Expansion and Storage of Insect Cells and Virus Stocks.

Healthy insect cell cultures are critical for any method described in this book, including making productive baculovirus banks, protein or AAV expression, and determining viral titers. This chapter describes cell maintenance in shake flasks using serum-free conditions and the expansion of virus stocks from a single plaque purified virus. Insect cells can be passaged over multiple generations, but as the cells may undergo changes over multiple passages, limiting the use of your cells to a defined number of passages such as 50 passages is recommendable. Baculovirus stocks once created using serum-free media are not very stable at 4-8 °C. This chapter also includes a simple method to store cells from an early cell passage and your virus stock in liquid nitrogen.

Innovations in the Insect Cell Expression System for Industrial Recombinant Vaccine Antigen Production.

The insect cell expression system has previously been proposed as the preferred biosecurity strategy for production of any vaccine, particularly for future influenza pandemic vaccines. The development and regulatory risk for new vaccine candidates is shortened as the platform is already in use for the manufacturing of the FDA-licensed seasonal recombinant influenza vaccine Flublok®. Large-scale production capacity is in place and could be used to produce other antigens as well. However, as demonstrated by the 2019 SARS-CoV-2 pandemic the insect cell expression system has limitations that need to be addressed to ensure that recombinant antigens will indeed play a role in combating future pandemics. The greatest challenge may be the ability to produce an adequate quantity of purified antigen in an accelerated manner. This review summarizes recent innovations in technology areas important for enhancing recombinant-protein production levels and shortening development timelines. Opportunities for increasing product concentrations through vector development, cell line engineering, or bioprocessing and for shortening timelines through standardization of manufacturing processes will be presented.

Glycobiotechnology of the Insect Cell-Baculovirus Expression System Technology.

The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed. Graphical Abstract.

Randomized Comparison of Immunogenicity and Safety of Quadrivalent Recombinant Versus Inactivated Influenza Vaccine in Healthy Adults 18-49 Years of Age.

Background: Seasonal influenza vaccines are transitioning to quadrivalent formulations including the hemagglutinins of influenza A subtypes H1N1 and H3N2 and B lineages Yamagata and Victoria. Methods: A new quadrivalent recombinant influenza vaccine (RIV4) was compared directly with a standard-dose, egg-grown, quadrivalent-inactivated influenza vaccine (IIV4) for immunogenicity and safety in adults 18-49 years of age. The coprimary endpoints for noninferiority were hemagglutination inhibition seroconversion rates and postvaccination geometric mean titer ratios for each antigen using US regulatory criteria. Reactogenicity solicited for 7 days, other safety events collected for 28 days, and serious or medically attended adverse events collected for 6 months after vaccination comprised the safety evaluation. Results: The immunogenicity of RIV4 was comparable to that of IIV4; the coprimary noninferiority criteria were met for 3 antigens, and the antibody responses to the fourth antigen, influenza B/Brisbane/60/2008, were low in each group, making comparisons uninterpretable. Systemic and injection site reactions were mild, transient, and similar in each group, whereas none of the spontaneously reported adverse events, serious or nonserious, were considered related to study vaccine. Conclusions: This first head-to-head comparison of recombinant versus inactivated quadrivalent influenza vaccines in 18-49 year old adults showed comparable immunogenicity, safety, and tolerability for both vaccines.

Efficacy of Recombinant Influenza Vaccine in Adults 50 Years of Age or Older.

BACKGROUND: Improved influenza vaccines are needed to control seasonal epidemics. This trial compared the protective efficacy in older adults of a quadrivalent, recombinant influenza vaccine (RIV4) with a standard-dose, egg-grown, quadrivalent, inactivated influenza vaccine (IIV4) during the A/H3N2-predominant 2014-2015 influenza season, when antigenic mismatch between circulating and vaccine influenza strains resulted in the reduced effectiveness of many licensed vaccines. METHODS: We conducted a randomized, double-blind, multicenter trial of RIV4 (45 µg of recombinant hemagglutinin [HA] per strain, 180 µg of protein per dose) versus standard-dose IIV4 (15 µg of HA per strain, 60 µg of protein per dose) to compare the relative vaccine efficacy against reverse-transcriptase polymerase-chain-reaction (RT-PCR)-confirmed, protocol-defined, influenza-like illness caused by any influenza strain starting 14 days or more after vaccination in adults who were 50 years of age or older. The diagnosis of influenza infection was confirmed by means of RT-PCR assay and culture of nasopharyngeal swabs obtained from participants with symptoms of an influenza-like illness. The primary end point was RT-PCR-confirmed, protocol defined, influenza-like illness between 14 days or more after vaccination and the end of the influenza season. RESULTS: A total of 9003 participants were enrolled and underwent randomization; 8855 (98.4%) received a trial vaccine and underwent an efficacy follow-up (the modified intention-to-treat population), and 8604 (95.6%) completed the per-protocol follow-up (the modified per-protocol population). Among RIV4 recipients, the RT-PCR-confirmed influenza attack rate was 2.2% (96 cases among 4303 participants) in the modified per-protocol population and 2.2% (96 cases among 4427 participants) in the modified intention-to-treat population. Among IIV4 recipients, the attack rate was 3.2% (138 cases among 4301 participants) in the modified per-protocol population and 3.1% (138 cases among 4428 participants) in the modified intention-to-treat population. A total of 181 cases of influenza A/H3N2, 47 cases of influenza B, and 6 cases of nonsubtypeable influenza A were detected. The probability of influenza-like illness was 30% lower with RIV4 than with IIV4 (95% confidence interval, 10 to 47; P=0.006) and satisfied prespecified criteria for the primary noninferiority analysis and an exploratory superiority analysis of RIV4 over IIV4. The safety profiles of the vaccines were similar. CONCLUSIONS: RIV4 provided better protection than standard-dose IIV4 against confirmed influenza-like illness among older adults. (Funded by Protein Sciences; ClinicalTrials.gov number, NCT02285998 .).

Panblok-H1+advax H1N1/2009pdm vaccine: Insights into rapid development of a delta inulin adjuvanted recombinant pandemic influenza vaccine.

Timely vaccine supply is critical during influenza pandemics but is impeded by current virus-based manufacturing methods. The 2009 H1N1/2009pdm 'swine flu' pandemic reinforced the need for innovation in pandemic vaccine design. We report on insights gained during rapid development of a pandemic vaccine based on recombinant haemagglutinin (rHA) formulated with Advax™ delta inulin adjuvant (Panblok-H1/Advax). Panblok-H1/Advax was designed and manufactured within 1 month of the pandemic declaration by WHO and successfully entered human clinical testing in under 3 months from first isolation and sequencing of the novel pandemic virus, requiring several major challenges to be overcome. Panblok-H1/Advax successfully induced neutralising antibodies against the pandemic strain, but also induced cross-neutralising antibodies in a subset of subjects against an H1N1 strain (A/Puerto Rico/8/34) derived from the 1918 Spanish flu, highlighting the possibility to use Advax to induce more broadly cross-protective antibody responses. Interestingly, the rHA from H1N1/2009pdm exhibited variants in the receptor binding domain that had a major impact on receptor binding and hemagglutination ability. We used an in silico structural modeling approach to better understand the unusual behavior of the novel hemagglutinin, thereby demonstrating the power of computational modeling approaches for rapid characterization of new pandemic viruses. While challenges remain in ensuring ultrafast vaccine access for the entire population in response to future pandemics, the adjuvanted recombinant Panblok-H1/Advax vaccine proved its utility during a real-life pandemic situation.

Randomized comparison of the safety of Flublok(®) versus licensed inactivated influenza vaccine in healthy, medically stable adults ≥ 50 years of age.

BACKGROUND: The safety and tolerability of Flublok(®), a purified recombinant hemagglutinin seasonal influenza vaccine, was compared to AFLURIA(®) in a randomized, blinded clinical trial in adults ≥ 50 years of age with attention to hypersensitivity reactions. METHODS: This blinded, randomized trial of healthy adults ≥ 50 years of age compared safety of Flublok vs. AFLURIA with respect to pre-specified possible hypersensitivity: "rash," "urticaria," "swelling" and "non-dependent edema;" solicited reactogenicity and unsolicited adverse events. Subject-reported outcomes were collected for 30 days after vaccination. All adverse event terms were reviewed by physicians blinded to vaccine group, who added other terms possibly reflecting hypersensitivity. Case records of subjects with possible hypersensitivity were adjudicated by independent experts blinded to treatment assignment to identify likely hypersensitivity reactions. Non-inferiority of the incidence of hypersensitivity in the two vaccine groups was pre-defined as an absolute difference with an upper bound of 2-sided 95% confidence limits ≤ 0.015. RESULTS: A total of 2640 subjects were enrolled, evenly split in age cohorts of 50-64 and ≥ 65 years. Fifty-two subjects reported at least one term possibly representing hypersensitivity, with a slight imbalance of 31 on Flublok and 21 on AFLURIA. The adjudicators determined that six and four subjects on Flublok and AFLURIA, respectively, likely met clinical criteria for hypersensitivity, yielding a difference in incidence between the two vaccine groups of 0.15% (upper bound of 2-sided 95% CI=0.9%). Reactogenicity and overall adverse event profiles were similar across both vaccines. CONCLUSIONS: Flublok was non-inferior to AFLURIA in adults ≥ 50 years of age with respect to expert-adjudicated events of likely hypersensitivity during 30 days following vaccination (Sponsored by Protein Sciences Corporation; ClinicalTrials.gov number NCT01825200).

Safety, efficacy, and immunogenicity of Flublok in the prevention of seasonal influenza in adults.

Flublok is the first recombinant hemagglutinin (HA) vaccine licensed by the US Food and Drugs Administration for the prevention of influenza in adults aged 18 and older. The HA proteins produced in insect cell culture using the baculovirus expression system technology are exact analogues of wild type circulating influenza virus HAs. The universal HA manufacturing process that has been successfully scaled to the 21,000L contributes to rapid delivery of a substantial number of doses. This review discusses the immunogenicity, efficacy and safety data from five pivotal clinical studies used to support licensure of trivalent Flublok for adults 18 years of age and older in the United States. The trial data demonstrate that the higher antigen content in Flublok results in improved immunogenicity. Data further suggest improved efficacy and a slightly lower local reactogenicity compared with standard inactivated influenza vaccine, despite the presence of more antigen (statistically significant). Flublok influenza vaccine can include HAs designed to mimic 'drift' in influenza viruses as the process of predicting antigenic drift advances and, at a minimum, could address late appearing influenza viruses. The implementation of the latter will require support from regulatory authorities.

Improved stability of recombinant hemagglutinin using a formulation containing sodium thioglycolate.

This study was designed to improve the stability of liquid formulations of recombinant influenza hemagglutinin (rHA) and to understand the mechanism of early loss of potency for rHA. The potency of rHA derived from several influenza strains was determined using single radial immunodiffusion (SRID), and the structure of the rHA was characterized using SDS-PAGE and dynamic light scattering. rHA formed disulfide cross-linked multimers, and potency decreased during extended storage. To reduce disulfide-mediated cross-linking and early potency loss, rHA was formulated with sodium thioglycolate (STG) and citrate. Addition of 80 mM STG and 55 mM sodium citrate inhibited disulfide-mediated cross-linking without affecting protein function for each rHA tested. The shelf life of the rHA formulation with STG-citrate, based on potency as determined by SRID, was extended as much as 20-fold, compared to a control formulation without STG-citrate. STG-citrate did not have a significant effect on the immunogenicity of H1 A/California/7/2009 rHA in mice.

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.

Titer on chip: new analytical tool for influenza vaccine potency determination.

Titer on Chip (Flu-ToC) is a new technique for quantification of influenza hemagglutinin (HA) concentration. In order to evaluate the potential of this new technique, a comparison of Flu-ToC to more conventional methods was conducted using recombinant HA produced in a baculovirus expression system as a test case. Samples from current vaccine strains were collected from four different steps in the manufacturing process. A total of 19 samples were analysed by Flu-ToC (blinded), single radial immunodiffusion (SRID), an enzyme-linked immunosorbent assay (ELISA), and the purity adjusted bicinchoninic acid assay (paBCA). The results indicated reasonable linear correlation between Flu-ToC and SRID, ELISA, and paBCA, with regression slopes of log-log plots being 0.91, 1.03, and 0.91, respectively. The average ratio for HA content measured by Flu-ToC relative to SRID, ELISA, and paBCA was 83%, 147%, and 81%, respectively; indicating nearly equivalent potency determination for Flu-ToC relative to SRID and paBCA. These results, combined with demonstrated multiplexed analysis of all components within a quadrivalent formulation and robust response to HA strains over a wide time period, support the conclusion that Flu-ToC can be used as a reliable and time-saving alternative potency assay for influenza vaccines.

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.

Innovations in vaccine development: can regulatory authorities keep up?

Vaccine Production Summit San Francisco, CA, USA, 4-6 June 2012 IBC's 3rd Vaccine Production Summit featured 28 presentations discussing regulatory challenges in vaccine development, including the use of adjuvants, vaccine manufacturing and technology transfer, process development for vaccines and the role of quality by design, how to address vaccine stability, and how vaccine development timelines can be improved. The conference was run in parallel with the single-use applications for Biopharmaceutical Manufacturing conference. Approximately 250 attendees from large pharmaceutical companies, large and small biotech companies, vendors and a more limited number from academia were allowed to access sessions of either conference, including one shared session. This article summarizes the recurring themes across various presentations.

Pandemic influenza vaccine: characterization of A/California/07/2009 (H1N1) recombinant hemagglutinin protein and insights into H1N1 antigen stability.

BACKGROUND: The recent H1N1 influenza pandemic illustrated the shortcomings of the vaccine manufacturing process. The A/California/07/2009 H1N1 pandemic influenza vaccine or A(H1N1)pdm09 was available late and in short supply as a result of delays in production caused by low yields and poor antigen stability. Recombinant technology offers the opportunity to shorten manufacturing time. A trivalent recombinant hemagglutinin (rHA) vaccine candidate for seasonal influenza produced using the baculovirus expression vector system (BEVS) was shown to be as effective and safe as egg-derived trivalent inactivated vaccine (TIV) in human clinical studies. In this study, we describe the characterization of the A/California/07/2009 rHA protein and compare the H1N1 pandemic rHA to other seasonal rHA proteins. RESULTS: Our data show that, like other rHA proteins, purified A/California/07/2009 rHA forms multimeric rosette-like particles of 20-40 nm that are biologically active and immunogenic in mice as assayed by hemagglutination inhibition (HAI) antibody titers. However, proteolytic digest analysis revealed that A/California/07/2009 rHA is more susceptible to proteolytic degradation than rHA proteins derived from other seasonal influenza viruses. We identified a specific proteolytic site conserved across multiple hemagglutinin (HA) proteins that is likely more accessible in A/California/07/2009 HA, possibly as a result of differences in its protein structure, and may contribute to lower antigen stability. CONCLUSION: We conclude that, similar to the recombinant seasonal influenza vaccine, recombinant A(H1N1)pdm09 vaccine is likely to perform comparably to licensed A(H1N1)pdm09 vaccines and could offer manufacturing advantages.

Randomized clinical trial of immunogenicity and safety of a recombinant H1N1/2009 pandemic influenza vaccine containing Advax™ polysaccharide adjuvant.

BACKGROUND: Timely vaccine supply is critical during influenza pandemics. A recombinant hemagglutinin (rHA)-based vaccine could overcome production hurdles of egg-based vaccines but has never previously been tested in a real-life pandemic setting. The primary aim was to determine the efficacy of a recombinant pandemic vaccine and whether its immunogenicity could be enhanced by a novel polysaccharide adjuvant (Advax™). METHODS: 281 adults aged 18-70 years were recruited in a randomized, subject and observer blinded, parallel-group study of rHA H1N1/2009 vaccine with or without adjuvant. Immunizations were at 0 and 3 weeks with rHA 3, 11 or 45 µg. Serology and safety was followed for 6 months. RESULTS: At baseline, only 9.1% of subjects (95% CI: 6.0-13.2) had seroprotective H1N1/2009 titers. Seroconversion rates varied by rHA dose, presence of adjuvant, subject age and number of immunizations. Eighty percent (95% CI: 52-96) of 18-49 year olds who received rHA 45 µg with adjuvant were seroprotected at week 3, representing a 11.1-fold increase in antibody titers from baseline. Advax™ adjuvant increased seroprotection rates by 1.9 times after the first, and 2.5 times after the second, immunization when compared to rHA alone. Seroprotection was sustained at 26 weeks and the vaccine was well tolerated with no safety issues. CONCLUSIONS: The study confirmed the ability to design, manufacture, and release a recombinant vaccine within a short time from the start of an actual influenza pandemic. Advax™ adjuvant significantly enhanced rHA immunogenicity.

Recombinant protein vaccines produced in insect cells.

The baculovirus-insect cell expression system is a well known tool for the production of complex proteins. The technology is also used for commercial manufacture of various veterinary and human vaccines. This review paper provides an overview of how this technology can be applied to produce a multitude of vaccine candidates. The key advantage of this recombinant protein manufacturing platform is that a universal "plug and play" process may be used for producing a broad range of protein-based prophylactic and therapeutic vaccines for both human and veterinary use while offering the potential for low manufacturing costs. Large scale mammalian cell culture facilities previously established for the manufacturing of monoclonal antibodies that have now become obsolete due to yield improvement could be deployed for the manufacturing of these vaccines. Alternatively, manufacturing capacity could be established in geographic regions that do not have any vaccine production capability. Dependent on health care priorities, different vaccines could be manufactured while maintaining the ability to rapidly convert to producing pandemic influenza vaccine when the need arises.

Influenza viral neuraminidase: the forgotten antigen.

Influenza is the most common cause of vaccine-preventable morbidity and mortality despite the availability of the conventional trivalent inactivated vaccine and the live-attenuated influenza vaccine. These vaccines induce an immunity dominated by the response to hemagglutinin (HA) and are most effective when there is sufficient antigenic relatedness between the vaccine strain and the HA of the circulating wild-type virus. Vaccine strategies against influenza may benefit from inclusion of other viral antigens in addition to HA. Epidemiologic evidence and studies in animals and humans indicate that anti-neuraminidase (NA) immunity will provide protection against severe illness or death in the event of a significant antigenic change in the HA component of the vaccine. However, there is little NA immunity induced by trivalent inactivated vaccine and live-attenuated influenza vaccine. The quantity of NA in influenza vaccines is not standardized and varies significantly among manufacturers, production lots and tested strains. The activity and stability of the NA enzyme is influenced by concentration of divalent cations. If immunity against NA is desirable, a better understanding of how the enzymatic properties affect the immunogenicity is needed.

A fast track influenza virus vaccine produced in insect cells.

The viral surface protein hemagglutinin (HA) has been recognized as a key antigen in the host response to influenza virus in both natural infection and vaccination because neutralizing antibodies directed against HA can mitigate or prevent infection. The baculovirus-insect cell system can be used for the production of recombinant HA molecules and is suitable for influenza vaccine production where annual adjustment of the vaccine is required. This expression system is generally considered safe with minimal potential for growth of human pathogens. Extensive characterization of this novel cell substrate has been performed, none of which has revealed the presence of adventitious agents. Multiple clinical studies have demonstrated that the vaccine is safe, well-tolerated and immunogenic. The baculovirus-insect cell system could, therefore, be used for the expedited production of a safe and efficacious influenza vaccine. As a result, this technology should provide a fast track worldwide solution for newly emerging influenza strains or pandemic preparedness within a few years.