Insect cell expression system: advances in applications, engineering strategies, and bioprocess development

Abstract

As technology and regulatory guidelines continue improve, insect cell expression system has gained increasing attention as promising and versatile platforms for production of biopharmaceuticals. This review provides a comprehensive overview of recent advances in this platform, with a focus on their applications in human and veterinary vaccines, therapeutic proteins, and structurally complex proteins for biomedical research. Special emphasis is placed on emerging strategies such as larval expression systems, baculovirus-mediated antigen displaying technologies, and the development of gene therapy vectors. Despite their growing utility, insect cell systems face critical technical bottlenecks that limit scalability, productivity, and regulatory compliance. We discuss recent innovations aimed at addressing these challenges, including improvements in baculovirus infection dynamics, the genome engineering, and bioprocess optimization at both upstream and downstream levels. By synthesizing current knowledge and technological trends, this review outlines future directions for unlocking the full potential of insect cell platforms in next generation biomanufacturing.

Introduction

With continuous advancements in biotechnology and evolving regulatory guidelines, interest in the insect cell expression system has grown significantly among academic researchers and industry professionals. The COVID-19 pandemic brought recombinant protein vaccines, such as Novavax’s NVX-CoV2373, into the spotlight due to their proven safety and efficacy. Novavax COVID-19 Vaccine, produced using this platform, has been authorized globally, including an updated 2024–2025 formulation in the United States [1]. China has also approved two insect cell-based COVID-19 vaccines for emergency use, marking a major achievement in biopharmaceutical innovation. These developments emphasize the critical role of insect cell platforms in combating emerging infectious diseases [2–4]. Originally developed for recombinant protein expression, this system has become a versatile biomanufacturing platform with broad applications across biomedical fields—including vaccine antigens, therapeutic proteins, gene delivery and therapy, and tissue engineering (Fig. 1).

Fig. 1.

Overview of the insect cell expression system and its applications. The schematic illustrates the production and application of recombinant proteins, as well as the use of baculovirus vectors in cell, tissue engineering, and gene delivery. (A) Recombinant Proteins: The process begins with the design and construction of recombinant DNA, which is subsequently expressed in insect cells. Post-translational modifications (PTMs) enable the production of membrane proteins, subunit vaccines, complex proteins, virus-like particles (VLPs), antibodies, nanoparticles, and therapeutic proteins (e.g., enzymes, cytokines, or growth factors). These recombinant products are formulated for applications in human and animal vaccines, immunotherapy, and protein-based therapies. (B) Gene Delivery: Recombinant baculovirus (rBAC) DNA is utilized to transduce mammalian cells, generating transduced cell lines and transduced mouse models for research. Purified budded viruses are utilized for viral vaccines, gene transfer, and adeno-associated virus (AAV)-based gene therapy, with downstream applications in immunization and disease modeling. (C) Cell and Tissue Engineering: Baculovirus display systems enable the presentation of pathogen antigens on budded viruses or insect cells, supporting vaccine development and cell-based detection systems. Additionally, these systems are applied in tissue engineering, such as bone tissue research and other biomedical applications. BioRender software (Biorender.com) was used to create figure

This review systematically explores key benefits, recent advancements, bioprocess development and remaining challenges of insect cell expression system-based biomanufacturing, providing a comprehensive overview to guide future innovations and broader applications in biomedical research and therapeutics.

Expanding applications of insect cell expression system in biomedicine

A recent survey study, “A concise guide to choosing suitable gene expression systems for recombinant protein production” reported that the insect cell expression system was rated slightly superior to mammalian stable expression platforms in ease of use, cost-efficiency, and speed, underscoring its value for early-stage or rapid-response production [5]. Beyond these practical advantages, the insect cell expression system is well-regarded for its safety. Baculoviruses infect only insect cells and do not replicate, transmit, or integrate in vertebrate cells; this was reaffirmed by the OECD Committee on Chemicals and Biotechnology in 2023, which concluded that baculoviruses pose no risk to human health [6]. Table 1 summarizes approved and in-development pharmaceuticals produced using this platform.

Table 1.

Pharmaceuticals produced with insect cell–baculovirus expression systems: approved products and candidates in development

Types	Protein	Application	Band	Sponsor	Status	Year
Human vaccines	HA protein	Influenza virus	FluBlok	Sanofi Pasteur	FDA approved	2013
	HA protein	Influenza virus	Flublok Quadrivalent	Sanofi Pasteur	FDA approved	2016
	HPV16/18 L1 protein	HPV	Cervarix	GSK	FDA approved	2007
	Spike (S) protein	COVID-19	NVX-CoV2373	Novavax	FDA approved EUA	2020
	CoV2 preS dTM	COVID-19	VidPrevtyn Beta	Sanofi-GSK	EMA Approved	2022, Withdrawal (2024)
	Recombinant RBD monomer	COVID-19	Weikexin	Westvac	NMPA approved EUA	2022
	Recombinant RBD monomer	COVID-19	Trivalent Weikexin	Westvac	NMPA approved EUA	2023
	Recombinant RBD monomer	COVID-19	WSK-V102D	Westvac	NMPA approved EUA	2023
	S protein extracellular domain	COVID-19	SpikoGen®	Vaxine/CinnaGen Co.	Iran approved EUA	2021
Animal vaccines	E2 protein	Classical swine fever	Porcilis Pesti	MSD Animal Health	Approved	2000,Withdrawn (2011)
	E2 protein	Classical swine fever	BAYOVAC CSF E2	Bayer AG	Approved	2001
	PCV2 ORF2 protein	Porcine circovirus-2	CircoFLEX	Boehringer Ingelheim	Approved	2005
	PCV2 ORF2 protein	Porcine circovirus-2	Cirumvent PCV	MSD Animal Health	Approved	2008
	PCV2 ORF2 protein	Porcine circovirus-2	Porcilis PCV	MSD Animal Health	Approved	2009
Human therapeutics	PAP-GM-CSF fusion protein	Prostate cancer	Provenge	Dendreon	Approved	2011
	Type I diabetes	GAD	Diamyd	Diamyd Medical AB	Phase III (NCT05018585)	2021

Abbreviations: HA, hemagglutinin; HPV, human papillomavirus; GSK, GlaxoSmithKline Biologicals; EUA, emergency use authorization; GAD, Type I diabetes Glutamate decarboxylase; VLP, virus-like particles

Human vaccines

The flexibility of the insect cell expression system plays a pivotal role in the rapid development and timely updating of vaccines, particularly in response to emerging pathogens and continuously evolving viral variants. For COVID-19, several insect cell expression system-derived vaccines have been developed, including Novavax’s NVX-CoV2373, WestVac’s Convince, Sanofi/GSK’s VidPrevtyn Beta, and Vaxine’s SpikoGen^®. Among these, NVX-CoV2373, which incorporates recombinant spike protein expressed in Spodoptera frugiperda 9 (Sf9) insect cells, achieved an efficacy rate of 89.7% in clinical trials [7]. WestVac BioPharma’s recombinant receptor-binding domain (RBD) vaccine also produced using insect cell-baculovirus expression system, induced strong antibody responses and effective viral neutralization in animal models [3]. As SARS-CoV-2 continued to evolve, WestVac developed a trimeric RBD vaccine by fusing the Delta variant RBD with the heptad repeat 1 (HR1) and HR2. This design enhanced immunogenicity and conferred broad protective efficacy in preclinical studies [4]. Building upon this platform, they advanced a trivalent-vaccine (Tri-Vac), comprising RBD trimers derived from Delta, BA.5, and XBB.1.5 variants. Tri-Vac demonstrated potent cross-variant neutralizing activity, sustained immune responses, and a favorable safety profile, highlighting its potential as a broad-spectrum SARS-CoV-2 vaccine (ChiCTR2200067245) [8]. Notably, both the recombinant RBD vaccine and the trimeric RBD-based vaccines have been granted emergency use authorization in China, reinforcing the clinical and regulatory value of insect cell-based vaccine platforms. Furthermore, SpikoGen^® [9] and VidPrevtyn Beta [10] also showed robust immunogenicity in clinical trials, further highlighting the advantages of insect cell expression system-derived vaccines. Overall, the pandemic significantly accelerated the development and approval of insect cell–derived vaccines.

In addition, this platform demonstrates considerable potential for the development of vaccines targeting a wide array of infectious diseases. Novavax developed a respiratory syncytial virus (RSV) F nanoparticle vaccine, which was evaluated in a Phase III trial (NCT02624947). It provided partial protection (39.4–58.8%) against severe outcomes, particularly in low- and middle-income countries (LMICs) where RSV burden is highest. Furthermore, its favorable safety profile in pregnant women highlights its potential for maternal immunization programs [11]. Novavax’s Ebola virus glycoprotein (EBOV GP) nanoparticle vaccine showed strong immunogenicity in a Phase I trial (NCT02370589). The vaccine elicited durable antibody titers that persisted for up to one year, demonstrating its promise as a preventive measure against Ebola virus outbreaks, where long-lasting immunity is critical [12]. Another notable application is the bivalent norovirus virus-like particle (VLP) vaccine HIL-214, jointly developed by Takeda and HilleVax. As one of the most advanced norovirus vaccine candidates currently in clinical development, HIL-214 exhibited significant efficacy in preventing moderate-to-severe acute gastroenteritis (AGE) in a phase II trial involving 4,712 adult participants (NCT02669121) [13]. In China, Sinocelltech Co., Ltd has advanced a recombinant 14-valent human papillomavirus (HPV) VLP vaccine (SCT1000) using this system platform. This candidate is currently undergoing phase III evaluation (NCT06041061), with preliminary data indicating strong immunogenicity and an excellent safety profile, offering a promising approach for broad-spectrum HPV prevention [14]. Similarly, CanSino Biologics has leveraged the Sf-RVN insect cell system to develop a recombinant trivalent poliovirus VLP vaccine (VLP-Polio), now in phase I clinical trials (NCT06101173) [15]. This effort represents a critical step toward safer and more sustainable polio vaccine solutions.

Collectively, the insect cell expression system enabling the rapid, precise, and safe production of innovative vaccine candidates, this platform not only accelerates the development of countermeasures against a diverse array of infectious diseases but also plays an increasingly vital role in the global response to emerging and re-emerging viral threats.

Animal vaccine productions

The need for scalable and cost-effective vaccine platforms has become increasingly urgent in veterinary medicine. The insect cell expression system has emerged as a leading platform for veterinary vaccine development, offering an optimal balance between eukaryotic protein processing capabilities and industrial scalability. This system has already been applied to subunit vaccines targeting classical swine fever virus (CSFV) and VLP-based vaccines against porcine circovirus type 2 (PCV2)-both of which have reached commercial implementation. Recent studies have validated the robust protective efficacy of vaccines produced using these vaccines [16, 17].

VLP-based next-generation subunit vaccines are gaining momentum due to their high immunogenicity, structural mimicry of native viruses, and excellent safety resulting from the absence of genetic material. The insect cell system is well-suited for VLP production, supporting proper protein folding and post-translational modifications critical for self-assembly and antigenicity. Bovine viral diarrhea virus (BVDV) VLPs composed of Pestivirus A (BVDV-LC) E0 and E2 proteins, expressed in insect cells, successfully self-assemble into spherical particles and elicit robust immune responses in animal models [18, 19]. In the case of Porcine Delta coronavirus (PDCoV), insect cell–derived VLPs have effectively stimulated specific immune responses in mice, highlighting their potential as PDCoV vaccine candidates [20]. H5N1 influenza VLPs produced using the insect cell have demonstrated strong immunogenicity. A single immunization with an H5N1 VLPs formulation at a low antigen dose was sufficient to induce high hemagglutination inhibition antibody titers and conferred protective immunity against challenge with a homologous highly pathogenic avian influenza virus (HPAIV) [21]. Macrobrachium rosenbergii nodavirus (MrNV) capsid protein VLPs have been expressed, purified, and administered to freshwater shrimp, resulting in significantly enhanced survival rates following viral challenge [22].

Collectively, these findings underscore the versatility and effectiveness of the insect cell expression system to produce veterinary VLP-based vaccines. Its ability to support multivalent antigen expression, structural integrity, and strong immunogenicity makes it an ideal platform for next-generation vaccines targeting both terrestrial and aquatic species.

Therapeutic Recombinant proteins

The approval of Provenge (Sipuleucel-T) by the U.S. Food and Drug Administration (FDA) marked a pivotal milestone in applying biotechnology to cancer therapy. Researchers have extensively explored insect cell expression system as an innovative system for therapeutic recombinant protein production, achieving significant advances in recombinant protein expression, scalable production, and process development.

Insect cells are capable of correctly folding antibody molecules and introducing specific glycosylation modifications, which are critical for enhancing the activity and stability of antibodies. However, the bottlenecks caused by viral infection significantly affect antibody production, and the relatively low final yield has restricted the broader application of this platform in antibody expression. Despite these challenges, researchers have continued to explore innovative solutions. For example, a baculovirus-free insect cell system has been employed to achieve high-yield antibody expression [23], and strong insect cell promoters have been used to establish transient gene expression systems or develop stable transgenic cell lines for the efficient production of the HIV-1 broad-spectrum neutralizing antibody b12 [24]. Furthermore, the silkworm-baculovirus expression system has successfully produced scFv, Fab, and IgG of the CR3022 antibody against SARS-CoV-2 [25]. These advancements indicate that insect cell expression system holds great potential as an alternative platform for producing bioactive monoclonal antibodies.

More broadly, in the field of recombinant protein therapeutics, the system has been successfully employed to produce structurally complex and functionally active proteins and VLPs, which often require disulfide bond formation or glycosylation to achieve proper folding and biological functionality. Representative examples include Lucilia sericata collagenase (MMP-1) [26], snake venom L-amino acid oxidase (SR-LAAO) [27], Fc fusion proteins [23], and Bone Marrow Tyrosine Kinase on Chromosome X (BMX) [28]. These studies have established scalable production processes and developed safe, multifunctional insect cell expression system-based production platforms. In the area of therapeutic VLP development, the insect cell platform has likewise exhibited remarkable potential. For example, canine parvovirus-VLPs specifically target transferrin receptors (TFRs) on cancer cells [29]. Additionally, Additionally, HER2-targeted VLPs produced in insect cells-bearing insect-type glycosylation-ave shown significant protective effects in HER2-positive breast cancer mouse models [30].

Taken together, these findings highlight the insect cell expression system as a powerful and adaptable platform for recombinant protein production. Its unique capacity to support proper protein folding, post-translational modifications, and VLP assembly makes it particularly well-suited for generating complex therapeutic proteins and bio-nanocarriers, thereby facilitating advances in biologic drug development and precision-targeted therapies.

Basic research on complex structural proteins

Achieving high yield, high purity and activity in protein production remains a significant challenge in the analysis of protein structure, function, and interactomes. The insect cell expression system has emerged as a versatile platform, particularly suitable for the simultaneous expression of multiple proteins and the efficient production of multi-subunit protein complexes. To further optimize this process, several advanced baculovirus vector platforms have been developed. These include HR-bac, which utilizes homologous recombination to efficiently assemble and express multiple gene expression cassettes [31]; PluriBAC, which employs Golden Gate assembly to enable modular combination of more than four promoters, genes of interest, and terminator sequences for flexible multi-gene expression [32]; and MoClo-Baculo, a yeast-compatible alternative expression system that supports efficient construction of multigene expression cassettes using the modular cloning (MoClo) strategy [33]. These tools enable multi-gene expression and high-level production of protein complexes, facilitating the rapid assembly of functional systems for structural studies.

Insect cell expression system-based platforms overcome common challenges associated with producing high-yield, active proteins. For instance, Sousa et al. produced 100 mg of purified BMX from 5 L suspension culture in 34 days, enabling potential high-throughput screening of BMX inhibitors [28]. Recombinant aldehyde oxidase (AOX), a complex molybdopterin-containing flavoprotein, has traditionally been difficult to produce. This allows for the efficient production and isolation of recombinant human AOX dimers with high purity through a streamlined two-step process. This method offers a reliable alternative to liver extracts, ensuring accurate assessment of AOX metabolism in drug development [34]. Wording with Nipah virus (NiV) in BSL-4 facilities limits the practicality of using wild-type virus for drug screening. To address this, an insect cell-based platform expressing NiV-F, NiV-G, and EphrinB2 was developed to reconstruct NiV-induced syncytium formation. This platform enables the screening of fusion inhibitory compounds with potential to block viral entry [35].

Beyond protein expression, in-cell crystallizing recombinant proteins within production cells is a rare but valuable strategy for structural studies. Utilizing baculovirus-based cloning systems, researchers optimized cellular environments to improve in-cell crystallization efficiency. Tang et al. developed a library of gateway-compatible baculovirus vectors and established a scalable pipeline that integrates Second Order Nonlinear Imaging of Chiral Crystals (SONICC) and transmission electron microscopy (TEM) techniques to screen for microcrystal formation in living insect cells [36]. Building on this, Schonherr et al. developed the InCellCryst method, which integrates high-throughput expression, enhanced crystal cell enrichment, and direct X-ray diffraction in insect cells [37]. These advancements highlight the potential of the insect cell expression system in enabling structural biology to capitalize on in-cell crystallization for protein structure analysis. Furthermore, selenomethionine (SeMet)-labeled proteins can be conveniently detected intracellularly. A novel approach combining SeMet labeling with the Titer Estimation for Quality Control (TEQC) method allows precise control of infection levels, thereby reducing SeMet-associated toxicity and significantly improving the production efficiency of labeled recombinant protein [38].

Collectively, these advances underscore the versatility and scalability of the insect cell expression system as a robust platform for producing structurally and functionally relevant proteins, paving the way for its broader application in drug discovery, functional proteomics, and structural biology.

Emerging technologies driving platform innovation

Insect larvae as scalable systems for recombinant protein bioproduction

The insect cell expression system is not limited to insect cell culture; it can also utilize insect larvae or pupae for producing recombinant proteins, establishing itself as an important and versatile biological production system. Using insect larvae as bioreactors offers high cost-efficiency and facilitates large-scale recombinant protein production with ease of scalability. Representative products approved by regulatory authorities include Virbagen^®Omega, developed using silkworm larvae, and Fatrovax^®, a rabbit hemorrhagic disease virus (RHDV) vaccine produced from the pupae of Trichoplusia ni (T. ni). In recent years, the low production cost of silkworm larvae has further promoted their extensive application in animal vaccine development, including vaccines targeting PCV2 [39], porcine rotavirus A [40], porcine epidemic diarrhea virus [41], and avian influenza virus [42].

The integration of CrisBio technology significantly enhances the performance of recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in T. ni, enabling the use of insect pupae as natural biocapsules for subunit vaccine production. This scalable platform combines single-use devices and robotic systems for efficient rearing, handling, and inoculation, while achieving milligram-level recombinant protein yields per infected pupa [43–45]. Its high yield and cost efficiency make it an innovative solution for large-scale protein production.

Insect larvae and pupae have garnered attention for oral vaccine development, with silkworms being explored as potential delivery systems [46]. However, their downstream processing is generally more complex than that of cultured cells, and the absence of standardized upstream protocols may hinder their broader application in human healthcare. Moreover, societal concerns-similar to those surrounding transgenic animals-may limit the acceptance of insect-based bioproduction platforms in certain markets.

rAAV manufacturing

Recombinant adeno-associated viruses (rAAVs) are widely utilized in gene therapy due to their in vivo stability and low toxicity, with mammalian cells (e.g., HEK293) and insect cells (e.g., Sf9) serving as primary production platforms. Although functionally similar, the insect cell expression system typically offers higher yields, improved full-to-empty capsid ratios, lower residual host DNA, and greater scalability compared to HEK293-based methods (Table 2). Reflecting its growing importance, the use of this system for rAAV production rose from approximately 5.6% before 2007 to over 20% by 2022 [47]. As of April 2025, a total of nine recombinant adeno-associated virus (rAAV) gene therapy products has received regulatory approval. Among them, Glybera, Hemgenix, and Roctavian are manufactured using insect cell expression systems, underscoring the industrial feasibility and regulatory acceptance of this platform for rAAV production.

Table 2.

Comparison of HEK293-based and insect cell expression ystem-based rAAV production manufacturing systems

Parameter	HEK293-based	Insect cell expression system-based	Refs
Scalability	Moderate (needs larger volumes, 50–200 L, 1–3 × 10^6 cells/mL)	Excellent (high density 8–10 × 10^6 cells/mL, ~ 10× higher yield; 50 L ≈ 200 L HEK293)	[48]
Vector genome yield	Moderate (e.g., 1.3 × 10¹⁴ vg @ 50 L)	High (e.g., 2.98 × 10¹⁵ vg @ 50 L)
Full/Empty capsid ratio	~ 70.8% full	~ 93.2% full
Host cell DNA impurities	Higher: 0.38% with 48 DNA-host-vector chimeras	Lower: 0.03% with 1 chimera detected
In vitro infectivity (TCID₅₀/vg)	Lower	Higher
In vitro expression	HEK293: 7.3 ± 0.3 mg/mL; ARPE19: 1.2 ± 0.1 mg/mL	Sf9: 13.6 ± 0.6 mg/mL; ARPE19: 2.1 ± 0.2 mg/mL
Capsid aggregation	Higher aggregation, larger particle size	Lower aggregation, smaller particles
In vivo expression (CNV model)	Comparable, dose-dependent	Comparable, more stable for large genes
VP1: VP2: VP3 composition	Higher VP1 and VP2 content	Lower proportion of VP1	[49]
miRNA concentration	Low (1.85 ± 1.39 ng per 1E10 vg)	High (6.23 ± 3.03 ng per 1E10 vg)	[50]
PTMs	Fewer (e.g., minor phosphorylation)	More (e.g., phosphorylation, methylation, ubiquitination)	[51]
Clinical Safety	Comparable	Comparable	[47]

In recent years, further optimizations have been implemented to enhance yield, improve the full/empty capsid ratio, and increase packaging efficiency. To improve the yield and consistency of rAAV produced in Sf9 insect cells, several key innovations have been developed. A novel directed evolution protocol was established to evaluate the structural integrity and biological fitness of Sf9-derived rAAV vectors, enabling the selection of high-performance capsid variants [52]. In parallel, a Lac repressor-inducible expression system was engineered to empirically regulate the stoichiometric balance between VP1, VP2, and VP3 capsid proteins within the baculovirus expression vector (BEV) framework, optimizing capsid assembly and genome packaging efficiency [53]. Additionally, the use of the SGMO Helper system was found to enhance rAAV production by improving replication and helper function coordination [54].

Efforts are also underway to develop scalable upstream processes for high-density Sf9 cell culture and efficient rAAV packaging, laying the groundwork for industrial-scale manufacturing [55]. To streamline quality control in viral vector production, a rapid, affinity-based high-performance liquid chromatography method was introduced for capsid assessment, offering a faster and more efficient method for capsid quantification [56]. In addition, an F-TCID₅₀ assay was established to accurately determine the infectious titer of recombinant baculovirus stocks expressing GFP (rBV-GFP), providing a reliable and quantitative method for monitoring viral infectivity during upstream process development [57]. To gain deeper insight into the underlying biology of rAAV production in insect cells, gene expression in Sf9 cells infected with recombinant baculovirus at low multiplicity of infection (MOI) was analyzed. Transcriptomic profiling revealed key transcriptional changes that occur throughout the course of baculovirus infection, offering valuable information on host cell responses and their impact on rAAV vector assembly and genome packaging [58]. Furthermore, a mechanistic model was developed to describe the critical extracellular and intracellular phenomena that govern baculovirus infection dynamics and rAAV maturation within the insect cell expression platform [59]. This model integrates parameters such as baculovirus entry, replication kinetics, capsid protein expression, and vector genome amplification, providing a systems-level framework for process optimization and predictive control.

Surface display platform for antigen presentation

The Baculovirus/insect cell system uniquely enables the display of exogenous proteins on viral or cellular membranes, expanding their applications to recombinant vaccines, protein therapeutics, diagnostic platforms, and advanced cellular analysis tools [60].

Baculovirus display technology has notable applications in studying human-infecting viruses or pseudoviruses, particularly their structural proteins. Its safety in humans allow baculoviruses to function as recombinant vaccine delivery platforms by displaying heterologous antigens on their surface or capsid. Examples include the hemagglutinin (HA) protein of the H5N2 avian influenza virus [61], the RBD fragment of the spike protein from SARS coronavirus [62], the glycoprotein Gn of Crimean-Congo hemorrhagic fever virus [63], and glycoprotein E of the varicella-zoster virus [64].

When a target protein contains a transmembrane (TM) domain or is fused with the TM domain of the AcMNPV surface glycoprotein GP64, baculovirus facilitates the display of recombinant proteins on the plasma membrane of insect cells [65]. This cell-based antigen display approach allows antigens to be presented on the cell surface while maintaining the native epitope conformation, effectively overcoming issues such as epitope loss during soluble expression, hydrophobic aggregation during purification, and complex experimental workflows. Cell-based ELISA systems enable rapid and straightforward detection of serum antibodies, eliminating the need for labor-intensive protein purification and antigen coating steps, while offering high accuracy and specificity [66–68].

Gene transfer and therapy

The baculovirus itself shows promise as a versatile gene transfer vector, given its large genetic payload capacity and inability to replicate or integrate into mammalian genomes. Baculovirus-based viral vector vaccines, such as FrC-OVA-BV, have demonstrated significant activation of CD8⁺ T cell-mediated immune responses through the expression of tetanus toxin fragment C (FrC) and ovalbumin (OVA) peptides, exhibiting potent antitumor activity in the EG7-OVA tumor mouse model [69]. Engineered baculovirus vectors displaying the envelope of human endogenous retrovirus (HERV) have significantly enhanced gene transfer efficiency in human cells compared to wild type baculovirus. Based on this platform, a recombinant vaccine encoding the Zika virus (ZIKV) prM/E genes (AcHERV-ZIKV) was developed, inducing stronger immune responses, including higher levels of IgG, neutralizing antibodies, and IFN-γ [70]. Additionally, vaccines targeting middle east respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2, and ZIKV were successfully developed by inserting DNA encoding the full-length spike protein, its S1 subunit, or the RBD into the AcHERV genome. These vaccines elicited robust humoral and cellular immune responses and provided complete protection in animal models [71].

Moreover, recombinant baculoviruses displaying decay-accelerating factor (DAF, CD55) on their envelopes have effectively addressed key limitations of gene transfer, such as transient transgene expression and inactivation by the serum complement system in vivo. This vector significantly enhanced the anti-angiogenic and antitumor effects of the human endostatin-angiostatin fusion protein (hEA) in a mouse model [72]. Baculoviruses have also been utilized to deliver gene-editing tools, successfully correcting a genetic defect in Steroid-Resistant Nephrotic Syndrome (SRNS) patient derived podocytes via CRISPR-Cas9 [73]. Furthermore, combining magnetic nanoparticles with recombinant baculovirus vectors (MNP-BVs) has enabled localized CRISPR-Cas9-mediated genome editing under magnetic field guidance, achieving precise tissue-specific gene delivery [74]. insect cell expression system has also demonstrated its potential role in both gene delivery and viral vector-based vaccine development, making significant contributions to the advancement of these fields.

Strategies for unlocking the full potential of insect cell expression system platform

Insect cell expression system continues to face several technical challenges that limit improvements in production efficiency and product quality. Key bottlenecks include apoptosis of infected cells, incomplete or non-humanized glycosylation patterns, and the risk of residual viral contamination, all of which significantly impact system performance. In recent years, advancements in biotechnology have led to breakthroughs in areas such as viral genome modifications, vector engineering, and host cell optimization. These innovations have not only enhanced system stability but also improved the quality, consistency, and efficiency of recombinant protein production.

Revealing novel mechanisms

Variations in insect cells during baculovirus infection remain one of the least understood phenomena in virology. Advances in next-generation sequencing (NGS) and bioinformatics have significantly driven research in insect cell expression system response, providing critical insights for rational design and process engineering strategies. For example, Bruder et al. utilized transcriptomic data to identify a series of natural AcMNPV promoters (e.g., polh, p6.9, vp39, and 39k) with distinct expression characteristics, which were subsequently validated using GFP and SEAP expression models. This study was the first to apply bioinformatics analysis to identify key promoter sequence features that influence late-stage gene transcription and translation efficiency. The findings not only expanded the catalog of baculovirus promoters but also enabled precise co-expression of exogenous genes in polycistronic structures [75]. Although population-level transcriptomic studies have revealed host responses to infection, they often overlook cellular heterogeneity during protein production. The application of single-cell RNA sequencing (scRNA-seq) has allowed researchers to dissect viral infection dynamics and identify cell subpopulations associated with efficient protein production. For instance, scRNA-seq analysis of High Five cells post-infection provided a resolution unattainable with traditional bulk RNA-seq methods. These studies opened new possibilities for genetic engineering applications, such as developing cell lines specialized for viral replication or exogenous protein expression, establishing inducible systems, or synchronizing infection across cultures to better control production uniformity [76]. Additionally, a novel directed evolution protocol was developed by integrating NGS and computational simulation. A combinatorial capsid library was packaged in Sf9 cells and screened in C12 cells expressing Cre recombinase to identify highly infectious variants. This approach successfully identified a small subset of Kozak sequences encoding optimized AAV capsid VP1 translation start sites. These optimized capsids demonstrated not only higher production yields but also superior transduction efficiency [52]. In conclusion, omics-guided studies have significantly improved our understanding of the cellular responses in insect cell expression systems. These insights provide a foundation for the rational engineering of host cell lines, synchronization of infection processes, and fine-tuning of expression cassettes to enhance the yield and consistency of complex biologics.

Genetic engineering approaches

Optimizing the insect cell expression system in terms of viral genome engineering for enhancing recombinant protein production. Recent advancements in CRISPR-Cas9 technologies have provided innovative solutions to overcome system bottlenecks. Pazmiño et al. first demonstrated the feasibility of CRISPR-Cas9 for AcMNPV gene knockout [77]. Building on this, Bruder et al. established insect cell lines stably expressing Cas9 and infected them with baculoviruses engineered to produce guide RNAs targeting critical viral genes (e.g., vp80, iE1, gp64), which reduced budded virus production to below 10% of wild-type levels [78]. Subsequent studies further expanded CRISPR applications, including the introduction of indel mutations in gp64 or simultaneous targeting of gp64 and vp80, resulting in markedly reduced baculovirus release and enhanced VLP yields [79, 80]. In parallel, non-CRISPR strategies have also contributed to system improvement. For instance, vector stability has been enhanced by shortening homologous regions and introducing point mutations to reduce recombination [81], Additionally, systematic deletion of non-essential AcMNPV genes using lambda red recombination has been shown to improve recombinant protein yields [82]. Collectively, these genome engineering strategies hold strong potential to advance the industrial and pharmaceutical applications of insect cell expression systems.

Genomic engineering also plays a crucial role in host insect cell modification to further improve insect cell expression system performance. The CRISPR-Cas9 site-specific genome editing tool has been employed to modify protein glycosylation patterns in baculovirus-insect cell systems. For instance, CRISPR-Cas9-mediated knockout of the Dicer-2 gene in Sf9 cells enhanced the replication of Spodoptera frugiperda rhabdovirus (Sf-RV) and, under certain conditions, increased baculovirus replication efficiency. These findings suggest that targeting the Dicer-2 gene could optimize the production environment of baculovirus, with significant implications for genetic engineering and large-scale production [83]. Similarly, CRISPR-Cas9 mediated functional knockout of the Sf-Caspase-1 gene successfully abolished apoptotic features, such as membrane bubbling and caspase activity, in Sf9 cells. In another study, short hairpin RNA (shRNA) targeting conserved regions of caspase-1 was inserted into a baculovirus vector with non-essential genes deleted, effectively inhibited apoptosis in both Sf9 and High Five cells, thereby improving protein expression efficiency, and broadening applications in both research and pharmaceutical industries [84, 85].

Innovations in expression vectors

Currently, the Bac-to-Bac system is widely used for stable single-gene expression, while FlashBAC improves efficiency by accelerating the expression process. MultiBac is designed for multi-gene expression, and both pTri and pQE-Trisystems are compatible with prokaryotic, insect, and mammalian cells, fulfilling diverse expression needs. The ongoing optimization of vector systems is driving continuous advancements in expression technologies.

Recent advancements in vector system modification include the development of an insect cell expression system-based protein production pipeline utilizing the pIEx/BacMagic 3 expression vector system and Gateway cloning technology, enabling large-scale parallel expression of recombinant proteins and the rapid screening of in vivo microcrystals [36]. To enhance promoter activity, a modified expression vector was developed incorporating Litopenaeus vannamei Yin Yang1 (Lv YY1), which was identified as a functional enhancer of the ie1 promoter. The presence of LvYY1 significantly promoted transcriptional activation, thereby increasing the yield of expressed antigens [86]. To improve biosafety and protein yields, a lef5-deficient baculovirus expression system was developed to suppress late-stage gene expression and prevent baculovirus particle formation. Compared to conventional systems, this approach improved protein secretion efficiency by 1.8-fold, extended infection duration, and optimized cellular conditions [87]. Moreover, De novo designed proteins with novel functionalities—such as recombinant proteins containing non-canonical amino acids—can be generated using an engineered baculovirus shuttle vector that delivers an orthogonal pyrrolysyl-tRNA/aminoacyl-.

tRNA synthetase pair (tRNA^Pyl/PylRS). This system enables the construction of a diverse library of amino acid derivatives with novel chemical properties for site-specific incorporation into target proteins [88]. The genetic code expansion strategy within the Bac-to-Bac system has expanded the possibilities for recombinant protein engineering, paving the way for the development of proteins with novel functions using unnatural amino acids. To facilitate recombinant protein production in the pupae of Antheraea pernyi multicapsid nucleopolyhedrovirus (AnpeNPV), researchers developed the AnpeNPV expression vector, creating a linear derivative AnpeNPVPhEGFP-AvrII. This approach significantly improved recombination efficiency and reduced the proportion of non-recombinant progeny [89]. Notably, synthetic biology holds great potential for the rational modification of viral genomes. Using Transformation-Associated Recombination (TAR) technology, researchers successfully synthesized the complete genome AcMNPV and rescued the recombinant virus in insect cells while preserving its native biological characteristics. On this basis, a novel baculovirus vector, AcBac-Syn, was constructed to enable high-efficiency expression of exogenous genes, providing a powerful tool for recombinant protein production and gene expression system development [90].

These advancements demonstrate the vast potential of insect cell expression system in vaccine development, virus assembly studies, and protein engineering, establishing it as a powerful tool for the next generation of viral vaccines and therapeutic protein production.

Establishment and optimization of production processes

The insect cell expression system still faces limitations, including relatively low expression in some contexts and downstream processing challenges, particularly the removal of residual baculoviruses and host cell impurities. Given these strengths and challenges, it is important to understand the core workflow of the insect cell expression system, which consists of baculovirus production, insect cell infection, and protein purification (Fig. 2). The process begins with the creation of recombinant baculoviruses (rBVs) containing foreign genes via recombination or transposition. These rBVs are then amplified through multiple passages to generate a high-titer viral stock. Next, insect cells are infected at an optimized multiplicity of infection (MOI) and incubated for an appropriate duration to maximize target protein production. Finally, the target protein is isolated cells and/or supernatants are collected and purified using optimal techniques.

Fig. 2.

The insect cell expression system production workflow and corresponding challenges are summarized as follows. (A) Insect Cells: Sf9, Sf21, and Hi5 cell lines are used, and optimized vectors (e.g., CRISPR-Cas9 and sRNAi) facilitate clonal selection and cell banking. (B) Generation of rBEV: Recombinant baculovirus expression vectors are created by transforming competent Bac cells with helper and donor plasmids containing the gene of interest (GOI). (C) Fermentation: Cell cultures are expanded and scaled up in wave bioreactors and bioreactors, with optimization of Multiplicity of Infection (MOI), Cell Culture Integrity (CCI), Time of Harvest (TOH), and temperature to improve yield. (D) Purification: The downstream process includes clarification filtration, ultrafiltration concentration, and chromatography to purify the final product. (E) Characterization: Analytical techniques such as LC-MS, Cryo-EM, DLS, and others are used to assess structural, molecular, and biophysical properties of products like VLPs, antibodies, nanoparticles, and rAAV. The workflow addresses several challenges, including (1) improving transfection efficiency through baculovirus-free systems and transient gene expression; (2) employing advanced screening technologies, such as flow micrometry and super-resolution fluorescence microscopy, for real-time insights into VLP formation; (3) developing novel chromatography techniques, including Fractogel-TMAE and Heparin-affinity methods; (4) optimizing fed-batch processes with mathematical modeling-driven feed strategies; and (5) adopting innovative characterization methods, such as MALDI-TOF MS, Cryo-EM, and CD/DSC, to enhance product yield, quality, and stability. Biorender software (Biorender.com) was used to create figure

Optimization key 1: fermentation and infection

Given the inherent limitations in insect cell proliferation and maximum cell density, optimizing parameters such as infection timing, MOI, pH, temperature, dissolved oxygen, and shear stress is essential for enhancing viral amplification efficiency and yield. To address transfection inefficiencies, Aline et al. developed an improved methodology that increased transfection efficiency from 2 to 20% (traditional methods) to 45–57%, independent of genetic insert size, thereby significantly enhancing large-scale heterologous protein production in insect cells [91]. Similarly, pre-incubation of insect cells with a low concentration of MβCD (0.25 mM) significantly enhanced baculovirus infection rates and foreign protein expression [92].

Fed-batch cultivation strategies, guided by mathematical modeling, have revolutionized insect cell-based systems by enabling precise control of feed rates and improving productivity compared to traditional batch cultures [93]. In the optimization and quality control of bioprocesses, the development of global predictive models for the offline biochemical monitoring of Sf9 cell cultures has become increasingly important. Leveraging chemometric techniques in combination with mid-infrared (MIR) spectral data acquired via attenuated total reflectance (ATR) mode enables the construction of robust models with adequate predictive capacity [94].

Meanwhile, transient gene expression (TGE) using polyethyleneimine (PEI)-mediated transfection, has demonstrated scalability for VLP production in bioreactors with High Five cells, achieving a 1.8-fold yield increase over traditional shake flask cultures [95]. To overcome the limitations of baculovirus-based systems, Puente-Massaguer et al. developed a non-baculovirus TGE system employing the pIZTV5 vector and PEI-mediated transfection, enabling rapid VLP production in insect cells [96].

Co-expression of baculovirus particles during VLP production complicates purification due to their similar physicochemical properties. To address this challenge, Puente-Massaguer et al. systematically evaluated the interplay between cell concentration at infection (CCI), MOI, and time of harvest (TOH). Their findings revealed that high CCI generally maximized VLP productivity, while high MOI and short TOH (~ 54 h) optimized immediate yields. However, the best overall production efficiency was achieved with low MOI and extended TOH (~ 84 h) [97]. Furthermore, super-resolution fluorescence microscopy offered new insights into VLP formation dynamics within live cells [98]. Advanced analytical methods, including confocal microscopy, flow micrometry, and nanoparticle tracking analysis, enabled comprehensive optimization of VLP production processes [99].

Optimization key 2: harvesting and purification processes

Given the significant risks of viral contamination, regulatory agencies mandate viral clearance studies to ensure product safety by assessing the purification process’s ability to eliminate both endogenous and exogenous viruses [100]. Downstream processing of VLPs expressed in insect cells is technically challenging and requires efficient chromatographic strategies. Among these, ion exchange chromatography (IEC) is commonly employed for capturing and concentrating VLPs. However, when anion exchange chromatography (AEX) was used for large-scale purification, overlapping elution conditions between baculoviruses and norovirus VLPs resulted in decreased recovery yields [101]. To address this issue, Katrin et al. developed Fractogel^®-TMAE, a polymer-grafted ion exchange medium with an increased surface area and ligand density, facilitating rapid and efficient purification of enveloped VLPs produced [102]. Another widely adopted approach is affinity chromatography (AC), which offers high specificity in VLP and virus purification [103]. However, additional polishing steps are often required following the primary capture step. Techniques such as size exclusion chromatography (SEC) and ultrafiltration/diafiltration (UF/DF) are commonly utilized to enhance product purity and quality.

In addition to optimizing downstream purification, upstream strategies to reduce baculovirus contamination have also been explored. Notably, van Oosten et al. developed a temperature-dependent control system for baculovirus production by engineering a thermosensitive switch in the expression vector. This approach enables tight regulation of baculovirus replication, thereby minimizing contaminating baculovirus particles during VLP manufacturing and simplifying downstream processing [104]. To further streamline downstream processing and mitigate contamination risks, researchers have developed baculovirus-free insect cell expression systems, which provide a viable alternative to produce monoclonal antibodies, recombinant antigens, and VLPs [23, 96, 101].

To ensure viral safety in insect cell–based expression systems, several complementary analytical methods are routinely employed to monitor and mitigate potential risks. Next-generation sequencing (NGS) enables comprehensive screening for endogenous viral elements and adventitious agents at the genomic level [105]. As encouraged in the recent revision of the ICH Q5A viral safety guideline, NGS is increasingly being adopted as a replacement for animal-based testing methods [106]. In parallel, quantitative PCR (qPCR) provides highly sensitive detection of specific viral sequences [107], reverse transcriptase (RT) assays are used to assess retroviral activity, and transmission electron microscopy (TEM) allows direct visualization of viral particles in cells [106]. Collectively, these approaches establish a robust safety framework that supports regulatory compliance and ensures the reliability of insect cell–derived biopharmaceuticals.

Regulatory guidelines

During the COVID-19 pandemic, emergency use authorization (EUA) mechanisms established by major regulatory agencies such as the WHO, FDA, the European Medicines Agency (EMA), and the National Medical Products Administration (NMPA) of China created accelerated pathways for vaccine development, which significantly promoted the rapid application of the insect cell expression system platform. For example, NVX-CoV2373/Covovax was included in the WHO’s emergency use listing.

Furthermore, according to the FDA’s draft guidance on Platform Technology Designation released in May 2024, once an insect cell–baculovirus expression product is approved, its manufacturing platform may be recognized as a reproducible, well-characterized, and standardized process. This designation allows subsequent biologics developed using the same platform to benefit from streamlined development, manufacturing, and regulatory review. For instance, requalification of cell banks may be waived, and only the recombinant baculovirus seed used for antigen expression would require testing and characterization. This provides substantial advantages for rapidly responding to public health emergencies or seasonal vaccine production (e.g., influenza, COVID-19).

In terms of viral safety, the 2024 revision of ICH Q5A (R2) affirms that although rodent-derived cell lines such as CHO contain retro-VLPs, their long-standing use and the current body of evidence indicate these particles are non-replicative and non-infectious to humans or animals. As such, CHO cells are considered well-characterized and equipped with effective viral clearance strategies, enabling certain viral safety tests to be waived for products derived from them. In contrast, insect cell lines may harbor non-rodent endogenous viral elements. Although there is no current evidence of their infectivity in humans, regulatory expectations still emphasize validation of virus removal and inactivation using the known or related viruses.

Viral safety concerns for insect cell–based platforms primarily involve retro-VLPs and rhabdoviruses. Early detection of reverse transcriptase (RT) activity in Sf9 supernatants raised theoretical concerns about endogenous retroviral elements. In 2025, a landmark study was the first to identify extracellular retro-VLPs in Spodoptera. These particles, detected in Sf9 cells-particularly after induction-were found to exhibit low buoyant density and heterogeneous morphology, with no evidence of replication or infectivity [108].

As for rhabdoviruses, some licensed insect cell expression system-derived products originated from insect cell lines containing rhabdovirus-related endogenous viral elements (EVEs). Although no infectivity has been demonstrated in humans, the potential for cross-species transmission associated with certain rhabdoviruses has led some regulatory agencies to require the use of rhabdovirus-free cell lines in new product applications. However, even among rhabdovirus-negative Sf9 clones, certain clones have shown susceptibility to rhabdovirus strains, indicating the importance of careful clone selection during master cell bank establishment and continued monitoring for potential recontamination [109].

Overall, the viral safety risks associated with insect cells are considered low and manageable with appropriate viral testing and clearance strategies. As the use of insect cell platforms expands in biologics development, both the characterization of these systems and the associated regulatory frameworks are expected to become increasingly mature.

Conclusions and perspectives

Preserving the core strengths in biomanufacturing

Insect cell expression system remains a cornerstone in biomanufacturing, offering unparalleled scalability and versatility, which makes it indispensable for addressing challenges in complex vaccine development. From expression design to large-scale production, this platform enables rapid, high-yield vaccine manufacturing while maintaining efficacy and quality. Although the emergence of mRNA vaccines has challenged its speed advantage, protein subunit vaccines retain unique strengths. Specifically, the structural characterization conducted during the clinical development of protein subunit vaccines provides researchers with a deeper understanding of the product’s fine structure and function, significantly reducing clinical trial risks. Furthermore, even with the inclusion of strong adjuvants, protein subunit vaccines exhibit superior safety profiles compared to mRNA-based platforms.

A key advantage of this system lies in its remarkable flexibility, enabling the precise design of vectors to achieve the desired size, structural presentation, and oligomeric configuration of the expressed target protein. One of the platform’s most notable achievements is the efficient assembly of VLPs, which structurally mimic native viruses, enhancing immunogenicity and substantially improved vaccine efficacy. With advances in protein structure prediction and design, an increasing number of VLPs or artificial protein cage structures have progressed to clinical trials or gained regulatory approval [110]. Artificial protein cages not only function as vaccines but also serve as carriers as therapeutic protein complexes. Looking ahead, the system’s ability to express complex proteins is expected to play an increasingly significant role, driving its broader application. However, challenges associated with the production process-such as cell lysis during viral infection and baculovirus residue complicating purification-necessitate intensified research into virus-free production systems and their commercial viability.

Advancing with cutting-edge biotechnologies and AI

Recent breakthroughs in single-cell sequencing, gene editing, and synthetic biology provide new avenues to overcome existing insect cell expression system limitations. scRNA-seq offers critical insights into cellular heterogeneity during production, enabling researchers to tailor expression systems to individual cellular responses, thereby maximizing production efficiency. Similarly, advanced gene editing techniques facilitate precise genome modifications, enhancing both vector and host cell systems to improve production characteristics, ultimately driving the comprehensive upgrade of system platforms.

Artificial intelligence (AI) is increasingly revolutionizing biomanufacturing by providing disruptive capabilities in production control and system optimization. Mathematical modeling and machine learning have demonstrated significant value in vaccine manufacturing. For instance, mathematical models can simulate operational parameters, such as nutrient supply and environmental conditions, to identify optimal production conditions, therapy accelerating process development and yield. Meanwhile, machine learning algorithms analyze vast datasets generated during production, uncovering patterns and insights that enhance efficiency. By enabling real-time adjustments to production protocols, AI substantially improves both productivity and quality control [111]. Furthermore, AI-driven omics analysis offers valuable strategies for optimizing vectors and cell lines, enhancing their performance in insect cell–based systems [112]. More recently, AI is increasingly recognized as a powerful driver of innovation across the vaccine lifecycle, with the potential to accelerate development, optimize manufacturing, and strengthen supply chains, thereby enhancing global vaccine effectiveness and accessibility. In the discovery phase, such as AlphaFold [113] and RoseTTAFold [114] enable accurate prediction of protein and protein–protein complex structures, facilitating the design of stable conformations, immunogenic sequences, and even de novo protein scaffolds. For immune-response modeling and infectious disease control, AI systems improve the ability to identify target antigens, predict epidemic trajectories, and simulate immune responses in diverse populations; notably, the EVEscape platform can anticipate viral variants before they emerge [115]. In manufacturing, digital twin technologies provide virtual simulations of production processes, significantly increasing efficiency and product quality [116]. At the clinical stage, AI-driven trial design streamlines protocol development and shortens development timelines [117]. Meanwhile, regulatory authorities are actively considering the use of artificial intelligence to support regulatory decision-making for drugs and biological products [118, 119]. These advances illustrate how AI can transform vaccinology by accelerating antigen design, improving predictive capacity, and optimizing downstream processes, ultimately promoting safer, more effective, and more accessible vaccines worldwide.

In summary, insect cell expression system continues to demonstrate immense potential as a next-generation biomanufacturing platform. Its proven capacity to efficiently produce complex biomolecules, combined with its rich history of innovation, makes it an invaluable tool in vaccine development and beyond. By integrating cutting-edge technologies such as scRNA-seq, gene editing, synthetic biology, and AI, this platform can overcome current limitations and cement its role as a fast, scalable, and efficient platform for vaccine production and other biomanufacturing applications.

Abbreviations

VLPs

Virus-like particles

Sf9

Spodoptera frugiperda 9

rAAV

Recombinant adeno-associated virus

PTMs

Post-translational modifications

GOI

Gene of interest

AEX

Anion exchange chromatography

AI

Artificial intelligence

Author contributions

LJ initiated the idea, guided the article structure, and improved the final manuscript. HNY reviewed the published studies and composed the draft of the manuscript. WYQ guided the article structure on drafts of the manuscript. All authors read and approved the final manuscript.

Funding

This work is supported in part by the Natural Science Foundation of Sichuan Province (2025ZNSFSC0663); the National Natural Science Foundation of China (82371852).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

This work was supported by the WestVac Biopharma Co. Ltd. Jiong Li and Yuquan Wei are also working at the WestVac Biopharma Co. Ltd. The remaining authors declare no competing interests.