Large-scale insect cell culture Mattheus F.A. Goosen

Queen's University, Kingston, Ontario, ~Zanada

Significant advances have been made over the past year in our understanding of the protective mechanisms, both fluid-mechanical and biological, of media additives on suspended animal cells. The degree of protection offered by different additives, such as pluronic polyol, appears to be cell-type dependent, varying quite dramatically not only between insect and mammalian cells, but

also between different insect cell lines themselves.

Current Opinion in Biotechnology 1992, 3:99-104

Introduction

The insect cell/baculovirus system has been the object of intense investigation since the discovery that bac- ulovirus vectors may be employed for the high, yet regulated, expression of many foreign genes whose products are then correctly processed in the insect cell host. Large-scale, efficient (i.e. simple, inexpen- sive and reliable) insect cell cultivation methods are a key factor in the commercial exploitation of this technology. The expression of heterologous genes in insect cells with baculovirus vectors has been recently and fully reviewed by Luckow [1"]. Two general re- views on animal cell culture scale-up have been pro- vided by Agathos [2] and Hu and Peshwa [3]. Im- mobilized animal cell culture technology is at a rel- atively early stage of development and is therefore beyond the scope of our paper.

Although it is generally recognized that conventional aerated stirred tank and air-lift bioreactors may be employed for insect cell suspension cultures, and re- combinant protein production, the key limitations of these systems still appear to be the need to protect the cells from fluid-mechanical damage associated with bursting bubbles and high agitation rates. An excellent in-depth discussion on media additives for protecting freely suspended animal ceils against agitation and aer- ation damage has been presented by Papoutsakis [4"]. Two mechanisms, one physical and the other biologi- cal, have been put forward to explain more fully the protective effects of various additives, such as pluronic polyols and polyethylene glycol (PEG), on suspended cells. A comparison of recent papers indicates that the degree of protection offered by different additives is cell-type dependent, varying quite significantly not only be tween insect and mammalian ceils, but also a m o n g the different insect cell lines themselves. This makes it difficult to draw general conclusions. The past year, however, has seen dramatic advances in our

understanding of the protective mechanisms of media additives on suspended animal ceils. This review will focus on these advances.

Media additives for protecting suspended animal cells

Initial reports suggest that damage to suspended cells in aerated and agitated bioreactors was primarily due to interactions of ceils with bubbles [4"']. Cell injury occurred apparently as a direct result of shear forces generated by film drainage around bubbles in the sur- face foam and by bubble breakup. The bubbles are produced either by direct sparging or as a result of gas entrainment. In the absence of a gas headspace and bubbles, cell damage only occurred at very high agitation rates as a result of stresses in the turbulent liq- uid. Cell damage in this case was correlated with Kol- mogorov eddy sizes similar to, or smaller than the cell size (i.e. relatively large portions of fluid which move rapidly from one position to another in turbulent flow).

Michaels et al. [5"q have suggested a further two mech- anisms (one physical and the other biological) that explain the protective effects of additives in mam- malian cells. In the physical protection mechanism the resistance of the cell to shear forces remains un- changed, but the frequency of transmitted shear forces that the cells experience decreases, so less cell dam- age is observed. In the case of the biological protec- tion mechanism, the additive changes the cell itself in order to make it more shear resistant. This effect may be fast-acting, because it only needs the addi- tive to be incorporated into the cell membrane (as suggested by Murhammer and Goochee [6]), or it may be slow-acting, requiring changes in the cells metabolic 'machinery'. Since mammalian ceils and insect cells dif-

Abbreviations CAT--chloramphenicol acetyl transferase; DIP~defective interfering particle; FBS fetal bovine serum;

HRI--helical ribbon impeller; MOI--multiplicity of infection; PEG--polyethylene glycol.

© Current Biology Ltd iSSN 0958-1669 99

1 O0 Biochemical engineering

fer in their metabolism, cell membrane and cell size, we would expect the additives to be cell-type dependent.

Several additives have been assessed for their ability to protect animal (i.e. mammal ian and insect) cells dur- ing suspension culture. These include serum, pluronic polyols, polyethylene glycol and various derivatized celluloses, as well as lesser known additives, such as starches, dextrans and polyvinyl-pyrrolidones.

Insect cell lines appear to be more sensitive than mam- malian cell lines to the protective effects of additives. For example, Hink [7"] deve loped a new serum-free medium (SFM-LP) for the culture of insect cells. He found that a low-protein aqueous lipid supplement (Ex-Cyte VLE), in combination with pluronic polyol, was an effective replacement for fetal bovine serum for Sf-9 cells. In fact, Hink's serum-free medium sup- ported higher cell viability, greater max imum cell den- sities and more recombinant protein production than serum-supplemented medium. The effectiveness of the same serum-free medium, however, was found to vary from good to unacceptable for three other insect cell lines (IPLB-SF-21-AE, TN-368 and IZD-Mb0503). After 10 passages the TN-368 line was in good morphologi- cal condition and it was replicating at a reasonable rate, whereas the other two cell lines grew slowly and were in poor condition even after 15 passages. To determine whether lipids in addition to pluronic polyol could replace serum on other media, Hink added them to IPL-41. Neither Sf-9 cells, nor IPLB-SF-21-AE cells would grow. This apparent sensitivity of insect cell lines to media additives was confirmed in our o w n laboratory: working with (6.5%) serum-supplemented Grace's medium in shake flasks, we demonstrated that the addition of 0.2% pluronic F-68 had a very beneficial effect on growth of Sf cells (designated IPLB-Sf-21) but a negligible effect on TN-368 cells [8].

Goldblum et a l . [9] used a cone and plate viscome- ter to assess the effect of protective additives on the shear resistance of Sf-9 and TN-368 insect cells. They also found a cell-type dependence. Cell damage was quantified by looking at the rates of lactate dehydro- genase release per unit of shear stress. The ratio of this rate with and without an additive gave an indi- cation of the degree of protection. Sf-9 cells grown in the presence of 0.1% pluronic F68 were found to be 15 times more resistant to shear. The same concen- tration of pluronic F68 made the TN-368 cells only six times more resistant. Increasing pluronic F68 concen- tration enhanced the shear resistance of both insect cell types.

In another viscometric study, Michaels et al. [5"] work- ing with hybridoma (CRL 8018) cells, obtained quite different results. In both short and long exposure vis- cometric experiments, pluronic F68 and PEG had no beneficial effects on the shear sensitivity of the cells, whereas Goldblum et al. [9] found that F68 did have a protective effect on insect cells. Conflicting results have also appeared on the protective effects of pluronic polyols on animal cell cultures in airlift and agitated

bioreactors. One report [6], using insect cell cultures, showed that pluronic F68 has no protective effect, whereas another [5"'], performed with hybridoma cells, showed that F68 does protect freely suspended animal cells. These apparently conflicting results suggest that the shear protection mechanisms (i.e. physical and bi- ological) of insect and mammalian cells may be differ- ent (Table 1). Furthermore, Michaels et al. [5"'] con- clude that in the case of pluronic F68, their findings (with hybridoma cells) do not support the p roposed (biological) protection mechanisms whereby pluronic polyol makes animal cells more resistant to shear by incorporation into the cell membrane (as suggested by Murhammer and Goochee [6]) from their insect cell studies.

The results from the study of Michaels et al., how- ever, are in agreement with the theory put forward by Handa and co-workers [10] in which the protective ef- fect of pluronic F68, PEG, and serum is probably due to changes in the properties of the gas-liquid interfaces. Both groups in this case worked with mammalian cells.

Let me finish this section by looking at serum as an additive. The protective effect of serum on suspended animal ceils has been suggested to be largely physical, al though it could also be explained by a fast-acting bio- logical mechanism (i.e. by incorporation of serum into the cell membrane) [5"', 11]. It was recently shown, for example, that progressively higher concentrations (up to 10%) of fetal bovine serum (FBS) not only reduce mammal ian (i.e. hybridoma) cell death but also allow cell growth at higher agitation rates. The protective ef- fect of se rum on the cells was measurable even after short exposure, when serum was added just before, or just after, exposure of the ceils to lethal agitation [11]. This suggests both a physical and/or a fast-acting bio- logical mechanism. Perhaps the same types of mecha- nisms may also be true for suspended insect cells.

Serum can also protect insect ceils in cultures main- tained at high temperature (e.g. 33°C). King et al. [12"], for example, cultured Sf cells in serum free me- dia (Sf-900) and two kinds of serum-supplemented (10% fetal calf-serum) media (IPL-41 and Grace's) using a temperature-sensitive recombinant chloramPhenicol acetyl transferase (CAT) baculovirus. In shake-flask cul- ture, the ceils were infected with a low multiplicity of infection (MOI) at day zero and then grown for sev- eral days at 33°C (to allow for cell growth but not virus replication). The temperature was then lowered to 27°C to initiate virus growth and recombinant pro- tein production. King et al. found that with the serum- supplemented media, good cell and virus growth and CAT expression were obtained. In contrast, the serum- free med ium gave poor results throughout. By running the entire serum-free culture at 27°C, however, excel- lent cell growth, virus growth and recombinant protein product ion were observed. This suggests that infected cells, in the absence of serum, are 'damaged ' at the higher temperature (33°C) and thus unable to grow and produce virus and recombinant protein in a reg- ular fashion.

Large-scale insect cell culture Goosen 101

Table 1. Cell-type dependence of media additives for protecting suspended animal cells.

Suspended animal Apparent level Protection Media additive* Medium cell system Cell type of protectiont mechanism~ Reference

Lipids/pluronic polyol (0.1%) Serum-free modified Grace's Spin flasks Insect Sf-9 High - - [7 °] Serum-free modified Grace's Spin flasks TN-368 Low - - Serum-free modified Grace's Spin flasks IPLB-SF-21AE None - - Serum-free modified Grace's Spin flasks IZD-Mb0503 None - -

Lipids/pluronic (0.1%) Serum-free IPL-41 Spin flasks Sf-9 None - - Serum-free IPL-41 Spin flasks IPLB4F-21AE None

Pluronic F68 (0.2%) Serum supplemented Grace's Shake flasks Insect IPLB-SF-21 High - - [8] TN-368 None - -

Pluronic F68 0.1% Serum supplemented TNM-FH Cone and plate viscometer Insect Sg9 Low Biological [9] 0.2%-0.5% Serum supplemented TNM-FH Cone and plate viscometer insect Sf-9 High Biological

Pluronic 0.2% Serum supplemented TNM-FH Cone and plate viscometer TN-368 Low

Methyl-cellulose (E4M) (0.5%) Serum supplemented TNM-FH Cone and plate viscometer Sf-9 High Biological Serum supplemented TNM-FH Cone and plate viscometer TN-368 High Biological

Dextran (4.5%) Serum supplemented TNM-FH Cone and plate viscometer Sf-9 Low Biological Serum supplemented TNM-FH Cone and plate viscometer TN-368 Low Biological

Pluronic F68 0.1% Serum-free Couette viscometer Hybridoma (CRL8018) None - - [5°',11]

PEG 0.1% Serum-free Couette viscometer Hybridoma (CRL8018) None - -

FBS (5%-10%) Serum-free Couette viscometer Hybridoma (CRL8018) None-Low Biological

Pluronic F68 0.1% Serum-free Agitated bioreactor with Hybridoma (CRL8018) High Physical surface aeration

PEG 0.1% Serum-free Agitated bioreactor with Hybridoma (CRL8018) High Physical surface aeration

FBS Serum-free Agitated bioreactor with Hybridoma (CRLB018) High Biological and surface aeration physical

Pluronic F68 0.2% TNM-FH with 5% FBS Sparged agitated bioreactor Insect Sf-9 High Biological [6] Airlift bioreactor Insect Sf-9 None - -

FCS 2% RPMI Bubble column Mammalian BMK-21 Low Physical [10] 5% RPMI Bubble column Mammalian BMK-21 Low Physical

10% RPMI Bubble column Mammalian BMK-21 High Physical FCS 5% + pluronic 0.1% RPMI Bubble column Mammalian BMK-21 High Physical

FCS 10% IPL-41 and Grace's Shake flask 33°C Insect IPLB Sf-21 High Thermal buffer [12 °] IPL-41 and Grace's Shake flask 27°C Insect IPLB Sf-21 High Biological

Pluronic F86 0.1% Serum-free Sf-900 Shake flask 33 ~C Insect IPLB Sf-21 None - - Serum-free Sf-900 Shake flask 27°C Insect IPLB Sf-21 High - -

*All percentages are quoted as weight per volume, tApparent level of protection refers to improvement in cell growth (none to high) in bioreactor studies or to retention of cell viability (none to high) in viscometer experiments. ~Biological refers to an increase in the cells resistance to shear as a result of changes in the cell itself. Physical refers to fluid-mechanical effects (i.e. decrease in shear forces transmitted by the medium). PEG, polyethylene glycol; FBS, fetal bovine serum; FCS, fetal calf serum.

Recent advances in low shear and continuous bioreactors

There are alternatives to media additives for protect- ing suspended animal cells. Mechanical shear within a stirred bioreactor, for example, may be reduced through the use of a helical ribbon impeller (HRI) which provides low-speed, low-shear mixing. Kamen et al. [13"] recently tested an 11-L HRI bioreactor on a culture of'Sf-9 cells using serum-supplemented (TN-

MFH) and serum-free (IPL/41) media. The combina- tion of HRI and surface baffling ensured homogenous mixing and high oxygen transfer through surface aer- ation (and surface-induced bubble generation) with- out conventional air sparging. Heterologous protein production of 35 btg per 106 ceils was reported. This is comparable with yields obtained in spinner flasks. When the HRI bioreactor was used for insect cell cul- tures, mixing and oxygenation were not found to be limiting factors. The deve lopment of an air-lift/fibre-

102 Biochemical engineering

bed bioreactor for anchorage-dependent animal ceils has been reported by Murakami et al. [14]. Such a system may be of use in the future for large-scale in- sect cell cultivation. Agathos [2] and Hu and Peshwa [3] have published reviews on the state of the art in insect cell culture and animal cell bioreactors.

There have been on-going efforts to develop contin- uous bioreactors for large-scale production of bac- uloviruses and proteins. Continuous production has been achieved by Kool et al. [15"], in a system consist- ing of one bioreactor producing insect cells in series with a second bioreactor for virus infection and pro- tein production. Due to the passage effect, however, the productivity decreases with time. After prolonged passage through muliple infection cycles the ability of a virus to infect insect cells diminishes. Kool et al. have worked on the elucidation of the mechanisms respon- sible for the decrease in baculovirus productivity dur- ing continuous operation. Working with Sf-AE-21 insect cells in TNM-FH medium supplemented with 10% fe- tal calf serum and 0.1% w / v methyl cellulose, they showed that defective interfering particles (DIP) were generated upon continuous production of baculovirus (AcNPV) in stirred (11) bioreactors.

These DIPs lacked 43% of the genetic information of the standard virus, including the polyhedrin and DNA polymerase genes. The existence of DIPs was con- firmed by electron microscopy. Wild-type virus was needed for DIPs to replicate, which explains the re- duction in productivity of a continuous culture system. Kool et al. suggest that DIPs function by competing for enzymes essential for DNA replication. In order to develop optimal growth protocols for large-scale production, the kinetics and growth characteristics of the different insect cell lines need to be determined. In the case of the si lkworm ( B o m b y x mori, BM-5) cell line, for instance, it has recently been demon- strated that glutamine can be a limiting nutrient and that lactate has an inhibiting effect on cell growth in batch culture [16].

Products: a problem of scale and the effects of multiplicity of infection

Insect cells perform many of the post-translational modifications of higher animal cells, including phos- phorylation and glycosylation. The recombinant pro- teins can undergo oligomeric assembly in the cell, where appropriate, which makes the insect-cell/ baculovirus expression system ideal for the production of biologically active proteins. Over the past decade, more than two hundred proteins have been expressed (mainly at a laboratory scale) by recombinant bac- uloviruses. In his excellent review, Luckow [1"] noted that careful analysis of glycoproteins expressed in in- sect cells by baculovirus vectors has revealed differ- ences in size and structure compared with the same glycoproteins present in mammalian ceils. However,

although mammalian glycoproteins produced in Sf-9 insect ceils, for example, were found to be slightly smaller than their counterparts in mammalian cells, this had little, if any, effect on the in vitro biologi- cal activities of the protein. Two recent examples of recombinant products produced in the insect system are the secretion of biologically active leech hirudin, a potent anticoagulant [17], and the production and characterization of Rift Valley fever virus expressed in baculovirus [18]. Mthough these papers are well written f rom a genetic engineering standpoint, they suggest a weak understanding of cell culture and scale- up technology. This demonstrates the gap that appar- ently still needs to be filled be tween laboratory-scale systems and large-scale protein product ion systems.

One of the factors influencing recombinant protein yields in an insect cell-baculovirus expression system is the MOI (which corresponds to the number of infec- tious units per cell). Licari and Bailey [19"] employed the Sf-9 cell-AcNPV baculovirus expression system for the synthesis of a model heterologous protein, 13- galactosidase. The relationship be tween final product titer and the MOI was found to be dependent on the growth phase of the ceils prior to infection. In the early exponential cell growth phase, for example, the final product titer was independent of the MOI, whereas in the late-exponential phase there was a logarithmic re- lationship be tween the final ~-galactosidase titer and the MOI used. The highest MOI resulted in the great- est protein synthesis. The authors suggest that by us- ing a lower MOI and relying on secondary infection of the culture (i.e. employ early-exponential phase in- fection), the amount of virus stock needed would be substantially reduced. A lower MOI may also result in reduced production of DIPs in infected cells.

The complex interaction between MOI, cells, medium additives and culture temperature was demonstrated by King et al. [12.]. Using a temperature-sensitive baculovirus, they assessed virus production and CAT expression by insect cells using serum-free medium (Sf-900) and two serum-supplemented media (IPL-41 and Grace's). In temperature shift down experiments (growth of infected cells at 33°C followed by a tempera- ture drop to 27°C to switch on virus growth) the lowest virus and CAT titers were obtained with the serum-free medium. In the temperature shift-down experiments, the cells were always infected at day zero into the culture. Surprisingly, with all three media, the low- est MOI always gave the highest virus and CAT titers. These results were always contrary to those normally obtained at constant-temperature (27°C) infection and culture. As a control, when the cultures were all run at 27°C, the effect of MOI concentration was reversed, with the highest MOI giving the highest titers. Under these conditions the serum-free medium performed as well as the serum-supplemented media. These results suggest that serum may play a protective role by acting as a ' thermal buffer' during high temperature culture. In addition, the results of the temperature shift-down experiments indicate that infected cells may be more susceptible to ' thermal damage ' than uninfected ceils.

Large-scale insect cell culture Goosen 103

Accordingly, cultures with a high MO1 would exhibit little cell growth during the culture stage and little virus or recombinant protein would be produced. Less susceptible, low-MOI cultures, on the other hand, did grow at 33"C. After temperature shift-down to 27°C, and presumably as a result of secondary infection, the cul- ture recovered and was capable of supporting regular virus replication and recombinant protein expression.

Conclusion

Our understanding of the protective mechanisms of media additives, Such as pluronic polyols, polyethy- lene glycol, and various derivatized celluloses, on sus- pended animal ceils, has significantly advanced over the past year. The protection mechanism for insect cells in sparged or agitated bioreactor and viscomet- ric studies is mainly biological (that is, due to an increase in the cells resistance to shear). In mam- malian cells, however, the mechanism appears to be a combination of biological forces and physical forces (due in part to a decrease in shear forces transmitted by the medium). The degree of protection offered by different additives is cell-type dependent. In vis- cometric studies, for example, while methyl cellulose (0.5%) provides a high level of protection to both TN-368 and Sf-9 ceils, pluronic (0.2 %) only provides a high level of protection to the latter cell line.

Acknowledgements

The insect cell culture project at Queen ' s University is run in col- laboration with Andrew J. Daugulis, Peter Faulkner, J ianyong Wu and Glenn King. The financial support of the Natural Sciences and Engineering Research Council of Canada, through a Strategic Grant, is gratefully acknowledged.

References and recommended reading

Papers of particular interest, publ ished within the annual period of review, have been highlighted as:

of special interest • . of outstanding interest

1. LUCKOW v a : C l o n i n g and Expression of Heterologous • . G e n e s i n I n s e c t Cel ls w i t h Bacu lov i ru s Vectors. In Re-

combinanl DNA Technology and Applications. Edited by Prokop A, Bajpai RK, Ho SC. McGraw H~I Inc., 1991, pp 97-152.

A comprehensive review containing 327 references and a list of over 230 proteins expressed by the baculovirus-insect cell system. Covers a wide variety of topics, including characterization of recombinant baculovirus vectors, insect cell lines, media, scale-up and post-trans- lational modifications of expressed proteins.

2. AGATHOS SN: P r o d u c t i o n Scale In sec t Cei l Cul tu re . Biotech Adv 1991, 9:51~58.

3. Hu WS, PESHWA MV: A n i m a l Cell B io reac to r s - - Recent Advances and Challenges to Scale-up. Can J of Chem Eng 199"2, 69:409-420.

4. PAPOUTSAKIS ET: Media Additives for Protecting Freely • . S u s p e n d e d A n i m a l Cells Against Agitation and Aera-

tion D a m a g e . Trends Biotechnol 1991, 9:316-324. An excellent review paper that covers both hybridoma and insect cells. Protective media additives that are discussed include pluronic polyols, various derivatized celluloses and starches, protein mixtures, polyvinyi-pyrrolidones, dextrans, polyethylene glycol and polyvinyl alcohol. The protective mechanisms of these additives are not fully unders tood but fluid-mechanical and biological mechanisms of pro- tection are suggested.

5. MICI-tAELS JD, PETERSON JF, MCINTIRE LV, PAPOUTSAKIS ET: • . Protection Mechanisms of Freely Suspended A~imal

Cells (CRL 8018) from Fluid-mechanical Injury. Visco- metric and Bioreactor Studies Using Serum, Pluronic F68 and Polyethylene Glycol. Biotechnol Bioeng 1991, 38:169-180.

A high quality experimental s tudy of the effect of additives on hy- br idoma cell culture. Protection mechanisms (physical and biologi- cal) are discussed with comparisons being made be tween insect and mammal ian cells.

6 M ~ E R DW, GOOCHEE CF: Scale-up of Insect Cell Cultures: Protective Effects of Pluronic F68. Biotech- nology 1988, 6:1411-1418.

7. HINK WF: A Sertmx-free Medium for the Culture of In- sec t Cells and Production of Recombinant Proteins. In Vitro Cell Dev Biol 1991, 27:397-401.

A new serum-free medium, containing a low protein aqueous lipid supplement in combination with pluronic polyol, appears to be highly cell-line dependent . The med ium is good for growing Sf-9 cells, adequate for the TN-368 cell line but is unacceptable for IPLB- Sf-21AE and IZD-Mb 0503 cell lines.

8. Wu J, KING F, DAUGULIS AJ, FAULKNER P, BONE DH, GOOSEN MFA: Adaptation of I n s e c t Cel ls to Suspension Cul ture . J Fermentation Bioeng 1990, 70:90-93.

9. GOLDBLUM S, BAE YK, HINK Xyc'F, CHALMERS J: P ro t ec t i ve Ef- fec t of Methylcellulose and Other Polymers o n I n s e c t Cel ls Subjected to Laminal Shear Stress. Biotechnol Prog 1990, 6:383-390.

10. HANDA A, EMERY AM, SPIER RE: Effect of Gas- l iqu id In ter - f aces on the Growth of Suspended M a m m a l i a n Cells: Mechanism o f Cell Damage by Bubbles. Enzyme Microb Technol 1989, 11:230-235.

11. KUNAS KT, PAPOUTSAKIS ET: Damage Mechanisms of Sus- p e n d e d A n i m a l Cel ls i n Agitated Bioreactors With and Without B u b b l e E n t r a i n m e n t . Biotechnol Bioeng 1990, 36:476-473.

12. KING G, KUZlO J, DAUGULIS A, FAULKNER P, ALLEN B, Wu J, GOOSEN M: Assessment of Virus Production and Chlor - a x n p h e n i c o l Aeetyl Transferase Expression b y In sec t Cells i n Seruna-free and Serum-supplemented Media using a Temperature-sensitive Bacu lov i rus . Biotechnol Bioeng 1991, 38:1091-1099.

Insect ceils infected with a temperature-sensitive baculovirus have difficulty in growing in serum-free med ium at 33 ° compared with 27°C. Control exper iments with se rum-supplemented media showed that serum may play a protective role by acting as a ' thermal buffer'. In temperature shift-down experiments, where infected ceils are grown at 33"C followed by a temperature drop to 27"C to switch on virus growth, higher virus titers and recombinant protein expression were obtained at lower MOI, which is the direct opposi te to results observed at constant temperature (27°C), infection and culture.

13. KAMEN AA, TOM RL, CARON AW, CHAVARIE C, MASSIE B, ARCHAMBAULT J: Cu l t u r e o f I n s e c t Cel ls i n a Heli- cal R i b b o n h n p e l l e r Bioreactor. Biotechnol Bioeng 1991, 38:619-628.

An 11-L HRI bioreactor rested on a culture of Sf-9 cells. Heterolo- gous protein production (Bac-BRV6L) was comparable with yields obtained in spinner flasks.

14. MURAKAMI S, CHIOU TW, W~NO DIC: A F ibe r -bed Biore- ac to r for Anchorage-Dependent A~im:~l Cel l Cultures:

104 Biochemical engineering

Part II. Scale Up Poten t ia l . Biotechnol Bioeng 1991, 37:762-769.

15. KOOL M, VONCKEN jx~, VAN LIER FLJ, TRAMPER J, VLAK JM: Detection and A n a l y s i s o f A u t o g r a p h a cal i fornica Nu- c l ea r P o l y h e d r o s i s V i ru s M u t a n t s w i t h Defec t ive Inter- f e r i n g P rope r t i e s . Virology 1991, 183:739-746.

This paper discusses the generat ion of DIPs u p o n continuous pro- duction of AcNPV baculoviruses in bioreactors. The DIPs lacked 43% of the genetic infornlation of the standard vires, including the poly- hedrin and DNA polymerase genes . These DIPs could not be plaque- purified and ne eded the s tandard virus to replicate, which explained the reduced productivity of an AcNPV expression vector/insect ceil system in cont inuous bioreactor operation.

16. STAVROULAKIS DA, KALOGERAKIS N, BEHIE LA: Kine t i c Data for the BM-5 I n s e c t Cel l Line i n R e p e a t e d - b a t c h Sus- p e n s i o n Cu l tu re s . Biotechnol Bioeng 1991, 38:116-126.

17. BENATTI L, SCACHERI E, BISHOP DHL, SARMIENTOS P: Se- c r e t i o n of Biolog ica l ly Active L e e c h H i r u d i n f r o m B a c u l o v i r u s - i n f e c t e d I n s e c t Cells. Gene 1991, 101:255- 260.

18. TAKEHARA K, MORIKAWA S, BISHOP DML: C h a r a c t e r i z a t i o n of Baculovirus-expressed Rift Val ley Fev e r Vi rus Gly- coproteins Synthesized i n I n s e c t Cells . Virus Res 1990, 17:173-190.

19. LICARI P, BAILEY JE: Fac to r s I n f l u e n c i n g R e c o m b i n a n t Protein Yields in an Insect Cell-baculovirus Expres- s ion System: Multiplicity of Infection and In t r ace l lu l a r P r o t e i n D e g r a d a t i o n . Biotechnol Bioeng 1991, 37:238-246.

The relationship be tween final product titer and the MOI was found to be dependen t on the growth phase of the cells prior to infection. In the early exponential phase, the final product titer was indepen- dent of MOI, whereas in the late-exponential phase the highest MOI resulted in the greatest protein synthesis. The authors suggest that for large-scale applications, a low M01 should be employed early in the exponential phase , thus allowing for secondary infection of the cul- ture. The amoun t of virus stock needed would thus be substantially reduced.

MFA Goosen, Department of Chemical Engineering, Queen ' s Univer- sity, Depuis Hall, Kingston, K7L 3NE, Ontario, Canada.