Investigation of the Electrokinetic Potential of Granules and Optimization of the Pelletization Method Using the Quality by Design Approach.

The preparation of pellets using a high-shear granulator in a rapid single-step is considered a good economic alternative to the extrusion spheronization process. As process parameters and material attributes greatly affect pellet qualities, successful process optimization plays a vital role in producing pellet dosage forms with the required critical quality attributes. This study was aimed at the development and optimization of the pelletization technique with the Pro-CepT granulator. According to the quality by design (QbD) and screening design results, chopper speed, the volume of the granulating liquid, binder amount, and impeller speed were selected as the highest risk variables for a two-level full factorial design and central composite design, which were applied to the formula of microcrystalline cellulose, mannitol, and with a binding aqueous polyvinylpyrrolidone solution. The design space was estimated based on physical response results, including the total yield of the required size, hardness, and aspect ratio. The optimized point was tested with two different types of active ingredients. Amlodipine and hydrochlorothiazide were selected as model drugs and were loaded into an optimized formulation. The kinetics of the release of the active agent was examined and found that the results show a correlation with the electrokinetic potential because amlodipine besylate can be adsorbed on the surface of the MCC, while hydrochlorothiazide less so; therefore, in this case, the release of the active agent increases. The research results revealed no significant differences between plain and model drug pellets, except for hydrochlorothiazide yield percent, in addition to acceptable content uniformity and dissolution enhancement.

Automated radiosynthesis of mGluR5 PET tracer [18F]FPEB from aryl-chloro precursor and validation for clinical application.

The radioligand [18F]FPEB, used for PET imaging of the brain's metabotropic glutamate receptor subtype 5 (mGluR5), undergoes a thorough validation process to ensure its safety, efficacy, and quality for clinical use. The process starts by optimizing the synthesis of [18F]FPEB to achieve high radiochemical yield and purity. This study focuses on optimizing the radiolabeling process using an aryl-chloro precursor and validating the GMP production for clinical applications. Fully automated radiolabeling was achieved via one-step nucleophilic substitution reaction. [18F]FPEB was produced and isolated in high radioactivity and radiochemical purity. Throughout the validation process, thorough quality control measures are implemented. Radiopharmaceutical batch release criteria are established, including testing for physical appearance, filter integrity, pH, radiochemical purity, molar activity, radiochemical identity, chemical impurity, structural identity, stability, residual solvent, sterility, and endotoxin levels. In conclusion, the validation of [18F]FPEB involved a comprehensive process of synthesis optimization, quality control, which ensure the safety, efficacy, and quality of [18F]FPEB, enabling its reliable use in clinical PET. Here, we successfully radiolabeled and validated [18F]FPEB using aryl-chloro precursor according to GMP production for clinical application.

Biomimetic leaves with immobilized catalase for machine learning-enabled validating fresh produce sanitation processes.

Washing and sanitation are vital steps during the postharvest processing of fresh produce to reduce the microbial load on the produce surface. Although current process control and validation tools effectively predict sanitizer concentrations in wash water, they have significant limitations in assessing sanitizer effectiveness for reducing microbial counts on produce surfaces. These challenges highlight the urgent need to improve the validation of sanitation processes, especially considering the presence of dynamic organic contaminants and complex surface topographies. This study aims to provide the fresh produce industry with a novel, reliable, and highly accurate method for validating the sanitation efficacy on the produce surface. Our results demonstrate the feasibility of using a food-grade, catalase (CAT)-immobilized biomimetic leaf in combination with vibrational spectroscopy and machine learning to predict microbial inactivation on microgreen surfaces. This was tested using two sanitizers: sodium hypochlorite (NaClO) and hydrogen peroxide (H2O2). The developed CAT-immobilized leaf-replicated PDMS (CAT@L-PDMS) effectively mimics the microscale topographies and bacterial distribution on the leaf surface. Alterations in the FTIR spectra of CAT@L-PDMS, following simulated sanitation processes, indicate chemical changes due to CAT oxidation induced by NaClO or H2O2 treatments, facilitating the subsequent machine learning modeling. Among the five algorithms tested, the competitive adaptive reweighted sampling partial least squares discriminant analysis (CARS-PLSDA) algorithm was the most effective for classifying the inactivation efficacy of E. coli on microgreen leaf surfaces. It predicted bacterial reduction on microgreen surfaces with 100% accuracy in both training and prediction sets for NaClO, and 95% in the training set and 86% in the prediction set for H2O2. This approach can improve the validation of fresh produce sanitation processes and pave the way for future research.

Roadmap for Drug Product Development and Manufacturing of Biologics.

Therapeutic biology encompasses different modalities, and their manufacturing processes may be vastly different. However, there are many similarities that run across the different modalities during the drug product (DP) development process and manufacturing. Similarities include the need for Quality Target Product Profile (QTTP), analytical development, formulation development, container/closure studies, drug product process development, manufacturing and technical requirements set out by numerous regulatory documents such as the FDA, EMA, and ICH for pharmaceuticals for human use and other country specific requirements. While there is a plethora of knowledge on studies needed for development of a drug product, there is no specific guidance set out in a phase dependent manner delineating what studies should be completed in alignment with the different phases of clinical development from pre-clinical through commercialization. Because of this reason, we assembled a high-level drug product development and manufacturing roadmap. The roadmap is applicable across the different modalities with the intention of providing a unified framework from early phase development to commercialization of biologic drug products.

A comprehensive assessment of the applicability of RoboColumn as a chromatography scale-down model for use in biopharmaceutical process validation.

High-throughput process development has become a standard practice in the biopharmaceutical industry to enable time, cost, and material savings. In downstream biopharmaceutical process development, miniaturized, parallelized chromatography columns, known as RoboColumn, have become the standard for process development, as RoboColumn have shown generally comparable performance to bench and manufacturing scale columns. However, RoboColumn have yet to be widely implemented in process validation and characterization, where many multifactor experiments are typically executed, and there is a strong value proposition for performing high-throughput experiments. The hesitancy to utilize RoboColumn in process validation arises from scale differences that result in exacerbated peak broadening at RoboColumn scale relative to traditional bench or manufacturing scales. Thus, to support reliable application of RoboColumn in process validation, the present study provides a comprehensive investigation to understand how scale differences affect chromatographic performance by comparing RoboColumn, bench, and manufacturing scales using seven different production processes covering three different antibody formats, five different resin types, and three chromatographic modes of operation. RoboColumn chromatographic performance was compared at target and off-target conditions to emulate scale-down model qualification and multifactor studies, respectively. RoboColumn demonstrated good comparability at both target and off-target process conditions. To further demonstrate an understanding of comparability, a study was performed to show a rare case in which product quality offsets may occur as a result RoboColumn scale differences. By showing scale comparability and an understanding of potential offsets, this work demonstrates that RoboColumn can be used in any stage of process development, including process validation and characterization.

SeDeM as a Tool to Validate Drug Substance Manufacturing Processes and Assess Scalability and Suitability for Direct Compression: Supplier Screening.

During the development of an oral solid form of a drug substance, a thorough understanding of the critical material attributes is necessary, as the physical properties of the active pharmaceutical ingredient (API) can profoundly influence the drug product's manufacturability, critical quality attributes, and bioavailability. The objective of this study was to validate the manufacturing process of the drug Linezolid from three different sources at both the pilot and industrial scale and to identify differences in critical material attributes between the API manufacturers. Furthermore, the scalability factor between the pilot and industrial scale and the suitability of a process for direct compression were also evaluated. In the present study, the different sources of API were characterized by SeDeM methodology, particle size distribution, and scanning electron microscopy determinations. The statistical analysis revealed that no statistically significant differences were found for any of the parameters under study for the same API source analyzed on both scales. On the other hand, for most of the parameters evaluated, statistical differences were observed between the different sources. It was concluded that SeDeM was able to successfully validate the API manufacturing process, assess scalability, and distinguish between sources. Therefore, it could be highly valuable in the formulation phase to select the best API source.

Validação de instrumento para Registro do Processo de Enfermagem no atendimento pré-hospitalar móvel de urgência / Validación de un instrumento para el Registro del Proceso de Enfermería en la atención móvil de emergencia prehospitalaria / Validation of an instrument for the Nursing Process Record in prehospital mobile emergency care

Objetivo: validar a aparência e o conteúdo de um instrumento para Registro do Processo de Enfermagem no Serviço de Atendimento Móvel de Urgência. Método: estudo de abordagem quantitativa, em que o instrumento foi submetido à validação de aparência e conteúdo por comitê de 21 experts na área de atendimento pré-hospitalar móvel de urgência nacionalmente. Um Índice de Validade de Conteúdo (IVC) igual ou superior a 0,80 estabeleceu a validação. Resultados: obteve-se um IVC de 0,94. Apenas o item facilidade de leitura, relacionado à aparência, teve um índice abaixo do estabelecido. Foi possível avaliar as 99 intervenções de Enfermagem elencadas. Conclusão: o instrumento para Registro do Processo de Enfermagem no Serviço de Atendimento Móvel de Urgência foi considerado válido e pode possibilitar a documentação manual da prática do enfermeiro neste cenário.

Objective: face and content validation of an instrument for Recording the Nursing Process in the Mobile Emergency Care Service. Method: quantitative study of face and content validation of the instrument by a committee of 21 experts in the field of prehospital mobile emergency care nationwide. A Content Validity Index (CVI) equal to or greater than 0.80 determined validation. Results: a CVI of 0.94 was obtained. Only the item ease of reading, related to appearance, had an index below the established. It was possible to evaluate the 99 nursing interventions listed. Conclusion: the instrument for the Nursing Process Record in the Mobile Emergency Care Service was considered valid and can enable the manual documentation of nursing practice in this setting.

Objetivo: validación de apariencia y contenido de un instrumento para el Registro del Proceso de Enfermería en el Servicio de Atención Móvil de Emergencia. Método: estudio cuantitativo de validación facial y de contenido del instrumento por un comité de 21 expertos en el campo de la atención prehospitalaria móvil de emergencia a nivel nacional. Un Índice de Validez de Contenido (IVC) igual o superior a 0,80 determinó la validación. Resultados: se obtuvo un IVC de 0,94. Únicamente el ítem facilidad de lectura, relacionado con la apariencia, presentó índice por debajo de lo establecido. Fue posible evaluar las 99 intervenciones de enfermería listadas. Conclusión: el instrumento para el Registro del Proceso de Enfermería en el Servicio de Atención Móvil de Emergencia se consideró válido y puede posibilitar la documentación manual de la práctica de enfermería en este escenario.

Holistic Design of Experiments Using an Integrated Process Model.

Statistical experimental designs such as factorial, optimal, or definitive screening designs represent the state of the art in biopharmaceutical process characterization. However, such methods alone do not leverage the fact that processes operate as a mutual interplay of multiple steps. Instead, they aim to investigate only one process step at a time. Here, we want to develop a new experimental design method that seeks to gain information about final product quality, placing the right type of run at the right unit operation. This is done by minimizing the simulated out-of-specification rate of an integrated process model comprised of a chain of regression models that map process parameters to critical quality attributes for each unit operation. Unit operation models are connected by passing their response to the next unit operation model as a load parameter, as is done in real-world manufacturing processes. The proposed holistic DoE (hDoE) method is benchmarked against standard process characterization approaches in a set of in silico simulation studies where data are generated by different ground truth processes to illustrate the validity over a range of scenarios. Results show that the hDoE approach leads to a >50% decrease in experiments, even for simple cases, and, at the same time, achieves the main goal of process development, validation, and manufacturing to consistently deliver product quality.

Specification-driven acceptance criteria for validation of biopharmaceutical processes.

Intermediate acceptance criteria are the foundation for developing control strategies in process validation stage 1 in the pharmaceutical industry. At drug substance or product level such intermediate acceptance criteria for quality are available and referred to as specification limits. However, it often remains a challenge to define acceptance criteria for intermediate process steps. Available guidelines underpin the importance of intermediate acceptance criteria, because they are an integral part for setting up a control strategy for the manufacturing process. The guidelines recommend to base the definition of acceptance criteria on the entirety of process knowledge. Nevertheless, the guidelines remain unclear on how to derive such limits. Within this contribution we aim to present a sound data science methodology for the definition of intermediate acceptance criteria by putting the guidelines recommendations into practice (ICH Q6B, 1999). By using an integrated process model approach, we leverage manufacturing data and experimental data from small scale to derive intermediate acceptance criteria. The novelty of this approach is that the acceptance criteria are based on pre-defined out-of-specification probabilities, while also considering manufacturing variability in process parameters. In a case study we compare this methodology to a conventional +/- 3 standard deviations (3SD) approach and demonstrate that the presented methodology is superior to conventional approaches and provides a solid line of reasoning for justifying them in audits and regulatory submission.

Example for process validation in biobanking: Fit for purpose testing of a cryopreservation method without isopentane.

Background: The freezing process of tissue samples is crucial for the preservation of morphological and molecular features. Several biobanking guidelines describe freezing techniques for optimal outcomes. As the Vetbiobank standard freezing protocol does not comply with those recommendations in detail, a process validation was performed to demonstrate that samples are suitable for downstream applications. Here we give a formal example of a process validation in the biobanking setting, as required by the biobanking guideline ISO 20387 (2018). Methods: Three different freezing protocols, freezing in liquid nitrogen, freezing via isopentane precooled on dry ice and freezing via liquid nitrogen vapor, were assessed based on morphological integrity of mouse liver and muscle tissue samples. Samples were either frozen in cryotubes (without Optimal Cutting Temperature compound, OCT) or in cryomolds (with OCT). The protocol providing the best results was validated for reproducibility and robustness in terms of defined acceptance criteria for morphological evaluability, A260/A280 ratio, and RNA integrity number values (RIN). In addition, performance tests were run by gene expression analyzes of selected, tissue specific biomarkers to confirm that processed samples are fit for purpose. Results: From the three applied freezing protocols, freezing in liquid nitrogen generated best results. Reproducibility acceptance criteria were met for both, morphological integrity and RNA quality. The freezing method was robust for the tested tissue types and the application of OCT, with exception of liver tissue, where it led to a significant decrease of the RIN value. Gene expression analyzes showed good comparability of results regardless of the applied freezing method. Conclusion: Freezing of tissue samples in liquid nitrogen provides samples of adequate quality for subsequent RNA investigations. A negative impact of OCT on the RIN value of liver samples was observed, which was independent from the applied freezing protocol and showed no impact on subsequent gene expression analysis.

FDA Warning Letters: A Retrospective Analysis of Letters Issued to Pharmaceutical Companies from 2010-2020.

Purpose: To launch a pharmaceutical product in the US market, approval from the FDA is required. Pharmaceutical companies undergo FDA pre-approval inspection (PAI for small molecule products) or pre-license approval (PLI for biological products) at their manufacturing sites (including contract development and manufacturing organization, testing laboratories, and packaging labelling facilities) prior to approval. After the products are approved by the FDA, surveillance inspections are performed by the FDA which are risk based as which company and which site will be inspected. The present study examines the causes of warning letters issued by the Center for Drug Evaluation and Research (CDER), FDA to the pharmaceutical companies after post-approval inspections. Methods: Warning letters issued from the time period 2010 to 2020 were obtained from the FDA website, and information about date of issuance, company, and type of violations was extracted for the study. Results: Poor compliance to CGMP and misbranding were the most common reasons for the warning letters. Detailed analysis of CGMP warning letters elucidated three major types of violations, namely deficiencies in process validation, documentation practices (data integrity), and quality control corresponding to 26%, 21%, and 15% warning letters, respectively. Conclusion: Review of the analysed letters demonstrates that the FDA's major concern is over CGMP compliance. To avoid these warning letters, pharmaceutical manufacturers need to improve their quality compliance and focus on creating effective quality management systems that govern the entire manufacturing process, quality control, employee training, and documentation practice. Companies should develop an internal compliance check list and also be ready for corrective measures as and when required. Supplementary Information: The online version contains supplementary material available at 10.1007/s12247-022-09678-2.

Thermal inactivation kinetics of Salmonella enterica and Enterococcus faecium NRRL B-2354 as a function of temperature and water activity in fine ground black pepper.

Fine ground black pepper generally consumed as a seasoning without any further processing has been associated with Salmonella enterica outbreaks. Thermal inactivation kinetics data is necessary to develop a pasteurization process for fine ground black pepper. This study investigates the influence of temperature and water activity on thermal inactivation kinetics of Salmonella in fine ground black pepper. It also assesses the suitability of Enterococcus faecium as a surrogate for Salmonella. Fine ground black pepper of varying water activities, aw (0.40, 0.55, 0.70) was subjected to isothermal treatments at different temperatures (65-80 °C) for five equidistant time points with intervals ranging from 18 s to 250 min. The survival data were used to fit two primary models (log-linear and Weibull) and two secondary models (response surface and Modified Bigelow). Results indicated that among the two primary models, the Weibull model explained the thermal inactivation kinetics better with lower RMSE (0.24 - 0.56 log CFU/g) and AICc values at all aw and temperatures. Water activity and treatment temperature significantly enhanced the thermal inactivation of Salmonella. E. faecium NRRL B-2354 was found to be a suitable surrogate for Salmonella in fine ground black pepper at all tested treatment conditions. The developed modified Bigelow model based on the Weibull model could be applied to predict the inactivation kinetics of Salmonella in black pepper and would benefit the spice industry in identifying process parameters for thermal pasteurization of fine ground black pepper.

Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product.

BACKGROUND: A growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). RESULTS: The GMP-compliant protocol defined in this work allows at least 4.11 × 109 Treg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function. CONCLUSIONS: These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases.

Survival of Escherichia coli O157:H7 during Moderate Temperature Dehydration of Plant-Based Foods.

The effect of moderate-temperature (≤60 °C) dehydration of plant-based foods on pathogen inactivation is unknown. Here, we model the reduction of E. coli O157:H7 as a function of product-matrix, aw, and temperature under isothermal conditions. Apple, kale, and tofu were each adjusted to aw 0.90, 0.95, or 0.99 and inoculated with an E. coli O157:H7 cocktail, followed by isothermal treatment at 49, 54.5, or 60.0 °C. The decimal reduction time, or D-value, is the time required at a given temperature to achieve a 1 log reduction in the target microorganism. Modified Bigelow-type models were developed to determine D-values which varied by product type and aw level, ranging from 3.0-6.7, 19.3-55.3, and 45.9-257.4 min. The relative impact of aw was product dependent and appeared to have a non-linear impact on D-values. The root mean squared errors of the isothermal-based models ranged from 0.75 to 1.54 log CFU/g. Second, we performed dynamic drying experiments. While the isothermal results suggested significant microbial inactivation might be achieved, the dehydrator studies showed that the combination of low product temperature and decreasing aw in the pilot-scale system provided minimal inactivation. Pilot-scale drying at 60 °C only achieved reductions of 3.1 ± 0.8 log in kale and 0.67 ± 0.66 log in apple after 8 h, and 0.69 ± 0.67 log in tofu after 24 h. This illustrates the potential limitations of dehydration at ≤60 °C as a microbial kill step.

Review: Continuous Manufacturing of Small Molecule Solid Oral Dosage Forms.

Continuous manufacturing (CM) is defined as a process in which the input material(s) are continuously fed into and transformed, and the processed output materials are continuously removed from the system. CM can be considered as matching the FDA's so-called 'Desired State' of pharmaceutical manufacturing in the twenty-first century as discussed in their 2004 publication on 'Innovation and Continuous Improvement in Pharmaceutical Manufacturing'. Yet, focused attention on CM did not really start until 2014, and the first product manufactured by CM was only approved in 2015. This review describes some of the benefits and challenges of introducing a CM process with a particular focus on small molecule solid oral dosage forms. The review is a useful introduction for individuals wishing to learn more about CM.

Guidance on validation of lethal control measures for foodborne pathogens in foods.

Food manufacturers are required to obtain scientific and technical evidence that a control measure or combination of control measures is capable of reducing a significant hazard to an acceptable level that does not pose a public health risk under normal conditions of distribution and storage. A validation study provides evidence that a control measure is capable of controlling the identified hazard under a worst-case scenario for process and product parameters tested. It also defines the critical parameters that must be controlled, monitored, and verified during processing. This review document is intended as guidance for the food industry to support appropriate validation studies, and aims to limit methodological discrepancies in validation studies that can occur among food safety professionals, consultants, and third-party laboratories. The document describes product and process factors that are essential when designing a validation study, and gives selection criteria for identifying an appropriate target pathogen or surrogate organism for a food product and process validation. Guidance is provided for approaches to evaluate available microbiological data for the target pathogen or surrogate organism in the product type of interest that can serve as part of the weight of evidence to support a validation study. The document intends to help food manufacturers, processors, and food safety professionals to better understand, plan, and perform validation studies by offering an overview of the choices and key technical elements of a validation plan, the necessary preparations including assembling the validation team and establishing prerequisite programs, and the elements of a validation report.

Process Humidity Affects Salmonella Lethality at the Surface and Core of Impingement-Cooked Meat and Poultry Products.

ABSTRACT: Recent revisions to U.S. Department of Agriculture, Food Safety and Inspection Service (FSIS) compliance and safe harbor guidelines for ready-to-eat meat and poultry products addressed process humidity requirements. Given the lack of prior data for impingement-cooked products, the present study was conducted to evaluate the impact of process humidity on Salmonella lethality at the product core and surface and compliance of the results with FSIS lethality performance standards. Whole muscle beef strips, ground beef patties, whole muscle chicken breast fillets, and breaded ground chicken patties were inoculated with an eight-serovar cocktail of Salmonella. Beef and chicken samples were cooked in a pilot-scale moist-air impingement oven to a core temperature of 70.0 and 72.8°C, respectively, immediately quenched in liquid nitrogen, and dissected to obtain core and surface samples. Variables included oven temperature (218 and 232°C), air velocity (0.7 and 2.8 m/s), and oven humidity (0.7, 15, 30, or 70% moisture by volume [%, v/v]). Additional treatments were performed to examine the impact of supplemental critical control processes such as increased endpoint temperature, postoven carryover time, and pre- or postoven steam treatments. Salmonella reductions of >7 log units were reliably achieved in chicken patties regardless of the processing variables; however, none of the treatments reliably ensured >6.5-log reductions of Salmonella in ground beef. A majority of whole-muscle samples failed to meet the required performance lethality when processed at 0.7% (v/v) humidity; however, Salmonella inactivation was significantly improved (P < 0.05) at oven humidities of ≥30% (v/v). Dry oven conditions achieved greater Salmonella lethality at the core than at the surface for multiple products (P < 0.05). The efficacies of minimal and supplemental critical controls were dependent on product, process, and humidity (P < 0.05). Overall, process humidity and product variability should be considered in regulatory requirements and process validations.

Evaluation of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella in ground black pepper at different water activities.

Thermal inactivation kinetics of Salmonella in low moisture foods are necessary for developing proper thermal processing parameters for pasteurization. The effect of water activity on thermal inactivation kinetics of Salmonella and Enterococcus faecium NRRL B-2354 in ground black pepper has not been studied previously. Identification of a suitable surrogate assists in conducting in-plant process validations. Ground black pepper was inoculated with a 5-serotype Salmonella cocktail or E. faecium NRRL B-2354, equilibrated to water activities of 0.25, 0.45 or 0.65 in a humidity-controlled chamber, and isothermally treated at different temperatures. The survivor data were used for fitting the log-linear models to obtain the D and z-values of Salmonella and E. faecium in ground black pepper. Modified Bigelow models were developed to evaluate the effects of temperature and water activity on the thermal inactivation kinetics of Salmonella and E. faecium. Water activity and temperature showed significant negative effects on the thermal resistance of Salmonella and E. faecium in ground black pepper. For example, significantly higher D values of Salmonella were observed at water activity of 0.45 (D70°C = 20.5 min and D75°C = 7.8 min) compared to water activity of 0.65 (D70°C = 3.9 min and D75°C = 2.0 min). D-values of E. faecium were significantly higher than those of Salmonella at all three water activities, indicating that E. faecium is a suitable surrogate for Salmonella in thermal processing validation.

Impact of Preanalytical Factors During Histology Processing on Section Suitability for Digital Image Analysis.

Digital image analysis (DIA) is impacted by the quality of tissue staining. This study examined the influence of preanalytical variables-staining protocol design, reagent quality, section attributes, and instrumentation-on the performance of automated DIA software. Our hypotheses were that (1) staining intensity is impacted by subtle differences in protocol design, reagent quality, and section composition and that (2) identically programmed and loaded stainers will produce equivalent immunohistochemical (IHC) staining. We tested these propositions by using 1 hematoxylin and eosin stainer to process 13 formalin-fixed, paraffin-embedded (FFPE) mouse tissues and by using 3 identically programmed and loaded immunostainers to process 5 FFPE mouse tissues for 4 cell biomarkers. Digital images of stained sections acquired with a commercial whole slide scanner were analyzed by customizable algorithms incorporated into commercially available DIA software. Staining intensity as viewed qualitatively by an observer and/or quantitatively by DIA was affected by staining conditions and tissue attributes. Intrarun and inter-run IHC staining intensities were equivalent for each tissue when processed on a given stainer but varied measurably across stainers. Our data indicate that staining quality must be monitored for each method and stainer to ensure that preanalytical factors do not impact digital pathology data quality.

Evaluation of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella enterica in milk powders at different storage times and temperatures.

While the increase in thermal resistance of microorganisms at reduced water activity is demonstrated for low-moisture food products, the effect of storage time on the thermal resistance of microorganisms in low-moisture foods is not well established. As low-moisture foods are stored for long periods and are used as ingredients, cross-contamination can occur at any time period before the lethality step. Therefore, this study was designed to investigate the effect of storage time (30, 60, and 90 d) on the thermal resistance of Salmonella and Enterococcus faecium NRRL B-2354 in milk powders at a low water activity of 0.10 (conservative level). In this study, 2 milk powders, whole milk powder (WMP) and nonfat dry milk (NFDM), were inoculated with a 5-serotype Salmonella cocktail or E. faecium and equilibrated to a water activity of 0.10. The thermal resistance of Salmonella and E. faecium in WMP and NFDM were determined at different storage times (30, 60, and 90 d) at 85°C. The storage time had no effect on the thermal inactivation kinetics of Salmonella within 90 d of storage at 85°C. In the second part of this study, isothermal treatments were also conducted at higher temperatures (90 and 95°C) to evaluate the suitability of E. faecium as a surrogate for Salmonella in milk powders. The D-values of Salmonella in WMP with 30 d of storage at 85, 90, and 95°C were 7.98, 3.35, and 1.68 min. The corresponding values for E. faecium were 16.96, 7.90, and 4.16 min. Higher D-values of E. faecium indicates that it is a conservative surrogate. Similar results were found for NFDM. In general, D-values of both microorganisms are slightly higher in NFDM than WMP. Two primary models (log-linear and Weibull) were compared for their goodness-of-fit. The Weibull model was found to be more appropriate than the log-linear model. This study provides valuable information for establishing process validation for the pasteurization of milk powders.