An Overview of Virus‐Free Protein Expression in Insect Cells: A Mode of Rapid Manufacturing Platform for Therapeutic Protein and Virus‐Like‐Particles

Production of therapeutic proteins, antibodies, and virus-like particles (VLP) using baculovirus expression systems (BEVS) has been explored for decades. However, we have realized an urgent need for accelerated production of recombinant proteins and VLPs to address critical situations in recent scenarios. In contrast to BEVSs, the virus-free method is significantly shorter as it bypasses the time-consuming process of infectivity monitoring and virus amplification. Moreover, in the virus-free method, complex steps of protein separation can be eliminated to ease downstream processing. Hence, we present a detailed review of the recent techniques for expressing recombinant proteins, therapeutics, and VLP in insect cells using virus-free methods. First, we focus on the specific methodologies used to optimize virus-free transfection. Here, we provide insight into the interplay between crucial factors, including concentration of transfection reagent, seeding density, and medium temperature. Secondly, we provide a structured review of the novel transfection reagents used for transient and stable transfection. Thirdly, we performed an assessment of the cell lines and plasmids used for virus-free expression and their evaluation based on corresponding protein yield. Finally, we provide the recent advancement in scaling up the transfection process from the shaker flask to the bioreactor level to achieve better yield. Various virus-free expression methodologies presented in this article are essential for evaluating the transfection processes toward improving protein yield. The readers can also use the information to design experiments and optimize process parameters for bioreactor operation.