Application of Instrumental Analysis Methods to Characterisation of Monoclonal Antibody-Based Formulations

Monoclonal antibodies (mAbs) are a dominant class of protein-based drugs used in multiple therapeutic contexts. These drugs have revolutionised the management of a number conditions and are positioned as an important strategy in tailored pharmaceutical approaches (targeted therapy) for immunomodulation and oncological management. The in vivo formulation of proteins, including antibodies, is a complex process tightly regulated through environmental factors and homeostatic mechanisms. Reproduction of these conditions is essential during ex vivo manufacture of mAbs to ensure the stability of proteins and to prevent aggregation, which may impact on the efficacy of the agent in biological tissue. Among these environmental factors, temperature pH, buffer characteristics, and the use of excipients, including salts, sugars, surfactants and amino acids are of paramount importance. The focus of the present paper was to provide an overview of contemporary literature, with a focus on key studies performed by the author and colleagues, focusing on how excipient use and instrumental monitoring of stability and aggregation in mAbs could have the potential to influence future drug design and protein formulation practice. Analysis of the data set suggested that the regulation of these features during the formulation of mAbs is a complex process and there is a need to ensure that all processes within the manufacture of mAbs are controlled according to the relevant environmental factors influencing protein stability and aggregation. This paper highlights the use of adherent and non-adherent cell lines to investigate the ability of excipients to maintain mAb stability. The specific excipients analysed including amino acids (arginine and glutamate), and combinations/comparison of ionic excipients in solution, pH and dynamics of solutions, including protein-protein interactions, concentrations and agglomeration. It was demonstrated in these studies that excipients have specific and contrasting effects on mAb stability and aggregation potential. Furthermore, characterisation of the effects of excipients and formulation techniques provided a key insight into the role of these factors in maintaining mAb stability, while instrumental techniques were evaluated during the characterisation of stability and agglomeration in a variety of contexts. The majority of this paper reflects the culmination of six published papers, in which the author has actively participated, focusing on the application of instrumental analysis methods to the characterisation of mAb formulations. These studies highlight a broad range of techniques used to characterise protein formulations and which may be applied to mAbs, including nuclear magnetic resonance (NMR) spectroscopy and orthogonal techniques. Characterisation of the size of protein/mAb aggregates has been demonstrated, with pharmaceutical relevance, including in the design of pro-drugs. Furthermore, key characteristics of mAb formulation, stability and aggregation have been characterised using multiple models, providing a comprehensive insight into this topic. The included studies indicate that additives that disrupt protein-protein interactions and prevent aggregation can influence mAb stability and that the stabilisation characteristics of these proteins can be measured using instrumental techniques. These studies have clear implications for the use of instrumental analysis in the development of therapeutic mAbs, highlighting the need for further research in this field, including further investigation of the value of NMR spectroscopy and orthogonal techniques

Empirical Evaluation of Pre-Trained Deep Learning Networks for Pneumonia Detection

Pneumonia is a significant global health issue, characterized by a substantial mortality risk, impacting a vast number of individuals on a global scale. The quick and precise identification of pneumonia is crucial for the optimal treatment and management of this condition. This research work aims to answer the pressing need for precise diagnostic methods by using two advanced deep learning models, namely VGG19 and ResNet50, for the purpose of pneumonia detection in chest X-ray pictures. Furthermore, the present area of research is on the use of deep learning methodologies in the domain of medical image analysis, namely in the identification of pneumonia cases via the examination of chest X-ray images. The study challenge pertains to the pressing need for accurate and automated pneumonia diagnosis to assist healthcare professionals in making timely and educated judgements. The VGG19 and ResNet50 models were trained and assessed using the comprehensive RSNA Pneumonia dataset. In order to enhance their performance in the classification of chest X-ray pictures as either normal or pneumonia-affected, the models underwent rigorous training and meticulous fine-tuning. Based on the results obtained from our investigation, it was seen that the VGG19 model exhibited a notable accuracy rate of 93\%, surpassing the ResNet50 model's accuracy of 84\%. Furthermore, it is worth noting that both models demonstrated a notable level of precision, recall, and f1-scores in the identification of normal and pneumonia patients. This indicates their potential for accurately classifying such instances. Furthermore, our research findings indicate that deep learning models, namely VGG19, have a high level of efficacy in reliably detecting pneumonia via the analysis of chest X-ray pictures. These models has the capacity to function as helpful tools for expediting and ensuring the precise identification of pneumonia by healthcare practitioners

Oral health related disorder among children with behavioral problems

Background: The best mouth function, the absence of disease, and oral health are achieved by maintaining the highest self-esteem possible. When someone has good dental health, they can communicate and connect with people without getting sick, feeling uncomfortable, or feeling embarrassed. This study aimed to evaluate the Oral health-related disorder specifically to assess dental caries and gingivitis among children with behavioral problems. Methods: This study was a prospective cross-sectional investigation carried out at Kalachandpur Government Primary School and Jashim Uddin Institute, Dhaka. The study was carried out between October 2021 and December 2022 and included 650 pediatric patients who were aged between 6 to 15 years. Results: Most 290 (44.62%) children were aged 6-9 years, 250 (38.46%) were 10-12 years, and the rest 110 (16.92%) were 13-15 years old. Patients with 11-15 years had lower dental caries. 310 (48%) were female, and 340 (52%) were male. Dental caries is associated with elevated risk. Children in the elevated caries risk group had a significantly higher mean value of conduct problems and Hyperactivity/inattention based on the SDQ compared to those with low caries risk. No statistically significant differences were found between the low caries risk group and the elevated caries group for the other subscales. Conclusions: There were statistically more children in the study group who had a high risk of getting caries. Behavior problems were observed while externalizing children who had a high risk of getting caries. Children with externalizing behavior also have more conduct problems and hyperactivity compared to children with reduced caries risk

Application of ER Stress Biomarkers to Predict Formulated Monoclonal Antibody Stability

For a therapeutic mAb to reach the clinic, the molecule must be produced at an appropriate yield and quality, then formulated to maintain efficacy and stability. The formation of sub‐visible particles (SVPs) can impact on product stability and is monitored during formulation development however, the potential of a mAb to form such species can be influenced throughout the whole bioprocess. We investigate levels of intracellular ER stress perceived by cells, day of mAb harvest and the relationship to subsequent product stability of two mAbs (denoted A and B), produced in CHO cell lines, as determined by SVP content after accelerated stability studies. We show the propensity of mAb A to form SVPs can be predicted by transcript expression of biomarkers of cellular ER stress, heavy/light chain transcript and polypeptide amounts, and harvest day. Further, mAb A material harvested on day 9 of culture was more stable, in terms of SVP formation, than material harvested on day 13. These data suggest that ER stress perceived by CHO cells during culture can reflect the stability of a mAb, and that biomarkers of such stress could help define culture harvest time as a tool to control or reduce SVP formation in formulated mAbs

An Intelligent Obstacle and Edge Recognition System using Bug Algorithm

Obstacle avoidance is an important task in robotics as the autonomous robot's aim is to reach the destination without collision.  One type of autonomous robot that can detect obstacles and edges and take alternative paths free of obstacles and edges is a real-time obstacle avoiding edge detection robot. This paper proposes a robotic Robot with an intelligence built into it that guides itself whenever an obstacle comes along its way by bug algorithm. This robotic Robot is constructed using AT mega 8 families’ micro-controller (Arduino Uno R3). The ultrasonic sensor is used to detect any obstacle with edges and sends a command to the microcontroller. The micro-controller, based on the received input signal, redirects the robot to push in an alternative direction by actuating the motors that are interfaced with it via a motor driver. Depending on the situation the robot is able to choose the correct path [1]. A decision making process of obstacle avoiding edge detection occurs spontaneously here. This robot was designed to think about its day-to-day potentialities

Redistribution of Flexibility in Stabilizing Antibody Fragment Mutants Follows Le Chatelier's Principle

Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc.), La Châtelier's principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect

Challenges and pitfalls in diagnosis of parosteal Osteosarcoma: a clinicopathologic study of 23 cases

Objective: Parosteal Osteosarcoma (PO) is an uncommon variant of osteosarcoma. Diagnosing PO is important due to its malignant nature but the diversity of histologic features makes it challenging by adding a number of soft tissue, bony and cartilaginous lesions into the list of differential diagnosis. Our aim was to study the clinicopathologic and histological features of PO with emphasis on features helpful in its discrimination from other mimicking lesions. Methods: We reviewed 23 cases of PO diagnosed in our institution between January 2001 and August 2015. Results: Femur was the most commonly involved bone (68.2%) along with other long bones and rib in a single case. Soft tissue component was graded as Grade1 in 9(39%), Grade2 in 8(34.7%) and Grade3 in 4(17.3%) cases. Bony component was seen either in combination of or exclusively as parallel streams and interconnected trabeculae (mosaic-pattern). Out of 9 cases with cartilage component, 3 showed a cartilage cap. 2(8.6%) cases showed dedifferentiation into osteosarcoma. Conclusion: PO should always be considered in the differential diagnosis of every lesion arising from the bone surface. Knowledge of the variations in histologic features helps to reach the correct diagnosis which should never be made without radiological correlation

Management of Primary Pterygium with Intralesional Bevacizumab (AVASTIN) Injection

Objective: To determine the management of primary Pterygium with intralesional Bevacizumab (AVASTIN) Injection. Study Design: Quasi-Experimental Study Place and Duration of Study: Armed Forces Institute of Ophthalmology, Rawalpindi Pakistan, from Oct 2019 to Mar 2020. Methodology: Sixty patients of Primary Pterygium with Grades 1, 2, and 3 were included. Pre-Intralesional injection evaluation includes the Ocular surface disease Index (OSDI), grading of Pterygium and ophthalmic examination, refraction,slit lamp bimicroscopy, fundoscopy, tonometry, and corneal topography. After four weeks of intralesional injection,reassessment was done. Results: A total of 60 participants with the mean age of the participants was 44.06±14.83 years were included in the study. In 26(43.3%) patients, grittiness, epiphora, redness, and photophobia were reported, and 16(26.6%) patients reported blurring of vision that improved in 100% of patients after intralesional injections. There was statistical significance (p-value ≤0.05) in means of K1, Sim K astigmatism, Surface asymmetry index, Surface Regularity Index, Grade of Pterygium, and Ocular surface disease index before and after the intralesional injection of Bevacizumab. However, no significant difference was recorded in Uncorrected Visual Acuity, Best Corrected Visual Acuity, and K2 parameters in pre and post-injection states (p-value ≥0.05).Only 7(11.6%) patients reported subconjunctival haemorrhage after the procedure. Conclusions: Treatment of Primary Pterygium with intralesional Bevacizumab injection successfully improves symptoms,Ocular Surface Disease Index, and reduces corneal astigmatism with minimum complications

The effect of lipidation on the self-assembly of the gut-derived peptide hormone PYY3–36

Lipidation is a powerful strategy to improve the stability in vivo of peptide drugs. Attachment of a lipid chain to a hydrophilic peptide leads to amphiphilicity and the potential for surfactant-like self-assembly. Here, the self-assembly and conformation of three lipidated derivatives of the gastrointestinal peptide hormone PYY3–36 is examined using a comprehensive range of spectroscopic, scattering, and electron microscopy methods and compared to those of the parent PYY3–36 peptide. The peptides are lipidated at Ser(11), Arg(17), or Arg(23) in the peptide; the former is within the β-turn domain (based on the published solution NMR structure), and the latter two are both within the α-helical domain. We show that it is possible to access a remarkable diversity of nanostructures ranging from micelles to nanotapes and fibrillar hydrogels by control of assembly conditions (concentration, pH, and temperature). All of the lipopeptides self-assemble above a critical aggregation concentration (cac), determined through pyrene fluorescence probe measurements, and they all have predominantly α-helical secondary structure at their native pH. The pH and temperature dependence of the α-helical conformation were probed via circular dichroism spectroscopy experiments. Lipidation was found to provide enhanced stability against changes in temperature and pH. The self-assembled structures were investigated using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). Distinct differences in nanostructure were observed for lipidated and unlipidated peptides, also depending on the position of lipidation. Remarkably, micelles containing lipopeptides with α-helical peptide conformation were observed. Gelation was observed at higher concentrations in certain pH intervals for the lipidated peptides, but not for unlipidated PYY3–36. Thus, lipidation, in addition to enhancing stability against pH and temperature variation, also provides a route to prepare PYY peptide hydrogels. These findings provide important insights into the control of PYY3–36 conformation and aggregation by lipidation, relevant to the development of future therapeutics based on this peptide hormone, for example, in treatments for obesity

Commonest cancers in Pakistan - findings and histopathological perspective from a premier surgical pathology center in Pakistan.

Abstract CONTEXT: There are no recent authoritative data about incidence and prevalence of various types of cancers in Pakistan. AIM: To determine the frequency of malignant tumors seen in our practice and provide a foundation for building a comprehensive cancer care strategy. MATERIALS AND METHODS: 10,000 successive cases of solid malignant tumors reported in 2014 were included. All cases had formalin fixed, paraffin embedded specimens available and diagnosis was based on histological examination of H and E stained slides plus ancillary studies at the Section of Histopathology, Department of Pathology and Laboratory Medicine, Aga Khan University Hospital, Karachi. The latest WHO classifications were used along with the latest CAP protocols for reporting and the most updated TNM staging. RESULTS: There were 9,492 (94.9%) primary tumors while 508 (5.1%) were metastatic. Some 5,153 (51.5%) were diagnosed in females and 4,847 (48.5%) in males. The commonest malignant tumors in females were breast (32%), esophagus (7%), lymphomas (6.8%), oral cavity (6.7%) and ovary (4.8%), while in males they were oral cavity (13.9%), lymphomas (12.8%), colorectum (7.9%), stomach (6.9%) and esophagus (6.6%). Malignant tumors were most common in the 5th, 6th and 7th decades. About 8% were seen under 20 years of age. CONCLUSIONS: Oral cavity and gastrointestinal cancers continue to be extremely common in both genders. Breast and esophageal cancers are prevalent in females. Lung and prostate cancer are less common than in the west. Ovarian cancer was very common but cervix cancer was less so