Mass spectrometry study on SARS-CoV-2 recombinant vaccine with comprehensive separation techniques to characterize complex heterogeneity.

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation and recombinant vaccine has gained much attention thanks to its potential for greater response predictability, improved efficacy, rapid development and reduced side effects. Recombinant vaccines are designed and manufactured using bacterial, yeast cells or mammalian cells. A small piece of DNA is taken from the virus or bacterium against which we want to protect and inserted into the manufacturing cells. Due to the extremely complex heterogeneity of SARS-CoV-2 recombinant vaccine, single technology platform cannot achieve thorough and accurate characterization of such difficult proteins so integrating comprehensive technologies is essential. This study illustrates an innovative workflow employing multiple separation techniques tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of SARS-CoV-2 recombinant vaccine, including ultra-high performance liquid chromatography (UHPLC), ion exchange chromatography (IEX) and imaged capillary isoelectric focusing (icIEF). The integrated methodology focuses on the importance of cutting-edge icIEF-MS online coupling and icIEF fractionation applied to revealing the heterogeneity secret of SARS-CoV-2 recombinant vaccine.

Using capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and liquid chromatograph mass spectrometry (LC-MS) to identify glycosylated heavy chain heterogeneity in the anti-VEGFR-2 monoclonal antibody.

The size variant, which can be measured by capillary electrophoresis sodium dodecyl sulfate (CE-SDS), is a critical quality attribute of monoclonal antibodies (mAbs). The CE-SDS size heterogeneity can hardly be identified by tandem mass spectrometry, which is an intractable obstacle of mAb development and quality control across the industry. We analyzed the purity of an anti-vascular endothelial growth factor receptor 2 (VEGFR-2) mAb, an antagonist of the human VEGFR-2, through reduced CE-SDS and observed glycosylated heavy chain heterogeneity. The heterogeneity has potential impact on safety, efficacy, and stability of drugs for clinical use. Therefore, it should be characterized so as to evaluate its potential risk. In order to identify the heterogeneity, we used mass spectrometry to confirm that the molecular size heterogeneity was not due to peptide bond cleavage in the heavy chain. Subsequently, we employed mass-spectrometry-glycosylation profiling and CE-SDS analysis of various glycosidase-treated samples, in addition to the preparation of mAb references with different glycoforms. Ultimately, we demonstrated that the heavy chain heterogeneity was induced by different levels of galactosylation modifications which will potentially impact the efficacy of antibody drugs (i.e., complement-dependent cytotoxicity). In this study, potential risk caused by heavy chain size heterogeneity was evaluated, which addressed the obstacle of mAb development and quality control. Therefore, this study offers a feasible approach for the investigation and identification of heavy chain heterogeneity in reduced CE-SDS, providing a novel strategy for mAb quality control and evaluation.

A novel alternative for pyrogen detection based on a transgenic cell line.

Pyrogen, often as a contaminant, is a key indicator affecting the safety of almost all parenteral drugs (including biologicals, chemicals, traditional Chinese medicines and medical devices). It has become a goal to completely replace the in vivo rabbit pyrogen test by using the in vitro pyrogen test based on the promoted 'reduction, replacement and refinement' principle, which has been highly considered by regulatory agencies from different countries. We used NF-κB, a central signalling molecule mediating inflammatory responses, as a pyrogenic marker and the monocyte line THP-1 transfected with a luciferase reporter gene regulated by NF-κB as an in vitro model to detect pyrogens by measuring the intensity of a fluorescence signal. Here, we show that this test can quantitatively and sensitively detect endotoxin (lipopolysaccharide from different strains) and nonendotoxin (lipoteichoic acid, zymosan, peptidoglycan, lectin and glucan), has good stability in terms of NF-κB activity and cell phenotypes at 39 cell passages and can be applied to detect pyrogens in biologicals (group A & C meningococcal polysaccharide vaccine; basiliximab; rabies vaccine (Vero cells) for human use, freeze-dried; Japanese encephalitis vaccine (Vero cells), inactivated; insulin aspart injection; human albumin; recombinant human erythropoietin injection (CHO Cell)). The within-laboratory reproducibility of the test in three independent laboratories was 85%, 80% and 80% and the interlaboratory reproducibility among laboratories was 83.3%, 95.6% and 86.7%. The sensitivity (true positive rate) and specificity (true negative rate) of the test were 89.9% and 90.9%, respectively. In summary, the test provides a novel alternative for pyrogen detection.

Qingchang Wenzhong Decoction ameliorates intestinal inflammation and intestinal barrier dysfunction in ulcerative colitis via the GC-C signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) is an idiopathic, chronic inflammatory disorder of the colonic mucosa, accompanied with abdominal pain, and bloody diarrhea. Currently, clinical treatment options for UC are limited. Qingchang Wenzhong Decoction (QCWZD) is an effective prescription of traditional Chinese medicine for the treatment of UC. However, the mechanism of QCWZD in alleviating intestinal barrier dysfunction in UC has not been clearly explained. AIM OF THE STUDY: To determine the mechanism whereby QCWZD promotes the recovery of intestinal barrier dysfunction in UC. MATERIALS AND METHODS: A secondary analysis of colonic mucosa from UC patients acquired from a prior RCT clinical trial was performed. The effects of QCWZD on intestinal mucus and mechanical barriers in UC patients were evaluated using colon tissue paraffin-embedded sections from UC patients. The mechanism was further investigated by in vivo and in vitro experiments. UC mice were established in sterile water with 3.0% dextran sodium sulfate (DSS). Meanwhile, mice in the treatment group were dosed with QCWZD or mesalazine. In vitro, an intestinal barrier model was constructed using Caco-2 and HT29 cells in co-culture. GC-C plasmid was used to overexpress/knock down GC-C to clarify the target of QCWZD. HE, AB-PAS, ELISA, immunohistochemistry and immunofluorescence assays were used to assess the level of colonic inflammation and intestinal barrier integrity. Rt-qPCR, Western Blot were used to detect the expression of genes and proteins related to GC-C signaling pathway. Molecular docking was used to simulate the binding sites of major components of QCWZD to GC-C. RESULTS: In UC patients, QCWZD increased mucus secretion, goblet cell number, and promoted MUC2 and ZO-1 expression. QCWZD accelerated the recovery of UC mice from DSS-induced inflammation, including weight gain, reduced disease activity index (DAI) scores, colon length recovery, and histological healing. QCWZD promoted mucus secretion and increased ZO-1 expression in in vivo and in vitro experiments, thereby repairing mucus mechanical barrier damage. The effects of QCWZD are mediated through regulation of the GC-C signaling pathway, which in turn affects CFTR phosphorylation and MUC2 expression to promote mucus secretion, while inhibiting the over-activation of MLCK and repairing tight junctions to maintain the integrity of the mechanical barrier. Molecular docking results demonstrate the binding of the main components of QCWZD to GC-C. CONCLUSION: Our study demonstrated that QCWZD modulates the GC-C signaling pathway to promote remission of mucus-mechanical barrier damage in the UC. The clarification of the mechanism of QCWZD holds promise for the development of new therapies for UC.

[Practical Optimization and Application of Time-delay Exposure System for Mobile Digital Radiography Equipment].

This study introduced a time-delay exposure system independent of the mobile digital radiography equipment. The system consisted of lithium battery, delay control circuit, micro electric motor and related auxiliary facilities. When the starting time was reached through the delay circuit, the motor pushed out the rod to squeeze the exposure button and completed the exposure. The accessories used in this system were easy to purchase and cheap. At the same time, the technology was mature and had good compatibility. The exposure success rate was high and the exposure effect was satisfactory. This time-delay exposure system had good practicability and popularization value.

Climate Change Potentially Leads to Habitat Expansion and Increases the Invasion Risk of Hydrocharis (Hydrocharitaceae).

Climate change is a crucial factor impacting the geographical distribution of plants and potentially increases the risk of invasion for certain species, especially for aquatic plants dispersed by water flow. Here, we combined six algorithms provided by the biomod2 platform to predict the changes in global climate-suitable areas for five species of Hydrocharis (Hydrocharitaceae) (H. chevalieri, H. dubia, H. laevigata, H. morsus-ranae, and H. spongia) under two current and future carbon emission scenarios. Our results show that H. dubia, H. morsus-ranae, and H. laevigata had a wide range of suitable areas and a high risk of invasion, while H. chevalieri and H. spongia had relatively narrow suitable areas. In the future climate scenario, the species of Hydrocharis may gain a wider habitat area, with Northern Hemisphere species showing a trend of migration to higher latitudes and the change in tropical species being more complex. The high-carbon-emission scenario led to greater changes in the habitat area of Hydrocharis. Therefore, we recommend strengthening the monitoring and reporting of high-risk species and taking effective measures to control the invasion of Hydrocharis species.

Vacuum-assisted dedusting lithotripsy in the treatment of kidney and proximal ureteral stones less than 3 cm in size.

PURPOSE: This study aimed to compare the outcomes of vacuum-assisted dedusting lithotripsy (VADL) using flexible vacuum-assisted ureteral access sheath (FV-UAS) versus traditional flexible ureteroscopic lithotripsy (fURL) in patients with kidney or proximal ureteral calculi less than 3 cm in size. METHODS: A total of 371 patients who successfully underwent fURL treatment were enrolled. These patients were divided into traditional fURL group and VADL group. Outcomes of both groups were compared using 1:1 propensity score-matched analysis. Stratified analyses based on stone size and location were also conducted. RESULTS: Finally, 103 well-matched patients in each group were identified. No septic shock or death occurred. The immediate stone-free rate (SFR) and follow-up SFR of VADL group were significantly higher (78.6% vs. 50.5%, p < 0.001; 94.2%% vs. 75.7%, p < 0.001). No difference was observed in postoperative fever rate (2.9% vs. 3.9%, p = 1.000) and duration of lithotripsy (37.7 ± 20.1 min vs. 40.3 ± 18.9 min, p = 0.235). For patients with stones ≤ 2 cm in size, the immediate SFR and follow-up SFR in VADL group were higher (86.7% vs. 60.6%, p < 0.001; 96.0% vs. 83.1%, p = 0.010). The same trend was observed in the 2-3 cm subgroup (57.1% vs. 28.1%, p = 0.023; 89.3% vs. 59.4%, p = 0.009). Although the in situ fragmentation strategy was employed more frequently in VADL group for lower pole stones, the SFR was still higher. Subgroup analyses did not reveal any significant differences in either infectious complications or duration of lithotripsy. CONCLUSION: VADL technique can significantly improve the postoperative SFR for the patients with kidney or proximal ureteral stones less than 3 cm in size treated by flexible ureteroscope.

Experimental Study on the Effect of CO2 Injection Pressure on the Migration Characteristics and Extraction Effects of Replacement CH4.

To study the effect of CO2 injection pressure on gas migration characteristics and coalbed methane (CBM) extraction, a platform for the experimental replacement of CH4 with CO2 was used to conduct experiments on the replacement of CH4 under different CO2 injection pressures and analyze the gas transport characteristics and CH4 extraction during the experiment. The results reveal that the rate of gas migration out of the coal seam accelerates with increasing gas injection pressure, as determined by comparisons of the migration rates between adjacent monitoring points. The change trend of the CH4 desorption rate under different gas injection pressures is divided into slow decline, sharp decline, and stability stages, and the maximum value of the effective diffusion coefficient increases from 2.3 × 10-5 to 3.4 × 10-5 and 4.6 × 10-5 cm2/s as the gas injection pressure increases from 0.6 to 0.8 and 1.0 MPa. Similarly, the change pattern of coal seam permeability can be divided into slow decline, sharp decline, and stability stages. After the gas injection pressure was increased from 0.6 to 0.8 and 1.0 MPa, the CH4 desorption volume increased from 90.2 to 94.1 and 97.8 L, whereas the coal seam CO2 sequestration volume increased from 269.2 to 274.2 and 322.8 L, respectively. In contrast, the CH4 extraction efficiency increased from 76.9 to 80.2 and 82.9%, respectively. The research results have important reference value and practical significance for optimizing the CO2 injection pressure and improving the CBM extraction.

Establishing a novel and sensitive assay for bioactivity determination of anti-CD25 antibodies.

Anti-CD25 antibodies have been approved for renal transplantation and has been used prior to and during transplantation by the Food and Drug Administration (FDA). However, no reported bioassays have been reflected the mechanism of action (MOA) of anti-CD25 antibodies. Here, we describe the development and validation of a reporter gene assay (RGA) based on the engineered C8166-STAT5RE-Luc cells expressing endogenous IL-2 receptors and a STAT5-inducible element-driven firefly luciferase in C8166 cell lines. The RGA was fully validated according to the International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for the Human Use-Q2 (ICH-Q2). After optimization, the assay showed excellent specificity, linearity, accuracy, precision, and robustness. Due to the MOA relatedness and the excellent assay performance, the RGA is suitable for exploring the critical quality attributes (CQAs), release inspection, comparability and stability of anti-CD25 mAbs.

Parallel interrogation of the chalcogenide-based micro-ring sensor array for photoacoustic tomography.

Photoacoustic tomography (PAT), also known as optoacoustic tomography, is an attractive imaging modality that provides optical contrast with acoustic resolutions. Recent progress in the applications of PAT largely relies on the development and employment of ultrasound sensor arrays with many elements. Although on-chip optical ultrasound sensors have been demonstrated with high sensitivity, large bandwidth, and small size, PAT with on-chip optical ultrasound sensor arrays is rarely reported. In this work, we demonstrate PAT with a chalcogenide-based micro-ring sensor array containing 15 elements, while each element supports a bandwidth of 175 MHz (-6 dB) and a noise-equivalent pressure of 2.2 mPaHz-1/2. Moreover, by synthesizing a digital optical frequency comb (DOFC), we further develop an effective means of parallel interrogation to this sensor array. As a proof of concept, parallel interrogation with only one light source and one photoreceiver is demonstrated for PAT with this sensor array, providing images of fast-moving objects, leaf veins, and live zebrafish. The superior performance of the chalcogenide-based micro-ring sensor array and the effectiveness of the DOFC-enabled parallel interrogation offer great prospects for advancing applications in PAT.

Mass Spectrometry-Based Charge Heterogeneity Characterization of Therapeutic mAbs with Imaged Capillary Isoelectric Focusing and Ion-Exchange Chromatography as Separation Techniques.

Imaged capillary isoelectric focusing (icIEF) and ion-exchange chromatography (IEX) are two essential techniques that are routinely used for charge variant analysis of therapeutic monoclonal antibodies (mAbs) during their development and in quality control. These two techniques that separate mAb charge variants based on different mechanisms and IEX have been developed as front-end separation techniques for online mass spectrometry (MS) detection, which is robust for intact protein identification. Recently, an innovative, coupled icIEF-MS technology has been constructed for protein charge variant analysis in our laboratory. In this study, icIEF-MS developed and strong cation exchange (SCX)-MS were optimized for charge heterogeneity characterization of a diverse of mAbs and their results were compared based on methodological validation. It was found that icIEF-MS outperformed SCX-MS in this study by demonstrating outstanding sensitivity, low carryover effect, accurate protein identification, and higher separation resolution although SCX-MS contributed to higher analysis throughput. Ultimately, integrating our novel icIEF-HRMS analysis with the more common SCX-MS can provide a promising and comprehensive strategy for accelerating the development of complex protein therapeutics.

Development of a reporter gene assay for antibody dependent cellular cytotoxicity activity determination of anti-rabies virus glycoprotein antibodies.

Rabies is a viral disease that is nearly 100% fatal once clinical signs and symptoms develop. Post-exposure prophylaxis can efficiently prevent rabies, and antibody (Ab) induction by vaccination or passive immunization of human rabies immunoglobulin (HRIG) or monoclonal antibodies (mAbs) play an integral role in prevention against rabies. In addition to their capacity to neutralize viruses, antibodies exert their antiviral effects by antibody-dependent cellular cytotoxicity (ADCC), which plays an important role in antiviral immunity and clearance of viral infections. For antibodies against rabies virus (RABV), evaluation of ADCC activity was neglected. Here, we developed a robust cell-based reporter gene assay (RGA) for the determination of the ADCC activity of anti-RABV antibodies using CVS-N2c-293 cells, which stably express the glycoprotein (G) of RABV strain CVS-N2c as target cells, and Jurkat cells, which stably express FcγRⅢa and nuclear factor of activated T cells (NFAT) reporter gene as effector cells (Jurkat/NFAT-luc/FcγRⅢa cells). The experimental parameters were carefully optimized, and the established ADCC assay was systematically validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2 guideline. We also evaluated the ADCC activity of anti-RABV antibodies, including mAbs, HRIG, and vaccine induced antisera, and found that all test antibodies exhibited ADCC activity with varied strengths. The established RGA provides a novel method for evaluating the ADCC of anti-RABV antibodies.

Review on Drug Regulatory Science Promoting COVID-19 Vaccine Development in China.

Regulatory science is a discipline that uses comprehensive methods of natural science, social science, and humanities to provide support for administrative decision-making through the development of new tools, standards, and approaches to assess the safety, efficacy, quality, and performance of regulated products. During the pandemics induced by infectious diseases, such as H1N1 flu, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS), regulatory science strongly supported the development of drugs and vaccines to respond to the viruses. In particular, with the support of research on drug regulatory science, vaccines have played a major role in the prevention and control of coronavirus disease 2019 (COVID-19). This review summarizes the overall state of the vaccine industry, research and development (R&D) of COVID-19 vaccines in China, and the general state of regulatory science and supervision for vaccines in China. Further, this review highlights how regulatory science has promoted the R&D of Chinese COVID-19 vaccines, with analyses from the aspects of national-level planning, relevant laws and regulations, technical guidelines, quality control platforms, and post-marketing supervision. Ultimately, this review provides a reference for the formulation of a vaccine development strategy in response to the current pandemic and the field of vaccine development in the post-pandemic era, as well as guidance on how to better respond to emerging and recurring infectious diseases that may occur in the future.

Finger printing human norovirus-like particles by capillary isoelectric focusing with whole column imaging detection.

Owing to the limitation of in vitro culture of human noroviruses (HuNoVs), the development of HuNoV vaccines has to depend on the self-assembling virus-like particles (VLPs) with capsid protein expression. The heterogeneity of artificial VLPs exert an impact on the immunogenicity, and should be considered as one of the factors in vaccine evaluation. In this study, we biochemically finger print the HuNoV VLPs with different genogroups, genotypes and sub-genotypes which constitute for a candidate vaccine, by using capillary isoelectric focusing with whole column imaging detection (cIEF-WCID). The electropherograms of GI.1, GII.3, GII.4 and GII.17 VLPs in fluorescence signal were described in the monomer VP1 forms after degenerated by 8 M urea. The four HuNoV VLPs showed different properties in electropherogram finger prints. The finger prints were also reproducible within a certain concentration range (approx. 150 ∼ 20 ug/ml). This method can also tell the changes of pI finger-print patterns when the expired HoNoV VLPs were tested. In conclusion, cIEF-WCID shows great promise for evaluating the production consistency of HuNoV VLP vaccine.

Heptadecanoic acid and pentadecanoic acid crosstalk with fecal-derived gut microbiota are potential non-invasive biomarkers for chronic atrophic gastritis.

Background: Chronic atrophic gastritis (CAG), premalignant lesions of gastric cancer (GC), greatly increases the risk of GC. Gastroscopy with tissue biopsy is the most commonly used technology for CAG diagnosis. However, due to the invasive nature, both ordinary gastroscope and painless gastroscope result in a certain degree of injury to the esophagus as well as inducing psychological pressure on patients. In addition, patients need fast for at least half a day and take laxatives. Methods: In this study, fecal metabolites and microbiota profiles were detected by metabolomics and 16S rRNA V4-V5 region sequencing. Results: Alteration of fecal metabolites and microbiota profiles was found in CAG patients, compared with healthy volunteers. To identify the most relevant features, 7 fecal metabolites and 4 microbiota were selected by random forest (RF), from A and B sample sets, respectively. Furthermore, we constructed support vector machines (SVM) classifification model using 7 fecal metabolites or 4 gut microbes, or 7 fecal metabolites with 4 gut microbes, respectively, on C sample set. The accuracy of classifification model was 0.714, 0.857, 0.857, respectively, and the AUC was 0.71, 0.88, 0.9, respectively. In C sample set, Spearman's rank correlation analysis demonstrated heptadecanoic acid and pentadecanoic acid were signifificantly negatively correlated to Erysipelotrichaceae_UCG-003 and Haemophilus, respectively. We constructed SVM classifification model using 2 correlated fecal metabolites and 2 correlated gut microbes on C sample set. The accuracy of classification model was 0.857, and the AUC was 0.88. Conclusion: Therefore, heptadecanoic acid and pentadecanoic acid, crosstalk with fecal-derived gut microbiota namely Erysipelotrichaceae_UCG-003 and Haemophilus, are potential non-invasive biomarkers for CAG diagnosis.

Quality comparability assessment of a SARS-CoV-2-neutralizing antibody across transient, mini-pool-derived and single-clone CHO cells.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a serious public health crisis worldwide, and considering the novelty of the disease, preventative and therapeutic measures alike are urgently needed. To accelerate such efforts, the development of JS016, a neutralizing monoclonal antibody directed against the SARS-CoV-2 spike protein, was expedited from a typical 12- to 18-month period to a 4-month period. During this process, transient Chinese hamster ovary cell lines are used to support preclinical, investigational new drug-enabling toxicology research, and early Chemistry, Manufacturing and Controls development; mini-pool materials to supply Phase 1 clinical trials; and a single-clone working cell bank for late-stage and pivotal clinical trials were successively adopted. Moreover, key process performance and product quality investigations using a series of orthogonal and state-of-the-art techniques were conducted to demonstrate the comparability of products manufactured using these three processes, and the results indicated that, despite observed variations in process performance, the primary and high-order structures, purity and impurity profiles, biological and immunological functions, and degradation behaviors under stress conditions were largely comparable. The study suggests that, in particular situations, this strategy can be adopted to accelerate the development of therapeutic biopharmaceuticals and their access to patients.

TP53 Mutation Related and Directly Regulated lncRNA Prognosis Markers in Hepatocellular Carcinoma.

PURPOSE: TP53 mutation is the most common genetic variation type in Hepatocellular carcinoma (HCC). We aim to illustrate the landscape of genomic alterations and TP53 mutation related and directly regulated lncRNA prognosis markers. MATERIALS AND METHODS: Utilizing the clinical and transcriptome data from The Cancer Genome Atlas (TCGA) website, we present the landscape of genomic alterations and RNA differential expression profiles. By analyzing the ENCODE TP53 ChIP-seq data, we get the TP53 chromatin binding profiles. By Kaplan-Meier (KM) survival analysis and ROC analysis, we identify lncRNA prognosis markers. RESULTS: TP53 ranks the highest mutation frequency gene and the maximum mutation type of TP53 is Missense Mutation (> 2.5×104). TP53 mutation showed poor clinical outcome among the pathological Stage II and Stage III HCC patients. By differential expression analysis of the TP53 wild type and mutation HCC, we find thousands of misregulated genes, including 699 differential expression lncRNAs (p <0.05, |log2FC| ≥1). Functional enrichment analysis of the misregulated genes shows that TP53 mutation events mainly alter DNA replication, cell cycle and immune response signaling pathways. By estimation of tumor-infiltrating immune cells through CIBERSORT, we find that the TP53 mutation events are significantly correlated with the different proportions of nine immune cells. We then integratively analyze the differential expression lncRNAs in TP53 wild type and mutation groups and the TP53 ChIP-seq binding lncRNAs, and get 112 overlap lncRNAs. By Kaplan-Meier survival analysis and ROC analysis, we identify two lncRNAs (RP4-736L20.3 and SNRK-AS1) that show significant prognosis value. Using the collected HCC samples, we validate the misregulated expression of RP4-736L20.3 and SNRK-AS1. CONCLUSION: The work presents the landscape of genomic variations and two TP53 mutation related and directly regulated lncRNA prognosis markers of HCC.

Development of a robust bioassay of monoclonal antibodies and biosimilars against TNF-α by NF-κB-inducible lentiviral reporter gene.

The tumor necrosis factor alpha (TNF-α)/nuclear factor-kappa B (NF-κB) signaling pathway plays a crucial role in the pathogenesis of inflammatory diseases. Several therapeutic monoclonal antibodies (mAbs) and biosimilars against TNF-α have been developed to treat patients who suffer from inflammatory diseases caused by disordered expression of TNF-α. Hence, quality control of biopharmaceuticals is crucial during research and development. The high-order structure of these complex molecules cannot be entirely identified by physiochemical attributes; however, they can be inferred by observing biological activities. Thus, we developed a U937-based bioassay to determine the biological activities of mAbs and biosimilars against TNF-α using a low-basal NF-κB-inducible lentiviral reporter gene. The reporter gene assay (RGA) can be induced with a high signal-to-noise ratio (SNR) in a short time by TNF-α. Validation of the RGA showed accuracy (% relative standard deviation [RSD] = 4.64%), linearity (r2 = 0.9856), and precision (Interday RSD = 4.6%, between analysts RSD = 3.51%) as well as reasonable specificity and robustness. The measured potency values of a biosimilar to adalimumab were between 90% and 110%. Results showed our RGA is suitable for mAb quality control and lot release, and for evaluation of the biological activity similarity of the biosimilar.

A reporter gene assay for determining the biological activity of therapeutic antibodies targeting TIGIT.

T cell immunoglobulin and ITIM domain (TIGIT) is a novel immune checkpoint that has been considered as a target in cancer immunotherapy. Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable. Here, we designed a reporter gene assay comprising two cell lines, namely, CHO-CD112-CD3 scFv, which stably expresses CD112 (PVRL2, nectin-2) and a membrane-bound anti-CD3 single-chain fragment variable (scFv) as the target cell, and Jurkat-NFAT-TIGIT, which stably expresses TIGIT as well as the nuclear factor of activated T-cells (NFAT) response element-controlled luciferase gene, as the effector cell. The anti-CD3 scFv situated on the target cells activates Jurkat-NFAT-TIGIT cells through binding and crosslinking CD3 molecules of the effector cell, whereas interactions between CD112 and TIGIT prevent activation. The presence of anti-TIGIT mAbs disrupts their interaction, which in turn reverses the inactivation and luciferase expression. Optimization and validation studies have demonstrated that this assay is superior in terms of specificity, accuracy, linearity, and precision. In summary, this reliable and effective reporter gene assay may potentially be utilized in lot release control, stability assays, screening, and development of novel TIGIT-targeted therapeutic antibodies.

Characterization of a reliable cell-based reporter gene assay for measuring bioactivities of therapeutic anti-interleukin-23 monoclonal antibodies.

Environmental disturbances may result in dysregulation of interleukin-23 (IL-23), which is a crucial modulator of immunity. Several therapeutic monoclonal antibodies (mAbs) have been developed for treating IL-23-related autoimmune inflammation, such as ustekinumab, guselkumab, tildrakizumab, and risankizumab. Accurate bioactivity determination of therapeutic mAbs is essential for their quality control and clinical application. However, the current methods are tedious and complicated. In the present study, we employed low-background lentivirus carrying sis-inducible element (SIE)-driven firefly luciferase to generate a stable DB-SIE-Luc cell line that expresses endogenous IL-23 receptors and developed a sensitive and straightforward reporter gene assay (RGA) based on DB-SIE-Luc cells. After the optimization of various assay parameters, we set up a bioassay with the best fit of a four-parameter model and an appropriate signal-to-noise ratio (SNR) for bioactivity determination of guselkumab. We further verified the excellent assay performance characteristics of our RGA, including specificity, linearity, accuracy, precision, and stability, according to ICH-Q2. Taken together, we established a reliable and robust cell-based RGA, which potentially serves as a valubale alternative bioactivity determination assay for the release control and stability study of anti-IL-23 mAbs.