Background Genome editing technology heralds a new era for animal cell engineering. Programmable site-specific nucleases, such as transcription activator-like effector nucleases (TALENs) and clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9, enable to induce DNA double-strand breaks (DSBs) at any desired genomic loci, resulting in efficient gene knockout and knockin in broad range of cultured cells [1]. As for gene knock-in, homologous recombination (HR)-assisted method has generally been used for spontaneous or programmable nuclease-mediated donor DNA integration. It enables precise gene knock-in, but the labor for constructing targeting vector with long homology arms and limited applicability due to the lower HR activity have been technical hurdles to utilize this method.

Background: Expression vector engineering technology is one of the most convenient and timely method for cell line development to meet the rising demand of novel production cell line with high productivity. Destabilization of dihydrofolate reductase (dhfr) selection marker by addition of AU-rich elements and murine ornithine decarboxylase PEST region was previously shown to improve the specific productivities of recombinant human interferon gamma in CHO-DG44 cells. In this study, we evaluated novel combinations of engineered motifs for further selection marker attenuation to improve recombinant human alpha-1-antitrypsin (rhA1AT) production. Motifs tested include tandem PEST elements to promote protein degradation, internal ribosome entry site (IRES) mutations to impede translation initiation, and codon-deoptimized dhfr selection marker to reduce translation efficiency. Results: After a 2-step methotrexate (MTX) amplification to 50 nM that took less than 3 months, the expression vector with IRES point mutation and dhfr-PEST gave a maximum titer of 1.05 g/l with the top producer cell pool. Further MTX amplification to 300 nM MTX gave a maximum titer of 1.15 g/l. Relative transcript copy numbers and dhfr protein expression in the cell pools were also analysed to demonstrate that the transcription of rhA1AT and dhfr genes were correlated due to the IRES linkage, and that the strategies of further attenuating dhfr protein expression with the use of a mutated IRES and tandem PEST, but not codon deoptimization, were effective in reducing dhfr protein levels in suspension serum free culture. Conclusions: Novel combinations of engineered motifs for further selection marker attenuation were studied to result in the highest reported recombinant protein titer to our knowledge in shake flask batch culture of stable mammalian cell pools at 1.15 g/l, highlighting applicability of expression vector optimization in generating high producing stable cells essential for recombinant protein therapeutics production. Our results also suggest that codon usage of the selection marker should be considered for applications that may involve gene amplification and serum free suspension culture, since the overall codon usage and thus the general expression and regulation of host cell proteins may be affected in the surviving cells.

Background Mammalian fed-batch processes for monoclonal antibody (mAb) production rely on strategic feeding of several nutrients such as glucose, vitamins and amino acids to extend culture time and improve protein production [1]. In actual processes, L-cysteine and L-tyrosine are fed separately at alkaline pH due their low stability and low solubility at neutral pH, resulting in pH peaks and precipitations [2]. To simplify next generation processes, both amino acids have been chemically modified to enhance their respective stability and solubility profiles. Previous work has demonstrated that phosphotyrosine disodium salt (PTyr2Na) is a stable L-tyrosine derivative and can be used in neutral pH feeds without having a detectable impact on the culture performance or the mAb quality attributes [3]. Here, we present results obtained using a L-cysteine derivative in a neutral pH, single-feed system.

Background The Multi-Organ-Chip (MOC) is a new micro size bioreactor. It offers two tissue cultivation segments (e.g. skin model, liver cells), which are connected by a dynamic micro pump system. [1,2] One future application of this system is the dermal application of substances for drug testing in vitro. In this study a method to simulate and validate the permeation of fluorescein sodium salt through a barrier in the MOC was established. Fluorescein sodium salt has a molecular weight of 376.28 g/mol which is in the range of steroid hormones like testosterone (288.4 g/mol), aldesterone (360.4 g/mol), hydrocortisone (362.5.4 g/ mol) and corticosterone (346 g/mol) [3]. Furthermore, first permeation experiments through four cell-collagen models, as reference for skin models, were carried out.

Background Nowadays, stem cells draw great interest in regenerative medicine [1]. However, due to low occurrence in tissues, their in vitro expansion is required to gain access to therapeutic applications [2]. Furthermore, the existing culture systems often include xeno-derived components, present upscaling limitations [3,4] and do not necessary address important considerations, such as those related to biosafety, availability and homogeneity of (biological) raw material and process reproducibility. Moreover, the regulatory guidelines focus on the importance of defined environments, minimal handling and continuity between development and industrialization scales [5,6]. In this context, new scalable solutions a reactively considered in order to achieve production of stem cells at higher densities [7]. Among these, synthetic peptides are of particular interest [4] as potential alternatives to xeno-derived culture supports, able to mimic the physico-biochemical properties of natural cell matrices. In this way our work aims at assessing new xeno-free culture matrices based on short, synthetic and bioactive peptides and demonstrating the scalability, reproducibility and performance of such peptides-based matrices for human stem cells culture. This report is focused on a part of the work performed to study the stability of synthetic peptide coatings on polymer surfaces and the capability of such coatings to favour stem cells adhesion and growth.

Background Chinese hamster ovary (CHO) cells are the most widely used host for large scale production of recombinant therapeutic proteins. A combination of several gene editing approaches applied to Novartis proprietary CHO cell line resulted in a superior cell line with a significant increase of titer and improved product quality. Inter alia we have surprisingly identified a key protease responsible for proteolytic degradation of mainly non-antibody format therapeutic proteins. The recently published CHO genome in combination with screening methods and cell line engineering tools has enabled the development of this novel CHO cell line.

Strengthening capacity in poorer countries to generate multi-disciplinary health research and to utilise research findings, is one of the most effective ways of advancing the countries' health and development. This paper explores current knowledge about how to design health research capacity strengthening (RCS) programmes and how to measure their progress and impact. It describes a systematic, evidence-based approach for designing such programmes and highlights some of the key challenges that will be faced in the next 10 years. These include designing and implementing common frameworks to facilitate comparisons among capacity strengthening projects, and developing monitoring indicators that can capture their interactions with knowledge users and their impact on changes in health systems.

Materials and methods AmbrTM 48 and 80L stirred tank bioreactor were run for 14 days in a fed-batch mode in a chemically defined medium. A CHO cell line expressing a recombinant monoclonal antibody (MAb) was used. Feed was added daily from day 3 onwards. If required, antifoam C was added to the bioreactor. DO, pH, and temperature were controlled at setpoint. Viable cell concentration (VCC), cell viability, and average cell diameter were measured using a ViCell cell counter. Osmolality was obtained using an Osmometer (Advanced Instruments). On harvest day, MAb concentration of the supernatant samples was quantified using Protein A high performance liquid chromatography. For the minibioreactors run, triplicates were pooled on harvest day for product quality attributes analysis. For both scales cell culture fluid samples were centrifuged and filtered to remove cell debris. The monoclonal antibody was purified by AKTAXpress (GE Healthcare) Protein-A purification. The neutralized eluate was used for product quality analysis. Results High throughput technologies enable us to interconnect clone and process development thus reducing the risks associated with early-stage process development while reducing timelines and enabling us to achieve higher process robustness in a leaner manner. In one ambrTM48 run different feeding strategies were assessed on 4 preselected lead clones for selecting the final lead clone, the fed-batch feeding strategy and additional data with feed compositions all at once. The selected condition (clone, type of feed and feeding strategy) was based on the maximization of the MAb titer and target High Molecular Weight Species (HMWS) level and Acidic Peak Group (APG) level (data not shown). Scale up from ambrTM 48 to 80L stirred tank bioreactor was performed with the selected clone, feed and feeding strategy. Similar cell growth profiles were obtained at ambrTM scale and 80L scale for the selected condition (Figure 1). Monoclonal Antibody titers and percentages of Acidic Peak Group obtained at harvest day were comparable between scales (Table 1). Process robustness was shown by performing a number of 4 batches at 80L scale.

Most neglected tropical diseases (NTDs) have complex life cycles and are challenging to control. The “2020 goals” of control and elimination as a public health programme for a number of NTDs are the subject of significant international efforts and investments. Beyond 2020 there will be a drive to maintain these gains and to push for true local elimination of transmission. However, these diseases are affected by variations in vectors, human demography, access to water and sanitation, access to interventions and local health systems. We therefore argue that there will be a need to develop local quantitative expertise to support elimination efforts. If available now, quantitative analyses would provide updated estimates of the burden of disease, assist in the design of locally appropriate control programmes, estimate the effectiveness of current interventions and support ‘real-time’ updates to local operations. Such quantitative tools are increasingly available at an international scale for NTDs, but are rarely tailored to local scenarios. Localised expertise not only provides an opportunity for more relevant analyses, but also has a greater chance of developing positive feedback between data collection and analysis by demonstrating the value of data. This is essential as rational program design relies on good quality data collection. It is also likely that if such infrastructure is provided for NTDs there will be an additional impact on the health system more broadly. Locally tailored quantitative analyses can help achieve sustainable and effective control of NTDs, but also underpin the development of local health care systems.