Drosophila melanogaster S2 cells for expression of heterologous genes: From gene cloning to bioprocess development

Insect cells are used for the expression of complex proteins in products such as vaccines and biopharmaceuticals. Physiology of a Drosophila mel- anogaster (lineage S2), transfected to stably express rabies virus glycoprotein (RVGP), was studied in batch culture and in a chemostat with serum-free medium. In batch mode, the system reached 3 9 107 cells mL-1 with specific growth rate of 1.5 d-1 with RVGP at 2.50 lg L-1. When operated continuously, three well- defined steady states were achieved at dilution rates (D) of 0.8, 0.5 and 0.2 d-1. The residual glucose and glutamine concentrations and the cell yields on glucose and glutamine decreased at lower D. High values of substrate consumption for maintenance may explain this variation in yields. The results indicated that glucose is not the limiting substrate of this process.

In the present study, the growth and key metabolic features of a gene-transfected Drosophila melanogaster (fruitfly) S2 (Schneider 2) cell population (S2AcRVGP cells), cultured in Sf900-II medium, have been evaluated to provide substantial support for the development of a bioprocess to produce RVGP (rabies-virus glycoprotein). Experimental cultures were grown both in a 100 ml Schott flask incubated in a shaker at 28 degrees C and 100 rev./min and in a 3 litre stirred-tank bioreactor at 28 degrees C, with increasing agitation. In small-scale culture, S2AcRVGP cells reached a maximum cell concentration of 1.13 x 10(7) cell/ml, presented a mu(max) (maximum specific growth rate) of 0.037 h(-1) and the growth was limited by oxygen deprivation. An early and remarkably long stationary phase was observed under hypoxia. Cell cultures grown in the bioreactor without oxygen limitation exhibited a maximum cell concentration of 2.2 x 10(7) cells/ml and mu(max) values as high as 0.048 h(-1). The ...

The speeches of Achilles form a notable component of Graeco-Latin poetry, including epic, and are essential in defining his character and issues important to poets. This discussion not only traces the evolution of themes and emotions in the direct and embedded speeches of Achilles from Homer’s Iliad to the poetic texts of Late Antiquity, including epic, but also identifies the various poetic ‘swerves’ and ‘mini-swerves’ (or ‘refractions’ and ‘mini-refractions’) that occur before and during Late Antiquity in the representation and characterization of Achilles as revealed in his direct and embedded speeches. The basic themes and emotions of the speeches of Achilles in Homer’s Iliad are the focus of the first part of the discussion, after which the development of these aspects are examined and tracked in Achilles’ speeches in subsequent classical and late antique epics and non-epic texts. A diachronic analysis of Achilles’ direct and embedded speeches from Homer to Late Antiquity reveals that major swerves in Achilles’ characterization occur in his speeches in the Iphigeneia of Aulis and Hecuba of Euripides, Statius’ Achilleid, and Quintus Smyrnaeus’ Posthomerica, while there is a poetic mini-swerve in Achilles’ speeches in Seneca’s Troades that becomes a major swerve in the Achilleid. Notwithstanding an oratio obliqua of Achilles in Philostratus’ Vita Apollonii that presents the Achaean hero favourably through his claimed lack of culpability for the death of Polyxena, these swerves in Achilles’ direct and embedded speeches ultimately serve to bring into question his Homeric characterization as a hero and even to undermine the nature of the heroic ideal itself.

Biotechnology Advances 30 (2012) 613–628 Contents lists available at SciVerse ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv Research review paper Drosophila melanogaster S2 cells for expression of heterologous genes: From gene cloning to bioprocess development Ângela M. Moraes a,⁎⁎, Soraia A.C. Jorge b, Renato M. Astray b, Claudio A.T. Suazo c, Camilo E. Calderón Riquelme c, 1, Elisabeth F.P. Augusto d, Aldo Tonso e, Marilena M. Pamboukian e, Rosane A.M. Piccoli d, Manuel F. Barral d, 2, Carlos A. Pereira b, e,⁎ a Departamento de Engenharia de Materiais e de Bioprocessos, Faculdade de Engenharia Química, Universidade Estadual de Campinas, Campinas, Brazil Laboratório de Imunologia Viral, Instituto Butantan, São Paulo, Brazil c Departamento de Engenharia Química, Universidade Federal de São Carlos, São Carlos, Brazil d Laboratório de Biotecnologia Industrial, Centro de Tecnologia de Processos e Produtos, Instituto de Pesquisas Tecnológicas do Estado de São Paulo, São Paulo, Brazil e Laboratório de Células Animais, Departamento de Engenharia Química, Escola Politécnica, Universidade de São Paulo, São Paulo, Brazil b a r t i c l e i n f o Article history: Received 11 February 2011 Received in revised form 7 October 2011 Accepted 30 October 2011 Available online 4 November 2011 Keywords: Drosophila melanogaster Schneider S2 cells Gene expression Heterologous genes Recombinant proteins Rabies virus glycoprotein Bioprocess a b s t r a c t In the present review we discuss strategies that have been used for heterologous gene expression in Drosophila melanogaster Schneider 2 (S2) cells using plasmid vectors. Since the growth of S2 cells is not dependent on anchorage to solid substrates, these cells can be easily cultured in suspension in large volumes. The factors that most affect the growth and gene expression of S2 cells, namely cell line, cell passage, inoculum concentration, culture medium, temperature, dissolved oxygen concentration, pH, hydrodynamic forces and toxic metabolites, are discussed by comparison with other insect and mammalian cells. Gene expression, cell metabolism, culture medium formulation and parameters involved in cellular respiration are particularly emphasized. The experience of the authors with the successful expression of a biologically functional protein, the rabies virus glycoprotein (RVGP), by recombinant S2 cells is presented in the topics covered. © 2011 Elsevier Inc. All rights reserved. Contents 1. 2. 3. 4. Introduction: Drosophila melanogaster S2 cells as a platform for recombinant protein production Plasmid vectors for heterologous protein expression in S2 cells . . . . . . . . . . . . . . . . 2.1. Selection vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Expression vectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Inﬂuence of vector transfection versus co-transfection . . . . . . . . . . . . . . . . . Metabolism of S2 cells in culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Metabolism of carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Metabolism of amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Lactate formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Ammonium formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Formulation of culture media for S2 cells . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. S2 cell performance in serum-supplemented media and in serum-free formulated media. 4.2. Cell behavior in media with supplements other than serum . . . . . . . . . . . . . . 4.3. Protein expression by rS2 cells cultivated in media with different compositions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614 615 615 615 616 617 617 618 619 620 620 620 621 622 ⁎ Correspondence to: C.A. Pereira, Laboratório de Imunologia Viral, Instituto Butantan, Av. Vital Brazil, 1500, CEP 05533-900, São Paulo, SP, Brazil. Tel.: + 55 11 3726 7222. ⁎⁎ Correspondence to: A.M. Moraes, Departamento de Processos Biotecnológicos, Faculdade de Engenharia Química, Universidade Estadual de Campinas, Av. Albert Einstein, 500, 13083-852 Campinas, SP, Brazil. Tel.: + 55 19 3521 3920. E-mail addresses: ammoraes@feq.unicamp.br (Â.M. Moraes), grugel@butantan.gov.br (C.A. Pereira). 1 On leave from Departamento de Ciências Químicas e Farmacêuticas, Universidad Arturo Prat, Iquique, Chile. 2 Present address: Instituto Federal de Educação Ciência e Tecnologia, Suzano, Brazil. 0734-9750/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2011.10.009 614 5. Factors affecting S2 cell growth in culture 5.1. Cell line . . . . . . . . . . . . . 5.2. Inoculum concentration . . . . . 5.3. pH . . . . . . . . . . . . . . . 5.4. Temperature. . . . . . . . . . . 5.5. Hydrodynamic forces. . . . . . . 5.6. Dissolved oxygen concentration. . 6. Final remarks. . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . Â.M. Moraes et al. / Biotechnology Advances 30 (2012) 613–628 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction: Drosophila melanogaster S2 cells as a platform for recombinant protein production The last three decades have witnessed an astonishing improvement in animal cell culture technology, going from basic studies on cell growth, metabolism and gene regulation to the large-scale production of high-value molecules used as biopharmaceuticals or as components of diagnostic kits and vaccines. Bioreactors with capacities as large as 20,000 L have been used (De Jesus and Wurm, 2011). Heterologous gene expression by animal cells is of particular interest in this area, mostly when the synthesis of proteins requiring complex post-translational modiﬁcations is envisaged. In this perspective, some technologies have been analyzed by research laboratories and also by pharmaceutical companies to develop cell lines which are able to attain high density and to produce high levels of the targeted protein. Most animal cells grow after adhesion to an appropriate support, but some cell lines can grow in suspension. The latter can be more appropriate for recombinant protein production on an industrial scale, since it is easier to scale up processes that do not require cell supports such as microcarriers. Moreover, a cell line can be transiently modiﬁed to express the desired heterologous protein. Transient expression systems are often used to produce virus or recombinant proteins for short periods. However, cell lines can be modiﬁed to stably express a heterologous protein. In this case, after a selection process, recombinant cell lines can be obtained. It is possible to use different transcription promoters to control the expression. The gene expression inﬂuenced by these promoters can be either constitutive or inducible. Mainly by their capability of performing complex post-translational modiﬁcations in proteins (such as glycosylation), several expression systems were developed using animal cell lines which are able to produce heterologous proteins with their original characteristics of localization and activity in the cell. Mammalian cells have been used to express a wide variety of proteins, such as antibodies, antigens, hormones and enzymes (Sunley and Butler, 2010), and insect cells have been increasingly used for the same purpose in recent years, since they require milder culture conditions than mammalian cells, such as lower temperatures and no CO2 addition to the gas phase. Spodoptera frugiperda (Sf) and Trichoplusia ni (BTI-TN-5BI-4 or High-Five™) cell lines are used mainly to produce heterologous proteins upon recombinant baculovirus infection. An example of a product obtained via the use of the baculovirus expression system and recently licensed by the FDA (in 2010) is Cervarix® (Glaxo Smith Kline), a vaccine for cervical cancer. Many others, such as Provenge® (Dendreon) for prostate cancer treatment; FluBlok® (Protein Sciences Co.), an inﬂuenza vaccine; Glybera® (AMT) for lipoprotein lipase deﬁciency; and Diamyd® (Diamyd Medical), a vaccine for type I diabetes, are in the ﬁnal stages of development or approval. Although very efﬁcient, the baculovirus system is transient, which can be quite inconvenient for production scale-up (Pfeifer et al., 1997; Summers and Smith, 1987). Recombinant protein expression systems based on the use of D. melanogaster cells are being increasingly used lately. To date, about 100 cell lines derived from D. melanogaster have been obtained. Of these, 12 are easily cultivated; however, the only cell lines used for . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622 622 623 623 623 623 623 624 625 625 heterologous gene expression are Schneider's 2 and 3 (S2 and S3, respectively) (Cherbas et al., 1994). Both are derived from late embryonic stages of D. melanogaster (Schneider, 1972), consisting of semiadherent cultures. These cells can be genetically altered to express functional proteins independently of viral infection. Stably transfected S2 cells can grow in suspension, reaching high concentrations, and this not only allows the use of continuous culture approaches, but also eases process scale-up. In addition, these cells can be transfected with vectors carrying inducible promoters, which allow the design of high-performance bioprocess protocols for protein production. A Drosophila Expression System (DES®) consisting of S2 cell expression and selection vectors was established in the 90s by Invitrogen (Carlsbad, CA). The developed vectors contain the necessary elements for ampliﬁcation in bacteria and for gene expression in S2 cells. These vectors need to be integrated into the cell genome to allow the heterologous cDNA replication and expression by the cell machinery. This system combines advantages of both the mammalian and baculovirus systems, namely, to be able to perform complex posttranslational modiﬁcations and to attain high cell densities in suspension under conditions of low-cost culture (Ikonomou et al., 2003; Ivey-Hoyle, 1991). Of the insect cells, the S2 cell line also has advantages in terms of maximum speciﬁc cell growth rates (μmax), reaching up to 0.084 h − 1 (Haldankar et al., 2006), and maximum cell concentrations (Xmax), attaining around 5.0 × 10 7 cells mL − 1 (Pamboukian et al., 2008; Shin et al., 2003). Regarding recombinant protein expression, some valuable molecules have already been expressed in S2 cells, such as the truncated recombinant dengue virus envelope protein subunits DEN2-80E (Clements et al., 2010), currently in the preclinical test phase as a dengue vaccine by Merck & Co. Other viral glycoproteins, such as the HIV gp120 (Ivey-Hoyle, 1991) and the Japanese Encephalitis Virus (JEV) glycoprotein (Zhang et al., 2008), have been expressed at good levels with appropriate biological activity, but maybe the one most extensively studied is the rabies virus glycoprotein (RVGP) (Astray et al., 2008; Batista et al., 2009, 2011; Bovo et al., 2008; Galesi et al., 2008; Lemos et al., 2009; Mendonça et al., 2008; Santos et al., 2009; Swiech et al., 2008a,b,c; Ventini et al., 2010; Yokomizo et al., 2007). This particular protein can be useful as a component of rabies vaccines and also of rabies detection kits. Aiming to improve RVGP production, several culture conditions for speciﬁc and volumetric RVGP expression were studied by these authors. Conditions found to be suitable for RVGP production were a serum-free TC-100 or IPL-41 based culture medium (Batista et al., 2008; Galesi et al., 2007a,b), inducible metallothionein promoter (Lemos et al., 2009; Ventini et al., 2010), low cultivation temperature (22 °C) (Swiech et al., 2008b) and low oxygen concentration (≤40%) (Batista et al., 2009). Some other approaches have been shown to increase the period of expression during culture, such as the supplementation of culture medium with amino acids which are depleted at later culture times (Swiech et al., 2008c) and the addition of Lonomia obliqua hemolymph, which resulted in an increase in the number of RVGP expressing cells and in the amount of RVGP produced Â.M. Moraes et al. / Biotechnology Advances 30 (2012) 613–628 (Mendonça et al., 2008). The studies of RVGP expression showed that a lower RVGP cell content (2 μg 10 − 7 cells) was achieved in a bioreactor than in shake ﬂasks (5.5 μg 10− 7 cells). Nevertheless, the larger cell densities provided by bioreactor cultures resulted in a RVGP recovery of 3 mg L − 1 (Ventini et al., 2010). Technical approaches were also improved in order to quantify the S2 expressed RVGP by optimizing methods of cell lysis and extraction protocols, reﬂecting better RVGP detection and quantiﬁcation in S2 cell batches (Astray et al., 2008). In spite of the fact that S2 cell lines are difﬁcult to grow at low densities, which somehow limits the process to select the desired cell clone or even recombinant cell populations, good performances in terms of the desired heterologous protein production have been reported (Jorge et al., 2008; Lemos et al., 2009; Santos et al., 2009; Ventini et al., 2010). These high levels of heterologous protein production are probably a consequence of the strong promoters used and also of the large number of plasmid copies integrated into the cellular genome (Brighty et al., 1991; Buckingham et al., 1996; Lemos et al., 2009; McCarroll and King, 1997). Even though protein post-translational modiﬁcation pathways may be performed differently by S2 cells than by mammalian cells, speciﬁc protein activity is not only frequently maintained but can even be increased. Human dopamine β-hydroxylase, for instance, can be successfully expressed and secreted by recombinant S2 cells in concentrations as high as 16 mg L − 1 (Li et al., 1996). Despite having a molecular weight lower than that of the human native protein, probably due to differences in glycosylation, since it was veriﬁed that the deglycosylated enzymes from both sources were identical in size, preserved enzymatic activity was detected for the protein expressed by the S2 cells. Similarly, Chang et al. (2005) expressed the human βsecretase in S2 cells, an enzyme related to Alzheimer's disease evolution, and depending on the expression vectors employed, the production of proteins with different molecular weights was observed and attributed to different post-translational modiﬁcation patterns. However, an increase of up to 260% in enzyme activity was also noticed. According to Kim et al. (2005), most N-glycans in recombinant glycoproteins expressed by S2 cells consist of simple paucimannosidic structures, a mostly trimannosyl core with or without an α(1,6)linked fucose core, not including galactose or sialic acid in their constitution. Despite the fact that the N-glycan structure and composition can control protein secretion, biological function and its in vivo circulatory half-life, to a certain extent this problem can be circumvented by suppressing the enzyme β-N-acetylglucosaminidase in the N-glycosylation pathway of S2 cells (Kim et al., 2009). Other biologically active recombinant proteins obtained by the use of S2 cell cultures include human plasminogen (effectively secreted in concentrations from 10 to 15 mg L − 1, according to Nilsen and Castellino (1999)), the site-speciﬁc biotinylated human myeloid differentiation factor 88 (MyD88) (Basile et al., 2007), the E protein of the Japanese Encephalitis Virus (JEV) (Zhang et al., 2007) and ﬁsh gonadotropin (GtHs) subunits of luteinizing hormone (LH) and follicle stimulating hormone (FSH) (Zmora et al., 2007). 2. Plasmid vectors for heterologous protein expression in S2 cells Plasmid vectors used for S2 cell transfection and generation of cell populations expressing heterologous genes basically do not differ from plasmids used for mammalian cell transfection. The plasmids have essentially the following elements: a bacterial replication origin gene (pUC ori), an actin (pAc) or metallothionein promoter (pMt) used for the expression of the target gene; a gene coding for a peptide that can be produced as a marker of expression (V5), a Drosophila copia gene promoter (pCopia) used for the selection gene (hygromycin, puromycin etc.) expression, an ampicillin gene for selection in bacteria, the SV40 polyadenylation signal (SV40pA) and the secretion signal sequence of Drosophila immunoglobulin heavy chain binding protein (BiP). 615 2.1. Selection vectors To obtain a stable recombinant cell line, it is common to transfect the cells with the expression gene and a selection gene (often an antibiotic resistance gene). For S2 cells, three antibiotic resistance genes have been described: hygromycin, blasticidin and puromycin. The hygromycin gene is the most commonly used and acts as an aminocyclitol to inhibit protein synthesis by disrupting translocation and promoting mistranslation. The hygromycin selection vector pCoHygro contains the Escherichia coli hygromycin resistance gene (HPH) (Gritz and Davies, 1983) for selection of transfectants with hygromycin B (Palmer et al., 1987). The hygromycin resistance gene can also be inserted into the expression vector, avoiding co-transfection protocols (Jorge et al., 2008; Lemos et al., 2009; Santos et al., 2009). The resistance hygromycin gene codes a phosphotransferase that inactivates hygromycin. Blasticidin, on the other hand, is a nucleoside antibiotic isolated from Streptomyces griseochromogenes that inhibits protein synthesis in both prokaryotic and eukaryotic cells (Takeuchi et al., 1958; Yamaguchi et al., 1965). Resistance is conferred by expression of either one of two blasticidin S deaminase genes: bsd from Aspergillus terreus (Kimura et al., 1994a,b) or bsr from Bacillus cereus (Izumi et al., 1991). These deaminases convert blasticidin S to a nontoxic deaminohydroxy derivative. Puromycin, in turn, is an aminonucleoside antibiotic produced by Streptomyces alboniger that causes premature chain termination during translation in various cell types (de la Luna and Ortin, 1992; Vara et al., 1985). Puromycin N-acetyltransferase inactivates cytotoxic puromycin by acetylating the amino position of its tyrosinyl moiety. Iwaki and Castellino (2008) recently generated a co-transfection vector, pCoPURO, which allows the expression of puromycin Nacetyltransferase. This system minimizes transfected S2 cell selection time. The co-transfection vector was reported to be functional in S2 cells at concentrations of puromycin between 2 and 10 μg mL − 1, eliminating nontransfected cells within three days (Iwaki et al., 2003). 2.2. Expression vectors Different genes have been used to explore gene expression by S2 cells (Chang et al., 2005; Clements et al., 2010; Deml et al., 1999a; Jorge et al., 2008; Lemos et al., 2009; Li et al., 1996; Nilsen and Castellino, 1999; Santos et al., 2007, 2009; Valle et al., 2001; Ventini et al., 2010). The particular use of vectors for the expression of the enhanced green ﬂuorescent protein (EGFP) (Santos et al., 2007) and of the viral proteins applicable to the formulation of vaccines and as components of HBsAg test kits (Deml et al., 1999a; Jorge et al., 2008) and RVGP (Lemos et al., 2009; Santos et al., 2009; Ventini et al., 2010) have been studied in detail. Under the control of metallothionein promoter (pMt), for instance, S2 cells were co-transfected with plasmid vectors containing the EGFP gene and vectors containing the hygromycin selection gene pCoHygro, with a view to establishing parameters for optimized gene expression (Santos et al., 2007). A protocol for EGFP stable transfection was established by using a lipotransfection method taking into account cell density, DNA concentration and the amount of Cellfectin reagent. After hygromycin selection, recombinant S2 ﬂuorescent cells were above 90% of the total population. Conditions for inducible EGFP expression using the metallothionein promoter were established with CuSO4 as inducer and with sodium butyrate (NaBu) as expression enhancer. EGFP recombinant S2 cell cultures were shown to be quite heterogeneous regarding the intensity and cell localization of ﬂuorescence through analysis of EGFP-expressing cells by confocal microscopy. Spectroﬂuorimetric kinetic studies of CuSO4-induced recombinant S2 cells showed EGFP expression as soon as 5 h after induction, with a progressive increase in ﬂuorescence attaining high values (up to 10 5 counts/s) 72 h after the induction. The use of 700 μM of CuSO4 in the exponential phase led to a better 616 Â.M. Moraes et al. / Biotechnology Advances 30 (2012) 613–628 performance in terms of S2 cell growth, percentage of ﬂuorescent cells and ﬂuorescence intensity. Treatment of EGFP recombinant S2 cells with NaBu resulted in a decrease in cell multiplication and viability, which were accompanied not only by an enhancement of ﬂuorescence intensity of the cell population but also by an early increase in the percentage of ﬂuorescent cells in the induced cell population. Efﬁcient HBsAg accumulation in culture supernatant was attained by use of a constitutive expression plasmid in S2 cells (Jorge et al., 2008). These S2 cells were transfected with the pAcHBsAgHygro plasmid vector, which contained the S gene coding for the HBsAg, under control of the constitutive Drosophila actin promoter (pAc), and the hygromycin B (Hygro) selection gene. The vector was introduced into S2 cells by DNA lipotransfection and a cell population was selected by its resistance to hygromycin B. The pAcHBsAgHygro vector integrated into the S2 cell genome and approximately 1000 copies per cell were detected by Southern blot in a highly HBsAg-producing cell subpopulation. HBsAg production, however, varied in between different cell subpopulations. In the same recombinant subpopulation, higher speciﬁc HBsAg expression was found in cells cultured in Insect Xpress (13.5 μg 10 − 7 cells) and SFX (7 μg 10 − 7 cells) media than in Sf-900 II medium (0.6 μg 10 − 7 cells), although higher HBsAg volumetric production was noticed in Sf-900 II medium due to the higher cell density obtained. An increase in HBsAg of 40% was observed in a culture maintained under hygromycin selection pressure (Jorge et al., 2008). The expression of the same protein in S2 cells was analyzed by Deml et al. (1999a) using two vector systems. The ﬁrst was based on the co-transfection of an expression vector for the S gene under the control of an inducible Drosophila metallothionein (Mt) promotor and a resistance plasmid which carries a selectable marker dihydrofolate reductase (dhfr) gene under the control of a Drosophila actin 5C distal promoter. The second system was based on the transfection of a single plasmid including both expression units. Both vector systems were suitable for the generation of stably transfected DS-2 cell lines and recombinant subpopulations were cloned by using the soft agar technique. HBsAg concentrations in cell culture media reached 4.75 and 2.95 μg mL − 1, respectively, for the co-transfection and transfection procedures. The cDNA encoding the RVGP gene was cloned in expression plasmids under the control of the inducible metallothionein (pMt) or the constitutive actin promoter (pAc) (Lemos et al., 2009; Santos et al., 2009; Ventini et al., 2010). Inducible expression vectors were designed to either bear or not bear the BiP external secretion signal (Kirkpatrick et al., 1995) and a cDNA coding for the hygromycin selection (H). Constitutive expression vectors were designed without BiP, with or without a cDNA coding for hygromycin selection (H). The vectors without H were co-transfected with the pCo-Hygro. All vectors were transfected or co-transfected into S2 cells, the cell populations were selected and subpopulations were then obtained by reselection with hygromycin. All cell populations expressed the RVGPmRNA, and upon induction with CuSO4, a transfected cell subpopulation was shown to express RVGP speciﬁc values as high as 8.3 10 − 7 μg cell − 1 (Lemos et al., 2009), leading to RVGP concentrations around 3 mg L − 1 in bioreactors (Ventini et al., 2010). The importance of subpopulation selection was evidenced by the increasing RVGP yields during the procedures. Additionally, RVGP synthesis may be optimized by chromatin opening and culture medium selection. Another gene, coding for human plasminogen, has also been expressed in S2 cells under the inﬂuence of the BiP protein signal sequence, which allowed protein secretion into the medium (Nilsen and Castellino, 1999). After clonal selection of high-expressing cells, an expression level of 10 to 15 mg L − 1 of the protein in the culture medium was obtained. The human dopamine beta-hydroxylase (DBH) gene has been expressed in S2 cells with yields higher than 16 mg L − 1 (Li et al., 1996). The vector used contained the hygromycin resistance gene under the control of the Drosophila copia long terminal repeat. The recombinant protein was mainly found in the supernatant, similarly to what was observed for HBsAg. An inducible vector was successfully employed to obtain human menin from transfected S2 cells (Valle et al., 2001). Experiments in shake ﬂasks showed that the production of menin was improved when the induction was performed late in the exponential phase, reaching 1 mg of puriﬁed protein per liter of culture medium. The use of different expression vectors to analyze beta-secretase (betaSec) glycosylation by recombinant S2 cells with cDNAs encoding beta1,4-galactosyltransferase (GalT) and Galbeta1,4GlcNAc alpha2,6-sialyltransferase (ST) was reported by Chang et al. (2005). To express human GalT and ST in S2 cells, the respective cDNAs were subcloned into a constitutive expression plasmid, pZT, which contained the zeocin resistance gene. On the other hand, the human beta-secretase was inserted into an inducible vector. Three vectors were co-transfected in S2 cells: an inducible vector containing beta-secretase, a constitutive vector containing GalT and ST and a pCoHygro vector containing the hygromycin resistance gene. Also, as a control, S2 cells were co-transfected with inducible and selection vectors. Stably transformed polyclonal cell populations were obtained after selection with both hygromycin and zeocin or only hygromycin in control cells. Different molecular weights were observed for the target protein, indicating that the recombinant beta-secretase from S2betaSEC/GalT-ST cells (S2 cells co-transformed with cDNAs encoding beta-secretase, glycosyltransferases, GalT and ST) contained the glycan residues of beta1,4-linked galactose and alpha2,6-linked sialic acid. Interestingly, the enzyme activity of the recombinant beta-secretase from S2betaSEC/GalT-ST cells was enhanced by up to 260% that of the S2betaSEC cells control. Recently, genes coding for truncated recombinant dengue virus envelope protein subunits (80E) were efﬁciently expressed in S2 cells. Four inducible vectors were constructed using cDNAs coding for 80E subunits from each of the four dengue serotypes. These four vectors and the pCoHygro vector were co-transfected in S2 cells. Immunization of mice with a mixture of all four 80E subunits expressed in S2 cells and a speciﬁc adjuvant resulted in potent virus neutralizing antibody responses to each of the four serotypes. Moreover, the dengue serotype 2 (DEN2-80E) subunit, at low doses in the vaccine formulation, was able to protect mice and monkeys against viral challenge (Clements et al., 2010). 2.3. Inﬂuence of vector transfection versus co-transfection The DES® conceived and commercialized by Invitrogen, is based on the use of two separate plasmid vectors for co-transfection into S2 cells, an expression vector and a selection vector. For instance, the gene coding for human menin (a potential tumor suppressor of type 1 multiple endocrine neoplasia according to Chandrasekharappa and Teh, 2003) could be successfully expressed in S2 cells, leading to protein concentrations up to 2 mg L − 1 in the supernatant (Valle et al., 2001) employing a co-transfection process using a pCoHygro selection vector and a pMT/HisA-Menin inducible expression vector. Although some protocols have been developed for improving the efﬁciency of the co-transfection (Santos et al., 2007), the probability of having heterogeneous S2 cell populations containing cells which received only the pCoHygro selection vector and not the expression vector is not negligible and results in a less efﬁcient process to obtain highly productive and more homogeneous S2 cell populations. Successful improvements in this system, based on the use of a single plasmid vector for single transfection carrying both the gene of interest and the gene coding for selection, have been reported (Iwaki and Castellino, 2008; Jorge et al., 2008). The BiP external secretion signal, which is present in the DES system, was shown to be not always necessary for target gene expression. 617 Â.M. Moraes et al. / Biotechnology Advances 30 (2012) 613–628 For RVGP gene expression, a higher production was, in fact, obtained without BiP (Lemos et al., 2009). Regardless of the transfection or co-transfection process, the use of constitutive (pAc) or inducible (pMt) promoters has been shown to result in high levels of gene expression and heterologous protein expression, although higher productivity has been described for inducible expression (Lemos et al., 2009; Ventini et al., 2010). 3. Metabolism of S2 cells in culture Even though studies concerning genetic, physiologic and metabolic aspects of the fruit ﬂy D. melanogaster can easily be found in the literature, the metabolism of cell lines derived from this insect has seldom been addressed in recent publications. The work devoted to elucidating their behavior when cultivated in vitro mainly focuses on the S2 cells, referring mostly to aspects of nutrient uptake and production of byproducts by nonrecombinant S2 cells (S2) (Bovo et al., 2008; Sondergaard, 1996) as well as by recombinant ones (rS2) (Batista et al., 2008, 2009; Galesi et al., 2008; Lim and Cha, 2006; Swiech et al., 2008a,b,c; Valle et al., 2001). Data on maximum speciﬁc cell growth rates and maximum cell concentrations attained have frequently been are analyzed; however, none of the consulted references shows speciﬁc studies on S2 cell metabolic pathways. Correlations have commonly been established between data on their substrate uptake and byproduct generation and those available for other insect cell lines, more speciﬁcally S. frugiperda (Sf9) and T. ni (Tn) (Benslimane et al., 2005; Bernal et al., 2009; Doverskog and Haggstrom, 1998; Doverskog et al., 1997, 2000; Drews et al., 1995, 2000; Ferrance et al., 1993; Öhman et al., 1995; Rhiel et al., 1997). Representative metabolic results for rS2 cells (Batista et al., 2009; Galesi et al., 2008) are presented in Fig. 1. S2 cell lines, as most animal cells, utilize glucose (GLC) and glutamine (GLN) as the main sources for carbon and nitrogen, respectively. Even though ammonium (NH4+) and lactate (LAC) in addition to alanine (ALA) are the most important resulting byproducts, as also observed for other animal cells, their byproduct formation pattern is different (Batista et al., 2008, 2009; Bovo et al., 2008; Galesi et al., 2008; Swiech et al., a 2007a). Their behavior seems to be in between that of mammalian (Amable and Butler, 2008) and other insect cells (Benslimane et al., 2005; Doverskog et al., 1997; Drews et al., 2000; Öhman et al., 1995). S2 and rS2 cell lines can show different metabolic performances in different media, as shown in Table 1. The maximum values for byproduct formation (NH4+, LAC and ALA) and the calculated kinetic parameters (μmax and the yield factors YX/GLC, YX/GLN, YLAC/GLC and YNH4/GLN, the ﬁrst two being related to cell concentration, X) express in quantitative terms the metabolic response of these cells and seem to be much more related to the substrates' initial concentration values (GLC, GLN and some other amino acids) than to the different operation modes practiced. The cells have signiﬁcantly different μmax in different media, within a range of 0.019 to 0.04 h − 1, with an increase in this kinetic parameter from the IPL-41-modiﬁed formulation to the Sf-900 II medium. The richest medium formulations, such as Sf-900 II, should simply extend the exponential growth phase. Nonetheless, the observed variation in μmax values demonstrates that the pathways of the metabolic processing of different culture media are associated with different yields or different ﬂuxes through them (Bovo et al., 2008). 3.1. Metabolism of carbohydrates As in many other animal cell systems, glucose and glutamine are consumed continuously often until they are exhausted. Both substrates are capable of providing energy and carbon skeleton to build cell components (refer to Table 1 for their initial concentrations and for those of other substrates in the most frequently used culture media), and the lack of either of the substrates generally results in cell growth limitation (Bovo et al., 2008; Galesi et al., 2008; Swiech et al., 2008a). In spite of this, only glucose starvation can be related to loss of viability in S2 cell cultivation (Batista et al., 2009; Bovo et al., 2008). In most studies using S2 cells, GLC is only completely exhausted at the end of the stationary phase, when other medium components have already limited cell growth. Cultures of S2 and Sf9 cells in supplemented IPL-41 result in GLC depletion only for S2 cells (Batista b 2.5 100 4.0 60 1.0 40 Xv Viab 0.5 0 0 c 2 4 6 8 10 12 40 20 0.0 14 12 GLC NH 4+ 1.0 20 0.0 60 2.0 + 1.5 80 3.0 NH 4 (mg L-1) 80 GLN (g L-1) 2.0 Viab (%) Xv (107cell mL-1) 100 0 0 2 4 0 2 4 6 8 10 12 14 6 8 10 12 14 d 0.4 1.0 6 0.2 GLC LAC 4 0.1 2 0 0.0 0 2 4 6 8 Time (d) 10 12 14 GLU (g L-1) 0.3 8 LAC (g L-1) GLC (g L-1) 10 0.8 0.6 0.4 0.2 0.0 Time (d) Fig. 1. Representative results for a rS2 cell line cultivated in a stirred tank bioreactor with dissolved oxygen controlled at 30% (adapted from Galesi et al., 2008): a) viable cell concentration (XV) and viability (Viab); b) glutamine concentration (GLN) and ammonium concentration (NH4+); c) glucose concentration (GLC) and lactate concentration (LAC); d) glutamate concentration (GLU). 618 Â.M. Moraes et al. / Biotechnology Advances 30 (2012) 613–628 Table 1 Typical initial substrate concentrations, byproduct formation and kinetic parameters indicating cell growth phase in which the maximum values occurred (calculated from data in the literature). Measured variable Medium formulation used Serum-containing TC100 Initial values Maximum values [occurrence in cell growth phase] Serum-free SF900IIc Modiﬁed TC100c,d Modiﬁed IPL41c,e HyQ SFXf,g Bovo et al. (2008) Swiech et al. (2008a) Galesi et al. (2008) Batista et al. (2009) Valle et al. (2001) GLC (g L− 1) GLN (g L− 1) ALA (g L− 1) ASN (g L− 1) CYS (g L− 1) PRO (g L− 1) SER (g L− 1) NH4+ (g L− 1) 0.700 0.600 0.225 0.350 0.029 0.350 0.550 NA LAC (g L− 1) 0–0.06 [exponential] 1.00 [stationary] 0.020 [exponential] 361 960 NA ND 10.0 3.5 0.45 ND 0.05 0.70 0.35 0.50 [stationary] 1.00 [decline] 3.30 [stationary] 0.040 [exponential] 260–480 960–1500 0.003–0.20 60–180 10.0 3.5 ND ND ND ND ND 0.08 [stationary] 0.20 [stationary] ND 10.0 3.5 0.30 1.30 0.12 0.50 0.20 0.35 [stationary] 0.60 [exponential/stationary] 1.60 [stationary] 0.019 [exponential] 59.5 558 NA 70.8 9.60 NA NA NA NA NA NA 0.46 [stationary] 0.89 [stationary] NA ALA (g L− 1) μmax (h− 1) Yield factors (exponential cell growth phase) a,b YX/GLC (107 cells g− 1) YX/GLN (107 cell g− 1) YLAC/GLC (g g− 1) YNH4/GLN (mg g− 1) 0.026 [exponential] 197 121 NA 57.3 0.034 [exponential] 1100 NA 0.23 NA Glucose, GLC; glutamine, GLN and the amino acids alanine, ALA; asparagines, ASN; cysteine, CYS; proline, PRO and serine, SER; ammonium, NH4+; lactate, LAC. NA, not available, ND, not determined. a TC-100 medium (Sigma, EUA) + 10% FBS. b S2 cell line cultivated in shake ﬂasks. c rS2 cell line expressing rabies virus glycoprotein (RVGP). d TC-100 medium (Cultilab, Brazil) supplemented with glucose, glutamine, yeastolate and lipidic emulsion. e IPL-41 medium (Invitrogen, EUA) supplemented with glucose, glutamine, tyrosine, methionine, yeastolate, and lipid emulsion. f HyQ SFX medium (SH30278.02, Hyclone). g rS2 cell line expressing human menin. et al., 2009, 2010, 2011). This result suggests a more intense ﬂux of GLC through a glycolysis pathway in dipteran cell lines than in lepdopteran ones. Except for the studies on TC-100 culture medium (Bovo et al., 2008), in which the initial GLC concentration is low (around 1.0 g L − 1), all other medium formulations mentioned in Table 1 indicate excess GLC, characterized by high speciﬁc uptake rates (Batista et al., 2008, 2009; Galesi et al., 2008; Swiech et al., 2007a; Valle et al., 2001). Therefore, in all these cases, a high byproduct concentration should be expected. In addition, high oxygen availability stimulates an increase in speciﬁc glucose uptake (Batista et al., 2009). Using labeled substrates and nuclear magnetic resonance, Drews et al. (2000) elucidated the high metabolic ﬂexibility of Sf9 cells, and a similar behavior can be expected from S2 cells. Tn-5 cells, on the other hand, have a metabolism similar to that of mammalian cell lines, characterized by a high level of LAC production as a response to the excess GLC in the culture (Benslimane et al., 2005; Rhiel et al., 1997; Sugiura and Amann, 1996). In the case of mammalian cells, GLC is clearly transported by the aid of a speciﬁc protein located in the cell membrane, in a process classiﬁed as facilitated, saturable, bi-directional and proportional to the concentration gradient (Amable and Butler, 2008). Higher availability of GLC in the culture medium implies a higher speciﬁc GLC uptake. The energetic yield derived from GLC uptake depends on the activated metabolic pathway: from 2 mol of ATP (adenosine triphosphate) per mol of GLC if only glycolysis is activated and up to 38 mol of ATP per mole of GLC if glycolysis, the tricarboxylic acid (TCA) cycle pathway and the electron transport chain are all activated. There is a balance between glycolysis and the pentose phosphate pathway (PPP) that is mediated by the glucose-6P molecule, although the ﬂux through the PPP is lower in mammalian cell systems in comparison to insect cells (Beslimane et al., 2005). Finally, concerning the utilization of other sugars by S2 cell lines, similarly to that observed for Sf9 (Bedard et al., 1993; Ikonomou et al., 2003; Swiech et al., 2008a), uptake of GLC seems to be favored over that of maltose, sucrose and lactose (Batista et al., 2009). Bedard et al. (1993) pointed out that the uptake of other sugars starts after GLC reaches low concentrations (approx. 2 g L − 1). 3.2. Metabolism of amino acids Amino acids are supposed to participate in protein synthesis or be degraded to provide carbon and nitrogen skeletons for catabolic and anabolic reactions. Glutamine is probably the most important substrate for biosynthetic and energetic purposes in S2 cells. Studies with the rich culture medium Sf-900 II indicate that lack of GLN limits growth, characterized by a shortening of the exponential growth phase (Bovo et al., 2008; Swiech et al., 2008a), and causes the end of the cell growth phase as well as cell death (Swiech et al., 2008a). Nevertheless, studies employing other medium formulations (supplemented TC-100 and modiﬁed IPL-41) (Batista et al., 2009; Galesi et al., 2008) point to different results, corroborating the idea of high metabolic ﬂexibility of S2 cells. The partitioning and yield observed may depend on the cell line. In mammalian cells, GLN is initially converted into GLU and NH4+ in the mitochondria via the glutaminolysis pathway (Amable and Butler, 2008; Palomares et al., 2004). With excess GLN, a deregulated consumption of GLN, characterized by high speciﬁc uptake rates and therefore high levels of ALA and NH4+, is observed (Amable and Butler, 2008; Benslimane et al., 2005; Bernal et al., 2009; Drews et al., 2000; Elias et al., 2003; Mendonça et al., 1999). Unlike mammalian cell lines, with excess GLN and O2, Sf9 insect cells show a metabolic behavior characterized by a lack of NH4+ formation (Doverskog et al., 1997; Drews et al., 2000; Öhman et al., 1995). On the other hand, under the same cultivation conditions, Tn-5 cells show high Â.M. Moraes et al. / Biotechnology Advances 30 (2012) 613–628 levels of NH4+ production, comparable to those of mammalian cells (Benslimane et al., 2005; Rhiel et al., 1997; Sugiura and Amann, 1996). Studies conducted by L. Haggstrom and her team elucidated a large part of insect cell metabolism (Doverskog et al., 2000; Drews et al., 2000; Haggstrom, 2000). These studies identiﬁed the presence in lepidopteran cells (Sf9 among them) of a NADH-dependent enzyme (glutamine: 2-oxoglutarate aminotransferase, GOGAT, also known as glutamate synthase), which catalyzes the transfer of GLN amino groups to α-ketoglutarate (α-KG), forming GLU, without NH4+ release. ALA and α-KG are produced through the condensation of GLU and pyruvate (PYR). Thus, GLU enters the TCA cycle and the system is simultaneously detoxiﬁed. Therefore, the GOGAT activity explains the central metabolic behavior of Sf9 cells, in addition to their capacity to grow in the absence of GLN or GLU (Haggstrom, 2000), the lack of NH4+ formation and the release of large amounts of ALA with excess GLN or GLU. Doverskog et al. (2000) also detected glutamate dehydrogenase (GDH) and glutaminase in Sf9 cells. However, the ﬂux through glutaminase and GDH was much lower than the ﬂux through the GOGAT pathway (11 and 55%, respectively). Finally, the dependence on NADH instead of on reduced nicotinamide adenine dinucleotide phosphate (NADPH) suggests that the GOGAT activity is related more to energy generation than to biosynthesis. Another important difference between mammalian and insect cells is in ﬂexibility in amino acid metabolism. Insect cells are more ﬂexible, being able to synthesize a higher number of amino acids, such as GLN, GLU and aspartate (ASP) (Öhman et al., 1996), although this fact become less important when richer culture media, such as Sf900 II, are employed (Bernal et al., 2009). This versatility of amino acid synthesis may explain the cells' capability to grow in GLN-free culture media as well as in GLN and GLU-free culture media, provided that ammonium is supplied (Öhman et al., 1996). In mammalian cell cultures, the use of GLN-free media requires ASP and/or asparagine (ASN) supplementation (Bebbington et al., 1992; Keen and Hale, 1996) in order to provide precursors for GLN intracellular biosynthesis. Moreover, Sf9 cells can also grow in cysteine-free media, provided that a cell population in early exponential phase is used as inoculum (Doverskog and Haggstrom, 1998). Therefore, amino acid uptake and synthesis are dependent on culture medium formulation. In supplemented IPL-41 and in Sf-900 II media, S2 cell lines show high uptake of proline (PRO), cysteine (CYS), serine (SER) and asparagine (ASN), sometimes even leading to the complete exhaustion of these amino acids (Batista et al., 2009; Swiech et al., 2007a, 2008a). The detailed inﬂuence of these amino acids on cell growth is not yet clear, but media supplementation with PRO results in a signiﬁcant increase in μmax (Swiech et al., 2008a). Besides, GLU is consumed only after GLN becomes limited (at concentrations less than 400–600 mg L − 1) (Batista et al., 2009; Bovo et al., 2008; Galesi et al., 2008; Swiech et al., 2007a). In some cases, GLU production at the end of cultivation is also observed (Bovo et al., 2008). The consumption of the other amino acids is much lower. As indicated above, the production of ALA by insect cells is a consequence of GLN partial oxidation via alanine aminotransferase (AlaT). ALA production of approximately 1.4 g L − 1 in Sf-900 II and supplemented IPL-41 culture media is reported in the literature for S2 cells (Batista et al., 2009). Apparently the amino acid donor for ALA production depends on the culture medium formulation employed. In Sf-900 II medium, it seems to depend on PRO availability, so that PRO exhaustion implies reconsumption of ALA (Swiech et al., 2008a). Even so, it must be considered that other factors may affect ALA formation. For instance, under O2-limiting conditions, ALA release is higher (up to 2.7 g L − 1) than in situations not involving O2 limitation. In supplemented IPL-41 culture medium, ALA formation in S2 cell cultures seems to depend directly on GLN availability (Batista et al., 2009). In spite of this, when GLN concentration is maintained at approximately 2.5 g L − 1 in fed-batch mode, ALA production by these 619 cells is terminated, indicating that an amino acid other than GLN may also limit growth (Batista et al., 2009). Differently from S2 cell cultures in Sf-900 II medium, no reconsumption of ALA is observed in supplemented IPL-41 culture medium, even though GLN is completely exhausted (Batista et al., 2009). Interestingly, in serumsupplemented TC-100 medium, ALA is produced even after GLN is exhausted; however, the nitrogen source for its formation is not yet known (Batista et al., 2009). Many authors deﬁne ALA generation in animals as the most important destination for PYR resulting from GLC metabolism through glycolysis. Small amounts of LAC and NH4+ are then released into the environment, contributing to cell detoxication (Bernal et al., 2009; Drews et al., 2000; Ikonomou et al., 2003; Öhman et al., 1995). As already mentioned, Drews et al. (2000) proposed the existence of a GOGAT via in insect cells as a form of transferring amidic and aminic nitrogen from GLN to ALA. They also pointed out the low generation of ALA in GLN-free culture media, where GLU acts as a GLN-nitrogen donor. According to Drews et al. (2000), the majority of other amino acids (such as histidine, lysine, threonine, glycine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophane), despite being incorporated into cellular protein, are not relevant for purposes of energy generation. 3.3. Lactate formation S2 cell lines have different lactate formation patterns as a consequence of medium formulation or initial culture conditions (Table 1). Very low lactate concentrations were observed by Galesi et al. (2007a) and Swiech et al. (2008a), but this compound may reach values from 0.6 to 3.0 g L − 1 under different conditions (Batista et al., 2009; Bovo et al., 2008). According to Ikonomou et al. (2003), LAC production by insect cells such as the Sf9 cell line only occurs under O2-limiting conditions. In spite of this, while working with S2 cell cultures in supplemented IPL-41 medium, Batista et al. (2009) observed LAC formation even at 40% O2 partial pressure. In Sf-900 II medium, with excess GLC, a clear correlation seems to exist between the beginning of LAC formation by S2 cells and GLNlimiting conditions (Bovo et al., 2008) or the incapacity to produce ALA (Batista et al., 2009). According to Bovo et al. (2008), LAC production by S2 cells depends on the speciﬁc GLC uptake rate as well as on the initial GLN: GLC ratio, which assumes values from 1:5 in Sf-900 II or supplemented TC-100 and IPL-41 media to 1:2 in serum-containing TC-100 medium. If there is a high concentration of GLC in the system at the moment of exhaustion of the amino acid nitrogen donor, LAC is formed. Data reported by Batista et al. (2009) indicate that LAC release by S2 cells does not depend solely on GLN availability, but mostly on the incapacity to produce ALA as a strategy of sink of LAC and NH4+. In fact, in this study, LAC production by S2 cells began when ALA reached its maximum value As LAC formation also depends on excess GLC, sugar availability is of utmost importance. When GLC becomes limited (less than 1–2 g L − 1), less LAC is formed and even reconsumed, so LAC can be considered as an alternative carbon source for the maintenance of metabolic activities of S2 cells (Batista et al., 2009; Bovo et al., 2008) Thus, the measured LAC concentration is the result of formation and uptake processes. Mammalian cells in culture produce large amounts of LAC as a response to excess GLC, even under nonlimiting O2 conditions (Altamirano et al., 2006a,b; Amable and Butler, 2008; Cruz et al., 2000). It may be toxic at concentrations higher than 3.6 g L − 1, but the most common deleterious effect is the reduction in medium pH, which can easily be corrected by the addition of an alkali (Amable and Butler, 2008). Some suggestions to reduce the effect of lactate are the use of fed-batch operation mode, the substitution of GLC by 620 Â.M. Moraes et al. / Biotechnology Advances 30 (2012) 613–628 other sugars or the use of metabolic engineering to improve the ﬂux of PYR through the TCA cycle. In insect cell systems, LAC is also a byproduct of GLC anaerobic metabolism and depends directly on its availability in culture. Lactate formation is still a way to regenerate NADH when complete oxidation of GLC through the TCA cycle and the respiratory chain is not possible (Doverskog et al., 1997; Drews et al., 2000), and its production may vary with culture medium composition (Rhiel et al., 1997), as illustrated in Table 1. 3.4. Ammonium formation Differently from what is observed for LAC formation, the production of ALA does not assure a complete sink of the NH4+ generated from amino acid metabolism, and therefore some NH4+ is always found (typical results in Fig. 1). There is a continuous NH4+ release in quantities which vary depending on culture conditions and medium formulation (Table 1). In supplemented TC-100 medium, values of up to 0.080 g L − 1 were observed (Galesi et al., 2008), while higher values of 0.35 g L − 1 were detected in supplemented IPL-41 (Batista et al., 2009), and up to 1.1 g L − 1 in Sf-900 II (Swiech et al., 2008a). Apparently, the tolerance of S2 cells to ammonium depends on the culture conditions, especially the culture medium formulation. This high tolerance is not surprising, given that it has been demonstrated that the insect D. melanogaster was favored by the evolutionary process with a high tolerance to ammonium (Doverskog and Haggstrom, 1998). Strategies to reduce the negative effects of ammonium may consider the use of fed-batch operation mode and the choice of GLN-free media or media containing dipeptides of GLN in addition to the use of metabolic engineering approaches (Amable and Butler, 2008). In supplemented TC-100 medium, the rate of NH4+ formation by S2 cell cultures decreased as GLN became limited, demonstrating a direct relationship between the generation of the byproduct and the uptake of this amino acid. No direct relationship was observed in other medium formulations in which NH4+ might be formed by the metabolism of other amino acids (Batista et al., 2009). The pattern of NH4+ release by S2 cells is very similar to that observed in mammalian cells, suggesting the existence of similar metabolic pathways for both types of cells. NH4+ formation results mainly from GLN assimilation through glutaminolysis, which also produces GLU. The latter can further liberate another molecule of NH4+, giving rise to a molecule of α-KG (Amable and Butler, 2008). 4. Formulation of culture media for S2 cells Several classic culture media and new formulations have been evaluated for the growth of S2 cells. Depending on inoculum concentration and culture conditions, different cell performances can be observed in different media. Key aspects of culture medium formulation for S2 and other animal cells are appropriateness for speciﬁc cell lines or cell populations, efﬁciency in terms of high cell growth and complete nutrient usage as well as low byproduct formation and high accumulation of the target protein to reduce downstream processing costs. Validation of culture media for the production of immunobiologicals for human use is of upmost importance, mostly for formulation of media free from animal-derived components such as serum and proteins. These topics are covered in the following sections. 4.1. S2 cell performance in serum-supplemented media and in serumfree formulated media D. melanogaster cells can reach high concentrations (above 10 6 cells mL − 1) in basal media such as Schneider's. This medium was originally developed for the culture of Drosophila cells; however it is suitable for cultivating other dipteran cell lines. In fact, the frequent use of Schneider's medium for Drosophila cell culture is more probably attributable to the longer time it has been on the market (Echalier, 1997). In addition to this formulation, many other culture media can be employed; examples include D22 (Sondergaard, 1996), M3 (Benyajati and Dray, 1984; Culp et al., 1991; Gibson et al., 1993; Jeon et al., 2003; Kim et al., 2011; Lee et al., 2000a, b, 2011; Lim et al., 2004; Millar et al., 1995; Park et al., 1999; Park et al., 2001; Shin and Cha, 2002; Shin and Cha, 2003; Sondergaard, 1996), M3:D22 a 1:1 volume ratio (Joanisse et al., 1998), Schneider (Backovic et al., 2010; Bengali et al., 2011; Clements et al., 2010; Deml et al., 1999a; Hill et al., 2001; Jorge et al., 2008; Torfs et al., 2000; Tota et al., 1995; Van Poyer et al., 2001; Vanden Broeck et al., 1995), Sf-900 II (Batista et al., 2008, 2009, 2011; Benting et al., 2000; Bovo et al., 2008; Deml et al., 1999a,b; Galesi et al., 2007b; Jorge et al., 2008; Swiech et al., 2007b; Ventini et al., 2010), serumfree EX-CELL 400 (Li et al., 1996), DES expression (Denault et al., 2000; Jorge et al., 2008; Khaznadji et al., 2003; Lehr et al., 2000; Perret et al., 2003; Prosise et al., 2004), HyQ SFX (Banks et al., 2003; Jorge et al., 2008; Valle et al., 2001), Drosophila SFM (Kleditzsch et al., 2003; Pawar et al., 2011; Southon et al., 2004), IPL-41 (Bachmann et al., 2004; Batista et al., 2008, 2009, 2011; Galesi et al., 2007b), Grace's (Galesi et al., 2007b), TC100 (Batista et al., 2008; Bovo et al., 2008; Galesi et al., 2007a,b, 2008; Jorge et al., 2008; Swiech et al., 2007b), TNM-FH (Swiech et al., 2007b), TNM-FH:Sf900 II at a 1:1 volume ratio (Swiech et al., 2007b) and Insect Xpress (Jorge et al., 2008; Rasch et al., 2010). Successful cultivation in many of these media often requires supplementation with fetal bovine serum (FBS) at volume concentrations from 5 to 10% or with insect medium supplements (IMS), such as the one commercialized by Sigma-Aldrich Corp. However, a major area of concern is the presence of animal-derived components in media used to culture cells for recombinant protein expression (Moraes et al., 2008). Particularly, after the mad cow crisis, the regulatory agencies have pushed for serum-free and protein-free media. By using an animal component-free medium, the possible threat of adventitious agent contamination from animal-sourced material is eliminated. The term “animal origin” is not applicable to lower eukaryotic organisms, such as the higher plants, fungi, protozoa, and algae. It does not include prokaryotic organisms, such as bacteria or blue-green algae either. This means that hydrolysates obtained from these sources are generally regarded as safe for animal cell culture aiming at the production of therapeutic proteins. Serum-free media such as Drosophila SFM, Sf-900 II, HyQ SFX and EX-CELL 400 series formulations can also be employed, frequently resulting in higher reproducibility than with FBS-supplemented basal media. However, these media are normally more expensive than basal media and when their components are not described, studies on formulation optimization can only be performed by the addition of components. Nevertheless, no culture medium formulation is currently considered in the literature to be absolutely superior to the others. Many publications refer to the use of serum-free formulations for S2 cells (Batista et al., 2008; Bovo et al., 2008; Galesi et al., 2007a,b, 2008; Jorge et al., 2008), mostly based on Sf-900 II, TC-100 and IPL41 media, among others. The data in Table 2 refer to a compilation of average results regarding the use of different medium formulations for S2 and rS2 cells (TNM-FH; TC-100; Sf-900 II and IPL-41, both supplemented or not supplemented with FBS and other compounds). It can be observed that S2 and rS2 cells have quite different μmax and Xmax values in the tested culture media. S2 cells have lower speciﬁc growth rates in FBS-supplemented media than in Sf-900 II, reaching cell concentrations from 4 to 10 × 10 6 cells mL − 1 in the tested formulations. Similarly, rS2 cells have lower speciﬁc growth rates in FBSsupplemented media, but cell concentrations above 1 × 10 7 621 Â.M. Moraes et al. / Biotechnology Advances 30 (2012) 613–628 Table 2 Growth parameters of S2 cell lines (S2 and rS2) in different culture media. Culture medium TNM-FH + 10% FBS μmax (h− 1) Xmax (cells mL− 1) S2 rS2 S2 0.0078 – 8.5 × 106 Reference rS2 6 – TC-100 + 10% FBS 0.0112 0.0151 3.8 × 10 Supplemented TC-100 (serum-free medium) Sf-900 II (serum-free medium) IPL-41 + 10% FBS – 0.0260 – 16.0 × 106 0.0375 0.0407 9.8 × 106 14.1 × 106 Supplemented IPL-41 (serum-free medium) Grace's + 10% FBS – b 0.01 – 0.0190 – b 0.01 4.4 × 10 6 2 × 10 6 36.4 × 10 – 1 × 106 6 Swiech et al. (2007b) Swiech et al. (2007b) Galesi et al. (2008) Swiech et al. (2007b) Galesi et al. (2007b) Batista et al. (2009) Galesi et al. (2007b) Cells were inoculated at a concentration of 5 × 105 cells mL− 1, except for S2 cells in serum-supplemented TNM-FH, at a concentration of 106 cells mL− 1 and in IPL-41 and Grace's FBS-supplemented media, at a concentration of 7.5 × 105 cells mL− 1. cells mL − 1 were observed only in serum-free formulations with the target protein downstream processing offering a clear advantage. S2 cells cultured in a stirred bioreactor with Sf-900 II can reach up to 5 × 10 7 cells mL − 1 (Pamboukian et al., 2008; Shin et al., 2003). The maximum speciﬁc growth rate observed for different cell lines can vary signiﬁcantly, for instance, from 0.033 to 0.072 h − 1, equivalent to duplication times of 21 h and 9.6 h, respectively (Augusto et al., 2008). This suggests that the appropriated choice of medium can not only increase cell concentration in the stationary growth phase, but also improve the speciﬁc growth rate, as discussed below. 4.2. Cell behavior in media with supplements other than serum Considering that insect cells have nutritional needs that may be quite different from those of mammalian cells, hemolymph would potentially be a quite suitable basal medium supplement. The addition of 1% hemolymph may result in an increase of around 24% in Xmax for S2 cells cultivated in TC-100 supplemented with 10% FBS and close to 6% for rS2 cells grown in Sf-900 II (Mendonça et al., 2008). Similarly, RVGP production by rS2 cells in 10% FBSsupplemented TC-100 and in Sf-900 II increased 59 and 43% respectively with the addition of 1% hemolymph. The effects of the fractionation of this supplement in a Hi-Prep 26/60 Sephacryl 200 gel column were also evaluated, showing that a 35% improvement in RVGP production could be observed with a pool containing high molecular weight proteins. Further studies along this line would make possible the identiﬁcation and expression of the most active hemolymph proteins by microorganisms, allowing their use as deﬁned supplements in S2 cell culture media, similarly to what has been done for Sf9 cells (Souza et al., 2005). Typical growth parameter values are presented in Table 3 for different S2 cell culture conditions in Sf-900 II medium. Values of μmax and Xmax for several S2 cell lines in Sf-900 II medium using shake ﬂasks and a stirred tank bioreactor vary in the ranges of 0.030 to 0.084 h − 1 and of 0.98 to 2.57 × 10 7 cells mL − 1, respectively. In spite of the fact that Sf-900 II is a culture medium speciﬁcally developed for Sf9 cells, the values of μmax obtained with it for S2 cells are higher than those obtained in many other media (Table 2). One hypothesis is that the cell growth values observed for S2 cells are higher than those for Sf9 cells due to the smaller size of the former (8 μm) in comparison to the latter cell type (12 μm), thus suggesting a higher speciﬁc metabolic activity associated with the S2 cells. In Table 4 it is also shown that the growth capacity of S2 cells in Sf-900 II medium can still be increased to μmax above 0.08 h − 1 by supplementing it with Table 3 Growth parameters of S2 cell lines (S2, rS2I and rS2II) in Sf-900 II medium in shake ﬂasks and a bioreactor. Culture system Cell lines Xmax (107 cells mL− 1) μmax (h− 1) Shake ﬂasks, 100 mL, 100 rpm S2 0.98 1.20a 1.41 1.89 2.00 2.13b 2.57c 0.038 0.044a 0.037 0.030 0.048 0.084b 0.061c Bubble-free stirred tank bioreactor, 2 L, DO 40–50% and pH 6.2 rS2I rS2II rS2I rS2II Cells were cultured in shake ﬂasks and a bioreactor with supplemented and nonsupplemented Sf-900 II culture medium at 28 °C. In all cases initial cell seeding (Xino) was of 5 × 105 cells mL− 1 (adapted from Swiech, 2007b). Proline, PRO; glutamine, GLN; cysteine, CYS; aspartate, ASP. S2, nontransfected S2 cells; rS2I, S2AcRVGP cells; rSII, S2AcRVGP2K cells. a PRO 3 g L− 1. b PRO 1 g L− 1. c PRO 1.94 g L− 1, GLN 2.35 g L− 1, CYS 0.013 g L− 1, SER 0.13 g L− 1, and ASP 0.07 g L− 1. proline (PRO), an amino acid that S2 cells are able to use as a source of energy (Culp et al., 1991). Sf-900 II medium has been used for large-scale production of recombinant proteins expressed by lepidoptern cells using baculovirus, but its composition is not available in the literature. Therefore, there is an interest in alternative optimized formulations with accessible components that are more suitable for S2 cell adaptation and growth. Two supplemented basal culture medium formulations with excellent potential for substituting Sf-900 II medium for S2 cells, TC-100 (Galesi et al., 2007a) and IPL-41 (Batista et al., 2008) medium, are described. In the case of TC-100 medium, supplementation with concentrated milk whey, lipid emulsion, yeastolate, Hy Soy (a soybean hydrolysate), glucose, glutamine, Pluronic F68, hydrolyzed lactalbumin and hemolymph was evaluated. The use of hydrolyzed lactalbumin and concentrated milk whey did not give adequate results for cell growth and recombinant protein production (Galesi, 2007). In the particular case of supplementation with milk whey, morphological changes were observed in the S2 cells, which showed increased surface granulation as well as high cell death rates. Lipid emulsion concentrations above 1% (v/v) were also observed to be deleterious to the mentioned S2 cells, causing either reduction in the speciﬁc cell growth rates or cell death (Galesi et al., 2007a). Despite the fact that Hy Soy is reported to be effective in promoting the growth of animal cells (Donaldson and Shuler, 1998; Table 4 Inﬂuence of temperature on the growth of S2 cell lines (rS2I and rS2II) in Sf-900 II medium in shake ﬂasks and a bioreactor. Culture system Temperature (°C) Cell lines μmax (h− 1) Xmax (107 cells mL− 1) Shake ﬂasks (100 mL), 100 rpm 22 25 28 28 34a 28a 24a 20 16 22 28 28a 20a rS2I 0.015 0.032 0.034 0.042 0.022 0.036 0.040 0.020 0.009 0.026 0.049 0.061 0.027 1.39 1.43 1.78 2.48 1.20 2.08 2.48 1.87 0.07 1.61 2.00 2.57 2.97 Bubble-free stirred tank bioreactor (1 L), DO 40–50%, pH 6.2 rS2II rS2I rS2II In all cases initial cell seeding (Xino) was 5 × 105 cells mL− 1 (adapted from Swiech, 2007). Proline, PRO; glutamine, GLN; cysteine, CYS; serine, SER. rS2I, S2AcRVGP cells; rSII, S2AcRVGP2K cells. a Sf-900 II medium supplemented with PRO 1.94 g L− 1, GLN 1.35 g L− 1, CYS 0.007 g L− 1 and SER 0.07 g L− 1. 622 Â.M. Moraes et al. / Biotechnology Advances 30 (2012) 613–628 Heidemann et al., 2000), yeastolate was more effective than the soy hydrolysate in improving the S2 cell growth rate (Galesi et al., 2007a). The formulation containing 3 g L − 1 of yeastolate, 10 g L − 1 of glucose, 3.5 g L − 1 of glutamine, 1% (v/v) lipid emulsion and 0.1% (w/v) Pluronic F68 was pointed to as the most satisfactory of the tested media formulated with TC-100 as basal medium. However, the use of this culture medium in a stirred tank bioreactor at 28 °C and 90 rpm required supplementation with 0.6% (m/v) Pluronic F68 to properly protect cells from the shear stress associated with mechanical mixing. Concentrations of up to 1.6 × 10 7 cells mL − 1 could be achieved under these conditions in stirred bioreactors (Galesi et al., 2008). The synthetic nonionic surfactant Pluronic F68 is routinely used in large-scale mammalian cell culture processes and in addition to increasing cell membrane resistance to hydrodynamic shear forces (Chisti, 2000; Ghebeh et al., 2002; Palomares et al., 2000), it may also inﬂuence glucose and glutamine metabolism and improve the transport of medium components into the cell (Clincke et al., 2011). S2 cells modiﬁed to express RVGP do not lose their target protein expression ability in serum-free supplemented TC-100 formulation (Galesi et al., 2008). In this TC-100-based formulation, RVGP production is higher to that found with the commercial media Sf-900 II and TC-100 supplemented with 10% FBS. Studies on rS2 cell growth carried out with IPL-41 medium, which is free of animal-derived compounds and proteins in general, containing 10 g L − 1 of glucose, 0.5 g L − 1 of fructose, 2 g L − 1 of lactose, 3.5 g L − 1 of glutamine, 0.6 g L − 1 of tyrosine, 1.48 g L − 1 of methionine, 6 g L − 1 of yeastolate, 1% lipid emulsion and 0.05% Pluronic F68 gave maximum speciﬁc growth rates up to 0.025 h − 1 and maximum cell concentrations of around 1.9 × 10 7 cells mL − 1 in shake ﬂasks (Batista et al., 2008). This formulation, together with a variant containing 0.1% of Pluronic F68, allowed higher RVGP production than the serum-free formulation Sf-900 II in a bench bioreactor operating at a dissolved oxygen concentration of 40% air saturation and pH 6.3 (Batista et al., 2009). The effects of different culture medium compositions may undoubtedly vary for different cell lines. For instance, IPLB-LdFB and IPLB-LdEIta cell lines from gypsy moth fat body and embryos, respectively, and UFL-AG-286 from velvetbean caterpillar embryos were maintained for 1 to 12 years on two medium formulations, namely, modiﬁed TC-100 containing 9% fetal bovine serum and the commercial serum-free medium EX-CELL 400 (Lynn, 2006). LdFB cells had similar behavior in both media, while a better performance was observed for LdEIta cells in EX-CELL 400 and for AG-286 cells in TC-100. A further topic to be addressed regarding culture medium composition is S2 cell response to osmolality. rS2 cells, like other insect cells show good performance in culture media with high osmolality (Ikonomou et al., 2003; Olejnik et al., 2003; Tramper et al., 1992), and this supports their potential use as a platform for the production of biologicals. Success can be attained in the culture of rS2 cells, even at initial osmolality values of around 360 mOsm kg − 1 H2O for supplemented TC-100 formulations (Galesi et al., 2008) to approximately 460 mOsm kg − 1 H2O for supplemented IPL-41 media (Batista et al., 2009). 4.3. Protein expression by rS2 cells cultivated in media with different compositions Choice of culture medium may affect not only cell growth but also recombinant protein production. Recent data on culture of rS2 cells expressing HBsAg employing different cell culture media gave quite interesting results (Jorge et al., 2008). In FBS-supplemented Schneider medium, both target protein concentration and cell growth were low. High HBsAg expression was obtained in InsectXpress (up to 13.5 μg 10 − 7 cells) and SFX medium (7 μg 10 − 7 cells), possibly as a consequence of reduced cell growth. In Sf-900 II, TC-100 and DES media, the opposite is observed (low target-product concentration and high values for cell growth). Even though higher speciﬁc HBsAg expression was observed in InsectXpress medium, the cultures performed in the SFX formulation produced a much larger total amount of the target product (115 μg) than that in the InsectXpress medium (12 μg). Altogether, these results indicate that culture medium composition plays in important role on cell growth and protein synthesis, which must be worked out together in the design of the bioprocess. In comparison, rS2 cells expressing RVGP under the control of an inducible metallothionein promoter cultured in Sf-900 II medium produced up to 30 μg of product (around 3.5 μg 10 − 7 cells) (Lemos et al., 2009). Despite the fact that the speciﬁc RVGP expression was higher for cells cultivated in Insect-Xpress medium (around 5.5 μg 10 − 7 cells), volumetric RVGP productivity was lower in this medium formulation due to the limited cell growth. Similarly, two other recombinant proteins (the polypeptide VP1 of human hepatitis A virus and canstatin), also produced by rS2 cells in an inducible manner, attained maximum levels of 6.24 and 76 μg mL − 1, respectively (Lee et al., 2007, 2009). All mentioned product concentrations are of the order of magnitude veriﬁed for the production of other recombinant proteins by genetically modiﬁed S2 cell lines in different media, such as erythropoietin (Lee et al., 2000b), endostatin (Park et al., 2001), tumstatin (Jeon et al., 2003), hepatitis B antigen (Deml et al., 1999a), menin (Valle et al., 2001) and cyclooxygenase 2 (Chang et al., 2002), and are also in the range normally observed for monoclonal antibody production (from 20 to 100 μg mL − 1) by mammalian cells (Tamashiro and Augusto, 2008). 5. Factors affecting S2 cell growth in culture Like most insect cells, S2 cells are semi-adherent and their cultivation is commonly accomplished in suspension. Many factors affect their behavior in culture, such as cell line and passage number, inoculum concentration, pH, temperature, dissolved oxygen concentration, hydrodynamic forces, culture medium and toxic metabolites. Since the effects of culture medium formulation and cell metabolism have already been discussed, the inﬂuence of other factors on S2 cell growth will be analyzed here, focusing mainly on μmax and Xmax. Particular emphasis is given to a comparison of stirred S2 cell culture to static cultures, since this information is more useful for scaling up production systems based on rS2 cells. 5.1. Cell line The original sources of most insect cell lines are complex organs or embryos, the latter being the case for the S2 cell line, which originated from around 300 embryos of D. melanogaster (Schneider, 1972). With an already intrinsic genetic heterogeneity at origin, variations among cells of the same cell line are not unexpected. Another factor that may exert a strong inﬂuence on cell growth behavior is the genetic modiﬁcation procedure commonly performed when cells are transfected with heterologous DNA aiming at the expression of a recombinant protein. As a matter of fact, S2 cells and rS2 cells have some different cultivation characteristics. For instance, a μmax of 0.0523 h − 1 and an Xmax of to 1.65 × 10 7 cells mL − 1 were reported for S2 cells and a μmax of 0.0482 h − 1 and an Xmax of 2.59 × 10 7 cells mL − 1 for rS2 cells expressing RVGP (Yokomizo et al., 2007). This behavior is consisted with other results reported in the literature (Flick, 1995; Keith et al., 1999), with the lower growth rate of the rS2 cells attributed to the limited availability of energy (or nutrients) for biomass synthesis. This is a consequence of the use of part of the host cell resources to fulﬁll the additional energy demand for maintenance and expression of the heterologous DNA. Efﬁcient cell line cryopreservation and reactivation are also crucial to achieving reproducible results. Cryopreservation of S2 cells is successfully performed by suspending 10 7 cells in a mixture of fetal 623 Â.M. Moraes et al. / Biotechnology Advances 30 (2012) 613–628 S2 cells in culture are known by their limited growth when inoculated at low densities. For instance, for rS2 cells expressing RVGP, as the inoculum density (Xino) increases from 2.5 × 10 5 to 6 × 10 5 cells mL − 1, the length of the lag phase (tlag) decreases from 72 h to virtually zero, while the values of μmax remain practically constant at 0.03 h − 1 and the values of Xmax, slightly increase from 1.29 × 10 7 to 1.73 × 10 7 cells mL − 1 (Swiech, 2007). As for other animal cells showing this behavior, the addition of large amounts of growth factors is probably necessary for cell replication if the conditioned medium derived from the inoculum is discarded or if low cell concentrations are employed. If the conditioned culture medium is discarded, inocula at high cell concentrations can contribute to faster accumulation of the autocrin growth factors produced. 5.3. pH Control of pH is fundamental in cell culture. Insect cells show better proliferation at lower pH values, from 6.2 to 6.5 (Léo et al., 2008), but there is little data available correlating the inﬂuence of pH with S2 cell growth. rS2 cells have been successfully cultivated at pHs of 6.0, 6.2 and 6.3 (Batista et al., 2009; Swiech et al., 2008a,b). However, better cell growth seems to be achieved at higher pH values, around 6.8. The scarce information available in the literature indicates the need for data to determine an optimal pH value for the growth of S2 cells. 5.4. Temperature Insect cells grow at optimal temperatures of 26 to 28 °C and, like other animal cells, are generally sensitive to changes in temperature (Léo et al., 2008). rS2 cells have high μmax values at temperatures between 24 °C and 28 °C, as shown in Table 4. Nevertheless, these cells can grow at a μmax of 0.014 h − 1, reaching an Xmax of 2.39 × 10 7 cells mL − 1 at 20 °C in 100 mL shake ﬂasks in Sf-900 II medium supplemented with amino acids in fed-batch mode (unpublished data). 5.5. Hydrodynamic forces Traditionally, insect cells are known to be very sensitive to hydrodynamic forces (Swiech, 2007). This characteristic prevails for S2 cells, as demonstrated in experiments in which the protecting agent Pluronic F68® was added to cultures carried out in shake ﬂasks (Swiech, 2007) and in a bioreactor (Galesi et al., 2008). These results are summarized in Table 5. Studies performed with rS2 cells cultured in Sf-900 II medium in shake ﬂasks at 28 °C at stirring rates from 100 to 250 rpm (Swiech, 2007) revealed that agitation between 100 and 140 rpm generates the best results in terms of growth: μmax = 0.054 h − 1 and Xmax = 1.32 × 10 7 cells mL − 1. In the case of cultures in a bioreactor, mixing rates of up to 150 rpm have not been found to cause cell damage by hydrodynamic forces (Swiech, 2007). 5.6. Dissolved oxygen concentration Oxygen is often the ﬁrst component of the culture medium to become limited in animal cell cultures. However, despite being important for cell growth, it can also be toxic at high concentrations, so its supply should be carefully controlled. Aeration of S2 cell cultures can be successfully performed through the use of diffusion Culture system Pluronic Xino (106 μmax F68® cells mL−1) (h− 1) (% m/v) Xmax (107 Reference cells mL− 1) Shake ﬂasks (250 mL): 220 rpm, Sf-900 II medium Stirred tank bioreactor (2 L): 90 rpm, bubble free, DO 30%, supplemented TC-100 medium 0.0 0.1 0.5 0.5 0.034 0.039 1.62 1.64 Swiech et al. (2007a) 0.1 0.3a 0.6 0.75 0.75 0.75 0.024 0.033 0.027 0.55 0.90 1.60 Galesi et al. (2008) TC-100 medium was supplemented with 3 g L− 1 of yeastolate, 10 g L− 1 of glucose, 3.5 g L− 1 of glutamine and 0.1% V/V of lipid emulsion. a Stirring was progressively increased, being maintained at 50 rpm from 0 to 6.5 h, 75 rpm from 6.5 to 69.5 h and ﬁnally 90 rpm from 69.5 to 264 h. membranes, such as silicon or polytetraﬂuorethylene (Teﬂon) membranes, which have high permeability to oxygen and carbon dioxide, resulting in high transfer rates between the membrane and the cell culture medium in the bioreactor. In this case, supplying oxygen to the culture medium does not require the introduction of bubbles, diminishing shear forces and consequently reducing cell damage. Although the speciﬁc respiration rate (QO2) of animal cells may vary signiﬁcantly between different cell lines, it falls within ranges as wide as from 45 to 504.10 − 11 mmol O2 (cell h) − 1 (Fleischaker et al., 1981). Most mammalian cells have speciﬁc respiration rates from small to intermediate within this range. According to Kioukia et al. (1995), hybridoma cells have QO2 values that vary within a range of 60 to 120.10 − 11 mmol O2 cell − 1 h − 1. Insect cells typically have lower QO2 than mammalian cells, which is partly explained by the fact that they are smaller and therefore have smaller biovolumes. Sf9 cells, for instance, show QO2 values between 11 and 76.10 − 11 mmol O2 cell − 1 h − 1 according to Kioukia et al. (1995), and being quite small, S2 cells consume less oxygen than Sf9 cells and much less than mammalian cells. A typical proﬁle of QO2 and oxygen uptake rate (OUR) during S2 cell culture is shown in Fig. 2. These cells demand more oxygen during the lag phase, when they are adjusting to the new culture conditions, as also pointed out by Swiech and Suazo (2006). After that phase, QO2 remains virtually constant, but at a lower level. The OUR tends to increase when cell concentration increases, reaching its highest level during the exponential phase. The inﬂuence of different concentrations of dissolved oxygen (from 5% to 95% air saturation) on the growth of rS2 cells was analyzed at pH 6.3 by Batista et al. (2009) in 500 mL spinner ﬂasks and also in a stirred tank bioreactor for comparison purposes. The data obtained are summarized in Table 6, showing that in spinner ﬂasks, the maximum value of μmax was seen at around 60% air saturation. On the other hand, the maximum concentration of viable cells viable cells Q O2 OUR 30 2 1,5 20 1 10 0,5 0 0 1 2 3 4 5 6 7 8 9 10 11 QO2 (1E-10 mmol/cell h) OUR (mmol O2/L h) 5.2. Inoculum concentration Table 5 Inﬂuence of Pluronic F68 on growth parameters of rS2 cells cultured at 28 °C in shake ﬂasks and a bioreactor in Sf-900 II medium and supplemented TC-100 medium. viable cells (1E6 cells/mL) bovine serum (FBS), Sf-900 II medium and dimethylsulfoxide (DMSO) at a 5:4:1 volume ratio, and storing by immersion in liquid nitrogen (−198 °C), while adequate cell reactivation can be achieved by rapid immersion in a water bath at 37 °C and washing with fresh medium (Galesi, 2007). days Fig. 2. Proﬁle of QO2 and OUR during a representative experiment with S2 cells with C controlled at 40% air saturation. 624 Â.M. Moraes et al. / Biotechnology Advances 30 (2012) 613–628 Table 6 Growth parameters of rS2 cells cultured in spinner ﬂasks and a stirred tank bioreactor at various dissolved oxygen concentrations. DO (%) Xino (106 cells mL− 1) Spinner ﬂask (1 L), pH = 6.3, 5 0.75 20 0.75 40 0.75 60 0.75 95 0.75 Bioreactor (1 L), pH = 6.2 50 (28 °C) 0.5 50 (22 °C) 0.5 Xmax (107 cells mL− 1) μmax (h− 1) Reference 28 °C 2.98 1.48 3.68 2.10 1.60 0.030 0.031 0.034 0.038 0.029 Batista et al. (2009) 2.30 1.61 0.059 0.037 Swiech et al. (2007a) The spinner ﬂasks and stirred tank bioreactor were operated with pH and dissolved oxygen (DO) control. Cultures in spinner ﬂasks were performed with IPL-41 medium supplemented with 3 g L− 1 of yeastolate, 10 g L− 1 of glucose, 3.5 g L− 1 of glutamine and 0.1% v/v lipid emulsion and those in the bioreactor, with Sf-900 II medium. (3.68 × 10 7 cells mL − 1) was obtained at a dissolved oxygen concentration (C) of 40%. The cultures in a 2 L bioreactor (at C of 50%) showed that at 28 °C and 22 °C, Xmax reaches 2.30 and 1.61 × 10 7 cells mL − 1, respectively, while μmax values of 0.059 and 0.037 h − 1 were observed under the same conditions. At C values of 100% and higher, the growth of these rS2 cells was totally inhibited, indicating that such high concentrations may be toxic to these cells (Swiech, 2007). In Table 7 a series of representative values of QO2 are given for S2 and rS2 cells and other animal cells. The data show that the speciﬁc respiration rate of S2 cells is about ﬁve times lower than that of rS2 cells. Comparing data on S2 and rS2 cells to those on Sf9 cells, it can be observed that Sf9 cells have higher speciﬁc respiration rates (Pamboukian et al., 2008). In addition, when infected by viruses, Sf9 cells tend to respire even more intensively (Kioukia et al., 1995; Pamboukian et al., 2008). The critical oxygen concentration level that causes a reduction inQO2 (Ccrit), also shown in Table 7 for different S2 cells, varies between 5 and 12%. The resistance of rS2 cells to hypoxia has been observed in cell cultures carried out in shake ﬂasks, where very low concentrations of oxygen are available after four days. When cells taken from the hypoxic stationary phase were inoculated into fresh and oxygenated Sf-900 II medium, they were able to recover normal cell growth after a brief lag phase with little or no loss of viability. The behavior of S2 cells, as observed here and reported in studies presented in the literature, sustains the hypothesis that the metabolism of Drosophila is especially adaptable to hypoxic conditions without signiﬁcant cell damage (Swiech et al., 2008c). Dissolved oxygen concentration may inﬂuence the expression of heterologous proteins. According to Batista et al. (2009), the highest expression of RVGP by rS2 cells in supplemented IPL-41 culture medium was achieved with C controlled at 40% air saturation whereas with C of 5%, a reduction of 18% was observed. Under the other conditions studied by these authors (C of 20%, 60% and 95%), the recombinant protein expression dropped to an average of 22%. On the other hand, in Sf-900 II culture medium the highest level of synthesis of recombinant protein by rS2 cells was observed to be associated with cell growth (Swiech et al., 2008c) occurring at C controlled at 30% in comparison to the other conditions tested (5, 50 and 80%). Cell growth and metabolism were shown not to be affected by C at 50 or 10% (Aguiar, 2010). Nevertheless, cells grown with C at 10% air saturation achieved higher and more sustained recombinant protein synthesis than cells growing with C at 50% (Ventini et al., 2010). It is important to point out, however, that the above-mentioned authors did not always use the same medium to culture the rS2 cells. 6. Final remarks S2 cells are a promising platform for recombinant protein expression, being able to grow at μmax above 0.08 h − 1 and reaching cell concentrations up to 5 × 10 7 cells mL − 1. Genetic instability occurring at high frequency in animal cells (Calos et al., 1983; Razzaque et al., 1983) can explain to some extent the heterogeneity of protein production by different recombinant S2 cell populations. Moreover, given that the S2 cell line originated in a pool of embryos, the S2 cell population is inherently heterogeneous with undesirable consequences for establishing reproducible bioprocess protocols. Procedures to optimize the synthesis of heterologous protein in S2 cells could be carried out by selection of subpopulation. Nevertheless cell sorting and cell cloning technologies are hampered by the limited S2 cell growth at low densities. On the other hand, chromatin opening by means of sodium butyrate treatment and appropriate choice of culture medium has been shown to increase the recombinant protein recovered from S2 cell cultures. The metabolic pattern in S2 cell lines depends mainly on the culture medium employed and the cultivation conditions. S2 cell lines seem to show a behavior pattern intermediate between mammalian Table 7 Typical values of QO2 for different animal cell lines. Cell lines Medium and culture conditions QO2 max (10− 11.mmol O2 cell h− 1) Reference Mammalian CHO HeLa NS0 (myeloma) NA NA NA 20 40 22–41 Gray et al. (1996) Shuler and Kargi (2002) Yoon and Konstantinov (1994) Sf-900 II/TC100 NA Sf-900 II; 1 L bioreactor, C = 40% TNM-FH medium with 10% FBS TNM-FH medium with 10% FBS 15/17 38 26 20–40/20–76 28–54/32–58 Batista et al. (2009) Palomares and Ramírez (1996) Pamboukian et al. (2008) Rhiel et al. (1997) Rhiel et al. (1997) Sf-900 II; 1 L bioreactor, C = 40% 1.3 Sf-900 II; bioreactor 750 mL, C = 35–50% Supplemented IPL-41a; bioreactor 1 L, C = 5/20/40/60/95%, Ccrit = 14% Sf-900 II; 1 L bioreactor, C = 40%, Ccrit = 10% Sf-900 II; 1 L bioreactor, C = 40%, Ccrit = 5% Sf-900 II; 1 L bioreactor, C = 40%, Ccrit = 12% 5.1 4.2/6.4/3.7/3.6/7.4 4.9 9.23/4.7 12 Pamboukian et al. (2008) Swiech and Suazo (2006) Swiech and Suazo (2006) Batista et al. (2009) Pamboukian et al. (2008) Pamboukian et al. (2008) Pamboukian et al. (2008) Insect Sf9 Sf9 Sf9 Sf9 uninfected/infected BTI-Tn-5BL-4 uninfected/ infected S2 S2AcGPV S2AcGPV S2AcGPV S2MtEGFP (pre/post induction) S2AcHBsAgHy NA: information not available. a IPL-41 medium supplemented with 3 g L−1 of yeastolate, 10 g L−1 of glucose, 3.5 g L−1 of glutamine and 0.1% v/v lipid emulsion. 625 Â.M. Moraes et al. / Biotechnology Advances 30 (2012) 613–628 Table 8 Growth parameters of S2 cell lines compared with those of other animal cells. Cell/product Culture condition μmax (h− 1) Xmax (107 cells mL− 1) Reference S2/RVGPa Sf-900 II medium, glycerol 1% v/v, shake ﬂask (250 mL), 100 rpm, 28 °C, suspension culture, batch Sf-900 II medium, stirred tank bioreactor 2 L, 100–150 rpm, 28 °C, DO 50%, bubble free, suspension culture, batch Sf-900 II medium, shake ﬂask (100 mL), 100 rpm, 28 °C, suspension culture, batch M3c IMSd 9:1 medium, spinner ﬂask (500 mL), 27 °C, 80 rpm, suspension culture batch M3 medium, spinner ﬂask (500 mL), 27 °C, 80 rpm, suspension culture, batch Sf-900 II medium, shake ﬂask (100 mL), 28 °C, 100 rpm, suspension culture, batch IPL-41 medium IPL-41 medium Spinner ﬂask (125 mL), 100 rpm, 27.5 °C, suspension IPL-41 medium culture, batch TC-100 medium Grace medium Serum-free CD-CHO medium, wave bioreactor, (50 L), 37 °C, 5% CO2, suspension culture, fed-batch On Pronectin F microcarrier, DMEM medium, spinner ﬂask (500 mL), 60 rpm, 10% CO2, 37 °C, pH 7.4, batch Glasgow BHK 21 medium with 5% FCS,f spinner ﬂask (750 mL), 50 rpm, 37 °C, 5% CO2, pH 7.2, suspension culture, batch DMEM and RPMI medium (1:1) with additional amino acids, stirred tank bioreactor (4 L), 37 °C, 30 rpm, DO 10–50%, suspension culture, fed-batch 0.039 1.60 Swiech et al. (2007b) 0.044 2.00 0.014 1.70 Jorge et al. (2008) 0.020 6.10 Shin et al. (2003) 0.032 1.20 Lim and Cha (2006) 0.030 0.17 Batista et al. (2005) 0.018 0.026 0.027 0.016 0.029 0.022 0.41 0.34 0.33 0.07 0.12 1.22 Keith et al. (1999) Haldankar et al. (2006) 0.028 0.28 Swiech et al. (2007a) 0.015 0.11 Srcek et al. (2004) 0.037 0.11 Jang and Barford (2000) S2/HBsAg b S2/interleukin-2 S2/human transferrin Sf-9/baculovirus e Bm5/GM-CSF High Five/GM-CSF Sf-21/GM-CSF IPLB-LdFB/GM-CSF IZD-MB/GM-CSF CHO-S/IgG I CHO-K1/protease BHK-21/Aujeszky's virus Murine hybridoma AFP 27/IgG 1 a b c d e f Rabies virus glycoprotein. Hepatitis B virus surface antigen. M3 is a serum-free highly enriched culture medium, formulated to mimic the main properties of the hemolymph of speciﬁc insects. Insect medium supplement, a serum replacement for general cell growth and maintenance. Granulocyte-macrophage colony-stimulating factor. Fetal calf serum. and Sf9 cells. Glucose and glutamine are generally the most important substrates for providing energy and building blocks for biosynthesis, and are frequently consumed up to exhaustion. The lack of these substrates limits cell growth, ending the exponential growth phase, but only glucose can be related to loss of viability. Proline, cysteine, asparagine and serine uptake is also considerable, and at least proline can be considered as another limiting medium component. Lactate production is associated with the impossibility of synthesizing alanine, and with glucose availability, while its consumption is determined by glutamine or glucose exhaustion. Continuous release of ammonium by these cells does not depend on medium formulation, but rather on the availability of the substrate that gives rise to its production, normally glutamine. The success in culturing S2 cells aiming at heterologous protein production is intimately bound to aspects related to choice of medium and design of the plasmid vectors for protein expression. When appropriately supplemented, basal medium formulations such as TC-100 and IPL-41 can be as effective as commercially available serum-free media. Despite the fact that the speciﬁc respiration rate of S2 cells is lower than those of many other animal cells, designing, operating and controlling adequate oxygen transfer systems are of major importance not only for recombinant protein production on a large scale, but also for better understanding their metabolism. The expression of heterologous recombinant proteins using insect cell systems has potential advantages over expression using mammalian cells in terms of scaling up and cost of production. For this reason, the use of cells with a high speciﬁc growth rate is essential to achieving high productivities. S2 cells meet these requirements better than some of the commercially relevant transfected animal cells cultured in suspension. Table 8 shows a panel of representative conditions of animal cell culture for the synthesis of different recombinant proteins. Finally, in view of attaining high levels of recombinant biologically functional protein expression with S2 cells, the appropriate choice of vectors for cell modiﬁcation and of culture conditions is essential for the development of well-controlled and productive bioprocesses. We hope that this review brings valuable and up-to-date information on molecular biology and bioprocess engineering related to the D. melanogaster S2 cell system for the expression of recombinant proteins. Acknowledgments The authors wish to acknowledge the ﬁnancial support received from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (02/09482-3). 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Glossary a: gass-liquid interfacial area for mass transfer Bip: the secretion signal sequence of Drosophila immunoglobulin heavy chain binding protein C: concentration of dissolved O2 in the liquid Ccrit: critical oxygen concentration Cs: concentration of dissolved O2 in the liquid at saturation cDNA: complementary DNA DES®: Drosophila Expression System DMSO: dimethylsulfoxide DO: dissolved oxygen EGFP: enhanced green ﬂuorescent protein FACs: ﬂuorescence-activated cell sorting FSH: follicle-stimulating hormone gp120: viral protein gp120 Gal-β-1,4-GlcNac: sialyltransferase GDH: glutamate dehydrogenase GLC: glucose GOGAT: glutamine 2-oxoglutarate aminotransferase synthase or glutamate synthase GtHs: gonadotropins HBsAg: hepatitis B surface antigen hMOR: human mu opioid receptor IMS: insect medium supplement JEV: Japanese encephalitis virus kLa: volumetric coefﬁcient of oxygen transfer kL: liquid ﬁlm mass transfer coefﬁcient LAC: lactate LDH: lactate dehydrogenase LH: luteinizing hormone mo: speciﬁc rate of maintenance respiration MyD88: myeloid differentiation factor 8 NADH: nicotinamide adenine dinucleotide, reduced form NADPH: nicotinamide adenine dinucleotide phosphate, reduced form OUR: total oxygen consumption rate PPP: pentose phosphate pathway PYR: pyruvate pZT: constitutive expression plasmid containing the zeocin resistance gene QO2: speciﬁc respiration rate RT: reverse transcription Sf: Spodoptera frugiperda cells SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis TCA: tricarboxylic acid tlag: length of the lag phase Tn or Tn-5: Trichoplusia ni cells Viab: viability X: cell concentration Xmax: maximum cell concentration Xino: cell seeding concentration XV: viable cells YLAC/GLC: glucose-to-lactate yield factor YNH4/GLN: glutamine-to-ammonium yield factor YX/GLC: glucose-to-cell yield factor YX/GLN: glutamine-to-cell yield factor YX/O: oxygen-to-cell yield α-GP: α-glycoprotein hormone (heterodimeric structure of gonadotropins) α-KG: α-ketoglutarate μ: speciﬁc growth rate μmax: maximum speciﬁc growth rate

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