Sf9 Cell Metabolism Throughout the Recombinant Baculovirus and Rabies Virus-Like Particles Production in Two Culture Systems.

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10-12 mmol cell/h) in SF and for glutamine (30.8 × 10-12 mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10-10 mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.

Distinct anti-NP, anti-RBD and anti-Spike antibody profiles discriminate death from survival in COVID-19.

Introduction: Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces rapid production of IgM, IgA, and IgG antibodies directed to multiple viral antigens that may have impact diverse clinical outcomes. Methods: We evaluated IgM, IgA, and IgG antibodies directed to the nucleocapsid (NP), IgA and IgG to the Spike protein and to the receptor-binding domain (RBD), and the presence of neutralizing antibodies (nAb), in a cohort of unvaccinated SARS-CoV-2 infected individuals, in the first 30 days of post-symptom onset (PSO) (T1). Results: This study included 193 coronavirus disease 2019 (COVID-19) participants classified as mild, moderate, severe, critical, and fatal and 27 uninfected controls. In T1, we identified differential antibody profiles associated with distinct clinical presentation. The mild group presented lower levels of anti-NP IgG, and IgA (vs moderate and severe), anti-NP IgM (vs severe, critical and fatal), anti-Spike IgA (vs severe and fatal), and anti-RBD IgG (vs severe). The moderate group presented higher levels of anti-RBD IgA, comparing with severe group. The severe group presented higher levels of anti-NP IgA (vs mild and fatal) and anti-RBD IgG (vs mild and moderate). The fatal group presented higher levels of anti-NP IgM and anti-Spike IgA (vs mild), but lower levels of anti-NP IgA (vs severe). The levels of nAb was lower just in mild group compared to severe, critical, and fatal groups, moreover, no difference was observed among the more severe groups. In addition, we studied 82 convalescent individuals, between 31 days to 6 months (T2) or more than 6 months (T3), PSO, those: 12 mild, 26 moderate, and 46 severe plus critical. The longitudinal analyzes, for the severe plus critical group showed lower levels of anti-NP IgG, IgA and IgM, anti-Spike IgA in relation T3. The follow-up in the fatal group, reveals that the levels of anti-spike IgG increased, while anti-NP IgM levels was decreased along the time in severe/critical and fatal as well as anti-NP IgG and IgA in several/critical groups. Discussion: In summary, the anti-NP IgA and IgG lower levels and the higher levels of anti-RBD and anti-Spike IgA in fatal compared to survival group of individuals admitted to the intensive care unit (ICU). Collectively, our data discriminate death from survival, suggesting that anti-RBD IgA and anti-Spike IgA may play some deleterious effect, in contrast with the potentially protective effect of anti-NP IgA and IgG in the survival group.

Zika virus-like particles (VLPs) produced in insect cells.

Introdutcion: The Zika virus (ZIKV) infections are a healthcare concern mostly in the Americas, Africa, and Asia but have increased its endemicity area beyond these geographical regions. Due to the advances in infections by Zika virus, it is imperative to develop diagnostic and preventive tools against this viral agent. Virus-like particles (VLPs) appear as a suitable approach for use as antiviral vaccines. Methods: In this work, a methodology was established to produce virus-like particles containing the structural proteins, C, prM, and E of Zika virus produced in insect cells using the gene expression system derived from baculovirus. The vector pFast- CprME -ZIKV was constructed containing the gene sequences of Zika virus structural proteins and it was used to generate the recombinant bacmids (Bac- CprME -ZIKV) through transformation into DH10BacTM cells. The Bac- CprME -ZIKV was transfected in Spodoptera frugiperda (Sf9) insect cells and batches of BV- CprME -ZIKV were obtained by infection assays using a multiplicity of infection of 2. The Sf9 cells were infected, and the supernatant was collected 96 h post-infection. The expression of the CprME -ZIKV protein on the cell surface could be observed by immunochemical assays. To concentrate and purify virus-like particles, the sucrose and iodixanol gradients were evaluated, and the correct CprME -ZIKV proteins' conformation was evaluated by the Western blot assay. The virus-like particles were also analyzed and characterized by transmission electron microscopy. Results and discussion: Spherical structures like the native Zika virus from 50 to 65 nm containing the CprME -ZIKV proteins on their surface were observed in micrographs. The results obtained can be useful in the development path for a vaccine candidate against Zika virus.

Sf9 Cells Metabolism and Viability When Coinfected with Two Monocistronic Baculoviruses to Produce Rabies Virus-like Particles.

This work aimed to describe the dynamics of the Sf9 insect cells death and primary metabolism when this host is infected simultaneously by two recombinant baculoviruses (BV) expressing rabies glycoprotein (BVG) and matrix protein (BVM) genes to produce rabies virus-like particles (VLP) at different multiplicities of infection (MOI). Schott flasks essays covering a wide range of MOI for both BV were performed. Viable cell density, cell viability, glucose, glutamine, glutamate, lactate, ammonium, and rabies proteins concentrations were monitored over the infection phase. The expression of both recombinant proteins was not limited by glucose, glutamine, and glutamate in a broad MOI (pfu/cell) range of BVG (0.15-12.5) and BVM (0.1-5.0) using SF900 III serum free culture medium. Death phase initiation and the specific death rate depend on BV MOI. The wave pattern of nutrient/metabolite profiles throughout the viral infection phase is related to the baculovirus lytic cycle. The optimal MOIs ratio between BVG (2.5-4.5) and BVM (1.0-3.0) for maximum protein expression was defined. The produced rabies VLP sizes are close to 78 nm. In general, these work outputs bring a better understanding of the metabolic performance of Sf9 cells when infected by BV for producing VLP, and specifically, for progressing in a rabies VLP vaccine development.

Anti-SARS-CoV-2 equine F (Ab')2 immunoglobulin as a possible therapy for COVID-19.

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab')2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab')2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Performance Comparison of Recombinant Baculovirus and Rabies Virus-like Particles production Using Two Culture Platforms.

This work aimed to assess, following upstream optimization in Schott flasks, the scalability from this culture platform to a stirred-tank bioreactor in order to yield rabies-recombinant baculovirus, bearing genes of G (BVG) and M (BVM) proteins, and to obtain rabies virus-like particles (VLP) from them, using Sf9 insect cells as a host. Equivalent assays in Schott flasks and a bioreactor were performed to compare both systems and a multivariate statistical approach was also carried out to maximize VLP production as a function of BVG and BVM's multiplicity of infection (MOI) and harvest time (HT). Viable cell density, cell viability, virus titer, BVG and BVM quantification by dot-blot, and BVG quantification by Enzyme-Linked Immunosorbent Assay (ELISA) were monitored throughout the assays. Furthermore, transmission electron microscopy was used to characterize rabies VLP. The optimal combination for maximum VLP expression was BVG and BVM MOI of 2.3 pfu/cell and 5.1 pfu/cell, respectively, and 108 h of harvest time. The current study confirmed that the utilization of Schott flasks and a benchtop bioreactor under the conditions applied herein are equivalent regarding the cell death kinetics corresponding to the recombinant baculovirus infection process in Sf9 cells. According to the results, the hydrodynamic and chemical differences in both systems seem to greatly affect the virus and VLP integrity after release.

Production of Rabies VLPs in Insect Cells by Two Monocistronic Baculoviruses Approach.

Rabies is an ancient zoonotic disease that still causes the death of over 59,000 people worldwide each year. The rabies lyssavirus encodes five proteins, including the envelope glycoprotein and the matrix protein. RVGP is the only protein exposed on the surface of viral particle, and it can induce immune response with neutralizing antibody formation. RVM has the ability to assist with production process of virus-like particles. VLPs were produced in recombinant baculovirus system. In this work, two recombinant baculoviruses carrying the RVGP and RVM genes were constructed. From the infection and coinfection assays, we standardized the best multiplicity of infection and the best harvest time. Cell supernatants were collected, concentrated, and purified by sucrose gradient. Each step was used for protein detection through immunoassays. Sucrose gradient analysis enabled to verify the separation of VLPs from rBV. Through the negative contrast technique, we visualized structures resembling rabies VLPs produced in insect cells and rBV in the different fractions of the sucrose gradient. Using ELISA to measure total RVGP, the recovery efficiency of VLPs at each stage of the purification process was verified. Thus, these results encourage further studies to confirm whether rabies VLPs are a promising candidate for a veterinary rabies vaccine.

Strategies for the Production of Soluble Interferon-Alpha Consensus and Potential Application in Arboviruses and SARS-CoV-2.

Biopharmaceutical production is currently a multibillion-dollar industry with high growth perspectives. The research and development of biologically sourced pharmaceuticals are extremely important and a reality in our current healthcare system. Interferon alpha consensus (cIFN) is a non-natural synthetic antiviral molecule that comprises all the most prevalent amino acids of IFN-α into one consensus protein sequence. For clinical use, cIFN is produced in E. coli in the form of inclusion bodies. Here, we describe the use of two solubility tags (Fh8 and DsbC) to improve soluble cIFN production. Furthermore, we analyzed cIFN production in different culture media and temperatures in order to improve biopharmaceutical production. Our results demonstrate that Fh8-cIFN yield was improved when bacteria were cultivated in autoinduction culture medium at 30 °C. After hydrolysis, the recovery of soluble untagged cIFN was 58% from purified Fh8-cIFN molecule, fourfold higher when compared to cIFN recovered from the DsbC-cIFN, which achieved 14% recovery. The biological activity of cIFN was tested on in vitro model of antiviral effect against Zika, Mayaro, Chikungunya and SARS-CoV-2 virus infection in susceptible VERO cells. We show, for the first time, that cIFN has a potent activity against these viruses, being very low amounts of the molecule sufficient to inhibit virus multiplication. Thus, this molecule could be used in a clinical approach to treat Arboviruses and SARS-CoV-2.

A new class of antimicrobial molecules derived from kefir, effective against Pseudomonas aeruginosa and methicillin resistant Staphylococcus aureus (MRSA) strains.

Many studies have linked the antimicrobial properties of kefir with the presence of bacteriocins and organic acids. In the present work, results obtained from bacteriostatic and bactericidal studies, and from RP-HPLC, Mass Spectrometry and proton NMR analysis, show that a sample of milk kefir grains is able to produce an antimicrobial fraction, denoted FK-1000, composed of sugars and amino acids, predominantly polymers of alanine, doublets of tyrosine and phenylalanine. Since this fraction is a lyophilized product whose molecular profile is different from bacteriocins and simple carboxylic acids, its antimicrobial effect cannot be attributed to these molecules, or to alcohols or hydrogen peroxide. The fraction is bactericidal against weak-acid-resistant MRSA and weak-acid resistant P. aeruginosa at pH 5, and is bacteriostatic against both pathogens at pH 7. In combination formulation, the FK-1000 fraction is able to increase fivefold the effect of streptomycin against P. aeruginosa and it is not toxic to human epithelial cells at antimicrobial concentrations. 16 S rRNA microbiota analysis of antimicrobial-producing and non-producing kefir grains demonstrated that they are distinct. In summary, the results indicate that milk kefir grains can produce different classes of molecules with potent antibiotic activity against resistant bacteria.

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: An efficient immunoaffinity method.

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.

Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins.

The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.

An optimization study for expression of the rabies virus glycoprotein (RVGP) in mammalian cell lines using the Semliki Forest virus (SFV).

The Semliki Forest virus (SFV) viral vector has been widely used for transient protein expression. This study aimed to analyze comprehensively the capacity of SFV vector to express rabies lyssavirus glycoprotein (RVGP) in mammalian cells. The assessed parameters were transfection strategy, multiplicity of infection (MOI), harvest time and mammalian cell host. Two transfection approaches, electroporation and lipofection were evaluated to obtain the recombinant SFV, and the electroporation was found to be the most effective. Viral quantification by RT-qPCR was performed to elucidate the relation between the amount of recombinant virus utilized in the infection process and the production levels of the heterologous protein. Four different multiplicities of infection (MOIs = 1; 10; 15; 50) were evaluated using five mammalian cell lines: BHK-21, HuH-7, Vero, L929, and HEK-293T. Protein expression was assessed at two harvest times after infection (24 and 48 h). The recombinant protein generated was characterized by western blot, dot blot, and indirect immunofluorescence (IIF), while its concentration was determined by enzyme-linked immunosorbent assay (ELISA). Similar expression patterns were observed in cell lines BHK-21, HEK-293T, L929, and Vero, with higher RVGP production in the first 24 h. The BHK-21 cells showed yields of up to 4.3 µg per 106 cells when lower MOIs (1 and 10) were used. The HEK-293 T cells also showed similar production (4.3 µg per 106 cells) with MOI of 1, while the L929 and Vero cell lines showed lower expression rates of 2.82 and 1.26 µg per 106 cells, respectively. These cell lines showed lower expression levels at 48 h after infection compared to 24 h. Controversially, in the case of the HuH-7 cell line, RVGP production was higher at 48 h after infection (4.0 µg per 106 cells) and using MOIs of 15 and 50. This work may contribute to optimize the RVGP production using SFV system in mammalian cells. This study can also substantiate for example, the development of approaches that use of SFV for applications for other protein expressions and suggests values for relevant parameters and cell lines of this biotechnique.

DROSOPHILA S2 cell culture in a WAVE Bioreactor: potential for scaling up the production of the recombinant rabies virus glycoprotein.

The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal preventable infectious disease known. The objective of this work was to evaluate the potential of a bioreactor using wave-induced agitation in the initial steps of scaling up the rRVGP production process by a Drosophila melanogaster S2 cell line to produce rRVGP in sufficient quantities for immunization and characterization studies. Taking advantage of some remarkable features recognized in Drosophila S2 cells for scaling the culture process, a robust recombinant lineage (S2MtRVGPH-His) engineered by our group for the expression of rRVGP using a copper-inducible promoter was used in the bioreactor cultures. The WAVE Bioreactor was chosen because it represents an innovative approach to the cultivation of animal cells using single-use technology. For that purpose, we firstly established a procedure for culturing the S2MtRVGPH-His lineage in 100 mL Schott flasks. Using an inoculum of 5 × 105 cells/mL in culture medium (Sf900-III) induced with solution of CuSO4 (0.7 mM) and a convenient pH range (6.2-7.0), optimal parameter values such as time of induction (72 h) and temperature (28 °C) to increase rRVGP production could be defined. This procedure was reproduced in culture experiments conducted in a WAVE Bioreactor™ 2/10 using a 2 L Cellbag. The results in Schott flasks and in WAVE Bioreactor™ were very similar, yielding a maximum titer of rRVGP above of 1 mg.L-1. The immunization study showed that the rRVGP produced in the bioreactor was of high immunogenic quality.

Intracellular Delivery of HCV NS3p gene using vectored particles.

Viral hepatitis caused by the hepatitis C virus (HCV) affects millions of people worldwide. The non-structural protein 3 (NS3), one of the most conserved proteins in HCV, is the target of many therapeutic studies. The NS3 protease domain (NS3p) has a range of cytotoxic T lymphocyte (CTL) epitopes, and synthesizing the protein inside the cells is the most appropriate way to present it to the immune system. We developed a tool to study this kind of presentation, using two vectored particle (VP) systems, one based on the Semliki Forest virus (SFV) and the other on HCV pseudoparticles (HCVpp), both carrying the protease domain of the NS3 gene. In addition to producing the particles, we developed a method to quantify these VPs using qRT-PCR. We produced batches of approximately 2.4 × 104 SFV-NS3p/µL and 4.0 × 102 HCVpp-NS3p/µL. BHK-21 and HuH-7 cells treated with the VPs expressed the NS3 protein, thus showing the functionality of this system.

Rabies vaccine development by expression of recombinant viral glycoprotein.

The rabies virus envelope glycoprotein (RVGP) is the main antigen of rabies virus and is the only viral component present in all new rabies vaccines being proposed. Many approaches have been taken since DNA recombinant technology became available to express an immunogenic recombinant rabies virus glycoprotein (rRVGP). These attempts are reviewed here, and the relevant results are discussed with respect to the general characteristics of the rRVGP, the expression system used, the expression levels achieved, the similarity of the rRVGP to the native glycoprotein, and the immunogenicity of the vaccine preparation. The most recent studies of rabies vaccine development have concentrated on in vivo expression of rRVGP by viral vector transduction, serving as the biotechnological basis for a new generation of rabies vaccines.

Expression of Viral Envelope Glycoproteins in Drosophila melanogaster S2 Cells.

The expression of recombinant viral envelope glycoproteins in S2 (Drosophila melanogaster) has been performed with good results. This chapter contains protocols for the utilization of this system for the expression and analysis of proteins presented in cell plasma membrane.

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture.

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10-30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 10(9) BHK-21 cells from 4 × 10(6) cells in 13 day with 1,051 mL culture medium.

Transient expression of rabies virus glycoprotein (RVGP) in Drosophila melanogaster Schneider 2 (S2) cells.

The transient transfection process has been developed to allow rapid production of recombinant proteins. In this paper, we describe the transient expression of recombinant rabies virus glycoprotein (RVGP) in Drosophila melanogaster Schneider 2 (S2) cells. Different cell transfection reagents were evaluated, together with the effects of different cell cultivation procedures on RVGP expression. Yields of RVGP in the range 50-90ng/10(7) cells were obtained in multi-well plate transfection experiments, where it was observed that RVGP expression was linked to the DNA concentration. RVGP expression was 1.3 times higher using 10µg rather than 5µg of DNA. Inhibition of RVGP expression was observed at higher concentrations of DNA, with DNA concentrations above 15µg decreasing RVGP expression 1.5-fold for cells transfected with polyethylenimine (PEI) and 1.6-fold for cells transfected with cationic lipid. The results of shake flask transfection indicated that S2 cells were more effectively transfected in suspension than under static conditions. RVGP yields of 182.2ng/10(7) cells (PEI), 201ng/10(7) cells (calcium phosphate), and 215ng/10(7) cells (cationic lipid) were obtained for S2 cell suspension cultures. The highest volumetric RVGP concentration (309ng/mL) was found for cells transfected with cationic lipid. This value was 1.21 and 1.16 times higher, respectively, than for cells transfected with PEI (253.4ng/mL) and calcium phosphate (237.2ng/mL). There was little effect of transfection on the kinetics of cell growth, with growth rates of 1.12 and 1.19d(-1) for transfected and control cells, respectively. In spinner flasks, the expression of RVGP was 150 and 138ng/10(7) cells for transfection using PEI and calcium phosphate, respectively. A comparison of the different transfection reagents (calcium phosphate, cationic lipid, and cationic polymer) showed no significant differences in RVGP expression when shake flasks were used. Overall, the data indicated that transient expression in D. melanogaster S2 cells is a practical way of synthesizing RVGP for use in structural and functional studies.

Influence of aeration-homogenization system in stirred tank bioreactors, dissolved oxygen concentration and pH control mode on BHK-21 cell growth and metabolism.

This work focused on determining the effect of dissolved oxygen concentration (DO) on growth and metabolism of BHK-21 cell line (host cell for recombinant proteins manufacturing and viral vaccines) cultured in two stirred tank bioreactors with different aeration-homogenization systems, as well as pH control mode. BHK-21 cell line adapted to single-cell suspension was cultured in Celligen without aeration cage (rotating gas-sparger) and Bioflo 110, at 10, 30 and 50 % air saturation (impeller for gas dispersion from sparger-ring). The pH was controlled at 7.2 as far as it was possible with gas mixtures. In other runs, at 30 and 50 % (DO) in Bioflo 110, the cells grew at pH controlled with CO2 and NaHCO3 solution. Glucose, lactate, glutamine, and ammonium were quantified by enzymatic methods. Cell concentration, size and specific oxygen consumption were also determined. When NaHCO3 solution was not used, the optimal DOs were 10 and 50 % air saturation for Celligen and Bioflo 110, respectively. In this condition maximum cell concentrations were higher than 4 × 10(6) cell/mL. An increase in maximum cell concentration of 36 % was observed in batch carried out at 30 % air saturation in a classical stirred tank bioreactor (Bioflo 110) with base solution addition. The optimal parameters defined in this work allow for bioprocess developing of viral vaccines, transient protein expression and viral vector for gene therapy based on BHK-21 cell line in two stirred tank bioreactors with different agitation-aeration systems.

Semliki forest virus as a vector: pros and cons for its use in biopharmaceuticals production

The number of biopharmaceuticals for medical and veterinarian use produced in mammalian cells is increasing year after year. All of them are obtained by stable recombinant cell lines. However, it is recognized that transient gene expression produces high level expression in a short time. In that sense, viral vectors have been extensively used for producing recombinant proteins on lab-scale. Among them, Semliki Forest virus is commonly employed for this purpose. This review discusses the main aspects related to the use of Semliki Forest virus technology as well as its advantages and drawbacks which limit currently its utilization in biopharmaceutical industry on large-scale.