Production Process & Release Testing
The GMP-Ready AAV pHelper-Kan plasmid DNA was produced and released according to batch records production (e.g., documentation, material segregation, traceability, etc.). Although CGMP-Ready plasmids are not manufactured in a CGMP suite, GMP-Ready plasmids are manufactured in dedicated clean-rooms with mandated changeovers before new production cycles. The GMP-Ready pHelper-Kan plasmid can be used for AAV CGMP manufacturing.
Production Process of pHelper-Kan GMP-R Plasmid
|Controlled Aspects pHelper-Kan GMP-R Plasmid|
|Establishment of E. coli master & working cell banks|
|Full traceability of materials|
|Full room changeover prior to each production|
|Production in segregated and dedicated space|
|Process & change control|
|Aseptic fill/finish and vialing|
|Document support for IND and IMPD filing|
|Master batch records|
|Product release tests|
|pHelper GMP-R Plasmid Release Testing|
|Safety: Endotoxin, sterility|
|Identity: Restriction analysis & DNA sequencing|
|Purity: UV spec, OD260/280|
|DNA homogeneity: Percentage of supercoiled|
|Purity: Residual impurities, host cell DNA, host cell RNA, host cell protein|
|Purity: Cross batch contamination test by Next-Gen sequencing|
pHelper-Kan Plasmid Map
pHelper-Kan is Proven For Efficient AAV Packaging of Different Serotypes
The robust virus packaging productivity of the pHelper-Kan plasmid has been tested in almost all AAV serotypes. pHelper-Kan is being used in Vigene’s daily AAV production, for both CGMP grade and research grade batches. Please see the production data below for AAV2, AAV5, AAV6, AAV8, and AAV9 serotypes.
Fig. 1. pHelper-Kan was used in AAV production in adherent HEK293 cells. pHelper-Kan was cotransfected with the AAV transfer vector and pRepCap into HEK293 cells for AAV packaging. The productivity data for each AAV serotype is from 10 individual productions with different AAV transfer vectors containing unique genes of interest. The viral genome titers were measured using qPCR with primers targeting the ITRs.
The Diagram of AAV Production Using Triple Plasmid Transfection Method
Fig. 2. AAV virus production using triple plasmid transient transfection method. AAV viral vectors will be packaged and assembled when the AAV transfer vector, pHelper, and pRepCap are cotransfected into HEK293 cells. The AAV transfer vector contains the gene of interest, pHelper plasmid provides Ad helper function genes, and pRepCap encodes AAV Rep and Cap.
Need Custom Plasmid Production?
For AAV viral vector packaging, you might also need Rep-Cap plasmid and your transfer vector with your gene of interest flanked by AAV ITRs. Vigene can help you with both GMP-Ready and CGMP grade plasmid DNA production. Please contact us.