The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture

Dissolved oxygen tension and oxygen uptake rate are critical parameters in animal cell culture. However, only scarce information of such variables is available for insect cell culture. In this work, the effect of dissolved oxygen tension (DOT) and the utility of on-line oxygen uptake rate (OUR) measurements in monitoring Spodoptera frugiperda (Sf9) cultures were determined. Sf9 cells were grown at constant dissolved oxygen tensions in the range of 0 to 30%. Sf9 metabolism was affected only at DOT below 10%, as no significant differences on specific growth rate, cell concentration, amino acid consumption/production nor carbohydrates consumption rates were found at DOT between 10 and 30%. The specific growth rate and specific oxygen uptake rate followed typical Monod kinetics with respect to DOT. The calculated _max and max were 0.033 h^-1 and 3.82×10^-10 mole cell^-1h^-1, respectively, and the corresponding saturation constants were 1.91 and 1.57%, respectively. In all aerated cultures, lactate was consumed only after glucose and fructose had been exhausted. The yield of lactate increased with decreasing DOT. It is proposed, that an apparent DOT in non-instrumented cultures can be inferred from the lactate yield of bioreactors as a function of DOT. Such a concept, can be a useful and important tool for determining the average dissolved oxygen tension in non-instrumented cultures. It was shown that the dynamic behavior of OUR can be correlated with monosaccharide (fructose and glucose) depletion and viable cell concentration. Accordingly, OUR can have two important applications in insect cell culture: for on-line estimation of viable cells, and as a possible feed-back control variable in automatic strategies of nutrient addition.Abbreviations DOT Dissolved oxygen tension - OUR Oxygen uptake rate - specific oxygen uptake rate - specific growth rate - X_v viable cell concentration - C_L, C^*, and oxygen concentrations in liquid phase, in equilibrium with gas phase, and medium molar concentration, respectively - H Henry's constant - K_La volumetric oxygen transfer coefficient - P_T total pressure - oxygen partial pressure - oxygen molar fraction - i discrete element     

A tubular segmented-flow bioreactor for the infection of insect cells with recombinant baculovirus

A continuous process of insect cell (S f9) growth and baculovirus infection is tested with the sequential combination of a CSTR and a tubular reactor. A tubular infection reactor enables continuous introduction of baculovirus and therefore avoids the ‘passage effect’ observed in two-stage CSTR systems. Moreover, a tubular reactor can be used to test cell infection kinetics and the subsequent metabolism of infected insect cells. Unlike batch and CSTR culture, cells in a horizontally positioned tubular reactor settle due to poor mixing. We have overcome this problem by alternately introducing air bubbles and media and by maintaining a linear velocity sufficient to keep cells suspended. This article addresses the development of the tubular reactor and demonstrates its use as an infection system that complements the two-stage CSTR. This revised version was published online in July 2006 with corrections to the Cover Date.     

The indirect effects of multiplicity of infection on baculovirus expressed proteins in insect cells: secreted and non-secreted products

The baculovirus expression vector system was employed to produce human apolipoprotein E and β-galactosidase in order to study the effect of multiplicity of infection on secreted and non-secreted recombinant protein production. Prior knowledge of the influence of other cell culture and infection parameters, such as the cell density at time of infection and the time of harvest, allowed determination of the direct and indirect influences of multiplicity of infection on recombinant protein synthesis and degradation in insect cells. Under non-limited, controlled conditions, the direct effect of multiplicity of infection (10⁻¹−10 pfu/cell) on specific recombinant product yields of non-secreted β-galactosidase was found to be insignificant. Instead, the observed increased in accumulated product was directly correlated to the total number of infected cells during the production period and therefore ultimately dependent on an adequate supply of nutrients. Only the timing of recombinant virus and protein production was influenced by, and dependent on the multiplicity of infection. Evidence is presented in this study that indicates the extremely limited predictability of post-infection cell growth at very low multiplicities of infection of less than 0.1 pfu/cell. Due to the inaccuracy of the current virus quantification techniques, combined with the sensitivity of post-infection cell growth at low MOI, the possibility of excessive post-infection cell growth and subsequent nutrient limitation was found to be significantly increased. Finally, as an example, the degree of product stability and cellular and viral protein contamination at low multiplicity of infection is investigated for a secreted recombinant form of human apolipoprotein E. Comparison of human apolipoprotein E production and secretion at multiplicities of infection of 10⁻⁴−10 pfu/cell revealed increased product degradation and contamination with intracellular proteins at low multiplicities of infection. This revised version was published online in July 2006 with corrections to the Cover Date.     

A mathematical model of baculovirus infection on insect cells at low multiplicity of infection

The expression efficiency of the insect cells-baculovirus system used for insecticidal virus production and the expression of medically useful foreign genes is closely related with the dynamics of infection. The present studies develop a model of the dynamic process of insect cell infection with baculovirus at low multiplicity of     

Production of adeno-associated viral vectors in insect cells using triple infection: optimization of baculovirus concentration ratios

The production of viral vectors or virus-like particles for gene therapy or vaccinations using the baculovirus expression system is gaining in popularity. Recently, reports of a viral vector based on adeno-associated virus (AAV) produced in insect cells using the baculovirus expression vector system have been published. This system requires the triple infection of cells with baculovirus vectors containing the AAV gene for replication proteins (BacRep), the AAV gene for structural proteins (BacCap), and the AAV vector genome (BacITR). A statistical approach was used to investigate the multiplicities of infection of the three baculoviruses and the results were extended to the production of AAVs containing various transgenes. Highest AAV yields were obtained when BacRep and BacCap, the baculovirus vectors containing genes that code for proteins necessary for the formation of the AAV vector, were added in equal amounts at high multiplicities of infection. These combinations also resulted in the closest ratios of infectious to total AAV particles produced. Overexpression of the AAV structural proteins led to the production of empty AAV capsids, which is believed to overload the cellular machinery, preventing proper encapsidation of the AAV vector transgene, and decreased the viability of the insect cells. Delaying the input of BacCap, to reduce the amount of capsids produced, resulted in lower infectious AAV titers then when all three baculoviruses were put into the system at the same time. The amount of BacITR added to the system can be less than the other two without loss of AAV yield.     

Expression of active human beta-glucuronidase in Sf9 cells infected with recombinant baculovirus

Antibody directed enzyme prodrug therapy (ADEPT) using glucuronide prodrugs is an experimental approach to reduce systemic toxicity of anti-cancer agents. Bioactivation of such prodrugs is achieved by fusion proteins consisting of targeting moieties (e.g. ligands of tumor specific antigens) and human beta-glucuronidase. In order to test a large panel of possible beta-glucuronidase fusion proteins for their applicability in ADEPT, an easy, rapid and high-yield expression system like the baculovirus/insect cell expression system would be needed. A prerequisite for using such fusion proteins is functional and biochemical characterization of human beta-glucuronidase expressed in baculovirus-infected insect cells. Therefore, recombinant human beta-glucuronidase was expressed in Sf9 insect cells and characterized at the protein and functional level. As shown by Western blot analysis the recombinant enzyme consists of dimers with their monomers being linked via disulfide bonds. Posttranslational modifications of the monomers seem to be different as compared with mammalian cells or tissues. The enzyme is functionally active in cleaving the substrates 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, 4-methylumbelliferyl-beta-D-glucuronide and the glucuronide prodrug HMR 1826, respectively, with similar enzyme kinetic parameters as those found in human tissues. Our data demonstrate that beta-glucuronidase expressed in Sf9 cells displays the same enzymatic features as the protein expressed in mammalian cells. Therefore, we suggest that beta-glucuronidase fusion proteins produced in this cell line will be valuable tools for testing a large panel of various targeting moieties in human tumor xenograft models or may be used for ADEPT in man.     

昆虫细胞制备AAV-ITR基因表达微载体

AAV-ITR基因表达微载体是只含有腺相关病毒(Adeno-associated virus,AAV)倒置末端重复序列(Inverted terminal repeats,ITR)、基因表达顺式元件和目的基因,而不含有其他外源DNA序列的双链或单链DNA。本研究利用杆状病毒表达系统,制备得到两种重组杆状病毒Bac-ITR-EGFP和Bac-inrep,并将二者的P3代病毒共同感染昆虫细胞Spodoptera frugiperda(Sf9),抽提小分子量DNA,获得AAV-ITR-EGFP基因表达微载体,2×107的Sf9细胞抽提可以得到100μg AAV-ITR-EGFP基因表达微载体,核酸电泳显示AAV-ITR-EGFP基因表达微载体主要以单体和二聚体的形式存在。将AAV-ITR-EGFP基因表达微载体通过polyethylenimine(PEI)转染HEK 293T细胞,24 h后荧光显微镜观察有EGFP表达,48 h后达到高峰,转化效率达到65%。     

On-line oxygen uptake rate and culture viability measurement of animal cell culture using microplates with integrated oxygen sensors

O2 uptake rates of animal cells (Chinese hamster ovary-CHO) were measured in 96-well microtiter plates by integrating with fluorescent sensors thereby measuring fluorescence intensity ratios of an O2-sensitive and an insensitive fluorophor. O2 consumption rate was estimated from measured dissolved O2 and from O2 mass transfer coefficient determined in advance. Specific uptake decreased with time from 3.2 x 10(-13) mol O2 cell(-1) h(-1) at 15 h cultivation to 1.8 x 10(-13) mol O2 cell(-1) h(-1) at 48 h. Specific O2 uptake was also determined by sampling from a spinner-flask culture giving identical values. A cell viability assay for cultures based on O2 measurements is described in which cells are incubated outside the fluorescence reader and then the dissolved O2 is measured only once at a fixed time after the start of incubation. This protocol can be directly applied for high-throughput measurements.     

Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm

Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.     

Real‐time monitoring of specific oxygen uptake rates of embryonic stem cells in a microfluidic cell culture device

Oxygen plays a key role in stem cell biology as a signaling molecule and as an indicator of cell energy metabolism. Quantification of cellular oxygen kinetics, i.e. the determination of specific oxygen uptake rates (sOURs), is routinely used to understand metabolic shifts. However current methods to determine sOUR in adherent cell cultures rely on cell sampling, which impacts on cellular phenotype. We present real‐time monitoring of cell growth from phase contrast microscopy images, and of respiration using optical sensors for dissolved oxygen. Time‐course data for bulk and peri‐cellular oxygen concentrations obtained for Chinese hamster ovary (CHO) and mouse embryonic stem cell (mESCs) cultures successfully demonstrated this non‐invasive and label‐free approach. Additionally, we confirmed non‐invasive detection of cellular responses to rapidly changing culture conditions by exposing the cells to mitochondrial inhibiting and uncoupling agents. For the CHO and mESCs, sOUR values between 8 and 60 amol cell^?1 s^?1, and 5 and 35 amol cell^?1 s^?1 were obtained, respectively. These values compare favorably with literature data. The capability to monitor oxygen tensions, cell growth, and sOUR, of adherent stem cell cultures, non‐invasively and in real time, will be of significant benefit for future studies in stem cell biology and stem cell‐based therapies.     

Specific oxygen, ammonia, and nitrate uptake rates of a biological nutrient removal process treating elevated salinity wastewater

Anaerobic/anoxic/aerobic systems inoculated without and with NaCl acclimated cultures, i.e., Models A and B, respectively, were fed with a synthetic wastewater at various salinity levels. After achieving a steady state, the systems were shocked with 70 g/l NaCl for four consecutive days before returning to pre-shock conditions. At the steady-state, the specific oxygen uptake rates (SOURs) increased with an increase of sodium chloride concentration (from 5.40 to 9.72 mg O₂/g mixed liquor suspended solids (MLSS)-h at 0–30 g/l NaCl for Model A and from 6.84 to 17.64 mg O₂/g MLSS-h at 5–30 g/l NaCl for Model B). In contrast, the specific ammonia uptake rate (SAUR) and specific nitrate uptake rate (SNUR) decreased with increasing chloride concentration (from 4.76 to 2.14 mg NH₃–N/g MLSS-h and 2.50 to 1.22 NO3–N/g MLSS-h, for Model A, and from 3.84 to 2.71 mg NH₃–N/g MLSS-hr and 2.54 to 1.82 mg NO₃–N/g MLSS-hr, for Model B). During the shocked period, the SOUR in most scenarios increased whereas the SAUR and SNUR tended to decrease. The impact of the chloride shock on nitrifiers was more obvious than on denitrifiers; however, after a certain recovery period, the activities of both nitrifiers and denitrifiers in terms of SAUR and SNUR were approximately the same as those prior to shock.     

Modelling the growth and protein production by insect cells following infection by a recombinant baculovirus in suspension culture

A mathematical model has been developed to describe the growth and infection of insect cells by recombinant baculoviruses. The model parameters were determined from a series of independent experiments involving batch suspension culture. The profiles generated by the model for cell growth, virus production and protein production agree with those observed in experiments. Presently, the model simulates only systems where cells are not growth-limited. The model is useful in aiding the design and optimization of large-scale systems for production of biological insecticides as well as recombinant proteins and in delineating those areas which are limiting the process and require further, more fundamental, investigation.     

Analysis of noise and bias in fermentation oxygen uptake rate data

The calculation of many derived fermentation variables such as the respiratory quotient (RQ) and mass transfer coefficient (K(L)a) requires the differences between the molar percentages of oxygen and carbon dioxide in the fermentor inlet and exit gas, called the %OUR and %CER. Noise and bias in %CER data is of order that in the exit gas carbon dioxide analysis. However, the relative amount of noise in the %OUR is one to two orders of magnitude greater than the noise in the raw oxygen analyses because the %OUR is calculated as a small difference between two large quantities. The noise in the %OUR is white with a Gaussian amplitude probability distribution of absolute standard deviation 0.0145. A chi-square filter of the %OUR data is shown to considerably improve the quality of the calculated RQ and K(L)a for a fermentation of Streptomyces clavuligerus. (c) 1992 John Wiley & Sons, Inc.     

The response of virally infected insect cells to dissolved oxygen concentration: recombinant protein production and oxidative damage

The effects of dissolved oxygen (DO) concentration on virally infected insect cells were investigated in 3-L bioreactor culture. Specifically, cultures of Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) were infected with Autographa californica multiple nucleopolyhedrovirus expressing secreted alkaline phosphatase (SEAP). Following infection at a DO concentration of 50% air saturation, the DO concentration was adjusted to a final value of either 190%, 50%, or 10% air saturation. Recombinant SEAP production, cell viability, protein carbonyl content, and thiobarbituric acid reactive substances (TBARS) content were monitored. The increases in protein carbonyl and TBARS contents are taken to be indicators of protein oxidation and lipid oxidation, respectively. DO concentration was found to have no noticeable effect on SEAP production or cell viability decline in the Sf-9 cell line. In the Tn-5B1-4 cell line, cells displayed an increased peak SEAP production rate for 190% air saturation and displayed an increased rate of viability decline at increased DO concentration. Protein carbonyl content showed no significant increase in the Sf-9 cell line by 72 h postinfection (pi) at any DO concentration but showed a twofold increase at 10% and 50% DO concentration and a threefold increase at 190% DO concentration by 72 h pi in Tn-5B1-4 cells. TBARS content was found to increase by approximately 50% in Sf-9 cells and by approximately twofold in Tn-5B1-4 cells by 72 h pi with no clear relationship to DO concentration. It is hypothesized that oxygen uptake changes due to the viral infection process may bear a relation to the observed increases in protein and lipid oxidation and that lipid oxidation may play an important role in the death of virally infected insect cells.     

Strategies for manipulating the relative concentration of recombinant rotavirus structural proteins during simultaneous production by insect cells

Adequate production strategies of virus-like particles are among the challenges that must be addressed before such complex multimeric structures find practical applications as vaccines. Attainment of the correct stoichiometric relation between proteins that constitute virus-like particles should result in an increased productivity by maximizing the concentration of assembled proteins and preventing the accumulation of waste monomers. In this work, strategies for manipulating the relative concentration between two of the structural proteins that constitute rotavirus-like particles (VP2 and VP6) were explored using the insect cell baculovirus expression vector system. It was shown that multiplicity of infection is a useful tool for manipulating protein production rates and maximum concentrations in cultures expressing one or two recombinant proteins. Thus, multiplicity of infection can be employed for improving production of rotavirus-like particles. VP2 and VP6 production rates obtained during individual infections remained unchanged when both were simultaneously produced, indicating that such rates can be utilized for estimating protein concentrations during coexpression. Manipulation of the time of infection between the two recombinant baculoviruses, proposed here for the first time, also proved to be effective for controlling the relative protein concentrations. The use of such sequential infections constituted an effective production alternative that does not require high amounts of virus stocks and is easy to implement. In addition to VP2 and VP6, kinetic parameters for the individual production of the other two proteins (VP4 and VP7) that constitute rotavirus-like particles were also obtained.     

The use of oxygen uptake rate to monitor discovery of microbial and enzymatic biocatalysts

Arising from the requirement for discovery of novel biocatalysts with unusual properties, a process was developed which uniquely combines aspects of continuous culture with the measurement of oxygen uptake. This adaptation of the chemostat can be used to facilitate the isolation of a number of microorganisms with desirable properties, particularly those with useful metabolic capabilities and/or enzymes. The technique was also used to provide feedback on the metabolic status of a microbial population and increase the feed flow rate (i.e., dilution rate) thereby enabling the isolation of microorganisms with enhanced 1,3‐propanediol dehydrogenase activity. The use of oxygen uptake as an indicator of cellular activity enables indirect measurement of substrate utilization and provides a real‐time online assessment of the status of microbial enrichment or evolutionary processes and provides an opportunity, through the use of feedback systems, to control these processes. To demonstrate the utility of the technique, oxygen uptake rate (OUR) was compared with a range of conventional analytical techniques that are typically used to monitor enrichment/evolutionary processes and showed good correlation. Further validation was demonstrated by monitoring a characterizable microbial population shift using OUR. The population change was confirmed using off‐line analytical techniques that are traditionally used to determine microbial activity. OUR was then used to monitor the enrichment of microorganisms capable of using a solvent (1‐methyl‐2‐pyrrolidinone) as the sole source of carbon for energy and biomass formation from a heterogeneous microbial population. After purification the microorganisms taken from the enrichment process were able to completely utilize 1 g L^?1 1‐methyl‐2‐pyrrolidinone within 24 h demonstrating that the technique had correctly indicated the enriched population was capable of growth on 1‐methyl‐2‐pyrrolidinone. The technique improves on conventional microbial enrichment that utilizes continuous culture by providing a real‐time assessment of the enrichment process and the opportunity to use the OUR output for automated control and variation of one or more growth parameters. Biotechnol. Bioeng. 2009;102: 673‐683. © 2008 Wiley Periodicals, Inc.     

High-density cultivation of insect cells and production of recombinant baculovirus using a novel oscillating bioreactor

A novel two-compartment bioreactor, BelloCell^®, was used to cultivate insect cells and a maximum yield of 4.6 × 10⁹ cells was attained. The cells were immobilized in a packed bed fixed in the upper chamber, and the bellow in the lower chamber was compressed and released in an alternating fashion. The motion resulted in gentle, cyclic movement of the medium that was contained in the lower chamber and consequently exposed the cells to air in an oscillatory manner, thus rendering adequate aeration and uniform cell distribution in the bed. The baculovirus yield produced in BelloCell^® could amount up to 3.3 × 10¹⁷ pfu using as little as 1.1 l medium in the production run. Besides, BelloCell^® was extremely easy to handle and operate. These benefits underline the potential of BelloCell^® for simple, economical and high-density cell culture and protein/virus production.     

Modeling the population dynamics of baculovirus-infected insect cells: Optimizing infection strategies for enhanced recombinant protein yields

The insect cell-baculovirus model presented here is capable of simulating cell population dynamics, extracellular virion densities, and heterologous product titers in reasonable agreement with experimental data for a wide rang of multiplicities of infection (MOI) and times of infection. The model accounts for the infection of a single cell by multiple virions and the consequences on the time course of infection. The probability of infection by more than one virion was approximated using the Poisson distribution, which proved to be a refinement over second-order kinetics. The model tracks initiation and duration of important events in the progression of infected cell development (virus replication, recombinant protein synthesis, and cell lysis) for subpopulations delineated by the time and extent of their initial infection. The model suggests infection strategies, weighing the importance of MOI and infection time. Maximum product titers result from infection in the early exponential growth phase with low MOI.     

Purification and characterization of a recombinant rat prohaptoglobin expressed in baculovirus-infected Sf9 insect cells

To generate hemoglobin-free full-length haptoglobin the cDNA encoding rat haptoglobin alphabeta subunits was cloned into shuttle vector pVT-Bac-His and used to produce a recombinant baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV) as an expression vector, named HpAcNPV. Recombinant virus was used to infect Spodoptera frugiperda (Sf9) insect cells. The 50 kDa protein expressed was mostly secreted into the culture medium at relatively high titer (15 microg/mL) and was found to be rat prohaptoglobin having a vector-derived N-terminal extension of 37 amino acids, containing both a hexahistidine tag and an enterokinase recognition sequence. The protein was successfully purified by a three step procedure including nickel-linked agarose and DEAE-Sepharose chromatography steps. Hemoglobin was not detected in the purified preparations. Purified recombinant rat prohaptoglobin protein was also found to be glycosylated, and to be capable of forming a complex with rat hemoglobin in vitro.