Inter-batch Variability Research Articles

Variability of 7K and 11K SomaScan Plasma Proteomics Assays.

SomaScan is an aptamer-based proteomics assay designed for the simultaneous measurement of thousands of human proteins with a broad range of endogenous concentrations. The 7K SomaScan assay has recently been expanded into the new 11K version. Following up on our previous assessment of the 7K assay, here, we expand our work on technical replicates from donors enrolled in the Baltimore Longitudinal Study of Aging. By generating SomaScan data from a second batch of technical replicates in the 7K version as well as additional intra- and interplate replicate measurements in the new 11K version using the same donor samples, this work provides useful precision benchmarks for the SomaScan user community. Beyond updating our previous technical assessment of the 7K assay with increased statistics, here, we estimate interbatch variability, assess inter- and intraplate variability in the new 11K assay, compare the observed variability between the 7K and 11K assays (leveraging the use of overlapping pairs of technical replicates), and explore the potential effects of sample storage time (ranging from 2 to 30 years) in the assays' precision.

Untargeted Metabolomics for Inborn Errors of Metabolism: Development and Evaluation of a Sustainable Reference Material for Correcting Inter-Batch Variability.

Untargeted metabolomics has shown promise in expanding screening and diagnostic capabilities for inborn errors of metabolism (IEMs). However, inter-batch variability remains a major barrier to its implementation in the clinical laboratory, despite attempts to address this through normalization techniques. We have developed a sustainable, matrix-matched reference material (RM) using the iterative batch averaging method (IBAT) to correct inter-batch variability in liquid chromatography-high-resolution mass spectrometry-based untargeted metabolomics for IEM screening. The RM was created using pooled batches of remnant plasma specimens. The batch size, number of batch iterations per RM, and stability compared to a conventional pool of specimens were determined. The effectiveness of the RM for correcting inter-batch variability in routine screening was evaluated using plasma collected from a cohort of phenylketonuria (PKU) patients. The RM exhibited lower metabolite variability between iterations over time compared to metabolites from individual batches or individual specimens used for its creation. In addition, the mean variation across amino acid (n = 19) concentrations over 12 weeks was lower for the RM (CVtotal = 8.8%; range 4.7%-25.3%) compared to the specimen pool (CVtotal = 24.6%; range 9.0%-108.3%). When utilized in IEM screening, RM normalization minimized unwanted inter-batch variation and enabled the correct classification of 30 PKU patients analyzed 1 month apart from 146 non-PKU controls. Our RM minimizes inter-batch variability in untargeted metabolomics and demonstrated its potential for routine IEM screening in a cohort of PKU patients. It provides a practical and sustainable solution for data normalization in untargeted metabolomics for clinical laboratories.

An end-to-end deep learning method for mass spectrometry data analysis to reveal disease-specific metabolic profiles

Untargeted metabolomic analysis using mass spectrometry provides comprehensive metabolic profiling, but its medical application faces challenges of complex data processing, high inter-batch variability, and unidentified metabolites. Here, we present DeepMSProfiler, an explainable deep-learning-based method, enabling end-to-end analysis on raw metabolic signals with output of high accuracy and reliability. Using cross-hospital 859 human serum samples from lung adenocarcinoma, benign lung nodules, and healthy individuals, DeepMSProfiler successfully differentiates the metabolomic profiles of different groups (AUC 0.99) and detects early-stage lung adenocarcinoma (accuracy 0.961). Model flow and ablation experiments demonstrate that DeepMSProfiler overcomes inter-hospital variability and effects of unknown metabolites signals. Our ensemble strategy removes background-category phenomena in multi-classification deep-learning models, and the novel interpretability enables direct access to disease-related metabolite-protein networks. Further applying to lipid metabolomic data unveils correlations of important metabolites and proteins. Overall, DeepMSProfiler offers a straightforward and reliable method for disease diagnosis and mechanism discovery, enhancing its broad applicability.

Physicochemical profiling of nanomedicines using centrifugal field flow fractionation

Nanomedicines comprise multiple components, and particle density is considered an important property that regulates the biodistribution of administered nanomedicines. The density of nanoparticles is characterized by centrifugal methods, such as analytical ultracentrifugation. Particle size and distribution are key physicochemical and quality attributes of nanomedicines. In this study, we developed a novel profiling method applicable to liposomes and lipid nanoparticles (LNPs), based on particle size and density, using centrifugal field-flow fractionation (CF3). We evaluated the elution profiles of PEGylated liposomes of different sizes with various doxorubicin (DOX)-loading amounts using CF3. This method was applied to evaluate the drug release of DOX-loaded liposomes, intra- and inter-batch variability, reconstitution reproducibility of AmBisome®, and elution characteristics of LNPs in COVID-19 vaccines (Comirnaty® and SpikevaxTM). The data obtained in the present study underscore the significance of the proposed methodology and highlight the importance of profiling and characterizing liposomes and LNPs using CF3 fractograms and a multi-angle light-scattering detector.

Dissimilar ferric derisomaltose formulations – In vitro comparisons between an originator and its intended similars

BackgroundThe complex nature of intravenous (IV) iron formulations makes manufacturing and characterising similars challenging. This study examined whether simple in vitro tests can distinguish the high-dose IV iron formulation, Monofer® (ferric derisomaltose [FDI]), from the first intended copies of FDI, Rapifer® (FDI intended similar A [FDIIS-A]) and Tosiron® (FDI intended similar B [FDIIS-B]), approved in India and Pakistan, respectively. Neither intended similar is available in Europe or the United States. MethodsIron content, pH, density, non-volatile residue, carbohydrate content, molecular weight distribution, complex robustness (measured using acid hydrolysis half-life [t½]) and free (dialysable) iron content were examined. Mean results from three batches of FDIIS-A were compared with mean values calculated from three batches of Monofer®. Due to product withdrawal, only one batch of FDIIS-B was available for comparison with Monofer®. ResultsIron content was similar for all formulations (∼100 mg/mL). The chromatograms (obtained using gel permeation chromatography) of FDIIS-A and FDIIS-B differed from that of Monofer®. FDIIS-A was substantially less robust than Monofer® (t½: 15 h versus 40.3 h); t½ for FDIIS-B was not tested. Free iron content was substantially higher in FDIIS-A (0.091 % w/v) and FDIIS-B (1.0 % w/v) versus Monofer® (<0.003 % w/v). Where tested, remaining parameters varied between the formulations (insufficient sample quantities prevented all tests being conducted for all intended similars). For all tests, greater inter-batch variability was seen for FDIIS-A versus Monofer®. ConclusionsSimple in vitro tests demonstrated that, aside from total iron content, the first intended similars of FDI bear little resemblance to their originator drug. It is clear that the efficacy and safety profile of Monofer® cannot be extrapolated to the two intended similars. The results call for increased regulatory scrutiny of intended IV iron similars.

Manufacturing mesenchymal stromal cells in a microcarrier-microbioreactor platform can enhance cell yield and quality attributes: case study for acute respiratory distress syndrome

Mesenchymal stem and stromal cells (MSCs) hold potential to treat a broad range of clinical indications, but clinical translation has been limited to date due in part to challenges with batch-to-batch reproducibility of potential critical quality attributes (pCQAs) that can predict potency/efficacy. Here, we designed and implemented a microcarrier-microbioreactor approach to cell therapy manufacturing, specific to anchorage-dependent cells such as MSCs. We sought to assess whether increased control of the biochemical and biophysical environment had the potential to create product with consistent presentation and elevated expression of pCQAs relative to established manufacturing approaches in tissue culture polystyrene (TCPS) flasks. First, we evaluated total cell yield harvested from dissolvable, gelatin microcarriers within a microbioreactor cassette (Mobius Breez) or a flask control with matched initial cell seeding density and culture duration. Next, we identified 24 genes implicated in a therapeutic role for a specific motivating indication, acute respiratory distress syndrome (ARDS); expression of these genes served as our pCQAs for initial in vitro evaluation of product potency. We evaluated mRNA expression for three distinct donors to assess inter-donor repeatability, as well as for one donor in three distinct batches to assess within-donor, inter-batch variability. Finally, we assessed gene expression at the protein level for a subset of the panel to confirm successful translation. Our results indicated that MSCs expanded with this microcarrier-microbioreactor approach exhibited reasonable donor-to-donor repeatability and reliable batch-to-batch reproducibility of pCQAs. Interestingly, the baseline conditions of this microcarrier-microbioreactor approach also significantly improved expression of several key pCQAs at the gene and protein expression levels and reduced total media consumption relative to TCPS culture. This proof-of-concept study illustrates key benefits of this approach to therapeutic cell process development for MSCs and other anchorage-dependent cells that are candidates for cell therapies.

Development and application of a protocol for extractables profiling from central venous catheters in neonates

Peripherally inserted central catheters (PICC-lines) used in neonatology are made of thermoplastic polyurethane (TPU) or silicone. These materials usually contain substances that may leach into drug vehicles or blood. In this extractables study, we determined the optimal extraction conditions using TPU films containing defined amounts of butylhydroxytoluene (BHT) and then applied them on unused and explanted PICC-lines. Maceration and sonication tests were carried out with hexane, acetone and water as the extraction solvents. The analyses were performed using gas and liquid chromatography coupled with mass spectrometry detectors, as well as inductive coupled plasma optical emission spectroscopy to detect a wide range of extractables. We selected a limited list of substances to be sought from the usual adjuvants and monomers, related to their carcinogenic, mutagenic or reprotoxic properties and/or existence in endocrine disruptors lists. The TPU-film experiments showed that acetone was slightly better than hexane, and maceration better than sonication. When applied to PICC-lines, the extraction methods were almost similar but acetone was clearly better than hexane for TPU. From the 48 peaks initially observed in GC-MS, we ended up with 37 peaks to follow in TPU PICC-lines, among which were those of BHT and 4,4′-Methylenebis(cyclohexyl isocyanate) isomers. For silicone PICC-lines, out of 41 peaks initially observed in GC-MS, we followed 20 peaks, most of them being identified as cyclosiloxanes. Barium was the main inorganic element extracted for both PICC-lines. For TPU PICC-lines, the inter-batch variability was higher than for intra-batch, but in silicone devices both were similar. When compared to new PICC-lines, explanted TPU PICC-lines extracted peaks had a lower area under the curve (AUC), while the AUCs of the peaks were higher for the majority of silicone PICC-lines extract compounds. No identified substances were detected above their toxicological threshold, but isocyanates and cyclosiloxanes toxicity was mostly studied for other exposition routes than intravenous. The methods defined in this study were efficient in producing extractable profiles from both PICC-lines.

Exploring microbial diversity during the artisanal Salchichón production: Food safety in the consumer spotlight

A study was conducted to evaluate the microbiological quality, safety, and variability along the production chain of two artisanal salchichón factories in Andalusia. Aerobic mesophilic bacteria (AMB), total coliforms, coagulase-positive Staphylococcus spp. (CPS), lactic-acid bacteria, moulds and yeasts and the presence of bacterial pathogens were investigated in 3 batches of raw materials, finished products, food-contact surfaces (FCS) and indoor ambient air by using classical microbiological and PCR/PFGE techniques. Of the 171 samples analysed, high inter-batch variability in microbial loads was observed. The prevalence of Listeria monocytogenes and Staphylococcus aureus in samples was 7.01% and 4.67%, respectively. Raw meat and FCS were identified as the potential sources of contamination of the finished products, with 16.67% testing positive for L. monocytogenes and 10% for S. aureus. Diverse sources of L. monocytogenes and a wide range of high pulsotypes were identified in the smaller producer. Although the largest producer showed better hygiene control, L. monocytogenes was detected in salchichón samples with growth-supporting physicochemical values. Therefore, the implementation of food safety management systems, hygiene measures for raw materials and monitoring of hazards are encouraged. Finally, the findings of this study offer valuable and current empirical data that can be used in risk assessments.

Deciphering the growth responses and genotypic diversity of bioluminescent Photobacterium phosphoreum on chicken meat during aerobic refrigerated storage

The advent of high-throughput sequencing technologies in recent years has revealed the unexpected presence of genus Photobacterium within the chicken meat spoilage ecosystem. This study was undertaken to decipher the occurrence, the growth patterns and the genotypic biodiversity of Photobacterium phosphoreum on chicken breast fillets stored aerobically at 4 °C through conventional microbiological methods and molecular techniques. Samples were periodically cultured on marine broth agar (MA; supplemented with meat extract and vancomycin) for the enumeration of presumptive bioluminescent Photobacterium spp. In total, 90 bioluminescent bacteria were recovered from the initial (time of first appearance), middle and end stages of storage. Concomitantly, 95 total psychrotrophic/psychrophilic bacteria were isolated from the same medium to assess the presence and diversity of non-luminous photobacteria. Genetic diversity between bioluminescent isolates was assessed with two PCR-based DNA fingerprinting methods, i.e. RAPD and rep-PCR. Moreover, the characterization of selected bacterial isolates at the genus and/or species level was performed by sequencing of the 16S rRNA and/or gyrB gene. Bioluminescent bacteria were scarcely encountered in fresh samples at population levels of ca. 2.0 log CFU/g, whilst total psychrotrophic/psychrophilic bacteria were found at levels of ca. 4.4 log CFU/g. As time proceeded and close to shelf-life end, bioluminescent bacteria were encountered at higher populations, and were found at levels of 5.3 and 7.0 log CFU/g in samples from the second and third batch, respectively. In the first batch their presence was occasional and at levels up to 3.9 log CFU/g. Accordingly, total psychrotrophic/psychrophilic bacteria exceeded 8.4 log CFU/g at the end of storage, suggesting the possible underestimation of bioluminescent populations following the specific cultivation conditions. Sequence analysis assigned bioluminescent isolates to Photobacterium phosphoreum, while genetic fingerprinting revealed high intra-species variability. Respectively, total psychrotrophs/psychrophiles were assigned to genera Pseudomonas, Shewanella, Psychrobacter, Acinetobacter, Vibrio and Photobacterium. Non-luminous photobacteria were not identified within the psychrotrophs/psychrophiles. Results of the present study reveal the intra- and inter-batch variability on the occurrence and growth responses of P. phosphoreum and highlight its potential role in the chicken meat spoilage consortium.

Comparative Quality Analysis of Three Marketed Melatonin Containing Products in Spain for the Improvement of Sleep

Background: Exogenous melatonin may effectively improve the sleep-wake cycle and sleep quality in subjects with age or disease related decrease in endogenous melatonin production or response. A controlled release melatonin-based medication, Circadin® has been developed and licensed for insomnia; in addition to several food supplements available on the market. However, the quality and release profile of these melatonin containing products may vary.&#x0D; Aims: The current study aimed to compare three melatonin containing products, all of them marketed in Spain: a licensed medicine prolonged release tablet, Circadin® 2 mg, and two food supplements, Aquilea® 1,95mg and Oniria® 1,98 mg.&#x0D; Materials and Methods: Three batches of each product were analyzed for melatonin content, inter-batch variability and dissolution profile.&#x0D; Results: Our results confirm a large gap in quality control of the two melatonin containing food supplements, where Aquilea® and Oniria® showed poorer quality control as demonstrated by high variability between batches in melatonin content compared to Circadin®. Moreover, the extended release characteristics of Circadin® were evident in its profile, which exhibited an initial release of 70% at the 4-hour mark, followed by a consistent and sustained release lasting up to 8 hours.&#x0D; Conclusion: In terms of the dissolution profile, only Circadin® provides the prolonged release profile as indicated in the specifications, with proven efficacy and safety for the improvement of sleep-onset, sleep maintenance and quality throughout the whole night.

Intra- and inter-batch variability in raw pork challenge test studies and their consequences on model predictions: An intricate interplay between L. monocytogenes, the microbiome, and packaging atmosphere

The purpose of this study was to conduct challenge studies in raw pork by strictly following all aspects of the 2014 EURL technical guidance document for conducting shelf-life studies on Listeria monocytogenes. Growth potential was assessed on three batches of self-cut pork chops and one batch of in-house prepared pure minced pork without any additives in air and MAP (70 % O2/30% CO2) packaging. Pork chops did not support the growth of the pathogen throughout the shelf-life, given the specific conditions used in this study, with growth potential values of 0.28 and 0.46 log CFU/g, respectively, for both air and MAP. Substantial growth (>0.5 log CFU/g) was obtained in minced pork after investigating only one batch, with growth potential values of 1.69 and 0.80 log CFU/g, for air and MAP. However, both intra- and inter-batch variability for pork chops and intra-batch variability for minced pork was observed; with elevated growth being evened out by the way growth potential is calculated in the EURL 2014 document, leading to underestimations and posing a potential risk to public health. Maximum growth rate in minced pork at a constant temperature of 7 °C was estimated at μmax = 0.680 log CFU/day and μmax = 0.489 log CFU/day in air and MAP, respectively. Model predictions for the growth potential showed acceptable results for air-packed minced pork with better accuracy when the lag phase was implemented as indicated in the renewed protocol (CRL EU, 2021). In MAP, all models used, including the Combase Growth model and to a lesser extent the DMRI dynamic safety model, overestimate the growth potential probably due to a lack of integration of the changing CO2 levels in the packages. The predictive models used in this study do not adequately account for the dynamics in the raw pig matrix, which may have an inhibitory effect on the growth of L. monocytogenes, including interaction with the microbiome and CO2, and emphasize the importance of remaining critical of predictive model outcomes. In addition, the experimental intra- and inter-batch variability raise questions about the sense or nonsense of using predictive microbiology in these raw pork products.

Anabolic Peptide-Enriched Stealth Nanoliposomes for Effective Anti-Osteoporotic Therapy.

The objective of the present work was to develop PTH (1-34)-loaded stealth nanoliposomes (PTH-LPs) by employing the use of the Quality by Design (QbD) approach. Risk identification was carried out using the Ishikawa fishbone diagram. PTH-LPs were optimized using Box Behnken Design, a type of response surface methodology to examine the effect of independent variables on dependent variables such as particle size and percentage entrapment efficiency (%EE). Design space was generated for PTH-LPs to reduce interbatch variability during the formulation development process. Furthermore, a cytotoxicity assay, cell proliferation assay, calcium calorimetric assay, mineralized nodule formation, and cellular uptake assay were carried out on MG-63 osteoblast-like cells. The results obtained from these procedures demonstrated that lipid concentration had a significant positive impact on particle size and %EE, whereas cholesterol concentration showed a reduction in %EE. The particle size and %EE of optimized formulation were found to be 147.76 ± 2.14 nm and 69.18 ± 3.62%, respectively. Optimized PTH-LPs showed the sustained release profile of the drug. In vitro cell evaluation studies showed PTH-LPs have good biocompatibility with MG-63 cells. The cell proliferation study revealed that PTH-LPs induced osteoblast differentiation which improved the formation of mineralized nodules in MG-63 cells. The outcome of the present study conclusively demonstrated the potential of the QbD concept to build quality in PTH-LPs with improved osteoanabolic therapy in osteoporosis.

Sepax-2 cell processing device: a study assessing reproducibility of concentrating thawed hematopoietic progenitor cells

BackgroundAutologous hematopoietic progenitor cell (HPC) transplantation is currently the standard of care for a fraction of patients with newly diagnosed myelomas and relapsed or refractory lymphomas. After high-dose chemotherapy, cryopreserved HPC are either infused directly after bedside thawing or washed and concentrated before infusion. We previously reported on the comparability of washing/concentrating HPC post-thaw vs. infusion without manipulation in terms of hematopoietic engraftment, yet settled for the prior favoring cell debris and DMSO removal. For almost two decades, automation of this critical step of washing/concentrating cells has been feasible. As part of continuous process verification, we aim to evaluate reproducibility of this procedure by assessing intra-batch and inter-batch variability upon concentration of thawed HPC products using the Sepax 2 S-100 cell separation system.MethodsAutologous HPC collected from the same patient were thawed and washed either in two batches processed within a 3-4 h interval and immediately infused on the same day (intra-batch, n = 45), or in two batches on different days (inter-batch, n = 49) for those patients requiring 2 or more high-dose chemotherapy cycles. Quality attributes assessed were CD34+ cell recovery, viability and CD45+ viability; CFU assay was only performed for allogeneic grafts.ResultsIntra-batch and inter-batch median CD34+ cell recovery was comparable (75% vs. 73% and 77% vs. 77%, respectively). Similarly, intra-batch and inter-batch median CD45+ cell viability was comparable (79% vs. 80% and 79% vs. 78%, respectively). Bland-Altman analysis describing agreement between batches per patient revealed a bias close to 0%. Additionally, lower HPC recoveries noted in batch 1 were noted as well in batch 2, regardless of the CD34+ cell dose before cryopreservation, both intra- and inter-batch, suggesting that the quality of the collected product plays an important role in downstream recovery. Intrinsic (high mature and immature granulocyte content) and extrinsic (delay between apheresis and cryopreservation) variables of the collected product resulted in a significantly lower CD45+ viability and CD34+ cell recovery upon thawing/washing.ConclusionsAutomated post-thaw HPC concentration provides reproducible cell recoveries and viabilities between different batches. Implications of this work go beyond HPC to concentrate cell suspension/products during manufacturing of cell and gene therapy products.

Inter-batch variability of hemagglutinin content transiently expressed in &lt;i&gt;Nicotiana benthamiana&lt;/i&gt; plants grown under sole-source lighting and sunlight conditions before gene transfer

Inter-batch variability of hemagglutinin content transiently expressed in &lt;i&gt;Nicotiana benthamiana&lt;/i&gt; plants grown under sole-source lighting and sunlight conditions before gene transfer

Injectable Carrier-Free Hydrogel Dressing with Anti-Multidrug-Resistant Staphylococcus aureus and Anti-Inflammatory Capabilities for Accelerated Wound Healing.

Antibacterial hydrogels have gradually become a powerful weapon to treat bacterially infected wounds and accelerate healing. In this paper, we designed a small-molecule self-healing antibacterial hydrogel containing 100% drug-loaded benzyl 3β-amino-11-oxo-olean-12-en-30-oate (GN-Bn), which was governed by π-π stacking, hydrogen bonding, and van der Waals forces. Due to the carrier-free design concept, the problems of interbatch variability during sample preparation and carrier-related toxicity can be effectively avoided. Moreover, the GN-Bn hydrogel exhibited promising antibacterial activities against multidrug-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentration (MIC) of the GN-Bn hydrogel was 1.5625 nmol/mL, which was lower than those against clinical agents such as norfloxacin, penicillin, and tetracycline. This is attributed to its unique antibacterial mechanism that aims at killing bacteria or preventing their growth by regulating arginine biosynthesis and metabolism through both transcriptomic (RNA-seq) analysis and quantitative polymerase chain reaction (qPCR) analysis. In addition, the GN-Bn hydrogel can also inhibit proinflammatory cytokines (TNF-α, IL-1β, and IL-6) to promote wound healing. Collectively, the GN-Bn hydrogel elicited dual therapeutic effects on an MRSA-infected full-thickness skin wound model through its antibacterial and anti-inflammatory activities, which is attributed to the fact that the GN-Bn hydrogel has multiple advantages including sufficient mechanical stability, biocompatibility, and unique antibacterial mechanisms, making it significantly accelerate MRSA-infected full-thickness skin wound healing as a wound dressing. In a word, the GN-Bn antibacterial hydrogel dressing with an anti-inflammatory and antibacterial bifunctional material holds great potential in clinical application.

Occurrence of foodborne pathogens in Italian soft artisanal cheeses displaying different intra- and inter-batch variability of physicochemical and microbiological parameters.

Artisanal cheeses are produced in small-scale production plants, where the lack of full automation and control of environmental and processing parameters suggests a potential risk of microbial contamination. The aim of this study was to perform a longitudinal survey in an Italian artisanal factory producing a spreadable soft cheese with no rind to evaluate the inter- and intra-batch variability of physicochemical and microbial parameters on a total of 720 environmental and cheese samples. Specifically on cheese samples, the evaluation was additionally performed on physicochemical parameters. Cheese samples were additionally collected during 15 days of storage at constant temperatures of 2 and 8°C, as well as a dynamic profile of 2°C for 5 days and 8°C for 10 days. Furthermore, Enterobacteriaceae isolates were identified at species level to have a better knowledge of the environmental and cheese microbiota potentially harboring human pathogens. High inter-batch variability was observed for lactic acid bacteria (LAB) and total bacteria count (TBC) in cheese at the end of production but not for pH and water activity. A temperature of 8°C was associated with a significantly higher load of Enterobacteriaceae in cheeses belonging to batch 6 at the end of storage, and this temperature also corresponded with the highest increase in LAB and TBC loads over cheese shelf life. Results from generalized linear mixed models (GLMMs) indicated that drains in the warm room and the packaging area were associated with higher levels of TBC and Enterobacteriaceae in cheese. Regarding foodborne pathogens, no sample was positive for verotoxigenic Escherichia coli (VTEC) or Listeria monocytogenes, whereas six Staphylococcus aureus and one Salmonella pullorum isolates were collected in cheese samples during storage and processing, respectively. Regarding Enterobacteriaceae, 166 isolates were identified at species level from all batches, with most isolates belonging to Klebsiella oxytoca and pneumoniae, Enterobacter cloacae, Hafnia alvei, and Citrobacter freundii evidencing the need to focus on standardizing the microbial quality of cow milk and on hygienic procedures for cleaning and disinfection especially in warm and maturation rooms. Further studies should be performed to investigate the potential pathogenicity and antimicrobial resistance of the identified Enterobacteriaceae species in artisanal cheeses.

Spraying treatment of fresh-cut apples as a sustainable alternative to dipping for browning inhibition: a preliminary lab-scale study

Several fresh-cut apple snacks are available on the market, and their color is highly relevant for consumers. As apple slices brown very quickly after cutting, dipping them in an antioxidant solution for a few minutes is common to avoid this phenomenon. However, this procedure has some limitations. Using the same solution for several immersion cycles over several hours can lead to cross-contamination and microbial growth. In addition, osmotic exchange phenomena in the immersion tank lead to a reduction in the concentration of the active ingredients of the antioxidant solution over time, which requires continuous readjustment of the concentrations. These aspects, together with the large quantities of exhausted antioxidant solution to be disposed of, are limiting factors that drive the search for alternative and more sustainable methods than dipping. In this work, a comparison between a conventional dipping treatment of fresh-cut apples and spraying at increasing volume of antioxidant solution was performed. Color, considered as the quality reference parameter, was visually assessed (occurrance of dark spots) and CIE Lab colorimetric indices were measured over a five-day experimental period. Based on dark spots and the changes in brightness (Δ L*), hue angle (Δ h) and chroma (Δ C*), some of the spraying treatments applied were found to be comparable to dipping in controlling browning. For all treatments, high intra- and inter-batch variability and within slices of the same apple was observed concerning both instrumental data and dark spots development, indicating that further investigation is needed to validate this result and to apply this procedure at an industrial level.

Topical bioequivalence: Experimental and regulatory considerations following formulation complexity

Documenting topical bioequivalence can be an extremely complex process, which is intrinsically dependent on the formulation technological features. According to EMA guideline, for simple formulations, BE may be demonstrated by documenting the qualitative (Q1), quantitative (Q2), microstructure (Q3) and performance (Q4) equivalence. Nevertheless, when addressing complex semisolids, equivalence regarding local availability should also be demonstrated. The purpose of this study is to pursue this strategy using two opposite scenarios: a simple dimetindene maleate 1 mg/g gel formulation and a diclofenac diethylammonium 23.2 mg/g emulgel, representing a complex formulation. For both formulations, Q1/Q2 test (TP) and reference products (RP) were used. Rheology, in vitro release (IVRT) and in vitro permeation methods (IVPT) were developed and validated for both products.For the dimetindene formulation, equivalence pertaining to Q4 was established. However, high variability was observed for some rheology endpoints, especially for the different RP batches. Therefore, equivalence could not be established for Q3 as per EMA requirements. Can some rheology endpoints be waived? Can we establish reasonable criteria that are overall feasible for generic manufacturers and at the same time safe for the patient? An attempt was made to propose a wider acceptance range based on the inter-batch variability of the RP. For that, the rationale presented in the EMA guideline on bioequivalence for highly variable products was used.For the diclofenac formulation, Q3 equivalence was likewise not established. Q4 equivalence was only found for some batch combinations and when applying a wider acceptance criterion (75–133%). Furthermore, IVPT equivalence also failed to be demonstrated. Nevertheless, since the TP displays an equivalent pharmacokinetic profile compared to the RP, the observed Q3, Q4 and local availability differences are not expected to be clinically significant. This study draws attention to an effective search to determine the most appropriate strategy for assessing topical bioequivalence on a case-by-case basis.

Reproducibility of identical solid phantoms.

.SignificanceTissue-like solid phantoms with identical optical properties, known within tolerant uncertainty, are of crucial importance in diffuse optics for instrumentation assessment, interlaboratory comparison studies, industrial standards, and multicentric clinical trials.AimThe reproducibility in fabrication of homogeneous solid phantoms is focused based on spectra measurements by instrument comparisons grounded on the time-resolved diffuse optics.ApproachEpoxy-resin and silicone phantoms are considered as matrices and both employ three different instruments for time-resolved diffuse spectroscopy within the spectral range of 540 to 1100 nm. In particular, we fabricated two batches of five phantoms each in epoxy resin and silicone. Then, we evaluated the intra- and interbatch variability with respect to the instrument precision, by considering the coefficient of variation (CV) of absorption and reduced scattering coefficients.ResultsWe observed a similar precision for the three instruments, within 2% for repeated measurements on the same phantom. For epoxy-resin phantoms, the intra- and the interbatch variability reached the instrument precision limit, demonstrating a very good phantom reproducibility. For the silicone phantoms, we observed larger values for intra- and interbatch variability. In particular, at worst, for reduced scattering coefficient interbatch CV was about 5%.ConclusionsResults suggest that the fabrication of solid phantoms, especially considering epoxy-resin matrix, is highly reproducible, even if they come from different batch fabrications and are measured using different instruments.

Super-elasticity in vitro assessment of CuNiTi wires according to their Austenite finish temperature and the imposed displacement.

To assess the super-elasticity of CuNiTi wires (Ormco, Glendora, Calif) according to their Austenite finish temperature (Af) and to the imposed displacement. The secondary objective was to compare the wire dimensions with the stated measurements and to study interbatch variability. 10 types of CuNiTi wires (Ormco, Glendora, Calif) (n = 350) were investigated at 36 ± 1°C, with conventional brackets (Victory Series, 3M Unitek, Monrovia, Calif). Tensile test with coronoapical displacement ranging from 1 to 5 mm of the canine bracket was imposed. The wire dimensions were initially measured from two batches (n = 10). Dimensional heterogeneity varied by ± 2.00% compared to the manufacturer's data, and even up to 5.54% for 0.014-inch CuNiTi (P = .00069). However, all unloading forces were reproducible. In decreasing order, the forces delivered by a CuNiTi 27 were greater than those with CuNiTi 35 and 40. The super-elasticity was expressed only for displacements of 1 to 2 mm, at best up to 3 mm for 0.014-inch CuNiTi 27. The value of Af as well as the amount of imposed displacement seem to influence the expression of the super-elasticity of CuNiTi wires and the amount of corrected malocclusion. Among the tested wires, under these experimental conditions, 0.014-inch wire could be suitable as a first archwire. CuNiTi 35, therefore, seems to offer the best compromise among the force level, the expression of super-elasticity and the amount of malocclusion correction.