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Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time-consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non-clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP-toxicology and early-phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS-CoV-2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost-effective clinical evaluation of potentially life-saving vaccines.

Monitoring antibody response to SARS-CoV-2 is critical for assessing the humoral response, especially important considering the emergence of multiple SARS-CoV-2 variants of concern (VOCs).

Inactivated Foot-and-Mouth Disease (FMD) vaccine has proven to be effective in the control of the disease. However, its production has some disadvantages, including the costly biosafety facilities required for the production of huge amounts of growing live virus, the need of an exhaustive purification process to eliminate non-structural proteins of the virus in the final formulations in order to differentiate infected from vaccinated animals and variable local regulatory restrictions to produce and commercialize the vaccine. Thus, a novel vaccine against FMD that overcome these restrictions is desirable. Although many developments have been made in this regard, most of them failed in terms of efficacy or when considering their transferability to the industry. We have previously reported the use of transient gene expression in mammalian cells to produce FMD virus-like particles (VLPs) as a novel vaccine for FMD and demonstrated the immunogenicity of the recombinant structures in animal models. Here, we report the optimization of the production system by assaying different DNA:polyethylenimine concentrations, cell densities, and direct and indirect protocols of transfection. Also, we evaluated the reproducibility and scalability of the technology to produce high yields of recombinant VLPs in a cost-effective and scalable system compatible with industrial tech-transfer of an effective and safe vaccine. Estación Experimental Agropecuaria Bariloche Fil: Mignaqui, Ana Clara. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; Argentina Fil: Ferella, Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; Argentina Fil: Cass, Brian. Human Health Therapeutics Research Center, National Research Council Canada; Canadá Fil Mukankurayija, Larissa. Human Health Therapeutics Research Center, National Research Council Canada; Canadá Fil: L’Abbé, Denis. Human Health Therapeutics Research Center, National Research Council Canada; Canadá Fil: Bisson, Louis. Human Health Therapeutics Research Center, National Research Council Canada; Canada Fil: Sánchez, Cintia. Biogénesis-Bagó; Argentina Fil: Scian, Romina. Biogénesis-Bagó; Argentina Fil: Cardillo, Sabrina Beatriz. Biogénesis-Bagó; Argentina Fil: Durocher, Yves. Human Health Therapeutics Research Center, National Research Council Canada; Canadá Fil: Wigdorovitz, Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; Argentina

Transient gene expression in human embryo kidney 293 (HEK293) cells is an established approach for the rapid production of large amounts of recombinant proteins (r-proteins). Milligram to gram quantities of r-proteins can be typically obtained within less than 10 days following transfection. In this chapter, we describe a simple and robust transfection process of suspension-growing human embryo kidney 293 cells using two commercially available serum-free media and polyethylenimine as the transfection reagent. This chapter provides examples for the production and purification of a his-tagged recombinant protein and two monoclonal antibodies.

Transient gene expression in human embryo kidney 293 (HEK293) cells is an established approach for the rapid production of large amounts of recombinant proteins (r-proteins). Milligram to gram quantities of r-proteins can be typically obtained within less than 10 days following transfection. In this chapter, we describe a simple and robust transfection process of suspension-growing human embryo kidney 293 cells using two commercially available serum-free media and polyethylenimine as the transfection reagent. This chapter provides examples for the production and purification of a his-tagged recombinant protein and two monoclonal antibodies., Series: Methods in Molecular Biology; no. 1850

Cette étude des dépôts quaternaires dans la région de Chibougamau a deux objectifs: 1) décrire les principaux traits de la géomorphologie et les caractères des unités lithostratigraphiques; 2) discuter quelques concepts de la paléogéographie tardiglaciaire. Deux directions majeures d'écoulement glaciaire sont identifiées : l'une, ancienne, vers le SE (125°) attribuée à un centre de dispersion localisé vers la baie d'Hudson, mais d'âge inconnu; l'autre vers le SSO causée par la dernière glaciation du Wisconsinien. Ce dernier mouvement est identifié par des formes fuselées (045-225°), des stries (035-215°) et par des débris erratiques provenant du bassin du lac Misstassini, au NE. Lors du retrait de l'inlandsis, dans le secteur ouest de la région, la bordure du glacier s'écoulait vers le SO et l'OSO, influencée par la présence du lac Ojibway : le mode dominant des orientations des segments d'eskers (055-235°) est interprété comme une preuve complémentaire de cet écoulement tardif; dans le secteur E, par contre, près de la ligne de partage des eaux, les marques d'écoulement vers le S sont abondantes. Une seule nappe de till de fond est identifiée. Une séquence de faciès de fusion (till stratifié et d'ablation) lui succède. Des déformations glaciotectoniques démontrent que la glace est active lors du dépôt des faciès stratifiés. Les contraintes exercées sur le front glaciaire par les reliefs et les eaux du lac Ojibway commandent un réseau de crevasses conjuguées à 25°: des moraines « mineures » (de De Geer?) de dimensions et d'espacements très variables y sont mises en place. Leur espacement ne peut être utilisé qu'avec prudence pour évaluer le taux de retrait glaciaire de cette région. Le niveau relatif supérieur du lac Ojibway est réévalué à environ 445 m. Une tourbe datée à 7600 BP (à 420 m) indique une afforestation rapide après l'exondation., This Quaternary deposits study has two purposes: 1) a description of the main géomorphologie features and the lithostratigraphic units; 2) a discussion of some concepts regarding the paleogeography during the late stage of the déglaciation. Two main glacial flow directions are identified : an early movement, of unknown age, towards the SE is assigned to a centre of dispersion located in the area of Hudson Bay; another movement, towards the SSW is attributed to the latest Wisconsinian pleniglacial. Evidence for the latter include fluting (045-225°), striae (035°-215°) and the numerous erratics derived from the Mistassini Basin to the NE. During the retreat of the last ice sheet on the west side of the area, the ice-margin was spreading to the SW and WSW, towards Lake Ojibway: the predominant mode of the esker segments trends (055°-235°) is interpreted as additional evidence of this late flow; on the other hand, near the water divide in the eastern area abundant flow marks are in a southerly direction. Only one lodgement till sheet is recognized. A melt-out till sequence (stratified and ablation tills) overlaps the former. GIaciotectonic deformations demonstrate that the ice was active during the deposition of the stratified faciès. The stress exerted on the glacial front by the bottom topography and waters of the Lake Ojibway induced a conjugate system of crevasses at 25°, where minor (or De Geer?) moraines were emplaced. These moraines are of variable sizes and spacings. The spacing must be used with caution when evaluating the rate of glacial retreat in this area. An altitude of 445 m is proposed for the upper relative level of Lake Ojibway. Organic deposits started to accumulate as early as 7,600 BP (at + 420 m) a short time after exundation., Diese Studie der Quartâr-Ablagerungen in der Gegend von Chibougamau hat zwei Ziele: (1) eine Beschreibung der wichtigsten Merkmale der Géomorphologie und der Eigenschaften der Gesteinsschichtungs-Einheiten und (2) die Erôrterung einiger Kronzepte der Palâogeographie der Spàteiszeit. Zwei glaziale Hauptstrômungs-Richtungen werden identifiziert: eine friihe Bewegung nach SO (125°), die einem Streuugszentrum im Gebiet der Hudson Bay zugeschrieben wird, deren Alter aber nicht bekannt ist; die andere nach SSW, verursacht durch die letzte Eiszeit des Wisconsin. Diese letzte Bewegung ist durch geriefelte Formen (045'-225°), Kritzen (035°-215°) und durch erratisches Gerôll gekennzeichnet, das vom Becken des Mistassini-Sees im NO stammt. Wàhrend des Rùckgangs des lnlandeises auf der Westseite des Gebiets ergofS sich der Rand des Gletschers nach SW und WSW, unterdem Einflufi des Ojibway-Sees: die dominierende Weise der Orientierung der Esker-Segmente (055°-235°) wird als ein zusàtzlicher Beweis dieses spâten Flie[5ens interpretiert ; auf der Ost-Seite dagegen nahe der Wasserscheide, gibt es zahlreiche Zeichen fur ein Fliejien nach Suden. Eine einzige Till-Ablagerungsschicht wird festgestellt. Ihr folgt eine Sequenz von Fusions-Faszies (Schicht- und Ablations-Till). Glaziotektonische Deformierungen zeigen, dap das Eis wàhrend der Ablagerung der Schicht-Fazies aktiv war. Der Druck, der durch die Reliefs und durch das Wasser des Ojibways-Sees auf die Eisfront ausgeubt wird, fiihrte zu einem Netz von Spalten, die miteinanderverbundensind, bei 250: "kleinere" (De Geer?) Morànen von sehr unterschiedlicher GroBe und Verteilung finden sich dort. Ihre Verteilung dart nur mit Vorsicht benutzt werden, urn den Grad des Eisrùckgangs in diesern Gebiet zu bestimmen.Das relative Hôchst-Niveau des Ojibways-Sees wird auf etwa 445 m veranschlagt. Torf, der auf 7600 v.u.Z. datiert wird (in 420m Hôhe), weist auf eine schnelle Bewaldung nach der Trockenlegung.

Proteolytic processing of human plasminogen generates potent antiangiogenic peptides such as angiostatin. The plasminogen kringle 5 (K5) domain, which is distinct from angiostatin, possesses potent antiangiogenic properties on its own, which can be exploited in cancer therapy. It has been recently observed that antiangiogenic agents promote leukocyte-vessel wall interaction as part of their antitumor effect. Although we have previously shown that K5 suppresses cancer growth in tumor xenograft models, its modulation of inflammation in experimental mice with intact immune systems is unknown. To determine whether K5 possesses immune proinflammatory properties, we investigated the effects of K5 in an immune competent model of breast cancer and observed that tumor rejection is substantially reduced in nonobese diabetic/severe combined immunodeficient and BALB/c nude when compared with wild-type BALB/c mice, suggesting an important role for T-lymphoid cells in the antitumor effect of K5. Tumor explant analysis shows that K5 enhances tumor recruitment of CD3+ lymphoid cells, in particular, the NKT phenotype. We also observed a significant decrease in tumor-associated microvessel length and density consistent with antiangiogenic activity. Histologic analysis of K5 tumors also revealed a robust neutrophilic infiltrate, which may be explained by the neutrophil chemotactic activity of K5 as well as its ability to promote CD64 up-regulation within the CD11b+ adhesive neutrophil population. In sum, our findings confirm that the K5 protein acts as a potent angiostatic agent and possesses a novel proinflammatory role via its ability to recruit tumor-associated neutrophils and NKT lymphocytes, leading to a potent antitumor response. [Mol Cancer Ther 2007;6(2):441–9]

Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect (Trichoplusia ni High-Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed.

The main objective of this study is to propose a method for validating the daily net basin supply data to obtain the most reliable values for the management of water resource systems. Based on the regression and exponential smoothing methods, various validating procedures are developed and compared. Results of a numerical application using data from La Grande 3 basin have shown that the simple exponential smoothing method is the most suitable. Further, it has been found that the proposed exponential smoothing has the simplest structure and would require a simple parameter estimation method because it contains only one parameter.Key words: real time validation, net basin supply, forecasting, time series analysis, reservoir management.

Camelid single domain antibodies fused to noncamelid Fc regions, also called chimeric heavy chain antibodies (cHCAb), offer great potential as therapeutic and diagnostic candidates due to their relatively small size (80 kDa) and intact Fc. In this chapter, we describe two approaches, limiting dilution and minipools, for generating nonamplified Chinese hamster ovary cell lines stably expressing cHCAb in suspension and serum-free cultures using a stringent antibiotic selection. Neither of the protocols necessitates the acquisition or implementation of expensive automated infrastructures and thus could be applied in any lab with minimal cell culture setup. The given protocol allows the isolation of stable clones capable of generating up to 100 mg/L of antibody in batch mode performed in shaker flasks.

Camelid single domain antibodies fused to noncamelid Fc regions, also called chimeric heavy chain antibodies (cHCAb), offer great potential as therapeutic and diagnostic candidates due to their relatively small size (80 kDa) and intact Fc. In this chapter, we describe two approaches, limiting dilution and minipools, for generating nonamplified Chinese hamster ovary cell lines stably expressing cHCAb in suspension and serum-free cultures using a stringent antibiotic selection. Neither of the protocols necessitates the acquisition or implementation of expensive automated infrastructures and thus could be applied in any lab with minimal cell culture setup. The given protocol allows the isolation of stable clones capable of generating up to 100 mg/L of antibody in batch mode performed in shaker flasks.

INTRODUCTIONFast 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, 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 six-well 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.

Background: Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity. Results: We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNα2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNα2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNα2b with a recovery greater than 70%. Purified IFNα2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNα2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNα2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNα2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNα2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNα2b produced naturally by lymphocytes. Further, the HEK293-produced IFNα2b is biologically active as shown with reporter gene and antiviral assays. Conclusion: These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNα2b., available, unlimited, public

INTRODUCTIONFast 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.

INTRODUCTIONFast 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.

Continuous cell lines are not capable of complete oxidising glucose via the TCA cycle. Recently, we presented a strategy of inverse metabolic engineering to increase the glycolytic flux into the TCA. This work summarises the effect of yeast pyruvate carboxylase 2 (PYC2) expressed in the cytoplasm of different host systems: BHK-21, CHO-K1, CHO-DG44, HEK293 and BTI-Tn-5B1-4 (High Five™) cells.

INTRODUCTIONFast 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. The HEK293 cell line stably expressing the Epstein-Barr virus nuclear antigen-1 (HEK293-EBNA1, or 293E) is the most commonly used cell line for large-scale transfection. When using expression vectors bearing the Epstein-Barr virus origin of replication, oriP (such as the pTT vector), a threefold improvement in r-protein yield is generally obtained over a similar non-oriP vector. This protocol describes a method for culturing HEK293-EBNA1 cells which will then be used to produce recombinant proteins.