tetano
Editor, Senior Moderator
BMC Biotechnol. 2015 May 16;15(1):31. doi: 10.1186/s12896-015-0152-x.
[h=1]Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems.[/h] Thompson CM[SUP]1,[/SUP][SUP]2[/SUP], Petiot E[SUP]3,[/SUP][SUP]4[/SUP], Mullick A[SUP]5[/SUP], Aucoin MG[SUP]6[/SUP], Henry O[SUP]7[/SUP], Kamen AA[SUP]8,[/SUP][SUP]9[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] [h=4]BACKGROUND:[/h] Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus' fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen's eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers.
[h=4]METHODS:[/h] In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles.
[h=4]RESULTS:[/h] For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles.
[h=4]CONCLUSIONS:[/h] This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.
PMID: 25981500 [PubMed - in process] PMCID: PMC4432999 Free PMC Article
[h=1]Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems.[/h] Thompson CM[SUP]1,[/SUP][SUP]2[/SUP], Petiot E[SUP]3,[/SUP][SUP]4[/SUP], Mullick A[SUP]5[/SUP], Aucoin MG[SUP]6[/SUP], Henry O[SUP]7[/SUP], Kamen AA[SUP]8,[/SUP][SUP]9[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] [h=4]BACKGROUND:[/h] Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus' fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen's eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers.
[h=4]METHODS:[/h] In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles.
[h=4]RESULTS:[/h] For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles.
[h=4]CONCLUSIONS:[/h] This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.
PMID: 25981500 [PubMed - in process] PMCID: PMC4432999 Free PMC Article