Standards-Free Absolute Quantitation of Oxidizable Glycopeptides by Coulometric Mass Spectrometry.

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.

Identification and validation of regulatory T cell-associated gene signatures to predict colon adenocarcinoma prognosis.

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.

Factors affecting product association as a mechanism of host-cell protein persistence in bioprocessing.

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.

Identification and characterization of CHO host-cell proteins in monoclonal antibody bioprocessing.

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.

Dissecting the Heterogeneous Glycan Profiles of Recombinant Coronavirus Spike Proteins with Individual Ion Mass Spectrometry.

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.

Comprehensive analysis of the oncogenic and immunological role of SPON2 in human tumors.

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.

To identify biomarkers associated with the transfer of diabetes combined with cancer in human genes using bioinformatics analysis.

Currently, the incidence of diabetes mellitus is increasing rapidly, particularly in China, and its pathogenesis is still unclear. The goal of this study was to find meaningful biomarkers of metastasis in patients with diabetes and cancer using bioinformatic analysis in order to predict gene expression and prognostic importance for survival. We used the Differentially Expressed Gene, Database for Annotation Visualization and Integrated Discovery, and Gene Set Enrichment Analyses databases, as well as several bioinformatics tools, to explore the key genes in diabetes. Based on the above database, we ended up with 10 hub genes (FOS, ATF3, JUN, EGR1, FOSB, JUNB, BTG2, EGR2, ZFP36, and NR4A2). A discussion of the 10 critical genes, with extensive literature mentioned to validate the association between the 10 key genes and patients with diabetes and cancer, to demonstrate the importance of gene expression and survival prognosis. This study identifies several biomarkers associated with diabetes and cancer development and metastasis that may provide novel therapeutic targets for diabetes combined with cancer patients.

Network characterization linc1393 in the maintenance of pluripotency provides the principles for lncRNA targets prediction.

Long non-coding RNAs (lncRNAs) have been implicated in diverse biological processes. However, the functional mechanisms have not yet been fully explored. Characterizing the interactions of lncRNAs with chromatin is central to determining their functions but, due to precise and efficient approaches lacking, our understanding of their functional mechanisms has progressed slowly. In this study, we demonstrate that a nuclear lncRNA linc1393 maintains mouse ESC pluripotency by recruiting SET1A near its binding sites, to establish H3K4me3 status and activate the expression of specific pluripotency-related genes. Moreover, we characterized the principles of lncRNA-chromatin interaction and transcriptional regulation. Accordingly, we developed a computational framework based on the XGBoost model, LncTargeter, to predict the targets of a given lncRNA, and validated its reliability in various cellular contexts. Together, these findings elucidate the roles and mechanisms of lncRNA on pluripotency maintenance, and provide a promising tool for predicting the regulatory networks of lncRNAs.

Recent applications of quantitative mass spectrometry in biopharmaceutical process development and manufacturing.

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.

Transcriptome analysis of two bloom-forming Prorocentrum species reveals physiological changes related to light and temperature.

Temperature and light substantially influence red tide succession. However, it remains unclear whether the molecular mechanisms differ among species. In this study, we measured the variation in the physiological parameters of growth and pigments and transcriptional levels of two bloom-forming dinoflagellates, namely Prorocentrum micans and P. cordatum. This was undertaken in four treatments that represented two factorial temperature combinations (LT: 20 °C, HT: 28 °C) and light conditions (LL: 50 µmol photons m-2 s-1, HL: 400 µmol photons m-2 s-1) for 7-day batch culture. Growth under high temperature and high light (HTHL) was the fastest, while growth under high temperature and low light (HTLL) was the slowest. The pigments (chlorophyll a and carotenoids) decreased significantly in all high light (HL) treatments, but not in high temperature (HT) treatments. HL alleviated the low light-caused photolimitation and enhanced the growth of both species at low temperatures. However, HT inhibited the growth of both species by inducing oxidative stress under low light conditions. HL mitigated the HT-induced stress on growth in both species by upregulating photosynthesis, antioxidase activity, protein folding, and degradation. The cells of P. micans were more sensitive to HT and HL than those of P. cordatum. This study deepens our understanding of the species-specific mechanism of dinoflagellates at the transcriptomic level, adapting to the future ocean changes including higher solar radiation and higher temperatures in the upper mixed layer.

[18F]FDG PET/CT versus [18F]FDG PET/MRI for the diagnosis of colorectal liver metastasis: A systematic review and meta-analysis.

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).

RRP15 deficiency induces ribosome stress to inhibit colorectal cancer proliferation and metastasis via LZTS2-mediated ß-catenin suppression.

Ribosome biogenesis (RiBi) plays a pivotal role in carcinogenesis by regulating protein translation and stress response. Here, we find that RRP15, a nucleolar protein critical for RiBi and checkpoint control, is frequently upregulated in primary CRCs and higher RRP15 expression positively correlated with TNM stage (P < 0.0001) and poor survival of CRC patients (P = 0.0011). Functionally, silencing RRP15 induces ribosome stress, cell cycle arrest, and apoptosis, resulting in suppression of cell proliferation and metastasis. Overexpression of RRP15 promotes cell proliferation and metastasis. Mechanistically, ribosome stress induced by RRP15 deficiency facilitates translation of TOP mRNA LZTS2 (Leucine zipper tumor suppressor 2), leading to the nuclear export and degradation of ß-catenin to suppress Wnt/ß-catenin signaling in CRC. In conclusion, ribosome stress induced by RRP15 deficiency inhibits CRC cell proliferation and metastasis via suppressing the Wnt/ß-catenin pathway, suggesting a potential new target in high-RiBi CRC patients.

Characterization and implications of host-cell protein aggregates in biopharmaceutical processing.

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.

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

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

Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry.

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.

Improvements on sample preparation and peptide separation for reduced peptide mapping based multi-attribute method analysis of therapeutic monoclonal antibodies using lysyl endopeptidase digestion.

The mass spectrometry based multi-attribute method (MAM) has gained popularity in the field of biopharmaceutical analysis as it promises a single method for comprehensive monitoring of multiple product quality attributes (PQAs) and product purity. Sample preparation for protein digestion and peptide separation are critical considerations for a reduced peptide mapping-based MAM. To avoid desalting steps required in most tryptic protein digestion and in order to improve peptide separation for hydrophilic peptides, we developed an improved robust sample preparation using lysyl endopeptidase (Lys-C) for high-throughput MAM testing. Additionally, this method optimizes the peptide retention and separation of a stability-indicating VSNK peptide using a HSS T3 column for comprehensive PQA monitoring. A fully automated sample preparation had similar assay variations for PQAs monitoring compared to manual sample preparation. To the best of our knowledge, this is the first report of a high-resolution MS-based MAM using a streamlined Lys-C digestion without desalting with enhanced PQA monitoring for hydrophilic peptides. The improved, robust MAM workflow for protein digestion and peptide separation will pave the way for broader MAM qualification and its applications for the characterization and quality control of therapeutic monoclonal antibodies.

Holistic analytical characterization and risk assessment of residual host cell protein impurities in an active pharmaceutical ingredient synthesized by biocatalysts.

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.

The measurement and control of high-risk host cell proteins for polysorbate degradation in biologics formulation.

Nonionic surfactant polysorbates, including PS-80 and PS-20, are commonly used in the formulation of biotherapeutic products for both preventing surface adsorption and acting as stabilizer against protein aggregation. Trace levels of residual host cell proteins (HCPs) with lipase or esterase enzymatic activity have been shown to degrade polysorbates in biologics formulation. The measurement and control of these low abundance, high-risk HCPs for polysorbate degradation are an industry-wide challenge to achieve desired shelf life of biopharmaceuticals in liquid formulation, especially for high-concentration formulation product development. Here, we reviewed the challenges, recent advances, and future opportunities of analytical method development, risk assessment, and control strategies for polysorbate degradation during formulation development with a focus on enzymatic degradation. Continued efforts to advance our understanding of polysorbate degradation in biologics formulation will help develop high-quality medicines for patients.

A pan-cancer analysis of the oncogenic role of polypyrimidine tract binding protein 1 (PTBP1) in human tumors.

BACKGROUND: Polypyrimidine tract-binding protein 1 (PTBP1) is an RNA-binding protein that regulates several posttranscriptional events and is closely related to the development of multiple tumors. However, little is known about PTBP1. Thus, we carried out a systematic pan-cancer analysis to explore the relationship between PTBP1 and cancer. METHODS: We used The Cancer Genome Atlas, Gene Expression Omnibus, and Human Protein Atlas datasets, as well as several bioinformatics tools, to explore the role of PTBP1 in 33 tumor types. RESULTS: The expression of PTBP1 in most tumor tissues was higher than that in normal tissues. Survival analysis indicated that overexpression of PTBP1 generally predicted poor overall survival in patients with tumors such as adrenocortical carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, and skin cutaneous melanoma. In addition, we compared the phosphorylation and immune infiltration of PTBP1 in cancer-associated fibroblasts between normal and primary tumor tissues and explored the putative functional mechanism of tumorigenesis mediated by PTBP1. CONCLUSION: These results provide clues to better understand PTBP1 from the perspective of bioinformatics and highlight its importance in various human cancers.

Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling.

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.