processes
Article
Modeling and Optimization of Continuous Viral
Vaccine Production
Caitlin S. Morris and Seongkyu Yoon *
Citation: Morris, C.S.; Yoon, S.
Modeling and Optimization of
Continuous Viral Vaccine Production.
Processes 2022, 10, 2426. https://
doi.org/10.3390/pr10112426
Academic Editor: Tao Sun
Received: 21 October 2022
Accepted: 11 November 2022
Published: 17 November 2022
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Processes 2022, 10, 2426. https://doi.org/10.3390/pr10112426
Department of Chemical Engineering, University of Massachusetts Lowell, Lowell, MA 01854, USA
* Correspondence: seongkyu_yoon@uml.edu; Tel.: +1-978-934-4741
Abstract: A model that captures realistic viral growth dynamics has been developed based on a con-
tinuous and semi-continuous production model of an influenza A virus. This model considers viral
growth parameters such as viral latency. It also captures the lag observed during the early production
of viruses in a culture and explains later-phase growth dynamics. Furthermore, a sensitivity analysis
was performed to investigate the effects of each input on each output. This revealed that production
of defective interfering particles (DIPs) highly depends on the number of cells introduced to the viral
reactor. The rationale for this is, as per the model, that a reduction in number of cells to be infected
causes a reduction in DIPs formed as rate of viral infection decreases. Finally, a flowsheet model was
created to optimize the continuous platform, including number of cells supplied to the viral reactor.
From this, it was observed that the peak number of DIPs formed could be reduced by one-third.
Finally, this model is tailorable to different viral particles using parameter estimation. Therefore, the
proposed mathematical model provides a versatile, comprehensive platform that can be tailored to
various viral cultures with or without a latent phase.
Keywords: in silico platform; viral latent phase; continuous; semi-continuous; flowsheet
modeling; optimization
1. Introduction
Mathematical models are enabling technology that can be used to optimize and
understand a variety of platforms. Specifically, first-principle modeling is a widely accepted
technique for analysis of population dynamics of viruses [1]. Mathematical models allow for
study and analysis of potential experimental techniques with less investment in laboratory
equipment. First-principle modeling can also be used as a process optimization tool by
determining the most likely experiments to be successful [2,3]. Several mathematical models
have been presented for the batch-level design of a virus bioreactor [4–6]. These models
account for growth and infection of susceptible target cells and growth and production
of virus particles [1,7]. However, with the paradigm switch from batch-level design to
continuous platforms, developing continuous models that can accurately track the growth
dynamics within these newer cultures is of paramount importance [2].
Continuous models for production of viruses for viral vaccines have been proposed
in the literature. The difference between continuous and batch models is the inclusion of
dilution and feed terms [3,8,9]. Including a dilution term is essential for the liquid phase
of a continuous bioreactor. The liquid phase operates continuously in a perfusion (or
continuous) bioreactor, whereas cells are considered to be in batch mode [10]. The liquid in
a perfusion system is continually added to and drained from the reactor, so the dilution
term must account for these volume changes [8,10].
Literature models of continuous vaccine production address the continuity of the
liquid phase. Still, they do not address the possibilities of an immune response, a latent
phase, or other potential changes in the virus that may affect its production [9]. A latent
phase is a period during a virus’s life cycle when it remains dormant within the host
https://www.mdpi.com/journal/processes
Processes 2022, 10, 2426 2 of 13

cell [11]. This period can account for the beginning of replication (transcription, translation,
etc.), or it can simply be a period of no activity. Therefore, in the latent phase, the virus is
not infectious, nor does it contribute to production of newly infected cells [6,11,12]. Viral
latency affects some but not all viruses. Cori et al. showed that influenza exhibits a viral
latency period [12]. Other studies have shown the same and provided estimations for the
latency period parameter, k [13]. However, these literature models did not capture the
experimental results’ early-phase viral growth dynamics because they did not account for
a latent phase [12]. Therefore, latent-phase parameters must be added to address this lag
and accurately represent the growth dynamics observed in viral cultivation.
Literature models for continuous viral production include several ODEs [8]. The model
herein will analyze four separate cell classifications, including uninfected cells (T), infected
cells (Is ), cells that contain defective interfering particles (Id ), and cells co-infected with both
DIPs and virus (Ic ). Viral latency was introduced into the model via implementation of
new terms, including a latent-phase parameter and a population parameter that accounted
for latent viruses [12]. Furthermore, the versatility of this model was tested through use of
parameter estimation [5,14–20].
The newly developed model will be applied to various virus types that do or do not
present viral latent-phase characteristics [1,7]. As viral latency may or may not be present
in all viral cultures, it was essential to ensure that the structure of the model would enable
latent-phase terms to be canceled out [21–24]. The following sections will describe how
this model was developed and how latent-phase parameters were added, then present an
optimization study, a sensitivity analysis, and a flowsheet model.

2. Materials and Methods

The hypothesis for the following model development was that inclusion of latent-
phase parameters and equations would enable the model to accurately track growth
dynamics exhibited by viruses in continuous cultivation. This hypothesis was based on
experimental results presented in the literature, which showed a delay in viral growth [8].
It will be shown that the newly developed “latent-phase” model could more accurately
track experimental results found in the literature. The following subsections will outline the
methodology used to improve the model, as well as the parameter estimation, sensitivity
analysis, optimization procedure, and flowsheet modeling.

2.1. Experimental Results

The experimental data used to evaluate and train the first-principle model proposed
herein is based on literature sources. These literature sources included rabies, measles, and
human influenza A/Puerto Rico/8/34 H1N1 [8,12,14,16,17].

2.2. Continuous Vaccine Production Model with Viral Latency

Viral latency has been studied in batch-mode cultures. Given that several literature
models appear to miss early-phase growth patterns, it was hypothesized that viral la-
tency mechanisms could be present in continuous cultivation [8,12,14,16,17]. However,
to preserve model versatility for non-latent viruses, it was necessary to ensure that these
latent-phase terms could be canceled out.
Figure 1 shows a diagram of the proposed model. It was assumed that viruses
infected with standard virus particles (STVs) and co-infected cells enter an eclipse or latent
phase [1,5,12]. Also shown in the diagram is the addition of the latency time parameter, k.
In this figure, if “viruses with an eclipse phase” were to be removed, the remaining figure
would represent a model without viral latency, as presented in the literature [8]. However,
the inclusion of this portion of the model affects number and concentration of cells with
standard viruses and co-infected cells and thus significantly changes the overall model.
Processes 2022, 10, x FOR PEER REVIEW 3 of 13

Processes 2022, 10, 2426

the inclusion of this portion of the model affects number and concentration of cells3with
of 13

standard viruses and co-infected cells and thus significantly changes the overall model.

Figure 1. Model flow diagram: The chart above gives a schematic view of the mathematical model
that has
that has been
been proposed.
proposed. From
From left
left to
to right,
right, the
the diagram
diagram shows
shows the
the possible
possible progression
progression ofof aa healthy
healthy
cell infected by both standard viral particles and defective interfering particles (DIPs). The diagram
cell infected by both standard viral particles and defective interfering particles (DIPs). The diagram
also shows the addition of a viral latent or eclipse phase.
also shows the addition of a viral latent or eclipse phase.

The following
The following equations
equations summarize
summarize the
the newly
newly developed
developed latent-phase
latent-phase model.
model. Each
Each
parameter is defined in Table 1.
parameter is defined in Table 1.

dT= 𝜇𝑇 − 𝑘 (𝑉 + 𝑉 )𝑇 + 𝐷(𝑇 − 𝑇) (1)

= µT − k1 (Vs + Vd ) T + D ( Tin − T ) (1)
dt
= 𝑘 𝑉 𝑇 − (𝑘 𝑉 − 𝜇)𝐼 − 𝐷𝐼 (2)
dId
= k1 Vd T − (k1 Vs − µ) Id − DId (2)
dt
= 𝑘 𝑉 𝑇 − (𝑘 𝑉 + 𝑘 )𝐼 − 𝐷𝐼 + 𝑘𝐸𝐼 (3)
dIs
= k1 Vs T − (k1 Vd + k2 ) Is − DIs + kEIs (3)
dt
(𝑉 𝐼 + 𝑉 𝐼 ) − 𝑘 𝐼 − 𝐷𝐼 + 𝑘𝐸𝐼
dI=c 𝑘 (4)
= k1 (Vs Id + Vd Is ) − k2 Ic − DIc + kEIc (4)
dt
dV=s 𝑘 𝐼 − 𝑘 (𝑇 + 𝐼 )+𝑘 𝑉 (5)
= k3 Is − k3 ( T + Id ) + k4 Vs (5)
dt
dV= 𝑘 𝐼 + 𝑓𝑘 𝐼 − 𝑘 (𝑇 + 𝐼 )+𝑘 𝑉 (6)
d
= k3 Ic + f k3 Is − k3 ( T + Is ) + k4 Vd (6)
dt
= 𝑘 𝑇𝑉 − 𝑘𝐸(𝐼 + 𝐼 )
dE (7)
= k1 TVs − kE( Ic + Is ) (7)
dt
The number of uninfected cells, T (cells), was not affected by latency, and Equation
The number of uninfected
(1) summarizes the growth of the cellscells, T (cells), was notrate
at the growth affected by latency,
μ (hr-1). and Equation
The growth (1)
of cells was
summarizes − 1
also affectedthe bygrowth of theinfection
the virus cells at the growth
rate rate µ (h ). The
k1 (mL/virion/hr), growth
which of cells two
involved was also
cell
affected by the virus infection rate k 1 (mL/virion/h), which involved two
concentrations: standard virus particles, Vs (virions/mL); and DIP virus particles, Vd cell concentra-
tions: standard
(virions/mL). virus particles,
Finally, Vs (virions/mL);
it was necessary to includeand DIP virus
a dilution particles,
term, Vd to
D (hr-1), (virions/mL).
account for
Finally, it was necessary to include a dilution term, D (h −1 ), to account for entrance and
entrance and removal of cells (Tin) and media [10].
removal of cells (Tin ) and media [10].
The number of cells containing DIPs, Id (cells), was not affected by latency, as DIP
The number of cells containing DIPs, Id (cells), was not affected by latency, as DIP
particles enter no latent phase. Equation (2) is similar to the equation that accounts for the
particles enter no latent phase. Equation (2) is similar to the equation that accounts for the
number of infected cells including the use of the dilution term, D, in order to account for
number of infected cells including the use of the dilution term, D, in order to account for
the dilution effects of a continuous platform.
the dilution effects of a continuous platform.
In Equation (3), which represents the population of cells infected with STV, the final
term that accounts for virus particles that enter a latent phase, Is (cells), was added. The
last term contains the latent-phase constant, k (mL/virions/hr); the concentration of viral
particles in a latent phase, E (virions/mL); and the number of cells infected with a standard
Processes 2022, 10, 2426 4 of 13

Table 1. Estimated model parameters and initial conditions.

Parameter Definition Value

µ Specific Growth Rate (1/h) 0.027
D Dilution Rate (1/h) 0.0396
f Fraction of Produced DIPs 10−3
k1 Virus Infection Rate (mL/virion/h) 1 × 10−8
k2 Apoptosis Rate of Infected Cells (1/h) 8 × 10−11
k3 Virus Production Rate (virion/mL/cell) 5
k4 Virus Degradation Rate (1/h) 0.035
Tin Cell Concentration in the Feed (cells) 3 × 106
k Latent-Phase Constant (mL/virions/h) 8 × 10−13
T (initial) Target Cell Population (cells) 5 × 106
Is (initial) Cells Infected with STVs Population (cells) 0
Id (initial) Cells Infected with DIPs Population (cells) 0
Ic (initial) Co-infected Cell Population (cells) 0
Vs (initial) STV Concentration (virions/mL) 1.25 × 105
Vd (initial) DIP Concentration (virions/mL) 0
Concentration of Viruses in a Latent Phase
E (initial) 0
(virions/mL)

In Equation (3), which represents the population of cells infected with STV, the final
term that accounts for virus particles that enter a latent phase, Is (cells), was added. The
last term contains the latent-phase constant, k (mL/virions/h); the concentration of viral
particles in a latent phase, E (virions/mL); and the number of cells infected with a standard
virus, Is (cells). This final term estimated the number of cells containing viral particles in
latent phase per hour. These cells, while still infected, could not contribute to production of
more viral particles until the latent-phase period was complete [12]. Therefore, this final
term added a time-delay effect to the virus growth curve.
Equation (4) represents the number of co-infected cells, Ic (cells). These cells contained
both standard viral particles and DIP particles. Due to distribution of the cells, some
co-infected cells contained many viral particles and only a few DIP particles [19]. Other
cells contained many DIP particles and a few viral particles. Therefore, co-infected cells
may contribute slightly to production of virus particles and may contain viral particles in a
latent phase. The final term in this equation accounts for viral particles that enter a latent
phase from a number of co-infected cells.
The final equations in this model account for concentration of standard virus, Vs
(virions/mL); concentration of DIP particles, Vd (virions/mL); and the number of cells
containing viruses in a latent phase, E (cells). Equations (5) and (6) were adapted from the
revised three-dimensional model presented by Frensing et al. (2013), which simplified the
original equations presented in the six ordinary differential equations model. Additionally,
within these final equations, fraction of produced DIPs, f (unitless); virus production rate, k3
(virions/mL/cell); and virus degradation rate, k4 (1/h) are introduced. The final equation,
Equation (7), was a newly introduced equation to the model. This equation accounts for
viral particles entering a latent phase. All the parameters mentioned above, their values,
and their units are summarized in Table 1. The values of the initial conditions are also
given in Table 1 [3,8].

2.3. Transition to Semi-Continuous Modeling

As discussed, the transition to a continuous platform is a significant topic of interest
in the pharmaceutical industry. Due to concerns with batch labeling and investment in new
equipment, a transition to a semi-continuous platform may be more feasible for some com-
panies. This combination of a partially continuous platform and a partially batch platform
would enable pharmaceutical companies to utilize their present, inherent knowledge of
their current batch processes while reaping some of the benefits of a continuous platform.
Processes 2022, 10, 2426 5 of 13

The cell bioreactor would be a continuously operating perfusion bioreactor for this
setup, while viral bioreactors would be changed to batch reactors. The platform would
consist of the perfusion bioreactor, which would operate continuously while feeding cells
to batch-wise wave bags inoculated with viral particles. Those batch wave bags could serve
as the virus bioreactor.
To model the described semi-continuous setup, the continuous model needed to
be adapted. The equation for uninfected cells was adjusted to adapt the model, as
shown below.
dT
= µT + D ( Tin − T ) (8)
dt
This equation removed the terms accounting for cellular death related to viral particles.
The cell bioreactor was operated separately from the virus reactor. Then, the same model
equations were used to model batch reactors, as shown in Equations (8)–(14), but the
dilution terms were removed, as the viral reactors were then operating in batch mode.
These new equations are shown in Equations (8)–(15).

dT
= µT − k1 (Vs + Vd ) T (9)
dt
dId
= k1 Vd T − (k1 Vs − µ) Id (10)
dt
dIs
= k1 Vs T − (k1 Vd + k2 ) Is + kEIs (11)
dt
dIc
= k1 (Vs Id + Vd Is ) − k2 Ic + kEIc (12)
dt
dVs
= k3 Is − k1 ( T + Id ) + k4 Vs (13)
dt
dVd
= k3 Ic + f k3 Is − k1 ( T + Is ) + k4 Vd (14)
dt
dE
= k1 TVs − kE( IC + Is ) (15)
dt
With these new model equations, the semi-continuous platform could be simulated.
First, the model for the cell bioreactor was analyzed. The primary challenge was deter-
mination of the number of cells to dilute from the cell bioreactor in order to inoculate the
viral wave reactors. In this analysis, 5 million cells were assumed to be diluted from the
cell bioreactor at one time to inoculate a 5 L wave bioreactor for virus infection at 1 million
cells per milliliter. Therefore, this simulation was performed on a research-lab scale. This
simulation also assumed that cells would be able to enter exponential growth after they
were bled from the perfusion cell reactor following a brief lag phase.
Finally, once the continuous portion of the semi-continuous platform was simulated,
the batch-wise portion could be simulated. This portion of the simulation focused on
production of virus particles. As simulated by this model, production of viral particles was
comparable to that of other batch models for viral particles as shown by other researchers.

2.4. Parameter Estimation

The latent-phase model added one constant to the model, estimated from the experi-
mental data. Furthermore, the apoptosis rate of the infected cells needed to be re-estimated.
This is because, with a latent phase, fewer cells are killed by viruses per unit of time, as
viruses remain in cells longer before causing cell death. Additionally, virus production
and degradation rates had to be re-estimated due to the extended period that a latent
virus will spend in a cell. The software gPROMS carried out all parameter estimations
and model simulations [20]. Parameter estimation enabled this model to be more versatile.
Parameter estimation and model fitting were performed on data for the measles and rabies
vaccine [16,17].
Processes 2022, 10, 2426 6 of 13

2.5. Sensitivity Analysis

The sensitivity analysis performed was a “one-at-a-time” type of analysis. In this
analysis, one input variable was manipulated while the others were held at standard val-
ues [6,14,18]. The effect on the output was analyzed to determine which output parameters
were most sensitive to a given input parameter.

2.6. Optimization and Flowsheet Modeling

A preliminary optimization was completed. The objective function for this optimiza-
tion problem was to minimize concentration of DIPs, Vd , by manipulating the number of
cells entering the viral reactor, Tin . The objective function is shown in Equation (16), in
which J is the objective function and VD is concentration of DIP-infected cells. Concentra-
tion of DIP-infected cells was subjected to the constraint that it must have been a number
greater than or equal to zero.
J = min VD (16)
VD ∈[0,∞)

This objective function is based on the sensitivity analysis results, which showed that
the concentration of DIPs highly depended on the inlet number of cells. Because DIP
particles inhibit the formation of new viral particles, it is desirable to reduce the number of
DIP particles formed [3,8,19]. Furthermore, reducing the number of DIPs formed will lead
to lower downstream costs if removal of DIP particles is necessary. The optimization study
was also completed with gPROMS through a flowsheet model [20].
Flowsheet modeling provided a realistic platform for the simulation to be performed.
It is often possible to miss critical aspects of the actual operation of the equipment associated
with the model because the equations do not provide a dynamic picture of the actual process
that is being simulated [7]. Furthermore, process parameters that are easier to manipulate
could be readily observed, such as flow rates between reactors, inlets, outlet flow rates, and
other parameters. In this way, the optimization process became more straightforward.
For the latent-phase model, the variables that could be easily manipulated included
the flow rates into, out of, and between the reactors. Therefore, in the optimization study,
these variables were studied.

3. Results
3.1. Parameter Estimation Results
The methodology used to re-estimate the parameters (as found in the literature) and
estimate the latent-phase parameters was model fitting to the original data presented in
the literature [8]. A least-squares methodology was employed to minimize the difference
between experimental data and model-predicted results.
The apoptosis, virus infection, and virus production rates were estimated. The apop-
tosis rate; virus infection rate, k1 ; and virus production rate, k3 had to be re-estimated
because the viral particles used for our model were hypothesized to remain in the cells for
more extended periods due to latency and thus were different from the values predicted
by the literature. In the literature, the apoptosis rate of infected cells was 7.13 × 10−3 per
hour, whereas the newly estimated apoptosis rate was 8 × 10−11 per hour [8]. The newly
estimated viral infection rate in the new latent-phase model was 1 × 10−8 mL/virion/h.
Finally, the newly estimated virus production rate was 5 virions/cell/mL/h, whereas
literature sources estimated this value to be 168 virions/cell/mL/h [3,8].

3.2. Additional Parameter Estimations for Other Viral Vaccines

Parameter estimation was performed for data found in the literature for both a rabies
vaccine and a measles vaccine [16,17]. These vaccines were produced using batch-mode
production techniques. Figure 2 shows the results of this parameter estimation.
Processes 2022, 10, x FOR PEER REVIEW 7 of 13
Processes 2022, 10, x FOR PEER REVIEW 7 of 13

Parameter estimation was performed for data found in the literature for both a rabies
vaccine andParameter estimation
a measles was performed
vaccine [16,17]. for data found
These vaccines were in the literature
produced usingforbatch-mode
both a rabies
Processes 2022, 10, 2426 7 of 13
production techniques. Figure 2 shows the results of this parameter estimation. batch-mode
vaccine and a measles vaccine [16,17]. These vaccines were produced using
production techniques. Figure 2 shows the results of this parameter estimation.

Figure Figure
2. Parameter
Figure estimation
Parameter
2. Parameter results:
estimation
estimation The The
results:
results: figure above
The figure
figure shows
above
above the the
shows
shows results
the results
resultsof of
two
of two parameter
parameter esti-
parameter
estimation attempts to capture data data
fromfromthe literature regarding viral vaccine production. (A)
mation attempts to capture data from the literature regarding viral vaccine production. (A)(A)
estimation attempts to capture the literature regarding viral vaccine production. shows
shows shows
the results from from
the results parameter estimation
parameter for aformeasles
estimation vaccine.
a measles (B) (B)
vaccine. shows
shows thethe
results
resultsofof
the results from parameter estimation for a measles vaccine. (B) shows the results of parameter
parameter estimation
parameter for a for
estimation rabies vaccine.
a rabies BothBoth
vaccine. figures show
figures viral
show titertiter
viral results
resultsas as
given
givenbyby
estimation
experimental datafor
experimental and a rabies
datamodel
vaccine. Both
prediction
and model from
prediction
figures show viral
parameters
from
titer results
estimated
parameters
as given
by gPROMS
estimated by gPROMS
by
[20]. experimental data
[20].
and model prediction from parameters estimated by gPROMS [20].
3.3. Model
3.3. Model
3.3. Comparisons
Comparisons
Model Comparisons
Literature
Literature sources
Literature sources
werewere
sources wereused
used to totocompare
compare
used experimental
experimental
compare results
results
experimental with
with
results model-predicted
model-predicted
with model-predicted
results.
results. Data
Data were were extracted from literature sources regarding continuous cultivationofof
results. Data extracted from from
were extracted literature sources
literature regarding
sources continuous
regarding continuouscultivation
cultivation of an
an influenza
an influenza virus.virus.
The The experimental
experimental setup
setup for for
the the literature
literature sources
sources involvedtwo
involved two
influenza virus. The experimental setup for the literature sources involved two consecutive
consecutive
consecutive reactors.
reactors. The first reactor was a perfusion-based cell cultivation reactor,
reactors. The firstThe firstwas
reactor reactor was a perfusion-based
a perfusion-based cellreactor,
cell cultivation cultivation reactor,
whereas the second
whereas
whereas the the second
second reactor
reactor was awas a perfusion-based
perfusion-based viralviral production
production reactor.
reactor.
reactor was a perfusion-based viral production reactor.
Comparison of Literature Model, Latent-Phase Model, and Experimental Results
Comparison
Comparison of Literature
of Literature Model,Model, Latent-Phase
Latent-Phase Model, Model, and Experimental
and Experimental ResultsResults
Data from a TCID50 graph from the literature about continuous cultivation of an
Data afrom
Data from TCID a 50TCID 50 graph
graph from thefrom the literature
literature about about continuous
continuous cultivation
cultivation of an of an
influenza virus was extracted. [8] Engauge Digitizer software was used. Engauge
influenza
influenza virus was extracted. [8] Engauge Digitizer software was used.
virus was extracted. [8] Engauge Digitizer software was used. Engauge Engauge Digitizer
Digitizer is available on GitHub. Experimental results and model results are shown
is available
Digitizer on GitHub.
is available Experimental
on GitHub. resultsresults
Experimental and model
and results
model are shown
results aregraphically
shown in
graphically in Figure 3.
Figurein3.Figure 3.
graphically

Figure 3. Model comparisons: The figure above shows a comparison between experimental results
(green line), the latent-phase model with the literature parameters (black line), and the latent-phase
Figure Figure
3. Model
model with comparisons:
3. Model
the newly The figure
comparisons:
estimated The above
figure shows
parameters above a comparison
shows
(red line). between
a comparison experimental
between results results
experimental
(green (green
line), the latent-phase model with the literature parameters (black line), and the latent-phase
line), the latent-phase model with the literature parameters (black line), and the latent-phase
model 3.4.
withSemi-Continuous
the newly estimated parameters (red line).
model with the newlyFlowsheet
estimatedModeling
parameters (red line).
Through a semi-continuous setup, the findings of current platforms could be used to
3.4. Semi-Continuous Flowsheet
3.4. Semi-Continuous Modeling
Flowsheet Modeling
optimize batch setup of reactors. The semi-continuous model combines the benefits of the
fully Through
Through a semi-continuous
a semi-continuous
continuous setup,
platform and setup,
thethe the findings
findings of current
of current
semi-continuous and platforms
platforms
platform acould
could be
provides used be used
to
complete
to optimize
optimize batch
batch setup setup of reactors.
of reactors. The semi-continuous
The semi-continuous model combines
model combines theofbenefits
the benefits the of
the fully continuous
fully continuous platform platform
and the and the semi-continuous
semi-continuous platform platform and provides
and provides a complete
a complete
picture of viral production dynamics in cell culture. In the desired semi-continuous setup, a
cell culture reactor would be operated continuously as a perfusion reactor. Viral production
reactors would be operated as batch-wise wave bag reactors. Figure 4 shows a simulated
result of bleeding of cells from the cell cultivation reactor to batch-wise wave reactors for
viral production.
 picture of viral production dynamics in cell culture. In the desired semi-continuous setup,
picture of aviral
cellproduction
culture reactor
dynamicswould beculture.
in cell operated continuously
In the as a perfusion
desired semi-continuous reactor. Viral
setup,
a cell culture reactor would be operated continuously as a perfusion reactor. Viral 4 shows
production reactors would be operated as batch-wise wave bag reactors. Figure
productiona simulated resultbe
reactors would ofoperated
bleeding asof batch-wise
cells from the cellbag
wave cultivation
reactors.reactor
Figureto4 batch-wise
shows wave
Processes 2022, 10, 2426
a simulatedreactors forbleeding
result of viral production.
of cells from the cell cultivation reactor to batch-wise wave
8 of 13

reactors for viral production.

Figure 4. Semi-continuous bleed: The graph above shows the potential results of bleeding cells from
a perfusion cell culture reactor to batch-mode wave bag reactors for viral production.
Figure 4. Semi-continuous bleed: The graph
Figure 4. Semi-continuous bleed: above shows
The graph the potential
above shows theresults of bleeding
potential cells
results of from cells from
bleeding
a perfusionacell culture
perfusion reactor to
cell culture batch-mode
reactor wave bag reactors for viral production.
to batch-mode wave bag reactors for viral production.
3.5. Sensitivity Analysis and Optimization
3.5. Sensitivity Two processes
3.5. Sensitivity
Analysis were
Analysis
and andcompleted to further analyze the latency model created in this
Optimization
Optimization
paper.
Two processes First, a sensitivity
Two processes
were completed analysis
were completed was
to furtherto performed
further
analyze to assess
analyze
the latencythe input
latency
model and output
model
created thisparameter
increated in this
correlations.
paper.
paper. First, Second,
a sensitivity an
First, a sensitivity optimization
analysis analysis analysis
was performed
was performed was performed
to input
to assess assessand
input to improve
and output
output the platform
parameter
parameter cor-
process.
relations. Second, an optimization analysis was performed to improve
correlations. Second, an optimization analysis was performed to improve the platform the platform process.
process.
Sensitivity Analysis
Sensitivity Analysis Results
Results
A sensitivity
A sensitivity
Sensitivity Analysis analysis was
Results analysis was performed
performed for for the
thecontinuous
continuouslatent-phase
latent-phase model.
model. The
The
sensitivity
sensitivity analysis
analysis results are
results summarized
are summarized in Figure
in 5. The
Figure coloration
5.
A sensitivity analysis was performed for the continuous latent-phase model. The The in Figure
coloration 4
in indicates
Figure 4
whether
indicates or not a
whether50%
or change
not a in
50% a given
change input
in a had
given a positive
input had
sensitivity analysis results are summarized in Figure 5. The coloration in Figure 4or
a negative
positive impact
or on
negative a given
impact
output.
on a given
indicates whether Different
or output.
not shades
a 50% of red
Different
change in a indicate
shades of red
given adverse
had aeffects,
indicate
input adverse
positiveand different
effects,
or shades
andimpact
negative ofshades
different green
indicate
of green positive
indicate impacts.
positive DarknessDarkness
impacts. of a shadeof indicates
a shade magnitude
indicates of impact;
magnitude of darker
impact;
on a given output. Different shades of red indicate adverse effects, and different shades
shades
darker indicate
shades more significant
indicate more impacts.impacts.
significant
of green indicate positive impacts. Darkness of a shade indicates magnitude of impact;
darker shades indicate more significant impacts.

Figure 5. Sensitivity
Figure 5. Sensitivity analysis: The figure
analysis: The figure above
above shows
shows aa correlation
correlation heat
heat map
map representing
representing the
the
sensitivity
sensitivity analysis
analysis results.
results. The colors in the map indicate
indicate negative
negative (red)
(red) correlation
correlation or
or positive
positive
Figure 5. Sensitivity analysis: The figure above shows a correlation heat map representing the
(green) correlation. Darkness of a color indicates a stronger correlation. Therefore, for the growth
sensitivity analysis results. The colors in the map indicate negative (red) correlation or positive
rate, µ, a 50% change in input had a strong negative impact on the population of DIPs, Id .

3.6. Optimization and Flowsheet Modeling Results

The optimization study aimed to reduce the number of defective interfering particles
formed. The rationale for performing this study was that defective interfering particles
interfere with viral reproduction. Therefore, more viral particles per cell could be produced
 (green) correlation. Darkness of a color indicates a stronger correlation. Therefore, for the growth
rate, μ, a 50% change in input had a strong negative impact on the population of DIPs, Id.

Processes 2022, 10, 2426 9 of 13

3.6. Optimization and Flowsheet Modeling Results
The optimization study aimed to reduce the number of defective interfering particles
formed. The rationale for performing this study was that defective interfering particles
ifinterfere
the number
with of DIPs
viral could be reduced.
reproduction. Therefore, This study
more was
viral performed
particles utilizing
per cell could abeflowsheet
model
produced built with
if the gPROMS
number software
of DIPs could be[20].
reduced. This study was performed utilizing a
flowsheet model builtmodel
The flowsheet with gPROMS
created software [20].
for the “latent-phase” model is shown in Figure 6. The
The flowsheet
flowsheet model created
model utilized for theequations
the model “latent-phase”
givenmodel
in theis shown in Figure
previous section6. The
and built-in
flowsheet model utilized the model equations given in the previous section
functions and equations provided by gPROMS [20]. These built-in functions enabled a and built-in
functions and equations provided by gPROMS [20]. These built-in functions enabled a
connection between the model within the cell bioreactor and the model within the viral
connection between the model within the cell bioreactor and the model within the viral
reactor. In this way, the model could be simulated closer to reality. The flowsheet model
reactor. In this way, the model could be simulated closer to reality. The flowsheet model
enabled variationofofthethe
enabled variation number
number of cells
of cells introduced
introduced into
into the thereactor.
viral viral reactor.
The numberThe ofnumber of
DIPs formed could be minimized without significant impact
DIPs formed could be minimized without significant impact to the viral titer. to the viral titer.

Figure Continuous
6. Continuous
Figure 6. flowsheet
flowsheet model:
model: The The
figurefigure
aboveabove
showsshows the flowsheet
the flowsheet model developed
model developed for for
the continuous
the continuous platform
platform presented in this
presented paper.
in this The flowsheet
paper. was developed
The flowsheet in gPROMS.
was developed in gPROMS.

The results
The resultsofofthe
theoptimization
optimizationstudy are are
study shown in Figure
shown 7. There
in Figure was a was
7. There reduction
a reduction of
of one-third in number of DIPs formed, which was accomplished through
one-third in number of DIPs formed, which was accomplished through reduction reduction of of the
the number of cells introduced to the viral reactor by 10%. This was also met with
number of cells introduced to the viral reactor by 10%. This was also met with a slight a slight
Processes 2022, 10, x FOR PEER REVIEW
reduction ofof approximately
approximately 7.4% 10 of 13
reduction 7.4%inin the
the peak
peak number
numberof ofSTV
STV(viral
(viralparticles)
particles)formed.
formed. Finally,
Finally, there was a reduction of 42% in the number of co-infected cells.
there was a reduction of 42% in the number of co-infected cells.

Figure
Figure7.7.Model
Model optimization: The figures
optimization: above show
The figures abovetheshow
results of results
the the optimization performed onperformed
of the optimization
this model. (A) The optimization study (which involved reducing the number of cells being fed into
on this model. (A) The optimization study (which involved reducing the number of cells being
the viral reactor by 10%) showed a reduction in concentration of DIP particles formed by one-third.
fed This
(B) into reduction
the viral in
reactor by 10%)resulted
DIP particles showed in aa reduction in concentration
corresponding of DIP particles
reduction in co-infected cells by formed by
one-third. (B)42%.
approximately This reduction in DIP particles resulted in a corresponding reduction in co-infected
cells by approximately 42%.
4. Discussion
The results of the analyses presented herein indicate that the model developed is a
versatile model with enhanced accuracy. Several conclusions could be drawn through
parameter estimation, model fitting, and optimization.
Processes 2022, 10, 2426 10 of 13

4. Discussion
The results of the analyses presented herein indicate that the model developed is a
versatile model with enhanced accuracy. Several conclusions could be drawn through
parameter estimation, model fitting, and optimization.

4.1. Parameter Estimation for Other Viral Vaccines

Figure 2 shows the parameter estimation results for the rabies and the measles vaccine.
The gap seen between the modeled results and the experimental results is explained by
the fact that both of these experimental results were derived from batch-mode production
techniques. The model used was continuous; therefore, there were terms in the model that
caused a gap between the modeled and the experimental results. In Figure 2A, the tailing
observed was not captured by the model because the dilution term present in the model
caused a reduction in the modeled viral titer that was faster than the reduction observed
during the experiment. Overall, however, it can be clearly seen that the model can capture
general trends observed in different viral cultures. A similar phenomenon, which used a
single model of population balance modeling to track the production of viruses by cell lines
that are altered differently (via genetic engineering), has been published [25]. In general,
these universal models are commonly based on population balance models and can be very
useful in process optimization [26–30].

4.2. Comparison of Literature Model, Latent-Phase Model, and Experimental Results

As shown in Figure 3, the plaque-forming units from the experimental data peaked
around 4.4 × 106 PFUs. The latency model peaked at 4.56 × 106 PFUs. Furthermore,
there were several later peaks in the experimental data around days 7 and 15. Only the
latent-phase model can capture these late-phase dynamics.
As shown in Figure 3, neither model had the same peak plaque-forming units as the
experimental results. The experimental data showed a slight lag period before viruses
begin to accumulate. The latent-phase model can capture this lag time. This model peaked
after the experimental data but is closer in value. Therefore, the latent-phase model can
better capture experimental data trends.
Finally, under the curve, the latent-phase model has a more comparable area to the
experimental data. The approximate area under the curve for the experimental data from
the first two days was 3.3 × 106, and the area under the curve for the latent-phase model
from the first two days was approximately 3.4 × 106 . Therefore, the latent-phase model
accurately captures PFU-AUC (plaque-forming units—area under the curve in units of
plaque-forming units—days).

4.3. Semi-Continuous Modeling

Based on Figure 4, the bleeding from the perfusion cell reactor took approximately
2.5 days to return to the initial viable cell density. This enabled multiple wave bag reactors
to be inoculated in a short period. Additionally, each wave-bag viral reactor could be
designated as a batch, thus simplifying batch designation, which can be difficult with
continuous cultivation.

4.4. Sensitivity Analysis

The sensitivity analysis showed that most of the outputs were not dramatically affected
by an increase and/or a perturbation in the input parameters. It was discovered that
population and concentration of DIPs were affected by an increase in specific growth rate,
µ; virus production rate, k3 ; and cell concentration in the feed, Tin . The rationale is that if
the specific growth rate increases for cells, or the number of cells added to the cell feed
increases, then the number of cells will increase, and thus the number of cells to be infected
will also increase. This, in turn, will enable the number of DIPs formed to increase, as
more defective viral particles can be formed due to the increased amount of infection
occurring. Furthermore, if the viral production rate increases, the number of DIPs formed
Processes 2022, 10, 2426 11 of 13

must decrease, as viral particles cannot form in the presence of DIPs, and thus these two
must be inversely proportional.
In addition to number of DIPs formed being affected by changes in input parameters,
number of standard viruses formed was also more affected if viral production rate, k3,
was increased. This is rational, as number of viruses formed and viral production rate are
directly proportional.
In general, most of the output parameters were not sensitive to changes in the input
parameters. However, the revelation that production of DIPs is affected by specific growth
rate of cells is interesting. This suggests that cells with slower growth rates may not be
as affected by DIPs as are other cells, as concentration of DIPs formed tended to increase
with increasing specific growth rates per this simulation. Therefore, this information could
determine what cells to use to produce viral vaccines.

4.5. Optimization and Flowsheet Modeling

This optimization study successfully determined a more optimal process. This sim-
ulation demonstrated that the number of DIPs could be dramatically reduced through
reduction of the number of cells introduced into the viral reactor. Furthermore, if fewer cells
are required to be transferred from the cell bioreactor to the viral reactor, it may be possible
to reduce the size of the cell bioreactor. This reduction in the size of the cell bioreactor could
cause a smaller capital investment requirement if a bioreactor must be purchased, cause
a minor footprint requirement, and cause a smaller amount of media to be used during
the cultivation. Therefore, potential benefits of this optimization study include multiple
benefits for the proposed continuous process.

5. Conclusions
Modeling influenza A growth dynamics for vaccine production is critical to under-
standing and optimizing the process. This model’s goal was to better explain realistic
viral growth dynamics observed in continuous cultivation. The experimental data in the
literature showed a noticeable delay in viral reproduction in the early phases of viral pro-
duction [3,8]. Adding latent-phase effects to the continuous model improved the model’s
accuracy in tracking experimental results. Furthermore, it proved effective in capturing
fed-batch dynamics that literature sources explored.
Continuous production enables various benefits, including increased titer, faster pro-
duction time, and less downtime. The viral titer obtained via a continuous platform can be
100-fold greater than batch-wise derived titers [1,2,7,10]. The modeled titer for influenza A
studied in this report reached approximately 108 TCID50 /mL, whereas batch-wise viral
titers for influenza have been reported in the literature at approximately 106 TCID50 /mL.
Therefore, the obtained titer has a clear advantage when a continuous platform is used.
Finally, the developed latent-phase model was adapted to a flowsheet model used
in optimization studies. It was demonstrated that use of a flowsheet model enabled
optimization studies, which showed that there could be a reduction in the number of
DIPs through manipulation of the number of cells fed to the viral reactor. Reducing
the number of cells needed to inoculate the viral reactor makes it possible to require a
smaller reactor for the cell cultivation reactor, thus reducing overall capital and costs of
consumables. Additionally, reduction of DIP particles formed could positively impact
downstream purification. The model presented in this paper also provides a methodology
for modeling various viral vaccines. This is possible through parameter estimation, which
enables the model to be tailored to different viral vaccine data.

6. Future Directions
The model presented in this paper provides a robust model for continuous production
of a viral vaccine. Newly formulated influenza vaccines are developed to ensure that “flu
season” can be handled without widespread infection. Therefore, accurate models could be
critical in developing new formulations of the flu vaccine and could significantly impact
Processes 2022, 10, 2426 12 of 13

future research endeavors. Additionally, this model has proven versatile, so it may be used
to develop platforms for various viral vaccines.
Development of universal mathematical models for important production platforms
such as viral vaccines cannot be ignored. Universal models enable users to tailor each
model to suit their desired platform, enabling faster development. The model presented
herein is part of the first steps toward creating a universal vaccine-production model.
Future efforts to improve this model’s usefulness will be invaluable to the pharmaceutical
industry as a whole.

Author Contributions: C.S.M. developed and tested the gPROMS/gSOLIDS mathematical model.
C.S.M. performed all parameter estimation studies and optimization studies. C.S.M. drafted the
manuscript. S.Y. assisted in the manuscript review and revision. All authors have read and agreed to
the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: All gPROMS models generated in this manuscript are available upon
request from the corresponding author, Seongkyu Yoon.
Acknowledgments: The authors would like to acknowledge the University of Massachusetts Lowell
for its continued support and PSE Enterprises for providing the gPROMS/gSOLIDS software.
Conflicts of Interest: The authors declare no conflict of interest.

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