Effect of Partial Medium Replacement on Cell Growth and Protein Production for the High-Five™ insect cell line

The potential of spent medium to support the growth and recombinant protein production of High-Five? cells was investigated. Growth in medium consisting of three parts fresh and one part spent medium was comparable to that in fresh medium (maximal specific growth rates of 0.028 and 0.029 h^?1, and maximal cell densities of 4 and 4.5 × 10⁶ cells ml^?1, respectively). Glucose exhaustion coincided with an abrupt decrease of viability. Of 15 amino acids analyzed, not a single one was completely exhausted at the end of the growth phase. Growth in medium consisting of equal parts spent and fresh medium led to lower maximal cell concentration (2.9 × 10⁶ cells ml^?1) with a smoother death phase. Glucose supplementation at the beginning of the culture or at the end of the growth phase did not lead to an increase of either the maximal cell density or the specific growth rate. Infection of High-Five? cells at three different densities (1.4, 2.5 and 4.2 × 10⁶ cells ml^?1) without medium change led to monotonically decreased specific productions for β-galactosidase. Partial (75%) or total medium replacement at the higher infection density restored the specific production at the levels of the intermediate density infection (321, 292 and 389 U.(10⁶ cells)^?1, respectively).     

Recombinant Protein Production by the Baculovirus-Insect Cell System in Basal Media Without Serum Supplementation

The production of β-galactosidase by Sf9 cells infected with recombinant Autographa californica nucleopolyhedrovirus (AcNPV) was investigated in shake-flask culture using two serum-free basal media: Grace's medium and TNM-FH (Grace's medium supplemented with lactalbumin hydrolysate and yeast extract). At the time of infection, cells grown in serum-supplemented TNM-FH were transferred into fresh basal media without adaptation. The absence of serum depressed the β-galactosidase yield considerably in Grace's medium, but to a much lesser extent in TNM-FH, where it reached around 2/3 of the level obtained in TNM-FH supplemented with 10% fetal bovine serum (FBS). While both lactalbumin hydrolysate and yeast extract promoted β-galactosidase production, their removal by medium replacement on post-infection day 1 gave a β-galactosidase yield nearly equal to that obtained in their continuous presence. Supplementation of basal media with phosphatidic acid (PA) from egg yolk lecithin, which has been shown to enhance cell growth and recombinant protein production in serum-free culture of Chinese hamster ovary (CHO) cells, was also effective in increasing β-galactosidase yield. Elevating the multiplicity of infection (MOI) from 2 to 10 plaque-forming units per cell (pfu/cell) also resulted in an increase in product yield. These results provide information important to the development of cost-effective serum-free culture technology for use in large-scale production of recombinant proteins by the baculovirus-insect cell system.     

Protein production (β-galactosidase) from a baculovirus vector inSpodoptera frugiperda andTrichpolusia ni cells in suspension culture

Protein production capabilities ofTrichpolusia ni (TN 368) cells andSpodoptera frugiperda (Sf9) cells were compared in GTC100 medium in suspension culture using as a vector a genetically engineeredAutographa californica nuclear polyhedrosis virus. TN 368 produces more -galactosidase than Sf9, on a per cell basis (2.2×10⁵ and 1.7×10⁵ units/ 10⁶ cells¹ respectively). In growth experiments serum-free medium supported a higher maximum Sf9 cell density (4±1×10⁶ cells/ml) than the serum- based media (1.5±5×10⁶ cells/ml in GTC100 and 2±1×10⁶ cells/ml in TNM-FH). However, using a cell density of 5×0⁵ cells/ml, the productivity per cell varied, from a low of 4.5×10⁴ units in EX-CELL-400 medium to a high of 7.6×10⁴ units in TNM-FH. The TN 368 cells were twice a large as Sf9 cells and appeared to be more shear sensitive than Sf9 cells.     

Propagation ofSpodoptera frugiperda cells (Sf9) and production of recombinant proteins with the baculovirus expression system using improved spinner flasks with membrane aeration

Summary It has been shown that the growth of Spodoptera frugiperda cells is significantly reduced or ceased under oxygen limiting culture conditions. This paper describes the use of a new membrane-aerated spinner flask which was compared to conventional surface-aerated spinner flasks with regard to growth of the insect cell line Sf9 and recombinant protein production after infection with baculovirus. Using a commercially available serum-free culture medium Sf9 cells reached highest cell densities (3×10⁶ ml^–1) in the membrane-aerated spinner flask. Production of recombinant protein was also influenced by the oxygen supply. In the membrane-aerated spinner flask and in a surface-aerated spinner flask with reduced filling volume more than 20000 U ml^–1 of a recombinant interleukin-2 variant were accumulated whereas only 100 U ml^–1 were produced in a surface-aerated spinner flask with insufficient oxygen supply. Sufficient oxygenation appears to be essential for proliferation of Sf9 cells as well as recombinant protein production after infection with baculovirus. Membrane oxygenation allows sufficient oxygen supply at high cell density and an at least 2.5 fold higher filling volume per spinner unit.     

Enhancement of expression of an introduced gene by 5-azacytidine in mammalian cell lines

We transfected a mouse myeloma cell line, P3/NS1-Ag4-1 (NS-1), and a Chinese hamster ovary cell line, CHO-K1 with the β-galactosidase (β-Gal) gene of Escherichia coli, and isolated stable transformants designated as NS-1Z/gpt and CHO-Z/neo, respectively. When NS-1Z/gpt cells were incubated with 5–20 μM 5-azacytidine (5-azaC), the specific and total activity of β-Gal were enhanced 2-to 3-fold and 1.5- to 2-fold, respectively. In CHO-Z/neo cells, similar treatment resulted in a 3- to 5-fold increase in the specific β-Gal activity and about a 2-fold enhancement in total enzyme activity. The growth of both cell lines was inhibited by more than 80% in the presence of 10 μM 5-azaC. It was confirmed in immunotitration experiments that the enhancement of β-Gal activities was due to an increase in the enzyme protein. Northern blot analysis revealed that 5-azaC-treatment resulted in the enhanced expression of β-Gal mRNA. 5-AzaC also enhanced the production of human interleukin 2 (IL2) from CHO cells that were transfected with the IL2 gene. On the other hand, 5-azaC did not significantly affect the expression of an endogenous gene like lactate dehydrogenase or β-actin. These results suggest that 5-azaC is a useful agent for up-regulating the expression of introduced genes.     

Propagation of Bombyx mori Nucleopolyhedrovirus in nonpermissive insect cell lines

This study addresses the susceptibility of Spodoptera frugiperda (Sf9 and Sf21), Trichoplusia ni (Hi5), and S. exigua (Se301) cells to the Bombyx mori nucleopolyhedrovirus (BmNPV). Although these cells have classically been considered nonpermissive to BmNPV, the cytopathic effect, an increase in viral yield, and viral DNA synthesis by BmNPV were observed in Sf9, Sf21, and Hi5 cells, but not in Se301 cells. Very late gene expression by BmNPV in these cell lines was also detected via beta-galactosidase expression under the control of the polyhedrin promoter. Sf9 cells were most susceptible to BmNPV in all respects, followed by Sf21 and Hi5 cells in decreasing order, while the Se301 cells evidenced no distinct viral replication. This particular difference in viral susceptibility in each of the cell lines can be utilized for our understanding of the mechanisms underlying the host specificity of NPVs.     

Utilizing the macroporous packed bed for insect cell/baculovirus expression

Due to their high porosity and biocompatibility, polyurethane foam (PUF) and cellulose foam were adopted for insect cell immobilization and baculovirus expression. Spodoptera frugiperda (SF-21) cells were grown within the macroporous matrix and then infected by Autographa californica nuclear polyhedrosis virus (AcNPV) which was encoded with human interleukin-5 (hIL-5) gene. An appropriate initial cell loading density and medium circulation velocity determined from the previous study were applied in this actual cell cultivation experiments to obtain a uniform initial and final axial cell distribution. The growth of insect cells and the expression of baculovirus were successful in the macroporous packed bed systems used. The final average cell density in cellulose foam achieved was 5.2×10⁷?cells/cm³ and 4.3×10⁷?cells/cm³ in PUF. Under the conditions of sufficient nutrition and oxygen supplement, the average productivity of hIL-5 in cellulose foam packed bed bioreactor reached 7.2×10⁷ unit/l-day. With 50% fresh medium replacement after viral infection, the average productivity of hIL-5 in PUF packed bed reached 8.4×10⁷ unit/l-day, about two fold than that without any fresh medium replacement at infection.     

5-Azacytidine enhances transient expression of a transfected gene in cultured mammalian cells

The level of transient expression of the Escherichia coli β-galactosidase (β-Gal) gene transfected into three mammalian cell lines was enhanced by culturing the cells with 1–5 μM 5-azacytidine (5-azaC) after the transfection. This enhancement was similarly observed in four different transfection procedures tested. The enhancement of β-Gal expression was primarily due to the increase in the level of its mRNA. Since 5-azaC did not exhibit any obvious effect on the levels of endogenous gene products examined, it is suggested that 5-azaC may preferentially affect the transient expression of an exogenous gene.     

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake flasks to a 20-L bioreactor

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.     

Persistent RNA virus infection of lepidopteran cell lines: Interactions with the RNAi machinery

RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.     

Expression analysis of a modified factor X in stably transformed insect cell lines.

A modified Factor X protein was combined with a cellulose-binding domain tag and expressed in insect cell lines. The protein, CBDFX, was expressed and secreted into the medium. Stable, transformed Hi5 and Sf9 insect cell lines were generated and tested for production of secreted CBDFX. The highest Sf9 and Hi5 CBDFX-producing cell lines were scaled up to 2-liter fermentors to evaluate production of this recombinant protein. Secreted protein production levels reached 4 mg/liter for the stable, transformed Hi5 cell line and 18 mg/liter for the stable, transformed Sf9 cell line. The protein was properly processed as determined by amino terminal sequencing and bound well to the cellulose substrate Avicel. In addition the activated recombinant CBDFX(a) was capable of recognizing and efficiently processing a Factor X cleavage site.     

Stable cell lines expressing baculovirus P35: resistance to apoptosis and nutrient stress, and increased glycoprotein secretion

Summary The baculovirus P35 protein is a caspase inhibitor that prevents the induction of apoptosis during infection of Sf21 cells byAutographa californica multicapsid nucleopolyhedrovirus (AcMNPV). P35 inhibits the induction of apoptosis in a broad range of cells and circumstances. In this study, we examined the effects of constitutive cellular P35 expression on the response of cells to stressful culture conditions and on protein production in AcMNPV infected cells. Sf9 cell lines expressing AcMNPV P35 or an epitope-tagged P35 protein were generated using a double selection technique, involving selection in the antibiotic G418, followed by a second round of selection by exposure to actinomycin D, a potent inducer of apoptosis in Sf9 cells. Clonal cell lines were generated and examined for (1) resistance to actinomycin D induced apoptosis, (2) resistance to nutrient deprivation, and (3) baculovirus expression of intracellular and secreted proteins. When compared with Sf9 cells, two P35-expressing cell lines (Sf9^P35AcV5-1 and Sf9^P35AcV5-3) showed increased resistance to actinomycin D-induced apoptosis and a profound resistance to nutrient deprivation. When these cell lines were infected with a recombinant baculovirus expressing a secreted glycoprotein (secreted alkaline phosphatase), expression of the glycoprotein from these cells exceeded that from the parental Sf9 cells and was comparable to expression levels obtained from Tn5B1-4 cells, the best available cell line for high-level expression. Increased levels of protein secretion in Sf9^P35AcV5-1 and Sf9^P35AcV5-3 cells appear to result from a prolonged infection cycle and accumulation of the secreted glycoprotein.     

Large Scale RNAi Reveals the Requirement of Nuclear Envelope Breakdown for Nuclear Import of Human Papillomaviruses

A two-step, high-throughput RNAi silencing screen was used to identify host cell factors required during human papillomavirus type 16 (HPV16) infection. Analysis of validated hits implicated a cluster of mitotic genes and revealed a previously undetermined mechanism for import of the viral DNA (vDNA) into the nucleus. In interphase cells, viruses were endocytosed, routed to the perinuclear area, and uncoated, but the vDNA failed to be imported into the nucleus. Upon nuclear envelope perforation in interphase cells HPV16 infection occured. During mitosis, the vDNA and L2 associated with host cell chromatin on the metaphase plate. Hence, we propose that HPV16 requires nuclear envelope breakdown during mitosis for access of the vDNA to the nucleoplasm. The results accentuate the value of genes found by RNAi screens for investigation of viral infections. The list of cell functions required during HPV16 infection will, moreover, provide a resource for future virus-host cell interaction studies.     

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l⁻¹ glucose, 8 g l⁻¹ YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7×10⁶ cells ml⁻¹) when compared to Grace’s containing 10% (v/v) FBS (9.5×10⁵ cells ml⁻¹). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×10⁷ PIBs ml⁻¹) than in Grace’s medium with 10% FBS (0.6×10⁷ PIBs ml⁻¹). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium. C.A. Pereira is recipient of a CNPq fellowship.     

Synthesis of Avian Oncornavirus DNA in Infected Chicken Cells

The intracellular synthesis and integration of viral DNA (vDNA) into the host cell genome was studied in cultured chicken embryo fibroblasts infected with avian sarcoma or leukemia viruses. The newly synthesized vDNA was detected by hybridization with 70S viral RNA. Extraction of infected cell DNA by the selective procedure of Hirt resulted in the enrichment of newly synthesized vDNA in the low molecular weight supernatant fraction while leaving the bulk of cellular DNA containing integrated vDNA in the high molecular weight pellet fraction. This approach led to detection of intracellular vDNA synthesis within 1 h after infection and to vDNA integration into cellular DNA within 24 h. There was a several-fold increase in the vDNA content of infected cells during the initial phase of virus infection. But only a part of this newly synthesized vDNA appeared to become covalently linked with high molecular weight cellular DNA. Most of the remaining unintegrated vDNA gradually disappeared. The sedimentation profiles of minimally sheared cellular DNA in alkaline sucrose velocity gradients suggest that vDNA is synthesized as free linear molecules of approximately 3 x 10(6) daltons which subsequently are covalently linked to host cell DNA.     

Production of recombinant proteins in high-density insect cell cultures

The effect of the growth phase of Spodoptera frugiperda (Sf9) cells on the production of recombinant proteins (beta-galactosidase and glucocerebrosidase) was investigated. Cells infected with the recombinant Autographa californica nuclear polyhedrosis virus at the late exponential and stationary phases yielded low quantities of expressed protein. Highest enzyme yields were obtained using Sf9 cells from the early exponential phase (0.9 mg beta-galactosidase/10(6) cells and 1.7 mug glucocerebrosidase/10(6) cells). Infection of resuspension of cells collected from various phases of growth in fresh medium resulted in 75% restoration of maximal expression levels. This finding suggested either nutrient limitation or waste product accumulation as the cause of the decrease in productivity at the latter phases of growth. Further experiments revealed that the highest productivity levels could be obtained with cultures of Sf9 cells grown in a fermentor to a cell concentration of 4 x 10(6) mL(-1). The medium needed to be replaced prior to infection with the recombinant virus and supplemented with a mixture of glucose, L-glutamine, and yeastolate ultrafiltrate. (c) 1993 John Wiley & Sons, Inc.     

Efficient synthesis of octyl-β-d-galactopyranoside by Bacillus spore-displayed β-galactosidase using an amphiphilic 1,2-dimethoxyethane co-solvent

For enzymatic synthesis of octyl-β-d-galactopyranoside (octyl-gal) from lactose and n-octanol, Escherichia coli β-galactosidase (β-Gal) was expressed and displayed on the surfaces of Bacillus subtilis spores. The spore-displayed β-Gal was found to be stable when an amphiphilic 1,2-dimethoxyethane (DME) was used as a co-solvent; the transgalactosylation efficiency and octyl-gal conversion were optimal at 50% (v/v) DME. In addition, the product was maximally obtained from 100mM lactose in a phosphate buffer/n-octanol/DME (25/25/50, v/v) mixture. By increasing the agitation speed and the amount of spores displaying β-Gal, a yield of 33.7 mM octyl-gal was obtained over 24h in a batch mode, which is much higher than in other octyl-gal bioconversion processes, such as those involving lipid-coating, reverse micelles, or whole cells. On the other hand, intermittent addition of spore-displayed β-Gal and/or lactose in the reaction medium had no effect on the octyl-gal yield. The synthesized octyl-gal was hydrolyzed by the spore-displayed β-Gal, and a high concentration of octyl-gal competitively inhibited the enzymes (K(i) value of 10.8mM). In summary, we demonstrate that octyl-gal synthesis by spore-displayed β-Gal in non-aqueous medium can be significantly improved with the use of DME as a co-solvent.     

Reassessing culture media and critical metabolites that affect adenovirus production

Adenovirus production is currently operated at low cell density because infection at high cell densities still results in reduced cell‐specific productivity. To better understand nutrient limitation and inhibitory metabolites causing the reduction of specific yields at high cell densities, adenovirus production in HEK 293 cultures using NSFM 13 and CD 293 media were evaluated. For cultures using NSFM 13 medium, the cell‐specific productivity decreased from 3,400 to 150 vp/cell (or 96% reduction) when the cell density at infection was increased from 1 to 3 × 10⁶ cells/mL. In comparison, only 50% of reduction in the cell‐specific productivity was observed under the same conditions for cultures using CD 293 medium. The effect of medium osmolality was found critical on viral production. Media were adjusted to an optimal osmolality of 290 mOsm/kg to facilitate comparison. Amino acids were not critical limiting factors. Potential limiting nutrients including vitamins, energy metabolites, bases and nucleotides, or inhibitory metabolites (lactate and ammonia) were supplemented to infected cultures to further investigate their effect on the adenovirus production. Accumulation of lactate and ammonia in a culture infected at 3 × 10⁶ cells/mL contributed to about 20% reduction of the adenovirus production yield, whereas nutrient limitation appeared primarily responsible for the decline in the viral production when NSFM 13 medium was used. Overall, the results indicate that multiple factors contribute to limiting the specific production yield at cell densities beyond 1 × 10⁶ cells/mL and underline the need to further investigate and develop media for better adenoviral vector productions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010