Comparative stability study and aggregate analysis of Bevacizumab marketed formulations using advanced analytical techniques.

Bevacizumab (Bvz) is the most preferred recombinant humanized monoclonal antibody in biosimilar development due to its prominence as a standard treatment in the oncology space. Therapeutic monoclonal antibodies are typically more complex and unlikely to produce a replica. As a result, regulatory agencies allow approval of biosimilars that differ structurally and functionally from their reference product, but these differences should not have any clinical significance. To identify these significant discrepancies, it is essential to perform a thorough characterization of critical product attributes both in real-time and after storage until the product's expiration. In the present study, two Bvz biosimilar brands (Bio-1 and Bio-2) marketed in India were evaluated and compared with the reference product Avastin® to assess their degree of similarity. A comprehensive physicochemical characterization of biosimilars and reference product was performed using orthogonal techniques including LC-ESI-QTOF, MALDI-TOF, FTIR-ATR, iCIEF, rCE, nrCE, UV280, and RP-HPLC. Furthermore, Bvz formulations under study were subjected to various stress conditions of thermal (elevated temperature 50 ± 2 °C), chemical (acidic pH 3.0 ± 0.2, neutral pH 7.0 ± 0.2, and basic pH 10.0 ± 0.2), and mechanical (agitation 200 rpm) for comparative stability evaluation. Any alteration in the secondary structure of the native protein was detected and quantified using far-UV circular dichroism (CD), indicating an average of 15% and 11% loss in native antiparallel ß-sheet conformation respectively in Bio-1 and Bio-2 upon exposure to elevated temperature and high pH. Additionally, covalent or non-covalent aggregates formed as a function of elevated temperature and agitation were quantified using SEC-MALS.

Outcome of Second Allogeneic HSCT for Patients with Inborn Errors of Immunity: Retrospective Study of 20 Years' Experience.

A significant complication of HSCT is graft failure, although few studies focus on this problem in patients with inborn errors of immunity (IE). We explored outcome of second HSCT for IEI by a retrospective, single-centre study between 2002 and 2022. Four hundred ninety-three patients underwent allogeneic HSCT for severe combined immunodeficiency (SCID; n = 113, 22.9%) or non-SCID IEI (n = 380, 77.1%). Thirty patients (6.0%) required second HSCT. Unconditioned infusion or no serotherapy at first HSCT was more common in patients who required second transplant. Median interval between first and second HSCT was 0.97 years (range: 0.19-8.60 years); a different donor was selected for second HSCT in 24/30 (80.0%) patients. Conditioning regimens for second HSCT were predominately treosulfan-based (with thiotepa: n = 18, 60.0%; without, n = 6, 20.0%). Patients received grafts from peripheral blood stem cell (n = 25, 83.3%) or bone marrow (n = 5, 16.7%) with median stem cell dose 9.5 × 106 CD34 + cells/kilogram (range: 1.4-32.3). Median follow-up was 1.92 years (0.22-16.0). Overall survival was 80.8% and event-free survival was 64.7%. Four patients died, two of early-transplant related complications, and two of late sepsis post-second HSCT. Three patients required third HSCT; all are alive with 100% donor chimerism. Cumulative incidence of acute graft-versus-host disease was 28.4%, (all grade I-II). Viral reactivation was seen in 13/30 (43.3%) patients, including HHV6 (n = 6), CMV (n = 4), and adenovirus (n = 2). At latest follow-up, 25/26 surviving patients have donor chimerism ≥ 90% and 16/25 (64.0%) have discontinued immunoglobulin replacement. Second HSCT offers IEI patients with graft failure curative treatment with good overall survival and immunological recovery.

Ground-based selected ionizing space radiation effects on stability of APIs and their formulations.

Primary goal of any space mission is to keep astronauts healthy and fit during entire mission. One of the important challenges for that is providing a safe and effective pharmacy with the capabilities to address both scheduled and unanticipated medical conditions that may arise during spaceflight. Extended exposure to space environmental conditions may trigger drug deterioration, impacting quality, effectiveness, and safety. To assess the influence of space ionizing radiations on the stability of medicines launched aboard spacecraft, a preliminary ground-based ionizing radiation drug interaction study was carried out at different doses in the present work. Four different types of radiations - proton, neutron (thermal, fast), gamma and heavy ions (56Fe) were used to irradiate selected drugs namely diclofenac, ciprofloxacin and metoprolol along with their formulations. Chemical stability of each irradiated sample was checked using high performance liquid chromatographic method. Significant degradation was observed in even solid-state during proton irradiation in both Active Pharmaceutical Ingredient (API) and tablet dosage form. Solution of active pharmaceutical ingredient and liquid dosage forms were prone to degradation in presence of gamma radiation. No physical and chemical changes observed after neutron and 56Fe irradiation. Irradiation of the same drug with different radiations demonstrated varying HPLC chromatographic profiles in terms of %degradation as well as proportion of same degradation products. This indicates possibility of different degradation pathways to generate degradation products.

Physical Instabilities of Therapeutic Monoclonal Antibodies: A Critical Review.

The proteinaceous nature of monoclonal antibodies (mAbs) makes them highly sensitive to various physical and chemical conditions, thus leading to instabilities that are classified as physical and chemical instabilities. In this review, we are discussing in detail the physical instability of mAbs because a large number of articles previously published solely focus on the chemical aspect of the instability with little coverage on the physical side. The physical instabilities of mAbs are classified into denaturation and aggregation (precipitation, visible and subvisible particles). The mechanism involved in their formation is discussed in the article, along with the pathways correlating the denaturation of mAb or the formation of aggregates to immunogenicity. Further equations like Gibbs-Helmholtz involved in detecting and quantifying denaturation are discussed, along with various factors causing the denaturation. Moreover, questions related to aggregation like the types of aggregates and the pathway involved in their formation are answered in this article. Factors influencing the physical stability of the mAbs by causing denaturation or formation of aggregates involving the structure of the protein, concentration of mAbs, pH of the protein and the formulations, excipients involved in the formulations, salts added to the formulations, storage temperature, light and UV radiation exposure and processing factors are mentioned in this article. Finally, the analytical approaches used for detecting and quantifying the physical instability of mAbs at all levels of structural conformation like far and near UV, infrared spectroscopy, capillary electrophoresis, LC-MS, microflow imagining, circular dichroism and peptide mapping are discussed.

Recent updates in the clinical trials of therapeutic monoclonal antibodies targeting cytokine storm for the management of COVID-19.

Clinical studies have identified a cytokine storm in the third stage of disease progression in critical ill patients with coronavirus disease 2019 (COVID-19). Hence, effectively suppressing the uncontrolled immune response of the host towards the invaded viruses in a cytokine storm is a critical step to prevent the deterioration of patient conditions and decrease the rate of mortality. Therapeutic monoclonal antibodies (mAbs) are found to be effective for the management of acute respiratory distress syndrome in patients with COVID-19. In this review, we compiled all therapeutic mAbs targeting cytokine storm, which are in clinical trials for its repurposing in the management of COVID-19. Compilation of clinical trial data indicated that therapeutic monoclonal antibodies targeting interleukins (IL-6, IL-1ra, IL-8, IL-1ß, IL-17A, IL-33), interferon-gamma, tumor necrosis factor-alpha, P-selectin, connective tissue growth factor, plasma kallikrein, tumor necrosis factor superfamily 14, granulocyte macrophage colony stimulating factor, colony stimulating factor 1 receptor, C-C chemokine receptor type 5, cluster of differentiation 14 and 147, vascular endothelial growth factor, programmed cell death protein-1, Angiopoietin - 2, human factor XIIa, complementary protein 5, natural killer cell receptor G2A, human epidermal growth factor receptor 2, immunoglobulin-like transcript 7 receptor, complement component fragment 5a receptor and viral attachment to the human cell were under investigation for management of severely ill patients with COVID-19. Among these, about 65 clinical trials are targeting IL-6 inhibition as the most promising one and Tocilizumab, an IL-6 inhibitor is considered to be the potential candidate to treat cytokine storm associated with the COVID-19.

Liquid Chromatographic Method Development for Quantification of Inorganic Nitrite and Nitrate Impurities from Nitroglycerin Drug Substance by Using Ion-Pair Reagents with Liquid-Liquid Extraction Technique.

A large number of laboratory studies have reported Nitrite (NO2-) and Nitrate (NO3-) to be among the most common degradation products of the high-explosive Nitroglycerin drug substance. A novel, simple, robust and rapid reversed-phase high-performance liquid chromatography method has been developed for quantification of inorganic Nitrite and Nitrate impurities from Nitroglycerin drug substance. Successful separation was achieved in isocratic elution, using Inertsil C8-3, (250 × 4.6 mm, 5.0 µm) column, with mobile phase consisting of pH 7.0 tetrabutyl ammonium hydrogen sulfate buffer, methanol and acetonitrile (96:02:02, v/v/v). Flow rate was monitored at 2.0 mL min-1 and ultraviolet detection at 220 nm. The present work describes the role of an ion-pair reagent in the separation of polar compounds and liquid-liquid extraction technique for separation of polar and non-polar compounds. Nitroglycerin was subjected to various stress conditions to demonstrate the stability-indicating power of the method. The performance of the method was validated as per present International Council for Harmonisation (ICH) guidelines for specificity, linearity, accuracy, precision, ruggedness and robustness. The developed method can be a valuable alternative to the current ion-exchange chromatographic method mentioned in the literature. To the best of our knowledge, a rapid Liquid Chromatography (LC) method, which separates inorganic Nitrite and Nitrate impurities of Nitroglycerin, disclosed in this investigation was not published elsewhere.

Simulated space radiation: Investigating ionizing radiation effects on the stability of amlodipine besylate API and tablets.

Efficacious pharmaceuticals with the adequate shelf life are essential for the well-being of the space explorers and successful completion of a space mission. Space is brimming with different types of radiations, which penetrate inside the spacecraft despite the shielding material. Such radiations can alter the stability of the pharmaceuticals during long duration space missions. The literature reporting the space radiation effects on the pharmaceuticals is scarce in a public domain. Ground-based simulation studies can be useful to predict the influence of the space radiations on the stability of the pharmaceuticals. Based upon these facts, the main objective of the present preliminary work was to investigate the effect of different types of ionizing radiations on the stability of amlodipine besylate API and tablets. Amlodipine besylate samples were irradiated by protons, neutrons (thermal and fast), gamma and heavy ion (56Fe) radiations with their different doses. The samples were also irradiated with UV-visible radiation to compare the effect of selected ionizing radiations with photodegradation. The physical stability was examined through organoleptic evaluation and the chemical stability was evaluated by FTIR and HPLC. The results of the organoleptic evaluation showed colour changes from colourless to yellow in proton irradiated solid API and gamma irradiated API aqueous solution. The FTIR spectrum of proton irradiated API showed one additional absorption band at 1728 cm-1 due to degradation products. HPLC analysis revealed that amlodipine degraded up to 10% and 21% after the highest doses of proton and gamma irradiation, respectively. No physical or chemical changes were observed after neutron and 56Fe irradiation. The structures of major radiolytic products were elucidated using LC-MS/MS. Two new impurities were found in the API aqueous solution as a result of gamma irradiation. The drug degradation pathways were postulated by proposing the plausible mechanism of formation.

Natural Killer Cell Degranulation Defect: A Cause for Impaired NK-Cell Cytotoxicity and Hyperinflammation in Fanconi Anemia Patients.

Fanconi anemia (FA) is a rare inherited syndrome characterized by progressive bone marrow failure (BMF), abnormal skin pigmentation, short stature, and increased cancer risk. BMF in FA is multifactorial and largely results from the death of hematopoietic stem cells due to genomic instability. Also, inflammatory pathology in FA has been previously reported, however the mechanism is still not clear. In literature, decreased NK-cell count and/or impaired NK-cell activity, along with other immunological abnormalities have been described in FA-patients (1). However, to the best of our knowledge, this is the first report showing a defective degranulation mechanism leading to abnormal NK-cell cytotoxicity in FA-patients, which may explain the development of a hyperinflammatory response in these patients. This may predispose some patients to develop Hemophagocytic lymphohistiocytosis (HLH) which manifests with prolonged fever, progressive cytopenias and organomegaly. Early diagnosis and initiation of immunosuppressive therapy in these patients will help to better manage these patients. We also propose FA genes to be listed as a cause of familial HLH.

Long-Acting Injectables: Current Perspectives and Future Promise.

The parenteral route of administration is preferred over the oral route for treatment of many chronic and life-threatening diseases due to better patient compliance. Long-acting injectables/depot delivery systems are formulations intended for prolonged/sustained drug release over a long period of time ranging from a few days to months. Depot delivery systems enhance product quality by decreasing dosing frequency, simplifying the drug regimen. Parenteral depots reduce the relapse rate of disease and the maintenance phase of therapy, hence improving efficacy and treatment adherence. However, despite being extensively explored in the last seventy years, only a few depot products have been marketed or have reached commercial viability. The introduction of long-acting injectables of any drug took 9 to 10 years after approval of its oral formulation. Mainly the market has been conquered by long-acting injectables for antipsychotic, substance abuse, and hormonal therapy drugs. This article focuses on the preparation of long-acting injectables with special emphasis on challenges associated with formulation. The evolution and current global market trend of various depot formulations are also discussed. Insight is provided into the promising future of long-acting injectables of protein-based drugs as well as multidrug therapy, along with potential uses in the treatment of chronic diseases like HIV, Parkinson's, and Alzheimer's.

Impact of space environment on stability of medicines: Challenges and prospects.

To upkeep health of astronauts in a unique, isolated, and extreme environment of space is the primary goal for a successful space mission, hence, safe and efficacious medications are essential for the wellness of astronauts. Space medication has been challenged with problems related to efficacy. Along with altered physiology, one of the possible reasons could be instability of space medications in the presence of harsh spaceflight environmental conditions. Altered physical and chemical stability can result in reduced potency which can result in reduced efficacy. Right now, medicines from the International Space Station are replaced before their expiration. But, for longer duration missions to Mars or any other asteroid, there will not be any chance of replacement of medicines. Hence, it is desired that medicines maintain the shelf-life throughout the space mission. Stability of medicines used for short term or long term space missions cannot be judged by drug stability guidelines based on terrestrial environmental factors. Unique environmental conditions related to spaceflight include microgravity, excessive vibration, hard vacuum, humidity variation, temperature differences and excessive radiation, which may cause instability of medicines. This write-up provides a review of the problem and countermeasure approaches for pharmaceuticals exposed to the space environment. The first part of the article discusses thought processes behind outlining of International Conference on Harmonization drug stability guidelines, Q1A (R2) and Q1B, and its acceptance limits for accelerated stability study. The second part of the article describes the difference in the radiation environment of deep space compared to radiation environment inside the space shuttle based on penetration power of different types of radiation. In the third part of the article, various promising approaches are listed which can be used for assurance of space medicine stability. One of the approaches is the use of ground-based space simulation analogues and statistical treatment to data to calculate failure rate of drugs and probabilistic risk assessment. Another approach is to innovate storage and packaging technology using radiation harden polymer or using cryogenic temperatures.

Effect of Phyllostachys parvifolia leaf extract on ionizing radiation-induced genetic damage: A preliminary in vitro cytogenetic study.

The ionizing radiation is a known carcinogen as well as cancer therapeutic agent however, the side effect on normal tissue is a limiting factor and inadequate doses necessitates search for an ideal radioprotective agent. Bamboo species are rich source of antioxidants hence have therapeutic value in many free radical mediated diseases. This is the first report regarding in vitro protective effect of bamboo leaf extract against radiation induced genetic damage in human peripheral blood lymphocytes by cytokinesis blocked micronuclei (CBMN) assay. Fresh whole blood was exposed to 5Gy of cobalt-6o gamma radiation with or without 30 min pre-treatment with 3 µl and 5 µl of hydro alcoholic leaf extract of Phyllostachys parvifolia. In addition to whole extract the effect of potential active compound orientin was also assessed. The frequency of radiation induced micronuclei decreased significantly in a dose dependent manner following treatment with whole extract as well as orientin. The extent of reduction in micronuclei frequency was higher with whole bamboo leaf extract as compared to orientin alone.

Current approaches for in vitro drug release study of long acting parenteral formulations.

Long acting parenteral formulations are preferred over conventional formulations for the treatment of chronic diseases. Prevalence of such diseases provoked the interest of researchers and pharmaceutical industries in the development of long acting parenteral formulations. The regulatory guidelines and pharmacopoeia have remained silent on dissolution methods for long acting parenteral formulations due to their diverse nature. The lack of compendial method for dissolution testing increases the duration of approval process for long acting parenteral formulations. This article reviews various dissolution methods used to study in vitro drug release profile of long acting parenteral formulations. Compendial as well as noncompendial methods, such as- rotating dialysis cell, dialysis tube, rotating bottle, shaking flask, single drop, inverted cup and incubation, are used by researchers for drug release profile of long acting parenteral formulations. This review article also highlights the advantages and disadvantages of reported dissolution methods along with the suitability of these methods for particular type of long acting formulation. The compiled work will help the researchers in designing the biorelevant dissolution method and expedite the development of long acting parenteral formulations.

Simultaneous determination of atorvastatin calcium and olmesartan medoxomil in a pharmaceutical formulation by reversed phase high-performance liquid chromatography, high-performance thin-layer chromatography, and UV spectrophotometric methods.

Validated RP-HPLC, HPTLC, and UV spectrophotometric methods have been developed for the simultaneous determination of atorvastatin calcium (ATV) and olmesartan medoxomil (OLM) in a pharmaceutical formulation. The RP-HPLC separation was achieved on a Kromasil C18 column (250 x 4.6 mm, 5 microm particle size) using 0.01 M potassium dihydrogen o-phosphate (pH 4 adjusted with o-phosphoric acid)-acetonitrile (50 + 50, v/v) as the mobile phase at a flow rate of 1.5 mL/min. Quantification was achieved by UV detection at 276 nm. The HPTLC separation was achieved on precoated silica gel 60F254 plates using chloroform-methanol-acetonitrile (4 + 2+ 4, v/v/v) mobile phase. Quantification was achieved with UV detection at 276 nm. The UV-Vis spectrophotometric method was based on the simultaneous equation method that involves measurement of absorbance at two wavelengths, i.e., 255 nm (lambda max of OLM) and 246.2 nm (lambda max of ATV) in methanol. All three methods were validated as per International Conference on Harmonization guidelines. The proposed methods were simple, precise, accurate, and applicable for the simultaneous determination of ATV and OLM in a marketed formulation. The results obtained by applying the proposed methods were statistically analyzed and were found satisfactory.

Concurrent estimation of amlodipine besylate, hydrochlorothiazide and valsartan by RP-HPLC, HPTLC and UV-spectrophotometry.

Accurate, sensitive and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC), high-performance thin-layer chromatography (HPTLC) and ultraviolet (UV) spectrophopometric methods were developed for the concurrent estimation of amlodipine besylate (AMLO), hydrochlorothiazide (HCTZ) and valsartan (VALS) in bulk and combined tablet dosage forms. For the RP-HPLC method, separation was achieved on a C18 column using potassium dihydrogen orthophosphate buffer (50 mM, pH 3.7) with 0.2% triethylamine as the modifier and acetonitrile in the ratio of 56:44 (v/v) as the mobile phase. Quantification was achieved using a photodiode array detector at 232 nm over a concentration range of 2-25 µg/mL for AMLO, 5-45 µg/mL for HCTZ and 20-150 µg/mL for VALS. For the HPTLC method, the drugs were separated by using ethyl acetate-methanol-toluene-ammonia (7.5:3:2:0.8, v/v/v/v) as the mobile phase. Quantification was achieved using UV detection at 242 nm over a concentration range of 100-600 ng/spot for AMLO, 150-900 ng/spot for HCTZ and 1,200-3,200 ng/spot for VALS. The UV-spectrophotometric simultaneous equation method was based on the measurement of absorbance at three wavelengths; i.e., at 237.6 nm (λmax of AMLO), 270.2 nm (λmax of HCTZ) and 249.2 nm (λmax of VALS) in methanol. Quantification was achieved over the concentration range of 2-20 µg/mL for AMLO, 5-25 µg/mL HCTZ and 10-50 µg/mL for VALS. All methods were validated according to International Conference on Harmonization guidelines and successfully applied to marketed pharmaceutical formulations. Additionally, the three methods were compared statistically by an analysis of variance test, which revealed no significant difference between the proposed methods with respect to accuracy and precision.

Nondestructive and rapid concurrent estimation of paracetamol and nimesulide in their combined dosage form using Raman spectroscopic technique.

A rapid, nondestructive Raman spectroscopic method was developed for quantitative estimation of paracetamol and nimesulide in their combined dosage form. A Raman univariate calibration model was developed by measuring the peak intensities of paracetamol and nimesulide at 853 cm(-1) and 1336 cm(-1), respectively. The developed method was successfully applied for in situ, concurrent estimation of paracetamol and nimesulide in their combined dosage and method was also validated according to International Conference on Harmonisation guidelines. Thus, the developed Raman spectroscopic method can be applied for simultaneous estimation of paracetamol and nimesulide in their combined dosage form as a process analytical technology tool by pharmaceutical industries for routine quality control.

Comprehensive Assessment of Degradation Behavior of Aspirin and Atorvastatin Singly and in Combination by Using a Validated RP-HPLC Method.

A fixed-dose combination of atorvastatin and aspirin is widely used for the treatment of myocardial infarction. The present work describes a comprehensive study of the stress degradation behavior of atorvastatin and aspirin alone as well as in combination of 1:1 and 1:7.5 ratios, respectively, as per ICH guidelines. The degradation products of aspirin as well as atorvastatin were successfully separated by a developed simple, selective, and precise stability-indicating reversed-phase HPLC method. Chromatographic separation was achieved on the Phenomenex Luna analytical column, 150 mm × 4.6 mm, 5µm. The mobile phase consisted of 0.1% glacial acetic acid in water and acetonitrile in the ratio of 50:50 v/v at a flow rate of 1.0 ml/min. UV detection was performed at 246 nm. The extent of degradation was significantly influenced when both of the drugs were present in combination. Stress degradation behavior of atorvastatin was highly influenced by aspirin under acid hydrolysis, thermal degradation, and oxidative stress conditions. Similarly, the stress degradation behavior of aspirin was affected by atorvastatin especially under neutral hydrolysis, thermal degradation, and oxidative stress conditions. Additionally, the combination ratio of aspirin and atorvastatin also influenced the percentage degradation of each other. A mixture of aspirin and atorvastatin was also analyzed after a one-month stability study at 40 °C and 75% RH. All the results indicate chemical incompatibility of both aspirin and atorvastatin if present in combination.

Spectrofluorimetric estimation of salbutamol sulphate in different dosage forms by formation of inclusion complex with ß-cyclodextrin.

A simple, precise, reproducible and accurate spectrofluorimetric method for estimation of Salbutamol sulphate (SAL) in bulk drug and various dosage forms has been developed. This method is based on formation of inclusion complex of SAL in ß-cyclodextrin (BCD) which gives fluorescence at excitation wavelength of 279.6 nm and emission wavelength of 609.8 nm in water. Formation of inclusion complex of drug with BCD enhances fluorescence intensity of drug leads to increased sensitivity. The developed method was validated according to ICH guidelines with respect to accuracy, precision, linearity, limit of detection, limit of quantification. Linearity was observed in the range of 4-20 µg/ml with correlation coefficient of 0.9982. The simplicity of the method permitted rapid analysis suitable for routine control. The developed method was successfully applied for the estimation of SAL in different marketed dosage forms like tablets, syrup and aerosol.