Optimization of Virus Yield as a Strategy to Increase the Efficiency of Rabies Vaccine by BHK-21 Cells in a Bioreactor

3.1. Cell Line and Culture Media

The BHK-21 cell line (baby hamster kidney) and 5% fetal bovine serum (FBS) (Life Technologies, USA) with Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, USA) with 2.5 g/L bicarbonate were used for cell culture, which was obtained from the Cell Bank Department of Pasteur Institute of Iran. It was previously tested for the absence of mycoplasma. The inoculated cells were incubated at 37°C with 5% CO₂.

3.2. Growth Assay

In this study, 250 mL spinner flasks with 200 mL of cultured cells and an initial concentration of 3 × 10⁷ cells per mL were used for the cultures, which were carried out at 37°C in an incubator with 5% CO₂ and 37°C. The cells were grown in DMEM supplemented with 0.2 mM for each of serine and methionine and 10% FBS. Samples were taken daily to measure cell concentration. The stirring speed was maintained at 30 rpm.

3.3. Cell Counting

The BHK-21 cells grown in clumps were trypsinized and counted in the presence of 0.2% trypan blue. The exponential growth function was used to calculate the specific growth rate as follows:

Where X denotes the viable cell density per mL (sampling is taken every 1 hour), and t is the time points expressed in hours.

3.4. Virus Strain and Cell Infection

The PV-PARIS/BHK strain is used to create veterinary vaccines. It was developed from the PV-CEPANZO strain of stable rabies, which was first serially transmitted through young rabbits (PV-PARIS). The PV-PARIS fixed rabies virus 93127 FRA was obtained for this purpose from the Institut Pasteur in Paris, France, and used to infect BHK-21 C-13 cells. The PV-PARIS rabies virus strain was introduced into BHK-21 cells with a cell density of 1 × 10⁶ cells/mL and an infection multiplicity of 0.1 cells, and they passed the initial infection stage for 2 hours in a shaker incubator.

3.5. Bioreactor Cultures

For this purpose, a 10-liter bioreactor (Inceltech, Toulouse, France) was used, in which Spanner flask cells that have passed the initial stage of infection are injected into the bioreactor. The infection proliferation stage was carried out under the following conditions:

Temperature 37°C, pH 7.1, air saturation at 70%, and stirring conditions at 40 rpm

In this case, daily samples were collected to measure cell count and cell viability, virus titer, and infection.

3.6. Bioprocess Optimization Using Design-of-Experiments Methodology

The statistical design of the experiments was used to optimize operational parameters of viral yield for BHK cells producing rabies vaccine in a bioreactor where the parameters dissolved oxygen (DO), cell density, temperature, and pH are measured.

3.7. Rabies Virus Titration

In order to compare the production quantity of the modified virus, its titer is evaluated in comparison to the titer of the original virus. In this method, rabies virus suspension in cell culture can be measured by the focus forming assay. Therefore, in this method, the antibody attached to the fluorescent material is attached to each virus particle and can be detected within 24 hours, and the virus titer is expressed as Fluorescent Focus Units (FFU/mL). Briefly, several serial dilutions of the virus are inoculated into the cell culture in duplicate, and the number of FFU of each dilution is counted after 24 hours. In this method, first, in a cell culture flask, BSR cells are cultured, and the cell suspension is prepared with the accumulation of 1 million cells per milliliter. Additionally, successive one-tenth dilutions are prepared from the viral solution in test tubes. In an eight-well Lab-Tek slide, 200 microliters of cell suspension, equivalent to 200,000 cells, are added to each well, and then 100 microliters of each virus dilution are added to each well. The resulting slide is placed at 37 degrees for 24 hours. Then, the liquid on top of the cells is removed and poured into a waste container containing disinfectants. In the next step, Lab-Tek lamella is washed with phosphate-buffered saline (PBS). The slide is placed in a container containing cold pure acetone for 30 minutes. The amount of 25 microliters of rabies virus anti-nucleocapsid antibody conjugated with fluorescein isothiocyanate (FITC) is poured on all areas with cells and placed at 37 degrees for one hour. Finally, the slides are studied using an ultraviolet (UV) microscope. The calculation of initial virus concentration is done using the following formula:

The number of cells containing viral particles in the well × the dilution factor × 10 = The number of viruses (per milliliter of the initial solution)

3.8. Plaque Assay for Infectivity

To determine the number of plaque-forming units (PFUs), serially diluted in vitro reaction mixtures were used by standard methods (Rueckert and Pallansch, 1981). Briefly, first, BHK-21 cells are grown to confluency on 60-mm Petri dishes and, therefore, exposed to the serial dilutions of the virus that are made up of DMEM containing 2% FBS (0.25 mL) for 30 minutes at room temperature. Finally, the cells are covered with 2.5 mL of medium containing 0.6% Noble agar (prepared in a 44°C water bath), and plaques are allowed to grow at 37°C and 5% CO₂ for 26 hours. The cell monolayers are stained with 0.1% crystal violet in 20% ethanol, and after washing the dishes with water, the plaques appear as transparent spots (typically 1 to 2 mm in diameter) on a blue background of cells.

3.9. Virus Harvest Inactivation

After 72 hours, the bioreactor’s supernatant containing the grown virus was collected and cleared by low-speed centrifugation for 15 minutes at 3000 g, removing cell debris. This was subsequently deactivated by beta-propiolactone (BPL) (HiMedia, India). Because the complete inactivation of the rabies virus can be achieved at this dilution without lowering the antigenicity properties of the rabies vaccine, the final concentration of the BPL used was 1/3000. Additionally, the deoxyribonucleic acid (DNA) of the remaining cell is sufficiently broken and inactivated at this dilution (Laboratory techniques in rabies).

3.10. Statistical Analysis and Model Validation

The CCD experiment generated all of the data, which were then submitted to multiple regression analysis using least squares to create the regression models. To fit the data, a second-order (quadratic) function was applied. Utilizing Design Expert 13 statistical software, several tasks, including experimental design, data analysis, interaction plotting, and factor condition optimization, were completed. Predicted responses were plotted against experimentally derived data in Excel 2019 to confirm model fits. A P-value less than 0.05 was considered significant.

4.1. Central Composite Analysis

Several experiments were carried out using the CCD technique to examine the impact of process variables (i.e., pH, cell density, DO, and temperature) on virus yield optimization (Tables 1 and 2). According to the data in Tables 1 and 2, the amount of response PFU was also calculated, as shown in Table 3. A second-order polynomial equation of coded units was expressed based on the CCD and input variables to show relationships between the responses and independent parameters.

4.2. Cell Infection

To check cell infection, the immunofluorescence method was used, and by using rabies nucleocapsid, the ratio of infected to uninfected cells was observed and reported as a percentage (Figure 1).

Figure 1.

Infected cells observed in green color by immunofluorescence method.

4.3. Analysis of Variance Parameters: Cell Density, Dissolved Oxygen, pH, and Temperature

The results of the analysis of variance for the model are shown in Table 4. The importance of each parameter is determined by the F-value and P-value. In this model, all the parameters except X₁X₂, X₁X₃, X₂X₄, ${\displaystyle X_1^2}$, and ${\displaystyle X_2^2}$ have values less than 0.05 and are significant.

Lack of Fit is the other important parameter that expresses the fraction of the sum of squares remaining, which occurs due to the inefficiency of the model. A good non-fit model should have a low value. The difference with the response’s P-value of 0.7575 shows that the second-order equation suggested for this project is valid.

4.4. Regression Analysis

It is necessary to investigate some statistical parameters before introducing the fitting model. The correlation coefficient (R-squared [R²]) is a measure of the fitting of the predicted model with the data resulting from the experiments. The adjusted R-squared (R²_Adj) or the corrected correlation coefficient, such as R², is an indicator for showing the model’s usefulness; however, more precisely, it considers degrees of freedom. The strength of the model is predicted to be examined using the predicted residual error sum of squares (PRESS), where the lower the parameter, the more sensitive the model is to its positive data. The predicted R-squared (R²_Pred), or the predicted correlation coefficient, is a combination of the PRESS and R², and the higher the value is, the more powerful the model will be. The signal-to-noise ratio of the model, which should be greater than 4 for the model under consideration, indicates that the model is accurate enough (10).

A quadratic model has been selected to predict the response, as shown in Table 5. The high correlation coefficient (0.9278) in this model confirms that the predicted model closely matches the data.

The values of R2Adj and R2Pred were accurately obtained in this model, and their closeness to 1 indicates a good relationship between the experimental results and the expected results. The anticipated model has a low PRESS value (0.63), which represents the sensitivity and power of the model. In addition, the model’s adequate precision is 15.546 > 4, which denotes a favorable signal-to-noise ratio. On the other hand, the high accuracy of the expressed model is expressed by the low value for the model’s standard deviation (0.61).

4.5. Diagnostic Tools

In addition to statistical parameters, there are several diagnostic instruments to demonstrate the suitability of the anticipated model as follows:

(1) Typical layout: The assumption that the error follows a standard button is a fundamental one in statistics. In order to verify this presumption, the test data are fitted with the normal probability curve. The error follows the normal distribution if the distribution is far from the S-shaped distribution. A normal probability curve is depicted in Figure 2A, in which the distribution of the residuals has a normal distribution and is relatively close to the reference line.

Figure 2.

Checking statistical parameters: (A) Normal probability plot, (B) residuals versus prediction plot, (C) residuals versus run plot, and (D) predicted versus actual plot.

(2) The residuals versus expected plot aims to investigate the systematic nature or prevalence of errors. If the residuals are systematic, it is a warning to re-examine the trial and possibly repeat it. If the residuals are random, it indicates that the tests were correct. The presence of the remaining on both the positive and negative sides indicates that the experiment did not contain any systematic errors.

(3) If residuals versus run plot exhibits a specific trend (uptrend or downtrend), it is indicative of a mistake. As can be seen in Figure 1C, this plot lacks a clear pattern for the expected model.

(4) The predicted versus actual plot shows how experimental results and predicted model outcomes relate to one another; the closer the data are to the bisector model, the more significant the model’s influence is. This plot also adheres to the normal distribution, as seen in Figure 2D.

4.6. Interaction Between Parameters

The relationships between the factors are depicted via three-dimensional (3D) and contour charts. The relationship between temperature and pH parameters has an impact on the reaction (Figure 3).

Figure 3.

(A) Contour and (B) three-dimensional (3D) plot for the effects of pH and dissolved oxygen (DO) (%) parameters on the response.

When pH and DO are held constant at their midpoints, the effect of DO (%) and cell density (cell/mL) parameters on the response is shown in Figure 4. The parameters’ contour plot shows that the highest response occurs at a cell density of 2.30 × 10⁶ cells per milliliter.

Figure 4.

(A) Contour and (B) three-dimensional (3D) plot for the effect of dissolved oxygen (DO) (%) and cell density (cell/mL) on the response.

4.7. Optimization of Independent Variables and Validation Experiment

In Table 6, the ideal circumstances for a full response are listed. As can be seen, at pH 7.21, temperature 35.05ºC, DO 68.75%, and cell density 2.30 × 10⁶ cell/mL with desirability of 1, the maximum response of 4.7 × 10⁷ PFU/mL occurs (Table 6).

The response was carried out four times experimentally under these conditions to determine the model’s accuracy in predicting the ideal conditions, and a value of 4.7 × 10⁷ PFU/mL was obtained. It stands for this model’s strong capacity for estimation and prediction.

5.1. Conclusions

An appropriate cell substrate for producing robust rabies vaccines, which is the fundamental requirement for these vaccines’ protective effect in humans, is the permanent BHK-21/C13 cell line used to manufacture the animal rabies vaccine. The technologies for BHK-21 cell culture and production are rather simple, and a concentration phase is not necessary for the manufacturing process. Therefore, the current study’s data indicated that in order to maintain a high cell density of BHK-21 cells in the fermentor, the following parameters should be maintained:

pH = 7.21, temperature = 35.05ºC, DO = 68.75%, and cell density = 2.30 × 106 cells/mL