Large-scale insect cell culture: methods, applications and products

Mattheus F.A. Goosen

Queen's University, Ontar io, Canada

The primary development in large-scale insect cell culture over the past year has been the continuing accumulation of documented evidence (fundamental and applied) that conventional aerated stirred-tank and air-lift bioreactors may be employed for insect cell cultivation and recombinant protein production, provided that air sparging, agitation, and the addition to the medium of Pluronic F-68 and methyl cellulose polymers are carefully

controlled.

Current Opinion in Biotechnology 1991, 2:36.5-369

Introduction

The past 10 years has seen a sober reassessment of the capability of genetically modified bacteria to produce commercially valuable proteins. The most crucial factor behind this re-evaluation was probably the inability of the host cell to perform many of the post-translational mod- ifications required for the correct biological functioning of many animal proteins. It now appears that the majority of economically attractive animal proteins will have to be produced on an industrial scale using insect and mam- malian cells.

The earlier interest in insect cell cultivation stemmed from the prospects of mass-producing viral insecticides as an environmentally sound alternative to chemical pest control. The current excitement about insect cell culti- vation, on the other hand, results from the recent de- velopment of baculovims vectors for high-level expres- sion of many foreign genes whose products are correctly processed post-translationally in the insect host cell. The most visible application of this system is the development of the first candidate vaccine against the human immune deficiency virus, now undergoing clinical trials.

Efficient cultivation methods are a key factor in the com- mercial exploitation of insect cell systems. The most promising approach involves cultivation of insect cells in suspension using bioreactor technology that is cur- rently applied successfully to the manufacture of micro- bial and mammalian cell products. Besides suspension culture, insect cells can also be immobilized in a polymer matrix, adsorbed onto surfaces, or be entrapped behind a polymeric membrane. Although this is carried out to en- hance cell densities and to aid downstream processing, this technology entails overcoming a slightly different set

of problems (e.g. biocompatibility) and is therefore be- yond the scope of this review.

Because of the high oxygen demand of production sys- tems employing insect cells in suspension, mass transfer- assisting operations such as agitation and air sparging need to be employed. This results in hydrodynamic shear stress, causing cell damage and death, which has been considered as the key bottleneck in insect cell culture scale-up. The problem is aggravated by our limited un- derstanding and the dearth of published literature on the behaviour of insect cells growing in reactors. This review will focus on developments in the past year that have in- creased our understanding of the growth of insect cells in aerated and agitated bioreactors.

Development of large-scale insect cell cultivation methods

Reports have been published over the past year on the sensitivity of insect cell suspension culture to scale-up (for reviews see [1.o,2o-5.]). For example, Caron et al. [6 °°] have assessed several types of stirred bioreactors for the high level expression by a recombinant bac- ulovims of the VP6 bovine rotavims capsid protein, us- ing Sf-9 insect cells in suspension culture. Scaling up to a surface-aerated 4 litre marine impeller stirred-tank biore- actor, cell densities of 5 x 106 viable cells ml - 1 were rou- tinely achieved at 100 rpm, in medium (TNMFH) supple- mented with 10% fetal bovine serum and 0.1% Pluronic F-68. However, growth was not as successful in the 4 litre bioreactor at 100rpm when using serum-free medium (IPL/41). The sensitivity of the insect cell cultivation pro- cess is shown by the fact that when the concentration of

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366 Biochemical engineering

Pluronic F-68 is increased to 0.3% and the stir speed de- creased to 75 rpm, cell densities were obtained similar to those of the serum-supplemented culture.

Some effort was also directed at duplicating, in sparged bioreactors, the growth curves obtained using surface- aerated bioreactors. In one experiment in a 131itre stirred bioreactor with serum-free medium containing 0.3% Pluronic F-68, culture was initiated with surface-aer- ation and then switched to minimal pure oxygen sparg- ing after the cells reached a density of 106m1-1 with- out detrimental effects on cell viability. This is in agree- ment with recent reports [7,8,9°], as well as work per- formed in our own laboratory with a 5 litre air-lift reac- tor and serum-free medium (G King, unpublished data) showing that sparging can be used effectively in insect cell culture in both stirred and airlift bioreactors. In con- trast, there have also been reports of serious problems in the scale-up of insect cell suspension cultures (Table 1) [1.o,2°--4°10,11o]. Sparging and agitation appeared to be the key culprits in the loss of cell viability during culture. Let us therefore take a closer look at the mechanism of animal cell damage in bioreactors.

Mechanism of cell death in sparged and agitated bioreaclors

McQueen et at [12] have observed that a correlation seems to exist between cell death of freely suspended cells exposed to turbulent flow and the size of the small- est turbulent eddies (4 lam in length). KolmogoroCs the- t r y states that the smallest turbulent eddies are respon- sible for dissipation of turbulence energy. An increase in fluid viscosity increases the size of the smallest eddies. The improvement in cell growth and viability in suspen- sion culture rendered more viscous by the addition of methyl cellulose seems to be consistent with the eddy- length mechanism [1.°,llo,lB°.,14].

Cell damage caused by excessive mechanical agitation may be associated with bubble entrainment in the fluid as a result of vortexing or cavitation [15.°,16oo]. With in- sect cells in a 3 litre stirred bioreactor, Murhammer and Goochee [16 °,] found that moderate agitation at 300 rpm with a fiat-blade impeller or at 500 rpm with a marine impeller, had no adverse effect on insect cell growth as there was no bubble entrainment via cavitation or vor- texing. When bubble entrainment occurred at 850 rpm, cells died rapidly in the absence of Pluronic F-68, though they grew well in the presence of 0.2% Pluronic F-68. We can speculate that the cell damage, reported by other groups as being caused by mechanical agitation, may ac- tually have resulted from 'air-sparging' effects caused by the entrainment of air bubbles.

Murhammer and Goochee also looked at different air- lift reactors. They found that a high-pressure drop across the gas sparger had a detrimental effect on cell growth, even in the presence of Pluronic F-68. They postulated

that, as the bfibble detached from the orifice, cell dam- age was caused by turbulence and oscil/ations arising as fluid rushed into the region previously occupied by the bubble. In an interesting study, Kuna and Papout- sakis [15"'], working with a surface-aerated stirred reac- tor, showed that rapid movement of small bubbles within the medium (entrained bubbles) did not cause signifi- cant cell damage even at stirspeeds as high as 600 rpm. They further demonstrated that in the absence of a gas headspace (achieved, for example, by using silicone tub- ing to aerate the medium), cells could grow even at stir speeds of up to 800 rpm. At higher stir speeds cell dam- age was observed. This was attributed to stresses from the turbulent liquid and related to the Kolmogorov eddy size. In the presence of a gas headspace, on the other hand, cell damage occurred at much lower stir speeds ( < 600 rpm). Their results suggested that this was prob- ably caused by bubble disengagement (bursting) at the air-liquid surface.

It has been proposed that, in sparged suspension cul- ture, Pluronic acts as a cell protective agent by stabiliz- ing the foam layer on the liquid surface and thereby re- ducing film drainage and bubble bursting in the vicin- ity of cells [17]. Bursting bubbles would cause nearby cells to oscillate rapidly and the draining of liquid film between air bubbles would result in shear damage to any cells trapped between the bubbles. Protection may also arise from an interaction of surfactant and methyl cellu- lose polymers with the cell membrane, thus preventing direct cell-air interfacial contact [13°,,16..].

The passage effect

After prolonged passage through multiple infection cy- cles, the ability of a virus to infect insect cells diminishes according to the passage effect [4,,18.]. Mutant virus par- ticles that interfere with the formation of regular vires are produced. These so-called defective interfering par- ticles (de Goo~?er, personal communication), require the presence of wild-type vires as a helper for multiplication. The passage effect limits the useful productivity time of a continuous system to about 1 month [4"]. Van Her et at [18°], studying the continuous production of a bac- ulovims by Sf cells in a cascade of insect cell reactors, concluded that increasing the number of vessels acceler- ated the occurrence of the passage effect. These studies suggest that batch culture might be the preferred method of recombinant protein production.

How can we best develop a continuous, insect cell cul- ture system? Let us not forget that insect cell systems suit- able for large-scale applications are not limited to those that serve as hosts to baculoviruses (i.e. the lepidopteran species) but also include ceils of dipteran insects (e.g. mosquitoes) that transmit important diseases. In contrast to the lepidopteran cell-baculovirus vector system which leads to the lysis and eventual death of the host cells, dipteran cells appear to be prime candidates for stable, long-term heterologous gene product formation [1o.].

Large-scale insect cell culture: methods, applications and products Goosen 367

Table 1. Comparison of insect cell suspension culture methods.

Type of Impeller Medium serum- Pluronic F-68 Problems with bioreactor Aeration agitation supplemented? (%) cell growth References"

Stirred Sparging Yes Yes 0 Yes [1 "°] Surface Yes Yes 0 No

Air-lift Sparging No Yes 0 Yes

Stirred Surface Yes; 100 rpm Yes 0.1 No [6 "°] Surface 100 rpm No 0.1 Yes Surface 75 rpm No 0.3 No

Surface/sparging No No 0.3 No

Air-lift Sparging No No 0.1 No [7]

Stirred Sparging Yes Yes 0.2 No [8] Air-lift Sparging No Yes 0,2 No

Stirred Surface/sparging Yes No - - No [9 ° ]

Stirred Surface Yes Yes 0 Yes [10] Air-lift Sparging No Yes 0 Yes

Air-lift Sparging No Yes 0.1 Yes [11,]

Stirred Surface Yes Yes 0 Yes [15 °° ] Silicone tubing < 600 rpm Yes 0 No

> 600 rpm Yes 0 Yes (No entrainment) 800 rpm Yes 0 No

Stirred Surface Yes; 500 rpm Yes 0 No [16 °°] 850 rpm Yes 0 Yes

0.2 No Air-lift 5parging No Yes 0.2 Yes

Bubble column Sparging No Yes 0.1 No [17]

Stirred Surface Yes Yes 0 No [18 °1

Air-lift Sparging No No - - No (G King, unpublished data)

*Cell lines are: Aedes albopictus [1oo]; baby hamster kidney cells (BHK-21) [17]; Sf for all remaining references.

This makes the latter more amenable to continuous cul- ture production.

Serum-free medium

Typical insect cell culture media, such as Grace's, tend to be quite complex, partly as a result of the addition of fetal bovine or calf serum. The presence of animal serum makes the medium expensive, thus impeding the economic scale-up of insect cell cultivation. Therefore, one of the major research thrusts, in industry as well as academia, has been aimed at developing completely defined (i.e. serum-free) insect cell culture media. Elimi-

nating serum peptides will also enhance the downstream processing of recombinant products.

Over the past year we have seen an increase in the use of serum-free medium for insect cell cultivation [6",9",19",20]. Serum-free cultures, however, may be more sensitive to agitation than serum-supplemented cul- tures. Caron et a l [6. '] recommended increasing the total Pluronic F-68 concentration to 0.3% with IPI/41 serum-free medium. They observed a high cell-growth rate but a decrease in recombinant protein production measured in the absence of serum. This differs from pre: vious results reported by Maiorella et al. [7] and sup- ports studies in my own laboratory (M Goosen, unpub- lished data) that suggest that use of serum-free medium

368 Biochemical engineering

may sometimes result in significan@ lower product con- centrations. The high cell growth and comparatively low levels of recombinant protein production in serum-free medium points to major differences between factors pro- moting cell division and those involved in the late phase of infection with baculovimses when recombinant pro- teins are produced. Serum-free medium formulations may need further modifications to allow for expression of more recombinants.

Characteristics of the products

The insect cell-baculovirus expression system has its lim- itations. The expression of a foreign gene comes at the end of the infection cycle, at a time when the host cell is in the process of degradation. In a recent review, Bishop [21.], noted that it should not be surprising that process- ing of highly expressed recombinant products by the cell may be incomplete when 'very late' baculovirus promot- ers are employed. Incomplete glycosylation, for example, was found for the glycoprotein of rabies [22]. In another study, Zu Putlitz [23"] produced a monoclonal IgG anti- body in baculovirus-infected insect cells. Both the light- and the heavy-chain cDNAs were introduced into the bac- ulovirus in a single step. The apparent molecular'weight of the recombinant heavy chain decreased after treatment with the enzyme endoglycosidase F/gtycopeptidase F, in- dicating that the protein was in fact glycosylated. Of par- Ocular interest was the fact that Coomassie blue staining revealed a slight size difference between the gtycosylated. recombinant heavy chain, and the control isolated from mouse ascites. This suggests a different glycosylation pat- tern (i.e. high-mannose type) compared with the native protein. The antibody molecule's antigen-binding capa- bility was not impaired.

Two successful large-scale studies were reported re- cently. A recombinant vaccine VP6 [6..], produced in stirred-tank bioreactors, was found to be fully effective as a subunit vaccine against bovine rotavirus. In another study, recombinant erythropoietin, produced in serum- free medium in stirred tanks, was analyzed by Western blot and a specific bioassay, and found to be fully glyco- s~4ated and resembled native protein [9"].

Conclusion

Evidence is accumulating that sparging, rather than agita- tion, is the main cause of insect cell death in large-scale suspension culture. This problem can apparently best be overcome by the addition of polymers such as Pluronic F-68 to the medium and careful control of sparging and agitation.

Acknowledgements

The author would like to thank Brian J BeUhouse of the University of Oxford for providing o~ce and laboratory space during his sab-

batical. The insect ceil culture project at Queen's University is being performed in collaboration with Andrew J Daugulis, Peter Faulkner, Glenn King and Jianyong Wu.

References and recommended reading

Papers of special interest, published within the annual period of re, Sew, have been highlighted as: • of interest ,,, of outstanding interest

1. AGATHOS SN, JEONG Y-H, VENKAT K: Growth Kinetics of Free , , and Immobilized Insect Cell Cultures. Ann NY Acad Sci

1990, 589:372-398. A sober review and assessment of the status of insect cell culture tech- nology. The paper also describes experimental studies on the'scale-up of mosquito ceil cultures.

2. Wu J, K~G G, DAUGULIS AJ, FAULKNER P, BONE DH, ~ S E N , MFA: Engineering Aspects o f Insect Cell Suspension Cul-

ture: a Review. Appl Microbiol Bioteclmol 1989, 32:249-255. The development of insect cell suspension culture techniques with an emphasis on process scale-up is reviewed. The paper recommends that more fundamental studies need to be undertaken on the relationship be~aeen bioreactor hydrodynamics and ceil processes.

3. SHULER MI., CtlO T, WICKH&M T, OGONAH O, KOOL M, HA-t, LMER . DA, G ~ . ~ O S RR, WOOD MA: Bioreactor Development f o r

Production of Viral Pesticides or Heterologous Proteins in Insect Cell Cultures. Ann NY Acad Sci 1990, 589:399-421.

A summary of important characteristics of the insect cell-baculovims system and aspects o f relevant bioreactor technology. Results are pre- sented on attempts to construct an appropriate bioreactor s3~tem.

4. TRAMPER J, v&'q DEN END EJ, DE GOOIJER CD, KOMPIER l~ VAN . LIER FLJ, US.~LA, NY M, VLAK JM: Production of Baculovirus in

a Continuous Insect Cell Culture. Ann NYAcad Sci 1990, 589:423--430.

The passage effect resulting in virus degeneration limits the productivity time of a continuous system to about 1 month.

5. GOOSEN MFA: Insect Ceil Cultivation Techniques for the • Production of High-Valued Products. Can J Chem Eng

Biotechnol 1991, 69, in press. This re, Sew paper looks at the mechanism of cell death in sparged and agitated reactors, as weU as the use of serum-free medium.

6. CARON AW, ARCH&~mAULT J, /~L~SSlE B: High-Level Re- o. combinant Protein Production in Bioreactors Using the

Baculovk'us-Insect Cell Expression System. Biotechnol Bio~ eng 1990, 36:1133-1140.

An excellent paper describing the scale-up and fine tuning of Sf-9 sus- pension culture using several t~pes of surface-aerated stirred bioreac- tors.

7. /~L~ORELtA B, L\q-OW D, SHANGER A, HARANO D: Large-scale Insect Cell Culture for Recombinant Protein Production. Biotechnology 1988, 6:1406-1410.

8. MUVat,LXLXtER DW, GOOCHEE CF: Scale-up of Insect Cell Cul- tures: Protective Effects o f Pluronic F-68. Biotecbnology 1988, 6:1411-1418.

9. WEISS SA, GORr'mNS S, FtKE R, DlSORBO D, JxY~m D: Large- . Scale Production of Proteins Using Serum-Free Insect Cell

Culture. In Proceedings of the Ninth Australian Biotecbnology Conference, Queensland, Australia, 24-27 September 1990, pp 220-231.

This paper demonstrates the scaleability of Sf-9 cells in commercially applicable stirred bioreactors in serum-free medium.

Large-scale insect cell culture: m e t h o d s , applications and products G o o s e n 369

10. TRAMPER J, WRJ.t~ts JB, JOUSTRA D: Shear Sensitivity of Insect Cells in Suspension. Enz)Tne Microb Tecbnol 1986, 8:33--36.

11. Wu J, K.kNG G, DAUGULIS AJ, FAULKNER P, BOSE DH, GOOSEN , MFA: Adaptation of Insect Cells to Suspension Culture. J

Ferm Bioeng 1990, 70:90-93. The protec~'e effect of Pluronic F-68 on insect cells in shake flasks w-as confirmed. Problems were encountered in culturing cells in an air-lift bireactor.

12. MCQUEEN A, MEILHOC E, BAILEY JE: Flow Effects on the Via- bility and Lysis of Suspended Mammalian Cells. Biotecbnol Lett 1987, 9:832-836.

13. GOLDBLUM S, BAE YK, H~'K WF, CHALMERS J: Protective Ef- • o feet o f Methylcellulose and Other Polymers on Insect Cells

Subjected to Lamina/ Shear Stress. Biotechnol Prog 1990, 6:383-390.

This paper suggests that the protection mechanism of methyl cellu- lose pobrners for animal cells against mechanical damage is the re- sult of polymer adsorbing to the cell membrane, rather than enhanced medium viscosity.

14. CROUGHAN MS, SAYRRE ES, WANG DIC: Viscous Reduction of Turbulent Damage in Animal Cell Culture. Biotecbnol Bioeng 1988, 33:862-872.

15. KUNA KT, PAPotrrSAKIS ET: Damage Mechanisms o f Sus- ** pended Animal Cells in Agitated Bioreactors .with and

wi thout Bubble Entrainment. Biotechnol Bioeng 1990, 36:476--473.

An excellent paper, in these experiments the authors report that, in the absence of a gas headspace, animal cells can grow in an agitated bioreactor at speeds of up to 800 rpm. Bubble entrainment by" itself does not cause significant cell damage.

16. MURHAMMER DW, GOOCHEE CF: Sparged Animal Cell Biore- • , actors: Mechanism of Cell Damage and Pluronic F-68 Pro-

tection. Biotechnol Prog 1990, 6:391-397. Also a good paper. The earlier results of this group [8] are confirmed, and the observation is made that cell damage is associated vdth bub- ble entrainment (and bubble bursting) in stirred reactors, and bubble detachment in sparged reactors.

17. HANDA A, EMERY _AM, SPIER RE: Effect o f Gas-Liquid Interfaces on the Growth of Suspended Mammalian Cells: Mechanism of Cell Damage by Bubbles. Enz3~ne Microb Tecbnol 1989, 11:230-235.

18. VAN L1ER FLJ, VAN DEN END EJ, DE GOOIJER CD, VLAK JM, • TRAMPER J: Continuous Production of Baculo~Srus in a Cas-

cade of Insect-Cell Reactors. Appl Microbiol Biotedmol 1990, 33:43-47.

This is a relevant paper which demonstrates that, when the number of reactors in series is increased, the passage effect (i.e. reduced suscep- tibility to infection) occurs earlier.

19. ~R'¢ X, FI~DIERE G: A New Serum-Free Medium for Lepi- • dopteran Cell Culture. J Invertebr Patbol 1990, 55:342-349. Gives an apparent new medium, based on Grace's, from which calcium chloride, maltose and 13-alanine were omitted. Serum ,~ts replaced by egg yolk. Cell lines grew better in the new medium than in Grace's.

20. LI~RY X, FI~DmRE G: Effect o f Different Amino Acids and Vitamins on Lepidopteran Cell Cultures. J lnvertebr Pathol 1990, 55:47-51.

21. BISHOP DL: Gene Expression Using Insect Cells and Viruses. Curr Opin Biotecbnol 1990, 1:62-67.

Characteristics of the products from single-gene and multiple-gene ex- pression ~ t e m s are described as well as recent technical developmertts that may aid the preparation of expression vectors.

22. PREHAUD C, TAKErLM~A K, FLAMAND A, BISHOP DILL: Immuno- genie and Protective Properties of Rabies Virus Glyco- protein Expressed by Baculo,drus Vectors. Virology 1989, 173:390-399.

23. ZtJ PIM'LrIZ J, KUBASEK WI., DUCttf:NE M, 3LM~GET M, VON • SPECIIT BU, DOMDEY It: Antibody Production in Baculo'~Srus-

Infected Insect Cells. Biotecbnology 1990, 8:651-654. Both light- and heaw-chain c D N ~ were introduced into the baculovims in a single step. Infected insect ceLls stably secreted antigen-binding and gb'cos~Sated recombinant antibody molecules.

MFA Goosen, Department of Chemical Engineering, Queen's Universiq b Kingston, Ontario K7L 3N6, Canada.