Production of viral vaccines in suspension on avian embryonic derived stem cell lines
Inventors
Mehtali, Majid • Champion-Arnaud, Patrick • Leon, Arnaud
Assignees
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Publication Number
US-8148132-B2
Publication Date
2012-04-03
Expiration Date
Abstract
The present invention relates to the development and manufacturing of viral vaccines, particularly the industrial production of viral vectors and vaccines, and more particularly the use of avian embryonic stem cells, preferably the EBx cell line derived from chicken embryonic stem cells, for the production of viral vectors and viruses; the invention is particularly useful for the industrial production of viral vaccines to prevent viral infection of humans and animals.
Core Innovation
The invention relates to a process of infecting avian embryonic derived stem cells with a poxvirus and replicating the poxvirus in the avian embryonic derived stem cells, followed by harvesting the virus. The avian embryonic derived stem cells are obtained by culturing avian embryonic stem cells in a complete culture medium containing exogenous growth factors and an inactivated feeder layer, and then passaging by withdrawing the exogenous growth factors and serum and/or the inactivated feeder layer.
After passaging, an adherent or non-adherent cell culture is established that proliferates in a basal medium in the absence of exogenous growth factors, serum, and/or inactivated feeder layer. The infecting and replicating steps further comprise proliferating the avian embryonic derived stem cells in suspension as clumped cells in a first serum-free medium to allow aggregation into cell clumps, and transferring the culture by dilution without disrupting the cell clumps.
The process infects the clumped cells with the poxvirus when the cell density is at least 1.5 million cells/ml and adds a second serum-free medium shortly before, simultaneously to, or shortly after infection. The infected cells are then further cultured to allow virus replication and propagation while maintaining the cell clumps during the culture and infection-related steps, and the virus is harvested.
Claims Coverage
Independent claim clm-00001 covers an integrated process for producing poxvirus by infecting and replicating poxvirus in avian embryonic derived stem cells that are defined by a specific derivation and passaging scheme and that are expanded and infected as suspension clumps in serum-free media. The claim includes five main inventive features governing stem cell derivation to a basal, growth-factor-free state, suspension clump cultivation in serum-free medium, clump-preserving transfer by dilution, infection conditions for poxvirus replication, and maintaining clumps through replication and harvesting.
Stem cell derivation to a basal, growth-factor/serum-free proliferation state
Avian embryonic derived stem cells are obtained by culturing avian embryonic stem cells in a complete culture medium containing exogenous growth factors and an inactivated feeder layer, followed by passaging with withdrawal of the exogenous growth factors, serum, and/or the inactivated feeder layer, to establish an adherent or non-adherent cell culture capable of proliferating in a basal medium in the absence of exogenous growth factors, serum, and/or inactivated feeder layer.
Suspension clump proliferation in a first serum-free medium
Proliferating the avian embryonic derived stem cells in suspension as clumped cells in a first serum-free medium, allowing the cells to aggregate to form cell clumps.
Clump-preserving transfer by dilution without disrupting cell clumps
Transferring the cell culture by dilution without disrupting the cell clumps.
Poxvirus infection of clumped cells at a defined cell density with serum-free medium timing
Infecting the clumped cells with the poxvirus when the cell density is at least 1.5 million cells/ml, and adding a second serum-free medium shortly before, simultaneously to, or shortly after infection.
Maintaining clumps during replication and harvesting the virus
Further culturing the infected cells to allow virus replication and propagation and harvesting the virus, while maintaining the cell clumps during the culture and infection-related steps.
Overall, the independent claim combines a defined avian embryonic derived stem cell derivation to a basal proliferation state with a suspension clump-based, serum-free infection and replication workflow for a poxvirus, including clump-preserving transfer, clump infection at a minimum cell density, addition of a second serum-free medium around infection, maintenance of clumps during replication, and virus harvesting.
Stated Advantages
Documented Applications
Industrial production of viral vaccines/vectors in suspension cultures using an avian embryonic derived stem cell platform, exemplified by poxviruses including MVA and influenza virus production workflows with harvest and formulation/uses.
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