Recombinant Protein Production at High Cell Densities in Insect Cell Culture from the 3 to 150-L Scale at the BRI

NRC publication: Ye

Purification of his-tagged proteins using fractogel-cobalt

Fast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Such proteins often need to be as pure as possible before any characterization study can begin. Although many types of protein tag are available, histidine is the most popular. Although small-scale immobilized metal-affinity column (IMAC) purification of such proteins (e.g., <500 mL of culture medium) can easily be achieved using gravity chromatography columns, larger volumes can be processed with the aid of automated chromatography systems. This protocol describes an IMAC purification technique for secreted proteins using a cobalt-loaded resin. Preliminary small-scale trials using this technique can be used to determine the production scale that will be needed to provide enough pure material for a given study.Peer reviewed: YesNRC publication: Ye

Transient Expression in HEK293-EBNA1 Cells

NRC publication: Ye

Transfection of adherent HEK293-EBNA1 cells in a six-well plate with branched PEI for production of recombinant proteins

Fast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin due to their superior capability to conduct elaborate post-translational modifications. Largescale transfection of mammalian cells is now establishing itself as a \u201cmust-have\u201d technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, polyethylenimine (PEI) is much easier to use: It binds to and precipitates DNA efficiently and the resulting DNA-PEI complexes are suitable for efficient transfection of mammalian cells. In particular, the branched isoform of PEI works well for adherent cells, as it promotes their attachment to the plastic surface. It is thus very useful in experiments requiring multiple medium exchanges or washing steps following transfection. Also, when used in conjunction with sixwell CellBIND plates, branched PEI can be used to adhere transfected cells when establishing stable cell lines. This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in adherent culture using branched PEI.Peer reviewed: YesNRC publication: Ye

Transfection of HEK293-EBNA1 cells in suspension with linear PEI for production of recombinant proteins

Fast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a "must-have" technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, it is usually achieved in serum-containing medium under tightly controlled conditions that are difficult to achieve on a large scale. In contrast, polyethylenimine (PEI) is much easier to use: It binds to and precipitates DNA efficiently and the resulting DNA-PEI complexes are suitable for efficient transfection of mammalian cells. PEI has been used successfully on a large scale in serum-containing and serum-free cultures. In particular, the linear isoform of PEI is more effective for transfecting cells in suspension. This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in suspension culture using linear PEI.Peer reviewed: YesNRC publication: Ye

Transient Expression in HEK293-EBNA1 Cells

NRC publication: Ye

Life course transitions and changes in alcohol consumption among older Irish adults: results from the Irish Longitudinal Study on Ageing (TILDA).

OBJECTIVE: The objective of this study was to determine whether trajectories of older adults' alcohol consumption are influenced by the following life course transitions, relationship status, employment status, and self-rated health. METHOD: Volume and frequency of drinking were harmonized across first three waves of The Irish Longitudinal Study on Ageing (TILDA; N = 4,295). Multilevel regression models were used to model frequency, average weekly consumption, and heavy episodic drinking. RESULTS: Men and women drank more frequently over time, with frequency decreasing with age for women. Average weekly consumption decreased over time and with increasing age. Transitions in self-rated health, particularly those reflecting poorer health, were associated with lower frequency and weekly consumption. Heavy episodic drinking decreased with age. Men who were retired across all waves were more likely to engage in heavy episodic drinking at baseline. DISCUSSION: Despite the decline in average weekly consumption and heavy episodic drinking, the observed quantities consumed and the increase in frequency of consumption suggest that older Irish adults remain vulnerable to alcohol-related harms

Noninvasive probing of inhibitory effects of cylindrospermopsin and microcystin-LR using cell-based impedance spectroscopy

In an effort to develop a noninvasive method for assessment of cyanobacterial toxins in drinking water, plausible cytotoxicity/inhibition of microcystin-LR and cylindrospermopsin was evaluated by cell-substrate impedance sensing (ECIS) using three different cell lines. Sf9 insect cells were attached to concanavalin A coated gold electrodes, whereas Chinese hamster ovary (CHO) and human embryo kidney (HEK) cells were attached to a fibronectin or laminin coated gold surface. Cytotoxic or inhibitory effects were dependent upon the cell line and the extracellular matrix (ECM) coating. Neither toxin exhibited any appreciable effect on the insect cells. In contrast, cytotoxicity of cylindrospermopsin on CHO cells was attested by both ECIS and viability tests. The half-inhibition concentration (ECIS50) of cylindrospermopsin for CHO cells was ~2 \u3bcg/mL (ppm) after 20 h of exposure and 4 \u3bcg/mL (ppm) after 30 h of exposure for a laminin or fibronectin coated surface. ECIS confirmed no significant effect of cylindrospermopsin on HEK cells. Microcystin-LR was also tested with CHO cells, resulting in an ECIS50 value of ~12 \ub5g/mL (ppm) after 25 h of exposure for a laminin coated gold surface. The effect of microcystin-LR on CHO cells probed by ECIS was inhibitory rather than cytotoxic, as confirmed by cell viability assays.Dans le but de mettre au point une m\ue9thode non effractive pour \ue9valuer les toxines cyanobact\ue9riennes dans l\u2019eau potable, on a \ue9valu\ue9 la cytotoxicit\ue9 ou l\u2019inhibition probable de la microcystine-LR et de la cylindrospermopsine au moyen de l\u2019imp\ue9dance \ue9lectrique de substrats cellulaires [ECIS pour cell-substrate impedance sensing] en utilisant trois diff\ue9rentes lign\ue9es cellulaires. Des cellules d\u2019insecte Sf9 se sont fix\ue9es \ue0 des \ue9lectrodes en or recouvertes de concanavaline A, alors que des cellules ovariennes de hamster chinois (CHO) et des cellules embryonnaires de rein humain (HEK) se sont fix\ue9es \ue0 une surface en or recouverte de fibronectine ou de laminine. Les effets cytotoxiques ou inhibiteurs d\ue9pendent de la lign\ue9e cellulaire et de la matrice extracellulaire [ECM pour extracellular matrix] utilis\ue9e pour recouvrir les \ue9lectrodes. La toxine n\u2019a manifest\ue9 aucun effet appr\ue9ciable sur les cellules d\u2019insecte. En revanche, les tests d\u2019ECIS et de viabilit\ue9 cellulaire confirment la cytotoxicit\ue9 de la cylindrospermopsine sur les cellules CHO. La concentration de demi-inhibition (ECIS50) de la cylindrospermopsine dans le cas des cellules CHO \ue9tait d\u2019environ 2 \u3bcg/ml (ppm) apr\ue8s 20 heures d\u2019exposition et de 4 \u3bcg/ml (ppm) apr\ue8s 30 heures d\u2019exposition pour une surface recouverte de laminine ou de fibronectine. L\u2019ECIS a confirm\ue9 qu\u2019il n\u2019y a aucun effet important de la cylindrospermopsine sur les cellules HEK. On a \ue9galement \ue9valu\ue9 la microcystine-LR sur les cellules CHO. Les r\ue9sultats montrent une ECIS50 d\u2019environ 12 \u3bcg/ml (ppm) apr\ue8s 25 heures d\u2019exposition dans le cas d\u2019une surface en or recouverte de laminine. L\u2019effet de la microcystine-LR sur les cellules CHO, mesur\ue9 par l\u2019ECIS, est inhibiteur plut\uf4t que cytotoxique, comme l\u2019ont confirm\ue9 les analyses de viabilit\ue9 cellulaire.Peer reviewed: YesNRC publication: Ye

A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

Transient gene expression in mammalian cells is a valuable alternative to stable cell lines for the rapid production of large amounts of recombinant proteins. While the establishment of stable cell lines takes 2\u20136 months, milligram amounts of protein can be obtained within a week following transfection. The polycation polyethylenimine (PEI) is one of the most utilized reagents for small- to large-scale transfections as it is simple to use and, when combined with optimized expression vectors and cell lines, provides high transfection efficiency and titers. As with most transfection reagents, PEI-mediated transfection involves the formation of nanoparticles (polyplexes) which are obtained by its mixing with plasmid DNA. A short incubation period that allows polyplexes to reach their optimal size is performed prior to their addition to the culture. As the quality of polyplexes directly impacts transfection efficiency and productivity, their formation complicates scalability and automation of the process, especially when performed in large-scale bioreactors or small-scale high-throughput formats. To avoid variations in transfection efficiency and productivity that arise from polyplexes formation step, we have optimized the conditions for their creation directly in the culture by the consecutive addition of DNA and PEI. This simplified approach is directly transferable from suspension cultures grown in 6-well plates to shaker flasks and 5-L WAVE bioreactors. As it minimizes the number of steps and does not require an incubation period for polyplex formation, it is also suitable for automation using static cultures in 96-well plates. This \u201cdirect\u201d transfection method thus provides a robust platform for both high-throughput expression and large-scale production of recombinant proteins.Peer reviewed: YesNRC publication: Ye