Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6636-8

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells R. Castro 1 & P. Fernandes 1,2 & T. Laske 1,3 & M. F. Q. Sousa 1 & Y. Genzel 3 & K. Scharfenberg 4 & P. M. Alves 1,2 & A. S. Coroadinha 1,2

Received: 4 March 2015 / Revised: 19 April 2015 / Accepted: 22 April 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract The potential of adherent Madin Darby Canine Kidney (MDCK) cells for the production of influenza viruses and canine adenovirus type 2 (CAV-2) for vaccines or gene therapy approaches has been shown. Recently, a new MDCK cell line (MDCK.SUS2) that was able to grow in suspension in a fully defined system was established. In this work, we investigated whether the new MDCK.SUS2 suspension cell line is suitable for the amplification of CAV-2 under serumfree culture conditions. Cell growth performance and CAV-2 production were evaluated in three serum-free media: AEM, SMIF8, and EXCELL MDCK. CAV-2 production in shake flasks was maximal when AEM medium was used, resulting in an amplification ratio of infectious particles (IP) of 142 IP out/IP in and volumetric and cell-specific productivities of 2.1×108 IP/mL and 482 IP/cell, respectively. CAV-2 production was further improved when cells were cultivated in a 0.5L stirred tank bioreactor. To monitor infection and virus production, cells were analyzed by flow cytometry. A correlation between the side scatter measurement and CAV-2 productivity was found, which represents a key feature to determine the best harvesting time during process development of gene

* A. S. Coroadinha [email protected] 1

iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal

2

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal

3

Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106 Magdeburg, Germany

4

University of Applied Sciences Emden/Leer, Constantiaplatz 4, 26723 Emden, Germany

therapy vectors that do not express reporter genes. This work demonstrates that MDCK.SUS2 is a suitable cell substrate for CAV-2 production, constituting a step forward in developing a production process transferable to industrial scales. This could allow for the production of high CAV-2 titers either for vaccination or for gene therapy purposes. Keywords Canine adenovirus . MDCK . Serum-free media . Suspension cultures . Process monitoring

Introduction The potential of adherent Madin Darby Canine Kidney (MDCK) cell lines for the production of influenza vaccines has been intensively investigated in the past decades (Genzel and Reichl 2009; Hu et al. 2011; Liu et al. 2009; Merten et al. 1996; Tapia et al. 2014). Indeed, Optaflu® produced in MDCK cells by Novartis Vaccines was recently commercialized (Doroshenko and Halperin 2009). Moreover, the safety assessment of the Optaflu® production process concluded that the theoretical risks of using MDCK as producer cells are reduced to insignificant levels (Onions et al. 2010). Therefore, MDCK cells are promising cell substrates for the production of biopharmaceuticals, including canine adenoviruses (CAV). In fact, our group recently established a lab-scale process for the production of CAV serotype 2 (CAV-2) vectors using an adherent E1-transcomplementing MDCK cell line (Fernandes et al. 2013a, b). From an industrial point of view, the producer cell line should ideally grow in serum-free conditions as a suspension of cells. The advantages of using serum-free media for the industrial production of biopharmaceuticals have been intensively discussed and include the reduction of contamination risks with pathogenic agents present in the serum, the

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reduction of production costs, and the ease in downstream processing (Falkner et al. 2006). The establishment of two suspension-adapted MDCK cell lines, MDCK.SUS2 and MDCK-SFS, has been recently reported (Lohr et al. 2010; van Wielink et al. 2011), and their use as cell substrate for influenza virus production is also described. Here, we investigated whether the MDCK.SUS2 cell line was able to produce CAV-2 in serum-free suspension cultures. Besides CAV’s attractiveness for veterinary vaccines (Hu et al. 2006, 2007; Liu et al. 2008), CAV vectors have been developed for gene therapy as an alternative to human adenovirus vectors since they present lower immunogenicity. In fact, while antibodies for human adenovirus serotype 5 are common in adult individuals, the presence of CAV-2 neutralizing antibodies is rare (Perreau et al. 2007). Moreover, it has been reported that CAV-2 preferentially transduces neurons and is capable of retrograde transport, characteristics that make these vectors promising for the treatment of neurodegenerative disorders (Bru et al. 2010; Soudais et al. 2001b). In this work, three serum-free media were tested for cell growth and CAV-2 production. Optimization of the infection parameters (multiplicity of infection (MOI) and cell concentration at infection) was performed towards increased productivity. Finally, the best production parameters were validated in a stirred tank bioreactor, under controlled conditions of pH, pO2, and temperature. The results described herein show the suitability of MDCK.SUS2 to produce CAV-2, providing valuable information to streamline the development of a scalable production process of CAV-2 vectors in stirred tank bioreactors.

Materials and methods Cell lines and culture media DKCre cells derived from the dog kidney (DK) cell line as described before (Soudais et al. 2001a) were used for CAV-2 stock production and titration. MDCK cells (ECACC #841211903) were used for CAV-2 production studies in adherent cultures. DKCre and MDCK cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies Ltd, Paisley, UK) supplemented with 10 % (v/ v) of fetal bovine serum (FBS; Life Technologies Ltd, Paisley, UK) and maintained at 37 °C in humidified atmosphere containing 5 % of CO2. MDCK.SUS2, a new suspension cell line derived from adherent MDCK cells as described before (Lohr et al. 2010), was evaluated in growth and CAV-2 production studies. MDCK.SUS2 cells were subcultured twice a week in Adenovirus Expression Medium (AEM; Life Technologies Ltd, Paisley, UK) supplemented with 4 mM of L-glutamine (Life Technologies Ltd, Paisley, UK).

Cell growth assays MDCK.SUS2 cells were cultivated in shake flasks, at 37 °C in humidified atmosphere with 8 % of CO2 and a stirring rate of 130 rpm. The flasks were inoculated with 5×105 cells/mL in 25 mL of medium, and cell growth was followed for 6–7 days. For each subculture, cells were pelleted at 200g for 5 min and resuspended in fresh medium containing 10 % (v/v) of conditioned medium. The following serum-free media were tested for cell growth and CAV-2 production: AEM, SMIF8 (proteinand peptide-free chemically defined medium; powder was provided by K. Scharfenberg, University of Applied Sciences Emden/Leer, Emden, Germany, and liquefied in accordance with the provided description for SMIF8PGd), and EXCELL MDCK (Sigma-Aldrich, MO, USA). SMIF8 media contained 5 mM of L-glutamine, whereas AEM and EXCELL MDCK were supplemented with 4 and 6 mM of L-glutamine, respectively. For culturing MDCK.SUS2 cells in SMIF8 and EXCELL MDCK, cells were subcultured once in 50 % of AEM and 50 % of SMIF8 or EXCELL MDCK, respectively. Cell concentration determination Cell concentration and viability were determined by the trypan blue exclusion method using a 0.1 % (v/v) solution prepared in Dulbecco’s phosphate-buffered saline (DPBS; Life Technologies Ltd, Paisley, UK) and counting cells in a Fuchs-Rosenthal hemacytometer (Brand Wertheim, Germany) on an inverted microscope (Olympus, Tokyo, Japan). In static cultures, cells were counted directly in the cell suspension obtained after trypsin treatment. For MDCK.SUS2 cultures (shake flasks and bioreactor), samples of 0.5 to 1 mL were harvested, cells were pelleted at 300g for 10 min, and 90 % of the supernatant was removed. The cell pellet was treated with trypsin for 10 min at 37 °C, and cells were resuspended with DPBS containing 10 % (v/v) of FBS (for trypsin inactivation) and counted. CAV-2 stock preparation CAV-2-pIX-GFP, a replication competent virus with a Cterminal fusion of EGFP in protein IX (Le et al. 2005), was used for CAV-2 production studies reported here. For virus stock preparation, DKCre cells were seeded in 150-cm2 Tflasks with an initial concentration of 6×104 cells/cm2 and infected the next day using a multiplicity of infection (MOI) of five infectious particles per cell (infectious particles (IP)/ cell), with medium replacement and addition of 1 % (v/v) of nonessential amino acid solution (Sigma-Aldrich) at time of infection. Forty hours postinfection (hpi) cells were collected and lysed with 0.1 % (v/v) of Triton X (Sigma-Aldrich, MO, USA). Cell debris was removed by centrifugation at 3000g for 10 min at 4 °C and the supernatant containing the virus was

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purified by CsCl gradients as described previously (Kremer et al. 2000), with the following modification: removal of CsCl was performed by dialysis using a HiPrep 26/10 desalting c o l u m n w i t h Ä K TA e x p l o r e r ( G E H e a l t h c a r e , Buckinghamshire, UK). The purified viruses were stored in Tris-HCl 10 mM, pH 8 with 0.5 M of trehalose and 2 mM of MgCl2, in aliquots at −85 °C. CAV-2 virus production assays Adherent MDCK cells were seeded in 75 cm2 T-flasks with an initial concentration of 3×104 cells/cm2, and infected the next day using an MOI of 5 IP/cell, with culture medium replacement and addition of 1 % (v/v) of nonessential amino acid solution at time of infection. Forty hpi cells were collected and lysed with 0.1 % (v/v) of Triton X. Cell debris was removed by centrifugation at 3000g for 10 min at 4 °C, and the viral supernatant was aliquoted and stored at −85 °C. For determination of the percentage of infected cells (cells expressing GFP), the cell suspensions obtained from one infected T-flask and one mock-infected T-flask (control) were analyzed by flow cytometry (CyFlow Space, Partec, Germany). CAV-2 production in suspension cultures was performed by culturing MDCK.SUS2 cells in the different serum-free media as described above, and when cultures reached cell densities of 1×106 or 2.5×106 cells/mL, cells were infected with CAV-2-pIX-GFP at MOI of 1 or 5 IP/cell. The medium was exchanged, and 1 % (v/v) of nonessential amino acid solution was added at infection time. Virus harvest was performed at 40 hpi, as described previously for the production of adenoviruses (Kremer et al. 2000; Fernandes et al. 2013b). For virus production in a stirred tank bioreactor, MDCK.SUS2 cells were cultured in a 0.5-L bioreactor (Biostat Q-Plus, Sartorius Stedim Biotech, Goettingen, Germany) equipped with three-blade impellers, using AEM media supplemented with 4 mM of L-glutamine (working volume of 0.4 L). Cultivations were carried out at 37 °C, with an agitation rate of 95 rpm, and controlled pH and pO2 of 7.2 and 40 %, respectively. Forty-eight hours after inoculation (1×106 cells/mL), spent medium was replaced by culture medium supplemented with 1 % (v/v) of nonessential amino acid solution and cells were infected at an MOI of 1 IP/cell. Viruses were sampled every 12 h until 72 hpi. For both CAV-2 production systems in suspension cultures (shake flasks and bioreactors), virus sampling was performed as follows: 2 mL of suspension culture was harvested and centrifuged at 1000g for 10 min at 4 °C. The extracellular viruses were collected directly from the culture supernatant, and the pelleted cells were lysed by addition of a known volume of 0.1 % (v/v) Triton-X 100 in Tris-HCl 10 mM, pH 8.0. Cell debris was removed by centrifugation at 3000g for 10 min at 4 °C, and the clarified intracellular viral samples were aliquoted and stored at −85 °C. For each time point of sampling, additional 2 mL of

infected cultures and 2 mL of control cultures, grown in the same medium, were subjected to trypsin treatment (as described above for determination of MDCK.SUS2 cell concentration) and used for flow cytometry analysis. Flow cytometry analysis of MDCK.SUS2-infected cells The production of CAV-2-pIX-GFP in the stirred tank bioreactor was monitored by flow cytometry. Two milliliters of culture was harvested at each time point (12, 24, 36, 48, 60, and 72 hpi), cells were pelleted at 300g for 10 min, and 90 % of the supernatant was removed. The cell pellet was treated with trypsin for 10 min at 37 °C, and cells were resuspended with DPBS containing 10 % (v/v) of FBS (for trypsin inactivation) and analyzed by flow cytometry (CyFlow Space, Partec, Germany), as described previously (Ferreira et al. 2009). For each time point, 2 mL of mock-infected MDCK.SUS2 culture was treated as described above and was used as control. A minimum of 10,000 cells within the gated region was analyzed, and the mean fluorescence intensity, mean side scatter (SSC), and mean forward scatter distribution (FSC) within the gated region were collected. Determination of the relative SSC and FSC measurements was performed by dividing mean FSC and mean SSC by the corresponding values determined in the control cells at each sampling time. Titration of CAV-2 IPs For quantification of infectious CAV-2-pIX-GFP particles, DKCre cells were seeded at 8×104 cells/cm2 in 24-well plates and incubated at 37 °C in humidified atmosphere with 5 % of CO2. Twenty-four hours after inoculation, culture media were removed and cells were infected with 0.5 mL of viral dilutions prepared in fresh DMEM supplemented with 10 % (v/v) of FBS. Twenty-four hours post infection, cells were washed with PBS, harvested, and resuspended and the percentage of cells expressing GFP was determined by flow cytometry analysis. This percentage was used to calculate the IP titer. The specific viral infectious titer was determined by the ratio between the IPs produced and the infected cell number determined at the harvesting time. Determination of CAV-2 total particles by real-time PCR CAV-2-pIX-GFP DNA was extracted from intracellular viral samples with the BHigh Pure Viral Nucleic Acid Kit^ (Roche Diagnostics, Penzberg, Germany) and was used for real-time PCR. Amplifications were performed using BFast Start Master SYBR Green I Kit^ (Roche Diagnostics), with forward primer 5′-AATATACAGGACAAAGA GGTGTGG-3′ and reverse primer 5′-GAACTCGCCCTGTCGTAAAA-3′, as described elsewhere (Segura et al. 2010). The 20 μL of reaction volume

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included 10 μL of DNA sample and 10 μL of master mix containing 0.5 μM of each primer and 2 mM of MgCl2. The pJB19 plasmid containing CAV-2 genome was used as standard. The PCR program was run in the Light Cycler RT-PCR (Roche Diagnostics) and analyzed using the Light Cycler software. PCR efficiency was 1.754, and the error associated was 0.0175. Metabolite analysis Glucose, lactate, and glutamine were quantified in culture supernatants using an automatic analyzer (YSI Incorporated, Yellow Springs, OH, USA), and ammonia was quantified using an enzymatic kit (NZYTech Lda., Lisbon, Portugal). Calculations To determine cell-specific growth rates and metabolite uptake/ production rates, the Boltzmann equation was used: dP ¼ μp C dt

ð1Þ

where P represents the viable cell concentration or metabolite concentration, t is the time, μp is the cell-specific rate for each parameter, and C is the viable cell concentration.

Results Growth of MDCK.SUS2 cells in serum-free media The growth kinetics of MDCK.SUS2 cells in shake flasks with AEM, SMIF8, and EXCELL MDCK were evaluated ( F i g. 1 ) . T he t hr ee s e r u m - f r ee m ed i a s up p or t e d MDCK.SUS2 cell growth in suspension, showing similar maximum specific growth rates (Table 1). Cells cultivated in AEM and EXCELL MDCK achieved maximal densities of 5.0×106 cells/mL, with a mean viability of 76 and 80 %, respectively. The mean viability determined for SMIF8 cultures was slightly higher (83 %), and the maximal cell density was lower (2.7×106 cells/mL), which is in accordance with the values reported before (Lohr et al. 2010). The effect of culture media on cell metabolism was assessed by the analysis of glucose and glutamine consumption rates and the production rates of the corresponding byproducts, lactate, and ammonia, respectively (Table 1). Glucose consumption and lactate production rates were similar for MDCK.SUS2 cells grown in AEM and SMIF8. In EXCELL MDCK, cells showed a sugar consumption rate in the same range as for the other media, however, the lactate production rate was higher, resulting in a higher lactate/ glucose yield. Glutamine consumption rates were higher for

Fig. 1 Growth kinetics of MDCK.SUS2 cells in different serum-free media. Cells were cultivated in shake flasks at 37 °C, in humidified atmosphere with 8 % of CO2 and a shaking rate of 130 rpm. AEM (squares), SMIF8 (triangles), EXCELL MDCK (diamonds). Error bars represent a 10 % error of the cell counting method

SMIF8 and EXCELL MDCK; nevertheless, the same trend was not observed for ammonia production rate, which was higher for SMIF8 and AEM. MDCK.SUS2 suspension cultures are characterized by cell aggregation (Lohr et al. 2010). In this study, we compare the size and shape of the MDCK.SUS2 cell aggregates for the three chosen culture media (Fig. 2). Cells grown in AEM and EXCELL MDCK formed medium-sized round-shaped aggregates (with a diameter below 100 μm), whereas the aggregates formed by cells cultivated in SMIF8 had an irregular shape and were bigger. CAV-2 production in shake flasks The ability of MDCK.SUS2 cells to produce CAV-2-pIX-GFP in serum-free suspension cultures was analyzed in cells grown Table 1 Maximum specific cell growth (μ max ) and metabolic parameters obtained for MDCK.SUS2 cultures AEM

SMIF8

EXCELL MDCK

μmax (h−1) γglc (nmol/106 cells min) γlac (nmol/106 cells min)

0.015±0.001 0.015±0.002 0.016±0.003 0.69±0.03 0.65±0.04 0.70±0.05 0.90±0.05 0.88±0.05 1.33±0.20

γlac/γglc γgln (nmol/106 cells min) γamm (nmol/106 cells min) γamm/γgln

1.31 0.18±0.01 0.22±0.03 1.21

1.34 0.29±0.03 0.26±0.01 0.88

1.90 0.27±0.02 0.16±0.01 0.62

The errors in μmax and metabolite consumption/production rates correspond to a 95 % confidence interval obtained in the fitting of Eq. (1) γlac lactate production rate, γglc glucose consumption rate, γamm ammonia production rate, γgln glutamine consumption rate

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Fig. 2 Light microscopy pictures of MDCK.SUS2 cells cultivated in a AEM, b SMIF8, or c EXCELL MDCK, taken with a Leica DMI6000 microscope (Leica Microsystems, Wetzlar, Germany)

in the three media. The amplification ratio (IP out/IP in), volumetric titer (IP/mL), and cell-specific titer (IP/cell) determined for the different media 40 h post infection of cells with MOI 1 or 5 are presented in Fig. 3. As control, the productivity of MDCK.SUS2 cells was compared to that of adherent MDCK infected with MOI 5 in DMEM supplemented with 10 % (v/v) FBS. For all infections of suspension cells, viral titers were lower than for the control. Nevertheless, infection of MDCK.SUS2 cells with an MOI of 1 resulted in higher CAV-2 amplification ratios than with an MOI of 5 for the three serum-free media tested. The virus volumetric and cellspecific titers were not significantly affected by the different MOIs used. In all media tested, a 2-fold reduction in viable cell concentration from time of infection to 40 hpi with MOI 5 was observed, whereas for MOI 1, the viable cell concentration was maintained (Table 2). The results show that AEM cultures, infected with MOI 1, represent the best conditions for CAV-2 production in

MDCK.SUS2 suspension cultures. Nevertheless, the productivity obtained was still 3-fold lower than that of adherent MDCK cultures under static conditions (amplification ratio of 421 IP out/IP in and volumetric and cell-specific productivity of 5.2×108 IP/mL and 1724 IP/cell, respectively). AEM cultures can reach maximum cell densities of 5×106 cells/mL; therefore, aiming to further improve the volumetric virus titer, new CAV-2 production experiments were performed by infecting the cells at a density of 2.5×106 cells/ mL (mid-exponential growth phase) with an MOI of 1. Under these conditions, a strong reduction in the amplification ratio was observed (57 compared to 142). The cell-specific productivity and also volumetric productivity decreased to 394 IP/cell and 1.5×108 IP/mL, respectively. Overall, these preliminary experiments showed CAV-2 production in MDCK.SUS2 suspension cultures for the three serum-free media tested. AEM cultures presented the best CAV-2 productivities; therefore, this medium was chosen to proceed to a stirred tank bioreactor system, to show the

Fig. 3 CAV-2-pIX-GFP amplification ratios (a), volumetric titers (b), and cell-specific titers (c) in MDCK.SUS2 cultivated in shake flasks with AEM, SMIF8, and EXCELL MDCK and in adherent MDCK cells cultivated in DMEM supplemented with 10 % (v/v) FBS (static culture). MDCK.SUS2 suspension cells were infected at a concentration of 1×

106 cells/mL with a multiplicity of infection (MOI) of 1 or 5, and viruses were harvested 40 h postinfection (hpi). Adherent MDCK cells were infected with an MOI of 5, and viruses were harvested 40 hpi. IP infectious particles. Error bars represent SD of the mean values calculated with at least duplicate measurements

Appl Microbiol Biotechnol Table 2 Viable cell concentrations of MDCK.SUS2 cells when infected with CAV-2-pIX-GFP at MOI 5 and MOI 1

MOI 5 MOI 1

CCI (106 cells/mL) CCH (106 cells/mL) CCI (106 cells/mL) CCH (106 cells/mL)

AEM

SMIF8

EXCELL MDCK

1.2±0.1 0.7±0.1 1.3±0.1 1.6±0.2

1.2±0.1 0.6±0.1 1.1±0. 1 1.0±0.1

1.4±0.1 0.8±0.1 1.1±0. 1 1.0±0.1

One culture for each condition is shown exemplarily here. The errors represent a 10 % variability of the cell counting method CCI cell concentration at infection, CCH cell concentration at virus harvesting (40 hpi)

Table 3

Summary of CAV-2-pIX-GFP production in different systems

Static culturea Shake flaskb Bioreactorc

Amplification ratio (IP out/IP in)

Volumetric productivity (108 IP/mL)

Cell-specific productivity (IP/cell)

421±73 142±11 366±26

5.2±0.4 2.1±0.6 4.0±0.3

1724±82 483±55 444±29

a

Virus production in static cultures of MDCK cells in DMEM supplemented with 10 % (v/v) FBS; MOI=5, TOH=40 hpi

b

Virus production in suspension cultures of MDCK.SUS2 cells in shake flask with AEM medium, MOI=1, TOH=40 hpi c

feasibility of large scale CAV-2 production using MDCK.SUS2 cells. CAV-2 production in stirred tank bioreactor The best CAV-2 production parameters determined in the shake flasks cultures were applied for virus production in a stirred tank bioreactor. MDCK.SUS2 cell growth was slightly improved in the bioreactor as compared to the shake flask cultures (μmax of 0.021 h−1 instead of 0.015 h−1). For CAV-2 production, cells were infected with an MOI of 1 and virus samples were collected every 12 h. After infection, MDCK.SUS2 growth was arrested by the viral infection and the cellular density maintained constant at values around 1× 106 cells/mL. The CAV-2-pIX-GFP volumetric titer reached its maximal value of about 4.0×108 IP/mL at 36 hpi and kept constant until 72 hpi (Fig. 4a). This volumetric titer corresponded to a mean cell-specific productivity of 444 IP/ cell and a mean amplification ratio of 360 IP out/IP in Table 3. The cell-specific productivity obtained in the bioreactor was similar to the one from the shake flask cultures, though the amplification ratio and volumetric productivity showed a 2.6and 2-fold increase, respectively (Table 3). The profile of metabolite consumption and production in MDCK.SUS2 cells cultivated in the bioreactor is shown in Fig. 4b. A strong increase of glucose consumption and lactate

Fig. 4 CAV-2-pIX-GFP production in MDCK.SUS2 cells grown in stirred tank bioreactor, with AEM medium. a Time course for the virus volumetric titer postinfection. IP intra intracellular infectious particles, IP extra extracellular infectious particles. b Uptake and release of

Virus production in suspension cultures of MDCK.SUS2 cells in a bioreactor with AEM medium, data shown correspond to an average of the values obtained from 36 to 72 hpi

production was observed after infection, which corresponded to a lactate/glucose yield of 2.3. At 62 hpi, glucose was exhausted and lactate leveled out at a concentration of 39 mM. The total CAV-2-pIX-GFP particle concentration present in the samples collected from the stirred tank bioreactor is shown in Table 4. The average ratio between total particles and IPs was 31. Monitoring CAV-2 production by flow cytometry Flow cytometry was evaluated as a tool to monitor CAV-2pIX-GFP production in the stirred tank bioreactor. The data collected for each time point were compared to the data obtained with noninfected MDCK.SUS2 cells cultivated in AEM medium, using the same acquisition parameters. Figure 5 shows the kinetics obtained for the mean fluorescence intensity, relative mean forward scatter (FSC), and relative mean side scatter (SSC). The profile obtained for the mean fluorescence intensity and SSC resembles the profile shown above for the CAV-2-pIX-GFP volumetric titers (Fig. 4a), with maximal values between 24 and 36 hpi. In opposition, the relative FSC values presented a constant decrease from 12 to 72 hpi. Linear regressions of mean fluorescence intensity and relative SSC values against CAV-2-pIX-

metabolites by MDCK.SUS2 cells before and after infection. Glucose (diamonds), lactate (triangles), glutamine (squares), ammonia (circles). The dashed line indicates the time of infection

Appl Microbiol Biotechnol Table 4 Ratio between total CAV-2-pIX-GFP particles and infectious particles produced in the stirred tank bioreactor Time (hpi)

TP productivity (108 TP/ml)

IP productivity (108 IP/ml)

TP/IP ratio

24 36 48 60 72

76±21 107±27 127±31 151±34 103±27

3.1±0.4 3.8±0.2 3.9±0.2 3.8±0.3 3.5±0.1

24 28 33 40 29

Values of at least two measurements were averaged and reported±SD TP total particles, IP infectious particles

GFP titers reveal a positive correlation between the values (Table 5). A positive correlation was also found for mean fluorescence intensity and the relative SSC values (Table 5), indicating that a fluorescence marker can be replaced by a label-free parameter for monitoring of CAV-2 productivity in MDCK.SUS2 cultures.

Discussion MDCK cells have been used as host for the production of viruses for vaccination or gene therapy, including influenza virus and CAV-2. Additionally, while the interest of CAV-2 vectors for gene therapy increases (Ariza et al. 2014; Cubizolle et al. 2014), the development of scalable production processes using a suspension cell line is important to streamline the bioprocess for clinical-grade material. Here, we report the use of the new MDCK.SUS2 cell line for the production of CAV-2 in serum-free suspension cultures. Similarly to what was reported previously (Lohr et al. 2010), the maximal specific growth rate calculated for the three media (AEM, SMIF8, and EXCELL MDCK) was about 0.02 h−1 (Table 1), although this value was lower than that determined for adherent MDCK cells in serum-containing media (0.04–0.05 h−1) (Fernandes et al. 2013a; Janke et al. 2011a). The reason for this difference can be the lack of specific supplements in the serum, such as growth factors, serum albumin, and hormones. Maximal cell densities of about 1-2× 106 cells/mL have been reported for serum-free cultures of MDCK cells using microcarriers (Fernandes et al. 2013a; Genzel and Reichl 2009; Glacken et al. 1986; Hu et al. 20 11) . T h e m a x i m a l c o n c e n t r a t i o n a c h i e v e d fo r MDCK.SUS2 cells in SMIF8 was in the same range (Fig. 1). AEM and EXCELL MDCK cultures reached even higher maximal cell concentrations of 5×106 cells/mL, which are comparable with those described for other cell lines used for the production of viruses, such as PER.C6, HEK293, and AGE1.CR (Lohr et al. 2009; Maranga and Goochee 2006; Schoofs et al. 1998). These differences could be explained

Fig. 5 Monitoring of CAV-2-pIX-GFP production in stirred tank bioreactor by flow cytometry. Kinetic analysis of a mean fluorescence intensity, b relative mean side scatter (SSC), and c relative mean forward scatter distribution (FSC) in MDCK.SUS2-infected cells collected from the stirred tank bioreactor. Relative mean FSC and SSC were obtained by dividing mean FSC and mean SSC by the corresponding values determined in noninfected MDCK.SUS2 cells at each time point. Error bars represent SD of the mean value obtained from two independent stirred tank bioreactors operated in the same conditions

Table 5 Linear regression analysis between FACS parameters and CAV-2-pIX-GFP titers obtained in the stirred tank bioreactor y-variable

x-variable

r2

Mean fluorescence intensity Relative mean SSC Mean fluorescence intensity

CAV-2 titer CAV-2 titer Relative mean SSC

0.81 0.77 0.79

The values obtained for two replica stirred tank cultivations were averaged and used for the linear regressions as described in the BMaterials and methods^ section

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by the medium composition, which may impact cell growth. Indeed, the ammonia levels in SMIF8 cultures reached values above 6 mM, whereas in AEM and EXCELL MDCK cultures, the maximal concentration of ammonia was 3.7 and 4.5 mM, respectively, and such values may inhibit cell growth of such cells (Cruz et al. 2000; Schneider et al. 1996). The reported MOIs used for adenovirus production are between 1 and 100, with the MOIs of 5 and 10 being the most common (Nadeau and Kamen 2003; Schoofs et al. 1998; Silva et al. 2010). Ideally, one should select the lowest MOI that originates the highest percentage of infected cells in order to reduce the amount of virus stock to use without compromising productivity. A first screen for CAV-2 production was performed with an MOI of 5 in MDCK.SUS2 cells, given that it was enough to infect about 70 % of adherent MDCK cells (not shown). However, in contrast to the adherent MDCK cultures, a drastic reduction of viable cell numbers (∼2-fold) was observed for suspension cells after infection (Table 2). As discussed previously, MDCK.SUS2 cells are more sensitive to virus infection (Peschel et al. 2013). In consequence, they may be more prone to lysis upon infection due to the shear stress in a stirred system. In addition, high viral loads (high MOI) may cause a faster progression of virus-induced cell death, which shortens the productive phase of the cells, as shown for influenza viruses (Isken et al. 2012). This effect was reduced by using a lower MOI of CAV-2 to infect MDCK.SUS2 cells. Under these conditions, the volumetric productivity was maintained resulting in a 3- to 6-fold increase in the virus amplification ratio (Fig. 3). To increase volumetric productivity beyond that obtained with cells infected at 1×106 cells/mL, MDCK.SUS2 cultures in AEM medium were infected with cell densities 2.5-fold higher. However, this led to a decrease in CAV-2 production, even when replacing spent medium at time of infection. This is a clear example of the so-called cell density effect, where a drop in the productivity is observed for cells infected at high cell concentrations (Altaras et al. 2005; Nadeau and Kamen 2003; Bernal et al. 2009; Carinhas et al. 2010). When transferring the optimized CAV-2 production parameters to a stirred tank bioreactor, CAV-2 volumetric productivity and amplification ratios (Fig. 4a) were 2- to 2.5-fold higher than those obtained in shake flask cultures and closer to those obtained in static cultures (Fig. 3 and Table 3). This can be explained by the improved infection efficiency of MDCK.SUS2 cells observed for the bioreactor, where at 24 hpi, the percentage of GFP-positive cells was already 40 % and the value increased up to 80 % at 60 hpi. This may be due to different dynamics of the bioreactor cultures, which favor mass transfer events. In the bioreactor, the mean CAV-2-pIX-GFP titer was 4.0×108 IP/mL and the mean amplification ratio was 366 IP out/IP in, values close to those obtained for the same CAV-2 produced in adherent MDCK cultures (5.2×108 IP/mL and 421 IP out/IP in, Table 3) and

within the range of the reported titers for other replicationcompetent CAV-2 viruses (Hu et al. 2006, 2007; Liu et al. 2008). The increase in glucose consumption rate, lactate formation, and lactate/glucose yield is common for infected cultures of MDCK cells (Genzel et al. 2004; Janke et al. 2011b; Lohr et al. 2010). This characteristic could be the result of increased cellular metabolic activity thought to be necessary to compensate the imbalance caused by viral infection. For recombinant adenovirus gene therapy vectors, an FDA Advisory Committee recommended a targeted total particle (TP)/IP ratio below 30. Using this value as reference, the average TP/IP of 31 obtained in this work indicates that MDCK.SUS2 cells are suitable to produce CAV-2 with the quality close to that required for gene therapy. Small-scale processes use viruses with reporter genes such as GFP, to facilitate process development and virus manipulation. However, final viruses and viral vectors usually produced at larger scales do not possess these reporter genes, challenging process monitoring. Different reports in the literature show the potential of flow cytometry for the analysis of infected cells. Correlations between either the forward scatter (FSC) or side scatter (SSC) measurements with human adenovirus titers in HEK293 have been established (Sandhu and AlRubeai 2008). Other studies showed that the SSC parameter can be used to monitor baculovirus infection processes (Deparis et al. 2003; Nordstrom et al. 1999). In this work, a positive correlation was obtained between either the fluorescence intensity or the SSC measurement and maximum productivity (Fig. 5 and Table 5). This indicates that an intrinsic cellular parameter, e.g., SSC, can be used to monitor process performance and be used to decide on the time of harvest, without the need of a reporter gene such as GFP, which would greatly facilitate a large-scale production process of CAV-2 with MDCK.SUS2 cells. In conclusion, this work confirms the suitability of MDCK.SUS2 to produce CAV-2, providing valuable information to streamline the development of a scalable production process of CAV-2 vectors and surpass the need of adherent culture systems or the use of microcarriers in stirred tank bioreactors. Acknowledgments This work was supported by Fundação para a Ciência e a Tecnologia, Portugal (FCT), project PTDC/EBB-EBI/ 118615/2010, and by the European Commission (BrainCAV HEALTH HS_2008_222992 and CLINIGENE-NoE-LSHB-CT-2006-018933). R. Castro and P. Fernandes acknowledge FCT for the award of research fellowships SFRH/BPD/72523/2010 and SFRH/BD/70810/2010, respectively. The authors thank Dr. Cristina Peixoto (IBET, Portugal) for the purification of the virus stock and Dr. Eric. J. Kremer (Institut de Génétique Moléculaire de Montpellier, France) for kindly providing the CAV-2pIX-GFP virus. Conflict of interest The authors declare no conflict of interest.

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