Optimum Infection Conditions for Recombinant Protein Production in Insect Cell (Bm5) Suspension Culture

Zhang, Junli; Kalogerakis, Nicolas; Behie, Leo A.; Iatrou, Kostas

doi:10.1021/bp00030a008

Cited by 22 publications

(11 citation statements)

References 27 publications

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“…In the large-scale production of recombinant proteins, cultures infected at high-cell densities produce higher volumetric protein levels than those infected at low cell densities (Wong et al, 1996;Yang et al, 1996). However, high-cell density infections are limited by the nutrient supply from the medium (Radford et al, 1992;Radford et al, 1997b;Zhang et al, 1994). Recently, Tsao et al (1996) studied the production of a parvovirus B19 vaccine in insect cells coinfected with two baculoviruses.…”

Section: Discussionmentioning

confidence: 99%

Optimized production of recombinant bluetongue core-like particles produced by the baculovirus expression system

Zheng

Greenfield

Reid

1999

Biotechnol. Bioeng.

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The baculovirus‐expression vector system (BEVS) has been widely used for the experimental production of many human and animal single‐ and multi‐unit vaccines, heterologous proteins, and viral insecticides. In this work, the production of recombinant bluetongue virus core‐like particles (CLPs), using Sf9 cells in shaker‐suspension culture with the SF900 II medium (GIBCO, NY), has been studied. This system involved the simultaneous production of two proteins, VP7 and VP3, and was shown to achieve high volumetric productivities. The key parameters of the time of infection (TOI), and the multiplicity of infection (MOI) were studied. The results show that the peak‐volumetric yields and cell‐specific yields achieved using low MOIs at low‐cell densities were the same as those obtained following infections with a high MOI at high‐cell densities. This work establishes the feasibility of using low MOIs in the baculovirus system to produce complex multiprotein particles. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 600–604, 1999.

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Section: Discussionmentioning

confidence: 99%

Optimized production of recombinant bluetongue core-like particles produced by the baculovirus expression system

Zheng

Greenfield

Reid

1999

Biotechnol. Bioeng.

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“…The abrupt decline in product yield has been partly reversed by nutrient supplementation and medium renewal (10,18,19). Nevertheless, in other reports no direct effects of cell density levels or of nutrient replacement strategies on productivity have been observed (20,21). It was suggested that this may indicate the presence of other simultaneous influences that should be examined for their role on infectivity and production at key points in the development of the population (22).…”

Section: Introductionmentioning

confidence: 96%

Cell Cycle Progression in Serum-Free Cultures of Sf9 Insect Cells: Modulation by Conditioned Medium Factors and Implications for Proliferation and Productivity

et al. 2000

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Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G(2)/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G(1) and in particular into the S phase. Maximum rate of proliferation (micro(N, max)) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G(2)/M phase. The following declining micro(N) could be explained by a slow increase in the G(2)/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G(2)/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G(2)/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase micro(N,max), decrease the time to reach micro(N,max), and decrease the synchronization of cells in G(2)/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of beta-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean population cell volume had attained a minimum, and this occurred 24 h before the switch into the G(2)/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G(2)/M cell cycle arrest.

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“…Likewise, an issue often overlooked is how well a particular insect cell line functions under different cell culture conditions. Generally, suspension culture provides the best environment for the optimal growth of insect cells and the production of virus and/or recombinant protein (Kioukia et al, 1995; Zhang et al, 1994; Deutschmann and Jaeger, 1994). The selection of an appropriate growth medium can be equally as important for the success of many production schemes (Akhnoukh et al, 1996; Hennsler and Agathos, 1994).…”

Section: Introductionmentioning

confidence: 99%

Increased Virus Production in Suspension Culture by a Trichoplusia ni Cell Line in Serum-Free Media

1997

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Trichoplusia ni-derived BTI-Tn5B1-4 (Tn-5B1-4) cells were grown in 50 mL spinner flasks in four different formulations of serum-free medium (SFM). The objectives of this work were to determine the capacity of this cell line to grow and support virus replication in suspension culture in different SFM. Maximum cell densities exceeding 3.0 × 10 6 cells/mL were attained for all cell/medium combinations. Likewise, cell doubling times were all within 20-22 h. Infection studies with a wild-type isolate of Autographa californica nuclear polyhedrosis virus (AcMNPV) yielded between 156 and 187 occlusion bodies (OBs)/cell. The lethal effect of AcMNPV-OBs produced in this cell line in the various types of media was demonstrated in vivo using T. ni neonate larvae. The percent mortality at a single dose (1.0 × 10 6 OBs/mL) ranged from 40 to 60% at 4 days post-inoculation (p.i.). These results compared favorably with mortality rates found for AcMNPV-OBs produced in vivo. DNA quantification analysis, comparing polyhedra-derived viral DNA concentrations from an equal number of cell culture-and larval-derived OBs, was also determined. The larval-derived OB DNA concentration averaged 0.514 µg/10 8 OBs, whereas the cell culture-derived viral DNA concentrations ranged from 0.347 to 0.710 µg/10 8 OBs. These results suggest that no significant differences exist in DNA content between larvae-and cell culture-derived samples. Expression of the recombinant protein, secreted alkaline phosphatase (SEAP), ranged from 2.7 to 5.5 IU/mL for each cell/medium combination, which was consistent with previous static culture results for this same cell line in serumcontaining TNM-FH medium. Electron microscopic analysis of OBs produced in vivo or in vitro revealed no obvious differences in packaging or number of enveloped nucleocapsids per OB between samples. However, AcMNPV-OBs from infected larvae appeared physically smaller (1.28 ( 0.164 µm) than those produced in cell culture (1.72 ( 0.120). The Tn-5B1-4 cell line is adaptable to suspension culture in serumfree medium and is suitable for further development in the production of viruses and recombinant proteins on an industrial scale.

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Optimum Infection Conditions for Recombinant Protein Production in Insect Cell (Bm5) Suspension Culture

Cited by 22 publications

References 27 publications

Optimized production of recombinant bluetongue core-like particles produced by the baculovirus expression system

Optimized production of recombinant bluetongue core-like particles produced by the baculovirus expression system

Cell Cycle Progression in Serum-Free Cultures of Sf9 Insect Cells: Modulation by Conditioned Medium Factors and Implications for Proliferation and Productivity

Increased Virus Production in Suspension Culture by a Trichoplusia ni Cell Line in Serum-Free Media

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