RESUMO
Monitoring and quantifying host cell proteins (HCPs) in biotherapeutic production processes is crucial to ensure product quality, stability, and safety. Liquid chromatography-mass spectrometry (LC-MS) analysis has emerged as an important tool for identifying and quantifying individual HCPs. However, LC-MS-based approaches face challenges due to the wide dynamic range between HCPs and the therapeutic protein as well as laborious sample preparation and long instrument time. To address these limitations, we evaluated the application of parallel accumulation-serial fragmentation combined with data-independent acquisition (diaPASEF) to HCP analysis for biopharmaceutical process development applications. We evaluated different library generation strategies and LC methods, demonstrating the suitability of these workflows for various HCP analysis needs, such as in-depth characterization and high-throughput analysis of process intermediates. Remarkably, the diaPASEF approach enabled the quantification of hundreds of HCPs that were undetectable by a standard data-dependent acquisition mode while considerably improving sample requirement, throughput, coverage, quantitative precision, and data completeness.
Assuntos
Proteômica , Proteômica/métodos , Ensaios de Triagem em Larga Escala , Animais , Cromatografia Líquida/métodos , Cricetulus , Células CHO , Humanos , Espectrometria de MassasRESUMO
Product association of host-cell proteins (HCPs) to monoclonal antibodies (mAbs) is widely regarded as a mechanism that can enable HCP persistence through multiple purification steps and even into the final drug substance. Discussion of this mechanism often implies that the existence or extent of persistence is directly related to the strength of binding but actual measurements of the binding affinity of such interactions remain sparse. Two separate avenues of investigation of HCP-mAb binding are reported here. One is the measurement of the affinity of binding of individual, commonly persistent Chinese hamster ovary (CHO) HCPs to each of a set of mAbs, and the other uses quantitative proteomic measurements to assess binding of HCPs in a null CHO harvested cell culture fluid (HCCF) to mAbs produced in the same cell line. The individual HCP measurements show that the binding affinities of individual HCPs to different mAbs can vary appreciably but are rarely very high, with only weak pH dependence. The measurements on the null HCCF allow estimation of individual HCP-mAb affinities; these are typically weaker than those seen in affinity measurements on isolated HCPs. Instead, the extent of binding appears correlated with the initial abundance of individual HCPs in the HCCF and the forms of the HCPs in the solution, i.e., whether HCPs are present as free molecules or as parts of large aggregates. Separate protein A chromatography experiments performed by feeding different fractions of a mAb-containing HCCF obtained by size-exclusion chromatography (SEC) showed clear differences in the number and identity of HCPs found in the protein A eluate. These results indicate a significant role for HCP-mAb association in determining HCP persistence through protein A chromatography, presumably through binding of HCP-mAb complexes to the resin. Overall, the results illustrate the importance of considering more fully the biophysical context of HCP-product association in assessing the factors that may affect the phenomenon and determine its implications. Knowledge of the abundances and the forms of individual or aggregated HCPs in HCCF are particularly significant, emphasizing the integration of upstream and downstream bioprocessing and the importance of understanding the collective properties of HCPs in addition to just the biophysical properties of individual HCPs.
Assuntos
Anticorpos Monoclonais , Proteômica , Cricetinae , Animais , Cricetulus , Proteômica/métodos , Células CHO , Anticorpos Monoclonais/química , Cromatografia em Gel , Proteína Estafilocócica A/químicaRESUMO
Host-cell proteins (HCPs) are the foremost class of process-related impurities to be controlled and removed in downstream processing steps in monoclonal antibody (mAb) manufacturing. However, some HCPs may evade clearance in multiple purification steps and reach the final drug product, potentially threatening drug stability and patient safety. This study extends prior work on HCP characterization and persistence in mAb process streams by using mass spectrometry (MS)-based methods to track HCPs through downstream processing steps for seven mAbs that were generated by five different cell lines. The results show considerable variability in HCP identities in the processing steps but extensive commonality in the identities and quantities of the most abundant HCPs in the harvests for different processes. Analysis of HCP abundance in the harvests shows a likely relationship between abundance and the reproducibility of quantification measurements and suggests that some groups of HCPs may hinder the characterization. Quantitative monitoring of HCPs persisting through purification steps coupled with the findings from the harvest analysis suggest that multiple factors, including HCP abundance and mAb-HCP interactions, can contribute to the persistence of individual HCPs and the identification of groups of common, persistent HCPs in mAb manufacturing.
Assuntos
Anticorpos Monoclonais , Cricetinae , Animais , Humanos , Anticorpos Monoclonais/química , Reprodutibilidade dos Testes , Cricetulus , Espectrometria de Massas , Células CHORESUMO
In the production of biopharmaceuticals such as monoclonal antibodies (mAbs) and vaccines, the residual amounts of host-cell proteins (HCPs) are among the critical quality attributes. In addition to overall HCP levels, individual HCPs may elude purification, potentially causing issues in product stability or patient safety. Such HCP persistence has been attributed mainly to biophysical interactions between individual HCPs and the product, resin media, or residual chromatin particles. Based on measurements on process streams from seven mAb processes, we have found that HCPs in aggregates, not necessarily chromatin-derived, may play a significant role in the persistence of many HCPs. Such aggregates may also hinder accurate detection of HCPs using existing proteomics methods. The findings also highlight that certain HCPs may be difficult to remove because of their functional complementarity to the product; specifically, chaperones and other proteins involved in the unfolded protein response (UPR) are disproportionately present in the aggregates. The methods and findings described here expand our understanding of the origins and potential behavior of HCPs in cell-based biopharmaceutical processes and may be instrumental in improving existing techniques for HCP detection and clearance.
Assuntos
Produtos Biológicos , Agregados Proteicos , Cricetinae , Animais , Humanos , Cricetulus , Anticorpos Monoclonais , Proteômica/métodos , Células CHORESUMO
Proteomic absolute quantitation strategies mainly rely on the use of synthetic stable isotope-labeled peptides or proteins as internal standards, which are highly costly and time-consuming to synthesize. To circumvent this limitation, we recently developed a coulometric mass spectrometry (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides, followed by mass spectrometry measurement of the peptide oxidation yield. Previously, CMS was only applied for single-protein quantitation. In this study, first, we demonstrated absolute quantitation of multiple proteins in a mixture (e.g., ß-lactoglobulin B, α-lactalbumin, and carbonic anhydrase) by CMS in one run, without using any standards. The CMS quantitation result was validated with a traditional isotope dilution method. Second, CMS can be used for absolute quantitation of a low-level target protein in a mixture; for instance, 500 ppm of PLBL2, a problematic host cell protein (HCP), in the presence of a highly abundant monoclonal antibody (mAb) was successfully quantified by CMS with no use of standards. Third, taking one step further, this study demonstrated the unprecedented quantitative analysis of deamidated peptide products arising from the mAb heavy chain deamidation reaction. In particular, absolute quantitation of the deamidation succinimide intermediate which had not been performed before due to the lack of standard was conducted by CMS, for the first time. Overall, our data suggest that CMS has potential utilities for quantitative proteomics and biotherapeutic drug discovery.
Assuntos
Peptídeos , Proteômica , Anticorpos Monoclonais , Espectrometria de Massas/métodos , Peptídeos/química , Proteômica/métodos , Técnica de Diluição de RadioisótoposRESUMO
Host cell proteins (HCPs) are a significant class of process-related impurities commonly associated with the manufacturing of biopharmaceuticals. However, due to the increased use of crude enzymes as biocatalysts for modern organic synthesis, HCPs can also be introduced as a new class of impurities in chemical drugs. In both cases, residual HCPs need to be adequately controlled to ensure product purity, quality, and patient safety. Although a lot of attentions have been focused on defining a universally acceptable limit for such impurities, the risks associated with residual HCPs on product quality, safety, and efficacy often need to be determined on a case-by-case basis taking into consideration the residual HCP profile in the product, the dose, dosage form, administration route, and so forth. Here we describe the unique challenges for residual HCP control presented by the biocatalytic synthesis of an investigational stimulator of interferon genes protein agonist, MK-1454, which is a cyclic dinucleotide synthesized using Escherichia coli cell lysate overexpressing cyclic GMP-AMP synthase as a biocatalyst. In this study, a holistic characterization of residual protein impurities using a variety of analytical tools including nanoscale liquid chromatography coupled to tandem mass spectrometry, together with in silico immunogenicity prediction of identified proteins, facilitated risk assessment and guided process development to achieve adequate removal of residual protein impurities in MK-1454 active pharmaceutical ingredient.
Assuntos
Proteínas , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Preparações Farmacêuticas , Proteínas/análise , Medição de RiscoRESUMO
Polysorbate is widely used to maintain stability of biotherapeutic proteins in pharmaceutical formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role for polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using an optimized chemical probe, we established the first global profile of active serine hydrolases in harvested cell culture fluid (HCCF) for monoclonal antibodies (mAbs) production from two Chinese hamster ovary (CHO) cell lines. A total of eight known lipases were identified by ABPP with enzyme activity information, while only five lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study with downstream samples, phospholipase A2 group VII (PLA2G7) was only identified by ABPP and confirmed to contribute to polysorbate-80 degradation for the first time. The established ABBP approach is approved to be able to identify low-abundance host cell enzymes and fills the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.
Assuntos
Produtos Biológicos , Polissorbatos , Animais , Anticorpos Monoclonais , Células CHO , Cricetinae , Cricetulus , HumanosRESUMO
Multiple reaction monitoring (MRM) is a liquid chromatography-mass spectrometry (LC-MS) based quantification platform with high sensitivity, specificity, and throughput. It is extensively used across the pharmaceutical industry for the quantitative analysis of therapeutic molecules. The potential of MRM analysis for the quantification of specific host cell proteins (HCPs) in bioprocess, however, has yet to be well established. In this work, we introduce a multiplex LC-MRM assay that simultaneously monitors two high risk lipases known to impact biologics product quality, Phospholipase B-like 2 protein (PLBL2) and Group XV lysosomal phospholipase A2 (LPLA2). Quantitative data generated from the LC-MRM assay were used to monitor the clearance of these lipases during biologics process development. The method is linear over a dynamic range of 1 to 500 ng/mg. To demonstrate the fitness for use and robustness of this assay, we evaluate a comprehensive method qualification package that includes intra- and inter-run precision and accuracy across all evaluated concentrations, selectivity, recovery and matrix effect, dilution linearity, and carryover. Additionally, we illustrate that this assay provides a rapid and accurate means of monitoring high risk HCP clearance for in-process support and can actively guide process improvement and optimization. Lastly, we compare direct digestion platforms and affinity depletion platforms to demonstrate the impact of HCP-mAb interaction on lipase quantification.
Assuntos
Cromatografia Líquida/métodos , Contaminação de Medicamentos/prevenção & controle , Fosfolipases A2 do Grupo IV/análise , Espectrometria de Massas em Tandem/métodos , Animais , Anticorpos Monoclonais/isolamento & purificação , Células CHO , CricetulusRESUMO
BACKGROUND: Large-scale, placebo-controlled trials established that nonsteroidal anti-inflammatory drugs confer a cardiovascular hazard: this has been attributed to depression of cardioprotective products of cyclooxygenase (COX)-2, especially prostacyclin. An alternative mechanism by which nonsteroidal anti-inflammatory drugs might constrain cardioprotection is by enhancing the formation of methylarginines in the kidney that would limit the action of nitric oxide throughout the vasculature. METHODS: Targeted and untargeted metabolomics were used to investigate the effect of COX-2 deletion or inhibition in mice and in osteoarthritis patients exposed to nonsteroidal anti-inflammatory drugs on the l-arginine/nitric oxide pathway. RESULTS: Analysis of the plasma and renal metabolome was performed in postnatal tamoxifen-inducible Cox-2 knockout mice, which exhibit normal renal function and blood pressure. This revealed no changes in arginine and methylarginines compared with their wild-type controls. Moreover, the expression of genes in the l-arginine/nitric oxide pathway was not altered in the renal medulla or cortex of tamoxifen inducible Cox-2 knockout mice. Therapeutic concentrations of the selective COX-2 inhibitors, rofecoxib, celecoxib, and parecoxib, none of which altered basal blood pressure or renal function as reflected by plasma creatinine, failed to elevate plasma arginine and methylarginines in mice. Finally, plasma arginine or methylarginines were not altered in osteoarthritis patients with confirmed exposure to nonsteroidal anti-inflammatory drugs that inhibit COX-1 and COX-2. By contrast, plasma asymmetrical dimethylarginine was increased in mice infused with angiotensin II sufficient to elevate blood pressure and impair renal function. Four weeks later, blood pressure, plasma creatinine, and asymmetrical dimethylarginine were restored to normal levels. The increase in asymmetrical dimethylarginine in response to infusion with angiotensin II in celecoxib-treated mice was also related to transient impairment of renal function. CONCLUSIONS: Plasma methylarginines are not altered by COX-2 deletion or inhibition but rather are elevated coincident with renal compromise.
Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Arginina/análogos & derivados , Doenças Cardiovasculares/etiologia , Ciclo-Oxigenase 2/metabolismo , Animais , Anti-Inflamatórios não Esteroides/sangue , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Arginina/sangue , Pressão Sanguínea/efeitos dos fármacos , Nitrogênio da Ureia Sanguínea , Celecoxib/farmacologia , Creatinina/sangue , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/genética , Inibidores de Ciclo-Oxigenase 2/farmacologia , Humanos , Rim/metabolismo , Metaboloma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoartrite/tratamento farmacológico , Osteoartrite/patologia , Efeito PlaceboRESUMO
Objective- Evening but not morning administration of low-dose aspirin has been reported to lower blood pressure in hypertensive patients. The present study was designed to determine whether this phenomenon could be replicated in mice, and if so, whether a time-dependent effect of aspirin on blood pressure was because of alteration of circadian clock function. Approach and Results- We recapitulated the protective effect of aspirin (50 µg/d for 7 days) at zeitgeber time 0 (active-to-rest transit), but not at zeitgeber time 12, on a high-salt diet-induced increase of blood pressure. However, the time of aspirin administration did not influence expression of canonical clock genes or their acetylation. We used mouse Bmal1 and Per2-luciferase reporters expressed in U2OS cells to determine the real-time effect of aspirin on circadian function but found that the oscillation of bioluminescence was unaltered. Timing of aspirin administration also failed to alter urinary prostaglandin metabolites or catecholamines, or the acetylation of its COX-1 (cyclooxygenase-1) target in platelets. Conclusions- The time-dependent hypotensive effect of aspirin in humans has been recapitulated in hypertensive mice. However, this does not seem to reflect a direct impact of aspirin on circadian clocks or on acetylation of platelet COX-1.
Assuntos
Anti-Hipertensivos/administração & dosagem , Aspirina/administração & dosagem , Pressão Sanguínea/efeitos dos fármacos , Ritmo Circadiano , Hipertensão/prevenção & controle , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Linhagem Celular Tumoral , Relógios Circadianos/efeitos dos fármacos , Relógios Circadianos/genética , Ritmo Circadiano/efeitos dos fármacos , Ciclo-Oxigenase 1/sangue , Modelos Animais de Doenças , Cronofarmacoterapia , Humanos , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Proteínas de Membrana/sangue , Camundongos Endogâmicos C57BL , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Cloreto de Sódio na Dieta , Fatores de TempoRESUMO
The cardiovascular safety of nonsteroidal antiinflammatory drugs (NSAIDs) may be influenced by interactions with antiplatelet doses of aspirin. We sought to quantitate precisely the propensity of commonly consumed NSAIDsibuprofen, naproxen, and celecoxibto cause a drug-drug interaction with aspirin in vivo by measuring the target engagement of aspirin directly by MS. We developed a novel assay of cyclooxygenase-1 (COX-1) acetylation in platelets isolated from volunteers who were administered aspirin and used conventional and microfluidic assays to evaluate platelet function. Although ibuprofen, naproxen, and celecoxib all had the potential to compete with the access of aspirin to the substrate binding channel of COX-1 in vitro, exposure of volunteers to a single therapeutic dose of each NSAID followed by 325 mg aspirin revealed a potent drug-drug interaction between ibuprofen and aspirin and between naproxen and aspirin but not between celecoxib and aspirin. The imprecision of estimates of aspirin consumption and the differential impact on the ability of aspirin to inactivate platelet COX-1 will confound head-to-head comparisons of distinct NSAIDs in ongoing clinical studies designed to measure their cardiovascular risk.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Ciclo-Oxigenase 1/metabolismo , Acetilação , Plaquetas/enzimologia , Humanos , MicrofluídicaRESUMO
Resolvins, maresins, and protectins can be formed from fish oils. These specialized pro-resolving mediators (SPMs) have been implicated in the resolution of inflammation. Synthetic versions of such SPMs exert anti-inflammatory effects in vitro and when administered to animal models. However, their importance as endogenous products formed in sufficient amounts to exert anti-inflammatory actions in vivo remains speculative. We biased our ability to detect SPMs formed in healthy volunteers by supplementing fish oil in doses shown previously to influence blood pressure and platelet aggregation under placebo-controlled conditions. Additionally, we sought to determine the relative formation of SPMs during an acute inflammatory response and its resolution, evoked in healthy volunteers by bacterial lipopolysaccharide (LPS). Bioactive lipids, enzymatic epoxyeicosatrienoic acids (EETs), and free radical-catalyzed prostanoids [isoprostanes (iPs)] formed from arachidonic acid and the fish oils, served as comparators. Despite the clear shift from ω-6 to ω-3 EETs and iPs, we failed to detect a consistent signal, in most cases, of SPM formation in urine or plasma in response to fish oil, and in all cases in response to LPS on a background of fish oil. Our results question the relevance of these SPMs to the putative anti-inflammatory effects of fish oils in humans.
Assuntos
Suplementos Nutricionais , Óleos de Peixe/administração & dosagem , Mediadores da Inflamação/sangue , Inflamação/dietoterapia , Metabolismo dos Lipídeos/efeitos dos fármacos , Adulto , Anti-Inflamatórios/administração & dosagem , Antígenos CD59/administração & dosagem , Ácidos Graxos Ômega-3/sangue , Feminino , Voluntários Saudáveis , Humanos , Inflamação/sangue , Inflamação/induzido quimicamente , Mediadores da Inflamação/síntese química , Lipopolissacarídeos/administração & dosagem , MasculinoRESUMO
Cyclooxygenase-2 (COX-2) expression is associated with poor prognosis across a range of human cancers, including breast cancer. The contribution of tumor cell-derived COX-2 to tumorigenesis has been examined in numerous studies; however, the role of stromal-derived COX-2 is ill-defined. Here, we examined how COX-2 in myeloid cells, an immune cell subset that includes macrophages, influences mammary tumor progression. In mice engineered to selectively lack myeloid cell COX-2 [myeloid-COX-2 knockout (KO) mice], spontaneous neu oncogene-induced tumor onset was delayed, tumor burden reduced, and tumor growth slowed compared with wild-type (WT). Similarly, growth of neu-transformed mammary tumor cells as orthotopic tumors in immune competent syngeneic myeloid-COX-2 KO host mice was reduced compared with WT. By flow cytometric analysis, orthotopic myeloid-COX-2 KO tumors had lower tumor-associated macrophage (TAM) infiltration consistent with impaired colony stimulating factor-1-dependent chemotaxis by COX-2 deficient macrophages in vitro. Further, in both spontaneous and orthotopic tumors, COX-2-deficient TAM displayed lower immunosuppressive M2 markers and this was coincident with less suppression of CD8(+) cytotoxic T lymphocytes (CTLs) in myeloid-COX-2 KO tumors. These studies suggest that reduced tumor growth in myeloid-COX-2 KO mice resulted from disruption of M2-like TAM function, thereby enhancing T-cell survival and immune surveillance. Antibody-mediated depletion of CD8(+), but not CD4(+) cells, restored tumor growth in myeloid-COX-2 KO to WT levels, indicating that CD8(+) CTLs are dominant antitumor effectors in myeloid-COX-2 KO mice. Our studies suggest that inhibition of myeloid cell COX-2 can potentiate CTL-mediated tumor cytotoxicity and may provide a novel therapeutic approach in breast cancer therapy.
Assuntos
Ciclo-Oxigenase 2/fisiologia , Macrófagos/imunologia , Neoplasias Mamárias Animais/imunologia , Neoplasias Mamárias Animais/prevenção & controle , Células Mieloides/imunologia , Linfócitos T Citotóxicos/imunologia , Microambiente Tumoral/imunologia , Animais , Western Blotting , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Feminino , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Integrases/metabolismo , Ativação Linfocitária , Macrófagos/metabolismo , Macrófagos/patologia , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/metabolismo , Células Mieloides/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/patologia , Células Tumorais CultivadasRESUMO
Colon adenocarcinoma (COAD) is a common cause of cancer-related death. Due to the difficulty in early diagnosis and drug resistance, conventional treatments are difficult to be effective. Some studies have found that the functional recovery of T cells in the tumor microenvironment, especially regulatory T cells (Tregs), plays an important role in the progression of cancer. This study used the TCGA data set, clinical information and RNA-seq data of COAD patients to construct a Tregs-related risk score (TRS) through methods such as WGCNA, single-factor Cox, multi-factor Cox and random survival forest (RSF). Moreover, we also used the TCGA test set and internal validation set to verify the predictive ability of TRS, and used functional enrichment analysis and somatic mutation analysis to mine genes related to TRS, such as like thrombin/trypsin receptor 2 (F2RL2), inhibin subunit beta B (INHBB) and melanoma antigen family A12 (MAGEA12). Moreover, this study confirmed the expression of these prognostic genes using scRNA-seq data. We also performed qPCR analysis of various genes in normal and cancerous colon cancer cell lines to verify that these genes indeed play a role in CODA patients. We also constructed a mouse CODA model to study and evaluate the impact of key genes such as MAGEA12 on tumor growth in mice. This study explores the important role of Treg cells in the prognosis of COAD and discovers some potential biomarkers for the occurrence and development of COAD, which provides some new ideas for the treatment of COAD.
Assuntos
Adenocarcinoma , Neoplasias do Colo , Linfócitos T Reguladores , Humanos , Animais , Linfócitos T Reguladores/imunologia , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/mortalidade , Prognóstico , Camundongos , Microambiente Tumoral/imunologia , Microambiente Tumoral/genética , Regulação Neoplásica da Expressão Gênica , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Transcriptoma , Masculino , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Feminino , Perfilação da Expressão GênicaRESUMO
Currently, glycopeptide quantitation is mainly based on relative quantitation due to absolute quantitation requiring isotope-labeled or standard glycopeptides which may not be commercially available or are very costly and time consuming to synthesize. To address this grand challenge, coulometric mass spectrometry (CMS), based on the combination of electrochemistry (EC) and mass spectrometry (MS), was utilized to quantify electrochemically active glycopeptides without the need of using standard materials. In this study, we studied tyrosine-containing glycopeptides, NYIVGQPSS(ß-GlcNAc)TGNL-OH and NYSVPSS(ß-GlcNAc)TGNL-OH, and successfully quantified them directly with CMS with a discrepancy of less than 5% between the CMS measured amount and the theoretical amount. Taking one step further, we applied this approach to quantify glycopeptides generated from the digestion of NIST mAb, a monoclonal antibody reference material. Through HILIC column separation, five N297 glycopeptides resulting from NIST mAb tryptic digestion were successfully separated and quantified by CMS for an absolute amount without the use of any standard materials. This study indicates the potential utility of CMS for quantitative proteomics research.
Assuntos
Glicopeptídeos , Espectrometria de Massas , Oxirredução , Glicopeptídeos/análise , Glicopeptídeos/química , Espectrometria de Massas/métodos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/análise , Proteômica/métodos , Proteômica/normas , Tirosina/análise , Tirosina/química , Técnicas Eletroquímicas/métodosRESUMO
Surface-embedded glycoproteins, such as the spike protein trimers of coronaviruses MERS, SARS-CoV, and SARS-CoV-2, play a key role in viral function and are the target antigen for many vaccines. However, their significant glycan heterogeneity poses an analytical challenge. Here, we utilized individual ion mass spectrometry (I2MS), a multiplexed charge detection measurement with similarities to charge detection mass spectrometry (CDMS), in which a commercially available Orbitrap analyzer is used to directly produce mass profiles of these heterogeneous coronavirus spike protein trimers under native-like conditions. Analysis by I2MS shows that glycosylation contributes to the molecular mass of each protein trimer more significantly than expected by bottom-up techniques, highlighting the importance of obtaining complementary intact mass information when characterizing glycosylation of such heterogeneous proteins. Enzymatic dissection to remove sialic acid or N-linked glycans demonstrates that I2MS can be used to better understand the glycan profile from a native viewpoint. Deglycosylation of N-glycans followed by I2MS analysis indicates that the SARS-CoV-2 spike protein trimer contains glycans that are more difficult to remove than its MERS and SARS-CoV counterparts, and these differences are correlated with solvent accessibility. I2MS technology enables characterization of protein mass and intact glycan profile and is orthogonal to traditional mass analysis methods such as size exclusion chromatography-multiangle light scattering (SEC-MALS) and field flow fractionation-multiangle light scattering (FFF-MALS). An added advantage of I2MS is low sample use, requiring 100-fold less than other methodologies. This work highlights how I2MS technology can enable efficient development of vaccines and therapeutics for pharmaceutical development.
Assuntos
Glicoproteína da Espícula de Coronavírus , Vacinas , Humanos , Glicoproteína da Espícula de Coronavírus/química , Espectrometria de Massas/métodos , Polissacarídeos/análiseRESUMO
BACKGROUND: Microfluidic devices can create hemodynamic conditions for platelet assays. We validated an 8-channel device in a study of interdonor response to acetylsalicylic acid (ASA, aspirin) with whole blood from 28 healthy individuals. METHODS: Platelet deposition was assessed before treatment or 24 h after ingestion of 325 mg ASA. Whole blood (plus 100 µmol/L H-d-Phe-Pro-Arg-chloromethylketone to inhibit thrombin) was further treated ex vivo with ASA (0-500 µmol/L) and perfused over fibrillar collagen for 300 s at a venous wall shear rate (200 s(-1)). RESULTS: Ex vivo ASA addition to blood drawn before aspirin ingestion caused a reduction in platelet deposition [half-maximal inhibitory concentration (IC50) approximately 10-20 µmol/L], especially between 150 and 300 s of perfusion, when secondary aggregation mediated by thromboxane was expected. Twenty-seven of 28 individuals displayed smaller deposits (45% mean reduction; range 10%-90%; P < 0.001) from blood obtained 24 h after ASA ingestion (no ASA added ex vivo). In replicate tests, an R value to score secondary aggregation [deposition rate from 150 to 300 s normalized by rate from 60 to 150 s] showed R < 1 in only 2 of 28 individuals without ASA ingestion, with R > 1 in only 3 of 28 individuals after 500 µmol/L ASA addition ex vivo. At 24 h after ASA ingestion, 21 of 28 individuals displayed poor secondary aggregation (R < 1) without ex vivo ASA addition, whereas the 7 individuals with residual secondary aggregation (R > 1) displayed insensitivity to ex vivo ASA addition. Platelet deposition was not correlated with platelet count. Ex vivo ASA addition caused similar inhibition at venous and arterial wall shear rates. CONCLUSIONS: Microfluidic devices quantified platelet deposition after ingestion or ex vivo addition of aspirin.
Assuntos
Aspirina/farmacologia , Plaquetas/efeitos dos fármacos , Colágeno , Técnicas Analíticas Microfluídicas/instrumentação , Inibidores da Agregação Plaquetária/farmacologia , Administração Oral , Adolescente , Adulto , Aspirina/uso terapêutico , Plaquetas/fisiologia , Ciclo-Oxigenase 1/metabolismo , Feminino , Corantes Fluorescentes , Humanos , Masculino , Pessoa de Meia-Idade , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/uso terapêutico , Testes de Função Plaquetária , Valores de Referência , Fluxo Sanguíneo Regional , Transdução de Sinais , Estresse Mecânico , Tromboxano A2/metabolismo , Adulto JovemRESUMO
Biopharmaceutical products have seen rapid growth over the past few decades and continue to dominate the global pharmaceutical market. Aligning with the quality by design (QbD) framework and realization, recent advances in liquid chromatography-mass spectrometry (LC-MS) instrumentation and related techniques have enhanced biopharmaceutical characterization capabilities and have supported an increased development of biopharmaceutical products. Beyond its routine qualitative characterization, the quantitative feature of LC-MS has unique applications in biopharmaceutical process development and manufacturing. This review describes the recent applications and implications of the advancement of quantitative MS methods in biopharmaceutical process development, and characterization of biopharmaceutical product, product-related variants, and process-related impurities. We also provide insights on the emerging applications of quantitative MS in the lifecycle of biopharmaceutical product development including quality control in the Good Manufacturing Practice (GMP) environment and process analytical technology (PAT) practices during process development and manufacturing. Through collaboration with instrument and software vendors and regulatory agencies, we envision broader adoption of phase-appropriate quantitative MS-based methods for the analysis of biopharmaceutical products, which in turn has the potential to enable manufacture of higher quality products for patients.
Assuntos
Produtos Biológicos , Humanos , Espectrometria de Massas , Cromatografia Líquida , Controle de QualidadeRESUMO
Purpose: The purpose of our meta-analysis and systematic review was to compare the diagnostic performance of [18F]FDG PET/CT and [18F]FDG PET/MRI in colorectal liver metastasis. Methods: We searched PubMed, Embase, and Web of Science for eligible articles until November 2022. Studies focusing on the diagnostic value of [18F]FDG PET/CT or PET/MRI for colorectal liver metastasis were included. Using a bivariate random-effect model, the pooled sensitivity and specificity for [18F]FDG PET/CT and [18F]FDG PET/MRI were reported as estimates with 95% confidence intervals (CIs). Heterogeneity among pooled studies was assessed using the I2 statistic. The Quality Assessment of Diagnostic Performance Studies (QUADAS-2) method was used to evaluate the quality of the studies that were included. Results: There were a total of 2743 publications identified in the initial search, finally, a total of 21 studies comprising 1036 patients were included. The pooled sensitivity, specificity, and AUC of [18F]FDG PET/CT in were 0.86 (95% CI: 0.76-0.92), 0.89 (95% CI: 0.83-0.94), and 0.92(95% CI: 0.90-0.94). [18F]FDG PET/MRI were 0.84 (95% CI: 0.77-0.89), 1.00 (95% CI: 0.32-1.00), and 0.89(95% CI: 0.86-0.92), respectively. Conclusion: [18F]FDG PET/CT shows similar performance compared to [18F]FDG PET/MRI in detecting colorectal liver metastasis. However, pathological results were not obtained for all patients in the included studies and PET/MRI results were derived from studies with small sample sizes. There is a need for additional, larger prospective studies on this issue. Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier (CRD42023390949).
RESUMO
BACKGROUND: Sapiens spondin-2 (SPON2) is a protein found in the extracellular matrix that plays a role in a number of processes, including immune reactions and cell adhesion, and is closely linked to the emergence of a number of tumor types. However, we know very little about Sapiens spondin-2. Therefore, we performed a systematic pan-carcinogenic analysis to explore the relationship between Sapiens spondin-2 and cancers. MATERIALS AND METHODS: By comprehensive use of datasets from TCGA, GEO, GTEx, HPA, CPTAC, GEPIA2, TIMER2, cBioPortal, STRING, we adopted bioinformatics methods to dig up the potential carcinogenesis of SPON2, including dissecting the correlation between SPON2 and gene expression, prognosis, gene mutation, Immunohistochemistry staining, immune cell infiltration, and constructed the interaction network of a total of 54 SPON2-binding proteins as well as explored the enrichment analysis of SPON2-related partners. RESULTS: The expression of Sapiens spondin-2 in most tumor tissues was higher than that of normal tissues. In addition, SPON2 showed the early diagnostic value in 33 kinds of tumors and was positively or negatively associated with the prognosis of different tumors. It also validates that SPON2 is the gene associated with the majority of immune-infiltrating cells in pan-cancer. High SPON2 expression is associated with tumor progression related pathways. CONCLUSION: We found and validated the potential use of SPON2 in cancer detection for the first time through pan-cancer analysis. The expression levels of SPON2 in various tumors were quite different from those in normal tissues. Furthermore, the performance of SPON2 in tumorigenesis and tumor immunity verified our hypothesis. At the same time, it has high specificity and sensitivity in cancer detection. Therefore, SPON2 can be employed as an auxiliary index for the initial diagnosis of tumors and a prognostic marker for various types of tumors.