Overcoming key challenges during the upstream development of a continous manufacturing process at 500L scale

Please click Additional Files below to see the full abstract

Platform based screening strategies that deliver reliable and high quality continous biomanufacturing processes

The challenge during mammalian cell line and upstream process development is to identify and isolate stable, high expressing cell lines producing product with the appropriate critical product quality attributes rapidly, reproducibly and with relative ease. Current platform processes are based on a defined set of hierarchical screening strategies utilised to identify key cellular performance criteria required for fed-batch culture (Porter et al 2010a,b). The application of continuous biomanufacturing principles has introduced a paradigm shift, due to their inherent advantage of higher productivity which can facilitate the implementation of smaller process equipment and result in cost-effective, lean and agile manufacturing facilities. However, as we move from fed-batch to continuous manufacturing we must re-evaluate and leverage the correct platform technologies (host cell line, expression vector, cell line development process, cell culture media/feed, process control) to rapidly identify the correct cellular performance criteria that are important for continuous biomanufacturing processes. Furthermore, whereas the adoption of robust and reproducible platform processes have been widely adopted for fed-batch processes, optimal upstream continuous processes performance still largely relies on the optimisation of key bioprocess parameters which are optimised in an ad-hoc manner during process development. To increase speed-to-clinic we show the application of both a new cell line development and continuous upstream production platform methodology which has been successfully utilised to establish reliable and high quality continuous upstream biomanufacturing processes for multiple CHO-DG44 derived cell lines and recombinant monoclonal antibody products. References Porter AJ, Dickson AJ, Racher AJ. (2010a) Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: Realising the potential in bioreactors. Biotechnol Prog 26(5):1446-54 Porter AJ, Racher AJ, Preziosi R, Dickson AJ (2010b) Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: Improving the efficiency of cell line generation. Biotechnol Prog 26(5): 1455-64

HDAC-mediated control of ERK- and PI3K-dependent TGF-β-induced extracellular matrix-regulating genes

Histone deacetylases (HDACs) regulate the acetylation of histones in the control of gene expression. Many non-histone proteins are also targeted for acetylation, including TGF-ß signalling pathway components such as Smad2, Smad3 and Smad7. Our studies in mouse C3H10T1/2 fibroblasts suggested that a number of TGF-ß-induced genes that regulate matrix turnover are selectively regulated by HDACs. Blockade of HDAC activity with trichostatin A (TSA) abrogated the induction of a disintegrin and metalloproteinase 12 (Adam12) and tissue inhibitor of metalloproteinases-1 (Timp-1) genes by TGF-ß, whereas plasminogen activator inhibitor-1 (Pai-1) expression was unaffected. Analysis of the activation of cell signalling pathways demonstrated that TGF-ß induced robust ERK and PI3K activation with delayed kinetics compared to the phosphorylation of Smads. The TGF-ß induction of Adam12 and Timp-1 was dependent on such non-Smad signalling pathways and, importantly, HDAC inhibitors completely blocked their activation without affecting Smad signalling. Analysis of TGF-ß-induced Adam12 and Timp-1 expression and ERK/PI3K signalling in the presence of semi-selective HDAC inhibitors valproic acid, MS-275 and apicidin implicated a role for class I HDACs. Furthermore, depletion of HDAC3 by RNA interference significantly down-regulated TGF-ß-induced Adam12 and Timp-1 expression without modulating Pai-1 expression. Correlating with the effect of HDAC inhibitors, depletion of HDAC3 also blocked the activation of ERK and PI3K by TGF-ß. Collectively, these data confirm that HDACs, and in particular HDAC3, are required for activation of the ERK and PI3K signalling pathways by TGF-ß and for the subsequent gene induction dependent on these signalling pathways

Dissecting cell death pathways in fed-batch bioreactors

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors. However, these studies were undertaken when less was known about non-apoptotic cell death pathways. Here, we report the occurrence of non-apoptotic cell death in an industrial antibody-producing CHO cell line during fed-batch culture. Under standard conditions, crucial markers of apoptosis were not observed despite a decrease in viability towards the end of the culture; only by increasing stress within the system did we observe caspase activation indicative of apoptosis. In contrast, markers of parthanatos and ferroptosis were observed during standard fed-batch culture, indicating that these non-apoptotic cell death pathways contribute to viability loss under these conditions. These findings pave the way for targeting non-conventional cell death pathways to improve viability and biologic production in CHO cells

Microfluidic Microbioreactor to Reduce the Cost and Speed Up Optimisation of Protein Production

Here, we demonstrate a low-cost polymer microfluidic microbioreactor with a working volume of 1 mL, integrated with optical sensors for pH, oxygen and cell density, and maintained at constant set temperature for the optimisation of recombinant protein production from Pichia pastoris. Oxygen is transferred from a headspace enclosure, formed using inkjet 3D printing, through a gas-permeable membrane within the microbioreactor with a KLa of 90 at 1500 rpm. A pressurised fluid driving system is used with flow rates controllable to 0.7 µL/min with fluid switching from four reservoirs performed off the microfluidic microbioreactor element so that this can be produced at low cost using high replication techniques

Genomic epidemiology of SARS-CoV-2 transmission lineages in Ecuador

Characterisation of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic diversity through space and time can reveal trends in virus importation and domestic circulation and permit the exploration of questions regarding the early transmission dynamics. Here, we present a detailed description of SARS-CoV-2 genomic epidemiology in Ecuador, one of the hardest hit countries during the early stages of the coronavirus-19 pandemic. We generated and analysed 160 whole genome sequences sampled from all provinces of Ecuador in 2020. Molecular clock and phylogeographic analysis of these sequences in the context of global SARS-CoV-2 diversity enable us to identify and characterise individual transmission lineages within Ecuador, explore their spatiotemporal distributions, and consider their introduction and domestic circulation. Our results reveal a pattern of multiple international importations across the country, with apparent differences between key provinces. Transmission lineages were mostly introduced before the implementation of non-pharmaceutical interventions, with differential degrees of persistence and national dissemination.ISSN:2057-157

SARS-CoV-2 Variants of Interest and Concern naming scheme conducive for global discourse [comment].

International audienc