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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.

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

Be it to aerate a glass of wine before tasting, to accelerate a chemical reaction or to cultivate cells in suspension, the "swirling" (or orbital shaking) of a container ensures good mixing and gas exchange in an efficient and simple way. Despite being used in a large range of applications this intuitive motion is far from being understood and presents a richness of patterns and behaviors which has not yet been reported. The present research charts the evolution of the waves with the operating parameters identifying a large variety of patterns, ranging from single and multiple crested waves to breaking waves. Free surface and velocity fields measurements are compared to a potential sloshing model, highlighting the existence of various flow regimes. Our research assesses the importance of the modal response of the shaken liquids, laying the foundations for a rigorous mixing optimization of the orbital agitation in its applications. Copyright (2014) American Institute of Physics. This article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Institute of Physics. The following article appeared in Physics of Fluids 26, 052104 (2014) and may be found at http://dx.doi.org/10.1063/1.4874612, 19 pages, 7 figures

Transient gene expression (TGE) is the most commonly used technology for the rapid production of moderate quantities of recombinant proteins for preclinical studies or for analytical assay development [1,2]. Whereas TGE can provide milligram to gram amounts of recombinant proteins within a time period as short as few days, technical limitations and high costs of plasmid DNA hamper the application of this technology at larger scales. Here we describe an alternative method for the fast production of recombinant proteins from pools of mammalian cells stably transfected with the PiggyBac (PB) transposon. Expression from stably integrated transgene is highly dependent upon the chromatin structure surrounding the site of integration [3]. As a consequence stable cell populations generated by conventional transfection techniques generally show low productivity and reduced expression stability. Several studies showed that the PB transposase mediated transgene insertion predominantly into actively transcribed regions of the mammalian genome [4,5], moreover we recently demonstrated that PB transposition generates high-producing CHO cell lines at a higher frequency than conventional plasmid transfection [6]. These results prompted us to develop a technology based on the use of stable pools generated by PB transposition for the rapid and scalable production of recombinant proteins. Initial experiments were aimed at determining the proportion of transgene expressing cells as well as the level and stability of recombinant protein production in PB transposed pools. First, we analyzed transgene expression in 952 cell lines recovered from a pool of CHO cells generated by PB-transposition to express tumor necrosis factor receptor as an Fc fusion protein (TNFR:Fc). The cell lines were analyzed by ELISA for TNFR:Fc expression in 4-day batch cultures. The clones showed levels of TNFR:Fc expression up to 360 mg/L with an overall mean of 96 +/- 54 mg/L. PB-transposition resulted in a high percentage (more than 98%) of TNFR:Fc expressing clones (data not shown). Analyses on the stability of transgene expression over time were conducted using a bicistronic PB-donor plasmid allowing co-expression of TNFR:Fc and the enhanced green fluorescent protein (eGFP). Five independent pools of cells were generated by transposition and recovered after 10 days of selection in puromycin. The pools were cultivated for 3 months in the absence of selection and eGFP expression was monitored periodically by flow cytometry. For each pool, the percentage of eGFP-positive cells remained stable over time (Table ​(Table1).1). The stability of transgene expression was further confirmed by TNFR:Fc productivity studies performed in 50-ml cultures at different time points post-transfection. At one month post-transfection, the five pools showed comparable growth and production characteristics, reaching TNFR:Fc titers in the range of 350-500 mg/L in 14-day batch cultures. Similar results were obtained when productivity was tested 2 – 3 months post-transfection (Table ​(Table11). Table 1 Analysis of the stability or recombinant protein expression in 5 independent pools of CHO cells. We developed a protocol for protein production from transposed cell pools at the 0.5-L scale (Figure ​(Figure1A).1A). Starting at 2 d post-transfection cells were subjected to 10 days of puromycin selection during which cells were expanded from TubeSpin® Bioreactor 50 tubes into orbitally shaken 250-mL cylindrical bottles. The batch bioprocess was finally started at 12 d post-transfection using orbitally shaken TubeSpin® Bioreactor 600 tubes. Using stable cell pools expressing either an IgG antibody (Fig. ​(Fig.1B)1B) or two TNFR:Fc variants (Fig. 1C, D), we produced 500-750 mg of recombinant protein within a month after transfection. Figure 1 (A) Schematic representation of the protocol for the rapid production of recombinant proteins from pools of transposed cells. This protocol was successfully used to produce a recombinant monoclonal antibody (B) and two variants of TNFR:Fc (C and D). For ... Our results demonstrated an improved level and stability of transgene expression in transposed pools, indicating usefulness of PB transposed cell pools as a valuable alternative to TGE for the rapid production of recombinant proteins.

Cylindrical containers agitated by orbital shaking are being developed as simple and cost-effective bioreactor systems for the cultivation of mammalian cells. Here the oxygen transfer capacities of containers with nominal volumes from 50 mL to 2000 L were determined, and the operating parameters influencing oxygen transfer were investigated. In general, the shaking speed necessary for efficient oxygen transfer diminished as the container size increased. At shaking speeds suitable for the growth of shear-sensitive cells, kLa values between 10 and 30 h−1 were typically achieved in small-scale (

Background One of the most important unit operations in upstream animal cell bioprocesses at scales over 100 L is the preparation and sterilization of the medium. This complex, sensitive, and expensive process requires a considerable investment in both material and time [1]. Traditionally, large-scale medium sterilization is performed with costly single-use dead-end filters. To optimize and reduce the cost of this unit operation, we investigated the sterilization of mammalian cell culture medium at volumes larger than 100 L.

Background Transient gene expression (TGE) allows rapid protein production in mammalian cells and has become an important tool in the pharmaceutical product development pipeline [1]. Polyethylenimine (PEI)-mediated, high-density transfection allowed to express recombinant proteins at yields exceeding 1 g/L in only a few weeks [2]. Although highly efficient protocols are available, volumetric scale-up of TGE is still a challenge. A major issue is the need to perform the transfection in fresh medium rather than in conditioned (spent) medium. This implies a medium exchange step just before transfection. In CHO-DG44 [3] cells we observed up to a 100-fold decrease in volumetric protein production if transfections were performed in conditioned medium, compared to fresh medium. The reasons for such a negative effect of conditioned medium on transfectability and/or protein production expression are not yet known. To study this problem we transfected CHO cells at small-scale in TubeSpin bioreactor 50 tubes using 41 different commercially available serum-free media formulations in combination with different transfection parameters and culture conditions. By comparing the transient production of a recombinant IgG antibody among the different media, we observed variation of up to 400-fold when transfecting in fresh media and up to 20-fold when using conditioned medium. The optimization of the PEI:DNA ratio allowed a significant improvement in yields of transfection in conditioned medium. Methods Suspension-adapted CHO-DG44 cells [3] were routinely cultivated in TubeSpin bioreactor 50 tubes (TPP, Trasadingen, Switzerland) in ProCHO5 medium (Lonza, Vervier, Belgium) supplemented with 13.6 mg/L hypoxanthine, 3.9 mg/L thymidine, and 4 mM glutamine. The 38 media samples for transfection were provided by Excellgene SA. Conditioned medium is defined as a cell culture medium where cells have been growing for more than two days up to a density between 4-5 million cells/mL. Cell growth was accessed with the Packed Cell Volume method. The dual expression vector pXLGCHO-A3, containing the cDNAs coding for human anti-Rhesus D IgG1 heavy and light chain cloned in separate expression cassettes in a head-to-head orientation, was kindly provided by Excellgene SA [4]. Transfections are performed at a cell density of 5.5 million cells/mL at a volume of 5 mL by the direct addition of 15 μg of pDNA and 76 μg of linear 25 kDa Polyethylenimine (PEI, Polysciences, Eppenheim, Germany)

Disposable cylindrical shaken bioreactors using plastic bags or vessels represent a promising alternative to stainless steel bioreactors, because they are flexible, cost-effective and can be pre-sterilized. Unlike conventional well-established steel bioreactors, however, such disposable bioreactor systems have not yet been precisely characterized. Thus, the aim of this current work is to introduce a new power input correlation as a potential means to characterize the hydrodynamics of these new systems. A set of rel- evant power input variables was defined and transformed into dimensionless numbers by using the Buckingham’s pi-Theorem. These numbers were then experimentally varied to quantify the relationship among the numbers. A simple correlation was generated for the power input with seven variables. The application of this new correlation was validated using 200 L and 2000 L orbitally shaken bioreactors. In conclusion, the proposed correlation is a useful tool to predict the power input and hydrodynamics during cell cultivation in cylindrical shaken bioreactors of all scales.

In this study, a recombinant monoclonal IgG antibody was produced by transient gene expression (TGE) in suspension-adapted HEK-293E cells. The objective of the study was to determine the variation in recombinant IgG yield and glycosylation in ten independent transfections. In a ten-day batch process, the variation in transient IgG yield in the ten batches was less than 30% with the specific productivity averaging 20.2 +/- 2.6 pg/cell/day. We characterized the N-glycosylation profile of each batch of affinity-purified IgG by intact protein and bottom-up mass spectrometry. Four major glycans were identified at Asn(297) in the ten batches with the maximum relative deviation for a single glycoform being 2.5%. In addition, within any single transfection there was little variation in glycoforms over the ten-day culture. Our experimental data indicate that with TGE, the production of recombinant IgG with little batch-to-batch variation in volumetric yield and protein glycosylation is feasible, even in a non-instrumented cultivation system as described here.

A major bottleneck in the manufacture of recombinant therapeutic proteins is the time and effort needed for the generation of stable, high-producing mammalian cell lines. Conventional gene transfer methods for stable cell line generation rely on random transgene integration, resulting in unpredictable and highly variable levels of expression of the transgene in individual clones [1,2]. As a consequence, a large number of stably transfected cells must be analyzed to recover a few high-producing clones. Recently, we described the use of a PiggyBac (PB) transposon for the generation of high-producing mammalian cell lines [3]. The PB dual vector system consists of 1) a donor vector carrying an artificial transposon with a mammalian expression cassette for the recombinant transgene and puromycin selection marker and 2) a helper vector driving transient expression of the PB transposase (PBase). PB transposition mediates stable transgene integration via a “cut and paste” mechanism in which the PBase excises the artificial transposon sequence from the plasmid and catalyzes its insertion into the host cell genome. A main advantages of the PB system over conventional passive integration are an improved efficiency of transgene integration resulting in a more integration events and more stable clones. Furthermore, PB favors transgene integration into actively transcribed regions of the host genome [4]. The PB transposon has a high cargo capacity of up to 14 Kb and transposition results in the stable genomic integration of well-defined sequences, thus reducing the probability of integration of truncated, non-functional transgenes [5]. Finally, recent reports have demonstrated the feasibility of using the PB system to obtain persistent expression of multiple genes carried either on a single or on distinct donor vectors [6]. We initially determined the efficiency of the PB system in the generation of stable lines using suspension adapted mammalian cells. CHO and HEK 293 cells were co-transfected with an eGFP-bearing donor plasmid along with the PB helper plasmid. The cells were then grown in the absence of puromycin, and the percentage of GFP-expressing cells in each culture was determined by flow cytometry on a daily basis. By 21 days post-transfection, the remaining GFP-positive cells were assumed to be recombinant. Compared to conventional transfection of plasmid DNA, PB transposition resulted in an improvement in the efficiency of stable cell line generation up to 20-fold for both CHO and HEK 293 cells (Figure ​(Figure1A1A). Figure 1 A) Enhanced stable integration efficiency in CHO and HEK 293 cells by PB transposition. B) Productivity analysis of CHO clonal cell lines sorted from cell pools generated by PB transposition (PB) or conventional transfection (TX). C) Analysis of the stability ... To further evaluate the PB system, CHO cells expressing a tumour necrosis factor receptor:Fc fusion protein (TNFR:Fc) were generated either by PB-transposition or by conventional transfection. Clonal cell lines were recovered following selection in 50 or 10 µg/mL puromycin for two weeks. Recovered lines were grown in suspension culture for 7 days in 24-well plates after which the medium was analyzed by ELISA to determine TNFR:Fc productivity. Transposition increased the frequency of high-producing clones in the transfected population (Figure ​(Figure1B).1B). To further characterized for the level and stability of transgene expression the original cell pools generated by PB transposition or conventional transfection, as well as the top 4 producers from each transfection were cultivated in the absence of selection in serum-free suspension culture, over a period of 16 or 14 weeks, respectively. When compared to clones and cell pools generated by conventional transfection, PB-derived cell lines and cell pools produced up to 4-fold more recombinant protein and had greater transgene expression stability (Figure ​(Figure1C1C) In conclusion our results demonstrate that stable cell lines derived by PB transposition are efficiently generated and are more productive than cell lines generated by conventional transfection methods. Therefore, the PB system represents a valuable and practical alternative to standard plasmid transfection to efficiently generate cell clones with stable and enhanced transgene expression.

Background Transposon systems mediate stable integration of exogenous DNA elements into a host cell genome, and have been successfully used in mammalian cells for the generation of stable cell lines. The piggyBac (PB) transposon system has been shown to have several advantages over the other transposon system available [1-3]. It has also been shown to generate stable cell lines at significantly higher frequency than the conventional transfections [3]. Here, we investigated the efficiency of the piggyBac (PB) transposon to facilitate the co-expression of multiple artificial transposons, each bearing a single transgene and the puromycin resistance gene for selection. Green fluorescent protein (eGFP), red fluorescent protein (mKate), and a human IgG1 antibody were used as model proteins [4]. The effect of the stringency of selection on pool productivity was determined with increasing concentrations of puromycin. The duration of selection necessary for the generation of recombinant cell pools was also tested by selecting for a period of either 5 or 10 days.

BackgroundGlutamine is an essential component in culture mediafor most of the mammalian cell lines. It is often used asanalternativesourceofenergybycells,alongwithglu-cose. Glutamine metabolism induces ammonia accumu-lation in cell culture. Elevated ammonia concentrationabove 2 mM has been shown to have negative impacton both cell growth and recombinant protein productiv-ity [1-4]. In this study we investigated the effects ofdecreased glutamine concentration in the medium forCHO-DG44 and HEK-293E cells during transient geneexpression (TGE). The rationale was to reduce ammoniaaccumulation in the culture, and consequently, improvecell viability and recombinant protein productivity.Materials and methods