Expediting online liquid chromatography for real-time monitoring of product attributes to advance process analytical technology in downstream processing of biopharmaceuticals.

The application of Process Analytical Technology (PAT) principles for manufacturing of biotherapeutics proffers the prospect of ensuring consistent product quality along with increased productivity as well as substantial cost and time savings. Although this paradigm shift from a traditional, rather rigid manufacturing model to a more scientific, risk-based approach has been advocated by health authorities for almost two decades, the practical implementation of PAT in the biopharmaceutical industry is still limited by the lack of fit-for-purpose analytical methods. In this regard, most of the proposed spectroscopic techniques are sufficiently fast but exhibit deficiencies in terms of selectivity and sensitivity, while well-established offline methods, such as (ultra-)high-performance liquid chromatography, are generally considered as too slow for this task. To address these reservations, we introduce here a novel online Liquid Chromatography (LC) setup that was specifically designed to enable real-time monitoring of critical product quality attributes during time-sensitive purification operations in downstream processing. Using this online LC solution in combination with fast, purpose-built analytical methods, sampling cycle times between 1.30 and 2.35 min were achieved, without compromising on the ability to resolve and quantify the product variants of interest. The capabilities of our approach are ultimately assessed in three case studies, involving various biotherapeutic modalities, downstream processes and analytical chromatographic separation modes. Altogether, our results highlight the expansive opportunities of online LC based applications to serve as a PAT tool for biopharmaceutical manufacturing.

A novel clinician-delivered intervention to reduce fear of recurrence in breast cancer survivors: Results from a Phase I/II implementation study (CIFeR_2).

OBJECTIVE: Fear of cancer recurrence (FCR) is highly prevalent, however there is no formal training for clinicians to address FCR. A novel brief clinician intervention to help patients manage FCR (Clinician Intervention to Reduce Fear of Recurrence (CIFeR)) was shown to be feasible, acceptable, and reduced FCR in breast cancer patients in a pilot study. We now aim to explore the barriers and facilitators of implementing CIFeR within routine oncology practice in Australia. METHODS: This multicentre, single-arm Phase I/II implementation study recruited surgical, medical and radiation oncologists who treat women with early breast cancer. Participating clinicians completed online CIFeR training and were asked to use CIFeR for the next 6 months. Questionnaires were administered before (T0), immediately after (T1), then 3 (T2) and 6 months (T3) after training to assess confidence in addressing FCR and Proctor Implementation outcomes. The primary outcome was adoption at T2. Secondary outcomes were self-efficacy in FCR management, acceptability, feasibility, costs, barriers and facilitators of implementation. RESULTS: Fifty-two clinicians consented of whom 37 completed the CIFeR intervention training. Median age of participants was 41.5 (range 29-61), 73% were female and 51% were medical oncologists. The primary endpoint was met, with CIFeR adopted by 82%. Clinician intervention delivery took 7.4 min on average and was deemed acceptable, appropriate and feasible. Self-efficacy in managing FCR improved significantly across all domains (p < 0.001). Lack of time was the greatest barrier to routine CIFeR_2 implementation. CONCLUSIONS: A structured brief, low-cost clinician intervention to reduce FCR is useful, acceptable and improved self-efficacy with FCR management. Fear of cancer recurrence training should be incorporated into communication skills training of oncologists and surgeons. TRIAL REGISTRATION: Prospectively registered with the Australian New Zealand Clinical Trials Registry, ACTRN12621001697875. TRIAL SPONSOR: Chris O'Brien Lifehouse.

Protocol of an implementation study of a clinician intervention to reduce fear of recurrence in cancer survivors (CIFeR_2 implementation study).

BACKGROUND: Fear of cancer recurrence (FCR) affects 50-70% of cancer survivors with 30% reporting an unmet need for help with managing FCR. Patients indicate desire to discuss FCR with clinicians, however clinicians indicate discomfort with managing FCR and no formal educational interventions on how to discuss FCR or worry exists for oncology clinicians. Our team developed a novel clinician-driven brief education intervention to help patients manage FCR (the Clinician Intervention to Reduce Fear of Recurrence (CIFeR) intervention). In earlier work, we demonstrated the feasibility, acceptability, and efficacy of CIFeR in reducing FCR in breast cancer patients. We now aim to explore the barriers and facilitators to implementing this low-cost brief intervention within routine oncology practice in Australia. The primary objective is to assess the adoption of CIFeR in routine clinical practice. Secondary objectives are to identify the uptake and sustainability, perceived acceptability, feasibility, costs, barriers and facilitators of implementation of CIFeR in routine clinical practice, and to assess whether training in CIFeR increases clinicians' self-efficacy in managing FCR with their patients. METHODS: This multicentre, single-arm Phase I/II implementation study will recruit medical and radiation oncologists and oncology surgeons who treat women with early breast cancer. Participants will complete online CIFeR training. They will then be asked to use CIFeR with suitable patients for the next 6 months. Participants will complete questionnaires prior to, immediately after and 3 and 6 months after training to assess confidence addressing FCR, and 3 and 6 months after training to assess Proctor Implementation outcomes. At 6 months, they will also be asked to participate in a semi-structured telephone interview to elicit their feedback about barriers and facilitators to using CIFeR in routine clinical practice. DISCUSSION: This study will provide further data to support the routine use of an evidence-based, clinician-lead educational intervention to reduce FCR in breast cancer patients. Additionally, this study will identify any barriers and facilitators to implementing the CIFeR intervention in routine care and evidence for integration of FCR training into oncology communication skills education. TRIAL REGISTRATION: Prospectively registered with the Australian New Zealand Clinical Trials Registry, ACTRN12621001697875. TRIAL SPONSOR: Chris O'Brien Lifehouse. PROTOCOL VERSION: 2.6, Dated 28th February 2023.

A new approach for calculating microalgae culture growth based on an inhibitory effect of the surrounding biomass.

Ever since the potential of algae in biotechnology was recognized, models describing the growth of algae inside photobioreactors have been proposed. These models are the basis for the optimization of process conditions and reactor designs. Over the last few decades, models became more and more elaborate with the increase of computational capacity. Thus far, these models have been based on light attenuation due to the absorption and scattering effects of the biomass. This manuscript presents a new way of predicting the apparent growth inside photobioreactors using simple models for enzymatic kinetics to describe the reaction between photons and the photosynthetic unit. The proposed model utilizes an inhibition kinetic formula based on the surrounding biomass to describe the average growth rate of a culture, which is determined by the local light intensities inside the reactor. The result is a mixed-inhibition scheme with multiple inhibition sites. The parameters of the new kinetic equation are replaced by empirical regression functions to correlate their dependency on incident light intensity and reactor size. The calibrations of the parameters and the regression functions are based on the numerical solutions of the growth rate computed with a classical Type II model. As a final verification, we apply the new equation in predicting the growth behavior of three phototrophic organisms in reactors of three different sizes.

Concentrating Model Solutions and Fruit Juices Using CO2 Hydrate Technology and Its Quantitative Effect on Phenols, Carotenoids, Vitamin C and Betanin.

Fruits have an important economic impact in the context of plant-based food production. The consumption of fruit juices, mostly produced from concentrates, is particularly noteworthy. Conventional concentration methods do not always enable a sustainable and gentle concentration. The innovative gas hydrate technology addresses this point with its energy-saving, gentle character, and high concentration potential. In this study, the concentration of fruit juices and model solutions using CO2 hydrate technology was investigated. To find a suitable operating point for hydrate formation in the used bubble column, the hydrate formation in a water-sucrose model solution was evaluated at different pressure and temperature combinations (1, 3, 5 °C and 32.5, 37.5, 40 bar). The degrees of concentration indicate that the bubble column reactor operates best at 37.5 bar and 3 °C. To investigate the gentle processing character of the hydrate technology, its quantitative effects on vitamin C, betanin, polyphenols, and carotenoids were analyzed in the produced concentrates and hydrates via HPLC and UV/VIS spectrophotometry. The results for fruit juices and model solutions imply that all examined substances are accumulated in the concentrate, while only small amounts remain in the hydrate. These amounts can be related to an inefficient separation process.

Development of a Continuous Pulsed Electric Field (PEF) Vortex-Flow Chamber for Improved Treatment Homogeneity Based on Hydrodynamic Optimization.

Pulsed electric fields (PEF) treatment is an effective process for preservation of liquid products in food and biotechnology at reduced temperatures, by causing electroporation. It may contribute to increase retention of heat-labile constituents with similar or enhanced levels of microbial inactivation, compared to thermal processes. However, especially continuous PEF treatments suffer from inhomogeneous treatment conditions. Typically, electric field intensities are highest at the inner wall of the chamber, where the flow velocity of the treated product is lowest. Therefore, inhomogeneities of the electric field within the treatment chamber and associated inhomogeneous temperature fields emerge. For this reason, a specific treatment chamber was designed to obtain more homogeneous flow properties inside the treatment chamber and to reduce local temperature peaks, therefore increasing treatment homogeneity. This was accomplished by a divided inlet into the chamber, consequently generating a swirling flow (vortex). The influence of inlet angles on treatment homogeneity was studied (final values: radial angle α = 61°; axial angle ß = 98°), using computational fluid dynamics (CFD). For the final design, the vorticity, i.e., the intensity of the fluid rotation, was the lowest of the investigated values in the first treatment zone (1002.55 1/s), but could be maintained for the longest distance, therefore providing an increased mixing and most homogeneous treatment conditions. The new design was experimentally compared to a conventional co-linear setup, taking into account inactivation efficacy of Microbacterium lacticum as well as retention of heat-sensitive alkaline phosphatase (ALP). Results showed an increase in M. lacticum inactivation (maximum Δlog of 1.8 at pH 7 and 1.1 at pH 4) by the vortex configuration and more homogeneous treatment conditions, as visible by the simulated temperature fields. Therefore, the new setup can contribute to optimize PEF treatment conditions and to further extend PEF applications to currently challenging products.

Kinetic Modeling and Numerical Simulation as Tools to Scale Microalgae Cell Membrane Permeabilization by Means of Pulsed Electric Fields (PEF) From Lab to Pilot Plants.

Pulsed Electric Fields (PEF) is a promising technology for the gentle and energy efficient disruption of microalgae cells such as Chlorella vulgaris. The technology is based on the exposure of cells to a high voltage electric field, which causes the permeabilization of the cell membrane. Due to the dependency of the effective treatment conditions on the specific design of the treatment chamber, it is difficult to compare data obtained in different chambers or at different scales, e.g., lab or pilot scale. This problem can be overcome by the help of numerical simulation since it enables the accessibility to the local treatment conditions (electric field strength, temperature, flow field) inside a treatment chamber. To date, no kinetic models for the cell membrane permeabilization of microalgae are available what makes it difficult to decide if and in what extent local treatment conditions have an impact on the permeabilization. Therefore, a kinetic model for the perforation of microalgae cells of the species Chlorella vulgaris was developed in the present work. The model describes the fraction of perforated cells as a function of the electric field strength, the temperature and the treatment time by using data which were obtained in a milliliter scale batchwise treatment chamber. Thereafter, the model was implemented in a CFD simulation of a pilot-scale continuous treatment chamber with colinear electrode arrangement. The numerical results were compared to experimental measurements of cell permeabilization in a similar continuous treatment chamber. The predicted values and the experimental data agree reasonably well what demonstrates the validity of the proposed model. Therefore, it can be applied to any possible treatment chamber geometry and can be used as a tool for scaling cell permeabilization of microalgae by means of PEF from lab to pilot scale. The present work provides the first contribution showing the applicability of kinetic modeling and numerical simulation for designing PEF processes for the purpose of biorefining microalgae biomass. This can help to develop new processes and to reduce the costs for the development of new treatment chamber designs.

New lattice Boltzmann method for the simulation of three-dimensional radiation transfer in turbid media.

Based on the kinetic theory of photons, a new lattice Boltzmann method for the simulation of 3D radiation transport is presented. The method was successfully validated with Monte Carlo simulations of radiation transport in optical thick absorbing and non-absorbing turbid media containing either isotropic or anisotropic scatterers. Moreover, for the approximation of Mie-scattering, a new iterative algebraic approach for the discretization of the scattering phase function was developed, ensuring full conservation of energy and asymmetry after discretization. It was found that the main error sources of the method are caused by linearization and ray effects and suggestions for further improvement of the method are made.