Journal of Bioscience and Bioengineering VOL. 117 No. 4, 471e477, 2014 www.elsevier.com/locate/jbiosc

HEK293 cell culture media study towards bioprocess optimization: Animal derived component free and animal derived component containing platforms Leticia Liste-Calleja*, Martí Lecina, and Jordi Joan Cairó Chemical Engineering Department, Universitat Autònoma de Barcelona, Campus Bellaterra, Cerdanyola del Vallès, 08193 Barcelona, Spain Received 27 May 2013; accepted 25 September 2013 Available online 30 October 2013

The increasing demand for biopharmaceuticals produced in mammalian cells has lead industries to enhance bioprocess volumetric productivity through different strategies. Among those strategies, cell culture media development is of major interest. In the present work, several commercially available culture media for Human Embryonic Kidney cells (HEK293) were evaluated in terms of maximal specific growth rate and maximal viable cell concentration supported. The main objective was to provide different cell culture platforms which are suitable for a wide range of applications depending on the type and the final use of the product obtained. Performing simple media supplementations with and without animal derived components, an enhancement of cell concentration from 2 3 106 cell/mL to 17 3 106 cell/mL was achieved in batch mode operation. Additionally, the media were evaluated for adenovirus production as a specific application case of HEK293 cells. None of the supplements interfered significantly with the adenovirus infection although some differences were encountered in viral productivity. To the best of our knowledge, the high cell density achieved in the work presented has never been reported before in HEK293 batch cell cultures and thus, our results are greatly promising to further study cell culture strategies in bioreactor towards bioprocess optimization. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: HEK293; Suspension cell culture; Cell culture media; High cell density culture; Recombinant adenovirus]

Mammalian cells are often the preferred expression system for the production of biopharmaceuticals as they can perform critical post-transcriptional modifications which are important for protein stability, ligand binding and immunogenic responses (1,2). Some comprehensive reviews on the biopharmaceutical market reveal that more than half of biopharmaceutical approved from 2003 to 2010 were produced in mammalian cell cultures (3,4). Although murine myelomas (NS0 and SP2), Chinese hamster ovary (CHO) and Baby hamster kidney cells (BHK-21) account for nearly all commercial recombinant therapeutics, Human embryonic kidney cells (HEK293) have recently become more relevant. The increasing demand on biomolecules (not only as biopharmaceuticals but also for research purposes) and the emerging market of biosimilars (5) has pushed industries to increase productivity of bioprocesses. Volumetric productivity (P) of a given bioprocess can be enhanced either by increasing the specific production rate of the producer organism (qp) or by increasing the viable cell density (Xv). Several approaches have dealt with the increment of qp by genetically engineering producer cell strains (6,7) and by implementing High throughput screening (HTS) tools for high producer clones selection (8). In order to increment Xv in High cell density cultures (HCDC), different strategies should be emphasized: (i) genetic manipulation to improve the efficiency of

* Corresponding author. Tel.: þ34 93 581 4794; fax: þ34 93 581 2013. E-mail address: [email protected] (L. Liste-Calleja).

central carbon metabolism or to avoid apoptosis (9e11); (ii) optimization of cell media composition (12); (iii) application of different cell culture strategies (i.e., batch, fed-batch, perfusion), and (iv) improvement of monitoring and control strategies (13,14). The work presented in this paper is focused on cell culture media evaluation for HEK293 cell line. In recent years, research on cell culture media design has changed in two major aspects. On one hand, definition of media for a wide range of cells and applications has shifted to defining specific formulations for specific cell strain, clone or process (15). On the other hand, due to safety and reproducibility concerns, animal derived supplements such as serum have been replaced by animal derived component free supplements (ADCF supplements). Even though the higher cell-strain specificity of the media has lead to improvements on cell growth and productivity, the substitution of serum in many of the formulations resulted in a reduction of cell growth and productivity compared to those achieved with serum supplementation (16). Therefore, a large number of industrial recombinant protein production processes are currently performed with serum-supplemented media (17,18). Also, serum is regularly used in research (16,19). Regarding the applications of HEK293 cells, production of recombinant adenoviruses for their use as vaccines (20,21) or for genetic therapy (22,23), and transient transfection for protein expression (24,25) or virus-like particle (VLP) production (26,27) are the two major ones. Furthermore, new studies have been focused on establishing HEK293 recombinant protein expression

1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.09.014

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systems by stable integration of the transgenes (28). Depending on the product of interest (viruses, proteins, VLPs), the downstream processing and the final product application (e.g., human therapy, veterinary therapy, diagnosis, research), media requirements, and thus, media composition might differ. Taking into account all these circumstances, this work provides simple and efficient media supplementation approaches of commercial HEK293 cell culture media for two possible scenarios; ADCF required and ADC allowed bioprocesses. Outstanding results have been achieved in both strategies of nutrient supplementation, which enabled the elongation of the cell growth phase and allowed a substantial increment of maximal cell concentration in comparison to the original medium in batch cultures. MATERIALS AND METHODS Cell line and cell line maintenance Three times a week, cell passaging was routinely performed in 125-mL plastic shake flasks (Corning Inc.), seeding 15 mL of 6 culture at a cell density of 0.25  10 cell/mL. Cultures were grown up to 1  106 cell/ mL and then a new passage was performed. Flasks were shaken at 110 rpm on an orbital shaker (SSL1, Stuart) at 37  C in a humidified atmosphere with 5% CO2 incubator (Steri-cult 2000 incubator, Forma Scientific). Culture media and nutritional supplements Five cell media were evaluated in the present work: HyQ SFMTransFx-293, HyQ SFM4HEK293, HyQ CDM4HEK293 (HyClone, Thermo Scientific), CD293 (Gibco, Invitrogen) and DMEM High Glucose (Gibco, Invitrogen). All media except HyQ SFM4HEK293 were supplemented with 4 mM of GlutaMax (Gibco, Invitrogen). Cell media were supplemented with heat inactivated Fetal bovine serum (FBS) (Invitrogen, Lot 07F6605K) when desired. Knowing that FBS composition might differ from lot to lot, other serums were also tested in order to enhance significance of the results (SigmaeAldrich Inc., lot 058K3396; SigmaeAldrich Inc., lot 039K3396; Omicron, lot 5067 and Invitrogen, lot 462115F). No significant differences on cell growth or viability were detected (data not shown). Cell Boosts 1, 5 and 6 (HyClone) were evaluated as ADCF supplements. All Cell Boosts were dissolved in MilliQ-water to the desired concentration and sterilized by filtration through 0.22 mm filters (Millipore, Billerica, MA, USA). Except when specified, the desired concentration was the limit of solubility of the supplement (80 g/L) once it was ruled out a possible cell growth inhibition or impairment (data not shown). Cell culture conditions and cell growth assessment For all experiments, inocula were prepared by centrifugation of cells aliquot at 300 g for 5 min. Thereafter cells were gently resuspended in fresh media at an initial cell density of 0.25e0.3  106 in an initial volume of 20e25 mL. Cultures were carried out in 125 mL vented-tap shake flasks under the same culture conditions than maintenance passages. Cell number was determined by manual counting using a Neubauer hemocytometer and a phase contrast microscope (Nikon Eclipse, TS100). Viability was assessed using the Trypan blue dye exclusion method. Adenovirus and cell infection Type 5 replication-deficient (DE1/E3) adenovirus encoding green fluorescent protein (GFP) under the control of a cytomegalovirus (CMV) promoter was purchased from Center of Animal Biotechnology and Gene Therapy (CBATEG, UAB, Barcelona). Aliquots of viral storage stock corresponding to 3.45  1011 Infective particle units/mL (IPU/ml) were kept in storage buffer (PBS1; 10% glycerol; 0.5 mM MgCl2; 0.9 mM CaCl2) at 80  C. Initiation of cell cultures for infection was done as described in prior section. At desired time of infection (TOI) inoculation of viral dilution was performed without media replacement before or after infection. Aliquots of viral storage stock were diluted 1:1000 in cell media without supplements to produce the working viral stock at 3.45  108 (IPU/ml), which was used to perform cell cultures’ infection. The volume of working viral stock needed for each culture was calculated (Eq. 1) in order to reach the desired multiplicity of infection (MOI). This volume was never higher than 100 mL ensuring the minimal dilution of nutrients in cell culture media. Vwvs ¼

Vc $Xv $MOI ½WVS

(1)

where VWVS is the volume of working viral stock needed; Vc is the cell culture volume; Xv is the cell density; MOI is the multiplicity of infection (MOI ¼ 1 for all cases) and [WVS] is the concentration of the working viral stock ([WVS] ¼ 3.45  108 IPU/ml). Viral infection efficiency determination and viral titration Twenty-four hours post infection (24 hpi) 1 mL of infected cell culture was harvested and the percentage of fluorescent cells was analyzed by flow cytometry on a FACS Canto (Becton and Dickinson, Bioscience) to determine efficiency of infection. Efficiency of infection was determined from the percentage of HEK293 expressing the gene reporter (GFP) independently from signal intensity and was evaluated by comparison to the value reported for an effective infection (29). Considering that viral infection

process follows a Poison distribution (Eq. 2), at MOI ¼ x the probability that one cell became infected can be expressed as indicated below (Eq. 3)

PðkÞ ¼

  em $mk

(2)

k!

where P(k) is the probability than any cell is infected with k particles; m is the MOI and k is the number of viral particles in a given cell. Pðn>0Þ ¼ 1  Pð0Þ ¼ 1 

ex $10 0!

 (3)

Approximately 40 hpi cell cultures were harvested and lysed by three heat shock cycles as follows: cells were frozen for 20 min at 80  C followed by rapid thawing for 5 min at 37  C. Then cells were centrifuged at 13,300 g and supernatant was recovered for viral titration. HEK293 cells on exponential growth cultured in shake flask as described in the Cell line and cell line maintenance section were diluted with fresh media to a final concentration of 0.5  106 cell/mL and plated into 12 wells plate (Orange Scientific) immediately previous to sample titration. At least six dilutions of each sample were tested. 24 hpi cells were harvested from plates and analyzed by flow cytometry on a FACS Canto (BD Bioscience). Final titer was calculated as follows (Eq. 4): ½IPU=mL ¼

ð%positive GFP  %positive GFP Neg CtrlÞ=100 $TCN$DF viral volume

(4)

where Neg Ctrl is an uninfected cell culture under the same conditions (cell media, cell concentration and viability) as infected cell culture; TCN is the total cell number per well and DF is the dilution factor of the sample. Only percentages between 3% and 25% were considered valid for titration (30). Validation of the assay was routinely performed by including a sample with known viral concentration in the assay as positive control. Statistics Values of cell density and viability are given as the average of two counts of each sample from at least biological triplicates experiments (n  3). The error bars represent the standard error of the biological replicates. Values of infection efficiency are given as the average of triplicates of infection and error bars represent the standard deviation of calculated percentages. Values of viral titration are given as the average of at least three valid dilutions of titration assay of triplicates of infection. Error bars represent the standard deviation of the calculated viral titer. The significance of the results from the infections has been evaluated by running a Student’s t-test (p value set on 0.05).

RESULTS Cell culture media screening for HEK293 The first part of this work was focused on the evaluation of different cell media recommended for HEK293 cells as a first step for bioprocess optimization. From the five different commercial cell media initially selected (HyQ CDM4HEK293, HyQ SFM4HEK293, HyQ SFMTransFx293, CD293 and DMEM), the last two were discarded due to the poor cell growth achieved after several adaptation passages (data not shown). Fig. 1A shows viable cell density (Xv) and viability profiles for the three selected media. Cultures performed in HyQ SFM4HEK293 and HyQ SFMTransFx-293 showed better cell growth than HyQ CDM4HEK293, reaching maximum cell densities (Xvmax) of about 3.5  106 cell/mL, 2  106 cell/mL and less than 1  106 cell/mL respectively. Aside from cell concentration, the main difference between tested media was that viability was sustained longer over 85% when HyQ SFM4HEK293 medium was used. Maximum specific growth rates (mmax) were estimated and compiled in Table 1. The mmax value obtained for media HyQ SFM4HEK293 and HyQ SFMTransFx-293 was about 0.024 h1 (which corresponds to a doubling time of 29 h) as it was expected (31), and it was sustained for approximately 74 h after inoculation (this mmax period is expressed here as tm). However, mmax value for HyQ CDM4HEK293 medium was only about the half (0.011 h1) meaning that the media is not adequate for the HEK293 strain used in this work. Supplements evaluation for HEK293 cell media All media selected are described as serum free media. In order to evaluate whether the substitution of critical serum components have

VOL. 117, 2014 satisfactorily been performed without affecting cell growth, the addition of fetal bovine serum (FBS) was assessed. Once the FBS lot was selected (see Materials and methods), the effect of its addition (5% v/v) to cell media was studied. The concentration of FBS was chosen as a starting intermediate point based on previous work of the group with mammalian cell lines (32). As shown in Fig. 1B, FBS addition significantly increased maximal cell concentration in two cases: 7-fold increment for HyQ CDM4HEK293 (6  106 cell/mL) and 3-fold increment for HyQ SFMTransFx-293 (7.02  0.13  106 cell/mL). In HyQ CDM4HEK293 cultures, the positive effect of FBS on "mmax" was also remarkable. This value was more than doubled (from 0.011 h1 to 0.026 h1) becoming comparable to the other cell media (values compiled in Table 1). On the contrary, the addition of 5% (v/v) FBS had an unremarkable effect on HyQ SFM4HEK293 cell cultures in terms of mmax (0.028 h1), Xvmax (4.67  0.82  106 cell/mL) or viability profile. In parallel to FBS addition experiments, supplementation of media with ADCF nutritional supplements recommended for HEK293 cell culture was performed in order to define a cell culture platform for those applications in which absence of serum is preferable. Three different supplements, Cell Boosts 1, 5 and 6, were evaluated. Addition of Cell Boosts 1 and 6 did not provide significant differences in comparison to basal media (data not shown), whereas the addition of 5% (v/v) of Cell Boost 5 solution (onwards

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CB5) enhanced significantly cell growth (Fig. 1C). In particular, CB5 addition resulted into a 4-fold increment of Xvmax on HyQ CDM4HEK293 (4.11  0.58  106 cell/mL), 2-fold increment on HyQ SFM4HEK293 (7.29  0.32  106 cell/mL) and 5-fold increment on HyQ SFMTransFx-293 (9.75  0.44  106 cell/mL). Application case: recombinant adenovirus production with HEK293 cells (I) From the range of applications in which HEK293 can be used, the work carried out in this work was directed to recombinant adenovirus production. Hence, the influence of cell media on viral infection and productivity was studied before further studies on media supplementation were performed. Cultures in the middles of the log phase (TOI z 0.5  106 cell/mL) growing in the three media tested were infected at MOI z 1 with a reporter adenovirus (rAdV-GFP). The efficiency of infection is presented on the left bar chart of Fig. 2. The percentages of infection for all three media were in good correlation with the percentage predicted by Poisson distribution for an effective infection (see Materials and methods) which at MOI ¼ 1 is 63%. This percentage is similar to those obtained from all different media evaluated (Fig. 2) indicating that there was no negative effect of the media on infection efficiency. In terms of infective adenovirus production, significant differences were neither detected between HyQ SFM4HEK293 and HyQ SFMTransFx-293 cell media (9.23  0.80  107 and

FIG. 1. Viable cell density (Xv) (solid symbols) and viability profiles (open symbols) of HEK293 cell cultures. (A) Profiles obtained with the basal commercial media. (B) Profiles with the commercial media with 5% (v/v) FBS supplementation. (C) Profiles when the addition of 5% (v/v) CB5 was performed. (AeC) Commercial media depicted are HyQ CDM4HEK293, HyQ SFM4HEK293 and HyQ SFMTransFx-293 (from left to right). The average from two cell count of three biological replicates (n ¼ 3) are shown. Error bars represent the standard error of the replicates.

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TABLE 1. Kinetic parameters for HEK293 cell cultures corresponding to the profiles depicted in Fig. 1. HyQ CDM4HEK293

HyQ SFM4HEK293

HyQ SFMTransFx-293

No addition

Xvmax (106 cell mL1) mmax (102 h1) tm (h)

0.85 1.06  0.01 96

3.53  0.21 2.46  0.14 74

2.1  0.12 2.43  0.03 74

Xvmax (106 cell mL1)

5% FBS

mmax (102 h1)

6 2.61  0.04 95

4.67  0.48 2.8  0.05 71

7.02  0.06 2.67  0.01 72

4.11  0.33 2.1  0.06 92

7.29  0.18 2.06  0.03 69

9.75  0.25 2.17  0.03 116

tm (h) Xvmax (106 cell mL1)

5% CB5

mmax (102 h1) tm (h)

7.58  2.12  107) whereas the use of HyQ CDM4HEK293 resulted in a decrease of final titer (Fig. 2). The results obtained so far showed that supplementation of all cell media tested is recommended in order to achieve higher cell density cultures. Among all the conditions, HyQ SFMTransFx-293 was the media which supported the highest Xvmax with both supplements tested (FBS and CB5). Therefore, it was selected for further experiments. Study of FBS and CB5 concentration effect in HyQ SFMTransFx-293 media In order to tune the final concentration of each supplement with the aim of lowering the medium cost, a range from 2.5 to 10% (v/v) for FBS and 2.5 to 20% (v/v) for CB5 were evaluated using HyQ SFMTransFx-293 media. As shown in Fig. 3, the maximal viable cell density reached was approximately 7  106 cell/mL and mmax was in the range reported for HEK293 cell line (0.024e0.027 h1) (31) for all FBS concentrations tested. On one hand, no negative effects associated with toxicity were observed at any concentration assessed, but addition of higher concentrations than 5% did not contribute to improve cell growth. On the other hand, mmax and Xvmax were slightly lower when 2.5% (v/v) of FBS was added, meaning that probably this FBS concentration could have limiting effects. Thus, a concentration of 5% FBS was selected as the preferred concentration for HyQ SFMtransFx-293 medium. Differently to what was observed in the FBS experiment, a proportional positive effect on Xvmax was noticed with the addition of CB5 up to a concentration of 10% (v/v) (Fig. 4). At this concentration, mmax was sustained for longer tm (144 h) in comparison to lower concentrations (116 h), and consequently resulted in an increase on viable cell density reaching about 13  106 cell/mL. However, when cultures were supplemented with 20% (v/v) of CB5, no significant differences on mmax were detected at the beginning of the culture, but this value decreased substantially beyond 96 h, and

thus, maximal cell density reached was limited to only 8  106 cell/ mL. Synergic effect of supplements combination: CB5 and FBS The results presented hitherto indicated that CB5 and FBS addition affect dissimilarly the growth kinetic parameters mmax and tm. Therefore, it became interesting to assess the effect of combining both supplements in HyQ SFMTransFx-293 medium. Concentrations of 5% (v/v) FBS and 10% (v/v) for CB5 were selected in concordance to previous experiments. In Fig. 5, viable cell density and viability profiles are presented and compared to profiles obtained with sole supplement addition and non-supplemented cultures. A synergic effect of both additives combination was observed, achieving the highest Xvmax value (about 16.77  0.70  106 cell/mL). Supplementation with both components was also reflected on kinetic parameters; tm was maintained for 144 h and specific growth rate was increased in 15% in comparison to those cultures in which FBS was deprived. Application case: recombinant adenovirus production with HEK293 cells (II) As it was previously performed with basal media, possible interferences on adenovirus infection and production due to supplements addition were assessed. The experiment was carried out maintaining the infection parameters of previous experiment: TOI z 0.5  106 and MOI z 1. All the infected cell cultures stopped to grow 24 hpi as it was reported (31) and cytopathic effect was clearly observed approximately 36 hpi, before adenovirus harvesting was performed. In Fig. 6, the efficiency of infection and the infective adenovirus production for each cell culture condition tested are presented. As it can be observed on the left bar chart of Fig. 6, the expected percentage of infection (around 63%) was reached in all conditions, although the addition of FBS slightly increased this percentage up to 67.13  1.01% in comparison to the 58.93  1.13% obtained when FBS was deprived. The positive effect of FBS became more evident when

FIG. 2. (A) Efficiency of infection of the tree commercial media tested. Percentages of cells expressing the gene reporter (GFP) are given as the average of three replicates of infection (n ¼ 3). Error bars represent standard deviation of one measure of each replicate. (B) Viral titer obtained from the infection of HEK293 cultured in the three commercial media tested. Titers are calculated from at least three dilutions of the supernatant recovered from each replicate. Error bars indicate the standard deviation of the titers obtained. Specific values of averages are pointed out on the top of each bar.

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FIG. 3. Growth profiles of HEK293 cell cultures in HyQ SFMTransFx-293 cell media supplemented with different FBS percentages in the range between 0% and 10% (v/v) final concentration. The average of two cell count of three biological replicates (n ¼ 3) are shown. Error bars represent the standard error of the replicates. Relevant cell growth parameters are shown in the table.

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FIG. 5. Viable cell density (closed diamonds, straight line) and viability profiles (open diamonds, straight line) of HEK293 cell cultures in HyQ SFMTransFx-293 cell culture media supplemented with 5% (v/v) FBS and 10% (v/v) CB5 are represented. Reference cell cultures of HyQ SFMTransFx-293 with 5% (v/v) FBS (triangle down, dotted line) and HyQ SFMTransFx-293 with 10% (v/v) CB5 are also represented. All plots show the average from two cell count of three biological replicates (n ¼ 3). Error bars represent the standard error of the replicates. Relevant cell growth parameters are shown in the table.

focusing on the viral titer obtained. As presented on the right bar chart of Fig. 6, the maximal viral production was achieved when FBS was added solely to HyQ SFMTransFx-293 (4.13  108 IPU/mL). This viral titer represents an increment of 5.5 times the viral titer obtained in cell cultures without supplementation. Differently, the addition of CB5 had no significant effect in comparison to cell medium without supplementations. When FBS was added in combination with CB5, viral production was almost doubled with respect the basal medium. Nonetheless, the viral titer did not reach the value obtained on FBS sole supplementation condition. DISCUSSION

FIG. 4. Growth profiles of HEK293 cell cultures in HyQ SFMTransFx-293 cell media supplemented with different CB5 percentages in the range between 0% and 20% (v/v) final concentration. The average from two cell counts of three biological replicates (n ¼ 3) are depicted. Error bars represent the standard error of the replicates. Relevant cell growth parameters are shown in the table.

When defining a bioprocess for biopharmaceuticals production, the choice of a proper cell culture media becomes a key factor. From the 5 different commercial media initially selected for HEK293 cells culture, two of them (CD293 and DMEM) were discarded due to the low cell expansion factor achieved. In addition, when DMEM medium was used, cells aggregated to a high extent resulting in lower averaged viability and significant variability on cell counting among samples. Despite the fact that DMEM medium has been used for HEK293 culture, it has commonly been reported for attacheddependent cells lines based bioprocesses (33), but not for suspension cultures. Hence, media finally selected for further experiments were HyQ CDM4HEK293, HyQ SFM4HEK293 and HyQ SFMTransFx293. Once the different basal media were selected, the addition of nutritional supplements was assessed. On one hand, addition of FBS was evaluated as a source of growth factors and other important biomolecules for cell expansion, and on the other hand, ADCF

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FIG. 6. (A) Efficiency of infection of HyQ SFMTransFx-293 without supplementation and supplemented with one or both supplements (FBS and CB5). Percentages of cells expressing the gene reporter (GFP) are given as the average of three replicates of infection (n ¼ 3). Error bars represent standard deviation of one measure of each replicate. (B) Viral titer obtained from the infection of HEK293 cultured in the same conditions as panel A. Titers are calculated from at least three dilutions of the supernatant recovered from each replicate. Error bars indicate the standard deviation of the titers obtained. Specific values of averages are pointed out on the top of the bars.

supplementation was performed by adding commercial nutrient formulation, Cell Boosts 1, 5 and 6. Since the addition of Cell Boosts 1 and 6 did not shown significant increment on maximal cell density in comparison to basal media (data not shown), only Cell Boost 5 was chosen for further work. Even though the rough components of the different Cell Boosts could be alike, the exact composition and the components’ concentration are unknown. Potential differences in the specific components and its concentration would explain the results obtained. Focusing on estimated mmax and tm values (compiled in Table 1), it can be noticed that specific growth rate was higher when FBS was used as supplement, independently from cell media. Nevertheless, CB5 addition extended the exponential growth phase for 44 h when HyQ SFMTransFx-293 was used. This fact would explain the higher Xmax achieved with HyQ SFMTransFx-293 5% (v/v) CB5 in comparison to HyQ SFMTransFx-293 5% (v/v) FBS. In the case of HyQ SFM4HEK293 cell medium, tm values were comparable for the three conditions tested. However, only a slightly decrease on mmax was detected beyond tm (around 70 h) when CB5 was used as supplement whereas a dramatic drop of this value was noticed beyond tm for non-supplementation or FBS addition. These differences would explain the final cell concentration achieved with HyQ SFM4HEK293 5% (v/v) CB5. With the aim to select the best medium to carry on with the bioprocess development, it was important to ensure that there was no negative effect of the selected medium on the vaccine candidate production. Therefore, the efficiency of adenovirus infection and production was assessed in all three media initially selected. The results revealed that no significant differences were found, so the final media selection was made in terms of cell growth. After the screening experiments for media and supplements selection, the next step was focused on tuning the optimal supplements’ concentration with the aim of increasing cell density and adenovirus production. The results showed that when HyQ SFMTransFx-293 was supplemented with FBS mmax values were higher compared to basal media, independently from the concentration used, confirming the positive effect of FBS on this parameter previously encountered. CB5 addition did not significantly affect mmax, but it indeed allowed the enlargement of tm, when the addition of CB5 preparation was up to 10% (v/v). However, when cultures were supplemented with higher amount of CB5 (20% v/v), the mmax value decreased substantially beyond 96 h, limiting the maximal cell density reached to only 8  106 cell/mL. This fact may be explained because of by-products accumulation in the culture broth, which together with higher nutrients concentration may increase osmolarity, affecting negatively to cell growth. More interesting were the results obtained when combining both supplements. A synergic effect was observed in the cultures supplemented with 5% FBS and 10% of CB5 solution, reaching cell densities up to 17  106 cell/mL.

After exploring and defining the best concentration of the nutritional supplements used in this work, a final experiment of adenovirus infection and production was carried out in order to evaluate different platforms for the vaccine candidate production. The results showed similar infection’s efficiency but revealed distinct production yield depending on the supplements used. In general terms, the addition of FBS was positive for adenovirus production while CB5 did not affect significantly. Nonetheless the combination of both supplements had minor negative effect and slightly decreased the final adenovirus concentration in comparison to FBS solely addition. Although unraveling the basis of the results obtained is beyond the scope of this work, some hypotheses based on referenced work can be discussed. It is well-known that a great number of variables can interfere in the viral infection process (31,34,35). From those variables directly related to cell culture media, the most studied are nutrient deprivation, pH, interference of cell media components with viral receptor, and osmolarity. Nutrient deprivation and pH may have had a minor influence on the experiments presented due to the early TOI selected. Possible hostvirus receptor interference by supplement components was also discarded considering the infection efficiency determined at 24 hpi, which fitted perfectly the Poisson distribution. Finally, an increment of osmolarity due to the addition of both supplements might explain the slight reduction on adenovirus production in comparison to the addition of FBS only (36). All in all, after exploring different combinations of cell media and supplements for HEK293 cell cultures, two culture platforms are proposed for two possible scenarios: HyQ SFMTransFx-293 CB5 supplemented (10% v/v) for ADCF required bioprocesses and HyQ SFMTransFx-293 FBS and CB5 supplemented (5% and 10% v/v, respectively) for ADC containing bioprocesses. The basis of this selection relies on (i) the high cell densities achieved in both cases which largely overcome the Xvmax reached with basal media, (ii) the preservation or even improvement of mmax value with respect to basal media and (iii) the relative low increment on cell media cost (1.3-fold increment with dual supplementation) compensated by far by the higher cell density reached. The ongoing work of the research group is focused on bioprocess scaling up and on controlling and monitoring systems which will enable to study the best strategies in order to increase productivities of the particular bioprocess presented in this work.

ACKNOWLEDGMENTS The authors would like to thank Dr. A. Kamen (Biotechnology Research Council of Canada) for kindly providing HEK293 cells which this work was performed with. This research was supported by the Spanish State Secretary for Research, Development and Innovation (SEIDI).

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HEK293 cell culture media study towards bioprocess optimization: Animal derived component free and animal derived component containing platforms.

The increasing demand for biopharmaceuticals produced in mammalian cells has lead industries to enhance bioprocess volumetric productivity through dif...
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