Volker Jäger

Optimization of the growth conditions of Sf21 insect cells for high-density perfusion culture in stirred-tank bioreactors

Spodoptera frugiperda insect cells (IPLB-Sf21-AE) (Sf21), infected with baculovirus expression vectors during their exponential growth phase, are commonly used to produce a variety of heterologous recombinant proteins. In the present study the culture conditions of these insect cells were studied to establish high-density suspension cultures with prolonged exponential growth phases. The Sf21 cells were grown in 125-ml spinner flasks using five different culture media supplemented with 5% fetal calf serum and four protein-free or low-protein culture media. The best results were achieved in EX-CELL 401 (protein-free media) and in IPL-41 modified with 2.5 g l-1 tryptose phosphate broth (serum-supplemented media), respectively. The latter was used for further batch and continuous cultivation of Sf21 cells in a perfused 1.4-l stirred-tank bioreactor with special attention to the oxygen requirement of these cells. Optimal growth was found at an oxygen concentration of 70% air saturation, resulting in a prolonged exponential growth phase that could be maintained for more than 16 days. A maximum cell density of 5.5 x 10(7) viable cells ml-1 was achieved.

Perfusion culture of baculovirus-infected BTI-Tn-5B1-4 insect cells : A method to restore cell-specific β-trace glycoprotein productivity at high cell density

The impact of different cultivation-infection strategies on the productivity of baculovirus-infected BTI-Tn-5B1-4 (High Five) cells was investigated. Using beta-trace protein as the recombinant glycoprotein, the effects of multiplicity of infection (MOI) and time of infection (TOI) were studied on growth after infection as well as the degree of infection and recombinant protein productivity in batch culture. The highest productivities were found when infecting Tn5 cells at early exponential growth phase (EGP) (low cell density) using a high MOI. To increase the productive cell density of Tn5 cells after beta-trace-baculovirus infection, we performed studies infecting cells in the range of 1 to 5 x 10(6) cells/mL in fresh medium. Although the protein production was increased twofold, a strong negative cell density effect was still observed when maximal productive cell density exceeded 1 x 10(6) cells/mL. To verify whether the changing cell environment of the batch experiments was responsible for the decrease in protein productivity at increasing cell density at infection, several perfusion experiments were designed by infecting Tn5 cells at cell densities over 2 x 10(6) cells/mL under more steady-state conditions. The use of this experimental setup enabled successful infections at high cell densities with volumetric productivities of up to 1.2 g L(-1) day(-1) of beta-trace protein, which is very high for a glycoprotein expressed with the baculovirus expression vector system (BEVS). The cell specific protein productivity observed after infections at higher cell densities in perfusion mode was the same as in batch experiments at low cell concentrations, which clearly demonstrates that the cell density effect could be completely overcome with perfusion cultivation.

Molecular cloning, expression, purification, and characterization of soluble full-length, human interleukin-3 with a baculovirus–insect cell expression system

We report gene cloning, plasmid construction, baculovirus expression, purification, and biological activity testing of the human hematopoietic cytokine interleukin-3. cDNA was constructed from extracted total RNA of Jurkat cells. Both signal and structural fragment of interleukin-3 were cloned from this cDNA library, modified by adding a hexahistidine-tag at the C-terminus, and introduced into the pBacPAK9 transfer vector to generate recombinant baculoviruses. For protein expression High Five cells were infected either in spinner flasks or 2.5-L bioreactors in batch culture yielding levels of 1.5-3 mg L(-1) interleukin-3 in the cell culture supernatant. Interleukin-3 was purified by a single step chromatography using cobalt metal affinity resins, which yielded a highly stable and soluble protein. N-terminal amino acid sequencing of the purified interleukin-3 showed correct cleavage of the signal peptide during protein processing. The two N-glycosylation sites were found to be occupied by 100 and 35%, respectively, with an N-glycan pattern of paucimannosidic structures, which are typical for recombinant glycoproteins produced by High Five lepidopteran cells. The specific biological activity of purified interleukin-3 was several times higher when compared with different lots of commercially available material from Escherichia coli. The results indicate that the strategy we used in this experiment is a straightforward and convenient way for recombinant protein preparation and can be adapted to produce other recombinant cytokines.

Proteolytic Activities in Serum-Free Supernatants of Mammalian Cell Lines

[3H]-labelled Casein and chromogenic substrates were used for a quantitative determination of protease activity in cell culture supernatants during long-term cultivation of BHK and hybridoma cells. They produced recombinant human interleukin 2 and an IgG2a - antibody respectively in serum-or protein free medium. Characterization of protease activities was performed by inhibitor studies and specific p-NA derivates. Only 20% of the total protease activity in hybridoma and up to 50% in BHK cell supernatants is based on serine type proteases. Supplementation of supernatants with 2-mercaptoethanol or magnesiumchloride caused an increase of protease activities.

Measurements of Changes in Cell Size Distribution to Monitor Baculovirus Infection of Insect Cells

We have found that the cell size distributions measured by a CASYTM cell counter can be used to follow the infection process of various insect cell lines. After infection, cell size deviates from the typical distribution of exponentially growing cells, shifting to an increased amount of cells withbigger cell diameters. This deviation has proved to be dependent on the MOI as well as time post-infection. A method is proposed to estimate the degree of infection of a population of insect cells based on the cell size distribution (CSD). The potential of using this method for measuring the ratio of infected to non-infected cells is discussed.

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

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.

HIGH DENSITY INSECT CELL CULTURE FOR THE PRODUCTION OF RECOMBINANT PROTEINS WITH THE BACULOVIRUS EXPRESSION SYSTEM

Abstract IPLB-Sf21-AE cells (Spodoptera frugiperda) were propagated in stirred tank perfusion bioreactors. A maximum cell density of 5.5×107 viable cells ml−1 was achieved during continuous cultivation in the perfusion mode with medium exchange rates of up to 4 reactor volumes per day. Usually, cells were infected after reaching a concentration of 1 to 3×107 ml−1. The secreted product was harvested continuously, together with spent medium free of cells and cell debris. In addition, more than 99.9 % of the baculovirus was retained within the bioreactor and could be used for subsequent infection of cells. Therefore, cells can be infected with an extremely low multiplicity of infection (MOI) which was usually kept at 0.02. Maximum volumetric production of the recombinant proteins (e.g. human IL-2 glycoforms) was usually achieved 5 to 6 days post infection, one to two days before cell viability dropped drastically because of the virus induced cell lysis. Compared to batch cultures there was only a slight increase of the productivity. However, product was rapidly removed from the bioreactor, thus reducing product degradations by enzymes of lysed cells. In addition, it has been shown that the product was not uniform during the infection phase. In contrast to batch cultures, several harvests containing different microheterogeneous forms of the recombinant protein could be separated.

Variation in the Ratios and Concentrations of Nucleotide Triphosphates and Udp-Sugars During a Perfused Batch Cultivation of Hybridoma Cells

A murine hybridoma cell line, which produces a monoclonal murine IgG2a antibody, was cultivated in a stirred reactor equipped with bubble-free aeration and a continuous perfusion system. During growth of up to 3.1•107 viable cells per ml, intracellular amounts of ATP, ADP, AMP, NAD, GTP, UTP, UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-G1cNac), UDP-N-acetyl-galactosamine (UDP-GalNac) and CTP were measured by the ion-pair HPLC technique after perchloric acid extraction of sedimented cells. Very stable values of the adenylate energy charge were found whereas the ratio of trinucleotides of the purine pool to these of the pyrimidine pool and the ratio between UTP and UDP-N-acetyl-sugars changed rapidly during different growth phases.

CELL CYCLE ANALYSIS AS A TOOL FOR CONTROL AND REGULATION OF MAMMALIAN CELL CULTURES IN BIOREACTORS

Abstract The control of continuously perfused mammalian cell cultures has been performed by applying the intracellular ribonucleotide pool method. The cell growth was monitored by flow cytometry. Optimal growth has only been achieved by monitoring the intracellular parameters resulting in a strict control and adjustment of the perfusion rate. The cell cycle analysis confirmed the optimal physiological growth conditions. The portion of cells in the G 2 /M phase increased up to 60% of the total viable cell number in comparison to 30% when only environmental control has been performed. Additionally, the cell specific productivity increased in dependence on the mitotic rate.

Metabolic Demands of Bti-TN-5B1-4 (High FiveTM) Insect Cells During Growth and After Infection with Baculovirus

High Five cells are characterized by a very active metabolism, by which Asn and Gln are consumed at very high rates. The use of feeding strategies, aimed to keep the concentrations of these amino acids in a region of lower qAsn and qGln, is strongly recommended to avoid nutrient limitations without extensive medium exchange. Due to the high specific oxygen demand of High Five cells, the growth of this cell line up to high cell densities could be limited by the transfer capabilities of current animal cell bioreactor systems. An additional increase in the oxygen demand must be expected during the first 30 hours post infection.