TELMISARTAN AND AZELNIDIPINE QUANTIFICATION EMPLOYING HPLC STRATAGEM; STABILITY INVESTIGATION ON TELMISARTAN AND AZELNIDIPINE

Objective: The focus of our research was to create a fairly sensitive HPLC stratagem for determining telmisartan (TLM) and azelnidipine (AEL) in bulk and tablet types. Methods: Analysis of TLM and AEL was performed on a “C18 Kromasil stationary column (5 µm, 250 mm × 4.6 mm)”. The mobile phase was made of 0.1M NaH2PO4 solution (pH 3.5) and methanol at a comparative volume ratio of 50% each. The analysis of TLM and AEL was isocratic, with the flow velocity adjusted at 1.0 ml/min and indeed, the TLM and AEL analysis was done at 256 nm using a PDA device sensor. TLM and AEL were stressed with acid, peroxide, dry heat, alkali, and sunlight-induced settings. Results: The retention/elution periods for the TLM and AEL were observed at 2.225 min and 3.178 min, respectively. The HPLC stratagem developed have a straight-line relation with relative concentrations in the ranges of 20-60 µg/ml for TLM and 4-12 µg/ml for AEL. The LOQ’s for TLM and AEL were 0.2516 μg/ml and 0.0871 μg/ml, respectively. The validation investigational findings done for TLM and AEL with the established sensitive HPLC stratagem were passed out in conformity with the ICH standards. Conclusion: The established sensitive HPLC stratagem was shown as competent for the quality check of bulk samples of TLM and AEL throughout batch release as well as in the course of TLM and AEL stability investigations.

Recommendations for the Contents of the “Glycan Profile” Part of a Product Specification File

“Glycan Profile” is a necessary part of manufacturers’ product specification files for monoclonal antibody active ingredients or final products and erythropoietin active ingredients. The expert of the Federal State Budgetary Institution “Scientific Centre for Expert Evaluation of Medicinal Products” of the Ministry of Health of the Russian Federation provides recommendations for a step-by-step presentation of the test procedure, which will allow applicants to align product specification files for Russian- and foreign-produced medicinal products, help experts to minimise or eliminate the need to request additional information from applicants, and will contribute to timely batch release of medicinal products.

Recommendations for the Contents of the “Peptide Mapping” Part of a Product Specification File

‘Identification. Peptide mapping’ is a necessary part of manufacturers’ product specification files for therapeutic proteins (active ingredients and final products of monoclonal antibodies, filgrastims, erythropoietins). The expert of the Federal State Budgetary Institution “Scientific Centre for Expert Evaluation of Medicinal Products” of the Ministry of Health of the Russian Federation provides recommendations for a step-by-step presentation of the test procedure, which will allow applicants to align product specification files for Russian- and foreign-produced medicinal products, help experts to minimise or eliminate the need to request additional information from applicants, and will contribute to timely batch release of medicinal products.

S-Carvone Formulation Based on Granules of Organoclay to Modulate Its Losses and Phytotoxicity in Soil

Based on the effects that allelochemicals can exert over organisms, their use as alternatives to synthetic pesticides has been proposed. To this aim, it is important to understand their behavior in soils as allelochemicals can readily dissipate by different routes. In this work, novel granules based on the commercial organoclay Cloisite® 10A were prepared as a new strategy for the possible application of S-carvone as a bioherbicide, overcoming its rapid dissipation in the environment. Batch release, degradation, mobility, and phytotoxicity tests in soil were performed. Until now, the phytotoxicity of organoclay-based formulations of S-carvone in soil has not been studied. The release of S-carvone in water from the granules occurred slowly. There were no differences in the persistence of the allelochemical after its application to soil as a free compound (readily available form) or supported on granules. However, the granulated formulation reduced and delayed the leaching of S-carvone, thus controlling its downward movement in soil columns, as compared to the free S-carvone. Bioassays revealed that S-carvone supported on granules reduced the germination and aerial biomass of Lactuca sativa L. to a greater extent than the free compound. Our results demonstrated that the prepared formulation of S-carvone, based on granules of the commercial organoclay Cloisite® 10A, could be used to control transport losses, such as leaching or volatilization, increasing the bioefficacy of the allelochemical. These findings could inspire further investigations for the preparation of novel formulations of monoterpenes as potential bioherbicides.

Development of a Monocyte Activation Test as an Alternative to the Rabbit Pyrogen Test for Mono- and Multi-Component Shigella GMMA-Based Vaccines

Generalised modules for membrane antigens (GMMA)-based vaccines comprise the outer membrane from genetically modified Gram-negative bacteria containing membrane proteins, phospholipids and lipopolysaccharides. Some lipoproteins and lipopolysaccharides are pyrogens; thus, GMMA-based vaccines are intrinsically pyrogenic. It is important to control the pyrogenic content of biological medicines, including vaccines, to prevent adverse reactions such as febrile responses. The rabbit pyrogen test (RPT) and bacterial endotoxin test (BET) are the most commonly employed safety assays used to detect pyrogens. However, both tests are tailored for detecting pyrogenic contaminants and have considerable limitations when measuring the pyrogen content of inherently pyrogenic products. We report the adaptation of the monocyte activation test (MAT) as an alternative to the RPT for monitoring the pyrogenicity of Shigella GMMA-based vaccines. The European Pharmacopoeia endorses three MAT methods (A–C). Of these, method C, the reference lot comparison test, was identified as the most suitable. This method was evaluated with different reference materials to ensure parallelism and consistency for a mono- and multi-component Shigella GMMA vaccine. We demonstrate the drug substance as a promising reference material for safety testing of the matched drug product. Our results support the implementation of MAT as an alternative to the RPT and use of the defined parameters can be extended to GMMA-based vaccines currently in development, aiding vaccine batch release.

A cell-based in vitro assay for testing of immunological integrity of Tetanus toxoid vaccine antigen

Vaccines containing inactivated toxins confer protection by eliciting a neutralizing antibody response against bacterial toxins such as tetanus and diphtheria. At present, release of tetanus toxoid (TT) and diphtheria toxoid (DT)-containing vaccines relies on in vivo experiments showing the protective vaccine response. The aim of this study was to develop a reliable in vitro assay for TT vaccine antigen characterization with the potential of replacing in vivo potency experiments. To this end, we exploited that TT elicits a recall response in vaccinated donors: human peripheral blood mononuclear cells (PBMC) were stimulated with alum-adsorbed TT bulk antigen and low concentrations of TLR9 ligand; induction of TT-specific IgG was quantified via ELISpot after 5 days. Proof-of-concept was obtained using paired samples from donors before and after vaccination; anti-TT IgG was only detected in PBMC collected after booster vaccination; specificity was demonstrated with DT stimulation as control. Notably, when using PBMC from buffy coats, the specific response to TT was reproducible in 30% of cells; responsiveness correlated with higher numbers of switched memory B cells. Consecutive results showed that TT-specific IgG was also detectable when PBMC were stimulated with DTaP final vaccine product. Thus, the assay provides a viable means to test B-cell differentiation and induction of TT-specific IgG secretion using bulk antigen and final vaccine. However, prequalification of PBMC is required for reliable performance. Along with physicochemical and immunochemical methods, the functional assay could represent a complementary tool to replace in vivo potency assays in batch release of TT-containing vaccines.

THE MANAGEMENT OF PROCESS LEAD TIME IN THE RELEASE OF STERILE PHARMACEUTICAL BATCHES: A CASE STUDY OF OPTIMIZATION IN AN OFFICIAL BRAZILIAN PHARMACEUTICAL LABORATORY

Medicines must comply with quality, safety, and efficacy pillars. Nowadays, organizations seek to incorporate new management models encouraged by quality program following the world trend regarding the technological revolution. The present research aims to improve the sterile pharmaceutical product batches release process, using the Failure Mode Effects Analysis (FMEA) method. This study addresses the gap in literature on quality risk management during batch release. The methodology uses a form adapted to the process in order to systematize the information, improving its comparison and analysis, thus estimating, the identification of potential failure modes and their effects on their performance. Made it possible to assign values for the severity, occurrence, and failure modes detection, to then determine the risk level and the priority of risk level. The results obtained showed the mitigation and elimination of failures in the process, as well as opportunities for improvement and causes of failures identification, improvement in the process performance indicators, greater reliability, and reduction in batch release time. Keywords: good manufacturing practices pharmaceutical industry, risk management, risk management tools

Equine Mesenchymal Stem/Stromal Cells Freeze-Dried Secretome (Lyosecretome) for the Treatment of Musculoskeletal Diseases: Production Process Validation and Batch Release Test for Clinical Use

In the last decades, it has been demonstrated that the regenerative therapeutic efficacy of mesenchymal stromal cells is primarily due to the secretion of soluble factors and extracellular vesicles, collectively known as secretome. In this context, our work described the preparation and characterization of a freeze-dried secretome (Lyosecretome) from adipose tissue-derived mesenchymal stromal cells for the therapy of equine musculoskeletal disorder. An intraarticular injectable pharmaceutical powder has been formulated, and the technological process has been validated in an authorized facility for veterinary clinical-use medicinal production. Critical parameters for quality control and batch release have been identified regarding (i) physicochemical properties; (ii) extracellular vesicle morphology, size distribution, and surface biomarker; (iii) protein and lipid content; (iv) requirements for injectable pharmaceutical dosage forms such as sterility, bacterial endotoxin, and Mycoplasma; and (v) in vitro potency tests, as anti-elastase activity and proliferative activity on musculoskeletal cell lines (tenocytes and chondrocytes) and mesenchymal stromal cells. Finally, proteins putatively responsible for the biological effects have been identified by Lyosecretome proteomic investigation: IL10RA, MXRA5, RARRES2, and ANXA1 modulate the inflammatory process RARRES2, NOD1, SERPINE1, and SERPINB9 with antibacterial activity. The work provides a proof-of-concept for the manufacturing of clinical-grade equine freeze-dried secretome, and prototypes are now available for safety and efficacy clinical trials in the treatment of equine musculoskeletal diseases

Potency Assessment of Dendritic Cell Anticancer Vaccine: Validation of the Co-Flow DC Assay

For many years, oncological clinical trials have taken advantage of dendritic cells (DC) for the design of DC-based cellular therapies. This has required the design of suitable quality control assays to evaluate the potency of these products. The purpose of our work was to develop and validate a novel bioassay that uses flow cytometry as a read-out measurement. In this method, CD3+ cells are labeled with a fluorescent dye and the DC costimulatory activity is measured by the degree of T cell proliferation caused by the DC–T cell interaction. The validation of the method was achieved by the evaluation of essential analytical parameters defined by international guidelines. Our results demonstrated that the method could be considered specific, selective, and robust. The comparison between measured values and estimated true values confirmed a high level of accuracy and a lack of systematic error. Repeated experiments have shown the reproducibility of the assay and the proportionality between the potency and the DC amount has proven its linearity. Our results suggest that the method is compliant with the guidelines and could be adopted as a quality control assay or batch-release testing within GMP facilities.