Biotechnol. frog. 1994, 10, 636-643
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Optimum Infection Conditions for Recombinant Protein Production in Insect Cell (Bm5) Suspension Culture Junli ZhangJ,*Nicolas KalogerakisJ Leo A. Behie,',?and Kostas Iatrod Pharmaceutical Production Research Facility (PPRF), Faculty of Engineering, and Department of Medical Biochemistry, The University of Calgary, Calgary, Alberta, Canada T2N 1N4
The baculovirus/insect cell expression system is an efficient a n d practical method for the production of many active therapeutic proteins on a large scale. The advantages of suspension cultures have been demonstrated with the study of a baculovirudinsect cell (BmNPVBm5) expression system for the production of recombinant chloramphenicol acetyltransferase (CAT), a model heterologous protein. Key infection parameters such as infection time and multiplicity of infection were examined systematically for the maximization of protein production. Furthermore, emphasis was placed on the development of possible medium replenishment strategies, which were necessary to achieve higher volumetric protein production from the infection of high-density cell cultures without sacrificing specific protein productivity. The highest protein production was achieved with the infection of suspended cells in the mid to late exponential growth phase.
Introduction The baculovirus/insect cell expression system continues to be an efficient method for the production of many active recombinant proteins. In insect cell culture, the nonoccluded form of baculoviruses (NOVs) is infectious, while the polyhedrin gene within the virus is nonessential. Replacement of the polyhedrin structural gene sequences with others encoding foreign proteins under the control of a polyhedrin gene promoter allows highlevel target protein production (up to 50% of cellular proteins; Luckow and Summers, 1988). In addition to high expression levels, the baculovirushsect cell system offers safety advantages over mammalian cell/virus systems because insect cells and baculoviruses are not pathogenic to other organisms. Moreover, insect cells have the ability to carry out many of the complex posttranslational protein modifications that occur in mammalian cells, including secretion, signal peptide cleavage, phosphorylation, and N- and O-linked glycosylation (Luckow and Summers, 1988). To date, a large number of genes from various prokaryotic and eukaryotic organisms have been cloned into the baculovirus/insect cell system for the production of corresponding recombinant proteins (Maeda, 1989; Murhammer, 1991; O'Reilly et al., 1992; Summers, 1991). For the large-scale production of recombinant proteins from baculovirus/insect cell systems, different process protocols have been reported for cell cultivation and viral infection (Shuler et al., 1990; Tramper et al., 1990; Zhang et al., 1993). On the basis of the operation pattern, these include batch, repeated batch, and continuous processes. In terms of culturing methods, insect cells can be cultivated in suspension, attached to a stationary surface, immobilized, or encapsulated inside microcarriers. As most of the insect cell lines can be easily adapted to grow
* Author t o whom correspondence should be addressed. PPRF, Faculty of Engineering.
* Current address: MicroGeneSys, Inc., 1000 Research Park-
way, Meriden, CT 06450. Department of Medical Biochemistry.
in suspension culture, and key technical problems associated with large-scale suspension culture can be resolved, this type of culture is expected to represent the method of choice in many future production systems. However, the question does still remain as to whether or not suspension culture gives higher protein yields compared to stationary culture. Lanford (1988) reported that large suspension cultures yielded 3-5-fold lower levels of recombinant T-antigen than Petri dish cultures when an AcNPV/SB expression system was used. On the other hand, with the production of @-galactosidase using an AcNPV/Sf expression system, the protein yield achieved in a 14-L airlift bioreactor (13 000 units/l06 cells; Wu et al., 1992) was much higher than that produced in 25-mL stationary tissue culture flasks (169 units/106 cells; Licari and Bailey, 1991). It should be noted that &galactosidase activities were measured a t 27 and 37 "C, respectively, and different extinction coefficients were used in their assays. m e r adjustments were made for temperature and extinction coefficients, the @-galactosidase yield achieved in the 14-L airlift bioreactor was computed to be 34 times higher than that in the stationary tissue culture flasks. Whether such a large difference in protein production results from different culturing methods or from different viral vectors used still is not clear. Obviously, it is particularly important to study the effect of culturing methods on protein production a t the earlier stage in process development. As the reported yields of proteins produced in insect cells vary widely from 1.0 to 1220 mg/L (Caron et al., 1990; Maiorella et al., 1988; Reuveny et al., 1993), much of the current efforts has been orientated toward the identification of the factors that affect expression levels. The various expression levels achieved could be affected by host insect cell lines, the location of gene insertion in the virus, and the expressed product itself (Hink et al., 1991; Johnson et al., 1993; Matsuura et al., 1986; Wickham et al., 1992). Moreover, the expression levels could be influenced by cell culture and infection conditions, including culturing methods, cell density, nutrient levels, stage of growth, and virus quality and quantity (Caron
8756-7938/94/3010-0636$04.50/0 0 1994 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog,, 1994,Vol. IO, No. 6
et al.,1990; Kool et al.,1991; Licari and Bailey, 1991; Radford et al.,1992; Wickham et al.,1991; Zhang et al., 1993). Nevertheless, the possibility of generalizing the effect of each major variable on expression levels is unlikely because most of the previous work on identifylng such factors has been limited to the AcNPV/Sf9 expression system and different results have been reported. For example, in reviewing the effect of the multiplicity of infection (MOI), one report mentions that a semilogarithmic relationship existed between the final yield and the MOI when cells were infected in the mid to late exponential growth phase (Licari and Bailey, 1991))while others report that protein production was relatively insensitive to the MOIs in the range from 1.0 to 20 (Maiorella et al.,1988; Murhammer and Goochee, 1988; Neutra et al.,1992). Hence, before the optimization of recombinant protein production from any baculovirus/ insect cell system can be possible, further research is needed to identify key factors affecting the expression levels and to determine their relative importance with different baculovirus/insect cell expression systems under stringently designed experimental conditions. In this article, we report on how the culturing methods affect the production of a recombinant protein using a BmNPV/Bm5 expression system. We also explore the effects of key infection parameters on the expression levels in a system efficiently scalable to production facilities.
Materials and Methods Cells, Virus, and Medium. Bombyx mori (Bm5) cells, which were derived from ovarian tissue of the silkworm (Grace, 1967), were grown in IPL-41 medium (Sigma) supplemented with 10% heat-inactivated (30 min a t 56 "C) fetal bovine serum (FBS, Hazelton). The cells were routinely maintained in 25-cm2tissue culture flasks and 125-mL spinner flasks (Corning) in a 28 "C humidified incubator, as previously described (Zhang et al.,1993). The pH and osmolality of the complete medium were 6.30 f 0.05 and 370 f 5 mOsm, respectively. The media used in all viral experiments were supplemented with 50 ,ug/ mL gentamicin sulfate (Sigma). A recombinant baculovirus, BmNPVP5C.cat) expressing chloamphenicol acetyltransferase (CAT) (Johnson et al.,1993; Zhang et al.,1993) was used throughout this study. This virus contained a bacterial cat gene under the control of the polyhedrin gene promoter of the B. mori nuclear polyhedrosis virus (BmNPV). Previous studies have shown that the majority of the expressed protein existed in a n extracellualr form (Zhang et al.,1993).The virus was propagated in Bm5 cells in spinner flasks; the infectious supernatant was collected by centrifuging infected cultures a t 2000g in a GPR centrifuge (Beckman) and stored at -70 "C. The same pool of viral inocula was used throughout. Infection of Bm5 Cells in Different Culturing Vessels. Experiments were carried out to examine the potential differences in protein productivity obtained from the infection of suspension and stationary cultures. Cells from 25-cm2 tissue culture flasks and 125-mL spinner flasks in the mid-exponential growth phase were collected by centrifuging the cultures a t 200g for 5 min and subsequently replenishing 50% of the culture broth with fresh medium. The cell cultures were then infected with BmNPVP5C.cat a t a high MOI of 13plaque-forming units per cell (PFU/cell)in order to achieve synchronous infection of cells. Infection of stationary and suspended cells occurred in their corresponding vessels for the cell cultures.
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Infection of Cells with Different MOIs. To study the effect of MOI in suspension culture, exponentially growing Bm5 cells in 125-mLspinner flasks were infected with different amounts of recombinant virus in order to achieve the desired MOI levels. To eliminate any interfering effects of the CAT protein present in the virus stock solution (Johnson et al.,19921, especially at high MOIs, the spent culture medium was replenished completely with fresh medium after virus adsorption a t 2 h postinfection (PI). At the same time, all of the cultures were diluted to a cell density of about 1.0 x lo6 cells/mL with fresh medium to ensure sufficient nutrient supply, including oxygen, during infection. Infection of Cells at Different Ages. To assess the effect of cell age on the expression of recombinant CAT protein, the Bm5 cells in early, mid to late, and a t the end of exponential growth phases were infected in 125mL spinner flasks with recombinant virus a t a n MOI of 5.0 PFU/cell. To eliminate the possible interference of nutrient conditions and cell density with the effect of cell age on protein production, the culture broth was completely replenished with fresh medium after 2.0 h for virus adsorption, and all cultures were diluted subsequently to a cell density of about 1.0 x lo6 viable cells/ mL in 125-mL spinner flasks. Sample Preparation. During infection, samples were collected every 24 h PI until the viable cells were completely utilized by the virus. Except for the sample used for cell counts, the rest of each sample was centrifuged a t lOOOg for 10 min. The supernatant of the sample was used for extracellular CAT assays and glucose analysis. The cell pellets were washed twice with phosphate-buffered saline (PBS, pH 6.30) and lysed in 0.25 M Tris-HC1 (pH 7.80) by three cycles of freezingthawing between dry ice (-70 "C) and a water bath (37 "C). The supernatant of the extract solution was collected by centrifugation a t 2000g for 10 min and used for the intracellular CAT assay. Analytic Methods. Cells were counted using a hemocytometer, and cell viabilities were determined by the standard trypan blue exclusion test (0.1%) Sigma). The glucose concentration was measured using a n automatic analyzer (Model 2000, YSI). A two-step end-point titration method (Zhang, 1993) was used to determine the titer of the recombinant virus. In this method, the first step was a standard end-point titration assay (Summers and Smith, 19871, which was used to obtain a n order-of-magnitude estimate. Then, the second step determined the actual virus titer with reduced dilution intervals, within the two most significant dilutions determined from the first set of assays. In the assays, the infectious virus DNA was detected using a cat gene probe by hybridization (Zhang et al.,1993); the virus concentration was expressed as PFU/mL. The accuracy of this method has been described mathematically by Nielsen et al. (1992). The total protein content of samples was determined by the Bradford protein assay (Bio-Rad) using bovine serum albumin as the standard. The cell lysate or supernatant was diluted in a dilution buffer (5' 3') to a total protein concentration of about 200 ,ug/mL, and further dilutions were made as required. Finally, the intracellular and extracellular recombinant CAT proteins were quantified by an ELISA assay as described previously (Zhang et al.,1993). All data points were the averaged values of nine measurements from three repeat experiments, and standard deviations were calculated on the basis of these measurements.
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638 20 0 0
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Figure 1. Bm5 cell growth in 125-mLspinner flasks (Corning) at different stirring speeds. Working volume: 100 mL. Culturing medium: IPL-41 supplemented with 10% FBS.
Experimental Results and Discussion Bm6 Cell Growth in 126-mL Spinner Flasks. Dissolved oxygen (DO) an important process parameter that affects the growth of insect cells and the expression levels of recombinant proteins (Caron et al., 1900; Zhang et al., 1994). In a small volume culture, oxygen transfer through surface diffusion can be sufficient for cell growth as the surface area to volume (SN) ratio is quite large. For example, the ratio is 5.0 cm-l in a 25-cm2 tissue culture flask with a working volume of 5.0 mL. However, oxygen limitation could occur even in spinner flasks (Caron et al., 1990) because the S N ratio decreases dramatically with the incrase in culture volume. For example, the SN ratio in 125-mL spinner flasks with a working volume of 100 mL is 0.33 cm-', which is 15-fold lower than that in 25-cm2 tissue culture flasks. Hence, proper agitation is necessary in order to maintain adequate DO levels in spinner flasks. As the majority of the experiment in this study were carried out in 125-mL spinner flasks without on-line DO control or monitoring, it was important to establish the limitations of these vessels in supplying oxygen to a culture. Cell growth in IPL-41+ 10% FBS was assessed in 125-mL spinner flasks with a working volume of 100 mL at different stirring speeds. Figure 1 summarizes the results obtained from these experiments. Initially, cell growth at different agitation rates was essentially identical. Then, a difference in cell growth was observed a t a cell density of about 1.0 x lo6cells/mL. Apparently, the slower growth rate a t 40 rpm resulted from an oxygen mass transfer limitation, because other nutrient limitations never occurred in cell cultures in the early to midexponential growth phases. Thus, oxygen limitations can occur even at a low cell density if the agitation rates in 125-mL spinner flasks are not high enough. On the other hand, with stirring speeds of 60 and 80 rpm, identical cell growth rates and maximum attainable cell densities, comparable to these obtained from controlledbioreactor experiments (Zhang et al., 19941, were achieved. The number of viable cells reached a plateau late in the experiments simply because glucose became a limiting nutrient (data not shown). Hence, a stirring speed of 60 rpm was chosen for all experiments in 125mL spinner flasks. In addition, it is generally recognized that the oxygen requirement of infected cells is higher
o
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Figure 2. Profiles of viable cell density and glucose concentration of the cultures in stationary tissue culture flasks ( 0 )and
suspension spinner flasks (0)infected with BmNPVP5C.cat at an MOI of 13 PFU/cell.
than that of uninfected cells. Therefore, in the design of all infection experiments, cell cultures were diluted to a low density of about 1.0 x lo6 cells/mL to ensure a sufficient oxygen supply during infection. Effect of Culturing Methods on Recombinant Protein Production. To identify whether the culturing methods affect the expression level of recombinant proteins, stationary cells in 25-cm2tissue culture flasks and suspended cells in 125-mL spinner flasks were infected with a recombinant virus under identical infection conditions. The profiles of viable cell density and glucose concentration in infected stationary and suspension cultures are depicted in Figure 2. As shown in Figure 2A, a slight increase in cell number was observed in the first 48 h postinfection (PI) both in stationary tissue culture flasks and in suspension spinner flasks. A similar trend of increases in cell number was also observed when suspended Sf9 cells were infected with a n MOI of 5.0 (Murhammer and Goochee, 1988) and stationary cultured Sfs cells were infected with a n MOI of 10 (Licari and Bailey, 1991). This general phenomenon is probably due to the fact that the cells are at different stages in their cycle a t the time of infection, and consequently cells may not be synchronously infected even with a n MOI above 5. Beyond 48 h PI, a dramatic decrease in the viable cell concentration was observed, mainly due to the infection of the cells by the viruses. As shown in Figure 2B, no nutrient limitations were observed during the complete infection process because of the facts that 50%of the culture medium was replenished before the addition of viral inoculum and that lowdensity cell cultures were used for virus infection in these vessels. Under these conditions, the effects of culturing methods on cell infection and the glucose consumption rate were insignificant. The total CAT concentrations, including intracellular and extracellular CAT proteins, from infected stationary and suspended cells are shown in Figure 3. It should be
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Figure 3. Effect of culturing methods on recombinant CAT production.
noted that initial levels of CAT protein in the medium during the early infection period ( e48 h PI) were due to the presence of the protein in the viral inoculum (Johnson et al., 1992). Expression of the cat gene was initiated between 24 and 48 h PI in both cases, which is in agreement with the observation that the polyhedringene promoter becomes active around 36 h PI. A comparison of the data in Figures 2A and 3 illustrates that the most rapid production of CAT protein between 72 and 96 h PI corresponded with the most rapid rate of cell death, indicating that most of the protein was synthesized during the exponential infection phase of the cells. However, the CAT expression rate in suspended cells was much faster than that in stationary cells. To allow direct comparisons, a n average specific protein productivity based on infected cells a t the end of infection has been defined a s follows:
where qCAT is the average specific CAT productivity [mg/ ( lo9 cells-day)], C,,(tf) is the total CAT concentration a t the end of infection (mg/L),Xiinfededis the total number of infected cells (lo6 cells/mL), and tf is the postinfection time (days) when all cells are infected and viability is approximately zero. If a culture is under good nutrient supply conditions, natural cell death is negligible. Hence, the total number of infected cells is approximately equal to the maximum number of viable cells available during infection (i.e., Xinfededx Xv,ma).Hence, eq 1 becomes
- CCAT(0) X","tf
CCAT(tf)
qcAT =
(2)
The specific CAT productivities achieved from the infection of suspended cells and stationary cells were 20.6 f 0.6 and 6.2 f 0.3 mg/(109cells-day), respectively. This means that the specific CAT productivity from suspended cells was three times higher than that from stationary cells. Since all infection parameters, except cell culturing systems, were essentially identical, the differences in specific CAT productivity appeared to be the result of the different culturing methods that might affect the physiological states of the cells. This indicates that suspension
cultures favor CAT expression over stationary cultures for the Bm"V/Bm5 expression system. An important question that must be raised is as follows: What are the rational criteria for the development of a high-efficiency system for the production of recombinant proteins from this baculovirus/insect cell system that can be scaled up to a production facility? Such a system must be simple and reliable in operation and control, so that it can be easily validated in a production facility. Moreover, the system, must be reproducible and efficient in protein production. In all aspects, suspension cultures were superior to attached, immobilized, or encapsulated microcarrier cultures. With this in mind, we proceeded to explore the most efficient infection protocol for suspension cultures. Effect of MOI on Recombinant Protein Production. The multiplicity of infection (MOI) is one of the most important infection parameters for the production of recombinant proteins when the baculovirus/insect cell system is used. Licari and Bailey (1991) reported the effect of MOI on j3-galactosidase production in stationary culture. However, the effect of MOI on protein production in suspension culture has not yet been studied in detail. Whether the effect of MOI on protein production in suspension culture follows the same patterns as in stationary culture remains unresolved. We tested the effect of different MOI values on cell growth and CAT production in suspension cultures. It should be noted that the time of infection (TOI) has been reported to interact with the effect of MOI on recombinant protein production in stationary culture (Licari and Bailey, 1991). Because of tremendous advantages with the infection of high-density cell culture for recombinant virus and protein production, the emphasis in our study was placed on the effect of MOI on protein production in the mid to late exponential phase. Bm5 cells in the exponential growth phase (-2.67 x lo6 viable cells/mL) in 125-mL spinner flasks were infected with the virus a t different MOI values (0.1-20). Cell densities, glucose concentration, and CAT protein production were monitored daily (Figures 4 and 5). In the range of MOIs from 0.1 to 5.0, the higher the MOI used, the lower the cell growth rate and the glucose consumption rate, as shown in Figure 4. With MOIs larger than 1.0, however, the effect of MOI on both cell growth and the glucose consumption rate was insignificant. As shown in Figure 5, CAT expression rates and final CAT concentrations were essentially the same with MOIs from 5.0 to 20. A time shift of about 24 h in the rapid protein expression phase was observed with MOIs of less than 5.0. Also, the final CAT yield at a n MOI of 1.0 was lower than a t any MOI greater than 1.0. However, a surprisingly high value of the final CAT concentration was observed a t a n MOI of 0.1, which was not expected before the experiments. Most likely, the reason for this phenomenon was CAT production from the secondary infection of newly produced cells. To determine the effect of MOI on specific protein productivity in suspension cultures, the specific CAT productivity is plotted against MOIs (Figure 6). With MOIs from 0.1 to 1.0, the same productivities were obtained. However, their levels were lower compared to the productivities achieved with MOIs from 5.0 to 20. With suspended Sf9 cells in the mid to late exponential growth phase, the final protein yields were not significantly affected by MOIs in the range 1.0-20 (Maiorella et al., 1988; Murhammer and Goochee, 1988; Neutra et al., 1992). In view of our results, it appears that the effect of MOI on the final protein yield in suspension cultures follows a similar pattern with different bacu-
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Figure 6. Relationship between MOI and specific CAT pro-
ductivity in spinner flasks. The veritical error bars represent the standard deviations of nine measurements fkom three repeat experiments. 0
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Figure 4. Viable cell density and glucose concentration of the cultures infected with different MOIs in spinner flasks. The MOI values used are (0)0.1, (O), 1.0,(V), 5.0, (V) 10, and (0) 20. MOI values used: 0 0.1
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Figure 5. Effect of MOI on recombinant CAT production in
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lovirus/insect cell expression systems. Neverthless, different results were obtained by Licari and Bailey (1991)) who reported a semilogarithmic relationship between the final protein concentration and the MOI used for cells infected in the mid to late exponential growth phase. The higher the MOI, the higher the protein concentration. However, it should be noted that their experiments were carried out on Sf9 cells growing as a monolayer in stationary tissue culture flasks and that with MOIs in the range 0.01-10, a large fraction of viable cells was not utilized by the virus a t the end of infection (Licari and Bailey, 1991). When compared to suspension cultures, the lower mobility of newly produced viruses in stationary cultures may affect the degree of secondary
infection, which is especially significant a t lower MOI conditions. In addition, the contact inhibition (Wood et al., 1982) may affect the production of recombinant proteins in attached stationary cultures because of lower MOI conditions cells still replicate and may reach confluence before infection. Hence, the effect of MOI on protein production can vary with the culturing method (i.e., stationary or suspension cultures) when cells in the mid to late exponential growth phase are infected. Now, a n important question to be raised is as follows: What level of MOI should be used for the infection of high-density cells cultures in the production of recombinant proteins on a larger scale using a baculovirdinsect cell system? Our strategy is to use a n MOI that is as low as possible and a t the same time to achieve maximum protein productivity. From a n economic point of view, one should consider a low MOI such as 0.1 and, hence, rely on secondary infection for protein production. A major advantage of using a low MOI is that less virus inoculum is required, a costly material to produce. In some cases, the use of a low MOI may prevent the accumulation of defective interfering particles during virus propagation (Kool et al., 1991; Wickham et al., 1991). However, it should be noted that the same level of protein production from the secondary infection may not be achieved in the infection of high-density cell cultures compared to low-density cell cultures because cell aging and nutrient limitation may occur in the prolonged infection cultures a t a high density. On the other hand, one should consider a high MOI in order to achieve synchronous infection of high-density cell cultures. Previously, a n experimental study on initial viral adsorption indicated that, on an average, about three BmNPV particles could adsorb onto each Bm5 cell (Zhang, 1993). On the basis of our results, a n MOI betweeen 1 and 5 is recommended in order to achieve higher specific protein productivity from the synchronous infection of high-density cell cultures. Effect of Cell Age on Recombinant Protein Production. To examine which stage of cell growth offers maximum specific protein productivity, cells in the early, mid to late, and the end of exponential growth phases (Figure 7) were infected with the virus a t a n MOI of 5.0 PFUkell. I n these experiments, the possible effects of
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Time (h) Figure 7. Typical growth curve of Bm5 cells in spinner flasks. The arrows indicate the cell ages at which the cells were infected with BmNPVP5C.cat virus. nutrient level and cell density were eliminated as the spent media in all cultures were completely replenished following virus adsorption. Infected cultures with a cell density of about 1.0 x lo6 cells/mL were prepared. Cell growth and glucose consumption rates in cell cultures infected a t different ages were quite similar, as shown in Figure 8. Only a slight change in the viable cell number was observed with the infected cell cultures from the early exponential growth phase. However, a dramatic effect of cell age a t infection on CAT protein production was observed (Figure 9). The highest CAT expression rate and final concentration were achieved with the infection of cells in the mid to late exponential growth phase. The specific CAT productivities obtained from the infection of cells in the early, mid to late, and the end of exponential growth phases were 12.9 f 0.3, 19.7 f 0.5, and 9.3 f 0.3 mg/(109 cellsday), respectively. These results indicate that cells in the mid to late exponential phase should be infected with the recombinant virus to achieve the highest specific CAT productivity. Moreover, the specific CAT productivity obtained from the infection of cells at the end of exponential growth phase (or in other words, the onset of the stationary phase) was only 47% of that achieved from the infection of cells in the mid to late exponential growth phase. Previously, Maiorella et al. (1988) reported that the infection of cells in the stationary phase resulted in 60% of recombinant protein production as compared with the infection of cells in the exponential growth phase. This is in agreement with our results. In addition, others also observed the effect of cell age on recombinant protein production (Caron et al., 1990; Neutra et al., 1992). Neutra et al. (1992) reported that the best infection time for the production of P-galactosidase using an AcNPV/ Sf9 expression system in shake flasks was when the cell density was 0.7 x lo6 cells/mL. Either earlier or delayed infection times resulted in a sharp decrease in protein concentrations. Obviously, the infection of such a lowdensity culture (i.e., 0.7 x lo6 cells/mL) limits the volumetric productivity in bioreactors and hence is undesirable. For another AcNPVISfS expression system, Caron et al. (1990) found that equivalent VP6 production per cell was achieved in cell cultures with densities from 1.3 x lo6 to 4.6 x lo6 cells/mL with proper medium replenishment after infection, whereas infection a t a cell
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Time ( h PI) Figure 8. Profiles of viable cell density and glucose concentration of the cultures infected at the different ages. The symbols indicate that the cells growing in the early (O), mid to late (O), and the end of (v)the exponential phases were infected with the virus at an MOI of 5.0PFU/cell. -
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Time ( h PI) Figure 9. Effect of cell age on recombinant CAT production in spinner flasks. The cells growing in the early (day 4),mid to late (day 6), and the end of the exponential phase (day 8) were infected with the virus at an MOI of 5.0 PFU/cell. density of 3 x lo6 celldmL did result in a 10-fold decrease in the level of the VP6 protein if the culture medium was not replenished. We can, therefore, conclude that the infection of cells during the exponential growth phase produces more protein than the infection of cells in the stationary phase. Moreover, our results indicate that the infection of cells in the mid to late exponential growth phase is recommended for optimum protein production using the BmNPVBm5 expression system, which also yields higher volumetric protein productivity from high-density infected cultures.
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642 Table 1. Effect of Medium Replenishment Strategy on Specific CAT Productivity specific CAT productivity [mg/(109celladay)] infection of mid to infection of early
medium replenishment before infection after virus adsorption
exponentially growing cells 14.7 f 0.4 12.9 2~ 0.3
late exponentially growing cells 20.6 f 0.6 19.7 5 0.5
Effect of Medium Replenishment Strategy on Recombinant Protein Production. For the largescale production of recombinant proteins, infection of high-density suspension cultures is a superior method, generating higher volumetric protein productivities than the infection of low-density cultures. However, proper strategies for medium replenishment during infection should be developed in order to obtain high specific protein productivities (qcAT) from infected high-density cultures because the nutrient limitation problems in infected high-density cultures are more severe than those occurring in uninfected high-density cultures (Zhang et al., 1993). Spent culture medium in high-density suspension cultures can be replenished either before the addition of viral inocula or after virus adsorption. Thus far, the effect of virus removal from cells after virus adsorption on recombinant protein production remains unknown. Therefore, a study was carried out in 125-mL spinner flasks to identifjr how the removal of virus inocula from the cells affects protein production. Two sets of experiments were performed with infection of the cells in two different growth phases (i.e., the early exponential phase and mid to late exponential phase). Each set of experiments included two different cases. In one case, the spent culture medium was replenished before addition of the virus inoculum, and the viral inoculum was left in the cell culture during virus replication. In the second case, the culture medium was replenished completely with fresh medium after virus adsorption for 2.0 h; consequently, the extra virus particles from the viral inoculum were removed from the cell culture following virus adsorption. In all of these experiments, the multiplicity of infection and the cell density during infection were preset a t 5.0 PFU/cell and 1.0 x lo6 viable cells/ mL, respectively. Under no nutrient limitation conditions, the effect of medium replenishment strategy on specific CAT productivity from three repeat experiments is summarized in Table 1. In each set of experiments, equivalent levels of the specific CAT productivity were achieved with medium replenishment before infection or after virus adsorption. This indicates that the effect of medium replenishment on the specific CAT productivity is insignificant. These results also imply that the effect of viral removal following virus adsorption on the specific CAT productivity is minimal. Hence, spent culture medium can be replenished either before the addition of viral inocula or after virus adsorption. On the basis of our results, two different medium replenishment strategies can be used to overcome nutrient limitation problems during the infection of highdensity cultures in a production facility. It should be noted that a dilution factor of medium nutrient levels by viral inocula must be taken into account in selecting medium replenishment strategies because the volume ratio of the viral inoculum to cell culture in the infection of high-density cultures is about 20% if a n MOI of 5.0 PFU/cell is used (Zhang et al., 1993). One strategy can be the replenishment of spent culture medium with fresh
medium before the addition of viral inocula. In this strategy, a fresh medium fortified with glucose and glutamine, two major limiting nutrients in baculovirusinfected insect cell cultures, is recommended to account for the dilution factor. Hence, media with different nutrient concentrations are used for cell culture and viral infection. Another strategy can be the replenishment of spent culture medium following virus adsorption. In this case, the same medium formulation can be used for cell growth and viral infection. Using the latter strategy for medium replenishment in a newly developed two-stage bioreactor system, the infection of high-density Bm5 cell cultures with B"PVP5C.cat produced consistent yields of 250 mg/L CAT protein (Zhang et al., 1993).
Conclusions Many factors, including cell culture age and infection conditions, can affect the levels of recombinant proteins produced in insect cell cultures. However, the relative importance of each factor can vary widely. For the BmNPVBm5 expression system, cell culturing methods and cell age have major effect on the specific protein productivity. The highest protein production has been achieved with the infection of suspension cultures in the mid to late exponential growth phase. We believe that suspension culture is a superior choice as a highefficiency production method for the large-scale production of recombinant proteins from the infection of anchorage-independent insect cells with recombinant baculoviruses. Different culturing methods can play a key role in the effect of MOI on protein production. The effect of MOI on the specific protein productivity in suspension cultures can be different from that in stationary cultures because of different physical conditions of insect cells and different mobilities of newly produced viruses for secondary infection, especially a t lower MOI conditions. For the maximization of protein production from the synchronous infection of high-density suspension cultures, a n MOI of between 1 and 5 is recommended.
Acknowledgment The authors acknowledge financial support provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) and Insect Biotech Canada (one of the Federal Network of Centres of Excellence).
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Accepted May 18, 1994.@ @
Abstract published in Advance ACS Abstracts, September 1,
1994.
