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Viral Vaccine Manufacturing for COVID-19 and other Infectious Diseases –

Process Modeling and Techno-Economic Assessment (TEA) using SuperPro
Designer.

Preprint · January 2022

DOI: 10.13140/RG.2.2.24745.67687

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 Viral Vaccine Manufacturing
Process Modeling and Evaluation using
SuperPro Designer®
by

Rafael da Gama, Nikiforos Misailidis and Demetri Petrides

February 2022

This is the ReadMe file of a SuperPro Designer example that analyzes the manufacturing of whole virus
vaccines such as inactivated virus vaccines, live attenuated vaccines and viral vector vaccines (e.g., those
developed against COVID-19 by Oxford/AstraZeneca and Johnson & Johnson). This example comes with
a detailed and a simplified model of the process. The flowsheet of the simplified model is appended to the
bottom of this document. You may test-drive both the detailed and simplified models by downloading the
functional evaluation edition of SuperPro Designer from the downloads page of our website
(www.intelligen.com). All the files of this example can be found in the Examples \ Pharmaceuticals \ Viral
Vaccine folder. The default installation path of the SuperPro Designer Examples folder follows below.

C:\ Users \ Public \ Public Documents \ Intelligen \ SuperPro Designer \ v12 \ Process Library \ Examples

If you have any questions regarding this example or SuperPro Designer in general, please send an email
message to dpetrides@intelligen.com

INTELLIGEN, INC.
Simulation, Design, and Scheduling Tools
For the Process Manufacturing Industries
www.intelligen.com
Introduction

Vaccines are considered the most effective way to prevent infectious diseases [1], saving millions of lives
every year [2]. In the case of viral diseases, vaccines are even more crucial given that many of them cannot
be cured by antiviral drugs [3]. Examples of viral vaccines include those that immunize against influenza,
hepatitis A & B, poliomyelitis, measles, rubella, chickenpox, mumps [4], and, more recently, COVID-19 [2].
They may be categorized as follows:

• Live Attenuated Virus Vaccine: based on a weakened form of the virus, usually obtained by genetic
modification of the pathogen. Attenuated vaccines generally elicit strong immune responses but
may be dangerous for immunodeficient individuals [5].
• Inactivated Whole Virus Vaccine: based on distorting the native structure of the virus, rendering it
unable to replicate. Inactivation is usually carried out by chemical agents (typically formaldehyde
or β-propiolactone), although heat and radiation may be employed as well [6].
• Split Virus Vaccine: a vaccine obtained by breaking down the viral particle into multiple pieces using
a detergent, so that the virus is inactivated [6].
• Protein Subunit Vaccine: based on a specific protein of the virus. It may be produced by isolating
the protein from the virus or by recombinant DNA technology. Protein subunit vaccines are safer
and more stable than live attenuated vaccines; however, they usually elicit a weaker immune
response [7]. A vaccine of this type was developed by Novavax for COVID-19, and it has been
approved for emergency use in Indonesia and the Philippines [8].
• Conjugate Vaccine: based on the combination of a weak antigen and a strong antigen that are
joined together covalently. This type of vaccine is more common for bacterial pathogens; typically
a polysaccharide of the bacterial cell wall is linked with a strongly immunogenic protein such as the
tetanus toxoid [9]. A viral vaccine of this type was nonetheless developed in Cuba in 2020 for
COVID-19, joining a protein from SARS-CoV-2 with the tetanus toxoid [10].
• Virus-Like Particle (VLP) Vaccine: VLPs consist of structures made of proteins that resemble the
actual virus particle but lack its genetic material, and therefore are incapable of replicating. This
type of vaccine can be produced by synthesizing one or more recombinant proteins capable of self-
assembling into the desired VLP [11,12].
• Viral Vector Vaccine: based on a harmless virus that carries a gene coding for a protein of another
virus (the target pathogen). The carrier virus (“vector”) infects host cells and delivers that gene,
leading to the expression of the antigenic protein in the host. This type of vaccine is fairly new; the
first viral vector vaccines ever approved for human use were authorized in 2019 and 2020, both of
them against Ebola. Three major COVID-19 vaccines are also based on this technology: the
vaccines from Oxford/AstraZeneca, Johnson & Johnson and the Gamaleya Institute [13].
• Messenger RNA (mRNA) Vaccine: based on a messenger RNA molecule that contains a gene
coding for a viral protein. This type of vaccine was first approved in 2020, when two mRNA vaccines

1
 against COVID-19 (one developed by Pfizer/BioNTech and the other by Moderna) received
emergency use authorization [13]. The manufacturing of mRNA vaccines is analyzed in another
SuperPro Designer example, located in the Examples \ Pharmaceuticals folder.
• Plasmid DNA (pDNA) Vaccine: based on a plasmid DNA molecule that contains a gene coding for
a viral protein [14]. The first pDNA vaccine ever approved for human use was developed by Zydus
Cadila in India for COVID-19. It received emergency use authorization in India in 2021 [15]. The
manufacturing of pDNA is analyzed in another SuperPro Designer example, located in the
Examples \ Pharmaceuticals folder.

The various types of vaccines listed above may be further grouped into three main categories, depending
on their structure and mechanism: whole virus, subunit, or nucleic acid, as shown in Figure 1.

Figure 1: Types of viral vaccines grouped according to their structure and/or mechanism.

Whole virus vaccines, as their name implies, are those consisting of entire viral particles, and include
inactivated whole viruses and live attenuated viruses. Viral vector vaccines are technically based on whole
viruses as well, though they are not usually categorized as such. Nucleic acid vaccines, on the other hand,
are based on the delivery of a DNA or RNA sequence to the recipient cells so that they express an antigenic
protein; viral vector vaccines belong to this category along with pDNA and mRNA vaccines. Subunit
vaccines are those based on using a piece of the pathogenic virus (e.g. a surface protein) rather than the

2
entire virus; they include protein subunit vaccines, conjugate vaccines and VLPs. Split virus vaccines are
somewhere between whole virus and subunit vaccines, given that they are obtained by breaking down
whole viral particles into multiple pieces.

Manufacture of Whole Virus Vaccines

The manufacturing processes of the various types of whole virus vaccines (here including live attenuated,
inactivated whole virus, and viral vector vaccines) have many similarities among each other given that they
all involve the inoculation, replication, recovery, and purification of entire viral particles (VPs). VPs are
usually produced in one of three manners: (1) hen eggs; (2) adherent cell culture; or (3) suspension cell
culture [16]. Virus production in hen eggs is a traditional process dating back to 1931 still widely employed
today, especially for influenza vaccines [17]. However, it has several drawbacks: it is labor-intensive and
time-consuming [18]; and it requires virus adaptation to eggs, during which the virus can mutate and
become less effective. In addition, manufacturing in eggs is vulnerable to avian disease outbreaks, which
could wipe out the supply of eggs and thus jeopardize vaccine production. Besides, the supply of eggs
might be insufficient for vaccine production in the case of a pandemic [19]. For all these reasons, vaccine
manufacturing has been moving away from eggs to cell culture-based processes.

Cell culture (using either adherent or suspended cells) enables virus production in a closed and controlled
environment [1], using well-characterized substrates [4]. As such, it presents many opportunities for process
optimization and reduction of manufacturing costs and facility space. Moreover, it makes it possible to easily
increase production capacity to meet surges in demand [1], and to produce vaccines faster [4]. Besides, if
mammalian cells are used, the glycosylation profile of the virus is closer to that of humans and thus allergic
reactions are avoided [19,20]. In general, mammalian cells, avian cells or insect cell-baculovirus systems
are employed for virus production in cell culture. Most cell lines grow naturally in adherent conditions [21],
which is an advantage of this modality. Adherent culture may be carried out in static systems or bioreactors.
Static systems include roller bottles and multilayered systems such as Cell Factory (Thermo Fisher
Scientific), CellCube (Corning) or CellSTACK (Corning), which have larger surface areas than roller bottles.
Static systems are simple and robust, but require extensive manual/robot handling, entail a high sterility
risk, offer limited process control, and are not easily scalable. Nonetheless, they are appropriate for
producing VPs on laboratory scale or for generating virus seeds for bioreactors [1].

There are two types of bioreactors for adherent culture: microcarrier-based or packed-bed bioreactors. In
the first case, cells are grown on microcarriers which are typically porous or non-porous beads made of
glass, plastic or dextran. By the end of cell culture, the beads can be easily separated from the medium by
simple sedimentation; this can also be done in the middle of cell culture, enabling medium exchange or
multiple harvests during the cultivation process. However, microcarrier systems have several
disadvantages: inoculation is troublesome; use of serum-free media may hamper cell attachment;
microcarriers are expensive and usually cannot be recycled; and the recovery of intracellular viruses may

3
not be optimal, especially in the case of porous beads. In the case of packed-bed bioreactors, cells grow
on a porous matrix made of polyester microfibers. The major limitation of this system is oxygen transfer
which can curb cell growth. A notable advantage of cell culture in bioreactors is the greater control over
process parameters, which facilitates process optimization [1].

Animal cells can also be grown suspended in liquid medium, i.e. in suspension culture. Suspension culture
is typically conducted in shake-flasks for laboratory scale or in bioreactors (rocking bioreactors or stirred-
tank bioreactors) for large-scale production. A major advantage of suspension culture is the ease to scale
up; in addition, suspension culture in bioreactors offers considerable process control and optimization
options, making it possible to achieve very high cell densities and virus yields. Three basic modes are
employed for growing cells in suspension: batch, fed batch and perfusion, which tend to give increasing cell
and virus yields (at the expense of greater process complexity and risk of contamination). In the case of
perfusion, a crossflow filter with a microfiltration membrane is placed either inside or outside of the vessel;
medium is continuously fed to the vessel and removed through the filter which retains the cells. As a result,
very high cell densities can be achieved, as well as very high VP titers [1].

Nevertheless, suspension culture usually requires adaptation of adherent cells to suspension conditions,
which may be a cumbersome process and lead to undesired modifications in the cell (such as changes in
the surface receptors that reduce the cell’s susceptibility to viral infection). Moreover, not all cell lines can
be adapted to grow satisfactorily in suspension. For cells well-adapted to suspension culture, however, this
cultivation mode is the method of choice for large-scale production [1].

For both adherent and suspended cells, virus production typically starts with a cell growth phase before
any virus is introduced, so that a high number of cells can be achieved (after all, viral infection hampers cell
growth). After that phase, the virus of interest is added to the cell culture; this is called the infection step,
and the ratio of VPs to cells, multiplicity of infection (MOI). The MOI is an important parameter for virus
production [22]. The virus then enters the cells, seizes their metabolic machinery, and starts making copies
of itself, mimicking the process that happens when it infects a live organism; this stage is named virus
replication. By the end of this step, depending on the nature of the virus, the culture conditions and its
duration, cells may be lysed by the virus or not, and the VPs produced may be found outside cells, inside
cells, or both [17,21,23]. For instance, enveloped viruses are released to the extracellular environment by
budding from the cell membrane [21,23].

The first step after viral replication is the harvest of VPs. If a significant portion of VPs is found inside cells,
a cell disruption (lysis) step is required. This may be accomplished by a variety of methods, though most
often by chemical lysis using non-ionic detergents such as Triton X-100 [17,18,21,23]. Once VPs are
released to the extracellular medium, cell debris, cells, and other large impurities can be removed by
centrifugation, filtration (depth filtration, normal flow microfiltration or crossflow microfiltration) or, in the case
of microcarriers, sedimentation. This is the clarification step [16–18,21,23,24].

4
Upon cell lysis, a large quantity of proteins and nucleic acids are released to the medium along with the
VPs. Host cell proteins must be removed as they may elicit allergic reactions and other side effects to the
vaccine recipient [16,24]. Extensive removal of host cell DNA is also crucial due to safety and regulatory
reasons; for that purpose, nucleases such as Benzonase® are often added to the cell lysate at some point
in the downstream process [16,18,21,23,24]. An alternative method to reduce the amount of DNA in the
lysate is to selectively precipitate it with cationic agents such as domiphen bromide; this can be done before
the clarification step, so that the precipitate is removed together with cell debris and other large impurities
[16,23].

A concentration step usually follows clarification. Concentration of the virus bulk is typically accomplished
by crossflow ultrafiltration (UF) or chromatography (in bind-and-elute mode). UF is performed using flat
sheet or hollow fiber membranes with a nominal molecular weight limit (NMWL) that is comfortably smaller
than the VPs in question (i.e., the VPs are retained by the UF membrane). Chromatography, on the other
hand, is performed using packed-bed columns, membrane adsorbers or monoliths. Monoliths and
membrane adsorbers are particularly advantageous for VP purification as they have much larger pores
than packed-bed columns, and therefore have much larger binding capacities and enable higher flow rates
[16,18,21,23,24]. Chromatography is also useful for purification steps after concentration because, unlike
UF, it is capable of separating impurities that have approximately the same size as VPs. The most common
types of chromatography used in the purification of VPs are ion-exchange chromatography (IEC), affinity
chromatography (AC), size-exclusion chromatography (SEC), hydrophobic interaction chromatography
(HIC), and multimodal chromatography (MMC) [16,23]. It is worth noting that IEC, and anion-exchange
chromatography in particular, is the type of chromatography most widely used in virus purification given
that most viruses have isoelectric points below 6 (i.e. they have a net negative charge under neutral pH)
[24].

If UF is employed for concentration, a diafiltration (DF) step is usually performed right after concentration
to remove small molecule impurities and introduce the VP into an appropriate buffer for the next unit
operation, which is typically a chromatography step. Buffer exchange is also carried out towards the end of
the downstream process to introduce the VPs into an appropriate formulation buffer. This may be done
through UF/DF or SEC. SEC has a lower capacity, poor pressure resistance (leading to low flow rates), and
dilutes the product stream, whereas UF/DF can handle large feed volumes more easily and concentrate
VPs, as mentioned above [16,18,21,23,24].

In the case of inactivated vaccines, it is also necessary to render the virus incapable of replicating. Viral
inactivation is usually achieved by chemical agents, typically formaldehyde or β-propiolactone, although
heat and radiation methods may be employed as well. Inactivation may be conducted at different stages of
the process; however, in the case of chemical inactivation, it may be necessary to remove the inactivating
agent from the product [6].

5
A generic process flow chart for the manufacture of whole virus vaccines is presented in Figure 2. It is worth
noting that the downstream processing of VLP vaccines is substantially similar to that of whole virus
vaccines [4,21]. The upstream portion of the process may be significantly different, however, given that
VLPs are usually produced by recombinant expression systems such as baker’s yeast [14] and therefore
viral infection does not take place.

Figure 2: Generic flow chart for the manufacture of whole virus vaccines.

Process Description

The present example was adapted from an article that our team published in a peer-reviewed journal [25],
and it includes two SuperPro Designer files:

• ViralVaccine_Detailed
• ViralVaccine_Simplified

Both files model essentially the same process: the industrial production of whole virus vaccines based on
suspension culture of animal cells. The process starts with cell expansion in shake flasks, rocking

6
bioreactors, and a stirred tank bioreactor. Subsequently, viral production takes place in a stirred tank
bioreactor. After viral replication, cells are disrupted with the aid of a detergent, and DNA is selectively
precipitated with domiphen bromide. The resulting DNA precipitate and cell debris are then removed by a
combination of centrifugation and depth filtration. Finally, the clarified bulk is subjected to ultrafiltration-
diafiltration, anion-exchange chromatography, viral inactivation and a second ultrafiltration-diafiltration step.
The process generates 11 kg of viral particles = 2.2 × 1019 viral particles per year, which corresponds to
400 million vaccine doses per year.

The ViralVaccine_Detailed file models the vaccine manufacturing process comprehensively, including
media and buffer preparation as well as chromatography pre- and post-processing steps. The
ViralVaccine_Simplified file represents a simpler version of the same process lacking the media and
buffer preparation steps and chromatography pre- and post-processing steps. It should be noted that the
process description, scheduling, and cost analysis presented next are based on the ViralVaccine_Detailed
file. The ViralVaccine_Simplified version is recommended for students that have access to the academic
departmental license of SuperPro Designer which has a limit of 25 unit procedures per flowsheet.

For reporting and analysis purposes, the process has been divided into three sections:

• Cell Culture (dark red icons)

• Primary Recovery (green icons)
• Purification (blue icons)

Flowsheet sections in SuperPro are simply sets of related unit procedures (processing steps). For
information on how to specify flowsheet sections and edit their properties, please use the Help tool (Help
Index Section). The contents of each process section are described in greater detail next. The flowsheet
of the simplified model is appended to the bottom of this document.

Cell Culture

Cell Expansion

The process starts with the expansion of the animal cells in which the virus is going to replicate. Cell
expansion begins in shake flasks (up to 5 L of working volume), then passes to rocking bioreactors (between
5 L and 300 L of working volume), and finally takes place in stirred tank bioreactors (more than 300 L of
working volume), with a step expansion factor of 7. Every cell expansion step operates in batch mode,
starting with a cell concentration of 0.1 g/L = 0.2 × 106 cells/mL and ending with a concentration of 0.7 g/L
= 1.4 × 106 cells/mL after 87 h of culture, yielding a doubling time of 31 h [26]. Each expansion step is
initiated by adding 6 volumes of fresh medium to the whole cell culture of the previous step.

Cell growth is performed at 37 °C with an aeration rate of 0.05 VVM of sterile air. Serum-free cell culture
medium is used containing glucose (6.5 g/L), amino acids (3.0 g/L), and other components (10.5 g/L)

7
dissolved in WFI. Media are prepared in dedicated tanks, sterilized with 0.2 µm filters and stored in holding
tanks that feed the bioreactors.

Every cell expansion step follows exponential growth, which is modeled in SuperPro Designer by a Batch
Kinetic Fermentation operation with the following mass stoichiometry:

100 Glucose + 13 Amino Acids + 85 O2 → 116 CO2 + 30 Cells + 52 Water (1)

The stoichiometric coefficients above were determined by elemental balance, considering a yield of
biomass on glucose of 0.30 g/g; an average amino acid formula of C 5.37H11.06O2.23N1.99 (based on media
composition published in the patent literature [27]); and an empirical formula for cells (C1.00H1.61O0.56N0.16
[28]). The heat released by cell growth is assumed to be 3700 kcal/kg of consumed O2. More details on
kinetic fermentation models are provided at the end of this document.

To convert cell numbers into mass units, a conversion factor of 2 × 10 6 cells/mg of dry cell weight was
employed. This factor assumes that cells are spherical, with an average diameter of 14 µm [29], a specific
wet weight of 1.05 g/cm3 [30]; and a dry mass content of 30% w/w [31].

Viral Replication

Viral replication only occurs in the last stage of the Cell Culture section (P-21 / BR-201), and it is carried
out in a stirred tank bioreactor. This processing step comprises two distinct phases: a cell growth phase
and a viral replication phase:

• Cell Growth: this phase starts by adding a seed cell culture with a cell density of 0.7 g/L = 1.4
× 106 cells/mL to 6 volumes of fresh medium resulting in a cell density of 0.1 g/L = 0.2 × 106
cells/mL, identical to the cell expansion steps described earlier. Similarly, the cell growth phase
follows an exponential growth model with the same doubling time and stoichiometry as the cell
expansion steps.
• Viral Replication: after 64 h of cell growth (when the cell density reaches 0.45 g/L = 0.9 × 106
cells/mL) [32], the cell growth phase is deemed complete, and the viral replication phase is
triggered by infecting the cell culture with a concentrated virus suspension (0.5 g/L = 1 × 1012
VP/mL). The volume of suspension is such that the number of VPs per cell, called multiplicity
of infection (MOI), is equal to 280. The viral replication phase takes 48 h [26,32–34] and is
modeled in SuperPro Designer by a Batch Stoichiometric Fermentation operation with a
stoichiometry similar to that used to model cell growth:

100 Glucose + 13 Amino Acids + 85 O2 → 116 CO2 + 0.61 VPs + 29.39 Cells + 52 Water (2)

The conversion rate of the viral replication reaction is assumed to be 80%, and the
stoichiometric coefficient for the VPs is such that the VP concentration by the end of viral

8
 replication is 0.025 g/L = 5 × 1010 VP/mL. The other coefficients of the equation were
determined by elemental balance, similarly to eq. (1), assuming that VPs and dry cell weight
have the same empirical formula, and that the yield of biomass (cells + VPs) on glucose was
equal to 0.30 g/g. The heat released by viral replication is assumed to be 3700 kcal/kg of
consumed O2. VPs are treated as an extracellular, secreted product in this model, even though
a fraction (or all) of VPs may be found inside cells by the end of viral replication, depending on
the nature of the virus. In addition, as in the case of the cell density, VP numbers had to be
converted into mass units. A conversion factor of 2 × 1012 VP/mg was used, assuming that VPs
are spherical, with an average diameter of 90 nm and a specific weight of 1.34 g/cm 3 [35,36].

Primary Recovery

Chemical Lysis

Assuming that a significant portion of the virus is located inside cells by the end of replication, a cell
disruption step must be performed. In this example, cell disruption is achieved by chemical lysis: first, a
concentrated lysis buffer (MgCl2 10 mM, sucrose 50% w/w, Tris-HCl 500 mM, and polysorbate-80 1% w/w)
is added at a ratio of 1 volume of lysis buffer to 9 volumes of cell harvest in a mixing tank (P-31 / V-301).
Subsequently, a 10% w/w solution of detergent Triton X-100 is added to the mixture, to obtain a final
concentration of Triton X-100 equal to 0.1% w/w (1 volume of detergent solution : 99 volumes of mixture)
[33,37,38]. The resulting mixture is incubated at 37 °C [37,38] for 2 h under gentle agitation [33], leading to
complete cell lysis. This phenomenon is represented by the following mass stoichiometry:

100 Cells → 50 Cell Debris + 10 Impurities + 10 Nucleic Acids + 30 Proteins (3)

It should be stressed that VPs are not present in the equation above because they were previously
considered as extracellular entities in the model. Since cell lysis is assumed to be complete, the conversion
rate of the reaction above is 100%.

DNA Precipitation

After cell lysis, liberated DNA is removed by selective precipitation with domiphen bromide [33,39–41].
Domiphen bromide is a quaternary ammonium compound commonly used in cosmetics and oral care
products for its antimicrobial properties [41]. A solution of domiphen bromide (domiphen bromide 4% w/w
and NaCl 40 mM) is added to the cell lysate so that the final domiphen bromide concentration is 0.04 %
w/w. The mixture is incubated for 2 h under gentle agitation to maximize the precipitation of nucleic acids
[33]. This step is represented by the following mass stoichiometry:

1 Nucleic Acids (aq) → 1 Nucleic Acids (s) (4)

9
The conversion of this reaction is assumed to be 90% [33,37,38,40]. In addition, it was assumed that 1%
of VPs precipitate together with DNA.

Cell Lysate Clarification

The cell lysate is clarified by a combination of centrifugation (P-32 / DS-301), depth filtration (P-37 /
NFF-301) and membrane filtration (P-38 / NFF-302) [18]. Centrifugation is performed first in a disk-stack
centrifuge, removing most of the cell debris and precipitated nucleic acids. The centrifugation time was set
at 2 h, and the concentration of the heavy stream, at 300 g/L. A disposable depth filter with a pore size in
the range of 0.2 µm to 1 µm is then used to remove most of the remaining solid impurities. The depth filter
is loaded with 650 L/m2 of supernatant [33], and filtration takes 1 h. A disposable membrane filter with a
pore size of 0.2 µm is also included in-line with the depth filter to protect the ultrafiltration step that comes
next; the filtrate flux in the membrane filter is assumed to be 2000 L/m2/h. The percent removal for each
particulate component in the clarification steps is provided in Table 1.

Table 1: Removal of particulate components in the clarification steps.

Component Centrifugation Depth Filtration Membrane Filtration

Cells 95% 100% 100%
Cell Debris 90% 95% 100%
Nucleic Acids (s) 95% 100% 100%
Viral Particles 10% 10% 1%

After filtration, both filters are flushed with Buffer A (MgCl2 1 mM, Tris-HCl 50 mM, NaCl 0.4 M,
polysorbate-80 0.1% w/w and sucrose 5% w/w) to maximize VP recovery; a volume of 20 L/m2 of depth
filtration area under a flux of 200 L/m2/h is used. The clarified bulk with the flushed material is collected in
a holding tank (P-39 / V-303). The overall clarification yield is approximately 80% [39].

Ultrafiltration-Diafiltration #1

The clarified bulk is then sent to a tangential flow filtration system (TFF) with a 300-kDa ultrafiltration
membrane [33,39,42–44], such that VPs are retained by the membrane and smaller impurities are removed
in the permeate. The TFF system is composed of a filter (P-41 / DF-301) and a retentate tank (P-40 /
TDF-301). The clarified bulk is first concentrated by a factor of 20, and then diafiltered with 10 volumes of
Buffer A [33,43]. After diafiltration, a nuclease treatment is performed to facilitate the removal of residual
DNA from the product. A Benzonase® solution (250 U/µL) is added to the retentate to achieve a final activity
of 10 U/mL, and the mixture is incubated at room temperature for 2 h. In reality, the nuclease treatment is
performed between the concentration and diafiltration steps, so that the small DNA fragments produced by
the enzyme are removed during diafiltration [33]. The order of these operations was changed in the present
example for the sake of simplicity. It should be noted that the Benzonase® activity required would be higher

10
if no DNA precipitation had been performed earlier (50 – 150 U/mL [33,40–46]). In fact, nuclease treatment
may not even be necessary when DNA precipitation is done [33,41].

Subsequently, the TFF membrane is flushed with 10 L/m2 of Buffer A to maximize recovery. Table 2 displays
the rejection coefficients assumed for each component during concentration and diafiltration.

Table 2: Rejection coefficients for tangential flow filtration.

Component Concentration Diafiltration

Nucleic Acids 0.10 0.10
Proteins 0.10 0.10
Viral Particles 1.00 0.99

The average permeate flux during concentration and diafiltration is assumed to be 30 L/m 2/h. The overall
product recovery yield of this processing step is 90% [33,43].

Purification

Anion-Exchange Chromatography
The retentate from the ultrafiltration-diafiltration (UF-DF) step is subjected to anion-exchange
chromatography (AEX) (P-51 / C-401), operating in bind-and-elute mode, to remove residual protein and
DNA impurities. A pair of strong anion exchange monolithic columns of 8 L each is employed in this step.
Elution is conducted with a linear gradient, starting with 100% of Buffer A and ending with 100% of Buffer B
(which is identical to Buffer A but with NaCl 2 M). The buffers, flow rates and volumes employed in each
chromatography operation are provided in Table 3.

Table 3: Anion Exchange Chromatography Buffers, Volumes and Flow Rates.

Step Buffer Volume (BV*) Flow Rate (BV*/min)

Equilibration Buffer A 20 1.0
Loading Previous diafiltration Maximum volume 1.0
with Buffer A allowed by the
column binding
capacity
Wash Buffer A 10 1.0
Elution Buffer A + Buffer B 20 of which 2 are 0.5
(linear gradient) collected
Regeneration Regen Buffer 10 1.0
* BV = Bed Volume = 8 L for each column

A column binding capacity of 1.5 g/L = 3 × 1012 VP/mL was assumed. The degrees of retention and release
assumed for each component during loading and elution are summarized in Table 4.

11
Table 4: Retention and release parameters for anion exchange chromatography.

Component Retention (Loading) Release (Elution)

Viral Particles 80% 100%
Nucleic Acids 30% 100%
Proteins 5% 100%

The entire eluate is collected in a mixing tank (P-62 / V-402) and agitated for 15 minutes prior to the next
processing step.

Viral Inactivation
In this example, the vaccine is assumed to be inactivated. Viral inactivation is carried out by adding formalin
(a saturated solution of formaldehyde in water, i.e. formaldehyde 37%) to the eluate, at a volume ratio of
1:2000, and incubating the mixture for 120 h (5 days) [6]. It should be noted that live attenuated vaccines
and viral vector vaccines (such as the COVID-19 vaccines from Oxford/AstraZeneca or Johnson &
Johnson) do not go through viral inactivation.

Ultrafiltration-Diafiltration #2
A second ultrafiltration-diafiltration (UF-DF) step is employed to remove the formaldehyde added in the
inactivation step, and to exchange the AEX elution buffer with an appropriate formulation buffer. As an
added benefit, this step facilitates further concentration of the product solution.

A TFF system with a 300-kDa ultrafiltration membrane is employed in the second UF-DF step, like that
employed in the first UF-DF step. This TFF system is composed of a filter (P-67 / DF-401) and a retentate
tank (P-66 / TDF-401). The inactivated bulk is first concentrated by a factor of 3, then diafiltered with 10
volumes of Formulation Buffer (MgCl2 1 mM, NaCl 75 mM, Tris-HCl 10 mM, sucrose 5% w/w and
polysorbate-80 0.005% w/w), and finally flushed with the same buffer to maximize VP recovery and dilute
the product to a concentration of 1.1 g/L = 2.2 × 1012 VP/mL. The rejection coefficients for each component
during concentration and diafiltration are the same as those given in Table 2, and the average permeate
flux is assumed to be 30 L/m2/h. The overall product recovery yield of this processing step is 90%.

Sterile Filtration
The suspension is then filter-sterilized using a 0.22-µm disposable membrane filter (P-68 / NFF-401). The
filtrate flux is 2000 L/m2/h, and the loss of VPs during this step is assumed to be 5%. Next, the VPs are
adsorbed on aluminum hydroxide (P-69 / V-403). Aluminum hydroxide is a vaccine adjuvant, i.e. a
substance that enhances the immunological response to the vaccine antigen. Adjuvants are often added
to inactivated virus and subunit vaccines because these types of vaccine usually elicit weaker immune
responses by themselves (as opposed to live attenuated vaccines or viral vector vaccines such as the
COVID-19 vaccines from Oxford / AstraZeneca and Johnson & Johnson, which do not require adjuvants).
Lastly, the vaccine is filled into 20-L plastic bags (P-70 / V-404). Approximately 130 L of vaccine solution is

12
produced per batch, with a viral titer of 1 g/L = 2 × 10 12 VP/mL; each batch generates 132 g = 2.64 × 1017
VPs. The overall downstream yield is approximately 49%, as indicated in Table 5.

Table 5: Summary of downstream processing yields

Processing Step Yield

DNA Precipitation 99%
Clarification 80%
UF-DF #1 90%
AEX 80%
Viral Inactivation 100%
UF-DF #2 90%
Sterile Filtration 95%
Overall 49%

Process Scheduling and Cycle Time Analysis

The overall batch time for this process is approximately 30 days. This is the time elapsed from the start of
a given batch (i.e., cell culture in shake flasks) to the end of that batch (freezing of sterile-filtered vaccine
drug substance). However, the time between consecutive batches, so-called Recipe Cycle Time (RCT), is
only 3.5 days; this is possible because many individual procedures in this process are shorter than 3.5
days, and multiple (staggered) equipment units are used for those that are longer. The user can find the
Batch Time and the minimum RCT for the process in the Recipe Scheduling Information dialog (Tasks
Recipe Scheduling Information…). In this dialog, the user may also specify the RCT that he or she desires
(as long as it is larger than the minimum RCT, which is equal to 2.9 days for this process).

To visualize the process schedule, the user may click on Charts Equipment Occupancy Multiple
Batches. This will generate the Equipment Occupancy Chart (EOC) presented in Figure 3. This chart
displays the utilization of each equipment item over time. Eight consecutive batches of the viral vaccine are
shown, each one associated with a different color. The three process sections – Cell Culture, Primary
Recovery, and Purification – are also indicated in the chart. The Cell Culture section shows that two
equipment units are employed for each cell culture step, operating in an alternating fashion. For instance,
the viral production step is performed either by BR-201 or BR-201b, which operate out of phase with each
other. A similar scheme is observed for the viral inactivation step in the Purification section, which is carried
out in tanks V-402 and V-402b. All these equipment units are said to operate in Stagger Mode. This
configuration enables the plant to initiate a new batch every 3.5 days, even though these procedures take

13
longer than that. Further details on specifying equipment in Stagger Mode can be found in the Farnesene
example, located in the …Examples \ Bio-Materials folder.

Another view of the process schedule is provided by the Operations Gantt chart (in the MS Project style).
That chart displays detailed scheduling information for one or multiple batches. The Gantt chart for a single
batch is generated by selecting Charts Gantt Charts Operations GC. Figure 4 displays a portion of
that Gantt chart, illustrating the scheduling of operations in the Cell Culture. The golden bar indicates the
duration of the entire recipe, whereas the dark blue and cyan bars represent the duration of procedures
and operations, respectively.

The Gantt chart enables users to visualize the execution of a batch process in detail. It also facilitates
editing of batch recipes. Double-clicking on any of its bars brings up the dialog of the corresponding entity
(e.g., operation, procedure, recipe, etc.). The simulation calculations can then be redone, and the chart can
be updated by clicking on the refresh button of the chart ( ).

Furthermore, SuperPro can export its scheduling data to MS Project by selecting File Export to MS
Project XML File. Likewise, SuperPro can export its recipe data to SchedulePro by selecting File Export
to SchedulePro’s Recipe DB. SchedulePro is a resource management, production planning and
scheduling tool available from Intelligen. Please consult the Help facility for information on these two
exporting options (Help Index Exporting).

14
 Cell Culture

Primary Recovery

Purification

Figure 3: Equipment Occupancy Chart (EOC) for eight batches. Buffer and media preparation tanks have been omitted for the sake of clarity.

15
Figure 4: Operations Gantt Chart (part of one batch).

16
Material Balances

Table 6 presents overall process data such as batch size, annual production rate and number of batches
per year. This table was extracted from the RTF version of the Materials & Streams report, which can be
generated by selecting Reports Materials & Streams from the main menu bar of SuperPro. The format
of the report can be specified through the dialog that is displayed when you select Reports Options from
the main menu bar. Notice that the batch size is indicated as 125 g, slightly lower than the number
mentioned in the Process Description (132 g) because a batch failure rate of 5% was specified for this
process. Production failure rates can be defined by right-clicking on an empty space of the process flow
diagram and selecting Economic Evaluation Parameters Production Level. The total number of
batches per year is 88 which leads to a Unit Production Rate of 11 kg of VPs per year. Considering that
each vaccine dose corresponds to 25 µg of VPs = 5 × 1010 VPs (which is the actual dose of the COVID-19
vaccines from Oxford-AstraZeneca [47] and Johnson & Johnson [48]), and that the vaccine is filled into
multi-dose vials with an overfill rate of 10% [41], the current manufacturing process would be able to
produce 400 million vaccine doses per year.

Table 6: Overall process data.

OVERALL PROCESS DATA

Annual Operating Time 47.81 wk

Unit Production Ref. Rate 11,003.57 g MP/yr
Batch Size 125.04 g MP
Recipe Batch Time 30.30 day
Recipe Cycle Time 3.50 day
Number of Batches per Year 88.00
MP = Flow of Component 'Viral Particles' in Stream 'Final Product Solution'

Table 7, which was also extracted from the Materials & Streams report, displays the raw material
requirements in kg/year, kg/batch, and kg/g of MP (“MP” stands for main product, which is VPs in this case).
It shows that water for injection (WFI), Buffer A, chromatography buffers and CIP solutions are the main
materials used in this process. The consumption of cell culture media is also large: given that 1.70 kg of
medium solids are consumed per g of MP produced, and that medium solids correspond to 2% of medium
volume, a total of 85 kg of reconstituted medium is consumed per g of MP produced.

17
Table 7: Material requirements.

BULK MATERIALS (Entire Process)

Material kg/yr kg/batch kg/g MP

Acetate Buffer 14,150.00 160.80 1.29
AEX Regen Bfr 209,119.93 2,376.36 19.00
AEX San Buffer 22,722.89 258.21 2.07
AEX Storage Bfr 26,726.25 303.71 2.43
Air 452,095.82 5,137.45 41.09
Alum Gel 2% 764.01 8.68 0.07
Benzonase Stock 2.17 0.02 0.00
Buffer A 1,109,474.25 12,607.66 100.83
Buffer B 106,398.53 1,209.07 9.67
Cells 0.01 0.00 0.00
CIP-Acid 326,524.41 3,710.50 29.67
CIP-Caustic 534,258.62 6,071.12 48.55
Detergent Sln 10,695.48 121.54 0.97
Domiphen Br Sln 10,803.39 122.77 0.98
Formaldehyde 7.26 0.08 0.00
Formul. Buffer 76,013.74 863.79 6.91
Lysis Buffer 125,360.97 1,424.56 11.39
Medium Solids 18,683.63 212.31 1.70
TFF San Buffer 50,481.20 573.65 4.59
TFF Storage Bfr 50,481.20 573.65 4.59
Viral Particles 0.06 0.00 0.00
WFI 7,635,237.98 86,764.07 693.89
TOTAL 10,780,001.81 122,500.02 979.68

Cost Analysis

SuperPro Designer performs thorough cost analysis, estimating capital costs (CAPEX) as well as operating
costs (OPEX), and generates the following three pertinent reports (through the Reports menu): the
Economic Evaluation Report (EER), the Cash Flow Analysis Report (CFR), and the Itemized Cost Report
(ICR). Table 8 displays the Executive Summary of the Economic Evaluation Report.

The total capital investment for such a facility is approximately $300 million. This includes equipment
purchase and installation costs; other costs related to plant construction; startup and validation costs; and
the working capital required for this project. Plant construction costs such as the cost of buildings and piping
are estimated through multipliers in SuperPro Designer, and these were modified in this example to more
accurately represent the capital costs associated with a vaccine manufacturing facility. For more information
on capital cost estimations, please refer to the Industrial Enzymes example in the …Examples / Bio-
Materials folder or consult the Help tool (Help Search  Capital Investment Dialog: DFC Tab).

18
Table 8 also displays the annual operating cost (AOC) and the unit production cost, which are approximately
$110 million and $10/mg of VPs, respectively. Considering the vaccine dose and overfill rate mentioned
earlier, this unit production cost translates to $0.50/dose. Further assuming a selling price of $20/mg of VPs
(i.e., $1.00/dose) leads to a gross margin of 50%, a return on investment (ROI) of 35% and a Payback Time
of approximately 3 years.

Table 8: Executive summary.

EXECUTIVE SUMMARY (2021 prices)

Total Capital Investment 298,724,000.00 $

Capital Investment Charged to This Project 298,724,000.00 $
Operating Cost 110,773,000.00 $/yr
Revenues 220,071,000.00 $/yr
Batch Size 125.05 g MP
Cost Basis Annual Rate 11,004.00 g MP/yr
Unit Production Cost 10,067.04 $/g MP
Net Unit Production Cost 10,067.04 $/g MP
Unit Production Revenue 20,000.00 $/g MP
Gross Margin 49.66 %
Return On Investment 34.62 %
Payback Time 2.89 years
IRR (After Taxes) 32.04 %
NPV (at 10.0% Interest) 371,377,000.00 $
MP = Flow of Component 'Viral Particles' in Stream 'Final Product Solution'

Figure 5: Annual operating cost breakdown.

Figure 5 displays a breakdown of the AOC; it was also extracted from the Economic Evaluation Report.
Clearly, raw materials and facility-dependent costs are the main contributors to the AOC, accounting for

19
45% and 37%, respectively. The facility-dependent cost comprises equipment maintenance, depreciation,
and overhead costs.

Table 9, which was extracted from the Economic Evaluation Report, provides detailed information on the
amounts and cost of raw materials. It also presents a breakdown of raw material costs, showing that media
account for approximately three-quarters of this cost category (75.5%). The costs of Benzonase®, WFI,
Buffer A and Lysis Buffer are also noticeable, ranging from 3.7% to 5.5%.

Table 9: Breakdown of material costs.

MATERIALS COST - PROCESS SUMMARY

Unit Cost Annual Annual Cost

Bulk Material %
($) Amount ($)
Acetate Buffer 4.13 14,150.00 kg 58,447.00 0.12
AEX Regen Bfr 3.08 209,119.93 kg 643,229.00 1.30
AEX San Buffer 3.19 22,722.89 kg 72,406.00 0.15
AEX Storage Bfr 5.19 26,726.25 kg 138,692.00 0.28
Air 0.00 452,095.82 kg 0.00 0.00
Alum Gel 2% 1.29 764.01 kg 989.00 0.00
Benzonase Stock 1,165.00 2,166.00 mL(STP) 2,523,387.00 5.10
Buffer A 2.45 1,109,474.25 kg 2,723,440.00 5.51
Buffer B 4.12 106,398.53 kg 438,717.00 0.89
Cells 0.00 0.01 kg 0.00 0.00
CIP-Acid 0.69 326,524.41 kg 226,608.00 0.46
CIP-Caustic 0.69 534,258.62 kg 366,566.00 0.74
Detergent Sln 10.27 10,695.48 kg 109,843.00 0.22
Domiphen Br Sln 48.33 10,803.39 kg 522,172.00 1.06
Formaldehyde 0.00 7.26 kg 0.00 0.00
Formul. Buffer 1.51 76,013.74 kg 114,790.00 0.23
Lysis Buffer 14.49 125,360.97 kg 1,816,270.00 3.67
Medium Solids 2,000.00 18,683.63 kg 37,367,266.00 75.54
TFF San Buffer 0.68 50,481.20 kg 34,213.00 0.07
TFF Storage Bfr 0.38 50,481.20 kg 18,958.00 0.04
Viral Particles 0.00 62.20 g 0.00 0.00
WFI 0.30 7,635,237.98 kg 2,290,571.00 4.63
TOTAL 49,466,562.64 100.00
NOTE: Bulk material
consumption amount
NOTE: Bulk material consumption amount includes material used as: includes material
- Raw Material used as:
- Cleaning Agent - Raw Material
- Heat Transfer Agent (if utilities are included in the operating cost) - Cleaning Agent
- Heat Transfer
Agent (if utilities are
In conclusion, the results of the cost analysis suggest that the present vaccine production
included in process
the would
be an attractive investment. It should be noted, however, that many assumptions operating
underliecost)
this kind of

20
analysis, such as the market demand and selling price of the vaccine; the prices of raw materials
(particularly media); the yields of cell culture, primary recovery, and purification steps, etc. As a result, the
actual economics of such an investment may be substantially better or worse than the current projection.
A useful exercise is to perform “what-if” analyses with SuperPro to determine the impact of various changes
(e.g., a higher viral production yield, a lower medium cost, etc.). This allows the user to understand the
potential risks and rewards of a project under different sets of assumptions. What-if scenarios can be
evaluated individually (by simply changing parameter values manually and re-running the simulation to see
the results), or they can be automated through MS Excel. For information on how to drive SuperPro
Designer through MS Excel and automate sensitivity analysis, please consult the examples in the
…Examples \ COM folder. Related information is available in the Help facility of the tool, which can be
accessed by selecting Help  COM Interface and Library. Math optimization and Monte Carlo simulation
can be performed in a similar manner.

Modeling Tips

Kinetic Model of Cell Growth

It is possible to implement kinetic models of cell growth in SuperPro Designer. In the present example, all
the cell expansion steps exhibit exponential growth, which was modeled with a Batch Kinetic Fermentation
operation. To add such operation to a cell culture procedure, first right-click on the procedure’s icon and
click on Add / Remove Operations… to bring up the dialog window shown in Figure 6:

21
Figure 7: Dialog for adding, removing, or reordering operations in a procedure.

Next, on the list of Available Operations on the left, select Ferment (Kinetic), add it to the Operation
Sequence on the right and click OK. After that, right-click on the procedure’s icon again and click on
Operation Data…  FERMENT (Batch Kinetic Fermentation) to open the dialog shown in Figure 8:

22
Figure 9: Operation Data dialog for a Batch Kinetic Fermentation operation.

In the Oper.Cond.’s tab, the user must provide basic operating parameters such as the temperature and
aeration rate. The Reaction Time, which corresponds to the cell growth duration and therefore is relevant
to the kinetics of the process, must also be specified in this tab. To specify a kinetic model for cell growth,
however, the user must switch to the Reactions tab, which is displayed on Figure 10. Several fermentation
reactions may be included in the Reaction Scheme frame on the right; by default, however, a single
reaction, Fermentation #1, is present. The user must specify the stoichiometry of each reaction by clicking
on the shake-flask ( ) button, just like he or she would for a stoichiometric fermentation model. In
addition, the user must specify a kinetic model and its parameters by clicking on the rate ( ) button; this
brings up the dialog shown in Figure 11.

23
Figure 10: Operation Data dialog for a Batch Kinetic Fermentation operation, showing the Reactions tab.
The buttons for specifying the fermentation stoichiometry and kinetics are highlighted in yellow.

As mentioned earlier, cell culture follows exponential growth in the cell expansion steps. The cell growth
rate is therefore given by the simple equation below:

𝑑𝑋
= 𝜇𝑚𝑎𝑥 ∙ 𝑋
𝑑𝑡

Where 𝑋 represents cell concentration and 𝜇𝑚𝑎𝑥 the maximum specific growth rate. As such, there is no
dependence on substrate concentration, and the substrate terms (S1, S2, S3) in the SuperPro Designer
model should be specified as None (see Figure 11). The 𝜶 and 𝜷 parameters should be specified as 1 and
0, respectively; the value of 𝝁𝒎𝒂𝒙 should be specified as appropriate; and the B-term should be specified
as Cells and First Order.

24
Figure 11: Dialog for the specification of the rate equation in a Batch Kinetic Fermentation operation.

In the present example, the value of 𝜇𝑚𝑎𝑥 was specified as 0.02236 h-1, given that the doubling time (𝑡𝐷 ) of
cells was assumed to be 31 h. It is easy to demonstrate that these two parameters are related by the
equation below:

ln 2
𝜇𝑚𝑎𝑥 =
𝑡𝐷

As Figure 9 shows, SuperPro Designer allows the use of more sophisticated cell growth models such as
Monod and Haldane models, with up to three different substrates. For these models, additional kinetic
parameters must be specified.

It is also worth noting that, by default, SuperPro will calculate the final concentrations of substrates, cells
and fermentation products based on the Reaction Time specified in the Oper. Cond.’s tab. However, the
timeframe in which the kinetic model is applicable may be adjusted using the Start Criteria… and End
Criteria… buttons shown in Figure 11.

After simulation, the user may visualize the kinetics of a fermentation operation by right-clicking on the host
procedure, clicking on Dynamic Data Records… and selecting the fermentation operation of interest.

25
Kinetic data may be viewed in table form or linked to an Excel spreadsheet. For more details on this subject,
please consult the BatchKinFerm example in the …Examples \ Misc folder or refer to the Help facility
(Help Search  Kinetic Reaction Operations: Profiles Tab).

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