Pioneering Just-in-Time (JIT) Strategy for Accelerating Raman Method Development and Implementation for Biologic Continuous Manufacturing.

Raman spectroscopy is a popular process analytical technology (PAT) tool that has been increasingly used to monitor and control the monoclonal antibody (mAb) manufacturing process. Although it allows the characterization of a variety of quality attributes by developing chemometric models, a large quantity of representative data is required, and hence, the model development process can be time-consuming. In recent years, the pharmaceutical industry has been expediting new drug development in order to achieve faster delivery of life-changing drugs to patients. The shortened development timelines have impacted the Raman application, as less time is allowed for data collection. To address this problem, an innovative Just-in-Time (JIT) strategy is proposed with the goal of reducing the time needed for Raman model development and ensuring its implementation. To demonstrate its capabilities, a proof-of-concept study was performed by applying the JIT strategy to a biologic continuous process for producing monoclonal antibody products. Raman spectroscopy and online two-dimensional liquid chromatography (2D-LC) were integrated as a PAT analyzer system. Raman models of antibody titer and aggregate percentage were calibrated by chemometric modeling in real-time. The models were also updated in real-time using new data collected during process monitoring. Initial Raman models with adequate performance were established using data collected from two lab-scale cell culture batches and subsequently updated using one scale-up batch. The JIT strategy is capable of accelerating Raman method development to monitor and guide the expedited biologics process development.

Two-Dimensional SEC-SEC-UV-MALS-dRI Workflow for Streamlined Analysis and Characterization of Biopharmaceuticals.

The emergence of complex biological modalities in the biopharmaceutical industry entails a significant expansion of the current analytical toolbox to address the need to deploy meaningful and reliable assays at an unprecedented pace. Size exclusion chromatography (SEC) is an industry standard technique for protein separation and analysis. Some constraints of traditional SEC stem from its restricted ability to resolve complex mixtures and notoriously long run times while also requiring multiple offline separation conditions on different pore size columns to cover a wider molecular size distribution. Two-dimensional liquid chromatography (2D-LC) is becoming an important tool not only to increase peak capacity but also to tune selectivity in a single online method. Herein, an online 2D-LC framework in which both dimensions utilize SEC columns with different pore sizes is introduced with a goal to increase throughput for biomolecule separation and characterization. In addition to improving the separation of closely related species, this online 2D SEC-SEC approach also facilitated the rapid analysis of protein-based mixtures of a wide molecular size range in a single online experimental run bypassing time-consuming deployment of different offline SEC methods. By coupling the second dimension with multiangle light scattering (MALS) and differential refractive index (dRI) detectors, absolute molecular weights of the separated species were obtained without the use of calibration curves. As illustrated in this report for protein mixtures and vaccine processes, this workflow can be used in scenarios where rapid development and deployment of SEC assays are warranted, enabling bioprocess monitoring, purity assessment, and characterization.

Accelerating Pharmaceutical Process Development with an Acoustic Droplet Ejection-Multiple Reaction Monitoring-Mass Spectrometry Workflow.

Fast-paced pharmaceutical process developments (e.g., high-throughput experimentation, directed evolution, and machine learning) involve the introduction of fast, sensitive, and accurate analytical assays using limited sample volumes. In recent years, acoustic droplet ejection (ADE) coupled with an open port interface has been invented as a sampling technology for mass spectrometry, providing high-throughput nanoliter analytical measurements directly from the standard microplates. Herein, we introduce an ADE-multiple reaction monitoring-mass spectrometry (ADE-MRM-MS) workflow to accelerate pharmaceutical process research and development (PR&D). This systematic workflow outlines the selection of MRM transitions and optimization of assay parameters in a data-driven manner using rapid measurements (1 sample/s). The synergy between ADE sampling and MRM analysis enables analytical assays with excellent sensitivity, selectivity, and speed for PR&D reaction screenings. This workflow was utilized to develop new ADE-MRM-MS assays guiding a variety of industrial processes, including (1) screening of Ni-based catalysts for C-N cross-coupling reaction at 1 Hz and (2) high-throughput regioisomer analysis-enabled enzyme library screening for peptide ligation reaction. ADE-MRM-MS assays were demonstrated to deliver accurate results that are comparable to conventional liquid chromatography (LC) experiments while providing >100-fold throughput enhancement.

Robust platform for inline Raman monitoring and control of perfusion cell culture.

Perfusion cell culture has been gaining increasing popularity for biologics manufacturing due to benefits such as smaller footprint, increased productivity, consistent product quality and manufacturing flexibility, cost savings, and so forth. Process Analytics Technologies tools are highly desirable for effective monitoring and control of long-running perfusion processes. Raman has been widely investigated for monitoring and control of traditional fed batch cell culture process. However, implementation of Raman for perfusion cell culture has been very limited mainly due to challenges with high-cell density and long running times during perfusion which cause extremely high fluorescence interference to Raman spectra and consequently it is exceedingly difficult to develop robust chemometrics models. In this work, a platform based on Raman measurement of permeate has been proposed for effective analysis of perfusion process. It has been demonstrated that this platform can effectively circumvent the fluorescence interference issue while providing rich and timely information about perfusion dynamics to enable efficient process monitoring and robust bioreactor feed control. With the highly consistent spectral data from cell-free sample matrix, development of chemometrics models can be greatly facilitated. Based on this platform, Raman models have been developed for good measurement of several analytes including glucose, lactate, glutamine, glutamate, and permeate titer. Performance of Raman models developed this way has been systematically evaluated and the models have shown good robustness against changes in perfusion scale and variations in permeate flowrate; thus models developed from small lab scale can be directly transferred for implementation in much larger scale of perfusion. With demonstrated robustness, this platform provides a reliable approach for automated glucose feed control in perfusion bioreactors. Glucose model developed from small lab scale has been successfully implemented for automated continuous glucose feed control of perfusion cell culture at much larger scale.

Association Between Clostridium innocuum and Antibiotic-Associated Diarrhea in Adults and Children: A Cross-sectional Study and Comparative Genomics Analysis.

BACKGROUND: A recent study from Taiwan suggested that Clostridium innocuum may be an unrecognized cause of antibiotic-associated diarrhea (AAD) and clinically indistinguishable from Clostridioides difficile infection. Our objective was to compare C. innocuum prevalence and strain between those with AAD and asymptomatic controls. METHODS: In this cross-sectional study, we collected stool from 200 individuals with AAD and 100 asymptomatic controls. We evaluated the association between AAD and C. innocuum in stool using anaerobic culture and quantitative polymerase chain reaction (qPCR). To identify strain-specific associations with AAD, we performed whole-genome sequencing of C. innocuum isolates using Illumina MiSeq and constructed comparative genomics analyses. RESULTS: C. innocuum was isolated from stool of 126/300 (42%) subjects and more frequently from asymptomatic controls than AAD subjects (50/100 [50%] vs 76/200 [38%], respectively; P = .047). C. innocuum isolation frequency was not associated with AAD in either the adult or pediatric subgroups. C. innocuum and C. difficile were frequently co-prevalent in individuals with and without diarrhea. There were no phylogenetic differences or accessory genome associations between C. innocuum isolates from AAD subjects and asymptomatic controls. CONCLUSIONS: C. innocuum was frequently isolated and at a greater frequency in asymptomatic controls than those with AAD. We did not identify strain lineages or accessory genomic elements associated with AAD. These data highlight that differentiating C. innocuum-associated diarrhea from asymptomatic colonization, and differentiating diarrhea caused by C. difficile from C. innocuum, are clinical microbiology challenges that require additional investigation to identify host-specific factors and/or biomarkers that distinguish these conditions.

Automated Online-Sampling Multidimensional Liquid Chromatography with Feedback-Control Capability as a Framework for Real-Time Monitoring of mAb Critical Quality Attributes in Multiple Bioreactors.

Real-time monitoring of biopharmaceutical reactors is becoming increasingly important as the processes become more complex. During the continuous manufacturing of monoclonal antibodies (mAbs), the desired mAb product is continually created and collected over a 30 day process, where there can be changes in quality over that time. Liquid chromatography (LC) is the workhorse instrumentation capable of measuring mAb concentration as well as quality attributes such as aggregation, charge variants, oxidation, etc. However, traditional offline sampling is too infrequent to fully characterize bioprocesses, and the typical time from sample generation to data analysis and reporting can take weeks. To circumvent these limitations, an automated online sampling multidimensional workflow was developed to enable streamlined measurements of mAb concentration, aggregation, and charge variants. This analytical framework also facilitates automated data export for real-time analysis of up to six bioreactors, including feedback-controlling capability using readily available LC technology. This workflow increases the data points per bioreactor, improving the understanding of each experiment while also reducing the data turnaround time from weeks to hours. Examples of effective real-time analyses of mAb critical quality attributes are illustrated, showing substantial throughput improvements and accurate results while minimizing labor and manual intervention.

Remote Real-Time Training for Sustainable Cleft Operation in Rural Region of West Africa: Effective Webinar in the COVID-19 Pandemic.

INTRODUCTION: The surgical requirement for cleft lip and palate repair remains unmet in many developing areas of the world, including remote regions of Ghana. This article reviews the utilization of Internet education and online consultation for cleft lip and palate surgical training in Sunyani Regional Hospital (SRH), Ghana. METHODS: The cleft lip and palate treatment was promoted to patients in remote areas of Sunyani, Ghana region, through a charitable outreach program. These basic designs and settings were managed by local participants such as doctors, residents, nurses, and staff in SRH, Ghana. RESULTS: From November 2014 to December 2020, the authors collaborated in surgical treatment for 84 cases that were diagnosed with unilateral cleft lip, bilateral cleft lip, hard and soft palate cleft, and microstomia. The type of surgery has varied and has included cheiloplasty, palatoplasty, and others. The average scores of esthetic outcome evaluation were nasal form=2.4, symmetry of the nose=2.9, and vermillion border=2.9. Through the program, the surgeons and residents became significantly more proficient at cleft lip and palate surgery. The seminar topics have covered essential and sustainable topics based on SRH's current needs and showed the effectiveness in the current coronavirus disease-19 pandemic situation. CONCLUSIONS: The shortage of orofacial cleft surgeons working in rural areas like Sunyani, Ghana, remains an obstacle that poses a challenge to any effort to improve health care quality in these rural communities. Sustainable remote education is essential for the training of local cleft surgeons to fill this local need; our collaborative and charitable program could be a recommended education design for cleft surgeons and institutes for their sustainable education.

Craniofacial Epidermoid and Dermoid Cysts.

BACKGROUND: Epidermoid and dermoid cysts are benign developmental anomalies that can form anywhere in the body. Despite the rarity of incidence in the head and neck, they can arise at a variety of craniofacial locations. The purpose of this study was to analyze the clinical features of epidermoid and dermoid cysts arising in the craniofacial region with a literature review. METHODS: A retrospective study was designed, and clinical features and surgical considerations were investigated from a literature review. Cases of epidermoid cysts in the scalp, temporal area, glabellar area, mouth floor, and buccal mucosa were described. RESULTS: Dermoid cysts in more lateral regions of the scalp are rarely associated with intracranial extension. Because temporal dermoid cysts have a high rate of intracranial extension, radiological evaluation of the lesions in the temporal area is imperative. Epidermoid cysts in the glabellar area are usually superficial. Consideration of the surgical approach for an epidermoid cyst of the mouth floor is important. Because epidermoid cysts in the buccal mucosa are extremely rare, differential diagnosis was emphasized. Epidermoid cysts in the scalp, in the temporal intradiploic area, on the glabellar area in the periorbital region, in the mouth floor, and in the buccal mucosa were surgically excised considering the depth and location. Ten cases of epidermoid cysts in the buccal mucosa were retrieved from the literature review. CONCLUSIONS: Consideration of the anatomic locations of epidermoid and dermoid cysts in the craniofacial region might help facilitate accurate diagnosis and treatment.

Odontogenic Necrotizing Fasciitis of Face and Scalp.

ABSTRACT: Necrotizing fasciitis of the face and scalp is a severe bacterial infection that can result in long-term morbidity or even mortality if not properly managed. In a third-world country like Ghana, where most of the population relies on daily income for livelihood, citizens with diseases that have long-term morbidity suffer financial difficulty, particularly when the patient is the breadwinner.This brief clinical study demonstrates the severity of necrotizing fasciitis of the face and scalp originated from oral infection, and its capability to affect any part of the body if proper treatment is not established at early onset of disease. Correct diagnosis, patient education, early antibiotic treatment, and timely surgery are crucial for controlling infection and for preventing irreparable damage. Therefore, public education is crucial in preventing such infection by discouraging use of over-the-counter medication in such situations.

Clinical classification of cervical necrotizing fasciitis.

PURPOSE: Necrotizing fasciitis of the craniofacial region is a rare and potentially life-threatening bacterial infectious disease. Odontogenic infections primarily spread along facial fascia and subcutaneous tissues, resulting in upper chest skin or thoracic necrosis. The purpose of this clinical classification was to demonstrate clinically important guidelines for early diagnosis and prompt management of CNF. METHODS: Although the incidence of cervical necrotizing fasciitis (CNF) is very rare in many developed countries, prompt management with appropriate initial diagnosis is essential, especially in tropical low-economic rural regions of African countries. Over the last 12 years, our charitable team in West Africa made clinical classifications of CNF according to onset time and spreading pattern to thoracic extension. RESULTS: CNF patients could be divided into two primary types, limited to neck type and extended to upper chest type. We also further categorized from each type into three different groups according to the CNF onset and clinical characteristics, including acute type with hematogenous spread within 2 weeks, subacute type with suppuration over 2 to 4 weeks, chronic type without suppuration over 4 weeks, multiple type with partial skin necrosis, island type with necrotic skin coverage, and broad type with whole skin necrosis. CONCLUSIONS: These classifications will help decrease the mortality rate in severely infected patients.

Clinical Manifestation of a Patient With Forehead Sparganosis.

Human sparganosis is a parasite infection caused by the larva of a tapeworm of the genus Spirometra. Ocular, central nervous system, auricular, pulmonary, intraosseous, intraperitoneal, and subcutaneous manifestations of this infection in the neck or inguinal region have been described.The authors report the rare occurrence of cutaneous forehead sparganosis of a 19-year-old male who presented with a soft subcutaneous mass in the forehead, along with a related literature review.

Pediatric Glial Heterotopia in the Medial Canthus.

Glial heterotopias are rare, benign, congenital, midline, and nonteratomatous extracranial glial tissue. They may be confused as encephalocele or dermoid cysts and are mostly present in the nose.An 8-month-old African female child presented with a slow growing paranasal mass. The mass had been present at the left upper medial canthus since birth and had slowly and progressively enlarged. There was no communication between the mass and the cranial cavity during the operational procedure. The mass was immunohistochemically positive for S-100 protein as well as for glial fibrillary acidic protein, but negative for proliferating cell nuclear antigen. This suggested that the mass was composed of benign glial tissues with many astrocytes.The purpose of this report is to demonstrate the first patient with pediatric glial heterotopic tissue in the medial canthus and to report the clinical importance of its immunohistochemical findings.

Mandibular clear cell odontogenic carcinoma.

BACKGROUND: Clear cell odontogenic carcinoma (CCOC) is a rare intraosseous carcinoma of the jaw; only 81 cases have been reported in the English literatures. CASE PRESENTATION: We reported an additional case and reviewed the existing literature. A 70-year-old woman presented with a large painful radiolucent mandibular lesion from the right canine to the left angle area through the midline. No metastatic lymph nodes or distant metastases were detected. She underwent wide surgical resection and reconstruction with a composite fibula free flap. She had no recurrence or metastasis after 18 months. CONCLUSION: CCOC occurs predominantly in women in their 50s-70s in the mandible. Painless swelling is the most common symptom, followed by pain, teeth loosening, and paresthesia. CCOC has a good prognosis after surgery. In large mandibular CCOC, wide resection and composite fibula free flap reconstruction is the treatment of choice.

Simple Repair of a Blow-Out Fracture by the Modified Caldwell-Luc Approach.

Here we report a patient with a blow-out fracture of the orbital floor that was treated by an intraoral transmaxillary approach. This 38-year-old man suffered a sudden blow to the periorbital area, which caused prolapse of the orbital contents into the maxillary sinus. The modified Caldwell-Luc approach was used to repair the orbital blow-out fracture and the maxillary sinus during was packed with Frazin gauze for 7 days to prevent recurrence of the prolapse. This was an easy and minimally invasive technique for the management of a blow-out fracture of the orbital floor.

Automated multicolumn screening workflow in ultra-high pressure hydrophilic interaction chromatography for streamlined method development of polar analytes.

The pharmaceutical industry is rapidly advancing toward new drug modalities, necessitating the development of advanced analytical strategies for effective, meaningful, and reliable assays. Hydrophilic Interaction Chromatography (HILIC) is a powerful technique for the analysis of polar analytes. Despite being a well-established technique, HILIC method development can be laborious owing to the multiple factors that affect the separation mechanism, such as the selection of stationary phase chemistry, mobile phase eluents, and optimization of column equilibration time. Herein, we introduce a new automated multicolumn and multi-eluent screening workflow that streamlines the development of new HILIC assays, circumventing the existing tedious 'hit-or-miss' approach. A total of 12 complementary columns packed with sub-2 µm fully porous and 2.7 µm superficially porous particles operated on readily available ultra-high pressure liquid chromatography (UHPLC) instrumentation across a diverse set of commercially available polar stationary phases were investigated. Different mobile phases with pH ranging from pH 3 to 9 were evaluated using different organic modifiers. The gradient and column re-equilibration were judiciously set to ensure a reliable assay screening framework that indicates promising conditions for subsequent method optimization to achieve resolution of challenging mixtures. This UHPLC screening system is coupled with a diode array and charged aerosol detectors (DAD, CAD and mass spectrometry) to ensure versatile detection for a variety of compounds. This fast-screening platform lays the foundation for a convenient generic workflow, accelerating the pace of HILIC method development and transfer across both academic and industrial sectors.

Knowledge, perception, and pesticide application practices among smallholder cocoa farmers in four Ghanaian cocoa-growing regions.

Pesticides are widely used in Ghana, especially in cocoa farming. However, the practice is suboptimal and unsupervised. Incorrect use of these chemicals can seriously harm human health, the environment, and economies that rely on these farmers' output. The study assessed cocoa farmers' pesticide knowledge, practices, and risk perception. Four hundred and four cocoa farmers were chosen randomly from 26 communities in four cocoa-growing regions of Ghana to answer questions about their risk knowledge, awareness, and practices, including personal protective equipment, storage and disposal of leftover pesticides, and used containers. The study revealed that 87% of the respondents belonged to cooperatives and certification groups. There was a significant positive relationship between group membership and benefits derived from inputs and training in pesticide use. About 70% of insecticides used were approved by the Ghana Cocoa Board, with neonicotinoids and pyrethroids being the most highly used insecticide classes in cocoa farms. Although farmers claimed adequate pesticide knowledge, this did not translate into practice, with the majority exhibiting improper pesticide storage, application, and disposal practices. Farmers appeared to know a lot but lacked the skills and attitude to put their knowledge to use. The improper practices appear to manifest in a variety of health symptoms experienced by farmers as a result of chemical exposure. The findings from this study suggest that cocoa farmers in Ghana require adequate practical training and support on pesticide use to reduce their associated health risks, protect the environment and ensure sustainable cocoa production in the world's second-largest cocoa bean exporter.

Real-time in situ monitoring of CRM-197 and polysaccharide conjugation reaction by fluorescence spectroscopy.

Aims: Process analytical technology (PAT) is increasingly being adopted within the pharmaceutical industry to build quality into a process. Development of PAT that provides real-time in situ analysis of critical quality attributes are highly desirable for rapid, improved process development. Conjugation of CRM-197 with pneumococcal polysaccharides to produce a desired pneumococcal conjugate vaccine is a significantly intricate process that can tremendously benefit from real-time process monitoring. Methods: In this work, a fluorescence-based PAT methodology is described to elucidate CRM-197-polysacharide conjugation kinetics in real time. Results & conclusion: In this work, a fluorescence-based PAT methodology is described to elucidate CRM-197-polysacharide conjugation kinetics in real time.

Use of optimized 1D TOCSY NMR for improved quantitation and metabolomic analysis of biofluids.

One dimensional selective TOCSY experiments have been shown to be advantageous in providing improved data inputs for principle component analysis (PCA) (Sandusky and Raftery 2005a, b). Better subpopulation cluster resolution in the observed scores plots results from the ability to isolate metabolite signals of interest via the TOCSY based filtering approach. This report reexamines the quantitative aspects of this approach, first by optimizing the 1D TOCSY experiment as it relates to the measurement of biofluid constituent concentrations, and second by comparing the integration of 1D TOCSY read peaks to the bucket integration of 1D proton NMR spectra in terms of precision and accuracy. This comparison indicates that, because of the extensive peak overlap that occurs in the 1D proton NMR spectra of biofluid samples, bucket integrals are often far less accurate as measures of individual constituent concentrations than 1D TOCSY read peaks. Even spectral fitting approaches have proven difficult in the analysis of significantly overlapped spectral regions. Measurements of endogenous taurine made over a sample population of human urine demonstrates that, due to background signals from other constituents, bucket integrals of 1D proton spectra routinely overestimate the taurine concentrations and distort its variation over the sample population. As a result, PCA calculations performed using data matrices incorporating 1D TOCSY determined taurine concentrations produce better scores plot subpopulation cluster resolution.

Adverse reaction to Coartem (artemether/lumefantrine) resulting in oculogyric crisis.

BACKGROUND: Artemether/lumefantrine (AL), sold under the brand name Coartem, is the most common artemisinin-based combination therapy for the treatment of malaria. Drug-induced oculogyric crisis is a neurological disorder characterized by frequent upward deviations of the eye. In the literature, no cases of Coartem-induced oculogyric crisis have been reported in Ghana. CASE PRESENTATION: A 19-year-old male patient, who presented fever measuring 37.9 °C, general body pains, and weakness was prescribed with antimalarial therapy artemether/lumefantrine, Coartem®, from a local pharmacy. Just after initiation of treatment, the patient complained of double vision, involuntary upward eye deviation, and inability to close both eyes. The patient was diagnosed with Coartem-induced oculogyric crisis and was treated with the cessation of the causing agent and intramuscular injection of promethazine hydrochloride. CONCLUSIONS: When a patient exhibits a neurological disorder, such as oculogyric crisis, with normal conscious state and normal vital signs, special attention should be given to obtaining a history of recently administered medications. Clinicians should recognize adverse reactions to drugs based on a thorough patient history and examination. The goal of this report was to present Coartem-induced oculogyric crisis.

Incidence and management of mandibular fractures in a low-resource health facility in Ghana.

OBJECTIVES: The mandible and other parts of the maxillofacial region suffer significant morbid injuries following road traffic accidents. Our study gives epidemiological description of mandibular fractures in Ghana and also evaluates the relevance of closed reduction and indirect fixation for managing mandibular fractures in low-resource health facilities in low-income countries like Ghana. PATIENTS ANDMETHODS: This is a retrospective study involving 268 patients who reported to the Department of Oral and Maxillofacial Surgery of the Sunyani Regional Hospital with mandibular fractures from January 2010 to December 2019. Patient medical records were assessed for information on age, sex, fracture etiology, anatomic location of fracture, time of day of road traffic accident, and other associated injuries. RESULTS: A total of 268 patients were included in this study (males, 216 [80.6%]; females, 52 [19.4%]). Motor vehicular accident (MVA) was the leading cause of mandibular fractures (202 injuries, 75.4%). Other etiologies included assault (39, 14.6%), gunshot (13, 4.9%), falls (12, 4.5%), and industrial accidents (2, 0.7%). Of the 161 male cases caused by MVA, 121 (75.2%) occurred at night and in the evening while the remaining 40 (24.8%) occurred in the morning and afternoon. Among all managed 222 patients, 212 (79.1%) were treated with closed reduction and indirect fixation technique while 10 (3.7%) were treated with open reduction and direct fixation. CONCLUSION: Closed reduction with indirect fixation could successfully be used to manage mandibular fractures in low resourced health facilities, especially in low-income countries. The poor lightening system on roads in Ghana is a major contributory factor to motor vehicular accidents.