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Optimization of microcarrier cell culture process for the inactivated

Vaccine22(2004)

3858–3864

enterovirus type71vaccine development

Suh-Chin Wu a,b,?,Chia-Chyi Liu a,Wei-Cheng Lian c

a Department of Life Science,Institute of Biotechnology,National Tsing Hua University,Hsinchu,Taiwan

b Vaccine Research andDevelopment Center,National Health Research Institutes,Taipei,Taiwan

c Center for Disease Control,Taipei,Taiwan

Received24May2002;accepted21May2004

Available online22July2004

Abstract

Enterovirus71(EV71)is an enterovirus that could lead to severe neurological disorders and fatalities.The inactivated vaccine is an appropriate EV71vaccine format for meeting current https://www.wendangku.net/doc/ae3517352.html,rge-scale preparation of the inactivated EV71vaccine depends on a scalable cell culture system for industrial mass production.In this paper,Vero cells were found to produce higher titers of EV71than did MRC-5 and WI-38cells.High-density microcarrier Vero cell cultures were established using5g/L Cytodex1microcarriers and found to promote the release of EV71s from infected Vero cells.For the large-scale production of the inactivated vaccine antigen,the extracellular virus titers produced in the2L bioreactor were found to be10times lower than the spinner?ask culture but improved by30-folds using glucose/glutamine feedings during infection.A serum-free Vero cell microcarrier culture was also established in the bioreactor,yielding a high-titer of5.8×107 TCID50/mL for EV71production.The immunogenicity of the inactivated virions produced in serum-free culture elicited a slightly higher level of neutralizing antibody response in immunized mice.These results constitute valuable information on the development of a large-scale microcarrier cell culture process for producing inactivated EV71vaccine.

Keywords:Enterovirus71;Inactivated vaccine;Microcarrier cell culture

1.Introduction

Enterovirus71(EV71)is a non-enveloped,single-stranded RNA virus of the family Picornaviridae,genus Enterovirus.The EV71infection in humans manifests most commonly as a childhood exanthema known as hand-foot-and-mouth disease,but has a tendency to cause neurological disease during acute infection[1].EV71was?rst isolated in1969in the United States[2]and subsequently reported in Australia[3],Sweden[4],Japan[5],Bulgaria[6],and Hungary[7].Recently,EV71epidemics have occurred in Sarawak[8],Japan[9],peninsular Malaysia[10],Singapore [11],Taiwan[12],and Australia[13].EV71epidemics in Taiwan caused78deaths in1998,25deaths in2000,and26?Corresponding author.Fax:+88635715934.

E-mail address:scwu@https://www.wendangku.net/doc/ae3517352.html,.tw(S.-C.Wu).deaths in2002[12,14–15].Developing an effective vaccine against EV71infection is the best way to solve this public health problem.

The only vaccine available against one of a wide range of enteroviruses is that against poliovirus infection.The inactivated poliovirus vaccine(IPV)developed by Jonas Salk was highly effective in reducing the incidence rates of poliomyelitis in the1940s and1950s[16].Based on the same concept,an inactivated EV71vaccine was developed in response to the Bulgarian epidemic in1975[6],but no ef?-cacy data have been obtained since they did not occur again. Laboratory mice were recently demonstrated to be a suitable animal model of EV71infection[17],although Cynomolgus monkeys are also susceptible to infection[18].In laboratory mice,inactivated EV71vaccine has been demonstrated to elicit a stronger immune response than the recombinant VP1 protein or DNA vaccines[19],indicating the inactivated

S.-C.Wu et al./Vaccine22(2004)3858–38643859

vaccine is an appropriate EV71vaccine format for meeting current needs.Additionally,a Vero cell-adapted EV71strain was developed as a prototype vaccine with high viral yield and broad immunogenicity[20].However,the large-scale preparation of the inactivated EV71vaccine depends on a scalable cell culture system for industrial mass production.

Only a few current cell substrate systems are useable in manufacturing human vaccines.Acceptable cell substrates include diploid cell strains(MRC-5,WI-38,and FRhL-2)and continuous cell lines,such as Vero(African green monkey kidney)and MDCK(Modin-Darby canine kidney)cells[21]. However,the acceptability of these cells must be anchorage-dependent,so these cells must adhere to the surface of a matrix to facilitate cell growth.Microcarrier technology[22] was initially developed for culturing anchorage-dependent cells in suspension;it can be easily applied in stirred-tank bioreactors;it is scalable,and its effectiveness easily validated.Microcarrier cell culture systems offer many ad-vantages over conventional?ask or bottle cultures in growing anchorage-dependent cells and have been used for the indus-trial manufacture of human polio and rabies vaccines[23,24].

This work addresses the production yields of the EV71-1207strain in cultured Vero,MRC-5,and WI-38cells. High-density Vero cell cultures were derived by increasing the microcarrier concentration from2to5g/L and10g/L Cytodex 1.The speci?c titers of the intracellular and extracellular viruses were obtained to compare the vial productivity at various microcarrier concentrations.A2L, bioreactor cultivation of a Vero cell microcarrier culture was preformed in the large-scale production of the inactivated vaccine antigen.A serum-free Vero cell microcarrier culture was?nally established in the bioreactor.The immunogenic-ity of the inactivated virions produced in serum-free culture was examined for the elicitation of neutralizing antibody re-sponse in immunized mice.These results constitute valuable information on the development of a large-scale microcarrier cell culture process for producing inactivated EV71vaccine.

2.Materals and methods

2.1.Cells,media,and virus

Vero,WI-38,and MRC-5cells were obtained from the Culture Collection and Research Center,Food Industrial Re-search and Development Institute,Hsinchu,Taiwan.Vero cells were grown in M199medium supplemented with10% fetal bovine serum(FBS)(GIBCO,Life Technologies,US). WI-38and MRC-5cells were grown in DMEM medium with 10%FBS.Rhabdomyosarcoma(RD)cells were obtained from the Center of Disease Control,Taipei,Taiwan,and maintained in Eagle’s minimum essential medium(MEM) plus10%FBS.All of these cells were passaged twice weekly in T?asks.The EV71-1207strain was obtained from the Cen-ter of Disease Control,Taipei,Taiwan.Stocks of EV71-1207 were collected from the supernatants of the infected RD cells after three days post infection(p.i.).Virus stocks were stored at?80?C freezer.

2.2.Preparation of microcarriers

Cytodex1microcarrier was purchased from Amersham Biosciences and prepared following the manufacture’s in-structions[25].Cytodex1microcarrier was immersed in phosphate buffer saline(PBS)for more than3h before each experiment was performed.After it was autoclaved for 15min,the microcarriers at speci?ed concentrations were washed twice with the culture media.

2.3.Spinner?ask cultures

Hundred mL spinner?asks(Bellco)were used,with a working volume of100mL.The?asks were placed on a mag-netic stirrer at60rpm in a5%CO2incubator at37?C during the cell growth phase and at32?C during the infection phase. Cells were detached from T-?asks using trypsin-EDTA,and then transferred to the spinner?asks at the given cell and mi-crocarrier concentrations.Cells attached to the microcarriers were counted by using the nuclei staining method[25].The cell growth phase tested for approximately3days.The spin-ner?asks were removed to the laminar hood and settled for 3min to separate the microcarriers and medium through the gravitational force.Infection was then performed by replac-ing70%medium with the fresh medium that contained the stocks of EV71-1207in an amount that corresponded to the multiplicity of infection(MOI)used in the experiments.

2.4.Cultivation in bioreactor andserum free med ium

A BIOSTAT bioreactor(B.BRAUN,Germany),in which a silicon membrane was inserted to ensure that the gas sup-ply was free of bubbles,was used for bioreactor cultivation. The bioreactor culture(1L working volume)was stirred at a rate of60rpm at pH=7.2,controlled by the surface aera-tion of CO2through the gas mixer apparatus.The Vero cells used in the bioreactor experiments were collected from24T-150culture?asks.The medium and microcarrier were intro-duced initially to the vessel and then the inoculated cells were transferred to the bioreactor directly with a constant stirring (60rpm).The inoculation cell density in the bioreactor cul-ture was around5×105cells/mL and the cell density reached 1.2–1.5×106cells/mL following72h of cultivation.Infec-tion was then preformed by replacing70%of the medium with the fresh medium that contained the virus stock at MOI =1.In the bioreactor,the agitation was stopped for5min to separate the microcarriers and medium through the gravita-tional force.The incubation temperature was then changed to 32?C.Glucose/glutamine concentrates(?nal concentration of5.6mM glucose and1.0mM glutamine)were fed twice to at12h p.i.and24h p.i.

The modi?ed Vero serum-free medium(M-VSFM)was provided by Professor Mike Butler,University of Manitoba,

3860S.-C.Wu et al./Vaccine22(2004)3858–3864

Canada[26].Vero cells were passaged directly into M-VSFM from10%FBS-containing medium199without any adapta-tion.In serum-free culture,0.25%(w/v)trypsin inhibitor was used for detaching cells during cell passages and transferring to bioreactor.Vero cells were maintained in M-VSFM for at least two passages in T-150?asks before the bioreactor cultivation.

2.5.Determination of virus titer

Virus titers were determined from the median end point of the tissue culture’s infectious dose(TCID50).The intracelluar virus titer was determined by measuring the TCID50values of the infected cell lysates treated over three free-thaw cycles.The extracellular virus titer was measured directly in culture supernatants.Serially diluted virus samples from10?2to10?9were added to Vero cells in 96-well plates.Quadraduplicate samples were used at each dilution.The cultures in96-well plates were incubated for seven days at37?C,and the TCID50values were measured by counting the cytopathic effects in infected Vero cells.The TCID50value was calculated using the software,ID-505.0, developed by J.Spouge,National Center for Biotech-nology Information,Bethesda,MD,USA(http://www. https://www.wendangku.net/doc/ae3517352.html,/CBBresearch/Spouge/Virology/).

2.6.Preparation of inactivatedEV71s andmouse immunization

Virus stocks were mixed with a37%formaldehyde so-lution(Merck)at a volume ratio of4000:1ratio and stored at4?C for35to40days for virus inactivation.The inacti-vated virus stocks were absorbed on aluminum phosphate at room temperature for1h and at4?C for1day before immu-nization.Female ICR mice(12–15g,3–4weeks after birth) were immunized intraperitoneally(i.p.)with0.5mL of the alum-absorbed inactivated EV71s,and the same doses were boosted i.p.after2weeks or3weeks after?rst immuniza-tion.The immunized mice were bled1week after the boost, and serum samples were investigated for virus neutralization. The50%neutralization inhibition dose(Neut-ID50),which is the geometric reciprocal of the serum dilution yielding50% reduction in the virus titer was obtained using the software, ID-505.0.

3.Results and discussion

3.1.Selecting cell line for EV71production

Three mammalian cell lines(Vero,WI-38,and MRC-5) commonly used for preparing human vaccines were inves-tigated for their use in EV71production.These cells were grown to con?uence in6-well plates at37?C(cell growth phase)and infected with the EV71-1207strain at32?C(virus infection phase)at MOI=1.The intracelluar virus

titer Fig.1.The titers of extracellular and intracellular EV71s produced in three mammalian cell lines(Vero,WI-38,and MRC-5).Samples were collected from the culture supernatants and cell lysates at120h p.i.

was determined from the TCID50values of the infected cell

lysates treated in three free-thaw cycles.The extracellular

virus titer was directly measured in the culture supernatants.

The results indicated that the Vero cells generated higher titers

of intracellular and extracellular titers of EV71,ranging from

2–5×107TCID50/mL of their maximum titers collected at

72h p.i.,than did WI-38and MRC-5cells(Fig.1).Vero cells

were thus chosen as the cell line for producing EV-71.

3.2.Microcarrier cell culture for EV-71production

Microcarrier cell culture was then used for producing EV-

71.Vero cells were grown on Cytodex1microcarriers at

concentrations2,5,and10g/L in100mL spinner?asks.The

cell density for inoculation at higher microcarrier concen-

trations was increased from2×105cells/mL(2g/L)to5×105cells/mL(5g/L)and1×106cells/mL(10g/L).Un-der such conditions,the cell density of the Vero cells grown

on microcarriers reached1.1×106,1.2×106,and1.9×106cells/mL,respectively,following72h of inoculation (Fig.2).Microcarrier-grown cells were then infected with EV71(MOI=1)and coupled with a70%culture medium replacement.The results reveal that the extracellular virus titers of2and5g/L microcarrier cultures exceeded over1.0×108TCID50/mL after150h(or3days p.i.)(Fig.3A).The replication kinetics of the extracellular viruses in10g/L mi-crocarrier culture was slower,and the maximum extracellular virus titer was lower at3.0×107TCID50/mL.In replicat-ing the intracellular viruses,the maximum titers of2,5,and 10g/L microcarrier cultures were all similar(Fig.3B).

The speci?c titers measured per cell(TCID50/cell)

were obtained to compare the virus yields produced un-

der various experimental conditions.In extracellular virus

production,the speci?c yields obtained from the micro-

carrier cultures increased from70TCID50/cell(2g/L)to

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Fig.2.The growth curves for Vero cells on microcarriers using 2g/L (?),5g/L ( ),and 10g/L ( )Cytodex 1in 100mL spinner ?asks.The cell growth phase was cultured at 37?C then infected with the EV71strain 1207at 32?

Fig.3.The viral replication kinetics for (A)the extracellular virus and (B)the intracellular virus of EV71produced in Vero cell microcarrier cultures.Various concentrations of Cytodex 1microcarriers were used,including 2g/L (?),5g/L ( ),and 10g/L ( )in 100mL spinner ?asks.

120TCID50/mL (5g/L)but declined to 20TCID50/cell (10g/L)(Fig.4).In intracellular virus production,the spe-ci?c yields also declined as the microcarrier concentration in-creased from 5TCID50/cell (2g/L)to 3TCID50/mL (5g/L)and 2TCID50/cell (10g/L)(Fig.4).Additionally,a high-

density Vero cell culture of 7×106cells/mL was generated to produce EV-71using 15g/L microcarrier (data not shown),but the resultant speci?c yield of the extracellular virus titer declined further to 2TCID50/mL,about 60times lower than that obtained using 5g/L microcarrier.The decline in the spe-ci?c yields of EV-71for high-density microcarrier cultures may be related to the limited supply of nutrients in cultures or other factors,as stated elsewhere [27].Under the experimen-tal conditions herein,Vero cells grown on 5g/L microcarriers yielded the highest titers of EV-71production in spinner ?ask cultures.

Another interesting result was that microcarrier Vero cell cultures promoted the release of EV-71particles into culture supernatants.EV-71particles have been previously reported to accumulate mainly intracellularly as cell-associated viri-ons in a conventional ?ask or well-plate culture [20].The results obtained using a well-plate culture also revealed that a large proportion of EV-71particles were produced intracel-lularly in Vero cells (Fig.1).EV71is a non-enveloped RNA virus,so the production of a mature EV7virion is completed intracellularly without the acquisition of membranes from subcellular organelles or the cytoplasm of the host cells.The increase in the extracellular production of the virus in micro-carrier culture may follow simply from the greater damage to the integrity of the membrane by the hydrodynamic shear forces,which thus facilitate the release of EV-91virions.However,the triggering of other cellular factors by hydro-dynamic forces cannot be completely ruled out.Microcarrier Vero cell culture has the advantage of harvesting EV71par-ticles in the culture supernatants,instead of in the infected cells,simplifying downstream processing in the development of inactivated EV-71vaccines.3.3.Microcarrier bioreactor culture

3.3.1.Serum-containing medium without feeding during infection

A 2-L bench-top bioreactor was used to culture Vero cells grown on 5g/L microcarriers to scale-up the cell culture pro-cess for EV-71production.The growth curve of Vero cells on microcarriers in the bioreactor reached a maximum cell den-sity of 1.3×106cells/mL following 72h inoculation,and the cells were then infected with EV-71at MOI =1(coupled with a 70%medium exchange)(Fig.5A).The replication curves of EV-71produced in the bioreactor cultivation were plotted as titers of extracellular and intracellular viruses at each spec-i?ed time (Fig.5A).These results reveal that the extracelluar virus titers increased from 1×106TCID50/mL at 72h (or 0h post-infection)to 3.0×107TCID50/mL at 192h (or 120h post-infection)in the bioreactor culture.The maximum ex-tracellular virus titer was about then times lower than that of the 100mL spinner ?ask cultures at the same concentration of microcarriers (5g/L),although the intracellular virus titers (3.1×106TCID50/mL in the bioreactor culture versus 4.5×106TCID50in the spinner ?ask culture)and the cell densi-ties during infection were similar (1.3×106cells/mL in the

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3858–3864

Fig.4.The speci?c virus titers of EV71produced in Vero cell microcarrier cultures.The speci?c virus titers are indicated in the extracellular virus titer (?)and intracellular virus titer ( ).

bioreactor culture versus 1.2×106cells/mL in the spinner ?ask culture).

The low yield of extracellular virus produced in the biore-actor cultivation may be related to the stronger hydrodynamic shear forces in the bioreactor.Microcarrier-attached cells are believed to be more sensitive to hydrodynamic shear forces than single cells in suspension,because they are anchored to microcarrier surface and so cannot freely rotate or translate to reduce the net forces that exerted on them [28].The lack of a signi?cant decline in the intracellular virus production in the bioreactor indicates that the reduced extracellular virus pro-duction in the bioreactor cultivation is probably not related to damage to the integrity of the membrane of the infected cells,and its contribution to virus release,or to increase cul-ture volume in the bioreactor vessel and the consequently less ef?cient adsorption of the virus.

3.3.2.Serum-containing medium with feedings during infection

The infected cells were fed with a glucose/glutamine con-centrate during the infection phase to increase further the extracellular production of virus in the bioreactor culture (Fig.5B).Two feedings of glucose and glutamine at 12and 24h post-infection maintained >2–4mM glucose in the culture medium throughout the infection period (data not shown).The resulted reveal that the bioreactor culture,with the feeding strategy,exhibited an increased extracellular virus titer of 8.9×107TCID50/mL (Fig.5B).In intracellular EV71production,no signi?cant increase of the maximum titers was found in the bioreactor culture with glucose/glutamine feedings (3.2×106TCID50/mL with feedings verse 3.1×106TCID50/mL without feeding).One explanation is that

only a very small fraction by around 3%of the EV71s was produced intracellularly from the microcarrier-grown Vero cells,so the nutrient feedings did not yield a signi?cant in-crease of the intracellular virus production.

3.3.3.Serum-free medium with feedings during infection The serum-free microcarrier culture was also fed with glucose/glutamine using M-VSFM serum-free medium to grow Vero cells [26,27,29].The results indicate that the Vero cell growth on microcarriers using M-VSFM serum-free medium was similar to that using M199medium supple-mented with 10%FBS (Fig.5C).The maximum extracellular virus titer produced in the serum-free bioreactor culture was 5.8×107TCID50/mL,30%lower of the virus yield obtained in the serum-supplemented bioreactor culture with glucose/glutamine feedings.The maximum intracellular virus titer produced in the serum-free bioreactor culture was 9.0×106TCID50/mL that are around two-folds higher than the maximum titers obtained in serum-supplemented bioreactor culture.The differences may be due to some components in the serum free medium M-VSFM affecting the viral assembly and release in infected Vero cells.Further optimization of the feeding components and schedules will improve the production process in serum-free Vero cell culture.

3.4.Immunogenicity of formalin-inactivatedEV71s harvestedfrom serum-free andserum-supplemented cultures

To further determine whether serum-free culture affects the immunogencity of EV71virions,the virus stocks

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Fig.5.Vero cells cultured on5g/L microcarrier in2-L bioreactor and in-fected with EV-71at MOI=1.(A)Serum-supplemented culture without feeding during infection,(B)serum-supplemented culture with feedings dur-ing infection,and(C)serum-free culture with feedings during infection.Ki-netics of cell growth and virus replication are indicated in cell density(?), the extracellular virus titer( ),intracellular virus titer( ).The infection time was indicated as(↓).Two feedings were indicated as( )at12h p.i. and24h p.i.Table1

Immunogenicity of formalin-inactivated EV71-1207harvested from Vero cells in serum-free(M-VSFM)and serum-supplemented(M199+10%FBS) cultures

Medium Neutralization ID50value

Two-week immunization Three-week immunization M-VSFM10.7±1.811.6±0.6

M199+10%FBS 6.7±1.66.7±1.6 harvested from serum free culture(bulk titer=8.9×107pfu/mL)and from serum-supplemented culture(bulk titer= 1.1×108pfu/mL)were treated formaldehyde solution for virus inactivation(stored at4?C for35days). Our preliminary results showed no differences in their inactivation kinetics of EV71s produced in serum-free and serum-supplemented cultures(data not shown).ICR mice were immunized with the formalin-inactivated EV71virions absorbed on alum gel based on a two-week or a three-week immunization schedule.The results reveal that the inactivated EV71s prepared from serum-free cultures elicited slightly higher titer of neutralizing antibodies(Neut-ID50values) than those prepared from serum-supplemented cultures using both immunization schedules(Table1).Noticeably, the antigen content as determined by the initial EV71titers prepared from serum free culture was around20%lower than that prepared from serum-supplemented culture,the elicited immunity as determined by the PRNT50values could by increased from60%(two-week immunization schedule)or 73%(three-week immunization schedule)to over one-fold increase of immunigeneity by using serum-free culture.

In conclusion,Vero cells were found to produce higher titers of EV71than did MRC-5and WI-38cells.Microcarrier Vero cell cultures were established using three concentrations of the Cytodex1microcarrier in spinner?ask cultures;the optimal Vero cell growth on5g/L microcarriers generated the highest titers.A2L bioreactor cultivation of a Vero cell microcarrier culture was preformed in the large-scale pro-duction of the inactivated vaccine antigen.The extracellular virus titers produced in the2L bioreactor were ten times lower than the spinner?ask culture but improved by30-folds using glucose/glutamine feedings during infection.A com-pletely serum-free Vero cell microcarrier culture was?nally established in the2L bioreactor,yielding a high-titer of5.8×107TCID50/mL for EV71production.The immunogenic-ity of the inactivated virions produced in serum-free culture elicited a slightly higher level of neutralizing antibody re-sponse in immunized mice.

Acknowledgement

The authors would like to thank Professor Mike Bulter, University of Manitoba,Canada,for providing the serum-free medium M-VSFM in this study.This work was supported by the National Science Council,Taiwan(NSC90-2214-E-007-019,NSC91-3112-B-007-013,NSC92-3112-B-007-014).

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