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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5886_Библиотеки_им_академика_М_И_Перельмана.pdf
190
J. A. S. Romine etal.
proceeds until the nucleotides are consumed or purication steps are started and
nishes within hours. IVT reactions have been demonstrated at manufacturing
scales over 100L [48].
As mentioned above, a key aspect of the RNA is the 5′-cap. The 5′-cap can be
added co-transcriptionally, using a molecule that is added to the IVT reaction as the
rst step in building the mRNA chain, or post-transcriptionally, using enzymes to
modify the rst nucleotide on the 5′ end. For co-transcriptional capping, there are
several commercially available options. The two most common are the antireverse
cap analog (ARCA) and CleanCap. The ARCA produces a cap 0 structure, while the
CleanCap produces a cap 1 structure (Fig.8.3) [58]. These different structures interact differently with the immune system, with the cap 1 structure less likely to trigger
an innate immune response. Post-transcriptional enzymatic capping is commonly
performed by the Vaccinia capping enzyme system [59]. This requires Vaccinia
virus capping enzyme to achieve a cap 0 structure [60] and 2′-O-methyltransferase
if adding the cap 1 structure [61]. The methyl donors required for the process are
GTP and S-adenosylmethionine (SAM). While post-transcriptional capping adds an
additional unit operation and more raw materials, it can have benets over cotranscriptional capping for the capping efciency (percentage of capped mRNA
transcripts) and potential for reverse capping (capping in the wrong orientation) [62].
8.2.2.3 Purication
For the isolation process, also known as the downstream process (DSP), biologics
requiring cell culture for production often rely on complex purication trains
designed to remove host-cell contaminants that impact the safety and efcacy of the
product [63]. Because the production of mRNA-based vaccines doesn’t require cell
Fig. 8.3 Cap 0–2 structures
8 COVID-19 Vaccine Manufacturing Processes: Making the Molecules to Solve…
culture, the purication processes used are typically much simpler and of shorter
duration. Contaminants from the synthesis of mRNA consist of enzymes and unreacted feed materials such as plasmids or partially reacted species that don’t have the
size or structure of the target product. Thus, a combination of enzymatic, chromatographic, and UF/DF steps is sufcient for clearing these contaminants.
Once the IVT reaction is complete, the reaction can be stopped by the addition
of DNase [64]. This enzyme degrades the DNA template in the reaction, halting any
further transcription into mRNA and allows the DNA to be broken down into a
smaller size. This step can also be used if the purication process is unable to separate RNA from DNA [65].
After degrading the DNA, in the simplest cases, a UF/DF operation has the
potential to remove the smaller contaminants, concentrate the mRNA, and replace
the buffering species from the reaction with those needed for nal formulations. In
more complex processes, the UF/DF step would be preceded by orthogonal purication steps, such as reverse phase or ion exchange chromatography [66]. Regardless
of the approach, the material is sterile ltered a nal time before lling into the nal
drug substance container. The drug substance is then used for formulation in lipid
nanoparticles [48]. However, that process is out of scope for this review.
For situations where the contaminants are not easily separated based on steric
interactions, afnity capture media have been developed that selectively bind the
polyadenylated (polyA) tail of RNA using hydrogen bonding [67]. The high selectivity of this interaction allows all non-RNA contaminant species or RNA fragments
without a polyA tail to ow through during loading, regardless of size, while the
mRNA is retained for later elution. This more extensive clearance of contaminants
makes polyA afnity a useful platform for mRNA, although some form of UF/DF
may be useful to achieve the desired product concentration and buffer replacement
for nal formulation. Despite being an additional operation, this approach should be
universally applicable to other mRNA products and is less burdensome than the
multi-column purication trains used for the purication of recombinant proteins.
191
8.3 Viral Vectors
8.3.1 Background
Viral vector vaccines work by modifying an existing virus to delete essential viral
genes that prevent replication of the vector, and instead replace them with DNA
encoding for the gene of interest for immune stimulation. The viral vector vaccines
that have been approved for SARS-CoV-2 contain DNA encoding for the S protein.
These vectors then enter target cells and deliver the genetic material, resulting in
expression of the S protein and eliciting an immune response. Thus far, four viral
vector vaccines have been approved and can be seen in Table8.3.
192
Table 8.3 Details for approved viral vector vaccines against SARS-CoV-2
Vaccine name Developer Vector Cell line Reference
Ad5-nCoV,
Convidecia
Gam-Covid-Vac
Sputnik V
ChAdOx1 nCoV-19;
AZD1222; Vaxzevria
Ad26.COV2.S Johnson &
CanSino Biologics Adenovirus type 5 HEK293SF-3F6 [22, 23]
Gamaleya
Research Inst
Univ of Oxford/
AstraZeneca
Johnson
Adenovirus type 5
and 26
Chimpanzee
adenovirus
(ChAdOx1)
Adenovirus type 26 PER.C6 [28, 29]
J. A. S. Romine etal.
Not disclosed [24, 25]
HEK293 [26, 27]
For the approved adenoviral vector vaccines, the vectors chosen are human adenovirus serotype 5 (Ad5), serotype 26 (Ad26), and a chimpanzee adenovirus (specically, ChAdOx1). Ad5 is the most characterized adenoviral vector [68], making
it a common choice for vaccines. However, Ad5 produces a strong antivector immunity after dosing that may inhibit repeat dosing, and it is also limited by high seroprevalence in human populations, which can limit its effectiveness [69]. Both Ad26
and ChAdOx1 (and other simian adenoviruses) are chosen due to their low seroprevalence [70, 71]. With this benet, the clinical trial from Oxford, AstraZeneca,
and Vaccitech has shown promise for a two-dose, prime-boost delivery of the chimpanzee adenoviral vector [72]. Additionally, Gamaleya Research Institution has
chosen to use Ad26 to prime and Ad5 to boost in order to deliver two separate
serotypes for its vaccine [25].
8.3.2 Production Process
8.3.2.1 Overview ofSteps
All of the adenovirus processes start with human cell lines. However, for the
HEK293 and PER.C6 cell lines, the USP differs. For HEK293, the adenovirus is
produced by infection with the virus where the PER.C6 process can use either infection or transfection with plasmid and cosmid DNA.In either approach, the virus
stays contained within the living cell until release during apoptosis. For the manufacturing process, chemical or physical means can be used to cause cell lysis and the
release of the viruses into the bulk media for further purication [73]. In general,
cell disruption is followed by clarication to remove cell debris, further purication
using chromatography, lter-based buffer exchange, and nal ltration [73].
8 COVID-19 Vaccine Manufacturing Processes: Making the Molecules to Solve…
193
8.3.2.2 Production
HEK293 and PER.C6 cells are the most commonly used cell lines for adenovirus
production because they both contain two key adenoviral genes, E1A and E1B [74,
75]. This allows for the replication of adenoviral vectors in which those essential
genes have been deleted and replaced with the gene of interest [71]. Despite that
similarity, the process of introducing the DNA to make the product of interest is
different for both cell lines.
In the case of production in HEK293 cells, a fed batch process is typically used
to achieve cell densities on the order of 2–4e6 cells/mL, at which point the DNA is
introduced by adding puried recombinant viral seed stock to the bioreactor [76].
These viruses infect the cells and begin to make the product of interest. Depending
on the multiplicity of infection (MOI; the ratio of virus to cells in the culture), the
produced adenovirus from the initial infection may go on to infect additional cells
in the culture. For Oxford and AstraZeneca’s process, they evaluated both a high
MOI (>1) process where cells were infected at a higher density and infected with
high amounts of viral seed stock and a low MOI (<1) process with low cell density
and low viral seed stock where cells were cultured longer after infection to allow for
reinfection inside the bioreactor [76]. In the end, the low MOI process was chosen
because it generated comparable volumetric productivity but with lower demand for
viral seed stock. While HEK293 cells are capable of being grown to much higher
densities (greater than 80e6 cells/mL by perfusion processes) [77], the use of the
infection process leads to several challenges such as the necessity for large quantities of viral seed stock and the “cell density effect” wherein the viral particles produced per cell becomes progressively lower with higher cell densities [78, 79].
For the PER.C6 process, cells can be grown to higher densities, up to 100e6
cells/mL [71]. High densities for adenoviral production have been obtained through
fed batch and perfusion processes [80]. To introduce the DNA, cells can either be
infected similarly to the HEK293 process, as described above, or the DNA can be
introduced by transfection. The transfection method uses a cosmid that encodes
most of the adenovirus genome and an adapter plasmid that contains the left inverted
terminal repeat (ITR) of the adenovirus genome, a packaging signal, the transgene
expression cassette (in the place of the E1 gene), and a small portion of residual E1
sequence to support homologous recombination with the cosmid to produce the
full-length recombinant genome [71]. Janssen has demonstrated this process up to
1000L [71].
8.3.2.3 Purication
Virus purication will vary based on the specic viral vector of interest, as each
virus, even compared to those with similar size and structure, has unique characteristics that may make it behave differently. Accordingly, even when limited to adenoviruses, purication trains differ signicantly.
194
J. A. S. Romine etal.
For a prototypical adenovirus, the purication process begins with the lysis of
the host cells using a detergent such as polysorbate 20 (PS20) or Triton X-100 [81].
Benzonase, a highly active endonuclease, is also added to degrade any DNA or
RNA in free solution, as shown in Fig.8.4 [82]. Additional chemicals like sucrose
or magnesium chloride may also be added to increase recovery of the virus from the
cell debris and improve stability. The bulk harvest is then ltered using a depth lter, often with diatomaceous earth, to remove particulate cell debris [83]. Depending
on the pH and conductivity of the claried material, it can then either be loaded
directly onto the capture chromatography media, or buffer exchanged using UF/DF
to provide the proper binding conditions for capture. The capture media varies
widely, with resins (Poros 50 HQ, Phenyl Sepharose FF, Source 15Q), membranes
(Mustang Q, Sartobind Q), and monolithic materials (Cimmultus QA-1) nding use
in this application [84]. The eluted material is then concentrated and buffer
exchanged into the nal formulation buffer using UF/DF.
8.4 Whole Inactivated Virus Vaccines
8.4.1 Background
A more traditional approach to vaccine design is the use of a whole pathogen, and
many historical vaccines have been developed this way [12–14]. Whole-virus vaccines fall into one of two classes: live-attenuated and whole inactivated virus (WIV)
[12]. Live-attenuated virus vaccines consist of a weakened form of a virus that
Fig. 8.4 Example process ow diagram for adenovirus vaccine DSP—depending on the specic
project, the capture step may be completed using a monolith or membrane
8 COVID-19 Vaccine Manufacturing Processes: Making the Molecules to Solve…
195
stimulates the immune system without causing severe illness. Live-attenuated virus
vaccines can reproduce in the vaccinated subject, effectively increasing the amount
of antigen present [10]. WIV vaccines use a form of the virus which cannot reproduce in cells due to chemical or physical inactivation, yet still triggers an immune
response [10]. They are generally safe and effective because they cannot induce an
infection, but often require larger doses of viral antigen to elicit an immune response
[10]. As shown in Table8.4, four WIV vaccines against SARS-CoV-2 have gained
full approval in at least one country, and more than 10 additional WIVs candidates
are in clinical development [85]. While no live-attenuated SARS-CoV-2 vaccines
have been approved for human use at the time of authoring this manuscript,
Codagenix has developed a live-attenuated virus vaccine, CoviLiv, that is given
intranasally and has advanced to Phase 2 testing [86]. A second live-attenuated
SARS-CoV-2 vaccine is being developed at Speransa Therapeutics and is in a Phase
1 clinical trial [85]. Focusing on the approved vaccines, the manufacturing process
for WIV will be described in detail in this review.
8.4.2 Production Process
8.4.2.1 Overview ofSteps
The manufacture of WIV vaccines begins with selection of appropriate cell lines for
viral propagation of the viral pathogen, specically SARS-CoV-2 [88]. Once a large
population of cells susceptible to the virus is grown, virus is added to the reactor to
infect the cells and initiate viral replication. Once a large amount of virus is produced inside the cell, the cells may begin to lyse naturally, or a mechanical force is
applied to release the virus. Once cell disruption has occurred, the lysate is collected
and claried to remove cell debris. The whole virus is then inactivated physically or
chemically to ensure that no further replication can occur. Additional purication,
including chromatography, lter-based buffer exchange, and nal ltration can then
be performed to yield a nal inactivated viral drug substance. A generic process
ow for the production of WIVs is shown in Fig.8.5.
Table 8.4 Details for approved whole inactivated viral vaccines against SARS-CoV-2
Cell
Vaccine name (developer)
CoronaVac, PiCoVacc (Sinovac) Vero cells CN2 β-propiolactone [30]
BBIBP-CorV (Sinopharm/Bejing
Inst.)
Covaxin, BBV152 (Bharat Biotech) Vero cells NIV-
(Sinopharm/Wuhan Inst) Vero cells WIV-04 β-propiolactone [87]
culture Virus strain
Vero cells HB02 β-propiolactone [32]
2020- 770
Inactivation
agent Reference
β-propiolactone [34]
196
Fig. 8.5 Generic process ow diagram for whole inactivated virus vaccines
J. A. S. Romine etal.
8.4.2.2 Production
While whole virus vaccines have been historically produced in eggs, Vero cells have
become a common choice for culturing the viruses, with the list of approved
COVID-19 vaccines (Table8.4) all using this approach. The Vero cell line, derived
from the kidney of an African green monkey, is a well-characterized, continuous
cell line that was found to be nontumorigenic (below a specic passage number)
and free of adventitious viruses and proteins [88], and was shown to be particularly
susceptible to viral infection due to an impaired antiviral response [89]. Growth of
these cells has historically required an adherent cell culture platform. This requires
the use of surfaces to support cell growth, typically roller bottles, cell factories, or
microcarriers in a bioreactor. Microcarrier processes have been demonstrated up to
the 6000L scale; however, the process still achieves low cell densities (<3e6 cells/
mL) due to the surface area limitations of the microcarriers [90]. It has also been
shown that this cell line can be adapted to full suspension culture, without the need
for cell attachment, and able to achieve cell densities of up to 8e6 cells/mL in batch
and perfusion culture processes [91]. This allows for more cells to be produced and
in greater volumes [91]. Once the cells are grown to the intended target cell density,
the cells are then infected with the SARS-CoV-2 virus [91].
There are some details available regarding the viral replication step for the
approved WIV vaccines against SARS-CoV-2. In the case of CoronaVac, Vero cell
cultures were inoculated with the CN2 SARS-CoV-2 viral strain (obtained from
bronchoalveolar lavage of hospitalized patients) using a MOI of 0.0001–0.01,
reaching a peak titer by 3–4days postinfection [30]. After allowing adequate time
for the virus to replicate within the cells, the supernatant was separated by centrifugation and taken into the inactivation step [30]. For the BBIBP-CorV vaccine, the
MOI was 0.01–0.3, and the virus titer peaked at 48–72hours postinfection. The
entire bioreactor cell culture process lasted 4–8days [32]. The starting virus strain
8 COVID-19 Vaccine Manufacturing Processes: Making the Molecules to Solve…
197
was selected by cultivating throat swabs from several patients with COVID-19, and
the SARS-CoV-2 virus strain that was the highest yielding was chosen for production [33]. The viral strain used for Covaxin was NIV-2020-770, and virus harvest
occurred 36–72hours postinfection. Because of the presence of the SARS-CoV-2
virus itself, Bharat Biotech operated their manufacturing facilities under Biosafety
Level (BSL) 3 requirements [35].
8.4.2.3 Viral Inactivation
For WIVs, a viral inactivation step follows separation of cell debris to ensure that no
further replication can occur. Each of the four whole inactivated SARS-CoV-2 vaccines listed in Table 8.4 was chemically inactivated with β-propiolactone. This
reagent was rst reported by LoGrippo etal. in 1955, and it represented an improvement over the then current standard (formalin and phenol). With the updated
approach, inactivation occurs very fast (within 10–15minutes at 37°C versus days
for the formalin and phenol) with no loss of antigenicity [92]. Modern viral inactivation processes are typically performed at lower temperatures (2–8 °C) for
24–48hours, which was reported to preserve major neutralizing antigens due to
selective inactivation of viral RNA over protein [87].
The structure of β-propiolactone is shown in Fig.8.6, along with the proposed
reaction mechanism that leads to viral inactivation [93]. It is proposed that hard
nucleophiles (e.g., basic amines) react with β-propiolactone to yield the acylation
products 1, while soft nucleophiles (e.g., thiols) react to give the alkylation product
2 [93]. RNA and DNA (single and double stranded) were shown to be rapidly alkyl-
ated by β-propiolactone [94], as were peptides side chains [93]. While it is impossible to determine the precise chemical modications leading to viral inactivation
with current technology, these studies have illuminated possible adducts with biologically relevant nucleophiles producing potential molecules of interest.
In the case of the WIV vaccine developed by Wuhan Institute with Sinopharm, a
chemical inactivation with β-propiolactone was performed twice during the DSP
with a depth ltration between the two inactivations [87]. Inactivation of the SARSCoV- 2 virus by physical means has been extensively studied using heat, detergent
and UV treatment as well as methanol and paraformaldehyde, although antigenic
properties of the inactivated viruses were not evaluated [95].
Fig. 8.6 Structure of β-propiolactone and reaction with nucleophiles
198
8.4.2.4 Purication
The large-scale DSP for viruses can face a range of obstacles different from those
of many biotherapeutics. These challenges mostly arise from the size and complexity of the virus, which can affect product purity and recovery. Often the rst step
includes Benzonase treatment to reduce free DNA and RNA, as was described for
viral vector vaccines [82]. Subsequent downstream steps can involve ultracentrifugation, UF/DF, and different types of chromatography methods to purify the
virus. These are chosen based on the specic properties of the virus. For example,
the DSP for CoronaVac consisted rst of concentration and buffer exchange of the
inactivated material using UF/DF in preparation for downstream purication [30].
Following Benzonase treatment, the purication train proceeded to ion exchange
chromatography (IEX), followed by a second concentration using UF/DF, and
nally size-exclusion chromatography (SEC) [30]. For Covaxin, the DSP included
a column chromatography step followed by concentration using tangential ow ltration (TFF) [34], while purication of BBIBP-CoV-2 consisted of buffer exchange
of the inactivated virus supernatant by UF/DF followed by endonuclease digestion
and a chromatography step [32]. The doubly inactivated WIV vaccine from
Sinopharm/Wuhan Institute was puried by gel-chromatography and IEX chromatography followed by sterile ltration [87]. Once the virus is in the puried form,
the nal step is putting it in a matrix that keeps it stable until it is ready to be formulated into the nal drug product.
J. A. S. Romine etal.
8.5 Protein-Based Vaccines
8.5.1 Background
Protein-based vaccines contain puried protein antigens lacking DNA or RNA and
come in a variety of different presentations. The earliest protein-based vaccines
were chemically detoxied bacterial toxins, e.g., diphtheria toxoid (DT) and tetanus
toxoid (TT) [14]. Recombinant protein vaccines composed of surface antigens of
viruses and bacteria are considered component or subunit vaccines [96]. Some modern examples include Trumenba [97] and VLA15 [98]. Protein antigens can also be
presented on nanoparticles or virus-like particles (VLPs), such as human papillomavirus vaccines [99].
While protein vaccines are one of the most established vaccine modalities, they
also have the most diversity in approach [100]. This modality provides a unique
challenge compared to the other modalities: how do we make the protein in the right
conformation with all of the right post-translational modications? This can be seen
by the breadth of different constructs and the cell lines that have been chosen [101].
The constructs range from full S proteins (monomeric and trimeric) to portions of
the protein like the receptor binding domain, to virus like particles with S proteins
8 COVID-19 Vaccine Manufacturing Processes: Making the Molecules to Solve…
199
incorporated [101]. Some constructs also have modications of the sequence to
improve protease resistance and antigen stability. For protein production, the cell
lines chosen are important for not only getting sufcient yields, but also the posttranslational modications that ensure proper protein folding and contribute to protection from protease cleavage and recognition by neutralizing antibodies [39, 102].
While at least 16 different protein-based vaccines against SARS-CoV-2 have
advanced to clinical testing so far [96], only three have been fully approved for
human use (Table8.5). Both NVX-CoV2373 and Covifenz utilize the full-length
trimeric form of the S protein with sequence modications to increase metabolic
stability by removing a furin cleavage site and increase conformational stability of
the prefusion form [39, 42, 96, 103]. The design of EpiVacCorona is very different
in that it consists of three peptides from the S protein sequence conjugated to a carrier protein [45, 46]. The design was intended to prevent the potential for antibodydependent enhancement (ADE) of infection by avoiding epitopes thought to be
responsible for ADE on the SARS-CoV-2 S protein [104]. With many differences in
process for these vaccines, each will be discussed in its own subsection.
8.5.2 Production Processes
8.5.2.1 NVX-CoV2373 (Novavax)
The protein drug substance for NVX-CoV2373 is the trimeric full-length S protein
from SARS-CoV-2 with mutations of the furin cleavage site (682-RRAR-685 to
682-QQAQ-685) and two proline substitutions (K986P and V987P) in a nanoparticle form (27.2nm) [39]. A high level process ow diagram for NVX-CoV2373 is
shown in Fig. 8.7. The double mutant SARS-CoV-2 S-gene was cloned into
Table 8.5 Details for approved protein-based vaccines against SARS-CoV-2
Vaccine name
(developer) Construct/Modality Cell line
NVX-CoV2373,
Covovax,
Nuvaxovid
(Novavax)
CoVLP, Covifenz
(Medicago)
EpiVacCorona,
Aurora-CoV
(BEKTOP)
Full-length S protein
incorporated into
nanoparticles
Full-length S protein
incorporated into
VLPs
Three 21–30 residue
synthetic peptides
from the S protein
covalently bound to
MBP-6xHis-NCoV-2019
Spodoptera
frugiperda
(Sf9, insect
cells)
Nicotiana
benthamiana
(plant cells)
E. coli (carrier
protein)
Downstream process
(DSP) References
TMAE anion
exchange column,
lentil lectin afnity
column, hollow ber
TFF
Similar to
hemagglutinin based
VLPs
No details provided [46]
[39]
[42, 105]