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Viral vectors for gene therapy, such as recombinant Adeno-Associated Viruses (rAAV), are produced in Human Embryonic Kidney (HEK) 293 cells. However, the presence of the SV40 T-antigen-encoding CDS SV40GP6 and SV40GP7 in the HEK293T genome raises safety issues when these cells are used in manufacturing for clinical purposes. We developed a new T-antigen-negative HEK cell line from ExcellGene’s proprietary HEKExpress®, using the CRISPR-Cas9 strategy. We obtained a high number of clonally-derived cell populations and all of them were demonstrated T-antigen negative. Stability study and AAV production evaluation showed that the deletion of the T-antigen-encoding locus did not impact neither cell growth nor viability nor productivity. The resulting CMC-compliant cell line, named HEKzeroT®, is able to produce high AAV titers, from small to large scale.

For the treatment of severe COVID-19, supplementation with human plasma-purified α-1 antitrypsin (AAT) to patients is currently considered. AAT inhibits host proteases that facilitate viral entry and possesses broad anti-inflammatory and immunomodulatory activities. Researchers have demonstrated that an interaction between SARS-CoV-2 spike protein (S) and lipopolysaccharides (LPS) enhances pro-inflammatory responses in vitro and in vivo. Hence, we wanted to understand the potential anti-inflammatory activities of plasma-derived and recombinant AAT (recAAT) in a model of human total peripheral blood mononuclear cells (PBMCs) exposed to a combination of CHO expressed trimeric spike protein and LPS, ex vivo. We confirmed that cytokine production was enhanced in PBMCs within six hours when low levels of LPS were combined with purified spike proteins (“spike”). In the presence of 0.5 mg/mL recAAT, however, LPS/spike-induced TNF-α and IL-1β mRNA expression and protein release were significantly inhibited (by about 46–50%) relative to LPS/spike alone. Although without statistical significance, recAAT also reduced production of IL-6 and IL-8. Notably, under the same experimental conditions, the plasma-derived AAT preparation Respreeza (used in native and oxidized forms) did not show significant effects. Our findings imply that an early pro-inflammatory activation of human PBMCs is better controlled by the recombinant version of AAT than the human plasma-derived AAT used here. Considering the increasing clinical interest in AAT therapy as useful to ameliorate the hyper-inflammation seen during COVID-19 infection, different AAT preparations require careful evaluation.

Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained and cross-variant SARS-CoV-2 neutralising antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a TLR7/8 small-molecule agonist chemisorbed on aluminium hydroxide. Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titre NAbs in mice, with no decay in responses over an 8-month period. NAbs from sera of CoVac-II-immunized mice, horses and rabbits were broadly neutralising against SARS-CoV-2 variants. Boosting long-term CoVac-II-immunized mice with adjuvanted spike protein from the Beta variant markedly increased levels of NAb titres against multiple SARS-CoV-2 variants; notably high titres against the Delta variant were observed. These data strongly support the clinical assessment of Alhydroxiquim-II-adjuvanted spike proteins to protect against SARS-CoV-2 variants of concern.

The Spike protein of SARS-CoV-2 is essential for virus entry into human cells. In fact, most neutralizing antibodies against SARS-CoV-2 are directed against the Spike, making it the antigen of choice for use in vaccines and diagnostic tests. In the current pandemic context, global demand for Spike proteins has rapidly increased and could exceed hundreds of grams to kilograms annually. Coronavirus Spikes are large, heavily glycosylated, homotrimeric complexes, with inherent instability. Their poor manufacturability now threatens availability of these proteins for vaccines and diagnostic tests. Here, we outline a scalable, GMP-compliant, chemically defined process for production of a cell secreted, stabilized form of the trimeric Spike protein. The process is chemically defined and based on clonal, suspension-CHO cell populations and on protein purification via a two-step, scalable downstream process. The trimeric conformation was confirmed using electron microscopy and HPLC analysis. Binding to susceptible cells was shown using a virus-inhibition assay. The diagnostic sensitivity and specificity for detection of serum SARS-CoV-2 specific IgG1 was investigated and found to exceed that of Spike fragments (S1 and RBD). The process described here will enable production of sufficient high-quality trimeric Spike protein to meet the global demand for SARS-CoV-2 vaccines and diagnostic tests.

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.

Orbitally shaken bioreactors (OSRs) are commonly used for the cultivation of mammalian cells in suspension. Here we conducted a three-dimensional computational fluid dynamics (CFD) simulation to characterize the fluid field in the disposable 600-mL orbitally shaken bioreactor (OSR600), basically a cylindrical vessel with a conical bottom and a ventilated cap. The CFD models established for the OSR600 were validated by visual comparison of the liquid flow pattern in an experimentally agitated OSR600. In the model, both shear stress and energy dissipation rate (Φ) were calculated to evaluate the hydrodynamic stress environment for cell cultivation. The highest values of shear stress and Φ were localized along the lower part of the conical vessel wall. The effect of filling volume and shaking speed on kLa, Φ and shear stress were also analyzed. An increase of the percentage of the liquid affected by higher shear stress and Φ was observed at filling volumes of 300 mL and 400 mL compared to lower filling volumes. This may be due to the twisted curvature at the base of the liquid wave under these conditions. In conclusion, the CFD model provided a means to characterize the fluid dynamics of the OSR600 under various operating conditions to help identify those most suitable for cell cultivation.

Recombinant proteins have been produced for over 30 years. Applications range from enzymes used in laundry detergents to antigen-detecting antibodies in cancer therapy. Despite similarities in manufacturing, drastic differences in retail pricing between recombinant proteins used for industrial (non-medical) versus pharmaceutical purposes exist. Industrial proteins often have a retail price in the tens of dollars per kilogram while recombinant proteins for medical use may cost billions of dollars per kilogram. This manuscript will briefly review manufacturing techniques and contrast the differences between industrial versus pharmaceutical production. Maximizing manufacturing technologies to reduce cost-of-goods (CoG) is desirable. However, the major reason for the very high pricing of pharma protein products does not reflect CoG, but the financial obligations of clinical trials, research and development, patent constraints, marketing, and return on investment.

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells. Keywords: adeno-associated virus, AAV8, AAV9, suspension HEK293 cells, transient transfection, orbitally shaken bioreactors, immune affinity chromatography, CNS transduction

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.

Alzheimer's disease (AD)-associated gamma-secretase is a ubiquitously expressed multi-subunit protease complex embedded in the lipid bilayer of cellular compartments including endosomes and the plasma membrane. Although gamma-secretase is of crucial interest for AD drug discovery, its atomic structure remains unresolved. Gamma-secretase assembly and maturation is a multistep process, which includes extensive glycosylation on nicastrin (NCT), the only g-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 g-secretase as a functional complex. We purified glycosylation-deficient g-secretase from this recombinant cell line (CL1-9) and fully glycosylated g-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 g-secretase, which may be suitable for crystallization studies. We expect that these findings will contribute both to solving the high-resolution 3D structure of g-secretase and to structure-based drug discovery for AD. Biotechnol. Bioeng. 2015;112: 2516-2526. (c) 2015 Wiley Periodicals, Inc.

To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line. 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-DR12xHis) 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. 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.

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 x 10(6) cells/mL with 0.9 mu g DNA and 1.35 mu g of linear 25 kD polyethylenimine (PEI) per million cells. Following transfection, the culture was diluted to 4 x 10(6) 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 mu g/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. (C) 2013 Elsevier B.V. All rights reserved.

Highlights • A brief overview of principles of TGE using mammalian cells. • Description of TGE processes for HEK293 and CHO cells. • Description of orbitally shaken bioreactors for suspension cell cultivation. • Description of polyethylenime-based transfection processes., 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.

Orbitally shaken bioreactors (OSRs) support the efficient cultivation of mammalian cells in suspension at various scales of operation. Here we introduce a disposable culture vessel with a ventilated cap having a nominal volume of 600 mL (TubeSpin (R) bioreactor 600) for medium-scale cell cultivation. It has a conical bottom designed to fit into a standard swinging bucket rotor to facilitate cell/liquid separation by centrifugation. At shaking frequencies suitable for mammalian cell cultivation (140-220 rpm), the volumetric mass transfer coefficient (k(L)a) ranged from 15 to 40 h(-1), the mixing time was less than 20 s, and the specific power consumption ranged from 0.03 to 0.5 kW/m(3). To define the working conditions for the TubeSpin (R) bioreactor 600, we measured cell growth and environmental culture conditions such as pH and dissolved O-2 and CO2 concentrations over a range of shaking frequencies and working volumes using a stable CHO-derived cell line expressing a human IgG antibody. The optimal cultivation conditions were observed at working volumes of 300-500 mL and shaking frequencies of 180-220 rpm. In 300-mL batch cultures of the same cell line in a TubeSpin (R) bioreactor 600, 1-L cylindrical glass bottle, and 1-L Erlenmeyer shake flask, the maximum cell densities and recombinant antibody yields were similar. This study demonstrated the suitability of the TubeSpin bioreactor 600 for cell culture applications with suspension-adapted CHO cells at culture volumes up to 500 mL. (C) 2013 Elsevier B.V. All rights reserved.

We investigated the cultivation of bacterial, mammalian and insect cells in an orbitally-shaken 250-mL disposable tube that incorporates a helical track (HT) on its inside wall. The mass transfer coefficient of oxygen (kLa) was 200%–400% higher in the HT tube than in a shake flask. Bacterial growth and plasmid production were 30% higher in the HT tube than in a 1-L Erlenmeyer flask. Mammalian cell cultures achieved a 25% higher cell density in the HT tube as compared to a 250-mL square-shaped bottle while insect cells grew as well in HT tubes as in 250-mL shake flasks. Because of their performance, disposability, and compact size, we conclude that 250-mL HT tubes are a useful alternative to other shaken containers for the cultivation of bacterial, mammalian and insect cells.

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.

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. (C) 2016 Elsevier Inc. All rights reserved.

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.

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.

Orbitally shaken bioreactors are an emerging alternative to stirred-tank bioreactors for large-scale mammalian cell culture, but their fluid dynamics is still not well defined. Among the theoretical and practical issues that remain to be resolved, the characterization of the liquid free surface during orbital shaking remains a major challenge because it is an essential aspect of gas transfer and mixing in these reactors. To simulate the fluid behavior and the free surface shape, we developed a numerical method based on the finite element framework. We found that the large density ratio between the liquid and the gas phases induced unphysical results for the free surface shape. We therefore devised a new pressure correction scheme to deal with large density ratios. The simulations operated with this new scheme gave values of wave amplitude similar to the ones measured experimentally. These simulations were used to calculate the shear stress and to study the mixing principle in orbitally shaken bioreactors. Copyright (C) 2012 John Wiley & Sons, Ltd.

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.

Improving qualitative and quantitative characterization of monoclonal antibodies is essential, because of their increasing popularity as therapeutic drug targets. Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth characterization of post-translationally modified large peptides, small- and medium-sized proteins, and noncovalent protein complexes. Here, we describe the performance of ETD-based top-down mass spectrometry for structural analysis of intact 150 kDa monoclonal antibodies, immunoglobulins G (IgGs). Simultaneous mass analysis of intact IgGs as well as a complex mixture of ETD product ions at sufficiently high resolution and mass accuracy in a wide m/z range became possible because of recent advances in state-of-the-art time-of-flight (TOF) mass spectrometry. High-resolution ETD TOF MS performed on IgG1-kappa from murine myeloma cells and human anti-Rhesus D IgG1 resulted in extensive sequence coverage of both light and heavy chains of IgGs and revealed information on their variable domains. Results are superior and complementary to those previously generated by collision-induced dissociation. However, numerous disulfide bonds drastically reduce the efficiency of top-down ETD fragmentation within the protected sequence regions, leaving glycosylation uncharacterized. Further increases in the experiment sensitivity and improvement of ion activation before and after ETD reaction are needed to target S-S bond-protected sequence regions and post-translational modifications.

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.

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. Biotechnol. Bioeng. 2011;108:2141–2150. © 2011 Wiley Periodicals, Inc.

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.

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‚Äâmg/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.

Efficient mixing in bioreactors is essential in order to avoid concentration gradients which can be harmful for mammalian cells. To study mixing and its scalability in orbitally shaken cylindrical bioreactors, we measured mixing times in containers with nominal volumes from 2 to 1500 L with a colorimetric method using two pH indicators. Four operating parameters were tested: the liquid height, the shaking diameter, the agitation rate, and the inner diameter of the container. The mixing time decreased as the agitation rate increased until a minimal value was reached. As the shaking diameter was reduced, a higher agitation rate was needed to reach the minimal mixing time. The liquid height did not have a significant effect on the mixing time, but for a constant volume, an increase of the inner diameter slightly reduced the mixing time. The fastest mixed zones were close to the wall of the container while the zone in the center of the bulk liquid was the last to achieve homogeneity. Our study showed that the free-surface shape correlated with the mixing regime and that by keeping the inner-to-shaking diameter ratio as well as the Froude number (Fr) constant, the free-surface shapes and the mixing regimes of a 1500-L bioreactor could be mimicked in a 30-L bioreactor. We concluded that the mixing in orbitally shaken cylindrical bioreactors ensures homogeneity for mammalian cell cultures at scales up to 1500 L and that the inner-to-shaking diameter is a suitable scale-up factor for mixing.

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.

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.

Synthetic biomaterials play an important role in regenerative medicine. To be effective they must support cell attachment and proliferation in addition to being non-toxic and non-immunogenic. We used a suspension-adapted Chinese hamster ovary-derived cell line expressing green fluorescent protein (GFP) to assess cell attachment and growth on synthetic biomaterials by direct measurement of GFP-specific fluorescence. To simplify operations, all cell cultivation steps were performed in orbitally-shaken, disposable containers. Comparative studies between this GFP assay and previously established cell quantification assays demonstrated that this novel approach is suitable for rapid screening of a large number of samples. Furthermore the utility of our assay system was confirmed by evaluation of cell growth on three polyvinylidene fluoride polymer scaffolds that differed in pore diameter and drawing conditions. The data presented here prove the general utility of GFP-expressing cell lines and orbital shaking technology for the screening of biomaterials for tissue engineering applications.

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.

Recombinant proteins are of great commercial and scientific interest. However, most current production methods using mammalian cells involve the time- and labor-intensive step of creating stable cell lines. Although production methods based on transient gene expression could offer a significant improvement, transient transfection is currently still limited by low titers and low specific productivity compared to stable cell lines. To overcome these bottlenecks, we have explored the use of various growth factors to enhance specific productivity and titers in the context of transient gene expression. For that purpose, several growth factors were cloned and screened for their effect on transient gene expression in HEK293E and CHO-DG44 cells. In particular, acidic fibroblast growth factor (aFGF) was able to increase specific productivity by 60% and recombinant protein titers by 80% in HEK293E cells, while FGF9 increased titers by 250% in CHO-DG44 cells.

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.

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.

The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10 days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks. With "classical" approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies.