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In-Use Physicochemical Stability of Sandoz Rituximab Biosimilar in 0.9% Sodium Chloride Solution After Prolonged Storage at Room Temperature Conditions
No full textOften, stability studies do not cover all facets of ensuring patient safety for biologics, unless the impact of the in-use and out-of-fridge conditions is also assessed. This study investigated the physicochemical and biological stability of Sandoz rituximab biosimilar (SDZ-RTX).In a worst-case setting, two SDZ-RTX batches in vials were exposed to long-term conditions (5 ± 3 °C) for at least the shelf-life period (36 months). These batches were exposed to out-of-fridge conditions of up to 25 ± 2 °C/60 ± 5% relative humidity in total for 14 days, and subsequently to 30 ± 2 °C/75 ± 5% relative humidity for 7 days. Thereafter, these batches were diluted to 1 mg/mL in 0.9% NaCl in 250-mL polyethylene infusion bags and stored at either 25 ± 2 °C/60 ± 5% relative humidity for 30 days or 30 ± 2 °C/75 ± 5% relative humidity for 14 days, representing in-use conditions. The stability of SDZ-RTX was assessed using a variety of analytical methods, including size-exclusion chromatography, cation exchange chromatography, non-reducing capillary electrophoresis sodium dodecyl sulfate, complement-dependent cytotoxicity-bioactivity, and subvisible particle count by light obscuration.Results for all assessments were within the stringent shelf-life acceptance criteria for SDZ-RTX for both batches under both in-use conditions.These data show that the physicochemical and biological quality of SDZ-RTX diluted in 0.9% NaCl infusion bags is assured, even after prolonged worst-case (out-of-fridge and in-use) storage at elevated temperatures up to 30 °C, if the medication is prepared under aseptic conditions according to the Summary of Product Characteristics
Immunogenicity assessment strategy for a chemically modified therapeutic protein in clinical development.
No full textThe clinical immunogenicity assessment for complex multidomain biological drugs is challenging due to multiple factors that must be taken into consideration. Here, we describe a strategy to overcome multiple bioanalytical challenges in order to assess anti-drug antibodies (ADA) for a novel and unique chemically modified protein therapeutic. A risk-centered approach was adopted to evaluate the immunogenic response to a modified version of human growth differentiation factor 15 (GDF15) connected to an albumin-binding fatty acid via a polyethylene glycol (PEG) linker. Key steps include monitoring anti-drug antibodies (ADAs), using a standard tiered approach of screening and confirmation. To deepen our understanding of ADA response, as a third tier of immunogenicity assessment, novel extensive characterization using a set of assays was developed, validated, and used routinely in clinical sample analysis. This characterization step included performance of titration, mapping of ADA response including anti-GDF15 and anti-PEG-fatty-acid antibody characterization, and assessment of the neutralizing anti-drug antibodies (NAbs) using cell-based assays for immunogenicity in parallel. The analytical methods were applied during two clinical trials involving both healthy volunteers and overweight or obese patients. We observed low incident rates for ADA and no ADAs against the PEG linker with fatty acid conjugation. In one of the clinical studies, we identified neutralizing ADAs. The proposed novel strategy of extensive characterization proved effective for monitoring the presence of ADAs and NAbs and can be used to support clinical development of a broad range of chemically modified proteins and multidomain biotherapeutics
Exploring 99mTc-Labeled Iron-Binding Glycoprotein Nanoparticles as a Potential Nanoplatform for Sentinel Lymph Node Imaging: Development, Characterization, and Radiolabeling Studies
No full textLactoferrin, an iron binding glycoprotein-based nanoparticle, has emerged as a promising platform for drug delivery and imaging. This study presents the potential use of the protein nanocarrier in tracking sentinel lymph nodes for cancer staging. Lactoferrin nanoparticles (LF-NPs) were synthesized using a thermal treatment process and optimized to obtain 60–70 nm particle size with PDI less than 0.2. The NPs were characterized microscopically and spectroscopically, ensuring a comprehensive understanding of their physicochemical properties. The LF-NPs were found to be stable in different pH conditions. Their biocompatibility was confirmed through cytotoxicity assessments on RAW 264.7 cells, and hemolysis assay and in vivo toxicity study reveal their safe profile. Additionally, LF-NPs were successfully radiolabeled with technetium-99m (>90% labeling yield). Cell uptake studies with RAW 264.7 exhibited an uptake of ∼6%. Biodistribution studies in Wistar rats shed light on their in vivo behavior and suitability for targeted drug delivery systems. These findings collectively emphasize the multifaceted utility of LF-NPs, positioning them as a promising platform for diverse biomedical innovations
Wurster Technology-Assisted Step-by-Step Engineering of Multi-layered Pellets (Sprinkles): Microscopy, Micro-CT, and e-Tongue-Based Analysis
No full textThe advancement in the formulation and characterization techniques have paved the path for development of new as well as modification of existing dosage forms. The present work explores the role of micro-computed tomography (micro-CT) as advanced characterization technique for multi-layered-coated pellets to ascertain the quality of coated pellets. The work further explored in-house e-tongue technique for understanding palatability of formulation in early stages of development thus by reducing clinical taste evaluation time. The developed multi-layered-coated pellets were characterized using microscopy (optical and electron microscopy). The obtained results demonstrated formation of spherical-shaped pellets with uniform coating. The uniform coating was further confirmed by results obtained from scanning electron microscopy (SEM) and cross-sectional SEM analysis, which showed visible difference in pellet surface before and after multi-layered coating. The micro-CT results confirmed the visible demarcation of layers (drug and polymer, i.e., hydroxypropyl methylcellulose (HPMC) and eudragit (EPO)) along with uniform thickness of various layering. The dissolution study of developed pellets suggested the role of layering EPO on drug release from pellets. The e-tongue analysis proved to be an excellent tool for early prediction of taste masking of drug via multi-layered pellets and can serve as potential platform for taste masking with high specificity. The overall results suggest the suitability of developed multi-layered platform as efficient dosage form (sprinkle) in pediatric/geriatric product development
Proteomic associations with forced expiratory volume – a Mendelian randomisation study
No full textA decline in forced expiratory volume (FEV1) is a hallmark of obstructive respiratory diseases, an important cause of morbidity among the elderly. While some data exist on biomarkers that are related to FEV1, we sought to do a systematic analysis of causal relations of biomarkers with FEV1.
Data from the general population-based AGES-Reykjavik study were used. Proteomic measurements were done using 4,782 DNA aptamers (SOMAmers). Data from 1,648 participants with spirometric data were used to assess the association of SOMAmer measurements with FEV1 using linear regression. Bi-directional Mendelian randomisation (MR) analyses were done to assess causal relations of observationally associated SOMAmers with FEV1, using genotype and SOMAmer data from 5,368 AGES-Reykjavik participants and genetic associations with FEV1 from a publicly available GWAS (n = 400,102).
In observational analyses, 473 SOMAmers were associated with FEV1 after multiple testing adjustment. The most significant were R-Spondin 4, Alkaline Phosphatase, Placental Like 2 and Retinoic Acid Receptor Responder 2. Of the 235 SOMAmers with genetic data, eight were associated with FEV1 in MR analyses. Three were directionally consistent with the observational estimate, Thrombospondin 2 (THBS2), Endoplasmic Reticulum Oxidoreductase 1 Beta and Apolipoprotein M. THBS2 was further supported by a colocalization analysis. Analyses in the reverse direction, testing whether changes in SOMAmer levels were caused by changes in FEV1, were performed but no significant associations were found after multiple testing adjustments.
In summary, this large scale proteogenomic analyses of FEV1 reveals protein markers of FEV1, as well as several proteins that are potentially causally related with lung function
Tight-binding small molecule carboxylesterase 2 inhibitors reduce intracellular Irinotecan activation
No full textAs the primary enzyme responsible for activatable conversion of Irinotecan to SN-38, carboxylesterase 2 is a significant prognostic and predictive biomarker towards Irinotecan-based treatments for pancreatic ductal adenocarcinoma (PDAC). High SN-38 levels stemming from high activity levels of CES2 leads to harmful effects, including life-threatening diarrhea. While alternate strategies have been explored, inhibition of CES2 presents as an effective strategy to directly alter the pharmacokinetics of Irinotecan (CPT-11) conversion to ultimately control the amount of SN-38 produced. To address this, we conducted a high-throughput screen to discovery 18 small molecule inhibitors of CES2. The inhibitors are validated by dose-response and counter-screening and 16 of these inhibitors demonstrate selectivity for CES2. These 16 inhibitors inhibit CES2 in cells, indicating they are cell permeable. Furthermore, all 16 inhibitors showed inhibition of Irinotecan conversion with purified enzyme. The top five inhibitors prohibit cell death mediated by Irinotecan upon pre-incubation in PDAC cells. Three of these inhibitors were determined to display a tight-binding mechanism of action with strong binding affinity
Blockade of IL1β and PD1 with Combination Chemotherapy Reduces Systemic Myeloid Suppression in Metastatic Pancreatic Cancer with Heterogeneous Effects in the Tumor.
No full textInnate inflammation promotes tumor development, although the role of innate inflammatory cytokines in established human tumors is unclear. Herein, we report clinical and translational results from a phase Ib trial testing whether IL1β blockade in human pancreatic cancer would alleviate myeloid immunosuppression and reveal antitumor T-cell responses to PD1 blockade. Patients with treatment-naïve advanced pancreatic ductal adenocarcinoma (n = 10) were treated with canakinumab, a high-affinity monoclonal human antiinterleukin-1β (IL1β), the PD1 blocking antibody spartalizumab, and gemcitabine/n(ab)paclitaxel. Analysis of paired peripheral blood from patients in the trial versus patients receiving multiagent chemotherapy showed a modest increase in HLA-DR+CD38+ activated CD8+ T cells and a decrease in circulating monocytic myeloid-derived suppressor cells (MDSC) by flow cytometry for patients in the trial but not in controls. Similarly, we used patient serum to differentiate monocytic MDSCs in vitro and showed that functional inhibition of T-cell proliferation was reduced when using on-treatment serum samples from patients in the trial but not when using serum from patients treated with chemotherapy alone. Within the tumor, we observed few changes in suppressive myeloid-cell populations or activated T cells as assessed by single-cell transcriptional profiling or multiplex immunofluorescence, although increases in CD8+ T cells suggest that improvements in the tumor immune microenvironment might be revealed by a larger study. Overall, the data indicate that exposure to PD1 and IL1β blockade induced a modest reactivation of peripheral CD8+ T cells and decreased circulating monocytic MDSCs; however, these changes did not lead to similarly uniform alterations in the tumor microenvironment
The seventh blind test of crystal structure prediction: structure generation methods.
No full textA seventh blind test of crystal structure prediction was organized by the Cambridge Crystallographic Data Centre featuring seven target systems of varying complexity: a silicon and iodine-containing molecule, a copper coordination complex, a near-rigid molecule, a cocrystal, a polymorphic small agrochemical, a highly flexible polymorphic drug candidate, and a polymorphic morpholine salt. In this first of two parts focusing on structure generation methods, many crystal structure prediction (CSP) methods performed well for the small but flexible agrochemical compound, successfully reproducing the experimentally observed crystal structures, while few groups were successful for the systems of higher complexity. A powder X-ray diffraction (PXRD) assisted exercise demonstrated the use of CSP in successfully determining a crystal structure from a low-quality PXRD pattern. The use of CSP in the prediction of likely cocrystal stoichiometry was also explored, demonstrating multiple possible approaches. Crystallographic disorder emerged as an important theme throughout the test as both a challenge for analysis and a major achievement where two groups blindly predicted the existence of disorder for the first time. Additionally, large-scale comparisons of the sets of predicted crystal structures also showed that some methods yield sets that largely contain the same crystal structures
Reference Standards for Potency Assays
No full textThis paper presents an overview of current practices common in the biopharmaceutical industry for the development of in-house reference standards for use in bioassays to determine the potency of biopharmaceutical preparations. It does not necessarily represent the views of every contributor nor of the organizations to which they are affiliated. It is intended to highlight some of the most common issues encountered in the development of reference standards and the ways in which some organizations address these. It should not be interpreted as an instruction to adopt a particular procedure or methodology. Relevant current regulatory guidelines should always be consulted and discussion with regulatory authorities undertaken when necessary
Pyridine N-oxide mediators for electrochemical benzylic C–H oxygenation
No full textAbstract: The electrooxidation of ubiquitous benzylic C(sp3)–H bonds offers a sustainable pathway to synthesize value-added carbonyl compounds. However, the lack of a versatile electrocatalytic system to accommodate the structural diversity of compounds containing benzylic C-H bonds hinders the practical implementation of electrochemical benzylic C-H oxidation technology. Herein, we designed and evaluated a library of electrochemical hydrogen atom transfer (HAT) mediators, derived from pyridine N-oxides, for the selective carbonylation of benzylic C(sp3)–H bonds. The O-H bond dissociation energy and oxidation potential can be finetuned by modifying the electronic properties of functional groups on the pyridine N-oxide, thus offering a facile way to adjust HAT characteristics to suit the target benzylic C-H bonds. A predictive neural network model was constructed based on experimental high-throughput screening data to map the quantitative structure-activity relationship (QSAR) for catalyst-substrate pairs. In addition, the practicality of the developed electrocatalytic system was demonstrated with a good substrate scope and scalability to achieve gram scale synthesis using a flow electrochemical reactor