Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Más filtros




Base de datos
Intervalo de año de publicación
1.
J Chromatogr A ; 1730: 465114, 2024 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-38964160

RESUMEN

Protein chromatography is the dominant method of purification of biopharmaceuticals. Although all practical chromatography involves competitive absorption and separation of M. species, competitive protein absorption has remained inadequately understood. We previously introduced the measurement of equilibrium protein adsorption isotherms with all intensive variables held constant, including competitor concentration. In this work, we introduce isocratic chromatographic retention measurements of dynamic protein adsorption in the presence of a constant concentration of a competitor protein. These measurements are achieved by establishing a dynamic equilibrium with a constant concentration of competitor (insulin) in the mobile phase flowing through an ion exchange adsorbent column and following the behavior of a test protein (α-lactalbumin) injected into this environment. We observed decreased retention times for α-lactalbumin in presence of the competitor. The presence of competitor also reduces the heterogeneity of the sites available for adsorption of the test protein. This investigation provides an approach to fundamental understanding of competitive dynamics of multicomponent protein chromatography.


Asunto(s)
Insulina , Lactalbúmina , Cromatografía por Intercambio Iónico/métodos , Adsorción , Lactalbúmina/química , Lactalbúmina/aislamiento & purificación , Insulina/química , Insulina/aislamiento & purificación , Proteínas/aislamiento & purificación , Proteínas/química , Animales , Bovinos
2.
Aesthet Surg J ; 2024 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-38913383

RESUMEN

BACKGROUND: A delayed seroma around breast implants is the most common clinical presentation of BIA-ALCL. However, most seromas are due to benign causes. Therefore, it is essential to distinguish benign seromas from seromas due to BIA-ALCL. In a prior study mean concentrations of IL-9, IL-10 and IL-13 were found to be significantly higher in BIA-ALCL than in benign seromas. OBJECTIVES: The aim of this research was to test the ability to detect high concentrations of IL-9 rapidly with a lateral flow assay (LFA). Because we previously reported that a LFA for CD30 detected BIA-ALCL in seromas we compared CD30 and IL-9 LFAs in distinguishing BIA-ALCL from benign seromas. METHODS: Thirty microliter samples of 26 seromas (15 benign, 11 malignant) were tested on in-house prepared strips for IL-9 and CD30. Nanoparticle-conjugated antibodies specific to IL-9 and CD30 were used for detection. IL-9 was analyzed in undiluted samples and CD30 samples were optimized at 1:3 dilution. The dynamic range of detection was determined by spiking recombinant IL-9 into a benign seroma. Image analysis measured intensity of both test line (TL) and control line (CL) and a TL/CL ratio was calculated. IL-9 protein and IL-9 transcription factor PU.1 were stained in BIA-ALCL lines and clinical samples. RESULTS: The IL-9 LFA was reliable in distinguishing BIA-ALCL from benign seromas when the concentration of IL-9 was greater than 10 ng/ml. The CD30 LFA was positive in all 11 malignant cases. In one case with only faint CD30 and IL-10 test lines, the IL-9 LFA was clearly positive. Immunohistochemistry showed IL-9 and its essential transcription factor PU.1 were present in tumor cells in BIA-ALCL lines and clinical samples. CONCLUSIONS: IL-9 is a tumor cell biomarker of BIA-ALCL that can be detected by lateral flow assay and immunohistochemistry. Concentrations of IL-9 greater than 10 ng/ml reliably distinguished BIA-ALCL from benign seromas. Moreover, IL-9 LFA could detect BIA-ALCL when CD30 LFA was not definitive and IL-10 was of low concentration with a faint IL-10 TL, suggesting a multiplex LFA including IL-9, CD30 and IL-10 might be more effective in detecting BIA-ALCL in selected cases.

3.
Biotechnol Bioeng ; 121(8): 2524-2541, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38795025

RESUMEN

Governments and biopharmaceutical organizations aggressively leveraged expeditious communication capabilities, decision models, and global strategies to make a COVID-19 vaccine happen within a period of 12 months. This was an unusual effort and cannot be transferred to normal times. However, this focus on a single vaccine has also led to other treatments and drug developments being sidelined. Society expects the pharmaceutical industry to provide an uninterrupted supply of medicines. However, it is often overlooked how complex the manufacture of these compounds is and what logistics are required, not to mention the time needed to develop new drugs. The overarching theme, therefore, is patient access and how we can help ensure access and extend it to low- and middle-income countries. Despite unceasing efforts to make medications available to all patient populations, this must never be done at the expense of patient safety. A major fraction of the costs in biopharmaceutical manufacturing are for drug discovery, process development, and clinical studies. Infrastructure costs are very difficult to quantify because they often depend on whether a greenfield facility or an existing, depreciated facility is used or adapted for a new product. To accelerate process development concepts of platform process and prior knowledge are increasingly leveraged. While more traditional protein therapeutics continue to dominate the field, we are also experiencing the exciting emergence and evolution of other therapeutic formats (bispecifics, tetravalent mAbs, antibody-drug conjugates, enzymes, peptides, etc.) that offer unique treatment options for patients. Protein modalities are still dominant, but new modalities are being developed that can be learned from including advanced therapeutics-like cell and gene therapies. The industry must develop a model-based strategy for process development and technologies such as continuous integrated biomanufacturing must be adopted. The overall conclusion is that the pandemic pace was unsustainable, focused on vaccine delivery at the expense of other modalities/disease targets, and had implications for professional and personal life (work-life balance). Routinely reducing development time from 10 years to 1 year is nearly impossible to achieve. Environmental aspects of sustainable downstream processing are also described.


Asunto(s)
Productos Biológicos , COVID-19 , Humanos , Vacunas contra la COVID-19 , SARS-CoV-2 , Industria Farmacéutica
4.
iScience ; 27(6): 109817, 2024 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-38770139

RESUMEN

Although glutamine addiction in cancer cells is extensively reported, there is controversy on the impact of glutamine metabolism on the immune cells within the tumor microenvironment (TME). To address the role of extracellular glutamine, we enzymatically depleted circulating glutamine using PEGylated Helicobacter pylori gamma-glutamyl transferase (PEG-GGT) in syngeneic mouse models of breast and colon cancers. PEG-GGT treatment inhibits growth of cancer cells in vitro, but in vivo it increases myeloid-derived suppressor cells (MDSCs) and has no significant impact on tumor growth. By deriving a glutamine depletion signature, we analyze diverse human cancers within the TCGA and illustrate that glutamine depletion is not associated with favorable clinical outcomes and correlates with accumulation of MDSC. Broadly, our results help clarify the integrated impact of glutamine depletion within the TME and advance PEG-GGT as an enzymatic tool for the systemic and selective depletion (no asparaginase activity) of circulating glutamine in live animals.

5.
Acc Chem Res ; 57(9): 1372-1383, 2024 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-38590049

RESUMEN

The COVID-19 pandemic further demonstrated the need for usable, reliable, and cost-effective point-of-care diagnostics that can be broadly deployed, ideally for self-testing at home. Antigen tests using more-detectable reporter labels (usually at the cost of reader complexity) achieve better diagnostic sensitivity, supporting the value of higher-analytical-sensitivity reporter technologies in lateral flow.We developed a new approach to simple, inexpensive lateral flow assays (LFAs) of great sensitivity, based on the glow stick peroxyoxalate chemistry widely used in emergency settings and in children's toys. At the peak of the COVID-19 pandemic, we had the opportunity to participate in the pandemic-driven NIH Rapid Acceleration of Diagnostics (RADx) initiative aiming to develop a deployable lateral flow diagnostic for SARS-CoV-2 nucleoprotein based on our novel glow stick-inspired light-emitting reporter technology. During this project, we screened more than 250 antibody pairs for analytical sensitivity and specificity directly in LFA format, using recombinant nucleoprotein and then gamma-irradiated virions spiked into negative nasal swab extracts. Membranes and other LFA materials and swabs and extraction reagent components also were screened and selected. Optimization of conjugate preparation and spraying as well as pretreatment/conditioning of the sample pad led to the final optimized LFA strip. Technology development also included optimization of excitation liquid enclosed in disposable droppers, design of a custom cartridge and smartphone-based reader, and app development, even a prototype reader usable with any mobile phone. Excellent preclinical performance was first demonstrated with contrived samples and then with leftover clinical samples. Moving beyond traditional academic focus areas, we were able to establish a quality management system (QMS), produce large numbers of customized LFA cassettes by contract injection molding, build in-house facilities to assemble and store thousands of complete tests for verification and validation and usability studies, and source kitting/packaging services and quality standard reagents and build partnerships for clinical translation, regulatory guidance, scale up, and market deployment. We were not able to bring this early stage technology to the point of commercialization within the limited time and resources available, but we did achieve strong proof-of-concept and advance translational aspects of the platform including initial high-performance LFAs, reading by the iPhone app using only a $2 plastic dark box with no lens, and convenient, usable excitation liquid packaging in droppers manufacturable in very large numbers.In this Account, we aim to provide a concise overview of our 18-month sprint toward the practical development of a deployable antigen lateral flow assay under pandemic conditions and the challenges and successes experienced by our team. We highlight what it takes to coach a technically savvy but commercially inexperienced academic team through the accelerated translation of an early stage technology into a useful product. Finally, we provide a guided tutorial and workflow to empower others interested in the rapid development of translatable LFAs.


Asunto(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , COVID-19/virología , Humanos , SARS-CoV-2/aislamiento & purificación , Pruebas en el Punto de Atención , Prueba Serológica para COVID-19/métodos , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Proteínas de la Nucleocápside de Coronavirus/análisis , Prueba de COVID-19/métodos
6.
Front Microbiol ; 15: 1345617, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38525075

RESUMEN

Agonists of the stimulator of interferon genes (STING) pathway are being explored as potential immunotherapeutics for the treatment of cancer and as vaccine adjuvants for infectious diseases. Although chemical synthesis of 2'3' - cyclic Guanosine Monophosphate-Adenosine Monophosphate (cGAMP) is commercially feasible, the process results in low yields and utilizes organic solvents. To pursue an efficient and environmentally friendly process for the production of cGAMP, we focused on the recombinant production of cGAMP via a whole-cell biocatalysis platform utilizing the murine cyclic Guanosine monophosphate-Adenosine monophosphate synthase (mcGAS). In E. coli BL21(DE3) cells, recombinant expression of mcGAS, a DNA-dependent enzyme, led to the secretion of cGAMP to the supernatants. By evaluating the: (1) media composition, (2) supplementation of divalent cations, (3) temperature of protein expression, and (4) amino acid substitutions pertaining to DNA binding; we showed that the maximum yield of cGAMP in the supernatants was improved by 30% from 146 mg/L to 186 ± 7 mg/mL under optimized conditions. To simplify the downstream processing, we developed and validated a single-step purification process for cGAMP using anion exchange chromatography. The method does not require protein affinity chromatography and it achieved a yield of 60 ± 2 mg/L cGAMP, with <20 EU/mL (<0.3 EU/µg) of endotoxin. Unlike chemical synthesis, our method provides a route for the recombinant production of cGAMP without the need for organic solvents and supports the goal of moving toward shorter, more sustainable, and more environmentally friendly processes.

7.
Analyst ; 149(5): 1665, 2024 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-38348476

RESUMEN

Correction for 'Smartphone-read phage lateral flow assay for point-of-care detection of infection' by Maede Chabi, et al., Analyst, 2023, 148, 839-848, https://doi.org/10.1039/D2AN01499H.

8.
Commun Eng ; 22023.
Artículo en Inglés | MEDLINE | ID: mdl-38586601

RESUMEN

The COVID-19 pandemic has increased demand for point-of-care (POC) screening tests such as lateral flow assays (LFAs) and highlighted the need for sensitive and cost-effective POC diagnostic platforms. Here, we demonstrate an LFA platform using standard fluorescent nanoparticle reporters in which optical excitation is replaced by chemical excitation using the peroxyoxalate-based chemistry of inexpensive, shelf-stable glowsticks. The one-step chemi-excitation of fluorescent particles produces visible light readable by an unmodified smartphone, enhancing sensitivity while preserving simplicity and cost-effectiveness. Our Glow LFA detected the common model analyte human chorionic gonadotropin with a limit of detection (LoD) of 39 pg/mL-over ten times more sensitive than standard gold nanoparticles using the same antibodies. We also demonstrate its application to the detection of SARS-CoV-2 nucleoprotein at 100 pg/mL in nasal swab extract. Multiple fluorescent dyes can be chemi-excited by a single reagent, allowing for color multiplexing on a single LFA strip with a smartphone camera. The detection of three analytes on a single LFA test line was demonstrated using red, green, and blue fluorescent reporter particles, making glow LFA a promising platform for multiplexed detection.

SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA