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200
Fig. 8.7 High-level process ow diagram for NVX-CoV2372
J. A. S. Romine etal.
recombinant baculovirus (rBV) which was then used to infect Spodoptera frugi-
perda (Sf9) cells that had been expanded to 2–3e6 cells/mL density (MOI≤0.1 pfu/
cell). Cell culture was maintained at 27°C for 68–72hours after infection, and the
cells were harvested by centrifugation. Cell pellets were suspended in Tris buffer,
and S proteins were extracted into Tris buffer with NP-9 surfactant. The downstream purication process of the S protein included trimethylaminoethyl (TMAE)
anion exchange chromatography followed by lentil lectin afnity chromatography.
Buffer exchange with hollow ber TFF yielded the nal drug substance.
8.5.2.2 CoVLP (Medicago)
The protein antigen for CoVLP is the trimeric full-length S protein of SARS-CoV-2
with mutations of the furin cleavage site (R667G, R668S, and R670S) and mutations to stabilize the prefusion conformation (K971P and V972P) presented on a
VLP surface [42]. VLP-vaccines are manufactured in cell lines capable of producing particles that mimic viral structures and demonstrate increased immunogenicity
over puried recombinant protein vaccines. The CoVLP manufacturing process follows Medicago’s plant-based platform for VLP production, which is based on agroinltration, a method of inducing transient gene expression in plants [105]. The
overall process is represented in Fig.8.8. Briey, the USP starts with batches of
Nicotiana benthamiana plants grown for 5weeks prior to agroinltration with an
Agrobacterium tumefaciens vector coding for the full-length S protein. Harvest
occurs 6days after transfection using Medicago’s proprietary extraction method [42].
The purication process begins with removal of cellular debris by centrifugation
followed by clarication using chemical and physical methods [105]. Concentration
of the claried supernatant precedes several chromatographic steps including IEX
and afnity chromatography. The nal puried eluent is concentrated by TFF, formulated and sterile ltered prior to nal ll [105].
8 COVID-19 Vaccine Manufacturing Processes: Making the Molecules to Solve…
Fig. 8.8 High-level CoVLP process ow diagram
201
8.5.2.3 EpiVacCorona (Vector Institute)
The EpiVacCorona vaccine comprises three chemically synthesized peptide
sequences from the SARS-CoV-2 S protein conjugated to a carrier protein, which
was reported to be the N-protein of SARS-CoV-2 [45, 46]. While no details of the
synthetic methods utilized to produce the peptides were provided, the reader is
directed to several recent reviews on the subject [106, 107]. Likewise, the specic
chemistry employed for conjugation of the peptides to the N-protein was not
reported for EpiVacCorona. A similar conjugate vaccine against SARS-CoV-2 has
been developed at the Finlay Vaccine Institute (Soberana 1) which consists of the
SARS-CoV-2 S protein RBD conjugated to TT [108]. The RBD containing an
unpaired cysteine was produced in CHO cells and was conjugated to maleimidoactivated TT using known conjugation techniques [108]. Capsular polysaccharideconjugate vaccines utilize similar manufacturing techniques and have been reviewed
elsewhere [109–112].
8.6 Comparison ofManufacturing Processes
fortheDifferent Modalities
While each of the COVID-19 vaccine modalities has been highlighted individually,
Table8.6 shows a comparison of several manufacturing parameters for the mRNA,
viral vectors, WIV, and protein-based vaccines. Manufacturing processes for viral
vaccines vary greatly depending on the identity of the antigen drug substance for a
given modality.
The USP typically requires cell culture to produce the antigen, whether it is a
whole pathogen or a puried component. Both the viral vector and whole pathogen
202
Table 8.6 Comparison of different manufacturing parameters for viral vaccine modalities
Modality mRNA
Upstream
process (USP)
Approximate
upstream scale
Downstream
process (DSP)
Duration of
manufacturing
batch
Manufacturing
safety
considerations
Enzymatic Cell culture,
50 to >100L ~1000L Up to 6000L Up to 10,000L
Enzymatic
digestion,
chromatography,
UF/DF
~Hours-days for
enzymatic
reaction, several
days for
purication
(3–4 unit ops)
None—enzymes
don’t pose risk
Viral vectors
(adenovirus)
HEK293/PER.C6
Varies with
different products:
depth ltration,
UF/DF,
chromatography
Cell culture scale
up requires
several weeks,
several days for
purication
(5 unit ops)
Viral product,
infection process
requires viral seed
banks
Whole inactivated
virus (WIV) Protein-based
Cell culture, Vero Varies (insect
Inactivation,
depth ltration,
UF/DF,
chromatography
Cell culture scale
up requires
several weeks,
several days for
purication
(7 unit ops)
Intermediate is
infectious
SARS-CoV-2
virus, toxic
materials used for
inactivation
J. A. S. Romine etal.
cells, plant cells,
E. coli)
Depth ltration,
UF/DF,
chromatography,
viral ltration
(mammalian
culture only)
Cell culture scale
up requires several
weeks, several
days for
inactivation and
purication
Low risk:
endotoxin
(E. coli), viruses
(mammalian)
vaccines use mammalian cells (HEK293, PER.C6, and Vero) to produce the viruses,
while the protein-based vaccines use insect, plant, and microbial cells. With this, the
raw materials required for the cell culture are typically inexpensive and abundant
such as glucose in contrast to the less abundant and more expensive puried enzymes
and NTPs for the mRNA process. One exception to the cell culture process is mRNA
vaccine, where the DS is mRNA produced enzymatically from NTPs using a DNA
template. This approach affords multiple advantages over traditional cell culture
processes, including shorter production times. The scale of the USPs is also notable,
with the cell culture reactors requiring much larger volumes to produce large quantities of product as compared to the mRNA process.
The DSPs for the modalities may appear to employ similar unit ops, but each
have unique nuances. Most of the DSPs for cell culture processes start with a lysis
step since the product is contained within the cells. These are either chemical detergents (viral vectors, NVX-CoV2373) or mechanical (CoVLP). The exception to this
is WIVs, since the product is in the supernatant. Following lysis, depth ltration is
required to remove cell debris. Several of the processes also employ enzymatic
steps for digestion of nucleic acid impurities: Benzonase to remove free DNA and
RNA (viral vectors, WIVs) and DNase to remove template DNA (mRNA). After
depth ltration, chromatography is typically the next step. However, the WIVs add
in an additional inactivation step, which can be either chemical or physical. The
8 COVID-19 Vaccine Manufacturing Processes: Making the Molecules to Solve…
203
chromatography steps can take many different iterations, directed by qualities of the
modality: afnity, IEX, and SEC.Multiple chromatography steps may be required
to reach the purity needed. Notably, a viral ltration step at the end of the DSP is
used only for the mammalian culture for the protein-based vaccines. This step is not
performed for the viral vectors, WIVs, or the CoVLP process because the product is
a virus or virus-like particle that would be retained by the lter. Overall, a simplied
DSP once again benets the mRNA vaccines since there is no need to remove cellular debris and enzymatic degradation of process-based impurities can reduce the
separation to simple chromatography and UF/DF operations. Fewer unit operations
also mean shorter processing times overall compared to DSP for a conventional cell
culture process with multiple chromatography steps.
Considering safety in the manufacturing process, the production of WIVs necessitates the use of the pathogenic virus which requires special containment facilities
to ensure safety during manufacture. Furthermore, inactivation step for WIVs
requires the use of toxic chemicals like β-propiolactone and formalin. Vaccines that
utilize recombinant proteins as antigens avoid the use of highly infective viruses but
risk contamination with adventitious viruses when mammalian cell cultures are
required. Viral removal ltration methods have been developed to ensure the safety
of biotherapeutics derived from mammalian cell culture, but this adds to the complexity of process development and requires an additional step in the DSP.Enzymatic
production of mRNA circumvents exposure to viruses by avoiding the use of cell
culture in the drug substance process.
8.7 Conclusions
Overall, many skilled pharmaceutical development groups used the full range of
technologies and tools in their portfolios to answer the critical need for a vaccine
against SARS-CoV-2. To respond to this challenge, time was the most critical factor
for the manufacturing processes. Faster process development times meant more
material could be generated quickly for clinical trials to evaluate safety and efcacy
and, if successful, more material could be made to dose the population. With this,
advantages are given to shorter processes with fewer unit operations. The cell culture processes inherently add more time due to the need for cell expansion and
growth. These processes also require more complex harvest streams, adding additional unit operations and more manufacturing time. Additionally, more unit operations mean more development to make sure yield and product quality requirements
are met for each operation, while shorter manufacturing processes give the advantage of quicker process validation.
Based on the outlined constraints, mRNA had signicant advantages over the
other approaches. Despite the necessity for more complex feed materials, the enzymatic USP reduces time over the cell culture processes and fewer contaminants
reduce the number of DSP unit operations and shorten the manufacturing run time.
With these advantages combined with the high efcacy demonstrated in their
204
J. A. S. Romine etal.
clinical studies, mRNA vaccines were able to reach FDA Emergency Use
Authorization rst. Early availability of the vaccines was a crucial development in
amplifying their impact on human health. The lessons learned while searching for a
vaccine for SARS-CoV-2 should help make the response to any future pandemics
more effective.
Acknowledgements The authors thank Adam Burrell, Anna Chau, Rodney Combs, Nathaniel
Leachman, Chris Murphy, Lara Payne, and Joe Russo for their valuable comments on this chapter.
Author Disclosure All authors are employees of Pzer, Inc. and have no conicts of interest to
declare.
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