Dissolved carbon dioxide determines the productivity of a recombinant hemagglutinin component of an influenza vaccine produced by insect cells.

Dissolved carbon dioxide (dCO2 ) accumulation during cell culture has been recognized as an important parameter that needs to be controlled for successful scale-up of animal cell culture because above a certain concentration there are adverse effects on cell growth performance and protein production. We investigated the effect of accumulation of dCO2 in bioreactor cultures of expresSF+(®) insect cells infected with recombinant baculoviruses expressing recombinant influenza virus hemagglutinins (rHA). Different strategies for bioreactor cultures were used to obtain various ranges of concentrations of dCO2 (<50, 50-100, 100-200, and >200 mmHg) and to determine their effects on recombinant protein production and cell metabolic activity. We show that the accumulation of dCO2 at levels > 100 mmHg resulted in reduced metabolic activity, slowed cell growth, prolonged culture viability after infection, and decreased infection kinetics. The reduced rHA yields were not caused by the decrease in the extracellular pH that resulted from dCO2 accumulation, but were most likely due to the effect of dCO2 accumulation in cells. The results obtained here at the 2 L scale have been used for the design of large-scale processes to manufacture the rHA based recombinant vaccine Flublok™ at the 2500 L scale Biotechnol. Bioeng. 2015;112: 2267-2275. © 2015 Wiley Periodicals, Inc.

Modifications of cysteine residues in the transmembrane and cytoplasmic domains of a recombinant hemagglutinin protein prevent cross-linked multimer formation and potency loss.

BACKGROUND: Recombinant hemagglutinin (rHA) is the active component in Flublok®; a trivalent influenza vaccine produced using the baculovirus expression vector system (BEVS). HA is a membrane bound homotrimer in the influenza virus envelope, and the purified rHA protein assembles into higher order rosette structures in the final formulation of the vaccine. During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency. Potency is measured by the Single Radial Immuno-diffusion (SRID) assay to determine the amount of HA that has the correct antigenic form. RESULTS: The five cysteine residues in the transmembrane (TM) and cytoplasmic (CT) domains of the rHA protein from the H3 A/Perth/16/2009 human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants (SDVs). These SDVs have been evaluated to determine the impact of the TM and CT cysteines on potency, cross-linking, and the biochemical and biophysical properties of the rHA. Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25 °C. The strategy of substituting TM and CT cysteines to prevent potency loss has been successfully applied to another H3 rHA protein (from the A/Texas/50/2012 influenza strain) further demonstrating the utility of the approach. CONCLUSION: rHA potency can be maintained by preventing non-specific disulfide bonding and cross-linked multimer formation. Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.

High yield purification of functional baculovirus vectors by size exclusion chromatography.

Recombinant baculoviruses carrying mammalian expression cassettes or "BacMam" are promising gene delivery vehicles shown to transduce mammalian cells efficiently both in vitro and in vivo. These viruses are vectors of choice because they are non-pathogenic; able to accommodate large foreign DNA inserts and can be produced at high titers. Hence, the demand for pure and functional baculovirus vectors for gene delivery experiments is anticipated in the future. The main goal of this work is to develop a simple and efficient process to purify recombinant baculovirus derived from Autographa californica multiple nucleopolyhedrovirus from a culture supernatant by size exclusion chromatography. The final yields obtained for total and infectious particles were 1.39 x 10(11) and 1.02 x 10(10) and recoveries of 25% and 24%, respectively. The virus was purified from the majority of the protein contaminants as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Negative stain electron microscopy demonstrated that >95% of the purified virus was intact particles with shape like rod and average diameter and length of 60 and 266 nm, respectively. Transduction of 293 human embryonic kidney cells by a purified GFP-expressing BacMam at a multiplicity of transduction of 200 resulted in 36% positive cell population.

Virus-like particle and viral vector production using the baculovirus expression vector system/insect cell system: adeno-associated virus-based products.

The ability to make a large variety of virus-like particles (VLPs) has been successfully achieved in the baculovirus expression vector system (BEVS)/insect cell system. The production and scale-up of these particles, which are mostly sought as candidate vaccines, are currently being addressed. Furthermore, these VLPs are being investigated as delivery agents for use as therapeutics. Recently, adeno-associated viral (AAV) vectors, which can be potentially used for human gene therapy, have been produced in insect cells using three baculovirus vectors to supply the required genes. The use of host insect cells allows mass production of VLPs in a proven scaleable system. This chapter focuses on the methodology, based on the work done in our lab, for the production of AAV-like particles and vectors in a BEVS/insect cell system.

Analysis of baculovirus aggregates using flow cytometry.

Aggregation of viral particles represents a significant problem for baculoviral stock processing and storage. Aggregation may also affect the results of viral particle counting. A method using flow cytometry was previously developed in our lab to measure the concentration of baculovirus particles produced in insect cell cultures. In the present study, the use of the flow cytometry method was extended to the detection of baculovirus aggregates. Flow cytometry analysis of freshly prepared baculovirus stocks, stained with SYBR Green, generally exhibited a single unimodal distribution; while, baculovirus stocks stored at 4 degrees C for a few months exhibited a bimodal distribution of the fluorescent intensity signal. The bimodal distribution was associated with a decrease in the size of the original viral population and an emergence of a new viral population with a high fluorescence intensity. Treatment of these samples with an endonuclease (Benzonase) confirmed that the new population observed in the flow cytometry analysis is not free cellular DNA. Filtration through 0.22 and 0.45 microm membranes of the stored samples prior to flow cytometry analysis confirmed that the high fluorescence intensity population involved particles larger than a single baculovirus. Exposing freshly amplified baculovirus stocks with a unimodal distribution to a pH of 5.3, a condition known to induce aggregation, showed the emergence of a second population with a bimodal distribution. These results suggest that flow cytometry analysis could be used to detect baculovirus aggregates. The aggregates were associated with high fluorescence intensity populations and the mean green fluorescence intensity of these populations could be used as an indicator of the mean aggregate size.

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake flasks to a 20-L bioreactor.

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.

Quantitation of baculovirus particles by flow cytometry.

A method using flow cytometry (FCM) analysis was developed to quantitate baculovirus total particles produced in insect cell cultures. The method is a direct count of particles and involves staining of the baculovirus DNA with SYBR Green I, a highly fluorescent nucleic acid specific dye. Sample preparation of cell-free supernatant containing budded viral particles involves fixation with paraformaldehyde, freeze-thaw treatment, viral membrane permeabilization with Triton X-100, and sample heating to improve staining efficiency and enhance baculovirus particle green fluorescence intensities. In this study, the effects of the different treatment steps and medium composition on viral particle counts were examined in order to identify optimal preparation conditions. FCM analysis linearity was established over a viral concentration range of two logs with a lower detection limit at 10(5) viral particles per ml. Robustness and reproducibility of the method were assessed using samples from large-scale bioreactor cultures. The events (or virus particle counts) obtained by FCM analysis were usually higher than the titres obtained by end-point dilution assay (EPDA). Results from 16 different viral stocks showed an average ratio of 3.7 total particles (FCM) to infectious particles (EPDA). Essentially, the FCM analysis reported below shortens baculovirus quantitation time to 2 h and provides a good estimation of virus titers. It is believed that these findings will contribute to acceleration of process development in the area of baculovirus expression technology in general and specifically in process where stoichiometric multi-viral infections of cells are critical to the expression of complex products.

Technology transfer and scale-up of the Flublok recombinant hemagglutinin (HA) influenza vaccine manufacturing process.

Multiple different hemagglutinin (HA) protein antigens have been reproducibly manufactured at the 650L scale by Protein Sciences Corporation (PSC) based on an insect cell culture with baculovirus infection. Significantly, these HA protein antigens were produced by the same Universal Manufacturing process as described in the biological license application (BLA) for the first recombinant influenza vaccine approved by the FDA (Flublok). The technology is uniquely designed so that a change in vaccine composition can be readily accommodated from one HA protein antigen to another one. Here we present a vaccine candidate to combat the recently emerged H7N9 virus as an example starting with the genetic sequence for the required HA, creation of the baculovirus and ending with purified protein antigen (or vaccine component) at the 10L scale accomplished within 38 days under GMP conditions. The same process performance is being achieved at the 2L, 10L, 100L, 650L and 2500L scale. An illustration is given of how the technology was transferred from the benchmark 650L scale facility to a retrofitted microbial facility at the 2500L scale within 100 days which includes the time for facility engineering changes. The successful development, technology transfer and scale-up of the Flublok process has major implications for being ready to make vaccine rapidly on a worldwide scale as a defense against pandemic influenza. The technology described does not have the same vulnerability to mutations in the egg adapted strain, and resulting loss in vaccine efficacy, faced by egg based manufacture.

Development of a simple and high-yielding fed-batch process for the production of influenza vaccines.

A robust and reliable GMP-compatible fed-batch process was successfully developed for the production of recombinant hemagglutinin (rHA) proteins by expresSF cells. The feeding solution, feeding strategy as well as the cell density at infection were optimized to maximize the final rHA production yields without affecting the existing rHA recovery protocol and downstream process. A simple and stable feeding solution was formulated and a rational feeding regimen designed to yield, depending on the rHA baculovirus used, between 2- and 3-fold enhancements in volumetric rHA production with increased specific productivity compared to the batch culture. Recombinant HA from fed-batch cultures could be simply recovered following cell lysis and purified through chromatographic steps. Overall, the increased rHA yield was maintained throughout the whole process. The performance, reproducibility and scalability of the fed-batch process was successfully demonstrated in 12 bioreactor runs of 2- and 10-L working volume using five different rHA encoding baculoviruses.