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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.

Alpha-1-antitrypsin (A1AT) is a serine protease inhibitor which blocks the activity of serum proteases including neutrophil elastase to protect the lungs. Its deficiency is known to increase the risk of pulmonary emphysema as well as chronic obstructive pulmonary disease. Currently, the only treatment for patients with A1AT deficiency is weekly injection of plasma-purified A1AT. There is still today no commercial source of therapeutic recombinant A1AT, likely due to significant differences in expression host-specific glycosylation profile and/or high costs associated with the huge therapeutic dose needed. Accordingly, we aimed to produce high levels of recombinant wild-type A1AT, as well as a mutated protein (mutein) version for increased oxidation resistance, with N-glycans analogous to human plasma-derived A1AT. To achieve this, we disrupted two endogenous glycosyltransferase genes controlling core α-1,6-fucosylation (Fut8) and α-2,3-sialylation (ST3Gal4) in CHO cells using CRISPR/Cas9 technology, followed by overexpression of human α-2,6-sialyltransferase (ST6Gal1) using a cumate-inducible expression system. Volumetric A1AT productivity obtained from stable CHO pools was 2.5- to 6.5-fold higher with the cumate-inducible CR5 promoter compared to five strong constitutive promoters. Using the CR5 promoter, glycoengineered stable CHO pools were able to produce over 2.1 and 2.8 g/L of wild-type and mutein forms of A1AT, respectively, with N-glycans analogous to the plasma-derived clinical product Prolastin-C. Supplementation of N-acetylmannosamine to the cell culture media during production increased the overall sialylation of A1AT as well as the proportion of bi-antennary and disialylated A2G2S2 N-glycans. These purified recombinant A1AT proteins showed in vitro inhibitory activity equivalent to Prolastin-C and substitution of methionine residues 351 and 358 with valines rendered A1AT significantly more resistant to oxidation. The recombinant A1AT mutein bearing an improved oxidation resistance described in this study could represent a viable biobetter drug, offering a safe and more stable alternative for augmentation therapy.

Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC), strongly depend on the N-glycans linked to asparagine 297 of the Fc region of the protein. A single α-(1,6)-fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α-1,6-linked fucose residue to the first GlcNAc residue of the N-linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs. Several approaches have been developed to modify glycans and improve the biological functions of antibodies. Here, we demonstrate that expression of a membrane-associated anti-FUT8 intrabody engineered to reside in the endoplasmic reticulum and Golgi apparatus can efficiently reduce FUT8 activity and therefore the core-fucosylation of the Fc N-glycan of an antibody. IgG1-producing CHO cells expressing the intrabody secrete antibodies with reduced core fucosylation as demonstrated by lectin blot analysis and UPLC-HILIC glycan analysis. Cells engineered to inhibit directly and specifically alpha-(1,6)-fucosyltransferase activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected. The quick and efficient method described here should have broad practical applicability for the development of next-generation therapeutic antibodies with enhanced effector functions.

Alpha-1-antitrypsin (A1AT) is an abundant serum inhibitor of serine proteases. A1AT deficiency is a common genetic disorder which is currently treated with augmentation therapies. These treatments involve weekly injections of patients with purified plasma-derived A1AT. Such therapies can be extremely expensive and rely on plasma donors. Hence, large-scale production of recombinant A1AT (rA1AT) could greatly benefit these patients, as it could decrease the cost of treatments, reduce biosafety concerns and ensure quantitative and qualitative controls of the protein. In this report, we sought to produce α2,6-sialylated rA1AT with our cumate-inducible stable CHO pool expression system. Our different CHO pools could reach volumetric productivities of 1,2 g/L. The human α2,6-sialyltransferase was stably expressed in these cells in order to mimic elevated α2,6-sialylation levels of native A1AT protein. Sialylation of the recombinant protein was stable over the duration of the fed-batch production phase and was higher in a pool where cells were sorted and enriched by FACS based on cell-surface α2,6-sialylation. Addition of ManNAc to the cell culture media during production enhanced both α2,3 and α2,6 A1AT sialylation levels whereas addition of 2F-peracetylfucose potently inhibited fucosylation of the protein. Finally, we demonstrated that rA1AT proteins exhibited human neutrophil elastase inhibitory activities similar to the commercial human plasma-derived A1AT.

Mammalian-inducible expression systems are increasingly available and offer an attractive platform for the production of recombinant proteins. In this work, we have conducted process development for a cumate-inducible GS-CHO cell-line-expressing rituximab. To cope with the limitations encountered in batch when inducing at high cell densities, we have explored the use of fed-batch, sequential medium replacements, and continuous perfusion strategies applied during the pre-induction (growth) phase to enhance process performance in terms of product yield and quality. In shake flask, a fed-batch mode and a complete medium exchange at the time of induction were shown to significantly increase the integral of viable cell concentration and antibody titer compared to batch culture. Further enhancement of product yield was achieved by combining bolus concentrated feed additions with sequential medium replacement, but product galactosylation was reduced compared to fed-batch mode, as a result of the extended culture duration. In bioreactor, combining continuous perfusion of the basal medium with bolus daily feeding during the pre-induction period and harvesting earlier during the production phase is shown to provide a good trade-off between antibody titer and product galactosylation. Overall, our results demonstrate the importance of selecting a suitable operating mode and harvest time when carrying out high-cell-density induction to balance between culture productivity and product quality.

Foot and mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, which causes severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). Although inactivated virus vaccines have proved to be effective in FMD control, they have a number of disadvantages, including the need for high bio-containment production facilities and the lack of induction of immunological memory. Novel FMD vaccines based on the use of recombinant empty capsids have shown promising results. These recombinant empty capsids are attractive candidates because they avoid the use of virus in the production facilities but conserve its complete repertoire of conformational epitopes. However, many of these recombinant empty capsids require time-consuming procedures that are difficult to scale up. Achieving production of a novel and efficient FMD vaccine requires not only immunogenic antigens, but also industrially relevant processes. This review intends to summarize and compare the different strategies already published for the production of FMDV recombinant empty capsids, focusing on large-scale production.

Highlights • CHO cells are more efficient than 293 cells for expressing trimeric SARS-CoV-2 spike protein. • A trimerization domain and elimination of the S1/S2 furin site are essential to obtain spike trimers. • Unprecedented trimeric spike expression levels of 150 mg/L are obtained in CHO cells 7 days post-transfection., Recombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 spike ectodomain. A high-cell-density protocol using DXB11-derived CHOBRI/55E1 cells gave substantially better yields than the other methods. Different forms of the spike ectodomain were expressed, including the wild-type SARS-CoV-2 sequence and a mutated form (to favor expression of the full-length spike ectodomain stabilized in pre-fusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100-150 mg/L in the harvested medium. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.

Chinese hamster ovary (CHO) cell-based platforms are the most widely used for the biomanufacturing of complex therapeutic proteins, such as monoclonal antibodies (mAbs). The development of high-producing clones that are stable and amenable to large-scale cultures is essential to advance a molecule toward clinical evaluation. Nevertheless, the generation of such clones generally relies on random integration of an expression plasmid encoding the therapeutic protein gene into the host genome. The ensuing clone selection relying on empirical screens and cell line characterization is extensive and time-consuming. An emerging paradigm in CHO cell line development is the use of site-specific recombinases to enable the integration of therapeutic transgenes into pre-marked chromosomal locations with defined expression characteristics. Recombinase-mediated cassette exchange (RMCE) provides a sophisticated alternative to conventional CHO cell line development, leading to the generation of more consistent and reliable clones and may ultimately shorten the "time-to-clinic" of recombinant therapeutics. Herein, we review the recent advances in the use of site-specific recombination systems and their associated cassette exchange technologies for the rapid generation of stable CHO clones with predictable growth, stability, quality and productivity characteristics. Particular emphasis is placed on cassette exchange technologies currently used in the industry. We also discuss the technical hurdles associated with uses of site-specific recombinase systems in CHO cells, illustrate how these problems can be mitigated and provide a perspective on future work concerning these systems.

Protein A affinity chromatography, which is the standard method for monoclonal antibodies (mAbs) purification is expensive and involves elution at acidic pH, which could degrade the mAb. Moreover, it cannot be used to fractionate charge variants or to remove mAb aggregates. In this study, we examine the purification of Trastuzumab by cation-exchange z2 laterally-fed membrane chromatography (or z2LFMC). It has been shown that z2LFMC is suitable for carrying out high-speed, high-resolution protein purification. The results discussed in this paper demonstrate that the purity of Trastuzumab obtained by the z2LFMC process was comparable to that obtained by protein A chromatography (i.e., >95% with both methods), while the mAb recovery was significantly greater with z2LFMC (90.4% as opposed to 81.7%). Also, the z2LFMC process was faster, did not require acidic pH conditions for elution, and the mAb was eluted in a smaller pool (2.24 bed volumes as opposed to 2.74 bed volumes). The mAb productivity obtained with the z2LFMC process was more than 3 times higher than that obtained with the column-based purification processes. The z2LFMC device is significantly cheaper than its equivalent protein A affinity column, and earlier studies have shown to be suitable for separating mAb charge variants and aggregates. Therefore, the z2LFMC process could potentially be explored as a cheaper and more efficient alternative to protein A affinity chromatography.

Lactate and ammonia accumulation is a major factor limiting the performance of fed-batch strategies for mammalian cell culture processes. In addition to the detrimental effects of these by-products on production yield, ammonia also contributes to recombinant glycoprotein quality deterioration. In this study, we tackled the accumulation of these two inhibiting metabolic wastes by culturing in glutamine-free fed-batch cultures an engineered HEK293 cell line displaying an improved central carbon metabolism. Batch cultures highlighted the ability of PYC2-overexpressing HEK293 cells to grow and sustain a relatively high viability in absence of glutamine without prior adaptation to the culture medium. In fed-batch cultures designed to maintain glucose at high concentration by daily feeding a glutamine-free concentrated nutrient feed, the maximum lactate and ammonia concentrations did not exceed 5 and 1 mM, respectively. In flask, this resulted in more than a 2.5-fold increase in IFNα2b titer in comparison to the control glutamine-supplied fed-batch. In bioreactor, this strategy led to similar reductions in lactate and ammonia accumulation and an increase in IFNα2b production. Of utmost importance, this strategy did not affect IFNα2b quality with respect to sialylation and glycoform distribution as confirmed by surface plasmon resonance biosensing and LC-MS, respectively. Our strategy thus offers an attractive and simple approach for the development of efficient cell culture processes for the mass production of high-quality therapeutic glycoproteins. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:494-504, 2018.

Increasing recombinant protein production while ensuring a high and consistent protein quality remains a challenge in mammalian cell culture process development. In this work, we combined a nutrient substitution approach with a metabolic engineering strategy that improves glucose utilization efficiency. This combination allowed us to tackle both lactate and ammonia accumulation and investigate on potential synergistic effects on protein production and quality. To this end, HEK293 cells overexpressing the pyruvate yeast carboxylase (PYC2) and their parental cells, both stably producing the therapeutic glycoprotein interferon α2b (IFNα2b), were cultured in media deprived of glutamine but containing chosen substitutes. Among the tested substitutes, pyruvate led to the best improvement in growth (integral of viable cell density) for both cell lines in batch cultures, whereas the culture of PYC2 cells without neither glutamine nor any substitute displayed surprisingly enhanced IFNα2b production. The drastic reduction in both lactate and ammonia in the cultures translated into extended high viability conditions and an increase in recombinant protein titer by up to 47% for the parental cells and the PYC2 cells. Product characterization performed by surface plasmon resonance biosensing using Sambucus nigra (SNA) lectin revealed that the increase in yield was however accompanied by a reduction in the degree of sialylation of the product. Supplementing cultures with glycosylation precursors and a cofactor were effective at counterbalancing the lack of glutamine and allowed improvement in IFNα2b quality as evaluated by lectin affinity. Our study provides a strategy to reconcile protein productivity and quality and highlights the advantages of PYC2-overexpressing cells in glutamine-free conditions.

The accumulation of metabolic by-products remains a critical challenge in the development of mammalian cells culture processes as it impacts cellular growth, productivity and product quality. Although the overexpression of the PYC2 gene was shown to significantly improve the nutrient metabolism efficiency of mammalian cells, its impact on recombinant protein quality has not been investigated yet. In this study, we assess the effect of this metabolic engineering strategy on the quality of a recombinant therapeutic glycoprotein, the human interferon α2b (IFNα2b). As inferred from densitometry analysis of SDS-PAGE gels, PYC2-overexpressing cells sustained a higher percentage of intact glycosylated IFNα2b at the late stage of batch cultures, which was correlated with prolonged viability and reduced accumulation of waste metabolites. Contrarily to the IFNα2b produced by the PYC2 cells, LC–MS analysis confirmed the presence of less glycosylated IFNα2b as well as the occurrence of proteolytic cleavage in the IFNα2b produced in the parental cells. Taken together, these results indicate that PYC2-overexpression in mammalian cells leads to extended favorable conditions for glycosylation and offer an attractive approach to mass-produce high-quality recombinant proteins.

In order to maximize cell growth and productivity for an inducible CHO cell line expressing rituximab, various fed-batch culture strategies were investigated. In each case, the performance was evaluated for cultures induced at moderate and high cell density conditions (4 × 106 and 10 × 106 cells/mL) to assess the impact of the timing of induction. We first demonstrate the importance of starting the feeding process during the growth phase, as this translated into significantly improved integral of viable cells and antibody concentration, when compared to post-induction feeding only. Secondly, we investigated the impact of the feed rate by maintaining different levels of glucose (25, 35 and 50 mM) via a dynamic feeding strategy. The highest antibody concentrations were achieved under a moderate feeding regime for both cell densities at induction, highlighting the risks of under- or over-feeding the cultures. We then evaluated the impact of performing a temperature shift at induction by testing different mild hypothermia conditions. At small-scale, the highest production yields (1.2 g/L) were achieved when the temperature was reduced from 37 to 30 °C during the production phase of a culture induced at high cell density. When the strategy was applied in bioreactor, the better controlled conditions led to even greater product concentrations (1.8 g/L). Furthermore, this production protocol was shown to promote a more galactosylated glycan profile than a bioreactor culture initiated at 34 °C during growth and downshifted to 30 °C during the production phase.

Chinese hamster ovary (CHO) cells are the most widely used mammalian host for industrial-scale production of monoclonal antibodies (mAbs) and other protein biologics. Isolation of rare high-producing CHO cell lines from heterogeneous populations of stable transfectants is a daunting task and delays the process of manufacturing of novel biologics. A variety of factors that contribute to the low frequency of high-producing clones have been described; however, the impact of metabolic burden and other stresses (eg. ER stress) associated with sustained high-level expression of recombinant protein (r-protein) during selection of stable transfectants has not been fully appreciated. CHO cell line development has not traditionally received much optimization in this area because the vast majority of platforms use constitutive expression systems to produce biologics. Previously, we developed a cell line (CHOBRI/rcTA) containing a robust inducible expression system, based on the cumate gene switch, that allows r-protein expression to be down-regulated during selection. Using this switch, we generated inducible CHOBRI/rcTA pools expressing an Fc-fusion protein within two weeks of transfection with volumetric productivity of up to 1.1 g/L at 17 days post-induction in a fed-batch culture process. Herein, we show that the ability to regulate r-protein expression during pool generation confers a substantial advantage for selecting high-producing stable clones. Reducing expression levels (“off-state”) during pool selection dramatically enhances high-producer frequency compared to a pool in which expression was maintained at a high level during selection (“on-state”, mimicking a constitutive expression system). Overexpression of the r-protein during the pool selection process negatively affects pool recovery and is associated with subtle but significant increases in BiP expression and cell death compared to pool selection in the “off-state”. Our data shows that the cumate gene switch is a valuable platform for stable clone generation and supports the wider application of inducible systems for scalable production of biologics in CHO cells.

Background Dicer is a 219-kDa protein that plays key roles in gene regulation, particularly as the ribonuclease III enzyme responsible for cleaving precursor miRNA substrates. Its enzymatic activity is highly regulated by protein factors, and this regulation can impact on the levels of miRNAs and modulate the behavior of a cell. To better understand the underlying mechanisms of regulation, detailed enzymatic and structural characterization of Dicer are needed. However, these types of studies generally require several milligrams of recombinant protein, and efficient preparation of such quantities of pure human Dicer remains a challenge. To prepare large quantities of human Dicer, we have optimized transfection in HEK293-6E cells grown in suspension and streamlined a purification procedure. Results Transfection conditions were first optimized to achieve expression levels between 10 and 18 mg of recombinant Dicer per liter of culture. A three-step purification protocol was then developed that yields 4–9 mg of purified Dicer per liter of culture in a single day. From SEC-MALS/RI analysis and negative stain TEM, we confirmed that the purified protein is monomerically pure ( ≥ 98%) and folds with the characteristic L-shape geometry. Using an electrophoretic mobility shift assay, a dissociation constant (Kd) of 5 nM was measured for Dicer binding to pre-let-7a-1, in agreement with previous reports. However, when probing the cleavage activity of Dicer for pre-let-7a-1, we measured kcat (7.2 ± 0.5 min− 1) and KM (1.2 ± 0.3 μM) values that are much higher than previously reported due to experimental conditions that better respect the steady-state assumption. Conclusions The expression and purification protocols described here provide high yields of monomerically pure and active human Dicer. Cleavage studies of a pre-let-7 substrate with this purified Dicer reveal higher kcat and KM values than previously reported and support the current view that conformational changes are associated with substrate binding. Large quantities of highly pure Dicer will be valuable for future biochemical, biophysical and structural investigations of this key protein of the miRNA pathway. Electronic supplementary material The online version of this article (10.1186/s12896-018-0485-3) contains supplementary material, which is available to authorized users.

Expression of recombinant proteins exerts stress on cell culture systems, affecting the expression of endogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabolites. In this work, 2D-DIGE proteomics was employed to analyze differential expression of proteins following stable transfection of a Chinese Hamster Ovary (CHO) cell line to constitutively express a heavy-chain monoclonal antibody. Thirty-four proteins of significant differential expression were identified and cross-referenced with cellular functions and metabolic pathways to identify points of cell stress. Subsequently, 1D-(1)H NMR metabolomics experiments analyzed cultures to observe nutrient depletion and waste metabolite accumulations to further examine these cell stresses and pathways. From among fifty metabolites tracked in time-course, eight were observed to be completely depleted from the production media, including: glucose, glutamine, proline, serine, cystine, asparagine, choline, and hypoxanthine, while twenty-three excreted metabolites were also observed to accumulate. The differentially expressed proteins, as well as the nutrient depletion and accumulation of these metabolites corresponded with upregulated pathways and cell systems related to anaplerotic TCA-replenishment, NADH/NADPH replenishment, tetrahydrofolate cycle C1 cofactor conversions, limitations to lipid synthesis, and redox modulation. A nutrient cocktail was assembled to improve the growth medium and alleviate these cell stresses to achieve a ∼75% improvement to peak cell densities.

Three commercially available linear polyethylenimines (25kDa LPEI, 40kDa PEI"Max" and PEIpro™) were compared regarding their potency to transfect serum-free growing and suspension-adapted HEK293 and CHO cells. We determined the optimal DNA:PEI ratios for maximal expression of the reporter gene SEAP while monitoring cytotoxicity following transfection. PEIs acylation was determined by (1)H NMR and their apparent size and polydispersity assessed by size-exclusion chromatography. The propensity of PEIs to condense plasmid DNA was evaluated by agarose-gel electrophoresis. The zeta potentials and particle sizes at optimal DNA:PEI ratio were analyzed. Polyplex attachment to the cells and internalization kinetics were monitored. The quantity of PEIpro™ needed to efficiently transfect the cells was significantly lower than with LPEI and PEI"Max" and, interestingly, the maximal amount of internalized PEIpro™-based polyplexes was approximately half of that observed with its counterparts. PEIpro™ was the largest and least polydisperse polymer, but also the most cytotoxic. The optimal transfection conditions were subsequently used to express three monoclonal antibodies at larger-scale. The use of the deacylated PEI"Max" and PEIpro™ resulted in a significant increase of recombinant protein expression compared to LPEI. These findings demonstrate the importance of properly choosing the most suitable polymers to obtain optimal recombinant protein transient expression.

Recombinant yeast pyruvate carboxylase (PYC2) expression was previously shown to be an effective metabolic engineering strategy for reducing lactate formation in a number of relevant mammalian cell lines, but, in the case of CHO cells, did not consistently lead to significant improvement in terms of cell growth, product titer and energy metabolism efficiency. In the present study, we report on the establishment of a PYC2-expressing CHO cell line producing a monoclonal antibody and displaying a significantly altered lactate metabolism compared to its parental line. All clones exhibiting strong PYC2 expression were shown to experience a significant and systematic metabolic shift toward lactate consumption, as well as a prolonged exponential growth phase leading to an increased maximum cell concentration and volumetric product titer. Of salient interest, PYC2-expressing CHO cells were shown to maintain a highly efficient metabolism in fed-batch cultures, even when exposed to high glucose levels, thereby alleviating the need of controlling nutrient at low levels and the potential negative impact of such strategy on product glycosylation. In bioreactor operated in fed-batch mode, the higher maximum cell density achieved with the PYC2 clone led to a net gain (20%) in final volumetric productivity.

Inducible mammalian expression systems are becoming increasingly available and are not only useful for the production of cytotoxic/cytostatic products, but also confer the unique ability to uncouple the growth and production phases. In this work, we have specifically investigated how the cell culture state at the time of induction influences the cumate-inducible expression of recombinant rituximab by a GS-CHO cell line. To this end, cells grown in batch and fed-batch cultures were induced at increasing cell densities (1 to 10 × 10 6 cells/mL). In batch, the cell specific productivity and the product yield were found to reduce with increasing cell density at induction. A dynamic feeding strategy using a concentrated nutrient solution applied prior and postinduction allowed to significantly increase the integral of viable cells and led to a 3-fold increase in the volumetric productivity (1.2 g/L). The highest product yields were achieved for intermediate cell densities at induction, as cultures induced during the late exponential phase (10 × 10 6 cells/mL) were associated with a shortened production phase. The final glycosylation patterns remained however similar, irrespective of the cell density at induction. The kinetics of growth and production in a 2 L bioreactor were largely comparable to shake flasks for a similar cell density at induction. The degree of galactosylation was found to decrease over time, but the final glycan distribution at harvest was consistent to that of the shake flasks cultures. Taken together, our results provide useful insights for the rational development of fed-batch cell culture processes involving inducible CHO cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2742, 2019.

For pre-clinical evaluation of biotherapeutic candidates, protein production by transient gene expression (TGE) in Chinese Hamster Ovary (CHO) cells offers important advantages, including the capability of rapidly and cost-effectively generating recombinant proteins that are highly similar to those produced in stable CHO clones. We have established a novel CHO clone (CHO-3E7) expressing a form of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) with improved TGE productivity relative to parental CHO cells. Taking advantage of a new transfection-compatible media formulation that permits prolonged, high-density culture, we optimized transfection parameters (cell density, plasmid vector and polyethylenimine concentrations) and post-transfection culture conditions to establish a new, high-performing process for rapid protein production. The growth media is chemically defined, and a single hydrolysate feed is added post-transfection, followed by periodic glucose supplementation. This method gave significantly higher yields than our standard low-cell density, F17-based CHO-3E7 TGE method, averaging several hundred mg/L for a panel of recombinant proteins and antibodies. Purified antibodies produced using the two methods had distinct glycosylation profiles but showed identical target binding kinetics by SPR. Key advantages of this new protein production platform include the cost-effectiveness of the transfection reagent, the commercial availability of the culture media and the ability to perform high-cell-density transfection without media change.

Over the last years, the biopharmaceutical industry has significantly turned its biologics production towards mammalian cell expression systems. The presence of glycosylation machineries within these systems, and the fact that monoclonal antibodies represent today the vast majority of new therapeutic candidates, has largely influenced this new direction. Recombinant glycoproteins, including monoclonal antibodies, have shown different biological properties based on their glycan profiles. Thus, the industry has developed cell engineering strategies not only to improve cell's specific productivity, but also to adapt their glycosylation profiles for increased therapeutic activity. Additionally, the advance of "omics" technologies has recently given rise to new possibilities in improving these expression platforms and will significantly help developing new strategies, in particular for CHO (Chinese Hamster Ovary) cells.

The expression of recombinant yeast pyruvate carboxylase (PYC) in animal cell lines was shown in previous studies to reduce significantly the formation of waste metabolites, although it has translated into mixed results in terms of improved cellular growth and productivity. In this work, we demonstrate that the unique phenotype of PYC expressing cells can be exploited through the application of a dynamic fed-batch strategy and lead to significant process enhancements. Metabolically engineered HEK293 cells stably producing human recombinant IFNα2b and expressing the PYC enzyme were cultured in batch and fed-batch modes. Compared to parental cells, the maximum cell density in batch was increased 1.5-fold and the culture duration was extended by 2.5 days, but the product yield was only marginally increased. Further improvements were achieved by developing and implementing a dynamic fed-batch strategy using a concentrated feed solution. The feeding was based on an automatic control-loop to maintain a constant glucose concentration. This strategy led to a further 2-fold increase in maximum cell density (up to 10.7×106cells/ml) and a final product titer of 160mg/l, representing nearly a 3-fold yield increase compared to the batch process with the parental cell clone. © 2013.

To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHOBRI/rcTA) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHOBRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHOBRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins.

Mutations in leucine-rich glioma inactivated (LGI1) are a genetic cause of autosomal dominant temporal lobe epilepsy with auditory features. LGI1 is a secreted protein that shares homology with members of the SLIT family, ligands that direct axonal repulsion and growth cone collapse, and we therefore considered the possibility that LGI1 may regulate neuronal process extension or growth cone collapse. Here we report that LGI1 does not affect growth directly but instead enhances neuronal growth on myelin-based inhibitory substrates and antagonizes myelin-induced growth cone collapse. We show that LGI1 mediates this effect by functioning as a specific Nogo receptor 1 (NgR1) ligand that antagonizes the action of myelin-based inhibitory cues. Finally, we demonstrate that NgR1 and ADAM22 physically associate to form a receptor complex in which NgR1 facilitates LGI1 binding to ADAM22.

Mounting evidence indicates that mesenchymal stem cells (MSC) are pivotal to vascular repair and neointima formation in various forms of vascular disease. Yet, the mechanisms that allow MSC to resist apoptosis at sites where other cell types, such as endothelial cells (EC), are dying are not well defined. In the present work, we demonstrate that apoptotic EC actively release paracrine mediators which, in turn, inhibit apoptosis of MSC. Serum-free medium conditioned by apoptotic EC increases extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation and inhibits apoptosis (evaluated by Bcl-xL protein levels and poly (ADP-ribose) polymerase cleavage) of human MSC. A C-terminal fragment of perlecan (LG3) released by apoptotic EC is one of the mediators activating this antiapoptotic response in MSC. LG3 interacts with β1-integrins, which triggers downstream ERK1/2 activation in MSC, albeit to a lesser degree than medium conditioned by apoptotic EC. Hence, other mediators released by apoptotic EC are probably required for induction of the full antiapoptotic phenotype in MSC. Adopting a comparative proteomic strategy, we identified epidermal growth factor (EGF) as a novel mediator of the paracrine component of the endothelial apoptotic program. LG3 and EGF cooperate in triggering β1-integrin and EGF receptor-dependent antiapoptotic signals in MSC centering on ERK1/2 activation. The present work, providing novel insights into the mechanisms facilitating the survival of MSC in a hostile environment, identifies EGF and LG3 released by apoptotic EC as central antiapoptotic mediators involved in this paracrine response.

There are slightly over 165 recombinant pharmaceuticals currently approved for human use. Another 500 protein candidates are in preclinical and clinical development, about 70% of these being glycosylated proteins. The need for expression systems allowing the efficient manufacturing of high quality glycoproteins is thus becoming imperative. Recent developments with CHO cells, the predominant mammalian expression system, have focused on either increasing cell specific productivity or prolonging the life span of cells in culture that translates to high integrated viable cell densities. These two factors have allowed volumetric productivities in excess of 5 g/L under conditions of controlled nutrient feeding. In addition to glycoengineering strategies, which are offering considerable advantage in producing proteins with enhanced therapeutic properties, several alternative expression systems are being developed for their manufacture, each with their advantages and limitations.

Chitosan has been reported as a promising material for gene and drug delivery as well as for tissue engineering and regenerative medicine. We report here the conjugation of a de novo designed coil peptide (Kcoil) to chitosan ( M(n) = 200 kDa) to achieve a universal Kcoil-chitosan scaffold for subsequent immobilization of proteins tagged with the Kcoil partner, i.e., the Ecoil peptide. Kcoil-chitosan conjugate was synthesized using a tyrosinase-catalyzed protocol. Extensive UV/vis and IR characterization demonstrated that Kcoil peptide was covalently grafted to amines of chitosan. The ability of Kcoil-chitosan conjugate to recruit Ecoil tagged epidermal growth factor (EGF) was assessed by surface plasmon resonance measurements (SPR). Despite nonspecific interactions between chitosan and EGF, the specific formation of an E/K coiled coil complex was observed at slightly acidic pH and high salt concentration conditions, demonstrating that grafting to chitosan did not negatively impact binding characteristics of Kcoil peptide. Finally, the benefits of such bioconjugates for biomedical applications are discussed.

We have developed new packaging cell lines (293SFPacLV) that can produce lentiviral vectors (LVs) in serumfree suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2S-M2) of the tetracycline (Tet) switch, was established first. We next generated clones stably expressing the Gag/Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G). Expression of Rev and VSV-G was tightly regulated by the cumate and Tet switches. Our best packaging cells produced up to 2.6 × 10⁷ transducing units (TU)/ml after transfection with the transfer vector. Up to 3.4 × 10⁷ TU/ml were obtained using stable producers generated by transducing the packaging cells with conditional-SIN-LV. The 293SFPacLV was stable, as shown by the fact that some producers maintained high-level LV production for 18 weeks without selective pressure. The utility of the 293SF-PacLV for scaling up production in serum-free medium was demonstrated in suspension cultures and in a 3.5-L bioreactor. In shake flasks, the best packaging cells produced between 3.0 and 8.0 × 10⁶ TU/ml/day for 3 days, and the best producer cells, between 1.0 and 3.4 × 10⁷ TU/ml/day for 5 days. In the bioreactor, 2.8 liters containing 2.0 × 10⁶ TU/ml was obtained after 3 days of batch culture following the transfection of packaging cells. In summary, the 293SF-PacLV possesses all the attributes necessary to become a valuable tool for scaling up LV production for preclinical and clinical applications., available, unlimited, public

Recombinant adeno-associated virus (rAAV) has emerged in recent years as a promising gene therapy vector that may be used in the treatment of diverse human diseases. The major obstacle to broadening the usage of rAAV vectors remains the limited capacity of available production systems to provide sufficient rAAV quantities for preclinical and clinical trials. The impracticality of expanding commonly used adherent cell lines represents a limitation to large-scale production. This paper describes successful productions of rAAV type 2 using suspension-growing human embryonic kidney (HEK293) cells in serum-free medium. The developed process, based on triple transfection employing polyethylenimine (PEI) as DNA transporter, allowed for a serum-free production of AAV, yielding viral vector titer up to 4.5x10(11) infectious viral particles (IVP) in a 3.5-L bioreactor. A maximum ratio of VG:IVP in the order of 200:1 was obtained, indicating the efficient encapsidation of viral vectors in HEK293 cells. The effect of varying the ratio of three plasmids and the influence of cell density at transfection were studied. The conditioned medium did not limit or inhibit the rAAV production; therefore, the elimination of the medium exchange step before or after transfection greatly simplified the scale-up of rAAV production. The cell-specific viral titers obtained in bioreactor suspension cultures were similar or higher than those obtained with control adherent cell cultures which further supported the scalability of the process. From multiple aspects including process simplicity, scalability, and low operating costs, this transfection method appears to be the most promising technology for large-scale production of rAAV.

Transient gene expression in mammalian cells is intensively used for the rapid generation of recombinant proteins for biochemical, biophysical and pre-clinical studies. Still, the principles behind DNA transfer to the cells and the cellular cascade of events that ultimately dictate protein expression levels are not fully understood. Using polyethylenimine (PEI) mediated transfection of HEK293-EBNA1 cells, we sought to determine the most critical parameters that drive and limit recombinant protein production. Our results showed that a maximum of 65,000 plasmid copies/cell can be recovered in total extracts at 1 day post-transfection. Analyses performed after cell sorting revealed equal amounts of plasmid DNA in GFP-positive and -negative populations. However, nuclear plasmid content was three-fold higher in GFP-positive cells (1850 copies) than in GFP-negative cells (550 copies). The fact that significant amounts of plasmid DNA are found in the nucleus of GFP-negative cells suggests that its transcriptional competency is impaired. Interestingly, transfecting cells using a wide range of plasmid quantities at the optimal DNA:PEI ratio did not significantly affect the number of expressing cells. Thus, it appears that successful transgene expression is more likely to depend on a cellular "competent" state than to the quantity of plasmid DNA delivered per cell. Moreover, Northern blot analysis and SEAP/GFP measurement following plasmid titration experiments showed that transcriptional and translational processes are operating near to saturation under optimal transfection conditions. Overall, our results suggest that events that regulate nuclear translocation of plasmid DNA and its transcriptional competency as well as translational/post-translational limitations represent major bottlenecks in the success of a PEI-mediated protein production.

Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%. Crown copyright © 2004 Published by Elsevier Inc. All rights reserved.

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.

We describe the discovery of a novel, 20 kDa, secreted human protein named mesencephalic astrocyte-derived neurotrophic factor, or MANF. The homologous, native molecule was initially derived from a rat mesencephalic type-1 astrocyte cell line and recombinant MANF subcloned from a cDNA encoding human arginine-rich protein. MANF selectively protects nigral dopaminergic neurons, versus GABAergic or serotonergic neurons. The discovery of MANF marks a more systematic approach in the search for astrocyte-derived, secreted proteins that selectively protect specific neuronal phenotypes. Compared to glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), MANF was more selective in the protection of dopaminergic neurons at lower (0.05–0.25 ng/mL) and middle (0.5–2.5 ng/mL) concentrations: MANF>GDNF>BDNF. GDNF was more selective at higher concentrations (25–50 ng/ml): GDNF>MANF>BDNF. Two domains in MANF of 39-AA and 109-AA respectively, and eight cysteines are conserved from C. elegans to man. MANF is encoded by a 4.3 Kb gene with 4 exons, and is located on the short arm of human chromosome 3. The secondary structure is dominated by α-helices (47%) and random coils (37%). Studies to determine the localization of MANF in the brains of rat, monkey, and man, as well as the receptor, signaling pathways, and biologically active peptide mimetics are in progress. The selective, neuroprotective effect of MANF for dopaminergic neurons suggests that it may be indicated for the treatment of Parkinson’s disease., DA - 20030609IS - 0895-8696LA - engPT - Journal ArticleRN - 0 (Brain-Derived Neurotrophic Factor)RN - 0 (DNA, Complementary)RN - 0 (MANF protein, human)RN - 0 (Nerve Growth Factors)RN - 0 (Nerve Tissue Proteins)RN - 0 (Neuroprotective Agents)RN - 0 (glial cell-line derived neurotrophic factor)RN - 51-61-6 (Dopamine)SB - IM

Chimeric growth factors may represent a powerful alternative to their natural counterparts for the functionalization of tissue-engineered scaffolds and applications in regenerative medicine. Their rational design should provide a simple, readily scalable production strategy while improving retention at the site of action. In that endeavor, we here report the synthesis of a chimeric protein corresponding to human vascular endothelial growth factor 165 being N-terminally fused to an E5 peptide tag (E5-VEGF). E5-VEGF was successfully expressed as a homodimer in mammalian cells. Following affinity purification, in vitro surface plasmon resonance biosensing and cell survival assays confirmed diffusible E5-VEGF ability to bind to its receptor ectodomains, while observed morphological phenotypes confirmed its anti-apoptotic features. Additional surface plasmon resonance assays highlighted that E5-VEGF could be specifically captured with high stability when interacting with covalently immobilized K5 peptide (a synthetic peptide designed to bind to the E5 moiety of chimeric hVEGF). This immobilization strategy was applied to glass substrates and chimeric hVEGF was shown to be maintained in a functionally active state following capture. Altogether, our data demonstrated that stable hVEGF capture can be performed via coiled-coil interactions without impacting hVEGF bioactivity, thus opening up the way to future applications in the field of tissue engineering and regenerative medicine. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Targeted and simultaneous delivery VEGF165b and IFN alpha in anti-angiogenic and other applications could offer several advantages. For this a system was design using artificial cell alginate-poly-L-lysine- alginate (APA) microcapsules. Result confirms the ability of this system for simultaneous production of these proteins for 28-days. The IFN alpha on a 3 days period increased from 8 ± 0.36 μg/ml at day 10 to 27 ± 2.4 μg/ml at day 16 and then dropped to 6.5 ± 0.5 μg/ml. The VEGF165b on a 3 days period increased from 2.7 ± 0.7 μg/ml at day 10 to 6.9 ± 1 μg/ml at day 16.

There is an imperative need for expression systems allowing the efficient and robust manufacturing of high quality glycoproteins. In the present work, HEK-293 cells stably expressing interferon-+¦2b were further engineered with the insertion of the yeast pyruvate carboxylase 2 gene. In batch cultures, marked reductions in lactate and ammonia production were observed compared to the parental cell clone. Although the maximum specific growth rate remained unchanged, the altered metabolism led to a 2-fold increase in maximum cell density and 33% increase in the integral of viable cell concentration and interferon production yield. The underlying metabolic changes were further investigated using various 13C-labeled substrates and measuring the resulting lactate mass isotopomer distributions. Simultaneous metabolite and isotopomer balancing allowed the accurate determination of key intracellular fluxes. Such detailed and quantitative knowledge about the central carbon metabolism of the cells is instrumental to further support the development of high-yield fed-batch processes. -¬ 2011 Elsevier

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 2-6 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 "direct" transfection method thus provides a robust platform for both high-throughput expression and large-scale production of recombinant proteins.

Background Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C. Results We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Conclusion Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability.

Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His) 8 to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%. Crown Copyright © 2004.

The utility of porous metals for the integration of orthopaedic implants with host bone has been well established. Quantification of the tissue response to cementless implants is laborious and time consuming process requiring tissue processing, embedding, sectioning, polishing, imaging and image analysis. Micro-computed tomography (μCT) is a promising three dimensional (3D) imaging technique to quantify the tissue response to porous metals. However, the suitability and effectiveness of μCT for the quantification of bone ingrowth remains unknown. The purpose of this study was to evaluate and compare bone growth within porous titanium implants using both μCT and traditional hard-tissue histology techniques. Cylindrical implants were implanted in the distal femora and proximal tibiae of a rabbit. After 6 weeks, bone ingrowth was quantified and compared by μCT, light microscopy and backscattered electron microscopy. Quantification of bone volume and implant porosity as determined by μCT compared well with data obtained by traditional histology techniques. Analysis of the 3D dataset showed that bone was present in the pores connected with openings larger 9.4 μm. For pore openings greater than 28.2 μm, the size of the interconnection had little impact on the bone density within the porosity for the titanium foams.

In an effort to evaluate the impact of various epidermal growth factor (EGF) grafting strategies upon cell surface receptor activation and cell adhesion, we generated low-fouling surfaces by homogeneously grafting carboxymethylated dextran (CMD) on amino-coated glass substrate. By preventing nonspecific cell adhesion while providing reactive groups facilitating subsequent protein grafting, CMD allowed achieving specific cell/tethered EGF interactions and therefore deriving unambiguous conclusions about various EGF grafting strategies. We demonstrate here that A-431 cell response to immobilized EGF is highly dependent on the bioactivity of the tagged protein being tethered, its proper orientation, and its surface density. Among all the approaches we tested, the oriented tethering of fully bioactive EGF via a de novo-designed coiled-coil capture system was shown to be the most efficient. That is, it led to the most intense and sustained phosphorylation of EGF receptors as well as to strong A-431 cell adhesion, the latter being comparable to that observed with amino-coated surfaces in the absence of CMD.

The de novo designed heterodimeric E/K coiled-coil system has been previously demonstrated to be an excellent capture/dimerization system applicable to various needs in both biotechnology and pharmaceutical fields. Those include controlled protein dimerization, capture, purification and Western-blot detection. We here report the development of a new generation of ELISA test based on coiled-coil interactions for the direct quantitation of coil-tagged epidermal growth factor (EGF). The new approach was evaluated for its specificity, plate storability and reusability as well as for convenience when compared to commercially available systems. Our results show a similar affinity/sensitivity to standard capturing antibody-based ELISA systems and an improved affinity/sensitivity when compared to the commercially available Ni-NTA capture system. The E/K coiled-coil ELISA system was validated with respect to recovery, intra- and inter-assay variations. The practical working range was estimated to be between 5.2 and 34,000 pM. Furthermore, the storability and reusability of the plates was greater than the two aforementioned systems, suggesting that the E/K coiled-coil system is a good alternative to traditional tags such as poly-histidine for the development of ELISA tests aiming at quantitating coil-tagged proteins. -� 2010 Elsevier B.V

An important goal in modern biology is to understand how molecular processes commonly studied at the cellular level give rise to physiological functions in complex tissues and organisms. Non-invasive imaging of gene-expression patterns in whole animals could provide information critical to this end, but current methods lack sensitivity and spatiotemporal precision. Enzymatic reporter systems detectable by magnetic resonance imaging (MRI) address these limitations by combining the relatively high spatial and temporal resolution of MRI with the ability of each genetically expressed enzyme to generate many MRI-detectable product molecules.1, 2 A challenge with the imaging-based detection of some of the most popular reporter enzymes is the need to deliver MRI probes to their sites of action within cells. Herein we describe a new reporter-gene system for MRI that relieves this problem by harnessing an extracellular enzyme, the mammalian secreted alkaline phosphatase (SEAP)., National Institutes of Health (U.S.) (grant DP2-OD002114)

To investigate the antiangiogenic potential of encapsulated VEGF165b producing HEK293 cells, Human Embryonic Kidney 293 (HEK293) cells were stably transfected to produce VEGF165b. Then they were encapsulated in alginate - polylysine -alginate (APA) microcapsules. VEGF165b productivity and viability of encapsulated cells were analyzed and compared with the non-encapsulated cells. Results showed that encapsulated cells proliferated and remained viable within the microcapsules throughout the 28-day period of the experiment. The quantity of VEGF165b increased from6.5±1.2 μg/ml at day 13 to13±0.96 μg/ml at day 16. Then it gradually dropped to5±1.2 μg/ml for the last 3 days period as measured at day 28. Production of VEGF165b from encapsulated and non-encapsulated cells was similar. The effect of VEGF165b harvested from encapsulated cells on Human Umbilical Vein Endothelial cells (HUVECs) proliferation were also examined.The same inhibitory effects on HUVECs proliferation was seen when the cells were incubated with a mixture of VEGF165b and a 2-fold VEGF165b or with VEGF165b and 2-fold excess VEGF165b released from encapsulated cells. Subcutaneous injection of microencapsulated VEGF165b producing cells in tumor site of nude mice resulted in the reduction of the number of vessels around the tumors.

Multiplasmid transient transfection is the most widely used technique for the generation of lentiviral vectors. However, traditional transient transfection protocols using 293 T adherent cells and calcium phosphate/DNA co-precipitation followed by ultracentrifugation are tedious, time-consuming, and difficult to scale up. This chapter describes a streamlined protocol for the fast mass production of lentiviral vectors and their purification by affinity chromatography. Lentiviral particles are generated by transient transfection of suspension growing HEK 293 cells in serum-free medium using polyethylenimine (PEI) as transfection reagent. Lentiviral vector production is carried out in Erlenmeyer flasks agitated on orbital shakers requiring minimum supplementary laboratory equipment. Alternatively, the method can be easily scaled up to generate larger volumes of vector stocks in bioreactors. Heparin affinity chromatography allows for selective concentration and purification of lentiviral particles in a singlestep directly from vector supernatants. The method is suitable for the production and purification of different vector pseudotypes.

We propose an approach for the detection of proteins by Western blot that takes advantage of the high-affinity interaction occurring between two de novo designed peptides, the E and K coils. As a model system, K coil-tagged epidermal growth factor (EGF) was revealed with secreted alkaline phosphatase (SeAP) tagged with E coil (SeAP-Ecoil) as well as with biotinylated E coil. In that respect, we first produced purified SeAP-Ecoil and verified its ability to interact with K coil peptides by surface plasmon resonance biosensing. We demonstrated that protein detection with Ecoil-biotin was more specific than with SeAP-Ecoil. We then showed that our approach is as sensitive as conventional detection strategies relying on nickel-nitrilotriacetic acid-horseradish peroxidase (Ni-NTA-HRP), anti-His-HRP, or anti-EGF. Altogether, our results indicate that the E/K coiled-coil system is a good alternative for protein detection by Western blot.