Rapid identification of antibody impurities in size-based electrophoresis via CZE-MS generated spectral library.

Methods for the reliable and effective detection and identification of impurities are crucial to ensure the quality and safety of biopharmaceutical products. Technical limitations constrain the accurate identification of individual impurity peaks by size-based electrophoresis separations followed by mass spectrometry. This study presents a size-based electrophoretic method for detecting and identifying impurity peaks in antibody production. A hydrogen sulfide-accelerated degradation method was employed to generate known degradation products observed in bioreactors that forms the basis for size calibration. LabChip GXII channel electrophoresis enabled the rapid (< 1 min) detection of impurity peaks based on size, while capillary zone electrophoresis-mass spectrometry (CZE-MS) facilitated their accurate identification. We combine these techniques to examine impurities resulting from cell culture harvest conditions and forced degradation to assess antibody stability. To mimic cell culture harvest conditions and the impact of forced degradation, we subjected samples to cathepsin at different pH buffers or exposed them to high pH and temperature. Our method demonstrated the feasibility and broad applicability of using a CZE-MS generated spectral library to unambiguously assign peaks in high throughput size-based electrophoresis (i.e., LabChip GXII) with identifications or likely mass of the antibody impurity. Overall, this strategy combines the utility of CZE-MS as a high-resolution separation and detection method for impurities with size-based electrophoresis methods that are typically used to detect (not identify) impurities during the discovery and development of antibody therapeutics.

Necrotizing Fasciitis: When skin confuses - An autopsy case report.

Necrotizing Fasciitis (NF) is a severe life-threatening soft tissue infection characterized by the rapid destruction of muscle, fat and fascial layers. This report details an autopsy case report of a 40year old male, unclaimed body lacking the complete history except that given by the Police personnel accompanying in which there is no prior history of trauma. This person succumbed to septic shock secondary to NF, despite clinical interventions. This case emphasizes the importance of early diagnosis and the need for heightened clinical awareness to improve patient outcomes.

Standards-Free Absolute Quantitation of Oxidizable Glycopeptides by Coulometric Mass Spectrometry.

Currently, glycopeptide quantitation is mainly based on relative quantitation due to absolute quantitation requiring isotope-labeled or standard glycopeptides which may not be commercially available or are very costly and time consuming to synthesize. To address this grand challenge, coulometric mass spectrometry (CMS), based on the combination of electrochemistry (EC) and mass spectrometry (MS), was utilized to quantify electrochemically active glycopeptides without the need of using standard materials. In this study, we studied tyrosine-containing glycopeptides, NYIVGQPSS(ß-GlcNAc)TGNL-OH and NYSVPSS(ß-GlcNAc)TGNL-OH, and successfully quantified them directly with CMS with a discrepancy of less than 5% between the CMS measured amount and the theoretical amount. Taking one step further, we applied this approach to quantify glycopeptides generated from the digestion of NIST mAb, a monoclonal antibody reference material. Through HILIC column separation, five N297 glycopeptides resulting from NIST mAb tryptic digestion were successfully separated and quantified by CMS for an absolute amount without the use of any standard materials. This study indicates the potential utility of CMS for quantitative proteomics research.

Multifunctional siRNA/ferrocene/cyclodextrin nanoparticles for enhanced chemodynamic cancer therapy.

The therapeutic outcome of chemodynamic therapy (CDT) is greatly hindered by the presence of oxidative damage repair proteins (MTH1) inside cancer cells. These oxidative damage repair proteins detoxify the action of radicals generated by Fenton or Fenton-like reactions. Hence, it is extremely important to develop a simple strategy for the downregulation of MTH1 protein inside cancer cells along with the delivery of metal ions into cancer cells. A one-pot host-guest supramolecular approach for the codelivery of MTH1 siRNA and metal ions into a cancer cell is reported. Our approach involves the fabrication of an inclusion complex between cationic ß-cyclodextrin and a ferrocene prodrug, which spontaneously undergoes amphiphilicity-driven self-assembly to form spherical nanoparticles (NPs) having a positively charged surface. The cationic surface of the NPs was then explored for the loading of MTH1 siRNA through electrostatic interactions. Using HeLa cells as a representative example, efficient uptake of the NPs, delivery of MTH1 siRNA and the enhanced CDT of the nanoformulation are demonstrated. This work highlights the potential of the supramolecular approach as a simple yet efficient method for the delivery of siRNA across the cell membrane for enhanced chemodynamic therapy.

Smart Biomaterials: An Evolving Paradigm in Dentistry.

According to definition and general agreement, smart materials have properties that can be altered in a controlled fashion by stimuli including stress, temperature, moisture, pH, and electric or magnetic fields. Various recent materials in materials science are in working order, meaning they must achieve their tasks and should go through intentional modification. Smart materials change one or more of their characteristics in response to inputs. They can be called as responsive materials. As these materials have been available for so long, they are used for a wide range of purposes. These qualities have useful applications in many different industries, including dentistry. Zirconia, shape-memory alloys, and SmartSeal obturation system (Prosmart-DRFP Ltd., Stamford, United Kingdom) are a few examples of dental materials with intelligent behavior. The creation of novel materials is a major trend in materials science. These materials might make it possible to develop cutting-edge dental therapies with vastly improved clinical results. This article reviews the following: nickel-titanium smart alloy, smart composites, self-healing composites, smart ceramics, glass ionomer cement as a smart material, SmartSeal obturation system, and smart coatings for dental implants. We can better understand these biosmart materials with the aid of this review. The development of these newer and superior smart materials makes the outcome of the treatment far better for both the operator and the patient.

3D Technology Used for Precision in Orthodontics.

One of the most crucial technologies used by orthodontists to assess and document the dimensions of craniofacial features is imaging. Orthodontists frequently employ two-dimensional (2D) imaging methods, although 2D imaging cannot localize or determine the depth of structures. Early in the 1990s, three-dimensional (3D) imaging was invented, and it has since become a crucial part of dentistry, especially in orthodontics. One of the newest and most important breakthroughs in dentistry is 3D technology. Clinicians have been able to significantly improve patient care while also shortening the time spent on treatment planning due to these technologies, which include intra-oral scanning, 3D imaging, computed-axial tomography (CAT) scan, cone-beam computed tomography (CBCT), computer-aided design/computer-aided manufacturing (CAD/CAM), and 3D software. 3D models of maxillary and mandibular arches can take the place of conventional plaster casts and their limits for planning treatments, appliance production, and estimated treatment results as part of this continuous progress. Digital orthodontics procedures have become more popular in the recent past. The development of "personalized" orthodontic appliances makes use of technology. These technologies' overall improvement can increase clinicians' productivity and efficiency by simplifying traditional methods that are seen to be particularly laborious. The objectives of this review are to provide an overall description of the 3D technology nowadays and to assess its orthodontic applications.

Diagnostic utility of N-terminal TMPP labels for unambiguous identification of clipped sites in therapeutic proteins.

Protein therapeutics are susceptible to clipping via enzymatic and nonenzymatic mechanisms that create neo-N-termini. Typically, neo-N-termini are identified by chemical derivatization of the N-terminal amine with (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) followed by proteolysis and mass spectrometric analysis. Detection of the TMPP-labeled peptide is achieved by mapping the peptide sequence to the product ion spectrum derived from collisional activation. The site-specific localization of the TMPP tag enables unambiguous determination of the true N-terminus or neo-N-termini. In addition to backbone product ions, TMPP reporter ions at m/z 573, formed via collision-induced dissociation, can be diagnostic for the presence of a processed N-termini. However, reporter ions generated by collision-induced dissociation may be uninformative because of their low abundance. We demonstrate a novel high-throughput LC-MS method for the facile generation of the TMPP reporter ion at m/z 533 and, in some instances m/z 590, upon electron transfer dissociation. We further demonstrate the diagnostic utility of TMPP labeled peptides derived from a total cell lysate shows high degree of specificity towards selective N-terminal labeling over labeling of lysine and tyrosine and highly-diagnostic Receiver Operating Characteristic's (ROC) of TMPP reporter ions of m/z 533 and m/z 590. The abundant generation of these reporters enables subsequent MS/MS by intensity and m/z-dependent triggering of complementary ion activation modes such as collision-induced dissociation, high-energy collision dissociation, or ultraviolet photo dissociation for subsequent peptide sequencing.

Mobile Affinity Selection Chromatography Analysis of Therapeutic Monoclonal Antibodies.

Federal regulatory agencies require continuous verification of recombinant therapeutic monoclonal antibody (mAb) quality that is commonly achieved in a two-step process. First, the host-cell proteome and metabolome are removed from the production medium by protein A affinity chromatography. Second, following recovery from the affinity column with an acidic wash, mAb quality is assessed in multiple ways by liquid chromatography-mass spectrometry (LC-MS). However, lengthy sample preparation and the lack of higher-order structure analyses are limitations of this approach. To address these issues, this report presents an integrated approach for the analysis of two critical quality attributes of mAbs, namely titer and relative aggregate content. Integration of sample preparation and molecular-recognition-based analyses were achieved in a single step utilizing an isocratically eluted mobile affinity selection chromatography (MASC) column. MASC circumvents the protein A step, simplifying sample preparation. Within 10 min, (i) mAbs are fluorescently coded for specific detection, (ii) monomers and aggregates are resolved, (iii) the mAb titer is quantified, (iv) relative aggregate content is determined, (v) analytes are detected, and (vi) the column is ready for the next sample. It is suggested herein that this mode of rapid quality assessment will be of value at all stages of discovery (screening, clone selection, characterization), process R&D, and manufacturing. Rapid monitoring of variant formation is a critical element of quality evaluation.

An Observational Study on the Utility of Lab Parameters in Evaluating the Severity of Patients in South India with Covid-19.

Laboratory testing has been extremely helpful in determining the severity and determining the course of treatment for COVID-19 patients. Our aim has been to look for variables of patient's clinical and laboratory profile for two weeks and to observe their significance. Observational, Cross-sectional study. Data from the clinic and laboratory were compiled on Google form after informed consent from the patient. Statistical analysis was done using the Mann-Whitney U and unpaired t test. Population statistics included 202 patients (1st week) and 161 patients (2nd week), with the mean age of 61 ± 18 years. Most patients fell under the mild category (SPO2 >94%). High body mass index (n = 119) and hypertensive (n = 98) were the most common comorbidities observed. Diabetes, cardiovascular and respiratory diseases are the other comorbidities studied in this study. Hypoalbuminemia (n = 194) is the most deranged laboratory parameter in mild category, followed by lymphopenia (n = 109). In severe category also, hypoalbuminemia (n = 13) was deranged more. Other laboratory parameters included are CRP, D-Dimer, neutrophil and lymphocyte count. This study showed that albumin is a good predictor for estimating the severity of COVID-19 patients especially in the first week of their admission.

"Lab of the Future"─Today: Fully Automated System for High-Throughput Mass Spectrometry Analysis of Biotherapeutics.

Here we describe a state-of-the-art, integrated, multi-instrument automated system designed to execute methods involved in mass spectrometry characterization of biotherapeutics. The system includes liquid and microplate handling robotics and utilities, integrated LC-MS, along with data analysis software, to perform sample purification, preparation, and analysis as a seamless integrated unit. The automated process begins with tip-based purification of target proteins from expression cell-line supernatants, which is initiated once the samples are loaded onto the automated system and the metadata are retrieved from our corporate data aggregation system. Subsequently, the purified protein samples are prepared for MS, including deglycosylation and reduction steps for intact and reduced mass analysis, and proteolytic digestions, desalting, and buffer exchange via centrifugation for peptide map analysis. The prepared samples are then loaded into the LC-MS instrumentation for data acquisition. The acquired raw data are initially stored on a local area network storage system that is monitored by watcher scripts that then upload the raw MS data to a network of cloud-based servers. The raw MS data are processed with the appropriately configured analysis workflows such as database search for peptide mapping or charge deconvolution for undigested proteins. The results are verified and formatted for expert curation directly in the cloud. Finally, the curated results are appended to sample metadata in the corporate data aggregation system to accompany the biotherapeutic cell lines in subsequent processes.

Subcutaneous Microabscesses and Myositis as Part of Immune Reconstitution Inflammatory Syndrome due to Chronic Disseminated Candidiasis in a Child With Acute Lymphoblastic Leukemia.

BACKGROUND: Immune reconstitution inflammatory syndrome (IRIS) occurs when there is immune recovery after a prolonged period of leucopenia as a response to an underlying latent or chronic infection due to a proinflammatory cascade. It can occur in a child on chemotherapy for acute lymphoblastic leukemia (ALL) with underlying chronic disseminated candidiasis (CDC). OBSERVATION: We present a 7-year-old girl with pre-B ALL on chemotherapy who had prolonged febrile neutropenia and CDC with microabscesses in the liver, spleen, and kidney and a prolonged intensive care unit stay. Upon neutrophil recovery, she continued to have high-grade fever (blood and urine cultures negative). She also presented severe myositis of bilateral thigh muscles and developed unusual granulomas in the subcutaneous region of the lower back and right thigh. Although IRIS was suspected, she could not be initiated on steroids due to right upper lobe collapse consolidation due to multidrug-resistant Acinetobacter baumanni, which was treated with sensitive antibiotics. Treatment with steroids resolved her fever and normalized inflammatory markers. She is currently well on maintenance chemotherapy. CONCLUSIONS: IRIS can complicate the treatment of ALL in children. Diagnosing it while having a concurrent bacterial infection is challenging. Rarely CDC can present with subcutaneous granulomas. Treatment with steroids at the right time is very crucial.

"Stapling" scFv for multispecific biotherapeutics of superior properties.

Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.

Rapid Characterization of Antibodies via Automated Flow Injection Coupled with Online Microdroplet Reactions and Native-pH Mass Spectrometry.

Microdroplet reactions have aroused much interest due to significant reaction acceleration (e.g., ultrafast protein digestion in microdroplets could occur in less than 1 ms). This study integrated a microdroplet protein digestion technique with automated sample flow injection and online mass spectrometry (MS) analysis, to develop a rapid and robust method for structural characterization of monoclonal antibodies (mAbs) that is essential to assess the antibody drug's safety and quality. Automated sequential aspiration and mixing of an antibody and an enzyme (IdeS or IgdE) enabled rapid analysis with high reproducibility (total analysis time: 2 min per sample; reproducibility: ∼2% coefficient of variation). Spraying the sample in ammonium acetate buffer (pH 7) using a jet stream source allowed efficient digestion of antibodies and efficient ionization of resulting antibody subunits under native-pH conditions. Importantly, it also provided a platform to directly study specific binding of an antibody and an antigen (e.g., detecting the complexes mAb/RSFV antigen and F(ab')2/RSVF in this study). Furthermore, subsequent tandem MS analysis of a resulting subunit from microdroplet digestion enabled localizing post-translational modifications on particular domains of a mAb in a rapid fashion. In combination with IdeS digestion of an antibody, additional tris(2-carboxyethyl)phosphine (TCEP) reduction and N-glycosidase F (PNGase F) deglycosylation reactions that facilitate antibody analysis could be realized in "one-pot" spraying. Interestingly, increased deglycosylation yield in microdroplets was found, simply by raising the sample temperature. We expect that our method would have a high impact for rapid characterization of monoclonal antibodies.

3D-Printed Piezoelectric Porous Bioactive Scaffolds and Clinical Ultrasonic Stimulation Can Help in Enhanced Bone Regeneration.

This paper presents a comprehensive effort to develop and analyze first-of-its-kind design-specific and bioactive piezoelectric scaffolds for treating orthopedic defects. The study has three major highlights. First, this is one of the first studies that utilize extrusion-based 3D printing to develop design-specific macroporous piezoelectric scaffolds for treating bone defects. The scaffolds with controlled pore size and architecture were synthesized based on unique composite formulations containing polycaprolactone (PCL) and micron-sized barium titanate (BaTiO3) particles. Second, the bioactive PCL-BaTiO3 piezoelectric composite formulations were explicitly developed in the form of uniform diameter filaments, which served as feedstock material for the fused filament fabrication (FFF)-based 3D printing. A combined method comprising solvent casting and extrusion (melt-blending) was designed and deemed suitable to develop the high-quality PCL-BaTiO3 bioactive composite filaments for 3D printing. Third, clinical ultrasonic stimulation (US) was used to stimulate the piezoelectric effect, i.e., create stress on the PCL-BaTiO3 scaffolds to generate electrical fields. Subsequently, we analyzed the impact of scaffold-generated piezoelectric stimulation on MC3T3 pre-osteoblast behavior. Our results confirmed that FFF could form high-resolution, macroporous piezoelectric scaffolds, and the poled PCL-BaTiO3 composites resulted in the d33 coefficient in the range of 1.2-2.6 pC/N, which is proven suitable for osteogenesis. In vitro results revealed that the scaffolds with a mean pore size of 320 µm resulted in the highest pre-osteoblast growth kinetics. While 1 Hz US resulted in enhanced pre-osteoblast adhesion, proliferation, and spreading, 3 Hz US benefited osteoblast differentiation by upregulating important osteogenic markers. This study proves that 3D-printed bioactive piezoelectric scaffolds coupled with US are promising to expedite bone regeneration in orthopedic defects.

Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry.

Proteomic absolute quantitation strategies mainly rely on the use of synthetic stable isotope-labeled peptides or proteins as internal standards, which are highly costly and time-consuming to synthesize. To circumvent this limitation, we recently developed a coulometric mass spectrometry (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides, followed by mass spectrometry measurement of the peptide oxidation yield. Previously, CMS was only applied for single-protein quantitation. In this study, first, we demonstrated absolute quantitation of multiple proteins in a mixture (e.g., ß-lactoglobulin B, α-lactalbumin, and carbonic anhydrase) by CMS in one run, without using any standards. The CMS quantitation result was validated with a traditional isotope dilution method. Second, CMS can be used for absolute quantitation of a low-level target protein in a mixture; for instance, 500 ppm of PLBL2, a problematic host cell protein (HCP), in the presence of a highly abundant monoclonal antibody (mAb) was successfully quantified by CMS with no use of standards. Third, taking one step further, this study demonstrated the unprecedented quantitative analysis of deamidated peptide products arising from the mAb heavy chain deamidation reaction. In particular, absolute quantitation of the deamidation succinimide intermediate which had not been performed before due to the lack of standard was conducted by CMS, for the first time. Overall, our data suggest that CMS has potential utilities for quantitative proteomics and biotherapeutic drug discovery.

Nivolumab in Recurrent/Metastatic Squamous Cell Carcinoma of Head and Neck: A Tertiary Cancer Center Experience.

Apurva A. PatelAnanya PareekBackground Immunotherapy is a proven therapeutic option in recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) after platinum therapy. At present, there are no published Indian data regarding administration of nivolumab in this setting. Aim The aim of this study is to retrospectively evaluate the efficacy and toxicity of nivolumab in R/M HNSCC among Indian patients who progressed after one or more lines of chemotherapy, including platinum agents. Methods All patients of R/M HNSCC who received nivolumab between 2/6/2018 to 31/3/2020 were assessed retrospectively for the efficacy and toxicity of nivolumab therapy. Statistical Analysis All the data analysis was performed using IBM SPSS Statistics for Windows, version 25 (IBM Corp., Armonk, N.Y., USA). Descriptive analysis was performed to obtain baseline characteristic of the study sample. Survival analysis was done using the Kaplan-Meier method. Results Nivolumab therapy was tolerated well, with no new safety concerns, except one (8.3%) patient experienced grade ¾ toxicity (gastrointestinal). The clinical benefit rate (CBR) was found to be 66.7%. The median progression-free survival (PFS) was 3 months (95% CI; 2.093-3.907), and median overall survival (OS) was 8 months (95% CI; 3.731-12.269) from the date of first dose of nivolumab. Conclusions In our study, efficacy and toxicity were comparable with international data with no new safety concerns. Nivolumab emerged as an astonishing treatment option with tolerable toxicity profile in patients with R/M HNSCC postplatinum therapy, although limited treatment options are available at present.

Investigation of Tryptic Protein Digestion in Microdroplets and in Bulk Solution.

Recent studies have shown that ultrafast enzymatic digestion of proteins can be achieved in microdroplet within 250 µs. Further investigation of peptides resulting from microdroplet digestion (MD) would be necessary to evaluate it as an alternative to the conventional bulk digestion for bottom-up and biotherapeutic protein characterization. Herein we examined and compared protein tryptic digestion in both MD and bulk solution. In the case of MD of ß-lactoglobulin B, the preservation of long peptides was observed due to the short digestion time. In addition, MD is applicable to digest both high- and low-abundance proteins in mixture. In the case of digesting NIST 8671 mAb antibody containing a low level of commonly encountered host cell protein (HCP) PLBL2 (mAb:PLBL2 = 100:1 by weight), MD produced lower levels of digestion-induced chemical modifications of asparagine/glutamine deamidation, compared with overnight digestion. No significant difference between MD and bulk digestion was observed in terms of trypsin digestion specificity based on examination of semi- and unspecific-cleaved peptides. Our study suggests that MD, a fast digestion approach, could be adopted for bottom-up proteomics research and for peptide mapping of mAbs to characterize site-specific deamidation and glycosylation, for the purpose of development of biopharmaceuticals.

Knowledge, Attitude and Practices among Gynecologists Regarding Oral Health of Expectant Mothers of South Bengaluru, Karnataka.

Background: To evaluate the knowledge perspective and trainings among gynecologists' considering oral health of pregnant mothers of South Bengaluru city, by questionnaire. Materials and methods: A total of 60 gynecologists are included in the study. Prior to the study, the questionnaire was pretested by Pedodontist. The questionnaire was administered on the first day of visit, and on the next day it was collected back. Results: The research unveiled that a greater number of gynecologists had satisfactory knowledge attitude and training concerning oral health of expectant mothers. Conclusion: The predominance of gynecologists has satisfying knowledge perspective as well as practices, but still there is a demand for better effective attendance and involvement of medical specialists like gynecologists and pediatricians in continuing the education programs and forums on dentistry. How to cite this article: Popli HP, Kumar VD, Khatib MS, et al. Knowledge, Attitude and Practices among Gynecologists regarding Oral Health of Expectant Mothers of South Bengaluru, Karnataka. Int J Clin Pediatr Dent 2022;15(1):85-89.

The Retrograde Basilic Approach for Balloon-Assisted Maturation of Brachiocephalic Arteriovenous Fistulas.

PURPOSE: To assess the feasibility and outcomes of an approach utilizing transbasilic access for balloon-assisted maturation (BAM) of brachiocephalic arteriovenous fistulas (BCAVFs). MATERIALS AND METHODS: This retrospective analysis comprised 28 patients (mean age, 63 years ± 10.8) who underwent endovascular treatment of their immature BCAVFs via a basilic approach from December 2016 to December 2018. The mean age of the BCAVFs was 3.3 months ± 1.4 at the time of BAM. Other demographic data, vascular access characteristics, procedural data, technical and clinical success rates, and adverse events were also evaluated. RESULTS: All patients had inflow juxta-anastomotic stenoses, with 4 patients (14%) having concomitant outflow tract stenoses and 1 patient (4%) having a short-segment occlusion at the stenotic juxta-anastomotic segment. Technical success was achieved in 27 patients (96%). The mean diameter of the largest balloon used was 5.7 mm ± 0.6. Clinical success was achieved in 22 patients (79%), with 6 patients (21%) requiring a subsequent additional intervention before successful cannulation. No perioperative adverse events were observed. CONCLUSIONS: The retrograde basilic approach is feasible, safe, and effective for BAM of BCAVFs.