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\begin{document}

\title{Advancements in Insect-Cell Baculovirus Expression Vector Platform for
Production of Recombinant Adeno-Associated Virus-Based Gene Delivery
Vectors}



\author[1]{Pranav Joshi}%
\author[1]{Alina Venereo-Sanchez}%
\author[2]{Parminder Chahal}%
\author[1]{Amine Kamen}%
\affil[1]{McGill University}%
\affil[2]{National Research Council Canada}%


\vspace{-1em}



  
  \date{May 17, 2024}


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\selectlanguage{english}
\begin{abstract}
Despite rapid progress in the field, scalable high-yield production of
AAV is still one of the critical bottlenecks the manufacturing sector is
facing. The insect cell-baculovirus expression vector system (IC-BEVS)
has emerged as a mainstream platform for a scalable production of
recombinant proteins with clinically approved products for human use. In
this review, we provide a detailed overview on advancements in IC-BEVS
for rAAV production. Since the first report of baculovirus induced
production of rAAV in insect cells in 2002, this platform has undergone
significant evolutions. The original three baculovirus system was
improved to enhance expression stability and further streamlined to
reduce the number of baculoviruses to two and eventually one. The one
baculovirus system consisting of an inducible packaging insect cell line
was further improved to enhance the AAV vector quality and potency. In
parallel, implementation of advanced manufacturing approaches and
control of critical processing parameters have demonstrated promising
results with process validation in large scale bioreactor runs.
Moreover, optimization of molecular design of vectors to enable higher
cell-specific yield of functional AAV particles in combination with
bioprocess intensification strategies may further contribute to
addressing current and future manufacturing challenges.%
\end{abstract}%



\sloppy


\textbf{1 Introduction}

Since the establishment of the first successful continuous insect cell
line in 1959{[}1{]}, unending research and development led to the
establishment of insect cells as a workhorse for the production of
numerous recombinant proteins for research and clinical applications.
The discovery and isolation of Autographa californica multinuclear
polyhedrosis virus (AcMNPV) in 1971 was an important step in the
realization of the broad potential of the insect cell-baculovirus
expression vector system (IC-BEVS){[}2{]}. In the following years,
extensive studies related to the biology of the baculovirus, its
infection kinetics, genome sequences, and structural variants were
completed. The identification of the polyhedrin as a strong late
promoter, which was non-essential for the baculovirus replication, was
the breakthrough finding by Smith et al. in 1983 that first established
the IC-BEVS. Smith et al. demonstrated the expression of human
interferon-beta (INF-\selectlanguage{greek}β\selectlanguage{english}) and subsequently interleukin-2 (IL-2), using
IC-BEVS {[}3{]},{[}4{]}. Additionally, another strong late promoter,
p10, was discovered and was used in protein expression studies{[}5{]}.

The cell line derived from the fall armyworm: Spodoptera frugiperda
(Sf21 and derived clone Sf9) and the cabbage looper: Trichoplusia ni
(High five; Hi5) were established as continuous cell lines and were
extensively used due to their susceptibility to baculovirus infection
and good growth performances in adherent and thereafter in suspension
cell cultures{[}6{]}--{[}10{]}.

In the early 90's cell culture engineers took the lead in studying the
insect cell growth kinetics and metabolism in serum-supplemented and
serum-free culture medium. Rapidly, serum-free cell media with shear
protective properties were developed to enable suspension cell cultures
in shake flasks and bioreactors{[}11{]}--{[}14{]} demonstrating
scalability and robustness of the IC-BEVS process for protein
production.

Initially employed for the production of baculoviruses as biopesticides,
the IC-BEVS quickly gained popularity for the expression of a broad
spectrum of recombinant proteins, including enzymes, glycoproteins,
recombinant viruses and vaccines {[}10{]}, {[}15{]}. The IC-BEVS
platform found applications in the production of veterinary vaccines
such as Porcilis pesti, Circumvent PCV, CircoFLEX{[}16{]}, and human
vaccines such as Cervarix\selectlanguage{ngerman}\textsuperscript{®}{[}17{]}, and
Flublok\textsuperscript{®}{[}9{]}. Regulatory approval of
Cervarix\textsuperscript{®}, a virus-like particle-based vaccine against
cervical cancer, was a critical milestone as it was the first biologics
produced in insect cell and approved for human use. The ability of the
insect cells to grow in scalable serum-free suspension cultures, with a
demonstrated history of commercial scale manufacturing and a regulatory
portfolio, established the IC-BEVS as a platform of choice for research
and development and industrial manufacturing of biologics.

Currently, AAV is gaining widespread popularity in gene therapy
applications for the correction of monogenic disease conditions. In the
last decades, there has been a steady growth in AAV based gene therapy
clinical studies, which have been supported by the accelerated
development of the IC-BEVS scalable production systems for AAV
manufacturing. From the regulatory perspective, additionally to the
approval of Glybera\textsuperscript{®} by the European Medicines Agency
in 2012{[}18{]}, a more recent and significant milestone is the
breakthrough designation by USFDA of Biomarin`s Hemophilia A gene
therapy candidate, a recombinant adeno associated virus 5 (rAAV5) gene
delivery vector produced in insect cells using IC-BEVS{[}19{]}. This
further contributed to align the IC-BEVS manufacturing process of AAV
with the current standard of Good Manufacturing Practice (GMP).

In this review, focusing on the advancements of the IC-BEVS as a
platform for AAV production, we provide detailed insights on the
molecular design of baculovirus vectors developed for the IC-BEVS
platform and their key features. Importantly, advanced AAV manufacturing
technologies using IC-BEVS are reviewed and discussed from a process
developer standpoint. It is foreseeable, that further vector
optimizations combined with innovative process intensifications may
significantly contribute to addressing current and future manufacturing
challenges to enable higher cell-specific and total yields of functional
AAV gene delivery vectors of different serotypes.

\textbf{2 AAV biology}

The AAV belongs to the parvoviridae family: a family of single-stranded
DNA viruses that have evolved over the years, expanding a spectrum of
hosts{[}20{]}. The viruses of this family share some common structural
and functional features such as conserved phospholipase
A\textsubscript{2} (PLA\textsubscript{2})-like enzymatic domain and
identical replication components and pathways {[}20{]}, {[}21{]}. Based
on the host specificity, the parvoviridae family is divided into two
subfamilies: Parvovirinae and Densovirinae, which infect mammalian and
invertebrate hosts, respectively (Figure 1A). The Parvovirinae subfamily
is further divided into eight genera, including Dependovirus, into which
AAV is classified. The insect cells are a natural host of Densovirinae
viruses, and from a taxonomical standpoint, they should provide an
adequate cellular environment for replication of viruses from closely
related subfamily Parvovirinae, including AAV. This relation was first
recognized and later found to be applicable by Urabe et al., where the
group demonstrated the first IC-BEVS based rAAV production platform
{[}22{]}.

AAV virus particle comprises a vector genome: the functional element of
the virus, and the capsid, a metastable entity that preserves it. The
vector genome is a linear single-stranded DNA with a size of about
\textasciitilde{}4.7kb and a major coding region consisting of the
sequences for non-structural replicase proteins (Rep) and structural
capsid proteins (Cap or VP). This coding region is flanked by an
inverted terminal repeat sequences (ITR). The ITR is a segment of 145bp
with a T-shaped hairpin-like structure formed via complementary base
pairing of palindromic sequence of 125 bp. The remaining 20 bases stay
unpaired. The ITR region also encompasses the origin of replication
formed by the terminal resolution site and rep-binding element (RBE).
The free 3` hydroxyl region acts as an initiation sequence for DNA
replication.

AAV contains four non-structural Rep proteins (Rep 78, Rep 68, Rep 52,
and Rep40). Rep78 and Rep52 are expressed under the transcriptional
control of p5 and p19 promoters, respectively. The larger Rep78 and its
spliced variant Rep68 are crucial for- and participate in vector genome
integration, excision, rescue, and replication at various stages of AAV
life cycle{[}23{]}--{[}25{]}. The smaller Rep proteins Rep52 and its
spliced variant Rep40 play a critical role in the packaging of vector
DNA in preformed capsids and regulation of replication by repressing p5
in the absence of a helper virus{[}26{]}, {[}27{]}. The three structural
proteins (VP1, VP2, and VP3) expressed from the right open reading frame
(ORF) under p40 promoter form an icosahedron capsid that carries the
viral genome and delivers it to the host cell traversing through post
cell surface attachment. A representative genomic map of wtAAV2 {[}28{]}
and its transcriptional profile are shown in Figure 1B.

\textbf{3 Molecular Design of Baculovirus Expression Vectors and Cell
Lines For Production of rAAV}

In the last two decades, various molecular design approaches have been
used to achieve efficient AAV production employing IC-BEVS platform.
Building upon intrinsic advantages of this platform, the active efforts
were directed towards achieving higher expression stability, improved
yield, and streamlining manufacturing processes. Below, we summarize the
progressive development of various strategies and highlight features
that might be of consideration when using IC-BEVS for AAV production.

\textbf{3.1. Three Baculovirus (BV) System: Three-Bac}

The Three-Bac system achieved the production of the functional
recombinant AAV2 particles via co-infection of Spodoptera frugiperda
(Sf9) cells with three recombinant AcMNPV vectors {[}22{]}. In this
particular study, these baculovirus vectors were identified as Bac-VP
(Cap), Bac-Rep, and Bac-ITR, which harbored the AAV2 \emph{cap} gene,
AAV2 \emph{rep} gene, and ITR flanked, for example, by \emph{gfp} gene
sequence cassettes as a transgene, respectively (Figure 2A). In general,
the insect cell promoters in combination with cellular machinery are
crucial to drive the production of foreign proteins in insect cells.
During the development of the Three-Bac system, the main challenges
faced were related to the optimization of molecular expression cassettes
to achieve proportionate expression of capsid subunits and replicase
proteins{[}29{]}. The main challenge with replicase protein expression
was due to the colinear ORFs in AAV genome wherein Rep52 and its p19
promoter sequences are in-frame with the entire Rep78 sequence{[}28{]}.
This arrangement also led to the duplication of sequences and any
modifications such as insertion of insect promoter in the Rep52 sequence
to facilitate transcription in insect cell will result in an altered
sequence of entire Rep78 and its functional characteristics. Therefore,
Urabe et al{[}22{]}. separately arranged Rep78 and Rep52 expression
cassettes in transcriptionally opposite orientation (Bac-Rep) under
different insect promoters to achieve their desired expression level
(Figure 2A). On the other hand, the challenge with the capsid proteins
was achieving stoichiometric expression of three VP proteins in a
prototypic ratio of 1:1:10 for VP1:VP2: VP3. Sufficient expression and
incorporation of VP1 in AAV capsid is critical for efficient
transduction{[}30{]}, {[}31{]}. During AAV production in mammalian
cells, the intron splicing and leaky scanning of translation initiation
codon by ribosomal complex results in differential expression of three
VP proteins in the desired proportion which was not observed in insect
cell{[}22{]}. As a result, the Bac-VP expression cassette was designed
with a modified \emph{cap} sequence consisting of a weak VP1 translation
initiation codon-ACG in combination with a complex of nine nucleotide
sequence to achieve the stochastic ratio of VP proteins. The resulting
AAV2 particles demonstrated biophysical and functional characteristics
identical to that of mammalian-cells produced AAV2{[}22{]}.

An important finding by Urabe et al{[}22{]}. was that unlike mammalian
cells, AAV production in insect cells via BEVS does not require an
additional helper virus such as Adenovirus or Herpes simplex virus
(HSV), and the necessary helper function is provided by the baculovirus
in a fashion possibly distinct yet functionally comparable to that of
Adenovirus or HSV. It can also be inferred that the insect cell
adequately provided the components and cellular machinery necessary for
AAV protein production, capsid assembly, genome replication, and its
packaging.

The original Three-Bac system, although extremely promising and
flexible, found minimal application due to the operational complexity
from the bioprocessing standpoint and the instability of all
three-helper baculoviruses coding \emph{rep} , \emph{cap} , and
transgene cassette. Continuously generating, characterizing, and
validating three baculoviruses seed stocks during AAV production runs
was a significant undertaking to assure process consistency and poses a
significant burden. Moreover, Kohlbrenner et al. reported the passage
dependent loss of AAV protein expression in these BVs, notably the loss
of Rep proteins. The loss of expression of Rep proteins, which are
essential for genome replication and its encapsidation, resulted in an
overall reduced yield of packaged and functional AAV particles{[}32{]}.
Palindromic orientation with Rep78 and consequent excision of the entire
Rep 52 sequence was believed to be the reason for Bac-Rep instability
(Figure 2A). When isolated onto different baculoviruses, the stability
of Rep78 and Rep52 was restored over the extended BV passage, however,
requires quadruple BV co-infection further enhancing operational
complexity{[}32{]}.

\textbf{3.2 Two Baculovirus system: Two-Bac}

The next generation baculovirus constructs design was driven by two key
objectives: (1) the increase in expression stability of BV constructs,
and (2) the reduction in number of baculoviruses for process
simplification (Figure 3). In 2008, Chen proposed a strategy to improve
the baculovirus stability with the introduction of a synthetic intron in
the coding region of Rep (Bac-Intron-Rep: Bac\emph{In} Rep) and Cap
(Bac-Intron-Cap: Bac\emph{In} Cap) sequences{[}33{]}. The synthetic
intron consisted of a splice acceptor and a donor sequence flanking polh
promoter sequence, which, when placed in a specific position upstream to
Rep52 or VP2 stably expressed Rep and Cap over the extended passage
numbers. The placement of a synthetic intron in Bac\emph{In} Rep
facilitated the independent expression of Rep78 and Rep52 from two
different mRNA transcripts with improved stability. Similarly, in
Bac\emph{In} Cap, the synthetic intron drove independent expression of
VP1 and VP2/3 from two different mRNA transcripts, respectively offering
not only stability but also desired stoichiometry of all three VP
proteins. The system was further simplified by combining both expression
cassettes onto a single baculovirus: Bac\emph{In} RepCap now requiring
only two baculovirus co-infection for AAV production (Figure 2A).

Addressing the stability issue of Bac-Rep construct, in 2009, Smith et
al. reported a different strategy leveraging ribosomal leaky scanning
mechanism for stable expression of Rep proteins{[}34{]}. Here,
the\emph{rep} gene sequence was altered which consisted of (1) a
modified weak translational initiation codon for Rep78-CUG and (2)
Modification of nine downstream AUG codons without altering the
functionality of Rep proteins. This resulted in a non-duplication of
Rep78 and Rep52 sequences and circumvented the root cause of the genetic
instability of the original Bac-Rep construct. These modifications
resulted in a weak Kozak sequence for Rep78, and subsequent leaky
scanning of Rep78 and Rep52 codons. As a result, the stable expression
of both Rep78 and Rep52 in a desired proportion was achieved from a
single mRNA transcript. Next, to reduce the number of baculovirus
vectors from three to two, the original VP expression cassette was
combined and arranged in transcriptionally opposite direction with
reference to Bac-Rep cassette (Figure 2A). This combination resulted in
Bac-Rep2CapX recombinant baculovirus now requiring dual baculovirus
co-infection for AAV production.

\textbf{3.3 One Baculovirus System: One-Bac1.0}

The One-Bac system reported by Aslanidi et al. was a further adaptation
of the IC-BEVS platform for rAAV production{[}35{]}. This system
consists of two components: (1) A baculovirus-inducible stably
transformed packaging Sf9 cell line harboring the AAV2 \emph{rep}
and\emph{cap} sequences under the transcriptional control of polh
promoter and (2) a single recombinant baculovirus carrying AAV2 ITR
flanked transgene/gene of interest sequences (Bac-ITR). The inducible
cell line offers the advantage of regulated yet amplified expression of
AAV proteins resulting in a higher yield of AAV, whereas the stable
packaging cell line ensures consistent expression levels requiring only
a single baculovirus infection.

The development of this system provided answers to two key questions,
(1) how to devise a regulation switch for inducible expression of AAV
proteins in insect cells? and (2) how to achieve sustained and higher
expression of incorporated Rep and Cap proteins in the desired
proportion? The initial unsuccessful attempt of generating stable cell
line suggested an absolute requirement of baculovirus genomic elements
in \emph{cis} . One such element used in the final expression cassette
was a modified homologous region 2 (hr2-0.9), derived from A.
californica, which is inducible in the presence of immediate early
(IE-1) transcriptional \emph{trans} -regulator, herein provided by
Bac-ITR baculovirus. Related to the second question, the optimal
expression level of Rep78/Rep52 is essential and has a direct effect on
the overall yield. The lower expression ratio of Rep78:Rep52 is favored
for better AAV yield{[}29{]}. In order to further improve the system,
the rep-binding element (RBE) was included in the rep and cap expression
cassette. This was based on the findings that the ITR and a complex of
p5 and rep-binding element (RBE), alone or in combination, upregulate
p19 promoter activity in mammalian cells{[}36{]}, {[}37{]}. Further
studies reported the evidence of partial activity p19 promoter in the
insect cells{[}32{]}, {[}38{]}. Combining these findings, the final
version of \emph{rep} and \emph{cap} expression cassettes to generate a
stable cell line consisted of hr2-0.9 and RBE regulatory elements
derived from baculovirus and AAV2, respectively (Figure 2A). As a
result, lower Rep78:Rep52 expression ratio and sustained higher
expression of rep and cap proteins (Figure 2B) were obtained with up to
ten times higher cell-specific yield of AAV2 when compared to the
original Three-Bac system.

The modular construct design of this One-Bac was further explored for
the production of AAV serotypes 1-12 {[}39{]}. The AAV production yield
for all the serotypes was either comparable or higher than the original
Three-Bac system. Notable though was the deficient VP1 expression and
consequent reduction in transduction efficiency of some serotypes,
specifically of AAV5.

\textbf{3.4 Low VP1 Expression and Functional AAV yields in Insect
Cells}

In the mammalian cells, AAV's natural host, the combination of mRNA
splicing of p40 promoter-driven transcript and leaky ribosomal scanning
of VP2's weak translation of initiation codon-ACG result into the
expression of three VP subunits in a standard ratio of 1:1:10 for
VP1:VP2:VP3 from a mRNA transcript. The VP1 carries the sequence of
PLA\textsubscript{2}-like enzymatic domain and nuclear localization
signal, both of which are critical due to their postulated role in AAV
transduction{[}30{]}, {[}40{]}. Therefore, the expression of VP1 in an
appropriate proportion and its subsequent incorporation in the AAV
capsid is crucial to generate functional viral particles. Due to the
inherent difference of splicing mechanism in insect and mammalian cells,
in the original Three-Bac system, authentic start codon AUG of VP1 was
mutated to weak ACG codon facilitating leaky scanning via insect cell
ribosomal machinery achieving expression of all three VP proteins in a
proportion comparable to mammalian cells{[}22{]}. The Three-Bac though
originally successful for AAV2, was found to be non-versatile for other
serotypes such as AAV5 and AAV8{[}32{]}. AAV5 being the evolutionary
most distant serotype{[}41{]}, is of important clinical significance due
to its relatively better immuno-privileged characteristics compared to
other serotypes that are closely related to human origin serotype AAV2.
The AAV5, when produced in insect cell, exhibited no or lesser
transduction efficiency, which was found to be associated with
insufficient VP1 expression{[}32{]}, {[}39{]}, {[}42{]}. The deficient
VP1 expression was linked to the AAV5 VP1 sequence and the leaky
scanning efficiency in insect cells. The efficiency of ribosomal
scanning for recognizing AUG as a translation initiation codon relies on
Kozak sequence: a sequence context surrounding AUG, importantly
ACCAUG\emph{G,} where G at +4 position is crucial (A of AUG being +1) in
case of weak initiation codon such as ACG{[}43{]}, {[}44{]}. In contrast
to AAV2, the G of +4 position is U, in the case of AAV5, which is
believed to be responsible for deficient expression and subsequent
lesser incorporation of VP1 in AAV5 capsid{[}42{]}. Urabe et al.
addressed this challenge by domain swapping where 86 amino acid long
AAV5 VP1 sequence at specific positions was substituted with that of
AAV2 VP1 sequence resulting in AAV5 chimeric capsid with infectivity
comparable to that of AAV2{[}42{]}. Similarly, Kohlbrenner et al.
performed the domain swapping of an entire AAV5 and AAV8 VP1 sequences
with that of AAV2 VP1, resulting in increased VP1 proportion and
PLA\textsubscript{2} activity with restoration of transduction
efficiency{[}32{]}. In One-Bac2.0, the second generation One-Bac, the
mutated VP1 initiation codon (ACG) was replaced with an authentic VP1
strong codon (AUG), which in combination with synthetic intron placement
strategy as reported earlier by Chen{[}33{]}, demonstrated stronger
expression of all three VP proteins from two mRNA transcripts and
restored original composition of VP1 and AAV5 functionality. The RBE
negative version of this cell line (\selectlanguage{english}[?]RBE) also significantly reduced the
cross packaging of \emph{rep/cap} sequences and hence the proportion of
replication competent viral particles confirming the role of RBE as a
packaging signal{[}45{]}. Overall, these modifications improved the
final quality of the AAV5 serotypes produced using One-Bac2.0. Further
improved version was the third generation system:One-Bac3.0 where the
canonical VP1 initiation codon AUG was restored and was located in
serotype-specific strategically modified Kozak context sequence to
achieve optimal expression level of all three VP proteins{[}46{]}. Here,
the strong AUG codon was surrounded by a weak Kozak sequence, which led
to suboptimal translation initiation efficiency of 40\%-45\% compared to
optimal mammalian Kozak sequence. As a result, reduced yet sufficient
VP1 expression was achieved, and concomitant leaky scanning of following
VP2/VP3 start codons restored their optimal expression ratio with
respect to VP1. AAV5 and AAV9 serotypes produced using this system
demonstrated in-vivo transduction efficiency better than or comparable
to those produced in mammalian cells. Coupled with the \selectlanguage{english}[?]RBE strategy,
this system produced vectors with only marginal encapsidation of foreign
DNA sequences as reported with next-generation sequencing
analysis{[}46{]}.

Recently, in the Two-Bac system, the selection of optimal translation
initiation codon and associated downstream nucleotide sequence which
exhibited optimal stoichiometric expression of all three VP proteins of
AAV5 with improved functionality of the resultant vector was reported
{[}47{]}.

\textbf{4 Bioprocessing of rAAV Using The IC-BEVS Manufacturing
Platform}

Besides the molecular design of expression systems, the bioprocessing
aspect of IC-BEVS is equally essential for successful manufacturing of
AAV. In a typical IC-BEVS process, the cellular state depends on the
cell-virus interaction and infection kinetics, cellular energetics and
metabolism, and extracellular culture environment. The overall outcome
is a result of multifactorial interactions under a dynamic and
productive environment of the cell culture process. The following
section will discuss the effect of these parameters on the overall
production performances of rAAV vectors in insect cells.

\textbf{4.1 Insect Cell Lines}

Various insect-derived cell lines are currently being used for the
manufacturing of recombinant proteins. As previously discussed Sf9 cells
are predominantly used in AAV production process. Meghrous et al.
studied the AAV production in Sf9 and Hi5 cells using Three-Bac
system{[}48{]}. The study suggested that both cell lines offered
comparable cell-specific yields of functional AAV particles. Due to
better suitability of Sf9 for baculovirus stock generation over Hi5
cells{[}49{]} and no significant advantage of Hi5 over Sf9 in overall
AAV yield, Sf9 cells were selected for further process development work
(Table 1){[}48{]}. Another study involving AAV production in Sf21 using
a triple BV co-infection strategy also suggested better performance of
Sf9 for AAV production{[}50{]}.

\textbf{4.2 Multiplicity of Infection (MOI) of Baculovirus Vectors}

In the IC-BEVS, the MOI of the baculovirus has a critical impact on the
rate of cell growth and viability, the production kinetics, the quantity
and quality of the recombinant protein expressed, and the harvest time
of the culture. The low and high MOI infections are characterized by
their different rate of infection (synchronous Vs. asynchronous) and the
associated difference in kinetics of protein expression. Expression of
viral vector or virus-like particles, which typically involve
co-infecting cell cultures with more than one rBV carrying the coding
sequences of individual components adds another complexity to the
production process {[}51{]}, {[}52{]}. Altering the MOI of multiple
baculoviruses and their time of infection often result in altered capsid
compositions as reported by various groups{[}53{]}, {[}54{]}. AAV
production using triple baculovirus co-infection strategy is also an
example of such a complex and dynamic system where primary work has
relied on multicomponent system. Initial studies by Meghrous et al.
demonstrated the effect of Bac-Rep on overall Sf9 growth behavior and
AAV vector yield using Three-Bac system and its association with
encapsidation of vector DNA, and the transducing particle units{[}48{]}.
The Bac-Rep showed no notable effect on cell viability and growth
behavior irrespective of MOI used. The study focused on the
multidimensional interactive effect of multiple baculoviruses, each
expressing an essential protein of rAAV, suggested a significant effect
of Bac-Rep and Bac-Cap MOI on packaging efficiency and functional AAV
particles. A full factorial study conducted by Aucoin et al. provided an
in-depth analysis of single factor and multifactorial interaction
effects of MOI of the three baculoviruses on AAV yields {[}55{]}. This
study also suggested a self-sustaining nature of insect cell-baculovirus
infection process, where differing MOI of one baculovirus did not lead
to the drastic changes in overall AAV yield when expressed collectively
as relative proportions of total, genome containing and functional
particles.

\textbf{4.3 Effect of Cell Density at Infection}

The cell density over which the cell-specific yield of recombinant
protein expression drops, is an important indicator for selecting the
infection time and the cell density at infection for a productive batch.
This has been referred to in many studies as the cell density effect or
the time of infection. In the case of insect cell-baculovirus system,
this critical indicator is a result of the overall nutrient
availability, the cellular physiological state, and the ratio of cell
population infected versus total viable cells in the culture determined
by the MOI (i.e. synchronous Vs. asynchronous) during the production
phase. Commercially available serum-free media such as Sf
900\selectlanguage{english}\textsuperscript{}-II support a maximum cell density growth in batch
mode of around 10 million cells/mL{[}56{]}. However, the same medium
does not sustain protein expression in the post-infection phase at such
a cell density. When analyzed for AAV production using a triple BV
co-infection strategy at an overall MOI 5, the optimal cell density at
the time of infection was reported to be 1 million cells/mL in the
EX-CELL\textsuperscript{(r)} 420 medium{[}48{]}. Below this cell density,
the nutrient consumption was suboptimal, and above this cell density,
the cell-specific yield dramatically dropped. In another study, Cecchini
et al. reported that cell-specific yield of AAV production in Sf9 cells
was maintained up to the cell density of 3.5-4 million cells/mL in SFX
serum-free medium{[}57{]}. When analyzed for AAV5 production in One-Bac
Sf9 cells using Sf900\selectlanguage{english}\textsuperscript{}-II or
Sf900\selectlanguage{english}\textsuperscript{}-III medium by Joshi et al., the cell density
breakpoint was found to be 1.7-2.0 million cells/mL under synchronous
infection {[}58{]}. Any combination of cell density at the time of
infection and MOI that results in an overall cell density above 2
million cells/mL resulted in a drop in the cell-specific yield.

\textbf{4.4 Effect of Operation Mode}

The low cell density culture process operating under a batch mode of
cultivation generally results in a lower volumetric yield of AAV
necessitating large scale bioreactor production to meet exceptionally
high demand of the late clinical phase studies. When coupled with a
sustained cell-specific yield, the high cell density culture process
under fedbatch or perfusion mode of operation provide a mean to enhance
the volumetric yield. Both modes have been successfully implemented to
achieve a high cell density culture of Sf9 cells{[}58{]}, {[}59{]}. The
nutrient feeding strategy in a fedbatch process aims at maintaining the
cells in the mid-exponential phase for a sustained time to achieve a
high cell density before infection. When coupled with nutrient feeding
in the post-infection phase, metabolic limitations are alleviated.
Bedard et al. described an exemplary work where the nutrient feed
formulation was designed to meet metabolic need of Sf9 cells to support
the sustained expression of \selectlanguage{greek}β-\selectlanguage{english}galactosidase following BV
infection{[}60{]}. Building on this, Elias et al. reported
\selectlanguage{greek}β-\selectlanguage{english}galactosidase production at a cell density of 14 million cells/mL
without reduction of the cell-specific yield{[}61{]}. Meghrous et al.
reported that when applied at the time of infection for the triple BV
co-infection system, a complete medium replacement strategy that
practically mimics the perfusion at shake flask scale, resulted in a
linear increase in volumetric yield at various cell densities at the
time of infection without any loss of cell-specific yield of
AAV{[}48{]}. When further scaled up in a 3L bioreactor, the infection of
Sf9 cells at 2.5 million cells/mL in a fresh medium offered similar
cell-specific yield and a proportionate increase in volumetric yield
(Table 1). In a further study by Mena et al. the medium replacement
strategy coupled with infection at 5 million cells/mL with high MOI of
triple BVs (3 MOI of each) resulted in around three fold increase in
genomic AAV particles in comparison to the control, low cell density
batch process{[}59{]}. This increase in volumetric yield was
proportionate to the increase in cell density, which demonstrated
sustained cell-specific yield. Furthermore, when carefully tailored, the
fedbatch strategy in combination with low MOI applied to AAV production
in Sf9, resulted in almost 1 log increase in volumetric yield of
functional AAV particles (Table 1){[}59{]}. In this case, Sf9 cells were
infected at 5 million cells/mL at an MOI of 0.1 (of each BVs), which
resulted in cell density increase up to 9.6 million cells/mL at 48 hpi.
The nutrient supplementation was provided at the time of infection, and
24 h before and after infection. A more recent study by Joshi et al.
involving AAV5 production employing One-Bac under fedbatch mode of
operation also suggested improved yield of genomic and functional
particles of AAV5 (Table 1){[}58{]}. The high cell density at the time
of infection (\textasciitilde{}10 million cells/mL) was achieved via
bolus nutrient supplementation followed by high MOI infection, which
resulted in an immediate transition of cell culture from the growth
phase to the productive phase within 24hpi under synchronous infection
of baculovirus. Coupled with an additional bolus feeding, the process
exhibited sustained AAV production in post-infection phase{[}58{]}.

\textbf{4.5 Effect of Temperature}

The cell cultivation temperature is crucial for optimal cellular
metabolism, protein production, and processing. The temperature
modulation strategy is often applied to alter the cell metabolism,
production of glycoprotein variants, and their processing. The insect
cells are reported to best grow at temperature between 27\selectlanguage{ngerman}°C to
30°C{[}62{]}. In addition, no heat shock protein expression has been
reported at a temperature below 37°C{[}63{]}. However, there are reports
available with varying conclusions on protein expression yield at
various temperatures{[}64{]}--{[}67{]}. Some reports suggest better
protein processing at temperature lower than 27°C (20°C -24°C) without
any effect on the overall yield. Aucoin et al. reported AAV production
in Sf9 cells using a triple BV co-infection strategy at various
temperatures{[}68{]}. Across different temperature range tested (°C:21,
24, 27, 30 and 33), the highest yield of AAV functional particle was
achieved at 30°C which was 2.5X and 5X more than at 27°C and
temperatures below it, respectively. At the temperature below 27°C, the
cells show reduced growth rate and lower protein expression; however,
the cell viability was maintained in the post-infection phase. There was
no significant decrease in overall maximum cell density achieved at
various temperatures. Interestingly, the temperature above 27°C showed a
significant effect on the kinetics of AAV helper protein expression.

\textbf{4.6 High Cell Density-High MOI Production Process: A Case Study
of Three-Bac}

When various proteins are co-expressed via multiple baculoviruses, the
kinetic of expression of these proteins have an important effect on the
overall quantitative and qualitative yield of AAV. The optimal MOI (MOI
of 5 of each) of three baculovirus vectors for AAV production was very
high{[}55{]}. However, infection of Sf9 cells at such a high MOI in a
high cell density process poses an additional burden on baculovirus
stock preparation. Generally, the baculovirus stock with a concentration
of 1-3x10\textsuperscript{8} pfu/mL requires addition of a large volume
to achieve a high MOI, which often results in a substantial dilution of
the culture. For example, in an AAV production process involving
infection at 3-4 million cells/mL, the baculovirus co-infection at
individual MOI of 5 requires around 17\%-20\% v/v of each of three
baculoviruses stock (1x10\textsuperscript{8} pfu/mL) which results in an
overall dilution of the culture by around 50\%-60\% v/v which is not
desirable. The direct solution to this is to use either concentrated
stock of baculovirus, preparations of which has its challenges, or low
MOI process{[}59{]}, which results in asynchronous infection. An
alternative solution was reported by Cecchini et al., where the
researchers proposed the strategy that mimics low MOI infection culture
behavior and involves baculovirus-infected insect cell (BIIC){[}57{]}.
Here, the Sf9 cells were infected at low MOI of each of three
baculoviruses, which results in an asynchronous infection and infection
of the entire cell population within 48 hpi. Following this, the BIIC at
48 hpi were mixed with non-infected insect cells (NIIC) in a ratio of
1:10000 and frozen under liquid nitrogen. When thawed, the cells undergo
a secondary wave of baculovirus infection from the progeny generated
from BIIC and infect the rest of NIIC in the culture within 48-72 hours
post vial thaw. This strategy eliminated the baculovirus infection step
in the actual culture and offered consistency in baculovirus infection
kinetics, and protein expression since the relative proportion of BIIC
and NIIC is kept constant in the frozen cell bank. After the vial thaw,
the culture is volumetrically expanded by the addition of fresh medium
until the desired working volume in a bioreactor is achieved. When
assessed at different scales (shake flask to 200L bioreactor) for
different serotypes (AAV -6 and -9), a comparable yield of AAV serotypes
was reported (Table 1) {[}57{]}.

\selectlanguage{english}\textbf{4.7 AAV Production Yield in STB and Wave Bioreactors}

The favorable features of insect cell baculovirus platform are the
ability of insect cells to grow in suspension culture at a high cell
density, a proven linear scalability, and a history of regulatory
acceptance of the platform for the production of various
biologics{[}9{]}, {[}17{]}. Various reports of AAV production in either
conventional stirred-tank bioreactors (STB) (glass, stainless steel, or
disposable vessel){[}48{]}, {[}58{]}, {[}69{]} or Wave\selectlanguage{english}
bioreactors{[}69{]} suggest comparable yields and quality of rAAV vector
material (Table 1). The very first report of AAV production employing a
Three-Bac system in a 20L bioreactor by Meghrous et al. suggested AAV
material generation with around 3-fold improvement in cell-specific
yield of functional AAV particles in a controlled environment of
bioreactor compared to shake flask{[}48{]}. This production run
generated around 5x10\textsuperscript{12} AAV transducing units (Table
1). Subsequent reports of AAV production using modified Three-Bac system
in Wave\selectlanguage{english}\textsuperscript{} and stirred tank bioreactors suggest that AAV
volumetric yield was in the range of 5x10\textsuperscript{9} --
3x10\textsuperscript{10} VG/mL with a comparable consistency at various
scales of Wave\selectlanguage{english} and STB, respectively{[}69{]}. The lower volumetric
yield of AAV in Wave\selectlanguage{english} bioreactor was attributed to poor oxygen mass
transfer control in the culture. However, the vector quality, as
indicated by the ratio of genomic: transducing units, was consistent in
all production runs. Table 1 summarizes the AAV yield at various scales,
and in different IC-BEVS production reported. A schematic overview of
AAV production process using IC-BEVS platform is depicted in Figure 3.

\textbf{5 Conclusion and Future Perspectives}

From a molecular design standpoint, the Three-Bac system offers the
greatest flexibility by providing many possible combinations of
expression of VP proteins, replicase protein, and the gene of interest
independently. It is also a modular tool for pseudotyping various AAV
capsids{[}45{]},{[}32{]} allowing the production of mosaic AAV capsids
with different combinations of one or more VP proteins from other
serotypes. The Two-Bac system somewhat restricts this flexibility,
especially in a case where \emph{rep} and \emph{cap} sequences are
combined and inserted in a single baculovirus construct. The One-Bac
system, which involves Rep2CapX packaging cell line and a Bac-GOI,
offers restricted flexibility. The generation of the stable packaging
cell line represents a strategy, though effective and simple from a
processing standpoint, often complex and resource intensive. An
alternative and more flexible variant to existing One-Bac system
reported is the combination of RepX (X=Serotype dependent Rep) insect
cell line and Bac-CapY-ITR-GOI BV (Y=Serotype of interest) for AAV
serotypes production{[}70{]}. Overall, the modular Three-Bac system
provides the necessary flexibility at the early stage of development
that involves screening of multiple serotypes. However, at larger
production scales, it is the less attractive system due to inherent
manufacturing challenges, as discussed in previous sections. On the
other hand, One-Bac, though less flexible and potentially difficult to
implement at an early stage of development, offers a robust yet simple
manufacturing process for well-established clinically relevant serotypes
such as AAV5 or AAV9.

A recently added tool offers a number of advantages, the
Mono-Bac{[}71{]} or Bac-RepX/CapY/ITR (Figure 2A) combines all the AAV
elements in a single baculovirus construct. Upon infection with this
single rBV vector, the insect cells produce AAV. Yet the Mono-Bac system
has not been extensively used.

Although insect cells are being used for AAV production for more than
fifteen years, the first major question, yet unanswered, relates to the
uncertainty whether the maximum cellular protein production/processing
capacity has been reached. The second question is the efficiency of
vector genome packaging in insect cells. Recent reports suggested that
AAV serotypes produced in insect cells show more empty particles and
consequently less viral genome containing particles when compared
side-by-side to mammalian cell productions {[}72{]} -submitted{]}. This
necessitates active efforts towards engineering insect cells to produce
more genomic and functional particles. The production of wild type AAV
serotypes may provide a model for such study since nature has perfected
the process of generating 100\% genomic and functional particles in the
presence of helper virus function in mammalian cells, the natural host
of AAV{[}73{]}. Moreover, another important factor, requiring
significant consideration is the selection of appropriate combination of
ITR and Rep proteins for their optimal functionality in genome
replication and its packaging into pre-formed capsids. Typical
production protocols of different AAV serotypes production in insect
cells involves rep and ITR sequences from AAV2 and the cap sequence of
choice. The selection of appropriate combinations of serotype-specific
rep/ITR remains an open area of investigation to further support the
findings of a single report studying its effect on genome packaging
efficiency and genomic particle yield of AAV5{[}42{]}. In addition to
molecular and cellular engineering, the improvement in high cell density
production can also contribute to address the challenges of high-yield
productions taking advantage of advancements in the field of biologics
manufacturing. This involves and not limited to, optimization of medium
formulation, design and optimization of nutrient feed for insect cells,
and detailed understanding of the critical process parameters guided by
the critical quality attributes of AAV serotype therapeutic products.
However, increasing the cell density in a production process beyond a
point may turn counterproductive at a downstream process stage. Because
AAV is an inherently intracellular product, the primary recovery step
involves the cell lysis and release of AAV in lysis buffer. In the high
cell density culture process, the cellular components (host cells genome
and proteins) are co-extracted with AAV in the lysate during the harvest
and clarification process. Their removal exerts a significant burden on
downstream processing. It would be appealing to study if the AAV capsid
sequences can be modified, without altering the functionality, to direct
its secretion in the extracellular environment as is the case with
secretory proteins such as monoclonal antibodies. This way, a high cell
density-high volumetric yield process can be derived devoid of the
presence of major interfering cellular components. Such modification can
also be adopted for a perfusion production process with an ultra-high
cell density culture, which will ultimately lead to a very high
volumetric yield of AAV and reduce the production scale. In conclusion,
comforted by the increased regulatory acceptance, ease of scale-up, and
recent advancements in production technologies, insect cell baculovirus
system is being more broadly adopted for the production of multiple AAV
serotypes (Table 1). As a result, more insect cell produced AAV vectors
can be expected in clinical trials in the future.

\textbf{6 Acknowledgement}

P.R.H.J. is financially supported through a grant from the Natural
Sciences and Engineering Research Council (NSERC RGPIN-2015-05132) of
the government of Canada. A.A.K. is a recipient of a Canada Research
Chair (CRC/240394)

\textbf{7 Conflict of interest}

The authors declare no financial or commercial conflict of interest

\textbf{8 References:}

{[}1{]} Gaw, Z. Y., Liu, N. T., Zia, T. U., Tissue culture methods for
cultivation of virus grasserie. \emph{Acta Virol} .1959, 3, 55-60.

{[}2{]} Vail, P. V., Sutter, G., Jay, D. L., Gough, D., Reciprocal
infectivity of nuclear polyhedrosis viruses of the cannage looper and
alfalfa looper. \emph{J Invert Patho} . 1971, 17 (3), 383-388.

{[}3{]} Smith, G. E., Summers, M. D., Fraser, M. J., Production of human
beta interferon in insect cells infected with a baculovirus expression
vector. \emph{Mol. Cell. Biol} . 1983, 3, 2156-2165.

{[}4{]} Smith, G. E., Ju, G., Ericson, B. L., Moschera, J., Lahm, H. W.
et al., Modification and secretion of human interleukin 2 produced in
insect cells by a baculovirus expression vector.\emph{Proc Natl Acad Sci
U.S.A.} 1985, 82(24), 8404--8408.

{[}5{]} Kuzio, J., Rohel, D. Z., Curry, C. J., Krebs, A., Carstens, E.
B. et al., Nucleotide sequence of the p10 polypeptide gene of autographa
californica nuclear polyhedrosis virus. \emph{Viro} . 1984, 139,
414-418.

{[}6{]} Kirnbauer, R., Booy, F., Cheng, N., Lowry, D. R., Schiller, J.
T., Papillomavirus L1 major capsid protein self-assembles into virus
like particles that are highly immunogenic. \emph{Proc Natl Acad Sci
U.S.A.} 1992, 89(24) 12180-12184.

{[}7{]} Wickham, T. J., Davis, T., Granados, R. R., Shuler, M. L., Wood,
H. A., Screening of insect cell lines for the production of recombinant
proteins and infectious virus in the baculovirus expression
system.\emph{Biotechnol Prog} . 1992, 8(5), 391-396.

{[}8{]} Felberbaum, R. S., The baculovirus expression vector system: a
commercial

manufacturing platform for viral vaccines and gene therapy
vectors.\emph{Biotechnol J} .

2015, 10(5), 702-714.

{[}9{]} Buckland, B., Boulanger, R., Fino, M., Srivastava, I., Holtz, K
et al., Technology transfer and scale-up of the
Flublok\textsuperscript{(r)} recombinant hemagglutinin (HA) influenza
vaccine manufacturing process. \emph{Vaccine} . 2014, 32(42), 5496-5502.

{[}10{]} Van Oers, M. M., Oijlman, G. P., Vlak, J. M., Thirty years of
baculovirus-insect cell protein expression: from dark horse to
mainstream technology. \emph{J Gen Virol} . 2015, 96, 6-23.

{[}11{]} Kamen, A. A., Bedard, C., Tom, R., Perret, S., Jardin, B.,
On-line monitoring of respiration in recombinant-baculovirus infected
and uninfected insect cell bioreactor cultures. \emph{Biotechnol.
Bioeng} . 1996, 50(1), 36-48.

{[}12{]} Kamen, A. A., Tom, R. L., Caron, A. W., Chavarie, C., Massie,
B. et al.,Culture of insect cells in a helical ribbon impeller
bioreactor. \emph{Biotechnol. Bioeng.} 1991, 38, 619-628.

{[}13{]} Kamen, A. A., Chavarie, C., Andre, G., Archambault, J., Design
parameters and performance of a surface baffled hellicle ribbon impeller
bioreactor for the culture of shear sensitive cells. \emph{Chem Engg
Sci} . 1992, 47, 2375-2380.

{[}14{]} Maranga, L., Cunha, A., Clemente J., Cruz, P., Carrondo, M. J.,
Scale-up of virus-like particles production: effects of sparging,
agitation and bioreactor scale on cell growth, infection kinetics and
productivity, \emph{J Biotechnol.} 2004, 107(1), 55-64.

{[}15{]} Mena, J. A., Kamen, A. A., Insect cell technology is a
versatile and robust vaccine manufacturing platform. \emph{Expert
Rev.Vaccines.} 2011, 10, 1063-1081.

{[}16{]} Desrosiers, R., Clark, E., Tremblay, D., Tremblay, R., Polson,
D., Use of a one-dose subuit vaccine to prevent loss associated with
procine circovirus type 2. \emph{J Swin Health Prod.} 2009, 17, 148-154.

{[}17{]} Harper, D. M., Impact of vaccination with
Cervarix\textsuperscript{TM} on subsequent HPV-16/18 infection and
cervical disease in women 15-25~years of age. \emph{Gynecol. Oncol} .
2008, 110, S11-S17.

{[}18{]} Yla-Hertuala, S., Endgame: Glybera finally recommended for
approval as the first gene

therapy drug in the European Union. \emph{Mol. Ther} . 2012, 20,
1831-1832

{[}19{]} AAV5-hFVIII-SQ (BMN 270) Annex III A. BioMarin Pharmaceutical
Inc. 2017.

{[}20{]} Kailasan, A., Agbandje-McKenna, M. Parrish, C. R., Parvovirus
Family Conundrum: What Makes a Killer?\emph{Annu. Rev. Virol} . 2015, 2,
425-450.

{[}21{]} Adler, S. P., Koch, W. C., Human Parvovirus, Infect. Dis.
Fetus. Elsevier Inc.2011, 834--

860.

{[}22{]} Urabe, M., Ding, C., Kotin, RM., Insect cells as a factory to
produce adeno-associated

virus type 2 vectors. \emph{Hum. Gene. Ther} . 2012, 13, 1935-1943.

{[}23{]} Zarate-Perez, F., Mansilla-Soto, J., Bardelli, M., BurgnerII,
J. W., Villamil-Jarauta, M. et al., Oligomeric properties of
adeno-associated virus Rep68 reflect its multifunctionality. \emph{J
Viro} . 2013, 87 (3), 1232-1241.

{[}24{]} James, J. A., Escalante, C. R., Yoon-Robarts, M., Edwards, T.
A., Linden, R. M. et al., Crystal structure of the SF3 helicase from
adeno-associated virus type 2. \emph{Structure} . 2003, 11(8),
1025-1035.

25{]} Yoon-Robarts, M., Blouin, A. G., Blenker, S., Kleinschmidt, J. A.,
Aggarwal, A. K. et al., Residues within the ss-motif are critical for DNA
binding by the superfamily 3 helicase rep40 of adeno-associated virus
type 2. \emph{J Biol Chem} . 2004, 279(48), 50472-50481.

{[}26{]} King, J. A., Dubiezlig, R., Grimm, D., Kleinschmidt, J. A., DNA
helicase-mediated packaging of adeno-associated virus type 2 genomes
into preformed capsids, \emph{EMBO J} . 2001, 20(12), 3282--3291.

{[}27{]} Collaco, R. F., Kalman-Maltese, V., Smith, A. D., Dignam, J.
D., Trempe, J. P. A., Biochemical characterization of the
adeno-associated virus rep40 helicase. \emph{J Biol Chem} . 2003,
278(36), 34011-34017.

{[}28{]} Srivastava, A., Lusby, E. W., Benrs, K. I., Nucleotide sequence
and organization of the adeno-associated virus 2 genome. \emph{J Virol}
. 1983, 45(2), 555-564.

{[}29{]} Grimm, D., Kern, A., Rittner, K., Kleinschmidt, J. A., Novel
tool for production and purification of recombinant adeno-associated
virus vectors. \emph{Hum. Gene. Ther} . 1998, 9(18), 2745-2760.

{[}30{]} Girod, A., Wobus, CE., Zadori, Z., Ried. M., Leike, K.,
Tijssen, P., The VP1 capsid protein of adeno-associated virus type 2 is
carrying a phospholipase A\textsubscript{2} domain required for virus
infectivity. \emph{J Gen. Virol} . 2002, 83, 973-8.

{[}31{]} Vihinen-Ranta, M., Wang, D., Weichert, W. S., Parrish, C. R.,
The VP1 N-terminal sequence of canine parvovirus affects nuclear
transport of capsids and efficient cell infection. \emph{J Virol} .
2002, 76(4), 1884-1891.

{[}32{]} Kohlbrenner, E., Aslanidi, G., Nash, K., Shklyaev, S.,
Thompson, MC., Bryne, BJ.et al.,

Successful production of pseudotyped rAAV vectors using a modified
baculovirus

expression system. \emph{Mol. Ther} . 2005, 12, 1217-1225.

{[}33{]} Chen, H., Intron splicing-mediated expression of AAV rep and
cap genes and

production of AAV vectors in insect cells. \emph{Mol. Ther} . 2008, 16,
924-930.

{[}34{]} Smith, RH., Levy, JR., Kotin, RM., A simplified baculovirus-AAV
expression vector

system coupled with one-step affinity purification yields high-titer
rAAV stocks from

insect cells. \emph{Mol. Ther} . 2009, 17, 1888-1896.

{[}35{]} Aslanidi, G., Lamb, K., Zolotukhin, S., An inducible system for
highly efficient

production of recombinant adeno-associated virus (rAAV) vectors in
insect sf9 cells.

\emph{Proc Natl Acad Sci U.S.A} . 2009, 106, 5059-5064.

{[}36{]} Nony, P., Tessier, J., Chadeuf, G., Ward, P., Giraud, A. et
al., Novel cis-acting replication element in the adeno-associated virus
type 2 genome is involved in amplification of integrated rep-cap
sequences. \emph{J Virol} . 2001, 75(20), 9991-9994.

{[}37{]} Lackner, D. F., Muzyczka, N., Studies of the mechanism of
transactivation of the adeno-associated virus p19 promoter by Rep
protein., \emph{J Virol.} 2002, 76(18), 8225-8235.

{[}38{]} Savy, A., Kaikkonen, M. U., Leger, A., Dickx, Y., Galibert, L.
et al., Genetics instability of wtAAV2 genome and AAV promoter
activities in the Baculovirus / Sf9 cells system. \emph{PLoS One} .
2018, 13(7), pp. 1--18, 2018.

{[}39{]} Mietzsch, M., Grasse, S., Zurawski, C, Weger, S., Bennett, A.,
Agabandje-McKenna, M. et al., One Bac: platform for scalable and
high-titer production of adeno-associated virus serotype 1-12 vectors
for gene therapy. \emph{Hum. Gene Ther.} 2014, 25(3), 212--222.

{[}40{]} Grieger, J. C., Snowdy, S., Samulski, R. J. Separate basic
region motifs within the adeno associated virus capsid proteins are
essential for infectivity and assembly. \emph{J Virol} . 2006, 80(11),
5199-5210.

{[}41{]} Bantel-Schaal, U., Dellius, H., Schmidt, R., zur Hausen, H.,
Human adeno-associated virus type 5 is only distantly related to other
known primate helper-dependent parvoviruses. \emph{J Virol.} 1999.
73(2), 939-947.

{[}42{]} Urabe, M., Nakakura, T., Xin K. Q., Obara, Y., Mizukami, H.,
Kume A. et al., Scalable generation of high-titer recombinant
adeno-associated virus type 5 in insect cells. \emph{J Virol.} 2006,
80(4), 1874-1885.

{[}43{]} Kozak, M., Point mutations define a sequence flanking the AUG
initiator codon that modulates translation by eukaryotic
ribosomes.\emph{Cell.} 1986, 44(2), 283-292.

{[}44{]} Grunert, S., Jackson, R. J. The immediate downstream codon
strongly influences the efficiency of utilization of eukaryotic
translation initiation codons. \emph{EMBO} . 1994, 13(15), 3618-3630.

{[}45{]} Mietzsch, M., Casteleyn, V., Weger, S., Zolotukhin, S.,
Heilbronn, R., OneBac 2.0: sf9 cell

lines for production of AAV5 vectors with enhanced infectivity and
minimal

encapsidation of foreign DNA. \emph{Hum. Gene. Ther} . 2015, 26, 1-24.

{[}46{]} Kondratov, O., Marsic, D., Crosson, SM., Mendez-Gomez, HR.,
Moskalenko, O., Mietzsch,

M. et al., Direct head-to head evaluation of recombinant
adeno-associated viral

vectors manufactured in human versus insect cells. \emph{Mol. Ther}
.2017, 25, 2661-2675.

{[}47{]} Bosma, B., du Plessis, F., Ehlert, E., Nijmeijer, B., deHaan,
M. et al., Optimization of viral protein ratios for production of rAAV
serotype 5 in the baculovirus system. \emph{Gene Ther} . 2018, 25(6),
415-424.

{[}48{]} Meghrous, J., Aucoin, MG., Jacob, D., Chahal, P. S., Arcand,
N., Kamen, AA. Production

of recombinant adeno-associated viral vectors using baculovirus/insect
cell

suspension culture system: From shake flasks to a 20-L
bioreactor.\emph{Biotechnol. Prog} .

2005, 21, 154-160.

{[}49{]} Wilde, M., Klausberge, M., Palmberge, D., Ernst, W., Grabherr,
R., Tnao38, high five and Sf9-evaluation of host-virus interactions in
three different insect cell lines: baculovirus production and
recombinant protein expression. \emph{Biotechnol Lett} . 2014, 36(4),
743-749.

{[}50{]} Liu, Y. K., Yang, C. J., Liu, C. L., Shen, C. R., Shiau, L. D.,
Using a fed-batch culture strategy to enhance rAAV production in the
baculovirus/insect cell system. \emph{J Biosci Bioeng} . 2010. 110 (2),
187-193.

{[}51{]} Hu, C. Y., Bentley, W. E., Effect of MOI ratio on the
composition and yield of chimeric infectious bursal disease virus-like
particles by baculovirus co-infection: deterministic predictions and
experimental results. \emph{Biotechnol. Bioeng} . 2001, 75(1), 104-119.

{[}52{]} Jiang, B., Barnaik, V., Smith, R. P., Sharma, R., Corsaro, B.
et al., Synthesis of rotavirus-like particles in insect cells:
comparative and quantitative analysis. \emph{Biotechnol. Bioeng} . 1998,
60(3), 369-374.

{[}53{]} Palomares, L. A., Lopez, S., Ramirez, O. T., Strategies for
manipulating the relative concentration of recombinant rotavirus
structural proteins during simultaneous production by insect
cells.\emph{Biotechnol. Bioeng} . 2002, 78(6), 635-644.

{[}54{]} Mena, J. A., Ramirez, O. T., Palomares, L. A., Intracellular
distribution of rotavirus structural proteins and virus-like particles
expressed in the insect cell-baculovirus system. \emph{J Biotechnol} .
2006, 122(4), 443-452.

{[}55{]} Aucoin, M. G., Perrier, M., Kamen, A. A., Production of
adeno-associated viral vectors in insect cells using triple infection\selectlanguage{english} :
optimization of baculovirus concentration ratios. \emph{Biotechnol.
Bioeng} . 2006, 95(6), 1081-1092.

{[}56{]} Schmid, G. Insect cell cultivation: Growth and
kinetics.\emph{Cytotechnology} , 1996, 20, 43--56.

{[}57{]} Cechini, S., Virag, T., Kotin, M., Reproducible high yields of
recombinant adeno-associated virus produced using invertebrate cells in
0.02- to 200-liter cultures. \emph{Hum. Gene. Ther} . 2011. 22(8),
1021-1030.

{[}58{]} Joshi, P. R. H., Cervera, L., Ahmed, I., Kondratov, O,
Zolotukhin, S. et al., Achieving high-yield production of functional
AAV5 gene delivery vectors via fedbatch in an insect cell-one
baculovirus system. \emph{Mol Ther Methods Clin Dev} . 2019, 13,
279-289.

{[}59{]} Mena, J. A., Aucoin, M. G., Montes, J., Chahal, P.S., Kamen, A.
A., Improving adeno-associated vector yield in high density insect cell
cultures. \emph{J Gene Med} . 2010. 12(2), 157-167.

{[}60{]} Bedard C., Kamen A., Tom, R., Massie B., Maximization of
recombinant protein yield in the insect cell / baculovirus system by
one-time addition of nutrients to high-density batch
cultures.\emph{Cytotechnology} . 1994, 15, 129-138.

{[}61{]} Elias, C.B., Zeiser, A., Bedard, C., Kamen, A. A., Enhanced
growth of Sf-9 cells to a maximum density of 5.2 x
10\textsuperscript{7}cells per mL and production of ss-galactosidase at
high cell density by fed batch culture. \emph{Biotechnol. Bioeng} .
2000, 68, 381-388.

{[}62{]} Shuler, M. L., Cho, T., Wickman, T., Ogonah, O., Kool, M. et
al., Bioreactor development for production of viral pesticides or
heterologous proteins in insect cell cultures. \emph{Ann N Y Acad Sci} .
1990, 589, 399-422.

{[}63{]} Huhtala, A., Linko, P., Mutharasan, R., Protein response of
insect cells to bioreactor environmental stresses. \emph{J Biotechnol} .
2005, 118(3), 278-289.

{[}64{]} Cain, K. D., Byrne, K. M., Brassfield, A. L., LaPatra, S. E.,
Ristow, S. S., Temperature dependent characteristics of a recombinant
infectious hematopoietic necrosis virus glycoprotein produced in insect
cells. \emph{Dis Aquat Organ} . 1999, 36(1), 1-10.

{[}65{]} Donaldson, M., Wood, H. A., Kulakosky, P. C., Shuler, M. L.,
Glycosylation of a recombinant protein in the Tn5B1-4 insect cell line:
Influence of ammonia, time of harvest, temperature, and dissolved
oxygen. \emph{Biotechnol. Bioeng} . 1999, 63(3), 255-262.

{[}66{]} Hara, T., Nonaka, K., Kawaguchi, H., Ogata, S., Etou, N.,
Effects of temperature on escherichia coli beta-galactosidase expression
in baculovirus-insect cell system. \emph{Biosci Biotechnol Biochem} .
1993, 57(6), 996-997.

{[}67{]} Reuveny, S., Kim, Y. J., Kemp, C. W., Shiloach, J., Effect of
temperature and oxygen on cell growth and recombinant protein production
in insect cell cultures. \emph{Appl Microbiol Biotechnol} . 1993, 38(5),
619-623.

{[}68{]} Aucoin, M. G., Perrier, M., Kamen, A. A., Improving AAV vector
yield in insect cells by modulating the temperature after
infection.\emph{Biotechnol. Bioeng} . 2007, 97(6), 1501-1509.

{[}69{]} Negrete, A., Kotin, R. M., Production of recombinant
adeno-associated vectors using two bioreactor configurations at
different scales. \emph{J Virol Methods} . 2007, 145(2), 155-161.

{[}70{]} Wu, y., Ting, M., Jiang, L., Zengpeng, H., Dong, R. et al.,
Development of versatile and flexible Sf9 packaging cell line-dependent
OneBac system for large-scale recombinant adeno-associated virus
production. \emph{Hum. Gene. Ther. Methods} . 2019, 30(5), 172-183.

{[}71{]} Galibert, L., Merten, O. W., Jacob, A. Baculovirus system for
the expression of a gene therapy vector. US patent 2014/0349374A1, 2014.

{[}72{]} Rumachik, N. G., Malaker, S. A., Poweleit, N., Maynard, L. H.,
Adams, C. M. et al., Methods matter-standard production platform for
recombinant AAV can produce chemically and functionaliy distinct
vectors. doi:~https://doi.org/10.1101/640169

{[}73{]} Zeltner, N., Kohlbrenner, E., Clement N., Weber, T., Linden, R.
M., Near-perfect

infectivity of wild-type AAV as benchmark for infectivity of recombinant
AAV

vectors. \emph{Gene. Ther} . 2011, 17, 872-879.

\textbf{Table 1.} Summary of AAV Production in IC-BEVS Platform\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllllll@{}}
\toprule
\begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Author and year\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Production system\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Serotype\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Transgene\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Culture volume\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Cell density (cells/mL)\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
VG/cell\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
VG/mL\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
IVP or TU/ cell\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
IVP or TU/mL\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Cp/mL\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Cp: VG: IVP or TU\strut
\end{minipage} & \begin{minipage}[b]{0.07\columnwidth}\raggedright\strut
Specific notes\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Urabe et al{[}22{]}. (2002)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Three-Bac\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
200mL\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2x10\textsuperscript{6 a}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4.5x10\textsuperscript{4}\selectlanguage{ngerman}± 0.32x10\textsuperscript{4 b}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA: 1344:1\textsuperscript{c} \selectlanguage{ngerman}±283\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
First report of rAAV production in sf9 cells\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Meghrous et al{[}48{]}. (2005)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Three-Bac\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
3L 20L\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
3.08x10\textsuperscript{6 a} 1.7x10\textsuperscript{6 a}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
132 253\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4.09x10\textsuperscript{8 b d} 4.46x10\textsuperscript{8 b d}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
6.7x10\textsuperscript{11 b e} 3.71x10\textsuperscript{11 b e}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1640: NA: 1 830: NA: 1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Bac-Rep MOI optimization and demonstration of AAV production process
scalability in bioreactor\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Urabe et al {[}42{]}. (2006)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Three-Bac\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
hGFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
5.6x10\textsuperscript{4}\selectlanguage{ngerman}± 0.32x10\textsuperscript{4 f}
7.67x10\textsuperscript{4} ± 2.12x10\textsuperscript{4 g}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Bac-Cap modification, VP1 Domain swapping to produce functional AAV5
vector particle\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Aucoin et al{[}55{]}. (2006)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Three-Bac\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20mL\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2x10\textsuperscript{10} 2x10\textsuperscript{9}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
5x10\textsuperscript{7 d} 1.7x10\textsuperscript{8 d}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
3400:400:1 3000:12:1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
BacITR: BacRep: BacCap (1:9:1) BacITR: BacRep: BacCap (9:9:9)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Nagrete and Kotin{[}69{]} (2007)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Three-Bac\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
200mL 5L-Wave 20L-Wave 10L-STB 40L-STB\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2x10\textsuperscript{6 a}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textasciitilde{}3x10\textsuperscript{10 b} \textasciitilde{}
9x10\textsuperscript{9 b} \textasciitilde{}5x10\textsuperscript{9 b}
\textasciitilde{}2x10\textsuperscript{10 b}
\textasciitilde{}4.33x10\textsuperscript{10 b}
\textsuperscript{(}\selectlanguage{ngerman}±4.1x10\textsuperscript{10})\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textasciitilde{}1x10\textsuperscript{9 h}
\textasciitilde{}5x10\textsuperscript{8 h}
\textasciitilde{}3x10\textsuperscript{8 h}
\textasciitilde{}5x10\textsuperscript{8 h}
\textasciitilde{}7.5x10\textsuperscript{8 h}
(\selectlanguage{ngerman}±2.5x10\textsuperscript{8})\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA: 30:1 NA: 18:1 NA: 16:1 NA: 40:1 NA: 20:1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Three-Bac system with a modified Bac-Cap sequence. Demonstration of AAV
process scalability at large scale\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Aucoin et al {[}68{]}. (2007)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Three-Bac\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textasciitilde{}12000\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
200\textsuperscript{d}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4x10\textsuperscript{8}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
4600:79:1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Temperature modulation study and its effect on AAV yield\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Mena et al{[}59{]}. (2010)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Three-Bac\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20mL 3L\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
9.43x10\textsuperscript{6 i} 9.5x10\textsuperscript{6 i}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1.17x10\textsuperscript{11 b} 2.2x10\textsuperscript{11 b}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2.9x10\textsuperscript{9 b d} (\selectlanguage{ngerman}±0.32x10\textsuperscript{9})
2.35x10\textsuperscript{9 b d}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA: 40:1 NA: 94:1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Low MOI, Fedbatch production\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Liu et al {[}50{]}. (2010)\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Three-Bac\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
GFP\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
50mL\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
1x10\textsuperscript{6}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
240\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
2.4x10\textsuperscript{8}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
AAV production in Sf21 cells in fed-batch culture\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Cecchini et al{[}57{]}. (2011)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Three-Bac\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
9 8 6\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP U7smOPT GFP U7smOPT PLS\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
10L 20L 100L 200L 20L\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
3.55x10\textsuperscript{6 i} 3.23x10\textsuperscript{6 i}
3.88x10\textsuperscript{6 i} 4.29x10\textsuperscript{6 i}
(\selectlanguage{ngerman}±1x10\textsuperscript{6}) 4.41x10\textsuperscript{6 i}
3.44x10\textsuperscript{6 i}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2.24x10\textsuperscript{4} 2.41x10\textsuperscript{4}
1.48x10\textsuperscript{4} 2x10\textsuperscript{4}
(\selectlanguage{ngerman}±0.3x10\textsuperscript{4}) 1.82x10\textsuperscript{4}
2.07x10\textsuperscript{4}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
7.9x10\textsuperscript{10 b} 7.8x10\textsuperscript{10 b}
4.44x10\textsuperscript{10 b} 8.75x10\textsuperscript{10 b}
(\selectlanguage{ngerman}±4.04x10\textsuperscript{10}) 7.45x10\textsuperscript{10 b}
5x10\textsuperscript{11 b}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
AAV production employing BIIC strategy. Demonstration of AAV production
process robustness at bioreactor scale regardless of the type of
transgene\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Chen{[}33{]}. (2008)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Three-Bac Two Bac BacInRep/Cap\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2 6 8 1 2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP GFP GFP GFP GFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
5x10\textsuperscript{6 a} 5x10\textsuperscript{6 a}
5x10\textsuperscript{6 a} 5x10\textsuperscript{6 a}
5x10\textsuperscript{6 a}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1.37x10\textsuperscript{11 b} (\selectlanguage{ngerman}±0.34x10\textsuperscript{11})
3.53x10\textsuperscript{10 b} 9.65x10\textsuperscript{10 b}
4.41x10\textsuperscript{10 b} 2.2x10\textsuperscript{11}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Bac-Intron constructs of baculovirus vector Baculovirus with enhanced
passage dependent stability Bac-In-Rep/Cap and Bac-In-ITR\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Smith et al. {[}34{]}. (2009)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Two-Bac BacRep2CapX\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1 2\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP GFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
200mL 200mL\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2.4x10\textsuperscript{6 a} 1.2x10\textsuperscript{6 a}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1.07x10\textsuperscript{13 j} (\selectlanguage{ngerman}±0.46x10\textsuperscript{13})
6.5x10\textsuperscript{12 j}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
5.08x10\textsuperscript{10 j k} (\selectlanguage{ngerman}±5.9x10\textsuperscript{10})
1.9x10\textsuperscript{10 j k}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA: 200:1 NA: 350:1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Bac-Rep/Cap and Bac-ITR based two baculovirus vectors with improved
stability\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Aslanidi et al{[}35{]}. (2009)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
One-Bac 1.0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2 1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP GFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1.4x10\textsuperscript{5} 7.3x10\textsuperscript{4}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Inducible packaging Sf9 cell line for AAV production\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Mietszch et al{[}39{]}. (2014)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
One-Bac 1.0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1-11 12\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP GFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
\textasciitilde{}10\textsuperscript{4}-10\textsuperscript{5 l}
\textasciitilde{}10\textsuperscript{3 l}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Production of all AAV serotypes employing One-Bac system\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
Mietszch et al{[}45{]}. (2015)\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
One-Bac 2.0\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
GFP\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
1x10\textsuperscript{5 l}\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.05\columnwidth}\raggedright\strut
One-Bac system with VP ratio restoration to improve AAV5 vector
functionality\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Joshi et al {[}58{]}. (2019)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
One-Bac 3.0\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
25mL 3L\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
12x10\textsuperscript{6 i} 12x10\textsuperscript{6 i}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
3x10\textsuperscript{4}\selectlanguage{ngerman}± 0.4x10\textsuperscript{4}
2.7x10\textsuperscript{4}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2.7x10\textsuperscript{11 b} (\selectlanguage{ngerman}±0.37x10\textsuperscript{11})
3.8x10\textsuperscript{11 b} (±0.53x10\textsuperscript{11})\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
20\selectlanguage{ngerman}±5 22±5\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1.9x10\textsuperscript{8 b m} (\selectlanguage{ngerman}±0.43x10\textsuperscript{8})
2.1x10\textsuperscript{8 b m} (±0.48x10\textsuperscript{8})\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
ND\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
9600\textsuperscript{n}: 2400:1 9400\textsuperscript{n}: 2500:1\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Robust, High MOI, Fedbatch AAV5 production process. Process validation
at bioreactor scale. Improved volumetric yield of genomic and functional
AAV5 vector\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
Yang et al {[}70{]}. (2019)\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
One-Bac 3.0+\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2 8 9\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
GFP GFP GFP\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
2x10\textsuperscript{6} 2x10\textsuperscript{6}
2x10\textsuperscript{6}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
1.35x10\textsuperscript{5} \selectlanguage{ngerman}±0.46x10\textsuperscript{5} 2.16x105
±0.53x10\textsuperscript{5} 1.80x10\textsuperscript{5}
±0.39x10\textsuperscript{5}\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
NA\strut
\end{minipage} & \begin{minipage}[t]{0.07\columnwidth}\raggedright\strut
One-Bac based Rep2 expressing transformed Sf9 cell line. Offer more
flexibility for the production of multiple AAV serotypes
production\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a} Cell density at the time of infection

\textsuperscript{b} Value reported for per milliliter of cell culture

\textsuperscript{c} Determined by infecting HEK293 cells and subsequent
analysis under a fluorescence microscope for GFP positive cells.
Originally reported as

transducing units (TU).

\textsuperscript{d} Determined via infecting HEK293 EBNA cells with AAV
vector in the presence of a helper virus and subsequent analysis via
flow cytometry for

GFP positive cells

\textsuperscript{e} Total virus capsids (Cp) analyzed via ELISA

\textsuperscript{f} Value of rAAV5-hGFP using Rep52 of Serotype 1.

\textsuperscript{g} Value of rAAV5-hGFP generated via AAV2 VP1 domain
swapping (VP1 2/5) and Rep52 of serotype 1

\textsuperscript{h} Analyzed via infecting adherent HEK293 cells and
subsequent analysis via flow cytometry

\textsuperscript{i} The peak cell density during production run

\textsuperscript{j} Value reported per milliliter of AVB-Sepharose
affinity-purified eluate fraction

\textsuperscript{k} Determined via infecting HEK293A cells via AAV in
the absence of helper virus and subsequent analysis using a fluorescence
microscope

\textsuperscript{l} Titer value reported from the analysis of one-step
AVB-Sepharose Affinity purified sample

\textsuperscript{m} Analyzed via infecting suspension adapted HEK293
cells with AAV in the presence of helper virus and subsequent analysis
by flow cytometry

\textsuperscript{n} Determined via analytical ultracentrifugation
analysis of one-step AVB-Sepharose Affinity purified sample and
originally reported as enhanced

transduction units (ETU)

NA: Not available in the published report

ND: Not determined

GFP: Green Fluorescence Protein

hGFP: Humanized Green Fluorescence Protein

STB: Stirred-tank bioreactor

\textbf{Figure legends}

\textbf{Figure 1.} AAV Family Tree and wtAAV2 Genome Map and Expression
Profile

(A)~AAV family tree. The Dependovirus AAV mainly infects mammalian hosts
and replicates in the presence of a helper virus. Though evolutionary
distant yet related to Densovirinae subfamily, it shares common
structural and functional features with viruses from this subfamily,
which mainly infect invertebrates hosts such as insects. (B) wtAAV2
genome map and protein expression profile. The numbers represent the
nucleotide position. The viral genome is flanked by two ITRs, one on
each end. Two promoters in the Left ORF drive the expression four
functional Rep proteins, whereas a single promoter in the right ORF
drives the expression of three capsid protein subunits (VP1, VP2, and
VP3) from a single mRNA transcript. The intron splicing generates four
Rep proteins of different sizes from two mRNA transcripts, whereas the
leaky scanning of weak translational codons results in three VP proteins
production in a stochastic ratio of 1:1:10. The diamond shape end
represents the N-terminal of the peptide. Two polyadenylation (PA)
signal sequences are shown via orange line.

\textbf{Figure 2.} AAV Insect Cell Baculovirus Expression Systems for
AAV Production and Mechanism of Inducible Expression in One-Bac.

(A)~Four major systems for rAAV production using IC-BEVS. The initial
Three-Bac system consisted of three rBV vectors, each carrying specific
nucleotide sequence. Further study with this system identified critical
shortcomings, related explicitly to expression stability of rBVs and
mechanism behind it. The second-generation systems (Two-Bac) exhibited
better expression stability of rBVs over the extended passage numbers
and required only two rBV co-infection for AAV production.
Third-generation systems such as One-Bac or Mono-Bac further simplified
the manufacturing process requiring only single rBV co-infection. The
One-Bac consisted of a Rep2CapX packaging cell line and a Bac-GOI,
whereas in Mono-Bac, a single baculovirus carries all the necessary gene
(Bac-ITR-GOI-Rep2-CapX) sequences.~(B)~The postulated mechanism of
induction and amplification of \emph{rep/cap} genes in One-Bac. Bac-ITR
infection provides IE-1, which activates hr2-0.9 (1) and induces the
Rep78/52 or Cap expression (2). The expressed Rep78 further forms a
complex with RBE (3) and induces the second round of amplification of
Cap or Rep52 expression (4), resulting in a lower Rep78: Rep52 ratio.
(The artwork in the figure was created with Biorender.com)

\textbf{Figure 3.} An Overview of Process-Flow for rAAV Production
Employing IC-BEVS Platform

The AAV production using the IC-BEVS platform consists of two stages, 1.
Generation of recombinant baculovirus expression vectors (rBEVS) and
additionally Rep2CapX Sf9 packaging cells in case of One-Bac system (Top
section), and 2. AAV production at bioreactor scale in suspension
culture of insect cells followed by purification and formulation (Bottom
part). The first part requires molecular cloning, transient expression,
plaque purification, and characterization of rBVs. Similarly, the
Rep2CapX cells are generated via stable transfection of respective
plasmid vector and selection followed by characterization and master
cell bank preparation. During the production stage, the rBVES and insect
cells are (Sf9 or Hi5) sequentially expanded in quantity required for
stirred tank or Wave production scale bioreactor. At the production
stage, the insect cells are infected with rBEVS at an appropriate
multiplicity of infection (Low:\textless{}1 or High:1-10) and at optimal
cell density. Generally, under the batch mode of cultivation, the cells
are infected at low cell density around 2-4 million cells/mL, whereas
under fedbatch or perfusion mode, the cells reach higher peak cell
density before infection:7-12 million cells/mL. The cultivation
temperature for insect cells is generally set at 27\selectlanguage{ngerman}°C, although one
study reported AAV production at higher and lower temperatures and their
effect on yield and production kinetic. Post infection, at 72-96 hours,
the culture is harvested, and cells are lysed to recover AAV. Next, the
lysate is subjected to multistep purification process followed by
formulation in appropriate buffer as a final step. (The artwork in the
figure was created with Biorender.com)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

