BACKGROUND:While Polyethylene glycol 3350 (PEG 3350) is approved by the USFDA for short term use by adults, it is commonly recommended for use in constipated children. Multiple reports of adverse events in children taking PEG 3350 raised safety concerns suggesting that low molecular weight species of PEG 3350 might be absorbed from the gut and cause side effects such as ethylene glycol (EG), diethylene glycol (DEG), and triethylene glycol (TEG). RESEARCH DESIGN AND METHODS:This article documents the development, validation, and application of analytical methods using GC-MS and GC-MS/MS for the quantitation of EG, DEG, and TEG in human plasma, serum, and urine. RESULTS:The analytical range for EG, DEG, and TEG was 2-20 µg/mL. The sample preparation process involves derivatization using N,O-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane in each biological matrices. Deuterated internal standards for each of the analytes were included to provide accurate quantitation of the glycol analytes. CONCLUSIONS:The validated methods were applied to analyze samples a pilot study of children taking PEG 3350. DEG and TEG were detected at levels below the limit of quantitation. In summary, a platform of analytical methods was developed to evaluate glycol analogs in urine, serum, and plasma clinical samples.

Study uses kinetic modeling to optimize continuous synthesis, boosting yield and impurity control. It underscores the role of reaction conditions to enhance purity, complementing traditional purification strategies.

A scalable continuous manufacturing process for the synthesis and crystallization of form III carbamazepine (CBZ) from iminostilbene (ISB) has been established. A high-yielding synthesis was first obtained using a plug flow reactor (PFR) and then scaled up using a continuous oscillatory baffled reactor (COBR). A real-time in-line Raman spectroscopy method was implemented to ensure that the conversion of the starting material ISB to the product CBZ was maintained above 99.0%. The monitored product stream was telescoped into a mixed-suspension mixed-product crystallizer (MSMPR-1) and a filtration unit to isolate the preliminary CBZ form I polymorph. A cooling recrystallization process was designed by using a crystal growth model derived from microscopy measurements. The impurity purging capacities and polymorph attainments were compared for the batch and flow processes. This study outlines the role of process modeling and process analytical technology (PAT) for impurity purging in a telescoped continuous manufacturing process.

Background: Zoonotic diseases pose a significant public health challenge globally, with developing countries like Pakistan facing heightened risks due to various factors such as climate change, environmental degradation, and socio-economic disparities. These diseases, transmitted between animals and humans, have profound implications for healthcare systems, food safety, and public health policies. Objective: This study aims to identify the key risk factors associated with the prevalence of zoonotic diseases in Pakistan and evaluate the effectiveness of the 'One Health' approach in addressing these multifaceted challenges. Methods: A comprehensive review of existing literature was conducted, focusing on zoonotic disease outbreaks in Pakistan, their associated risk factors, and the impact of integrated health strategies. Data from government reports, peer-reviewed articles, and international health organization databases were analyzed to assess the current state of zoonotic diseases in Pakistan. Results: The findings indicate that climate change, environmental issues, lack of public awareness, and socio-economic inequities are the primary drivers of zoonotic disease transmission in Pakistan. Despite some progress in disease surveillance and public health education, gaps in food and water safety practices, vector control, and environmental management persist. The 'One Health' approach has shown potential in improving cross-sectoral collaboration and enhancing disease prevention measures, yet its implementation remains inconsistent. Conclusion: Effective management of zoonotic diseases in Pakistan requires a concerted effort that encompasses improved surveillance, enhanced public awareness, and robust environmental and food safety policies. The 'One Health' approach emerges as a critical framework for fostering collaboration across human, animal, and environmental health sectors to mitigate the risks and impacts of zoonotic diseases. Keywords: Zoonotic Diseases, One Health Approach, Public Health, Pakistan, Climate Change, Environmental Health, Disease Surveillance, Food Safety, Vector Control, Socio-economic Factors.

Metal ions may act as enzyme cofactors and influence the kinetics of biochemical reactions that may also influence the biological production of therapeutic proteins and quality attributes such as glycosylation. Because sample preparation is a significant step in the reliable analysis of metals, we compared two sample preparation procedures for metal analysis of bioreactor culture media samples by ICP-MS: (i) samples were diluted in 2 % nitric acid (treatment with nitric acid, TNA); and (ii) samples were mixed with equal volume of 5 % nitric acid and closed vessel digestion was performed in a microwave (closed vessel digestion, CVD). In the comparison of extraction efficiencies between TNA and CVD procedures, CVD showed better extraction for Ca and Cu among bulk metals (∼30%) and for Ni among the trace metals (∼65%) for the bioreactor broth supernatant samples. For the cell pellet samples, the CVD procedure was found to be better for extraction of Fe (∼65% more) among bulk metals, Zn (∼20% more) among minor metals and Co (∼60% more) and Ni (∼45% more) among trace metals. Differences between the two procedures were less than 10% and TNA was better for all other metals quantified from both supernatant samples and cell pellet samples. The current study helps bring more clarity to the methodology on comprehensive metal analysis to monitor and maintain trace metal content for biologics production.

Developing a crystallization model that accurately predicts crystal growth and nucleation has been an important topic in the pharmaceutical industry for the past few decades. Particularly, as the pharmaceutical industry shifts toward continuous manufacturing, modeling will both reduce the workload for experimental optimization and allow for the development of model-based control systems that yield more consistent quality output. In this work, a unique approach for modeling size-dependent growth was applied to a set of batch cooling crystallizations. The cooling crystallization of carbamazepine (CBZ) in ethanol was monitored for solute concentration measurement by in-line Raman spectroscopy as well as for seed and product crystal size distribution (CSD) measurement by off-line laser diffraction. Based on these data, modeling was performed with MATLAB software using a combined quadrature method of moments and a method of characteristics technique in conjunction with a modified Mydlarz and Jones (MJ3) expression for size-dependent growth. This work expands upon our past work on modeling the cooling crystallization of CBZ by evaluating the effect of variable seed CSD on crystal growth rates as well as the accuracy of the model-predicted product CSD. Using the MJ3 size-dependent growth expression, variation in seed CSD resulted in high prediction errors for product CSD especially for the D10 value [root-mean-square error (RMSE) = 29.8%]. The error was reduced by varying the size-dependent growth parameters as a function of the seed CSD (RMSE = 7.4%). This new technique provided a better understanding of how the overall CSD affects crystal growth rates. The improved model may reduce the time needed to optimize experiments and provide better control of the variation of the CSD of the system.

The mitigation of nitrosamine formation in drug products has been studied and approaches such as using formulations with pH modifiers and antioxidants have been shown to decrease the formation of nitrosamines. However, more studies are needed to explore the effectivness of mitigation strategies with different drug models and formulations. The primary objective of this work was to assess the role of different antioxidants and pH modifiers in tablet formulations to mitigate the formation of NDMA, prepared in-house, using metformin hydrochloride as a model drug. A study design for manufacturing metformin hydrochloride formulations was created to evaluate potential mitigation stratigies. The formulations were prepared by wet granulation that included a sodium nitrite spike and various antioxidants such as ascorbic acid, caffeic acid and ferulic acid at various concentrations that may inhibit nitrosamine formation. The study design also included pH modifiers such as hydrochloric acid and sodium carbonate. The metformin hydrochloride formulations were placed under stability conditions that included humidity, temperature and time over a six month period. NDMA inhibition was found to be most effective in formulations with basic pH, followed by the addition of tested antioxidants with 0.1% concentrations in the formulations. All tested antioxidants showed complete mitigation in formulations with 0.5% and 1% concentrations. In summary, basic pH and the inclusion of antioxidants exhibited the potential to mitigate the formation of NDMA in metformin hydrochloride tablets.

Endometrial stromal tumors are extremely rare uterine malignancy, mainly among young females. Herein, we report a case of low-grade endometrial stromal tumor in a 19-year-old women who presented with the non-specific symptoms of lower abdominal pain, swelling and vaginal bleeding similar to the uterine leiomyoma. Ultrasound finding showed bulky uterus with multiple fibroid and degenerative changes. Contrast enhanced computed tomography (CECT) displayed diffusely enlarged uterus with heterogenous enhancement of the solid cystic mass with normal bilateral tubes and ovary. Considering the patient in the reproductive age group and her fertility status, she was operated with surgical subtotal hysterectomy. Histopathological examination and immunohistochemistry confirmed the diagnosis of Low-Grade Endometrial Stromal Sarcoma (LG-ESS). Patient was closely followed up for recurrence. Endometrial Stromal Sarcoma (ESS) should be included in the differential diagnosis of uterine leiomyoma to avoid recurrence of the lesion.

Accurate kinetic models for reaction systems allow for improved process understanding and greater quality control, which is particularly beneficial as the pharmaceutical industry shifts from batch to continuous manufacturing (CM).

Nitrosamine compounds are classified as potential human carcinogens, the origin of these impurities can be broadly classified in two categories, nitrosamine impurity found in drug products that are not associated with the Active Pharmaceutical Ingredient (API), such as N-nitrosodimethylamine (NDMA) or nitrosamine impurities associated with the API, such as nitrosamine drug substance-related impurities (NDSRIs). The mechanistic pathway for the formation of these two classes of impurities can be different and the approach to mitigate the risk should be tailored to address the specific concern. In the last couple of years number of NDSRIs have been reported for different drug products. Though, not the only contributing factor for the formation of NDSIRs, it is widely accepted that the presence of residual a nitrites/nitrates in the components used in the manufacturing of the drug products can be the primary contributor to the formation of NDSRIs. Approaches to mitigate the formation of NDSRIs in drug products include the use of antioxidants or pH modifiers in the formulation. The primary objective of this work was to evaluate the role of different inhibitors (antioxidants) and pH modifiers in tablet formulations prepared in-house using bumetanide (BMT) as a model drug to mitigate the formation of N-nitrosobumetanide (NBMT). A multi-factor study design was created, and several bumetanide formulations were prepared by wet granulation with and without sodium nitrite spike (100 ppm) and different antioxidants (ascorbic acid, ferulic acid or caffeic acid) at three concentrations (0.1%, 0.5% or 1% of the total tablet weight). Formulations with acidic and basic pH were also prepared using 0.1 N hydrochloric acid and 0.1 N sodium bicarbonate, respectively. The formulations were subjected to different storage (temperature and humidity) conditions over 6 months and stability data was collected. The rank order of N-nitrosobumetanide inhibition was highest with alkaline pH formulations, followed by formulations with ascorbic acid, caffeic acid or ferulic acid present. In summary, we hypothesize that maintaining a basic pH or the addition of an antioxidant in the drug product can mitigate the conversion of nitrite to nitrosating agent and thus reduce the formation of bumetanide nitrosamines.

Background and Objective: Guillain-Barré syndrome (GBS) is an immune system-mediated polyradiculoneuropathy that accounts for approximately 100,000 new patients per year globally. GBS has also been linked to respiratory failure in 20% to 40% of cases. The objective of this study was to find an association between Erasmus GBS respiratory insufficiency score (EGRIS) and the need for mechanical ventilation. Methods: We conducted our cross-sectional study at department of Neurology, Pakistan Institute of Medical Sciences from November 2019 to February 2020. Sixty patients were selected for this study. Data including demographic profile, variant of GBS, and EGRIS was calculated from all the study participants. Data was entered and analyzed using SPSS version 25. Results: All 60 patients enrolled in the study underwent continuous monitoring during admission time. Out of 60 patients, three (5%) endured mechanical ventilation with a mean EGRIS of 4.5. Others who didn't need mechanical ventilation had mean EGRIS of 1.5. The three patients requiring mechanical ventilation were one each from Acute Inflammatory Demyelinating polyradiculoneuropathy, Acute Motor Axonal Neuropathy, and Acute Motor and Sensory Axonal Neuropathy variants of GBS. p-value was non-significant for the presented data. Conclusion: EGRIS could not meet statistical significance in identifying the requirement of mechanical ventilation for GBS in this study. This may be due to low sample size.

Mitigation of failure modes in the continuous synthesis (CS) of a drug substance (DS) has the potential to widen the adoption of continuous manufacturing (CM) technologies by the pharmaceutical industry.

Starting in July 2018, the FDA alerted patients and health care professionals to the recall of ARBs such as valsartan by several pharmaceutical companies because of their potential contamination with carcinogenic nitrosamine impurities, including: (1) N-nitrosodimethylamine (NDMA), (2) N-nitrosodiethylamine (NDEA), (3) N-nitrosoethylisopropylamine (NEIPA), (4) N-nitrosodiisopropylamine (NDIPA), (5) N-nitrosodibutylamine (NDBA) and (6) N-nitroso-N-methyl-4-aminobutyric acid (NMBA). The FDA initiated a laboratory investigation to develop analytical procedures to test multiple lots of marketed ARB drugs to determine the possible presence of carcinogenic impurities and, if present, quantitate the levels of these impurities. Here the FDA laboratory developed and validated an automated micro-solid phase extraction MS/MS method, where all the analytes are not separated prior to elution to the MS, to simultaneously quantify NEIPA, NDIPA, NDBA and NMBA in ARB drug substances with an instrument sample analysis time of 12 seconds. The method was validated according to the ICH Q2(R1) guideline, and was determined to be specific, accurate, precise and linear over the corresponding nitrosamine analytical ranges. The method has been successfully implemented to quantitate the four nitrosamine impurities in 129 generic losartan, valsartan, olmesartan, irbesartan and telmisartan drug substance samples from 32 lots; and 32 losartan and valsartan drug product samples from 6 lots.

Trace metals play a critical role in the development of culture media used for the production of therapeutic proteins. Iron has been shown to enhance the productivity of monoclonal antibodies during Chinese hamster ovary (CHO) cell culture. However, the redox activity and pro-oxidant behavior of iron may also contribute toward the production of reactive oxygen species (ROS). In this work, we aim to clarify the influence of trace iron by examining the relationship between iron supplementation to culture media, mAb productivity and glycosylation, and oxidative stress interplay within the cell. Specifically, we assessed the impacts of iron supplementation on (a) mAb production and glycosylation; (b) mitochondria-generated free hydroxyl radicals (ROS); (c) the cells ability to store energy during oxidative phosphorylation; and (d) mitochondrial iron concentration. Upon the increase of iron at inoculation, CHO cells maintained a capacity to rebound from iron-induced viability lapses during exponential growth phase and improved mAb productivity and increased mAb galactosylation. Fluorescent labeling of the mitochondrial hydroxyl radical showed enhanced environments of oxidative stress upon iron supplementation. Additional labeling of active mitochondria indicated that, despite the enhanced production of ROS in the mitochondria, mitochondrial membrane potential was minimally impacted. By replicating iron treatments during seed train passaging, the CHO cells were observed to adapt to the shock of iron supplementation prior to inoculation. Results from these experiments demonstrate that CHO cells have the capacity to adapt to enhanced environments of oxidative stress and improve mAb productivity and mAb galactosylation with minimal perturbations to cell culture.

Trace metal variation in culture media has the potential to affect CHO cell culture performance in an unpredictable manner. Previously, zinc variation in CHO cultures were shown to impact cell growth, viability, specific activity and apoptosis [1]. In mammalian cells, zinc deficiency is frequently tied to oxidative stress. Although recombinantly produced protein drugs are affected by oxidative stress, the relationship between cellular metal uptake and key oxidative stress defense mechanisms remains unclear in CHO cells. Here, we developed a strategy to assess the intracellular zinc and iron content of cultured CHO cells. Next, we supplemented a CHO cell line producing beta-glucuronidase (GUS) with 0, 50, 100, or 150 mu M zinc sulfate cultured in batch mode in spinner flasks and in fed-batch mode in parallel bioreactors. Zinc supplementation impact on GUS production was marginal (<= 6%), but, impacted glycan fucosylation by decreasing up to similar to 15 % compared to control cultures. Zinc depletion in the supplemented culture medium was as much as similar to 20 mu M under intense fed-batch cultures in parallel bioreactors. However, in less intense spinner flasks batch cultures zinc depletion was less profound at similar to 2-5 mu M over the course of the run. Intracellular zinc analysis showed that most zinc consumption occurs primarily during lag phase and minimal consumption thereafter. Harvest day analyses showed a five-fold increase in intracellular iron, higher peroxidase activity and lowered total superoxide dismutase activity in zinc-deficient control cells indicating enhanced oxidative stress defense activity correlated to zinc deficiency. Comparatively smaller impact on CHO culture performance in spinner flask cultures indicated a stronger need for zinc supplementation under intense cell culture conditions.

Pharmaceutical crystallization affects the properties of APIs as it determines the purity and crystal size distribution, among other attributes. This work presents two CLD–CSD models, theoretical and empirical, for a model compound.

The variability of trace metals in cell culture media is a potential manufacturing concern because it may significantly affect the production and quality of therapeutic proteins. Variability in trace metals in CHO cell culture has been shown to impact critical production metrics such as cell growth, viability, nutrient consumption, and production of recombinant proteins. To better understand the influence of excess supplementation, zinc and copper were initially supplemented with 50-μM concentrations to determine the impact on the production and quality of β-glucuronidase, a lysosomal enzyme, in a parallel bioreactor system. Ethylenediaminetetraacetic acid (EDTA), a metal chelator, was included as another treatment to induce a depletion of trace metal bioavailability to examine deficiency. Samples were drawn daily to monitor cell growth and viability, nutrient levels, β-glucuronidase activity, and trace zinc flux. Cell cycle analysis revealed the inhibition of sub-G0/G1 species in zinc supplemented cultures, maintaining higher viability compared to the control, EDTA-, and copper-supplemented cultures. Enzyme activity analysis in the harvests revealed higher specific activity of β-glucuronidase in reactors supplemented with zinc. A confirmation run was conducted with supplementations of zinc at concentrations of 50, 100, and 150 μM. Further cell cycle analysis and caspase-3 analysis demonstrated the role of zinc as an apoptosis suppressor responsible for the enhanced harvest purity of β-glucuronidase from zinc-supplemented bioreactors.

A mycoplasma contamination event in a biomanufacturing facility can result in costly cleanups and potential drug shortages. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and penetrate the standard 0.2-µm filters used in the clarification of harvested cell culture fluid. Previously, we reported a study regarding the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G 1 (IgG1) antibody. Our previous work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Careful evaluation of certain identified process parameters over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before detection from a traditional method. In this report, we studied the changes in the IgG1 product quality produced by CHO cells considered to be induced by the M. arginini contamination events. We observed changes in critical quality attributes correlated with the duration of contamination, including increased acidic charge variants and high mannose species, which were further modeled using principal component analysis to explore the relationships among M. arginini contamination, CHO cell growth and metabolites, and IgG1 product quality attributes. Finally, partial least square models using NIR spectral data were used to establish predictions of high levels (≥104 colony-forming unit [CFU/ml]) of M. arginini contamination, but prediction of levels below 104 CFU/ml were not reliable. Contamination of CHO cells with M. arginini resulted in significant reduction of antibody product quality, highlighting the importance of rapid microbiological testing and mycoplasma testing during particularly long upstream bioprocesses to ensure product safety and quality.