Optimization of insect cell based protein production processes - online monitoring, expression systems, scale-up

Due to the increasing use of insect cell based expression systems in research and industrial recombinant protein production, the development of efficient and reproducible production processes remains a challenging task. In this context, the application of online monitoring techniques is intended to ensure high and reproducible product qualities already during the early phases of process development. In the following chapter, the most common transient and stable insect cell based expression systems are briefly introduced. Novel applications of insect cell based expression systems for the production of insect derived antimicrobial peptides/proteins (AMPs) are discussed using the example of G. mellonella derived gloverin. Suitable in situ sensor techniques for insect cell culture monitoring in disposable and common bioreactor systems are outlined with respect to optical and capacitive sensor concepts. Since scale-up of production processes is one of the most critical steps in process development, a conclusive overview is given about scale up aspects for industrial insect cell culture processes

Complex dynamics of defective interfering baculoviruses during serial passage in insect cells

Defective interfering (DI) viruses are thought to cause oscillations in virus levels, known as the 'Von Magnus effect'. Interference by DI viruses has been proposed to underlie these dynamics, although experimental tests of this idea have not been forthcoming. For the baculoviruses, insect viruses commonly used for the expression of heterologous proteins in insect cells, the molecular mechanisms underlying DI generation have been investigated. However, the dynamics of baculovirus populations harboring DIs have not been studied in detail. In order to address this issue, we used quantitative real-time PCR to determine the levels of helper and DI viruses during 50 serial passages of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in Sf21 cells. Unexpectedly, the helper and DI viruses changed levels largely in phase, and oscillations were highly irregular, suggesting the presence of chaos. We therefore developed a simple mathematical model of baculovirus-DI dynamics. This theoretical model reproduced patterns qualitatively similar to the experimental data. Although we cannot exclude that experimental variation (noise) plays an important role in generating the observed patterns, the presence of chaos in the model dynamics was confirmed with the computation of the maximal Lyapunov exponent, and a Ruelle-Takens-Newhouse route to chaos was identified at decreasing production of DI viruses, using mutation as a control parameter. Our results contribute to a better understanding of the dynamics of DI baculoviruses, and suggest that changes in virus levels over passages may exhibit chaos.The authors thank Javier Carrera, Just Vlak and Lia Hemerik for helpful discussion. MPZ was supported by a Rubicon Grant from the Netherlands Organization for Scientific Research (NWO, www.nwo.nl) and a 'Juan de la Cierva' postdoctoral contract (JCI-2011-10379) from the Spanish 'Secretaria de Estado de Investigacion, Desarrollo e Innovacion'. JS was supported by the Botin Foundation. SFE was supported by grant BFU2012-30805, also from the Spanish 'Secretaria de Estado de Investigacion, Desarrollo e Innovacion'.Zwart, MP.; Pijlman, G.; Sardanyes Cayuela, J.; Duarte, J.; Januario, C.; Elena Fito, SF. (2013). Complex dynamics of defective interfering baculoviruses during serial passage in insect cells. Journal of Biological Physics. 39(2):327-342. doi:10.1007/s10867-013-9317-9S327342392Von Magnus, P.: Incomplete forms of influenza virus. Adv. Virus. Res. 2, 59–79 (1954)Huang, A.S.: Defective interfering viruses. Annu. Rev. Microbiol. 27, 101–117 (1973)Kool, M., Voncken, J.W., Vanlier, F.L.J., Tramper, J., Vlak, J.M.: Detection and analysis of Autographa californica nuclear polyhedrosis-virus mutants with defective interfering properties. Virology 183, 739–746 (1991)Wickham, T.J., Davis, T., Granados, R.R., Hammer, D.A., Shuler, M.L., Wood, H.A.: Baculovirus defective interfering particles are responsible for variations in recombinant protein-production as a function of multiplicity of infection. Biotechnol. Lett. 13, 483–488 (1991)Pijlman, G.P., van den Born, E., Martens, D.E., Vlak, J.M.: Autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells. Virology 283, 132–138 (2001)Giri, L., Feiss, M.G., Bonning, B.C., Murhammer, D.W.: Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k. J. Gen. Virol. 93, 389–399 (2012)King, L.A., Possee, R.D.: The Baculovirus Expression System. University Press, Cambridge (1992)Lee, H.Y., Krell, P.J.: Reiterated DNA fragments in defective genomes of Autographa californica nuclear polyhedrosis virus are competent for AcMNPV-dependent DNA replication. Virology 202, 418–429 (1994)Pijlman, G.P., Dortmans, J., Vermeesch, A.M.G., Yang, K., Martens, D.E., Goldbach, R.W., Vlak, J.M.: Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses. J. Virol. 76, 5605–5611 (2002)Pijlman, G.P., van Schijndel, J.E., Vlak, J.M.: Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells. J. Gen. Virol. 84, 2669–2678 (2003)Pijlman, G.P., Vermeesch, A.M.G., Vlak, J.M.: Cell line-specific accumulation of the baculovirus non-hr origin of DNA replication in infected insect cells. J. Invertebr. Pathol. 84, 214–219 (2003)Roux, L., Simon, A.E., Holland, J.J.: Effects of defective interfering viruses on virus-replication and pathogenesis in vitro and in vivo. Adv. Virus. Res. 40, 181–211 (1991)Grabau, E.A., Holland, J.J.: Analysis of viral and defective-interfering nucleocapsids in acute and persistent infection by Rhadoviruses. J. Gen. Virol. 60, 87–97 (1982)Kawai, A., Matsumoto, S., Tanabe, K.: Characterization of Rabies viruses recovered from persistently infected BHK cells. Virology 67, 520–533 (1975)Roux, L., Holland, J.J.: Viral genome synthesis in BHK-21 cells persistently infected with Sendai virus. Virology 100, 53–64 (1980)Palma, E.L., Huang, A.: Cyclic production of vesicular stomatitis virus cause by defective interfering particles. J. Infect. Dis. 129, 402–410 (1974).Stauffer Thompson, K.A., Yin, J.: Population dynamics of an RNA virus and its defective interfering particles in passage cultures. Virol. J. 7, 257–266 (2010)Szathmáry, E.: Cooperation and defection – playing the field in virus dynamics. J. Theor. Biol. 165, 341–356 (1993)Bangham, C.R.M., Kirkwood, T.B.L.: Defective interfering particles – effects in modulating virus growth and persistence. Virology 179, 821–826 (1990)Kirkwood, T.B.L., Bangham, C.R.M.: Cycles, chaos, and evolution in virus cultures – a model of defective interfering particles. Proc. Natl. Acad. Sci. USA 91, 8685–8689 (1994)De Gooijer, C.D., Koken, R.H.M., van Lier, F.L.J., Kool, M., Vlak, J.M., Tramper, J.: A structured dynamic model for the baculovirus infection process in insect-cell reactor configurations. Biotech. Bioeng. 40, 537–548 (1992)Van Lier, F.L.J., van der Meijs, W.C.J., Grobben, N.G., Olie, R.A., Vlak, J.M., Tramper, J.: Continuous beta-galactosidase production with a recombinant baculovirus insect-cell system in bioreactors. J. Biotechnol. 22, 291–298 (1992)Van Lier, F.L.J., van den Hombergh, J., de Gooijer, C.D., den Boer, M.M., Vlak, J.M., Tramper, J.: Long-term semi-continuous production of recombinant baculovirus protein in a repeated (fed-)batch two-stage reactor system. Enzyme Microb. Technol. 18, 460–466 (1996)Zwart, M.P., Erro, E., van Oers, M.M., de Visser, J.A.G.M., Vlak, J.M.: Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants. J. Gen. Virol. 89, 1220–1224 (2008)Luckow, V.A., Lee, S.C., Barry, G.F., Olins, P.O.: Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J. Virol. 67, 4566–4579 (1993)Vaughn, J.L., Goodwin, R.H., Tompkins, G.J., McCawley, P.: Establishment of 2 cell lines from insect Spodoptera frugiperda (Lepidoptera, Noctuidae). In Vitro 13, 213–217 (1977)Zwart, M.P., van Oers, M.M., Cory, J.S., van Lent, J.W.M., van der Werf, W., Vlak, J.M.: Development of a quantitative real-time PCR for determination of genotype frequencies for studies in baculovirus population biology. J. Virol. Meth. 148, 146–154 (2008)Zwart, M.P., Hemerik, L., Cory, J.S., de Visser, J.A.G.M., Bianchi, F.J.J.A., van Oers, M.M., Vlak, J.M., Hoekstra, R.F., van der Werf, W.: An experimental test of the independent action hypothesis in virus-insect pathosystems. Proc. R. Soc. B 276, 2233–2242 (2009)Olkin, I., Gleser, L.J., Derman, C.: Probability Models and Applications. Macmillan, New York (1994)Parker, T., Chua, L.: Practical Numerical Algorithms for Chaotic Systems. Springer-Verlag, Berlin (1989)Dieci, L., van Vleck, E.S.: Computation of a few Lyapunov exponents for continuous and discrete dynamical systems. J. Appl. Numer. Math. 17, 275–291 (1995)Matsumoto, T., Chua, L.O., Komuro, M.: The double scroll. IEEE Trans. Circuits Syst. 32, 797–818 (1985)Chua, L.O., Komuro, M., Matsumoto, T.: The double scroll family: rigorous proof of chaos. IEEE Trans. Circuits Syst. 33, 1072–1097 (1986)Ramasubramanian, K., Sriram, M.S.: A comparative study of computation of Lyapunov spectra with different algorithms. Phys. D: Nonlin. Phenom. 139, 72–86 (2000)Lee, H.Y., Krell, P.J.: Generation and analysis of defective genomes of Autographa californica nuclear polyhedrosis virus. J. Virol. 66, 4339–4347 (1992)Kovacs, G.R., Choi, J., Guarino, L.A., Summers MD: Functional dissection of the Autographa californica nuclear polyhedrosis virus immediate early 1 transcriptional regulatory protein. J. Virol. 66, 7429–7437 (1992)Legendre, P., Legendre, L.: Numerical Ecology. Elsevier, Amsterdam (1998)Schuster, H.G.: Deterministic Chaos: An Introduction. Wiley-VCH Verlag GmbH & Co. KGaA, Wienheim (2005)Strogatz, S.H.: Nonlinear Dynamics and Chaos: With Applications to Physics, Biology, Chemistry and Engineering. Westview Press, Cambridge (1994)Dennis, B., Desharnais, R.A., Cushing, J.M., Henson, S.M., Constantino, R.F.: Can noise induce chaos? Oikos 102, 329–339 (2003)Crutchfield, J.P., Huberman, B.A.: Fluctuations and the onset of chaos. Phys. Lett. A 77, 407–410 (1980)Crutchfield, J.P., Farmer, J.D.: Fluctuations and simple chaotic dynamics. Phys. Rep. 92, 45–82 (1982

Chemical Magnetoreception: Bird Cryptochrome 1a Is Excited by Blue Light and Forms Long-Lived Radical-Pairs

Cryptochromes (Cry) have been suggested to form the basis of light-dependent magnetic compass orientation in birds. However, to function as magnetic compass sensors, the cryptochromes of migratory birds must possess a number of key biophysical characteristics. Most importantly, absorption of blue light must produce radical pairs with lifetimes longer than about a microsecond. Cryptochrome 1a (gwCry1a) and the photolyase-homology-region of Cry1 (gwCry1-PHR) from the migratory garden warbler were recombinantly expressed and purified from a baculovirus/Sf9 cell expression system. Transient absorption measurements show that these flavoproteins are indeed excited by light in the blue spectral range leading to the formation of radicals with millisecond lifetimes. These biophysical characteristics suggest that gwCry1a is ideally suited as a primary light-mediated, radical-pair-based magnetic compass receptor

Evolutionary Trajectory of White Spot Syndrome Virus (WSSV) Genome Shrinkage during Spread in Asia

Background - White spot syndrome virus (WSSV) is the sole member of the novel Nimaviridae family, and the source of major economic problems in shrimp aquaculture. WSSV appears to have rapidly spread worldwide after the first reported outbreak in the early 1990s. Genomic deletions of various sizes occur at two loci in the WSSV genome, the ORF14/15 and ORF23/24 variable regions, and these have been used as molecular markers to study patterns of viral spread over space and time. We describe the dynamics underlying the process of WSSV genome shrinkage using empirical data and a simple mathematical model. Methodology/Principal Findings - We genotyped new WSSV isolates from five Asian countries, and analyzed this information together with published data. Genome size appears to stabilize over time, and deletion size in the ORF23/24 variable region was significantly related to the time of the first WSSV outbreak in a particular country. Parameter estimates derived from fitting a simple mathematical model of genome shrinkage to the data support a geometric progression (

Silkworm expression system as a platform technology in life science

Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3–6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the “Silkroad,” we expect that the “Bioroad” from Asia to Europe will be established by the silkworm expression system

The Gene Pool Concept Applied to Crop Wild Relatives: An Evolutionary Perspective

Crop wild relatives (CWR) can provide important resources for the genetic improvement of cultivated species. Because crops are often related to many wild species and because exploration of CWR for useful traits can take many years and substantial resources, the categorization of CWR based on a comprehensive assessment of their potential for use is an important knowledge foundation for breeding programs. The initial approach for categorizing CWR was based on crossing studies to empirically establish which species were interfertile with the crop. The foundational concept of distinct gene pools published almost 50 years ago was developed from these observations. However, the task of experimentally assessing all potential CWR proved too vast; therefore, proxies based on phylogenetic and other advanced scientific information have been explored. A current major approach to categorize CWR aims to comprehensively synthesize experimental data, taxonomic information, and phylogenetic studies. This approach very often ends up relying not only on the synthesis of data but also intuition and expert opinion and is therefore difficult to apply widely in a reproducible manner. Here, we explore the potential for a stronger standardization of the categorization method, with focus on evolutionary relationships among species combined with information on patterns of interfertility between species. Evolutionary relationships can be revealed with increasing resolution via next-generation sequencing, through the application of the multispecies coalescent model and using focused analyses on species discovery and delimitation that bridge population genetics and phylogenetics fields. Evolutionary studies of reproductive isolation can inform the understanding of patterns of interfertility in plants. For CWR, prezygotic postpollination reproductive barriers and intrinsic postzygotic barriers are the most important factors and determine the probability of producing viable and fertile offspring. To further the assessment of CWR for use in plant breeding, we present observed and predicted gene pool indices. The observed index quantifies patterns of interfertility based on fertilization success, seed production, offspring viability, and hybrid fertility. The predicted gene pool index requires further development of the understanding of quantitative and qualitative relationships between reproductive barriers, measures of genetic relatedness, and other relevant characteristics for crops and their wild relatives