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.