tricine electrolyte components were separated from the analytes in a second electrophoretic dimension prior to ESI-MS detection. The degradation products were identified as salicylic acid and mono- and diacetylated ascorbic acid. This setup is expected to be generally applicable for the mass spectrometric characterization of CZE separated analytes in highly ESI-interfering electrolyte systems. Graphical Abstract A CZE-UV method for the quantification of effervescent tablet ingredients and degradation products was developed and validated. In order to identify unknown degradation products separated in the CZE-UV, a 2D heart-cut approach was performed applying a mechanical 4-port-valve. The unknown substances were transferred from the 1st to the 2nd dimension followed by the separation of ESI-interfering tricine from the analytes prior to mass spectrometric detection.

Matrix-assisted ionization (MAI) mass spectrometry (MS) is a simple and sensitive method for analysis of low- and high-mass compounds, requiring only that the analyte in a suitable matrix be exposed to the inlet aperture of an atmospheric pressure ionization mass spectrometer. Here, we evaluate the reproducibility of MAI and its potential for quantification using six drug standards. Factors influencing reproducibility include the matrix compound used, temperature, and the method of sample introduction. The relative standard deviation (RSD) using MAI for a mixture of morphine, codeine, oxymorphone, oxycodone, clozapine, and buspirone and their deuterated internal standards using the matrix 3-nitrobenzonitrile is less than 10% with either a Waters SYNAPT G2 or a Thermo LTQ Velos mass spectrometer. The RSD values obtained using MAI are comparable to those using ESI or MALDI on these instruments. The day-to-day reproducibility of MAI determined for five consecutive days with internal standards was better than 20% using manual sample introduction. The reproducibility improved to better than 5% using a mechanically assisted sample introduction method. Hydrocodone, present in a sample of undiluted infant urine, was quantified with MAI using the standard addition method.

Background: Cancer-related disturbances in myeloid lineage development, marked by high levels of myeloid-derived suppressor cells (MDSC) and impaired dendritic cell (DC) development, are associated with poor clinical outcome due to immune escape and therapy resistance. Redressing this balance may therefore be of clinical benefit. Here we investigated the effects of combined Prostate GVAX/ipilimumab immunotherapy on myeloid subsets in peripheral blood of castration-resistant prostate cancer (CRPC) patients as well as the putative predictive value of baseline and on-treatment myeloid parameters on clinical outcome., Methods: Patients with CRPC (n = 28) received thirteen intradermal administrations of Prostate GVAX, consisting of two allogeneic GM-CSF-transduced and irradiated prostate cancer cell lines (LN-CaP and PC3) and six infusions of escalating doses of anti-CTLA4/ipilimumab. Frequencies and activation status of peripheral blood DC (PBDC) and MDSC were determined before, during and after treatment by flowcytometric analysis and related to clinical benefit., Results: Significant treatment-induced activation of conventional and plasmacytoid DC subsets (cDC and pDC) was observed, which in the case of BDCA1/CD1c(+) cDC1 and MDC8(+)/6-sulfoLacNAc(+) inflammatory cDC3 was associated with significantly prolonged overall survival (OS), but also with the development of autoimmune-related adverse events. High pre-treatment levels of CD14(+)HLA-DR(-)monocytic MDSC (mMDSC) were associated with reduced OS. Unsupervised clustering of these myeloid biomarkers revealed particular survival advantage in a group of patients with high treatment-induced PBDC activation and low pretreatment frequencies of suppressive mMDSC in conjunction with our previously identified lymphoid biomarker of high pretreatment CD4(+)CTLA4(+) T cell frequencies., Conclusions: Our data demonstrate that DC and MDSC subsets are affected by prostate GVAX/ipilimumab therapy and that myeloid profiling may contribute to the identification of patients with possible clinical benefit of Prostate GVAX/ipilimumab treatment.

Despite many advantages like high separation efficiency CE comprises the main limitation of low concentration sensitivity, when compared to HPLC. In-line SPE is an efficient way to increase concentration sensitivity. Here, a fritless in-line-SPE-CE-MS method was developed in order to analyze anions of strong acids. Mixed-mode (weak anion exchange and RP) particles were used for enrichment and an acidic BGE was applied for separation. Different particle and capillary sizes were tested. A novel bead string design with a 100 μm id column filled with particles of 90 μm followed by a separation capillary with 50 μm id was easy to prepare and showed the best performance with respect to separation efficiency and reproducibility. Three aromatic sulfonic acids were employed in an in-line SPE-CE-UV approach for method development. Method validation was performed with respect to reproducibility, robustness, and linearity. Thereafter the method was transferred to SPE-CE-MS and applied to the analysis of glycans labeled with 8-aminopyrene-1,3,6-trisulfonic acid. Lower limits of detection in the low nM range were achieved injecting about 10 μL of sample. This corresponds to an enrichment factor of more than 800 compared to the corresponding CE-MS method without preconcentration., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The complexity and multiscale nature of the mammalian immune response provides an excellent test bed for the potential of mathematical modeling and simulation to facilitate mechanistic understanding. Historically, mathematical models of the immune response focused on subsets of the immune system and/or specific aspects of the response. Mathematical models have been developed for the humoral side of the immune response, or for the cellular side, or for cytokine kinetics, but rarely have they been proposed to encompass the overall system complexity. We propose here a framework for integration of subset models, based on a system biology approach., Results: A dynamic simulator, the Fully-integrated Immune Response Model (FIRM), was built in a stepwise fashion by integrating published subset models and adding novel features. The approach used to build the model includes the formulation of the network of interacting species and the subsequent introduction of rate laws to describe each biological process. The resulting model represents a multi-organ structure, comprised of the target organ where the immune response takes place, circulating blood, lymphoid T, and lymphoid B tissue. The cell types accounted for include macrophages, a few T-cell lineages (cytotoxic, regulatory, helper 1, and helper 2), and B-cell activation to plasma cells. Four different cytokines were accounted for: IFN-γ, IL-4, IL-10 and IL-12. In addition, generic inflammatory signals are used to represent the kinetics of IL-1, IL-2, and TGF-β. Cell recruitment, differentiation, replication, apoptosis and migration are described as appropriate for the different cell types. The model is a hybrid structure containing information from several mammalian species. The structure of the network was built to be physiologically and biochemically consistent. Rate laws for all the cellular fate processes, growth factor production rates and half-lives, together with antibody production rates and half-lives, are provided. The results demonstrate how this framework can be used to integrate mathematical models of the immune response from several published sources and describe qualitative predictions of global immune system response arising from the integrated, hybrid model. In addition, we show how the model can be expanded to include novel biological findings. Case studies were carried out to simulate TB infection, tumor rejection, response to a blood borne pathogen and the consequences of accounting for regulatory T-cells., Conclusions: The final result of this work is a postulated and increasingly comprehensive representation of the mammalian immune system, based on physiological knowledge and susceptible to further experimental testing and validation. We believe that the integrated nature of FIRM has the potential to simulate a range of responses under a variety of conditions, from modeling of immune responses after tuberculosis (TB) infection to tumor formation in tissues. FIRM also has the flexibility to be expanded to include both complex and novel immunological response features as our knowledge of the immune system advances.