Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single Cell Level

Biopharmaceutical manufacturing using Chinese hamster ovary (CHO) cells requires the generation of high-producing clonal cell lines. During cell line development, cell cloning using fluorescence activated cell sorting (FACS) has the potential to combine isolation of single cells with sorting based on specific cellular attributes that correlate with productivity and/or growth, identifying cell lines with desirable phenotypes for manufacturing. This study describes the application of imaging flow cytometry (IFC) to characterize recombinant cell lines at the single cell level to identify cell attributes predictive of productivity. IFC assays to quantify organelle content, and recombinant heavy (HC) and light (LC) chain polypeptide and mRNA amounts in single cells were developed. The assays were then validated against orthogonal standard flow cytometry, western blot and qRT-PCR methods. We describe how these IFC assays may be used in cell line development and show how cellular properties can be correlated with productivity at the single cell level, allowing the isolation of such cells during the cloning process. Our analysis found HC polypeptide and mRNA to be predictive of productivity early in the culture, however specific organelle content did not show any correlation with productivity

Characterisation of Chinese Hamster Ovary (CHO) cells at the single cell level

Biopharmaceuticals are a class of biological macromolecules that include antibodies and antibody derivatives, generally produced from cultured mammalian cells line via secretion directly into the media. Manufacturing requires the generation of Chinese hamster ovary (CHO) clonal cell lines capable of expressing the biopharmaceutical product at commercially relevant quantities with desirable product quality. The isolation of cell clones based on random single cell deposition via fluorescence activated cell sorting (FACS) provides a heterogeneous panel of expressers. We hypothesize that the application of FACS to provide an additional sorting step based on cell characteristics that correlate with productivity, product quality or cell growth attributes could lead to the isolation of higher producing cell lines with enhanced product quality attributes. A panel of 20 cell lines expressing a model recombinant monoclonal antibody were characterised in terms of growth, productivity, and intracellular recombinant protein and mRNA amounts. Assays were also developed to investigate cell attributes and organelle content using the ImageStream instrument, an imaging flow cytometer, which enables the investigation of cellular characteristics that correlate with cell productivity at the single cell level. Characterisation revealed the cell lines exhibited a range of values for productivity, growth, and intracellular (IC) antibody mRNA and protein expression, ideal for further ImageStream characterisation. Western blot and qRT-PCR analysis demonstrated that final titre correlated with both IC heavy chain (HC) protein and mRNA amounts (Pearson Correlation Coefficient (R) = 0.70 and R = 0.80, respectively). To assess productivity at the single cell level, assays multiplexing IC HC protein and mRNA with organelles, such as mitochondria, endoplasmic reticulum and Golgi apparatus, were therefore developed. ImageStream quantification of HC mRNA and protein amounts also showed correlations between titre and IC HC protein and mRNA (R = 0.84 and R = 0.79, respectively), confirming results from western blots and qRT-PCR analysis. A cell attribute that correlates with specific productivity has been found, and current work is investigating whether this cell attribute could be used during cell sorting for the isolation of more productive clones. Future experiments will also look at cell attributes that could lead to improved product quality. The developed assays are expected to allow a greater understanding of the intracellular mechanisms underlying productivity and product quality in CHO cells. Moreover, outcomes from this study have the potential to not only integrate into the cell line development clonal selection process, shortening timelines and reducing cost and resource requirements, but also inform host cell engineering projects with the potential for the development of an improved CHO host

Controlling fab terminal sialylation of antibodies through culture conditions

Biologics are used for the treatment of a wide-range of diseases with specificity and minimal side effects. Safety and efficacy of the drugs has been linked to carbohydrate structures found on the antibody, termed N-linked glycans. These glycans are mainly found within the Fc-region of an antibody but 20% of all IgG antibodies also contain Fab glycans1. Glycans are composed of a range of sugars whose presence or absence affects the biological qualities of a drug. Sialic acid is one such sugar; its role is to “cap” the glycan chain, protecting the internal sugars, which when exposed are bound by receptors and cleared to host lysosomes2. The presence of sialic acid is linked to an increase in biologic half-life along with a reduced inflammatory response3. It has been established that bioprocess conditions such as cell culture temperature and pH directly impact glycan composition and site occupancy. A shift in the cell culture pH and its effect on the sialic acid content within the Fab region of an antibody product was examined. The product was produced in three CHO cell clones, each innately producing varying levels of sialic acid. An initial experiment utilized the Ambr 24® to run cultures at pH 7.1, 6.8 and with a shift from 7.1 to 6.8 on day 6. Data from this was used to establish a second experiment, utilizing an Ambr 48® system. This experiment looked at the supplementation of ManNAc, copper as well as the effect of pH and temperature shifts on sialylation. Glycan analysis was undertaken using a novel method for triple-quadrupole MS. A pH shift was found to produce overall more processed glycans. Although cell growth was negatively affected, antibody productivity and specific rate of sialylation were both increased at reduced culture pH (Figure 1). The extent of the effect differed between the clones and was correlated to how early the shift occurred. Due to the negative effects on growth, overall antibody yield was reduced with some clones having less than half that of the respective control. To determine the origin of the effect and the differences between the clones further analysis is being undertaken. The Ambr 48® experiment determined the effect of different supplements on sialylation, as well as the effect of a temperature shift and further understanding of the role of cell culture pH in increasing sialylation. Flux balance analysis and expression analysis of the enzymes involved in terminal glycosylation process is underway. Please click Additional Files below to see the full abstract

Metabolomics reveals the physiological response of Pseudomonas putida KT2440 (UWC1) after pharmaceutical exposure

Human pharmaceuticals have been detected in wastewater treatment plants, rivers, and estuaries throughout Europe and the United States. It is widely acknowledged that there is insufficient information available to determine whether prolonged exposure to low levels of these substances is having an impact on the microbial ecology in such environments. In this study we attempt to measure the effects of exposing cultures of Pseudomonas putida KT2440 (UWC1) to six pharmaceuticals by looking at differences in metabolite levels. Initially, we used Fourier transform infrared (FT-IR) spectroscopy coupled with multivariate analysis to discriminate between cell cultures exposed to different pharmaceuticals. This suggested that on exposure to propranolol there were significant changes in the lipid complement of P. putida. Metabolic profiling with gas chromatography-mass spectrometry (GC-MS), coupled with univariate statistical analyses, was used to identify endogenous metabolites contributing to discrimination between cells exposed to the six drugs. This approach suggested that the energy reserves of exposed cells were being expended and was particularly evident on exposure to propranolol. Adenosine triphosphate (ATP) concentrations were raised in P. putida exposed to propranolol. Increased energy requirements may be due to energy dependent efflux pumps being used to remove propranolol from the cell. © The Royal Society of Chemistry 2016

Effectiveness of cricoid pressure in preventing gastric aspiration during rapid sequence intubation in the emergency department: study protocol for a randomised controlled trial

<p>Abstract</p> <p>Background</p> <p>Cricoid pressure is considered to be the gold standard means of preventing aspiration of gastric content during Rapid Sequence Intubation (RSI). Its effectiveness has only been demonstrated in cadaveric studies and case reports. No randomised controlled trials comparing the incidence of gastric aspiration following emergent RSI, with or without cricoid pressure, have been performed. If improperly applied, cricoid pressure increases risk to the patient. The clinical significance of aspiration in the emergency department is unknown. This randomised controlled trial aims to; 1. Compare the application of the 'ideal" amount of force (30 - 40 newtons) to standard, unmeasured cricoid pressure and 2. Determine the incidence of clinically defined aspiration syndromes following RSI using a fibrinogen degradation assay previously described.</p> <p>Methods/design</p> <p>212 patients requiring emergency intubation will be randomly allocated to either control (unmeasured cricoid pressure) or intervention groups (30 - 40 newtons cricoid pressure). The primary outcome is the rate of aspiration of gastric contents (determined by pepsin detection in the oropharyngeal/tracheal aspirates or treatment for aspiration pneumonitis up to 28 days post-intubation). Secondary outcomes are; correlation between aspiration and lowest pre-intubation Glasgow Coma Score, the relationship between detection of pepsin in trachea and development of aspiration syndromes, complications associated with intubation and grade of the view on direct largyngoscopy.</p> <p>Discussion</p> <p>The benefits and risks of cricoid pressure application will be scrutinised by comparison of the incidence of aspiration and difficult or failed intubations in each group. The role of cricoid pressure in RSI in the emergency department and the use of a pepsin detection as a predictor of clinical aspiration will be evaluated.</p> <p>Trial registration</p> <p>Australian New Zealand Clinical Trials Registry (ANZCTR): <a href="http://www.anzctr.org.au/ACTRN12611000587909.aspx">ACTRN12611000587909</a></p

Iconic dishes, culture and identity: the Christmas pudding and its hundred years’ journey in the USA, Australia, New Zealand and India

Asserting that recipes are textual evidences reflecting the society that produced them, this article explores the evolution of the recipes of the iconic Christmas pudding in the United States, Australia, New Zealand and India between the mid-nineteenth and the mid-twentieth centuries. Combining a micro-analysis of the recipes and the cookbook that provided them with contemporary testimonies, the article observes the dynamics revealed by the preparation and consumption of the pudding in these different societies. The findings demonstrate the relevance of national iconic dishes to the study of notions of home, migration and colonization, as well as the development of a new society and identity. They reveal how the preservation, transformation and even rejection of a traditional dish can be representative of the complex and sometimes conflicting relationships between colonists, migrants or new citizens and the places they live in

Electrophysiological measurements of peripheral vestibular function—A review of electrovestibulography

Electrocochleography (EcochG), incorporating the Cochlear Microphonic (CM), the Summating Potential (SP), and the cochlear Compound Action Potential (CAP), has been used to study cochlear function in humans and experimental animals since the 1930s, providing a simple objective tool to assess both hair cell (HC) and nerve sensitivity. The vestibular equivalent of ECochG, termed here Electrovestibulography (EVestG), incorporates responses of the vestibular HCs and nerve. Few research groups have utilized EVestG to study vestibular function. Arguably, this is because stimulating the cochlea in isolation with sound is a trivial matter, whereas stimulating the vestibular system in isolation requires significantly more technical effort. That is, the vestibular system is sensitive to both high-level sound and bone-conducted vibrations, but so is the cochlea, and gross electrical responses of the inner ear to such stimuli can be difficult to interpret. Fortunately, several simple techniques can be employed to isolate vestibular electrical responses. Here, we review the literature underpinning gross vestibular nerve and HC responses, and we discuss the nomenclature used in this field. We also discuss techniques for recording EVestG in experimental animals and humans and highlight how EVestG is furthering our understanding of the vestibular system

Modulation of transcription factor function by an amino acid: activation of Put3p by proline

Saccharomyces cerevisiae are able to convert proline to glutamate so that it may be used as a source of nitrogen. Here, we show that the activator of the proline utilization genes, Put3p, is transcriptionally inert in the absence of proline but transcriptionally active in its presence. The activation of Put3p requires no additional yeast proteins and can occur in the presence of certain proline analogues: an unmodified pyrrolidine ring is able to activate Put3p as efficiently as proline itself. In addition, we show that a direct interaction occurs between Put3p and proline. These data, which represent direct control of transcriptional activator function by a metabolite, are discussed in terms of the regulation of proline-specific genes in yeast and as a general mechanism of the control of transcription