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Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells are the two most important mammalian hosts for the production of recombinant proteins. In this chapter, the suspension cultivation and transfection of these cells in small-scale, single-use orbitally shaken bioreactors, TubeSpin™ bioreactor 50 [orbitally shaken reactor 50 (OSR50)], and TubeSpin™ bioreactor 600 [orbitally shaken reactor 600 (OSR600)] are described. These are conical centrifuge tubes with nominal volumes of 50 mL and 600 mL, respectively, that have been redesigned with a ventilated cap for the cultivation of animal cells in suspension at working volumes up to 20 mL and 400 mL, respectively., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In this study, the combination of dimensional analysis (DA) and analysis of variance (ANOVA) was used to predict the volumetric mass transfer coefficient ( k L a ) values under different operating conditions for orbitally shaken bioreactors (OSRs) with different filling volumes. It was found that Reynolds number and the interaction between Froude number and geometric number have the largest impact on k L a with impact indexes of 7.41 and 7.50, respectively. Moreover, the volume number has the largest negative impact on k L a , with an impact index of - 5.34. Thus, an effective way to increase the oxygen supply is by increasing the shaking speed and shaking diameter or decreasing the vessel diameter. However, cell cultivation with a higher filling volume will have an increased risk of oxygen scarcity. Therefore, with the help of the k L a prediction model, a suitable operating condition can be determined effectively and easily., Competing Interests: Conflict of interestThe authors confirm that there are no conflicts of interest., (© King Abdulaziz City for Science and Technology 2020.)

The presence of the proteins mouse R-Spondin1 (mRSpo1) and mouse Noggin (mNoggin) in a 3D-organoid culture allows for the maintenance of intestinal stem cells. Here, we describe a transient gene expression method for the production of these proteins from human embryo kidney 293 (HEK293) cells cultivated in suspension using orbitally shaken bioreactors. Plasmid DNA was delivered into cells using the cationic polymer polyethylenimine (PEI). The 7-day production cultures were performed in the presence of valproic acid (VPA), an enhancer of recombinant gene expression. Both proteins were secreted from the transfected cells. mRSpo1 was produced as a secreted Fc fusion protein (mRSpo1-Fc) and purified by protein A-based affinity chromatography. mNoggin was produced as a secreted histidine-tagged protein (mNoggin-His) and purified by immobilized metal affinity chromatography (IMAC). This transient transfection system supports a high production efficiency.

Objectives: To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line., Results: When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR1 2xHis ) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested., Conclusions: Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.

Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells are the two most popular mammalian hosts for the production of recombinant proteins. In this chapter the suspension cultivation and transfection of these cells in small-scale disposable bioreactors is described. The TubeSpin bioreactor 50 and TubeSpin bioreactor 600 are designed for the cultivation of suspension cells by orbital shaking and have maximum working volumes of about 15 mL and 400 mL, respectively.

We describe a one-liter transfection of suspension-adapted Chinese hamster ovary (CHO-DG44) cells using polyethyleneimine (PEI) for DNA delivery. The method involves transfection at a high cell density (5 × 10 6 cells/mL) by direct addition of plasmid DNA (pDNA) and PEI to the culture and subsequent incubation at 31 °C with agitation by orbital shaking. We also describe an alternative method in which 90% of the pDNA is replaced by nonspecific (filler) DNA, and the production phase is performed at 31 °C in the presence of 0.25% N, N-dimethylacetamide (DMA).

Orbitally shaken cylindrical bioreactors [OrbShake bioreactors (OSRs)] without an impeller or sparger are increasingly being used for the suspension cultivation of mammalian cells. Among small volume OSRs, 50-mL tubes with a ventilated cap (OSR50), originally derived from standard laboratory centrifuge tubes with a conical bottom, have found many applications including high-throughput screening for the optimization of cell cultivation conditions. To better understand the fluid dynamics and gas transfer rates at the liquid surface in OSR50, we established a three-dimensional simulation model of the unsteady liquid forms (waves) in this vessel. The studies verified that the operating conditions have a large effect on the interfacial surface. The volumetric mass transfer coefficient (k L a) was determined experimentally and from simulations under various working conditions. We also determined the liquid-phase mass transfer coefficient (k L ) and the specific interfacial area (a) under different conditions to demonstrate that the value of a affected the gas transfer rate more than did the value of k L . High oxygen transfer rates, sufficient for supporting the high-density culture of mammalian cells, were found. Finally, the average axial velocity of the liquid was identified to be an important parameter for maintaining cells in suspension. Overall these studies provide valuable insights into the preferable operating conditions for the OSR50, such as those needed for cell cultures requiring high oxygen levels. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:192-200, 2017., (© 2016 American Institute of Chemical Engineers.)

Orbitally shaken bioreactors (OSRs) support the suspension cultivation of animal cells at volumetric scales up to 200 L and are a potential alternative to stirred-tank bioreactors (STRs) due to their rapid and homogeneous mixing and high oxygen transfer rate. In this study, a Chinese hamster ovary cell line producing a recombinant antibody was cultivated in a 5 L OSR and a 3 L STR, both operated with or without pH control. Effects of bioreactor type and pH control on cell growth and metabolism and on recombinant protein production and glycosylation were determined. In pH-controlled bioreactors, the glucose consumption and lactate production rates were higher relative to cultures grown in bioreactors without pH control. The cell density and viability were higher in the OSRs than in the STRs, either with or without pH control. Volumetric recombinant antibody yields were not affected by the process conditions, and a glycan analysis of the antibody by mass spectrometry did not reveal major process-dependent differences in the galactosylation index. The results demonstrated that OSRs are suitable for recombinant protein production from suspension-adapted animal cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1174-1180, 2016., (© 2016 American Institute of Chemical Engineers.)

Heterogenous populations of recombinant cells (cell pools) stably expressing 1-4 transgenes were generated from Chinese hamster overy (CHO) cells with the piggyBac (PB) transposon system. The cell pools produced different combinations of three model proteins-enhanced green fluorescent protein (EGFP), secreted alkaline phosphatase (SEAP), and a monoclonal IgG1 antibody. Each transgene was present on a separate PB donor plasmid with either the same or a different selection gene. In both cases, we obtained PB-derived cell pools with higher recombinant protein yields than from cell pools generated by conventional gene delivery. In PB-derived cell pools generated using a single selection agent, both protein production and the number of integrated copies of each transgene declined as the number of transfected transgenes increased. However, the total number of integrated transgenes was similar regardless of the number of different transgenes transfected. For PB-derived cell pools generated by selection of each transgene with a different selection agent, the total number of integrated transgenes increased with the number of transfected transgenes. The results suggest that the generation of cell pools producing multiple recombinant proteins is feasible and that the method is more efficient when each individual transgene is selected with a different marker. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1308-1317, 2016., (© 2016 American Institute of Chemical Engineers.)

Disposable orbitally shaken TubeSpin bioreactor 600 tubes (TS600s) were recently developed for the bench-scale cultivation of animal cells in suspension. Here we compared batch cultures of Sf9 insect cells in TS600s, spinner flasks, and shake flasks. Superior cell growth was observed in TS600s and shake flasks as compared with spinner flasks, and more favorable oxygen-enriched cell culture conditions were observed in TS600s as compared with either spinner or shake flasks. The results demonstrated the suitability of TS600s as a disposable vessel for the cultivation of Sf9 cells in suspension., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Several naturally occurring vertebrate transposable elements have been genetically modified to enable the transposition of recombinant genes in mammalian cells. We compared three transposons-piggyBac, Tol2, and Sleeping Beauty-for their ability to generate cell pools (polyclonal cultures of recombinant cells) and clonal cell lines for the large-scale production of recombinant proteins using Chinese hamster ovary cells (CHO-DG44) as the host. Transfection with each of the dual-vector transposon systems resulted in cell pools with volumetric yields of tumor necrosis factor receptor-Fc fusion protein (TNFR:Fc) that were about ninefold higher than those from cell pools generated by conventional plasmid transfection. On average, the cell pools had 10-12 integrated copies of the transgene per cell. In the absence of selection, the volumetric productivity of the cell pools decreased by 50% over a 2-month cultivation period and then remained constant. The average volumetric TNFR:Fc productivity of clonal cell lines recovered from cell pools was about 25 times higher than that of cell lines generated by conventional transfection. In 14-day fed-batch cultures, TNFR:Fc levels up to 900 mg/L were obtained from polyclonal cell pools and up to 1.5 g/L from clonal cell lines using any of the three transposons. Biotechnol. Bioeng. 2016;113: 1234-1243. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

The High Five (H5) cell line, derived from the lepidopteran Trichoplusia ni, is one of the major insect cell hosts for the production of recombinant proteins using the baculovirus expression vector system (BEVS). Here, we describe a simple polyethylenimine (PEI)-based transient gene expression (TGE) process for the rapid production of recombinant proteins from suspension-adapted H5 cells. The method was optimized using two model proteins, enhanced green fluorescent protein (EGFP) and human tumor necrosis factor receptor-Fc fusion protein (TNFR-Fc). After screening several promoter and enhancer combinations for high levels of TNFR:Fc production, an expression vector containing the Autographa californica multicapsid nucleopolyhedrovirus immediate early 1 (ie1) promoter and homologous region 5 (hr5) enhancer was selected. Cells were transfected at a density of 2×10(6) cells/mL by direct addition of DNA and PEI. Under optimized conditions, a 90% transfection efficiency (percentage of EGFP-positive cells) was obtained. In addition, we observed volumetric TNFR-Fc yields over 150μg/mL within 4 days of transfection. The method was found to be reproducible and scalable to 300mL. This plasmid-based transient transfection process is a simple and efficient alternative to the BEVS for recombinant protein production in H5 cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD)-associated γ-secretase is a ubiquitously expressed multi-subunit protease complex embedded in the lipid bilayer of cellular compartments including endosomes and the plasma membrane. Although γ-secretase is of crucial interest for AD drug discovery, its atomic structure remains unresolved. γ-Secretase assembly and maturation is a multistep process, which includes extensive glycosylation on nicastrin (NCT), the only γ-secretase subunit having a large extracellular domain. These posttranslational modifications lead to protein heterogeneity that likely prevents the three-dimensional (3D) crystallization of the protease complex. To overcome this issue, we have engineered a Chinese hamster ovary (CHO) cell line deficient in complex sugar modifications (CHO lec1) to overexpress the four subunits of γ-secretase as a functional complex. We purified glycosylation-deficient γ-secretase from this recombinant cell line (CL1-9) and fully glycosylated γ-secretase from a recombinant CHO DG44-derived cell line (SS20). We characterized both complexes biochemically and pharmacologically in vitro. Interestingly, we found that the complex oligosaccharides, which largely decorate the extracellular domain of fully glycosylated NCT, are not involved in the proper assembly and maturation of the complex, and are dispensable for the specific generation, in physiological ratios, of the amyloid precursor protein (APP) cleavage products. In conclusion, we propose a novel bioengineering approach for the production of functional glycosylation-deficient γ-secretase, which may be suitable for crystallization studies. We expect that these findings will contribute both to solving the high-resolution 3D structure of γ-secretase and to structure-based drug discovery for AD., (© 2015 Wiley Periodicals, Inc.)

Although the protein yields from transient gene expression (TGE) with Chinese hamster ovary (CHO) cells have recently improved, the amount of plasmid DNA (pDNA) needed for transfection remains relatively high. We describe a strategy to reduce the pDNA amount by transfecting CHO-DG44 cells with 0.06 μg pDNA/10(6) cells (10% of the optimal amount) in the presence of nonspecific (filler) DNA and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide (DMA), and hexamethyl phosphoramide (HMP). All of the polar solvents with the exception of HMP increased the production of a recombinant antibody in comparison to the untreated control transfection. In the presence of 0.25% DMA, the antibody yield in a 7-day batch culture was 500 mg/L. This was fourfold higher than the yield from the untreated control transfection. Mechanistic studies revealed that the polar solvents did not affect polyethylenimine-mediated pDNA delivery into cells or nuclei. The steady-state transgene mRNA level was elevated in the presence of each of the polar solvents tested, while the transgene mRNA half-life remained the same. These results indicated that the polar solvents enhanced transgene transcription. When screening a panel of recombinant antibodies and Fc-fusion proteins for production in the presence of the polar solvents, the highest increase in yield was observed following DMA addition for 11 of the 12 proteins. These results are expected to enhance the applicability of high-yielding TGE processes with CHO-DG44 cells by decreasing the amount of pDNA required for transfection., (© 2015 American Institute of Chemical Engineers.)

Heterogeneous populations of stably transfected cells (cell pools) can serve for the rapid production of moderate amounts of recombinant proteins. Here, we propose the use of the piggyBac (PB) transposon system to improve the productivity and long-term stability of cell pools derived from Chinese hamster ovary (CHO) cells. PB is a naturally occurring genetic element that has been engineered to facilitate the integration of a transgene into the genome of the host cell. In this report PB-derived cell pools were generated after 10 days of selection with puromycin. The resulting cell pools had volumetric productivities that were 3-4 times higher than those achieved with cell pools generated by conventional plasmid transfection even though the number of integrated transgene copies per cell was similar in the two populations. In 14-day batch cultures, protein levels up to 600 and 800 mg/L were obtained for an Fc-fusion protein and a monoclonal antibody, respectively, at volumetric scales up to 1L. In general, the volumetric protein yield from cell pools remained constant for up to 3 months in the absence of selection. In conclusion, transfection of CHO cells with the PB transposon system is a simple, efficient, and reproducible approach to the generation of cell pools for the rapid production of recombinant proteins., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Transient gene expression (TGE) in human embryonic kidney (HEK-293) and Chinese hamster ovary (CHO) cells is a well-established technology for the rapid generation of recombinant proteins. Although the maximum TGE yields have reached 1 g/L or more, the amount of plasmid DNA (pDNA) required for transfection remains high. Although greater than 10(3) copies of pDNA are present per transfected cell, protein yields are still lower than those achieved in recombinant cell lines with only one or a few copies of the transgene. This indicates a clear limitation to TGE in terms of the maximum level of recombinant protein production. In this study, we investigated the limitations to high-yielding TGE processes with CHO and HEK-293E cells using a monoclonal antibody as a model protein. For either cell host, both the intracellular and intranuclear pDNA levels increased linearly with the amount of pDNA added to the culture. In contrast, transgene mRNA accumulation reached a plateau as the intranuclear pDNA amount increased, suggesting a limitation in pDNA transcription. A post-transcriptional limitation to TGE yields was revealed by calculating the amount of antibody produced per transgene mRNA (mRNA utilization). For both hosts the transgene mRNA utilization decreased dramatically when transfected pDNA amounts increased beyond the level giving the maximum protein yield. The post-transcriptional limitation did not appear to be due to bottlenecks in antibody assembly or secretion, suggesting that transgene mRNA translation may be limiting. The results show that TGE yields are not limited by pDNA delivery into the nuclei, but in pDNA and transgene mRNA utilization., (© 2014 American Institute of Chemical Engineers.)

A method for virus-free transient gene expression from suspension-adapted Sf9 insect cells was developed with the gene of interest being expressed from a plasmid carrying the homologous region 5 enhancer (hr5) and the immediate early 1 (ie1) promoter from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Under the optimal conditions described in the study, cells were transfected at a density of 30×10⁶ cells/mL with 0.9 μg DNA and 1.35 μg of linear 25 kD polyethylenimine (PEI) per million cells. Following transfection, the culture was diluted to 4×10⁶ cells/mL for the protein production phase. The volumetric yield of tumor necrosis factor receptor (ectodomain) fused to an Fc domain (TNFR-Fc) was about 100 μg/mL for cultures at volumes up to 300 mL. As expected, the molecular weight of the dimeric TNFR-Fc produced from Sf9 cells was about 6 kDa less than that produced from a recombinant Chinese hamster ovary (CHO) cell line due to differences in glycosylation between the two hosts. Transient transfection provides an alternative to the baculovirus expression vector system (BEVS) for the rapid production of recombinant proteins from Sf9 cells., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Transient gene expression (TGE) from mammalian cells is an increasingly important tool for the rapid production of recombinant proteins for research applications in biochemistry, structural biology, and biomedicine. Here we review methods for the transfection of human embryo kidney (HEK-293) and Chinese hamster ovary (CHO) cells in suspension culture using the cationic polymer polyethylenimine (PEI) for gene delivery., (Copyright © 2013 Elsevier Inc. All rights reserved.)

PEI and polylysine are among the most investigated synthetic polymeric carriers for DNA delivery. Apart from their practical use, these 2 classes of polymers are also of interest from a fundamental point of view as they both can be prepared in different architectures (linear and branched/dendritic) and in a wide range of molecular weights, which is attractive to establish basic structure-activity relationships. This manuscript reports the results of an extensive study on the influence of molecular weight and architecture of a library of polylysine variants that includes linear, dendritic and hyperbranched polylysine. Hyperbranched polylysine is a new polylysine-based carrier that is structurally related to dendritic polylysine but possesses a randomly branched structure. Hyperbranched polylysine is attractive as it can be prepared in a one-step process on a large scale. The performance of these 3 classes of polylysine analogs was evaluated by assessing eGFP and IgG production in transient gene expression experiments with CHO DG44 cells, which revealed that protein production generally increased with increasing molecular weight and that at comparable molecular weight, the hyperbranched analogs were superior as compared to the dendritic and linear polylysines. To understand the differences between the gene delivery properties of the hyperbranched polylysine analogs on the one hand and the dendritic and linear polylysines on the other hand, the uptake and trafficking of the corresponding polyplexes were investigated. These experiments allowed us to identify (i) polyplex-external cell membrane binding, (ii) free, unbound polylysine coexisting with polyplexes as well as (iii) polymer buffer capacity as three possible factors that may contribute to the superior transfection properties of the hyperbranched polylysines as compared to their linear and dendritic analogs. Altogether, the results of this study indicate that hyperbranched polylysine is an interesting, alternative synthetic gene carrier. Hyperbranched polylysine can be produced at low costs and in large quantities, is partially biodegradable, which may help to prevent cumulative cytotoxicity, and possesses transfection properties that can approach those of PEI., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Inefficient production of membrane-embedded multi-protein complexes by conventional methods has largely prevented the generation of high-resolution structural information and the performance of high-throughput drug discovery screens for this class of proteins. Not exempt from this rule is γ-secretase, an intramembrane-cleaving protease complex regulating a multitude of signaling pathways and biological processes by influencing gene transcription. γ-Secretase is also implicated in the pathogenesis of Alzheimer's disease and several types of cancer. As an additional challenge, the reconstitution of the protease complex in its active form requires an intricate assembly and maturation process, including a highly regulated endoproteolytic processing of its catalytic component. In this article we report the application of a transposon-mediated multigene stable integration technology to produce active γ-secretase in mammalian cells in amounts adequate for crystallization studies and drug screening. Our strategy is expected to help elucidate the molecular mechanisms of intramembrane proteolysis. It is further expected to be widely used for the production of other multi-protein complexes for applications in structural biology and drug development., (Copyright © 2013 Wiley Periodicals, Inc.)

We describe a rapid method for monitoring the cell growth and decline phases in suspension cultures of animal cells. During the cell growth phase, ultraviolet (UV)-absorbing components in the medium are consumed, but at later times as cells begin to die, UV-absorbing molecules such as proteins are released into the medium. Measuring the absorbance at 280nm (A(280)) with a NanoDrop spectrophotometer, an inverse correlation between the onset of the cell decline phase and A(280) was observed. This simple method can be applied to quickly determine the beginning of the decline phase of cultures of mammalian and insect cells in suspension., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the TGE volumetric productivity has improved significantly over the past decade, the amount of plasmid DNA (pDNA) needed for transfection remains very high. Here, we examined the use of non-specific (filler) DNA to partially replace the transgene-bearing plasmid DNA (coding pDNA) in transfections of Chinese hamster ovary (CHO) and human embryo kidney (HEK-293E) cells. When the optimal amount of coding pDNA for either host was reduced by 67% and replaced with filler DNA, the recombinant protein yield decreased by only 25% relative to the yield in control transfections. Filler DNA did not affect the cellular uptake or intracellular stability of coding pDNA, but its presence lead to increases of the percentage of transfected cells and the steady-state level of transgene mRNA compared to control transfections. Studies of the physicochemical properties of DNA-polyethyleneimine (PEI) complexes with or without filler DNA did not reveal any differences in their size or surface charge. The results suggest that filler DNA allows the coding pDNA to be distributed over a greater number of DNA-PEI complexes, leading to a higher percentage of transfected cells. The co-assembly of filler DNA and coding pDNA within complexes may also allow the latter to be more efficiently utilized by the cell's transcription machinery, resulting in a higher level of transgene mRNA., (Copyright © 2012 Wiley Periodicals, Inc.)

Three synthesis lots of linear poly(ethyleneimine) (PEI) are compared to a fully hydrolyzed linear PEI (commercially available as PEI "Max") regarding structure, polyplex formation with plasmid DNA, and transfection of suspension-adapted HEK-293E cells. PEI "Max" binds DNA more efficiently than the other PEIs, but it is the least effective in terms of transient recombinant protein yield. One PEI lot is fractionated by means of SEC. The fractions of high-M(n) PEI are the most efficient for complex formation and transfection. Nevertheless, the highest transient recombinant protein yields are achieved with unfractionated PEI. The results demonstrate that the polydispersity and charge density of linear PEI are important parameters for gene delivery to suspension-adapted HEK-293E cells., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

For most cultivated mammalian cells, glutamine is an essential medium component. However, glutamine consumption results in the production of ammonia, a cytotoxic byproduct. Here we investigated the effect of glutamine reduction on recombinant protein production and ammonia accumulation in transiently transfected CHO and HEK-293E cells maintained under conditions of growth arrest. Maximum transient recombinant protein yields were observed in HEK-293E cultures without glutamine and in CHO cultures with 2 mM glutamine. The initial concentration of glutamine correlated with the level of ammonia accumulation in each culture. For both a stable CHO-derived cell line and a polyclonal population of recombinant CHO cells grown under conditions of mild hypothermia, the highest volumetric protein productivity was observed in cultures without glutamine. Here, the level of ammonia accumulation also corresponded to the initial glutamine concentration. Our data demonstrate that reduction of glutamine in the medium is an effective approach to improve protein production in both transiently and stably transfected mammalian cells when applying conditions that reduce or arrest the growth of these cells.

The aim of this study was to gain a better understanding of orbitally shaken bioreactors (OSRs) operated without controllers for pH and dissolved oxygen (DO) concentration. We used cylindrical OSRs with working volumes ranging from 250mL to 200L to determine that the volumetric mass transfer coefficient of oxygen (k(L)a) is a good predictor of the performance of OSRs at different scales. We showed that k(L)a values of 7-10hour(-1) were required to avoid DO limitations and to prevent conditions of low pH during the cultivation of CHO cells. Overall, cell cultures in probe-independent OSRs of different nominal volumes ranging from 250mL to 200L achieved similar cell densities, recombinant protein concentrations, and pH and DO profiles when having the same k(L)a. We conclude that k(L)a is a key parameter for probe-independent bioprocesses in OSRs and can be used as a scale-up factor for their operation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The large-scale transfection of mammalian cells allows moderate (milligram to gram) amounts of recombinant proteins (r-proteins) to be obtained for fundamental or clinical research. In this article, we describe a one-liter transfection using polyethyleneimine (PEI) for DNA delivery into human embryonic kidney (HEK-293) cells cultivated in serum-free suspension to produce a recombinant human monoclonal antibody that yields up to about 1 g/L in a 10-day process. The method is based on a DNA delivery step performed at high cell density (20×10(6) cells/mL) by direct addition of DNA and PEI to the culture. Subsequently, the cells are diluted 20-fold for the 10-day production phase in the presence of valproic acid (VPA), a histone deacetylase inhibitor. The methods for plasmid purification, antibody quantification by enzyme-linked immunosorbent assay (ELISA), and affinity purification with protein A are also described.

Generating stable, high-producing mammalian cell lines is a major bottleneck in the manufacture of recombinant therapeutic proteins. Conventional gene transfer methods for cell line generation rely on random plasmid integration, resulting in unpredictable and highly variable levels of transgene expression. As a consequence, a large number of stably transfected cells must be analyzed to recover a few high-producing clones. Here we present an alternative gene transfer method for cell line generation based on transgene integration mediated by the piggyBac (PB) transposon. Recombinant Chinese hamster ovary (CHO) cell lines expressing a tumor necrosis factor receptor:Fc fusion protein were generated either by PB transposition or by conventional transfection. Polyclonal populations and isolated clonal cell lines were characterized for the level and stability of transgene expression for up to 3 months in serum-free suspension culture. Pools of transposed cells produced up to fourfold more recombinant protein than did the pools generated by standard transfection. For clonal cell lines, the frequency of high-producers was greater following transposition as compared to standard transfection, and these clones had a higher volumetric productivity and a greater number of integrated transgenes than did those generated by standard transfection. In general, the volumetric productivity of the cell pools and individual cell lines generated by transposition was stable for up to 3 months in the absence of selection. Our results indicate that the PB transposon supports the generation of cell lines with high and stable transgene expression at an elevated frequency relative to conventional transfection. Thus, PB-mediated gene delivery is expected to reduce the extent of recombinant cell line screening., (Copyright © 2011 Wiley Periodicals, Inc.)

Bioprocesses for recombinant protein production with mammalian cells are typically controlled for several physicochemical parameters including the pH and dissolved oxygen concentration (DO) of the culture medium. Here we studied whether these controls are necessary for efficient and reproducible bioprocesses in an orbitally shaken bioreactor (OSR). Mixing, gas transfer, and volumetric power consumption (P(V)) were determined in both a 5-L OSR and a 3-L stirred-tank bioreactor (STR). The two cultivation systems had a similar mixing intensity, but the STR had a lower volumetric mass transfer coefficient of oxygen (k(L)a) and a higher P(V) than the OSR. Recombinant CHO cell lines expressing either tumor necrosis factor receptor as an Fc fusion protein (TNFR:Fc) or an anti-RhesusD monoclonal antibody were cultivated in the two systems. The 5-L OSR was operated in an incubator shaker with 5% CO(2) in the gas environment but without pH and DO control whereas the STR was operated with or without pH and DO control. Higher cell densities and recombinant protein titers were obtained in the OSR as compared to both the controlled and the non-controlled STRs. To test the reproducibility of a bioprocess in a non-controlled OSR, the two CHO cell lines were each cultivated in parallel in six 5-L OSRs. Similar cell densities, cell viabilities, and recombinant protein titers along with similar pH and DO profiles were achieved in each group of replicates. Our study demonstrated that bioprocesses can be performed in OSRs without pH or DO control in a highly reproducible manner, at least at the scale of operation studied here., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Here we present the TubeSpin bioreactor 50 (TubeSpins) as a simple and disposable culture system for Sf-9 insect cells in suspension. Sf-9 cells had substantially better growth in TubeSpins than in spinner flasks. After inoculation with 10(6) cells/ml, maximal cell densities of 16×10(6) and 6×10(6) cells/ml were reached in TubeSpins and spinner flasks, respectively. In addition the cell viability in these batch cultures remained above 90% for 10 days in TubeSpins but only for 4 days in spinner flasks. Inoculation at even higher cell densities reduced the duration of the lag phase. After inoculation at 2.5×10(6) cells/ml, the culture reached the maximum cell density within 3 days instead of 7 days as observed for inoculation with 10(6) cells/ml. Infection of Sf-9 cells in TubeSpins or spinner flasks with a recombinant baculovirus coding for green fluorescent protein (GFP) resulted in similar GFP-specific fluorescence levels. TubeSpins are thus an attractive option for the small-scale cultivation of Sf-9 cells in suspension and for baculovirus-mediated recombinant protein production., (© Springer Science+Business Media B.V. 2011)

Here we describe a simplified method for transient gene expression (TGE) in suspension-adapted Chinese hamster ovary (CHO) cells using polyethylenimine (PEI) for DNA delivery. Both the transfection and production phases of the bioprocess were performed at a density of 4 × 10⁶ cells/mL at 31 °C. In addition, the amounts of both PEI and plasmid DNA were reduced up to 50% on a per cell basis compared to previously published protocols from this laboratory, resulting in higher cell viability after transfection and higher volumetric recombinant protein yields. In batch cultures of up to 14 days, reproducible recombinant antibody yields up to 300 mg/L were achieved at small scale (5 mL) and up to 250 mg/L at large scale (500 mL). The simplicity and improved yields are expected to increase the utility of CHO cells for the rapid production of recombinant proteins at larger scales by TGE., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Lentivirus-derived vectors (LVs) were studied for the generation of stable recombinant Chinese hamster ovary (CHO) cell lines. Stable pools and clones expressing the enhanced green fluorescent protein (eGFP) were selected via fluorescence-activated cell sorting (FACS). For comparison, cell pools and cell lines were also generated by transfection, using the LV transfer plasmid alone. The level and stability of eGFP expression was greater in LV-transduced cell lines and pools than in those established by transfection. CHO cells were also infected at two different multiplicities of infection with an LV co-expressing eGFP and a tumor necrosis factor receptor:Fc fusion protein (TNFR:Fc). At 2-day post-infection, clonal cell lines with high eGFP-specific fluorescence were recovered by FACS. These clones co-expressed TNFR:Fc with yields of 50-250 mg/L in 4-day cultures. The recovered cell lines maintained stable expression over 3 months in serum-free suspension culture without selection. In conclusion, LV-mediated gene transfer provided an efficient alternative to plasmid transfection for the generation of stable and high-producing recombinant cell lines., (Copyright © 2010 Wiley Periodicals, Inc.)

The effect of hyperosmolarity on transient recombinant protein production in Chinese hamster ovary (CHO) cells was investigated. Addition of 90 mM NaCl to the production medium ProCHO5 increased the volumetric yield of recombinant antibody up to 4-fold relative to transfection in ProCHO5 alone. Volumetric yields up to 50 mg l(-1) were achieved in a 6 day batch culture of 3 l. In addition, hyperosmolarity reduced cell growth and increased cell size. The addition of salt to cultures of transiently transfected CHO cells is a simple and cost-effective method to increase TGE yields in this host.

Valproic acid (VPA) is a small molecule that inhibits histone deacetylase activity. Here we report that VPA increases recombinant mRNA and protein levels in transiently transfected CHO DG44 cells. In the presence of VPA, transient recombinant antibody yields of up to 40 mg/L were achieved in simple batch cultures. The steady-state levels of the IgG light and heavy chain mRNAs were nearly 10 times higher than in the untreated control transfection even though the level of transfected plasmid DNA was the same in the presence or absence of VPA. The combination of VPA treatment and incubation of the transfected cells in mildly hypothermic conditions resulted in recombinant antibody yields of over 90 mg/L by 6 days post-transfection in batch cultures. The results demonstrated that the treatment of transfected CHO DG44 cells with VPA is a cost-effective strategy for enhancing transient gene expression by increasing the transgene mRNA levels., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The purpose of this review is to describe the current status and to highlight several emerging trends in the manufacture of recombinant therapeutic proteins in cultivated mammalian cells, focusing on Chinese hamster ovary cells as the major production host. Over the past 25 years, specific and volumetric productivities for recombinant cell lines have increased about 20-fold as the result of improvements in media and bioprocess design. Future yield increases are expected to come from further developments in gene delivery and genetic selection for more efficient recovery of high-producing cell lines and in high-throughput cultivation systems to simplify medium design and bioprocess development. Other emerging trends in protein manufacturing that are discussed include the use of disposal bioreactors and transient gene expression. We specifically highlight current research in our own laboratories.

Although respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in infants and adults at risk, no RSV vaccine is currently available. In this report, efforts toward the generation of an RSV subunit vaccine using recombinant RSV fusion protein (rRSV-F) are described. The recombinant protein was produced by transient gene expression (TGE) in suspension-adapted human embryonic kidney cells (HEK-293E) in 4 L orbitally shaken bioreactors. It was then purified and formulated in immunostimulating reconstituted influenza virosomes (IRIVs). The candidate vaccine induced anti-RSV-F neutralizing antibodies in mice, and challenge studies in cotton rats are ongoing. If successful in preclinical and clinical trials, this will be the first recombinant subunit vaccine produced by large-scale TGE in mammalian cells.

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.

The kinetics of polyethylenimine (PEI)-mediated gene transfer at early times after transfection of Chinese hamster ovary (CHO) cell in suspension were investigated using a novel in vitro assay. Addition of an excess of competitor DNA to the culture medium at various times after the initiation of transfection inhibited further cellular uptake of PEI-DNA particles. Using this approach, a constant rate of particle uptake was observed during the first 60 min of transfection at a PEI:DNA ratio of 2:1 (w/w) and a cell density of 2 x 10(6) cells/ml under serum-free conditions. The uptake rate declined considerably during the next 2 h of transfection. Both the rate and the level of PEI-DNA uptake in serum-free minimal medium were found to be dependent on the PEI-DNA ratio, the cell density at the time of transfection, and the extent of particle aggregation. These studies of the early phase of PEI-mediated transfection are expected to lead to further opportunities for optimization of gene transfer to suspension cultures of mammalian cells for the purpose of large-scale transient recombinant protein production.

Recombinant therapeutic proteins produced in mammalian cells represent a major class of biopharmaceuticals. In recent years, their demand has increased dramatically and is now driving the development of a variety of improvements to maximize their expression in mammalian cells. Advances in media- and process optimization have already resulted in more than 100-fold improvement in yield, but further insights and subsequent targeted modifications with respect to the general biology of cells (genomics, physiology, selection and adaptation) in bioreactors are hoped to further improve protein yields and quality in the near future.:, (© 2008 Elsevier Ltd . All rights reserved.)

Various DNA methyl transferase inhibitors (iDNMTs) and histone deacetylase inhibitors (iHDACs) were screened for their ability to enhance transient gene expression (TGE) in Human Embryonic Kidney 293-EBNA (HEK293E) cells. The effects in HEK293E cells were compared to those in Chinese Hamster Ovary DG44 (CHO-DG44) cells. The iDNMTs and iHDACs were chosen based on their different cellular activities and mechanisms of action. For each inhibitor tested, the optimum concentration was determined for both cell lines, and these conditions were used to evaluate the effect of each compound using a recombinant monoclonal antibody as a reporter protein. All the iHDACs increased transient antibody yield at least 4-fold in HEK293E and at least 1.5-fold in CHO-DG44. By comparison, the iDNMTs increased antibody yields by a maximum of approximately 2-fold. Pairwise combinations of iDNMTs and iHDACs had a linearly additive effect on TGE in CHO-DG44 but not in HEK293E. With valproic acid (VPA), volumetric and specific productivities of 200 mg/L and 20 pg/cell/day, respectively, were achieved in HEK293E cells with a 10-day process. As VPA is both FDA-approved and 5-fold less expensive than sodium butyrate (NaBut), we recommend it as a cost-effective alternative to this widely used enhancer of recombinant protein production from mammalian cells.

A new scalable reactor was developed by applying a novel mixing principle that allows the large-scale cultivation of mammalian cells simply with surface aeration using air owing to increased liquid-gas transfer compared to standard stirred-tank bioreactors. In the cylindrical vessels (50 mL-1500 L) with a helical track attached to the inside wall, the liquid moved upward onto the track as the result of orbital shaking to increase the liquid-gas interface area significantly. This typically resulted in a 5-10-fold improvement in the volumetric mass transfer coefficient (k(L)a). In a 1500-L helical track vessel with a working volume of 1000 L, a k(L)a of 10h(-1) was obtained at a shaking speed of 39 rpm. Cultivations of CHO cells in a shaken 55-L helical track bioreactor resulted in improved cell growth profiles compared to control cultures in standard systems. These results demonstrated the possibility of using these new bioreactors at scales of 1000 L or more.