ABSTRACT
While surface-enhanced Raman spectroscopy (SERS) has experienced substantial advancements since its discovery in the 1970s, it is an opportunity to celebrate achievements, consider ongoing endeavors, and anticipate the future trajectory of SERS. In this perspective, we encapsulate the latest breakthroughs in comprehending the electromagnetic enhancement mechanisms of SERS, and revisit CT mechanisms of semiconductors. We then summarize the strategies to improve sensitivity, selectivity, and reliability. After addressing experimental advancements, we comprehensively survey the progress on spectrum-structure correlation of SERS showcasing their important role in promoting SERS development. Finally, we anticipate forthcoming directions and opportunities, especially in deepening our insights into chemical or biological processes and establishing a clear spectrum-structure correlation.
ABSTRACT
Artificial intelligence has demonstrated notable advancements in the realm of visual inspection and defect detection in substations. Nevertheless, practical application presents challenges, with issues arising from the dynamic shooting environment and limited dataset resulting in suboptimal defect identification accuracy and instability. To address these concerns, a pioneering approach based on hybrid pruning YOLOv5 and multiscale data augmentation is proposed for enhancing defect detection in substations. Initially, an enhanced multiscale data augmentation method is proposed. The improved multiscale data augmentation mitigates the impact of the time-varying shooting environment on recognition accuracy and enhances defect detection precision. Subsequently, YOLOv5 is employed for training and detecting defects within multi-scale image data. To alleviate the potential destabilizing effects of YOLOv5's large-scale parameters on model stability, a new model pruning method is implemented. This method strategically prunes parameters to bolster the model's defect identification accuracy. The efficacy of the proposed methodology is evaluated through testing on substation defect images, confirming its effectiveness in enhancing defect detection capabilities.
ABSTRACT
BACKGROUND: Azvudine has been approved for the treatment of coronavirus disease 2019 (COVID-19) patients in China, and this meta-analysis aims to illustrate the safety of azvudine and its effectiveness in reducing mortality. METHODS: PubMed, Embase, Web of science, Cochrane Library and the Epistemonikos COVID-19 Living Overview of Evidence database (L.OVE) were searched to aggregate currently published studies. Cochrane risk of bias tool and ROBINS-I tool were used to assess the risk of bias of randomized controlled study and cohort study respectively. Odds radios (ORs) with 95% confidence interval (CIs) were combined for dichotomous variables. Publication bias was assessed by Egger's test and funnel plots. RESULTS: A total of 184 articles were retrieved from the included databases and 17 studies were included into the final analysis. Pooled analysis showed that azvudine significantly reduced mortality risk in COVID-19 patients compared with controls (OR: 0.41, 95%CI 0.31-0.54, p < 0.001). Besides, either mild to moderate or severe COVID-19 patients could benefit from azvudine administration. There was no significant difference in the incidence of ICU admission (OR: 0.90, 95%CI 0.47-1.72, p = 0.74) and invasive ventilation (OR: 0.94, 95%CI 0.54-1.62, p = 0.82) between azvudine and control group. The incidence of adverse events was similar between azvudine and control (OR: 1.26, 95%CI 0.59-2.70, p = 0.56). CONCLUSIONS: This meta-analysis suggests that azvudine could reduce the mortality risk of COVID-19 patients, and the safety of administration is acceptable. TRIAL REGISTRATION: PROSPERO; No.: CRD42023462988; URL: https://www.crd.york.ac.uk/prospero/ .
Subject(s)
Antiviral Agents , COVID-19 Drug Treatment , COVID-19 , SARS-CoV-2 , Humans , COVID-19/mortality , Antiviral Agents/therapeutic use , Antiviral Agents/adverse effects , SARS-CoV-2/drug effects , Treatment OutcomeABSTRACT
Denoising is a necessary step in image analysis to extract weak signals, especially those hardly identified by the naked eye. Unlike the data-driven deep-learning denoising algorithms relying on a clean image as the reference, Noise2Noise (N2N) was able to denoise the noise image, providing sufficiently noise images with the same subject but randomly distributed noise. Further, by introducing data augmentation to create a big data set and regularization to prevent model overfitting, zero-shot N2N-based denoising was proposed in which only a single noisy image was needed. Although various N2N-based denoising algorithms have been developed with high performance, their complicated black box operation prevented the lightweight. Therefore, to reveal the working function of the zero-shot N2N-based algorithm, we proposed a lightweight Peak2Peak algorithm (P2P) and qualitatively and quantitatively analyzed its denoising behavior on the 1D spectrum and 2D image. We found that the high-performance denoising originates from the trade-off balance between the loss function and regularization in the denoising module, where regularization is the switch of denoising. Meanwhile, the signal extraction is mainly from the self-supervised characteristic learning in the data augmentation module. Further, the lightweight P2P improved the denoising speed by at least ten times but with little performance loss, compared with that of the current N2N-based algorithms. In general, the visualization of P2P provides a reference for revealing the working function of zero-shot N2N-based algorithms, which would pave the way for the application of these algorithms toward real-time (in situ, in vivo, and operando) research improving both temporal and spatial resolutions. The P2P is open-source at https://github.com/3331822w/Peak2Peakand will be accessible online access at https://ramancloud.xmu.edu.cn/tutorial.
ABSTRACT
Surface-enhanced Raman spectroscopy (SERS) has been demonstrated as an ultrasensitive tool for various molecules. However, for the negatively charged molecules, the widely used SERS substrate [negatively charged Ag and Au nanoparticles (Ag or Au NPs (-)] showed either low sensitivity or poor stability. The best solution is to synthesize positively charged silver or gold nanoparticles [Ag or Au NPs (+)] with high stability and excellent SERS performance, which are currently unavailable. To this end, we revitalized the strategy of "charge reversal and seed growth". By selection of ascorbic acid as the reductant and surfactant, the surface charge of Ag or Au NP (-) seeds is adjusted to a balanced state, where the surface charge is negative enough to satisfy the stabilization of the NPs (-) but does not hinder the subsequent charge reversal. By optimization of the chain length and electric charge of polyamine molecules, the highly stable and size-controllable uniform Ag NPs (+) and Au NPs (+) were seed-growth synthesized with high reproducibility. More importantly, the SERS performance of both Ag NPs (+) and Au NPs (+) achieved the trace detection of negatively charged molecules at the level of 1 µg/L, demonstrating an improved SERS sensitivity of up to 3 orders of magnitude compared to the previously reported sensitivity. Promisingly, the introduction of polyamine-capped Ag NPs (+) and Au NPs (+) as SERS substrates with high stability (1 year shelf life) will significantly broaden the application of SERS.
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BACKGROUND: Severe acute respiratory syndrome coronavirus 2 will coexist with humans for a long time, and it is therefore important to develop effective treatments for coronavirus disease 2019 (COVID-19). Recent studies have demonstrated that antiviral therapy is a key factor in preventing patients from progressing to severe disease, even death. Effective and affordable antiviral medications are essential for disease treatment and are urgently needed. Azvudine, a nucleoside analogue, is a potential low-cost candidate with few drug interactions. However, validation of high-quality clinical studies is still limited. METHODS: This is a multicentre, randomized, double-blind, placebo-controlled phase III clinical trial involving 1096 adult patients with mild-to-moderate symptoms of COVID-19 who are at high risk for progression to severe COVID-19. Patients will be randomized to (1) receive azvudine tablets 5 mg daily for a maximum of 7 days or (2) receive placebo five tablets daily. All participants will be permitted to use a standard treatment strategy except antiviral therapy beyond the investigational medications. The primary outcome will be the ratio of COVID-19-related critical illness and all-cause mortality among the two groups within 28 days. DISCUSSION: The purpose of this clinical trial is to determine whether azvudine can prevent patients at risk of severe disease from progressing to critical illness and death, and the results will identify whether azvudine is an effective and affordable antiviral treatment option for COVID-19. TRIAL REGISTRATION: ClinicalTrials.gov NCT05689034. Registered on 18 January 2023.
Subject(s)
Azides , COVID-19 , Deoxycytidine/analogs & derivatives , Adult , Humans , Critical Illness , SARS-CoV-2 , Antiviral Agents/adverse effects , Randomized Controlled Trials as Topic , Multicenter Studies as Topic , Clinical Trials, Phase III as TopicABSTRACT
Reagent purity is crucial to experimental research, considering that the ignorance of ultratrace impurities may induce wrong conclusions in either revealing the reaction nature or qualifying the target. Specifically, in the field of surface science, the strong interaction between the impurity and the surface will bring a non-negligible negative effect. Surface-enhanced Raman spectroscopy (SERS) is a highly surface-sensitive technique, providing fingerprint identification and near-single molecule sensitivity. In the SERS analysis of trace chloromethyl diethyl phosphate (DECMP), we figured out that the SERS performance of DECMP is significantly distorted by the trace impurities from DECMP. With the aid of gas chromatography-based techniques, one strongly interfering impurity (2,2-dichloro-N,N-dimethylacetamide), the byproduct during the synthesis of DECMP, was confirmed. Furthermore, the nonignorable interference of impurities on the SERS measurement of NaBr, NaI, or sulfadiazine was also observed. The generality ignited us to refresh and consolidate the guideline for the reliable SERS qualitative analysis, by which the potential misleading brought by ultratrace impurities, especially those strongly adsorbed on Au or Ag surfaces, could be well excluded.
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With the widespread use of immune checkpoint inhibitors to treat various cancers, pulmonary toxicity has become a topic of increasing concern. Anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibodies are strongly associated with rapidly progressive interstitial lung disease (RP-ILD) in patients with clinically amyopathic dermatomyositis. However, anti-MDA5 antibody expression has not been reported in patients with immune-related adverse events. We present the case of a 74-year-old man with lung adenocarcinoma who developed RP-ILD after treatment with immune checkpoint inhibitors. Further investigation revealed multiple autoantibodies, including anti-MDA5 antibodies. He initially responded to systemic glucocorticoids, immunosuppressants, and tocilizumab but eventually died from worsening pneumomediastinum. This case is the first one to suggest that checkpoint inhibitor pneumonitis can present as RP-ILD with positive anti-MDA5 antibodies, which may be predictive of a poor prognosis.
Subject(s)
Lung Diseases, Interstitial , Lung Neoplasms , Pneumonia , Aged , Humans , Male , Autoantibodies , Immune Checkpoint Inhibitors/adverse effects , Lung Diseases, Interstitial/diagnosis , Lung Diseases, Interstitial/drug therapy , Lung Diseases, Interstitial/etiology , Lung Neoplasms/drug therapy , Lung Neoplasms/complications , Pneumonia/diagnosis , Pneumonia/drug therapy , Pneumonia/etiologyABSTRACT
A 56-year-old Chinese woman with previous disseminated mycobacterium avium complex infection and recurrent cervical abscesses from Burkholderia cepacia complex visited our hospital. She was diagnosed with adult-onset immunodeficiency (AOID) and tested positive for interferon-γ-neutralizing autoantibody. Ceftazidime was administered as the initial antimicrobial treatment, which was later combined with sulfamethoxazole-trimethoprim (SMZ-TMP). She developed drug rash with eosinophilia and systemic symptoms (DRESS) syndrome after SMZ-TMP administration and improved after withdrawal of the culprit antibiotic and systemic glucocorticoids treatment. Her cervical infection was eventually cured after combined therapy of long-term antibiotics and anti-IFN-γ autoantibodies (AIGA) titer-lowering treatments including glucocorticoids, rituximab, and plasmapheresis. This is the first case of DRESS syndrome in the setting of AIGA-induced AOID and is worthy of notice.
Subject(s)
Drug Hypersensitivity Syndrome , Eosinophilia , Exanthema , Immunologic Deficiency Syndromes , Mycobacterium Infections, Nontuberculous , Adult , Anti-Bacterial Agents/therapeutic use , Autoantibodies , Drug Hypersensitivity Syndrome/complications , Drug Hypersensitivity Syndrome/etiology , Eosinophilia/chemically induced , Eosinophilia/complications , Eosinophilia/diagnosis , Female , Humans , Interferon-gamma , Middle Aged , Trimethoprim, Sulfamethoxazole Drug Combination/adverse effectsABSTRACT
A new method for the determination of oxytetracycline (OTC) has been established by coupling the catalytic amplification reaction of copper nanoclusters (CuNCs) with the aptamer reaction. CuNCs prepared by a wet chemical method have the catalytic activity for the formation of gold nanoparticles (AuNPs) resulting from a HAuCl4-ethanol (En) reaction. The experimental results showed that OTC aptamer (Apt) can be adsorbed on the surface of CuNCs in a non-specific way, thus inhibiting its catalytic activity. When OTC was added to the solution, the OTC-Apt complex was generated by a specific reaction, which made the CuNCs desorb and restore their catalytic activity. With the increase of OTC, the recovery of the catalytic activity of CuNCs is strengthened, the reaction speed is accelerated, and the number of AuNPs is increased. The generated AuNPs exhibited surface enhanced Raman scattering (SERS) signals at 1615 cm-1 in the presence of Vitoria blue 4R (VB4R) molecular probes, and a resonance Rayleigh scattering (RRS) peak at 586 nm. There is a good linear relationship between the intensities of SERS, or RRS, and OTC concentration at the range of 37.5-300 ng/L or 37.5-225 ng/L, respectively. A new SERS and RRS assay for the determination of trace OTC based on the regulation of CuNCs catalysis was established.
ABSTRACT
Liquid crystals (LCs) have been applied for a long time in the field of analytical chemistry. To date, there are no reports about utilization of LCs as the catalyst to amplification analytical signal. In this article, three small molecules LCs in water aqueous solutions were characterized using molecular spectra and particle size analysis. The characterization indicated that there are nanoparticles in the system. Among the them, 4-heptylbenzoic acid (HPB) exhibits the most sensitive performance in the analytical system based on the reduction of HAuCl4 to gold nanoparticles (AuNPs) by NaH2PO2 by the spectrophotometric slope evaluation procedure. As the concentration of LCs catalyst increases, the AuNPs surface plasmon resonance (SPR) absorption peak at 550 nm increases linearly, that can be utilized to amply the absorption signal. Based on the LCs catalytic amplification reaction and immunoreaction, a new SPR spectrophotometric analysis method was developed for the label-free detection of oxytetracycline, with a detection limit of 0.50 ng/mL. The method was also successfully applied for the detection of oxytetracycline-spiked environmental water samples to demonstrate its practical usefulness.
Subject(s)
Aptamers, Nucleotide , Liquid Crystals , Metal Nanoparticles , Oxytetracycline , Gold , Limit of Detection , Surface Plasmon ResonanceABSTRACT
With the development of modern biomedicine, research on the molecular mechanism of tumors has developed gradually. The CD147 gene has been applied to tumor molecular targeted therapy, and significant differences were found in the expression of the CD147 gene in different tumor tissues and normal tissues. Many previous studies have also shown that the expression of the CD147 gene plays a crucial role in the development of tumors. To understand whether CD147 can be used as a therapeutic target for oral cancer, CRISPR/Cas9 gene-editing technology was used to knock out the CD147 gene in cal27 cells to obtain knockout cell lines. Using CCK-8, Transwell, RT-PCR, and Western blotting, the proliferation and invasion abilities of the knockout cell lines were decreased significantly, and the expression of matrix metalloproteinase was also inhibited. Next, a subcutaneously transplanted tumor model in nude mice was constructed to detect the effect of the CD147 gene on tumors. Subcutaneous tumor growth and immunohistochemistry results showed that the proliferation and doxorubicin resistance of knockout cell line were significantly inhibited compared with those in the wild-type group. These results indicated that knocking out CD147 significantly reduced the proliferation and invasion of cal27 cells, and CD147 may be a potential therapeutic target for oral cancer.
Subject(s)
Basigin/metabolism , Drug Resistance, Neoplasm , Gene Knockout Techniques , Mouth Neoplasms/drug therapy , Mouth Neoplasms/pathology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , CRISPR-Cas Systems/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Disease Models, Animal , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Drug Resistance, Neoplasm/drug effects , Humans , Matrix Metalloproteinases/metabolism , Mice, Nude , Neoplasm InvasivenessABSTRACT
As with excellent catalytic performance, palladium nanoclusters (PdNCs) have a wide range of applications. However, the traditional PdNCs are easy to agglomerate in the analysis system and lose their catalytic activity. A covalent organic framework (COF) has a definite structure, good stability, and easy surface functionalization. So, it is of great significance to develop stable PdNCs with high catalytic activity and then combine with advanced analysis techniques to analyze ultratrace small-molecule pollutants in the environment. In this research, a stable PdNC dispersed on a COF (PdTpPa) catalyst is prepared and we find it with strong catalysis for the NaH2PO2-HAuCl4 catalytic reaction. Furthermore, this nanocatalytic indicator reaction can be tracked by surface-enhanced Raman spectroscopy (SERS) and resonance Rayleigh scattering (RRS) dual-mode. Combined with a highly specific aptamer-modifying technique, a highly sensitive and selective SERS/RRS dimode assay platform for trace organic pollutants has been developed. The detection limits of oxytetracycline (OTC), glyphosate (GLY), tetracycline (TEC), and bisphenol A (BPA) are 0.64, 0.03, 6.2 × 10-3, and 0.53 × 10-3 ng/mL, respectively. This work also provides ideas for the application of COF materials and Pd nanocatalysts in the molecular spectral detection of trace pollutants.
Subject(s)
Aptamers, Nucleotide/chemistry , Biocompatible Materials/chemistry , Gold/chemistry , Metal Nanoparticles/chemistry , Metal-Organic Frameworks/chemistry , Palladium/chemistry , Benzhydryl Compounds/analysis , Biocompatible Materials/chemical synthesis , Catalysis , Environmental Monitoring , Glycine/analogs & derivatives , Glycine/analysis , Materials Testing , Metal-Organic Frameworks/chemical synthesis , Oxytetracycline/analysis , Particle Size , Phenols/analysis , Spectrum Analysis, Raman , Tetracycline/analysis , GlyphosateABSTRACT
Mitochondrial abnormalities accelerate the progression of ischemic brain damage. Sirtuin 3 (SIRT3) is mainly found in mitochondria and affects almost all major aspects of mitochondrial function. Luteolin, a flavonoid with diverse biological properties, including antioxidant activity, inhibition of cell apoptosis and regulation of autophagy. It also modulates the activity of AMP activated kinase and/or sirtuin 1 (SIRT 1) by regulating the expression of sirtuins. We investigated the protective effects of luteolin on cerebral ischemia-reperfusion. It was found through experiments that luteolin reduced the infarcted area of MCAO rat model, and based on the experimental results, it was inferred that luteolin affected the AMPK, mTOR and SIRT3 pathways, thereby protecting brain cells. As expected, we found that luteolin can reduce the neurological function score, the degree of cerebral edema, the cerebral infarction volume, alleviate morphological changes in the cortex and hippocampus, increase neuron survival and decrease the number of apoptotic neurons. At the same time, luteolin significantly reduced the number of GFAP and Iba-1 positive glial cells in the hippocampus while enhanced the scavenging of oxygen free radicals and the activity of SOD in mitochondria. Addtionally, it can also enhance antioxidant capacity via the reversal of mitochondrial swelling and the mitochondrial transmembrane potential. Moreover, luteolin can increase SIRT3-targeted expression in mitochondria, decrease the phosphorylation of AMPK, and increase phosphor-mTOR (p-mTOR) levels, which may have occurred specifically through activation of the SIRT3/AMPK/mTOR pathway. We speculate that luteolin reduces the pathological progression of CIRI by increasing SIRT3 expression and enhancing mitochondrial function. Therefore, the results indicate that luteolin can increase the transduction of SIRT3, providing a potential mechanism for neuroprotective effects in patients with cerebral ischemia.
Subject(s)
Brain/drug effects , Infarction, Middle Cerebral Artery/drug therapy , Luteolin/therapeutic use , Neuroprotective Agents/therapeutic use , Reperfusion Injury/drug therapy , Signal Transduction/drug effects , AMP-Activated Protein Kinases/metabolism , Animals , Anti-Inflammatory Agents/therapeutic use , Apoptosis/drug effects , Brain/pathology , Brain Edema/drug therapy , Brain Edema/epidemiology , Female , Infarction, Middle Cerebral Artery/complications , Male , Neurogenesis/drug effects , Rats, Sprague-Dawley , Reperfusion Injury/complications , Sirtuins/metabolism , TOR Serine-Threonine Kinases/metabolismABSTRACT
Co-delivery nanoparticles with characteristics of intracellular precision release drug have been generally accepted as an effective therapeutic strategy for eye diseases. In this study, we designed a new co-delivery system (miRNA/NP-BRZ) as a lasting therapeutic approach to prevent the neuro-destructive after the long-term treatment of glaucoma. Neuroprotective and intraocular pressure (IOP) response were assessed in in vivo and in vitro models of glaucoma. At the meaning time, we describe the preparation of miRNA/NP-BRZ, drug release characteristics, intraocular tracing, pharmacokinetic and pharmacodynamics study and toxicity test. We found that miRNA/NP-BRZ could remarkably decrease IOP and significantly prevent retinal ganglion cell (RGC) damages. The new formula of miRNA-124 encapsulated in PEG-PSA-BRZ nanoparticles exhibits high encapsulation efficiency (EE), drug-loading capacity (DC), and stable controlled-release efficacy (EC). Moreover, we also verified that the miRNA/NP-BRZ system is significantly neuroprotective and nontoxic as well as lowering IOP. This study shows our co-delivery drug system would have a wide potential on social and economic benefits for glaucoma.
Subject(s)
Drug Delivery Systems , Glaucoma/therapy , MicroRNAs/administration & dosage , Sulfonamides/administration & dosage , Thiazines/administration & dosage , Animals , Carbonic Anhydrase Inhibitors/administration & dosage , Carbonic Anhydrase Inhibitors/pharmacokinetics , Carbonic Anhydrase Inhibitors/pharmacology , Decanoic Acids/chemistry , Delayed-Action Preparations , Dicarboxylic Acids/chemistry , Drug Liberation , Gene Transfer Techniques , Glaucoma/physiopathology , Intraocular Pressure/drug effects , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , Nanoparticles , Neuroprotective Agents/administration & dosage , Neuroprotective Agents/pharmacokinetics , Neuroprotective Agents/pharmacology , Polyethylene Glycols/chemistry , Rabbits , Sulfonamides/pharmacokinetics , Sulfonamides/pharmacology , Thiazines/pharmacokinetics , Thiazines/pharmacologyABSTRACT
Covalent organic frameworks (COFs) and Ag-doped COFs (AgCOFs) are prepared by the polycondensation procedure and characterized by electron microscopy and molecular spectral techniques. Their catalysis of the Cu2O particle reaction of glucose (GL)-Cu(II) was examined by resonance Rayleigh scattering (RRS), and AgCOFs were found to exhibit the strongest catalysis. The melamine (ML) aptamers (AptML) can attach to the surface of AgCOF and inhibit its catalytic activity. When melamine (ML) is added to this reacting solution, AptML-ML complexes are formed and the Apts are desorbed from the surface of AgCOF. As the concentration of ML increased, the catalytic activity of AgCOF increased and the RRS signal enhanced due to the increase in Cu2O particles. When the ML concentration was in the range of 0.79-13.2 nmol/L, the RRS intensity increased linearly, with a detection limit of 0.72 nmol/L. When the Apts of urea and bisphenol A (BPA) were replaced by the AptML, 66.7-1333 nmol/L urea and 0.33-2.7 nmol/L BPA, respectively, could also be determined, with detection limits of 30.4 nmol/L urea and 0.15 nmol/L BPA. Based on this, a new AgCOF amplification RRS method was established.
ABSTRACT
An integrated continuous tubular reactor system was developed for processing an autoprotease expressed as inclusion bodies. The inclusion bodies were suspended and fed into the tubular reactor system for continuous dissolving, refolding and precipitation. During refolding, the dissolved autoprotease cleaves itself, separating the fusion tag from the target peptide. Subsequently, the cleaved fusion tag and any uncleaved autoprotease were precipitated out in the precipitation step. The processed exiting solution results in the purified soluble target peptide. Refolding and precipitation yields performed in the tubular reactor were similar to batch reactor and process was stable for at least 20 h. The authenticity of purified peptide was also verified by mass spectroscopy. Productivity (in mg/l/h and mg/h) calculated in the tubular process was twice and 1.5 times of the batch process, respectively. Although it is more complex to setup a tubular than a batch reactor, it offers faster mixing, higher productivity and better integration to other bioprocessing steps. With increasing interest of integrated continuous biomanufacturing, the use of tubular reactors in industrial settings offers clear advantages.
Subject(s)
Bioreactors , Biotechnology/methods , Inclusion Bodies/metabolism , Models, Chemical , Peptide Hydrolases/metabolism , Recombinant Fusion Proteins/metabolism , Chemical Precipitation , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Kinetics , Mass Spectrometry , Protein RefoldingABSTRACT
A two-step purification strategy comprising of polyethylene glycol (PEG) precipitation and anion-exchange chromatography was developed for a panel of monoclonal immunoglobulin M (IgM) (pI 5.5-7.7) produced from hybridoma cultures. PEG precipitation was optimized with regards to concentration, pH and mixing. For anion-exchange chromatography, different resins were screened of which Fractogel EMD, a polymer grafted porous resin had the highest capacity. Despite its significantly slower mass transfer, the binding capacity was still higher compared to a convection driven resin (monolith). This purification strategy was successfully demonstrated for all 9 IgMs in the panel. In small scale most antibodies could be purified to >95% purity with the exception of two which gave a lower final purity (46% and 85%). The yield was dependent on the different antibodies ranging from 28% to 84%. Further improvement of recovery and purity was obtained by the digestion of DNA present in the hybridoma supernatant using an endonuclease, benzonase. So far this strategy has been applied for the purification of up to 2l hybridoma supernatants.