Fed-Batch E. coli cultures in a shaken, single-use 24-well miniature bioreactor

At industrial scale, microbial cultivations are usually performed in fed-batch mode to allow for high cell density cost-effective processes. Miniature bioreactors are becoming widely used in the biopharmaceutical industry as a tool for high throughput strain evaluation and fermentation process development. However, there are relatively few examples of miniature bioreactors capable of fed-batch operation and of supporting the high oxygen demand. There are several challenges that need to be addressed to establish high cell density fed-batch cultivation at microscale: attaining high oxygen mass transfer rates, achieving good mixing for the duration of the culture and implementation of an industrially relevant feeding strategy requiring low volume additions. In this work a shaken, single-use 24-well miniature bioreactor (Pall, Micro 24 MicroReactor System) has been characterised in terms of volumetric oxygen mass transfer coefficient (kLa) and liquid phase mixing time (tm) to assess the feasibility of high cell density microbial cultures. The impact of shaking frequency, total gas flow rate and fill volume on oxygen transfer and fluid mixing were investigated and the optimum operating conditions were determined. To enable fed-batch cultivation in the miniature bioreactor system a bespoke feeding system for direct, continuous feed delivery has been developed that works at feed flow rates of 20μL h-1 and above. This feeding system allows for 24 fed-batch cultures to be run in parallel. Within the operating ranges of the miniature bioreactor system, it was found that oxygen transfer was dependant on both shaking frequency and gas flow rate, but was independent of fill volume; the oxygen mass transfer coefficient, kLa increased with both increasing shaking frequency and gas flow rate over the range 3-101h-1. The liquid phase mixing time, tm under non-aerated conditions increased with shaking frequency and decreased with fill volume over the range 0.8-15.3s. It has been demonstrated that the miniature bioreactor system is well mixed under the range of operating conditions evaluated. The bespoke feed delivery system was used to perform fed-batch cultures of an industrial E. coli strain producing an antibody fragment under operating conditions defined from the engineering characterisation studies. Fermentations were performed on a semi-complex medium containing glycerol with direct feeding of a glycerol solution initiated around 15 hours. It was found that direct feeding enhances biomass production by 30-40% and product expression by 45-65% in comparison to non-fed cultures. The feeding system developed in this work allows for industrially relevant microbial processes to be implemented at the microscale

Cell culture scale translation from a 24-well Single-Use miniature bioreactor and subsequent impact on product and broth quality

To accelerate cell culture process development, most companies have validated scale-down models of their pilot and manufacturing scale bioreactors. Advancing such mimics to even smaller scales requires the large scale engineering environment to be accurately recreated. Here we describe a single-use microwell methodology that accurately reproduces not only cell growth kinetics but also key attributes related to product quality and broth processability. The μ24 miniature bioreactor system enables system level control of agitation (by orbital shaking), with individual well control of pH, DO and temperature. Two distinct plate types are investigated, allowing for either headspace or direct gas sparging. An engineering characterisation was performed evaluating fluid mixing, gas transfer capacity and the dispersed gas phase. Cell culture is investigated using a model CHO DG44 cell line expressing a whole IgG1 mAb [1]. In addition, this work describes scale-up of μ24 results to conventional laboratory scale stirred tank bioreactors (2L) and use of the device for selection of robust and scaleable cell lines through evaluation of product quality. The ‘broth quality’ is also evaluated for primary clarification efficiency using an Ultra Scale-Down (USD) depth filtration rig that requires quantities of material compatible with those available from the miniature bioreactors. Apparent kLa values ranged between 3–22 hr-1 and 4–53 hr-1 for headspace aeration and direct gas sparging respectively. Mixing times were generally in the range 1–13 seconds and decreased with increasing shaking frequency (500–800 rpm). Direct gas sparging also helped to reduce tm values. Cultures performed with headspace aeration showed the highest VCD and antibody titres, whereas those operated with direct gas sparging showed cell growth kinetics and product titres that were more comparable to those found in a conventional 2L stirred bioreactor. Initial results also indicate that key product and broth processability attributes are maintained making the combination of μ24 and USD technologies useful tools in ‘Quality by Design’ driven cell culture process development

Use of AMBR250 as a small scale model for manufacturing-scale single-use bioreactors

Quality by Design (QbD) has become an integral part of biopharmaceutical process development and manufacturing. To gain the enhanced process understanding required by QbD, a well-designed small scale model that accurately predicts behavior at manufacturing scale is essential. This process understanding should ideally be achieved with rapid, efficient experimentation to decrease both the time and cost required for development. The ambr250 automated microscale bioreactor system has the potential to address all of these challenges. By embedding the ambr250 into the upstream process development workflow, throughput can be dramatically increased allowing for greater exploration of parameter operating ranges and more complete process understanding. However, the value of such microscale technologies hinges on their ability to accurately mimic manufacturing scale. We embarked on a study to demonstrate the applicability of the ambr250 (250 mL) as a small scale model for a 2000-L single-use bioreactor (SUB). We evaluated consistency of cell culture process performance from the ambr250 to 2000-L SUB scale along with intermediate scales such as our legacy small scale model (3-L glass stirred-tank reactors) and 50-L to 1000-L SUBs. Scalability was assessed using two monoclonal antibody molecules expressed from different CHO hosts (CHO K1 and DG44) and cultivated in different media platforms (chemically-defined and yeastolate-containing) to ensure broad applicability of the small scale model. Engineering principles were applied to develop appropriate agitation and gassing strategies at each scale to ensure comparability, with a power input based scaling strategy performing the best. Based on both univariate and multivariate data analysis methods the ambr250 behaved comparably to both our legacy small scale model and the SUBs for the assets evaluated. Areas of focus to further refine the ambr250 as a small scale model have also been identified

Multi-Object Tracking by Iteratively Associating Detections with Uniform Appearance for Trawl-Based Fishing Bycatch Monitoring

The aim of in-trawl catch monitoring for use in fishing operations is to detect, track and classify fish targets in real-time from video footage. Information gathered could be used to release unwanted bycatch in real-time. However, traditional multi-object tracking (MOT) methods have limitations, as they are developed for tracking vehicles or pedestrians with linear motions and diverse appearances, which are different from the scenarios such as livestock monitoring. Therefore, we propose a novel MOT method, built upon an existing observation-centric tracking algorithm, by adopting a new iterative association step to significantly boost the performance of tracking targets with a uniform appearance. The iterative association module is designed as an extendable component that can be merged into most existing tracking methods. Our method offers improved performance in tracking targets with uniform appearance and outperforms state-of-the-art techniques on our underwater fish datasets as well as the MOT17 dataset, without increasing latency nor sacrificing accuracy as measured by HOTA, MOTA, and IDF1 performance metrics

Otitis media in the Tgif knockout mouse implicates TGFβ signalling in chronic middle ear inflammatory disease

Otitis media with effusion (OME) is the most common cause of hearing loss in children and tympanostomy to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of OM are known to have a very significant genetic component, however, until recently little was known of the underlying genes involved. The identification of mouse models of chronic OM has indicated a role of transforming growth factor beta (TGFβ) signalling and its impact on responses to hypoxia in the inflamed middle ear. We have, therefore, investigated the role of TGFβ signalling and identified and characterized a new model of chronic OM carrying a mutation in the gene for transforming growth interacting factor 1 (Tgif1). Tgif1 homozygous mutant mice have significantly raised auditory thresholds due to a conductive deafness arising from a chronic effusion starting at around 3 weeks of age. The OM is accompanied by a significant thickening of the middle ear mucosa lining, expansion of mucin-secreting goblet cell populations and raised levels of vascular endothelial growth factor, TNF-α and IL-1β in ear fluids. We also identified downstream effects on TGFβ signalling in middle ear epithelia at the time of development of chronic OM. Both phosphorylated SMAD2 and p21 levels were lowered in the homozygous mutant, demonstrating a suppression of the TGFβ pathway. The identification and characterization of the Tgif mutant supports the role of TGFβ signalling in the development of chronic OM and provides an important candidate gene for genetic studies in the human population