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-β) 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®[17], and
Flublok®[9]. Regulatory approval of
Cervarix®, 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® 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.