Experimental and computational characterization of mass transfer in high turndown bioreactors

Single-use bioreactors (SUBs, or disposable bioreactors) are extensively used for the clinical and commercial production of biologics. Despite widespread application, minimal results have been reported utilizing the turndown ratio; an operation mode where the working range of the bioreactor can be expanded to include low fluid volumes. In this work, a systematic investigation into free surface mass transfer and cell growth in high turndown single-use bioreactors is presented. This approach, which combines experimental mass transfer measurements with numerical simulation, deconvolutes the combined effects of headspace mixing and the free surface convective mass transfer on cell growth. Under optimized conditions, mass transfer across the interface alone may be sufficient to satisfy oxygen demands of the cell culture. Within the context of high turndown bioreactors, this finding provides a counterpoint to traditional sparge-based bioreactor operational philosophy. Multiple monoclonal antibody-producing cell lines grown using this high turndown approach showed similar viable cell densities to those cells expanded using a traditional cell bag rocker. Furthermore, cells taken directly from the turndown expansion and placed into production showed identical growth characteristics to traditionally expanded cultures. Taken together, these results suggest that the Xcellerex SUB can be run at a 5:1 working volume as a seed to itself, with no need for system modifications, potentially simplifying preculture operations.     

Environmental sustainability of orthopedic devices produced with powder bed fusion

Additive manufacturing consists in melting metallic powders to produce objects from 3D data, layer upon layer. Its industrial applications range from automotive, biomedical (e.g., prosthetic implants for dentistry and orthopedics), aeronautics and others. This study uses life cycle assessment to evaluate the possible improvement in environmental performance of laser‐based powder bed fusion additive manufacturing systems on prosthetic device production. Environmental impacts due to manufacturing, use, and end of life of the designed solution were assessed. In addition, two powder production technologies, gas atomization (GA) and plasma atomization (PA), were compared in order to establish the most sustainable one. Production via traditional subtractive technologies and the additive manufacturing production were also compared. 3D building was found to have a significant environmental advantage compared to the traditional technology. The powder production process considerably influences on a damage point of view the additive manufacturing process; however, its impact can be mitigated if GA powders are employed.     

Source apportionment and health risk quantification for heavy metal sources in soils near aluminum-plastic manufacturing facilities in northeast China

Abstract

Identifying the source effect on heavy metals to human health risk is essential for devising and implementing restoration policies for polluted soils. For this purpose, eight heavy metals (As, Cd, Hg, Cr, Cu, Ni, Pb, and Zn) in soil profile samples (0–10, 10–20, 20–30, and 30–40?cm) collected in the area around aluminum-plastic manufacturing facilities (APMF) were determined. An absolute principal component score multiple linear regression (APCS-MLR) model supported by a health risk assessment (HRA) model was developed to determine the source apportionment of soil heavy metals and contribution rate of pollution sources to human health risk. Results showed significant accumulations of eight metals in the topsoil (0–20?cm), parent material, transportation, APMF, and agricultural practices were the four major contributing sources for heavy metals in soils, with average contribution percentages of 21.69%, 24.99%, 29.60%, and 14.25%, respectively. Carcinogenic risk factors for adults (1.23E-04) and children (1.32E-04) were found to be above the acceptable level (1E-06 to 1E-04). The health risk quantification results indicated that parent material, APMF, transportation, agricultural practices, and unidentified factors accounted for 55.76%, 14.48%, 12.09%, 10.13%, and 7.54% of the carcinogenic risk for children and adults. The adverse impacts of Cd, Zn, and Pb accumulations in soil coming from APMF activities were significant and need to receive more attention.     

Reduction of medium consumption in perfusion mammalian cell cultures using a perfusion rate equivalent concentrated nutrient feed

Media preparation for perfusion cell culture processes contributes significantly to operational costs and the footprint of continuous operations for therapeutic protein manufacturing. In this study, definitions are given for the use of a perfusion equivalent nutrient feed stream which, when used in combination with basal perfusion medium, supplements the culture with targeted compounds and increases the medium depth. Definitions to compare medium and feed depth are given in this article. Using a concentrated nutrient feed, a 1.8-fold medium consumption (MC) decrease and a 1.67-fold increase in volumetric productivity (PR) were achieved compared to the initial condition. Later, this strategy was used to push cell densities above 100 × 10⁶ cells/ml while using a perfusion rate below 2 RV/day. In this example, MC was also decreased 1.8-fold compared to the initial condition, but due to the higher cell density, PR was increased 3.1-fold and to an average PR value of 1.36 g L⁻¹ day⁻¹ during a short stable phase, and versus 0.46 g L⁻¹ day⁻¹ in the initial condition. Overall, the performance improvements were aligned with the given definitions. This multiple feeding strategy can be applied to gain some flexibility during process development and also in a manufacturing set-up to enable better control on nutrient addition.     

Dissolvable Gelatin-Based Microcarriers Generated through Droplet Microfluidics for Expansion and Culture of Mesenchymal Stromal Cells

Microcarriers are synthetic particles used in bioreactor-based cell manufacturing of anchorage-dependent cells to promote proliferation at efficient physical volumes, mainly by increasing the surface area-to-volume ratio. Mesenchymal stromal cells (MSCs) are adherent cells that are used for numerous clinical trials of autologous and allogeneic cell therapy, thus requiring avenues for large-scale cell production at efficiently low volumes and cost. Here, a dissolvable gelatin-based microcarrier is developed for MSC expansion. This novel microcarrier shows comparable cell attachment efficiency and proliferation rate when compared to several commercial microcarriers, but with higher harvesting yield due to the direct dissolution of microcarrier particles and thus reduced cell loss at the cell harvesting step. Furthermore, gene expression and in vitro differentiation suggest that MSCs cultured on gelatin microcarriers maintain trilineage differentiation with similar adipogenic differentiation efficiency and higher chondrogenic and osteogenic differentiation efficiency when compared to MSCs cultured on 2D planar polystyrene tissue culture flask; on the contrary, MSCs cultured on conventional microcarriers appear to be bipotent along osteochondral lineages whereby adipogenic differentiation potential is impeded. These results suggest that these gelatin microcarriers are suitable for MSC culture and expansion, and can also potentially be extended for other types of anchorage-dependent cells.     

Process intensification in fed-batch production bioreactors using non-perfusion seed cultures

ABSTRACT

Although process intensification by continuous operation has been successfully applied in the chemical industry, the biopharmaceutical industry primarily uses fed-batch, rather than continuous or perfusion methods, to produce stable monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells. Conventional fed-batch bioreactors may start with an inoculation viable cell density (VCD) of ~0.5 × 10⁶ cells/mL. Increasing the inoculation VCD in the fed-batch production bioreactor (referred to as N stage bioreactor) to 2–10 × 10⁶ cells/mL by introducing perfusion operation or process intensification at the seed step (N-1 step) prior to the production bioreactor has recently been used because it increases manufacturing output by shortening cell culture production duration. In this study, we report that increasing the inoculation VCD significantly improved the final titer in fed-batch production within the same 14-day duration for 3 mAbs produced by 3 CHO GS cell lines. We also report that other non-perfusion methods at the N-1 step using either fed batch or batch mode with enriched culture medium can similarly achieve high N-1 final VCD of 22–34 × 10⁶ cells/mL. These non-perfusion N-1 seeds supported inoculation of subsequent production fed-batch production bioreactors at increased inoculation VCD of 3–6 × 10⁶ cells/mL, where these achieved titer and product quality attributes comparable to those inoculated using the perfusion N-1 seeds demonstrated in both 5-L bioreactors, as well as scaled up to 500-L and 1000-L N-stage bioreactors. To operate the N-1 step using batch mode, enrichment of the basal medium was critical at both the N-1 and subsequent intensified fed-batch production steps. The non-perfusion N-1 methodologies reported here are much simpler alternatives in operation for process development, process characterization, and large-scale commercial manufacturing compared to perfusion N-1 seeds that require perfusion equipment, as well as preparation and storage vessels to accommodate large volumes of perfusion media. Although only 3 stable mAbs produced by CHO cell cultures are used in this study, the basic principles of the non-perfusion N-1 seed strategies for shortening seed train and production culture duration or improving titer should be applicable to other protein production by different mammalian cells and other hosts at any scale biologics facilities.     

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan δ solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials'' morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by Stöber''s methods, which have smooth surfaces.     

Leveraging flow mechanics to determine critical process and scaling parameters in a continuous viral inactivation reactor

A continuous viral inactivation (CVI) chamber has been designed to operate with acceptable residence time distribution (RTD) characteristics. However, altering the CVI's geometry and operation to accommodate the scale was not obvious. In this work, we elucidate the influence of Dean vortices and leverage the transition into the weak turbulent regime to establish relationships between input variables and process outputs. This study was targeted to understand and quantify the impact of viscosity, Dean number, internal diameter, and path length on the RTD. When the Dean number exceeds 70, radial mixing generated by the Dean vortices began to consistently alter the axial dispersive effects experienced by the pulse injection. Increasing to a Dean number of >100, the axial dispersive effects were dominated by the Dean vortices which allowed the calculation of the minimum and maximum residence time to be generated. This work provides a method to calculate operational solutions for a tubular incubation reactor in terms of path length, internal diameter, flow rate, and target minimum and maximum residence time specifications that assures both viral residence times while also establishing criteria to maximize product quality during continuous operation.