Monoclonal Antibody Production Via Fluidized Bioreactor Technology

Monoclonal antibodies (mAbs) are biologically identical antibodies created by homogenous immune cells originating from the same parent cell. MAbs target a specific epitope of an antigen on a cell’s surface, allowing it to neutralize the antigen. This unique characteristic has made them a key tool in the biopharmaceutical industry for the production of therapeutic drugs. One of these drugs is Rituxan® (rituximab), a mAb drug for the treatment of various cancers and autoimmune diseases. Currently, most mAb products are grown via cell suspension technology in stirred tank bioreactors. However, we have found that by using an integrated bioprocessing model, including conventional cell suspension culture tanks and fluidized bioreactor technology, overall product yield per day is increased by about 7-fold for the production of Rituxan®. Additionally, an economic analysis shows the fluidized bioreactor process is more profitable. Furthermore, though it requires a higher initial investment than the stirred tank process, the differential present worth of the fluidized bioreactor process in comparison to the stirred tank process is $13 billion. Overall, for the production of Rituxan®, the use of fluidized bioreactor technology is a more productive and lucrative process than the conventional stirred tank process

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Bioassay Development for Molecular Diagnostics on an Optofluidic Platform

Optofluidics, the science that integrates photonics and microfluidics, has produced a number of promising devices for biosensing and bioanalysis. In the past couple of years, several optofluidic approaches have demonstrated the capability to optically detect single biological microparticles. However, despite the sensitivity, many other requirements must be met to fully realize a molecular diagnostic device on a lab-on-a-chip platform. These requirements include target specificity, large dynamic range of detection, a low limit of detection, and multiplex differentiated diagnosis of many relevant biomarkers in a single sample. To this end, we have previously developed an optofluidic biosensor based on liquid-core antiresonant reflecting optical waveguides (LC-ARROWs). Orthogonally intersecting liquid and solid core ARROWs deliver the appropriate architecture for a highly sensitive, reconfigurable, and portable to the point-of-care device for ultra-sensitive detection of fluorescently labeled biomarkers in flow. We have recently introduced multiplexing capabilities into the ARROW-based optofluidic platform by integrating multi-mode interference (MMI) excitation waveguides, allowing for spectral and spatial multiplexing on a single compact chip. As a proof of concept, influenza virions were non-specifically labeled and simultaneously and differentially detected on chip. The goal of this thesis was to expand the detection capabilities of the MMI waveguide-detection platform via developing different specific bioassays to analyze different molecular biomarkers on chip and in clinically relevant environments and concentrations. First, a bead-based nucleic acid capture assay is described, which specifically captures nucleic acids onto an immobilization magnetic microsphere and functionalizes the complex with fluorescent dyes. The first simultaneous, multiplex differentiated detection of non-amplified total RNA samples from Viral Hemorrhagic Fever (VHF) infected cell cultures is reported. The four VHF samples detected are from the Zaire Ebolavirus (EBOV), Lake Victoria Marburgvirus (MARV), Ravn Marburgvirus (RAVN), and Crimean Congo Hemorrhagic Fever (CCHF). This assay is then expanded to specifically trap viral protein antigens, creating a specific bead-based protein antigen capture assay. This expansion enabled the target agnostic differentiated detection of both non-amplified Zika virus (ZIKV) genomic material and Non-structural protein (NS1) antigens. Target agnostic detection is a profound consequence for an optofluidic molecular diagnostic platform as few devices have the capability to detect the different viral biomarkers that may occur at different times after infection. However, this thesis continues to push the limits of sensitivity in the ARROW optofluidic detection platform, culminating in an ultra-sensitive single antigen detection assay enabled by a newly developed bright fluorescent probe. The probes were designed and synthesized in house from a fluorescently functionalized 1kB strand of double-stranded DNA. Their brightness and specificity to viral antigens enabled specific detection of single SARS-CoV-2 and Influenza A antigens from clinical nasopharyngeal swabs at a clinically relevant concentration of 30 ng/mL

Resistance of M. leprae to Quinolones: A Question of Relativity?

International audienceMultidrug resistant leprosy, defined as resistance to rifampin, dapsone and fluoroquinolones (FQ), has been described in Mycobacterium leprae. However, the in vivo impact of fluoroquinolone resistance, mainly mediated by mutations in DNA gyrase (GyrA 2 GyrB 2), has not been precisely assessed. Our objective was to measure the impact of a DNA gyrase mutation whose implication in fluoroquinolone resistance has been previously demonstrated through biochemical studies, on the in vivo activity of 3 fluoroquinolones: ofloxacin, moxifloxacin and garenoxacin. Methodology/Principal Findings: We used the proportional bactericidal method. 210 four-week-old immunodeficient female Nude mice (NMRI-Foxn1 nu /Foxn1 nu) were inoculated in the left hind footpad with 0.03 ml of bacterial suspension containing 5610 3 , 5610 2 , 5610 1 , and 5610 0 M. leprae AFB organisms of strain Hoshizuka-4 which is a multidrug resistant strain harboring a GyrA A91V substitution. An additional subgroup of 10 mice was inoculated with 5610 21 bacilli in the untreated control group. The day after inoculation, subgroups of mice were treated with a single dose of ofloxacin, moxifloxacin, garenoxacin or clarithromycin at 150 mg/kg dosing. 12 months later mice were sacrificed and M. leprae bacilli were numbered in the footpad. The results from the untreated control group indicated that the infective inoculum contained 23% of viable M. leprae. The results from the moxifloxacin and garenoxacin groups indicated that a single dose of these drugs reduced the percentage of viable M. leprae by 90%, similarly to the reduction observed after a single dose of the positive control drug clarithromycin. Conversely, ofloxacin was less active than clarithromycin. Conclusion/Significance: DNA gyrase mutation is not always synonymous of lack of in vivo fluoroquinolone activity in M. leprae. As for M. tuberculosis, in vivo studies allow to measure residual antibiotic activity in case of target mutations in M. leprae

Effect of Antiplatelet Therapy on Survival and Organ Support–Free Days in Critically Ill Patients With COVID-19

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