Under the ACE System Process, candidate cell lines are also subjected to a stability study as part of their performance testing.
The final candidate cell lines are selected, based on their stability over a minimum number of generations, as well as their
overall expression. Stability studies consist of maintaining cultures of the candidate cell lines in 125-mL shake flasks and
passaging them twice weekly to ~3 x 105 cell/mL. At specific times throughout the study, the cells are subjected to full productivity analysis in 250-mL shake flasks
to determine growth profiles, maximum titers, and specific productivities. The stability study showed that single-load and
double-load candidate cell lines were stable for over 60 generations. In addition, it was shown that these candidate cell
lines were stable for over 96 generations when subjected to fed-batch conditions in 250-mL shake flasks.
Rapid Generation of Candidate Cell Lines
In this case study, the goal was to generate as quickly as possible a cell line expressing a monoclonal antibody to support
pharmacological and toxicological testing, as well as early clinical development. It was estimated that a cell line expressing
greater than 500 mg/L would be sufficient for these purposes. A single-load process was selected with minimal clone screening
and an emphasis on generating a stable cell line in less than four months from transfection. Briefly, the Platform ACE Cell
Line was targeted with an ATV containing the sequences for the heavy and light chain of an IgG4 monoclonal antibody flanked
by cHS4 insulator sequences and the hygromycin resistance gene. After transfection, only 50 drug-resistant colonies were taken
forward to 96-well plates and only 10 primary transfectants expanded to 24-well plates. Finally, two primary transfectants
whose antibody titers were greater than 200 mg/L were selected for single-cell subcloning by limiting dilution. The resulting
candidate cell line had an antibody titer of approximately 430 mg/L and was generated in under three months from transfection.
This candidate cell line was shown to be stable for over 50 generations and that antibiotic selection was not required to
maintain their stability. This candidate cell line was then subjected to scale-up and simple fed-batch by supplementing the
basal CD-CHO medium with glucose and plant hydrolysates. Antibody titers were doubled (Table 2) in the 1.6-L fed-batch bioreactor
with a significant increase in specific productivity and culture time. The antibody titer fell slightly to 660 mg/L when scaled
up to the 15-L and 500-L fed-batch bioreactors. After purification, over 140 g of purified antibody was recovered from the
500-L fed-batch bioreactor and used to support the pharmacological and toxicological studies.
Auditioning of Cells for Expression
Among the key features of the ACE system is the ability to purify ACEs (loaded with the gene[s] of interest or unloaded) using
flow sorting and transfer of ACEs to a variety of mammalian cells, primary cells, or cell lines1, 2, 3. This feature enables the quick use of loaded ACEs to audition host cells for improved quality of the product (e.g., desired
glycosylation pattern) or enhanced quantity (e.g., improved growth or expression). To demonstrate this feature, a DG44 CHO
Platform ACE Cell Line was loaded with monoclonal antibody heavy and light chains2 and candidate cell lines were generated according to the single load process. The resultant candidate cell line had a specific
productivity of 12 pg/cell/day. The loaded ACE was then isolated by flow cytometry and transferred into the parental DG44
line (without the Platform ACE). The resulting cell lines containing the transferred ACE had comparable levels of antibody
expression with an average specific productivity of 11.4 pg/cell/day. This demonstrated that a loaded ACE could successfully
be transferred from one cell line to another without any loss of antibody expression. Furthermore, upon transfer of the same
loaded ACE to a CHOK1SV cell line, the average specific productivity increased to 55 pg/cell/day. In both transfers, FISH
analyses revealed intact single ACEs, demonstrating that the specific productivity differences were due to the host cell.
Beyond demonstrating the difference in expression in CHO lines for this monoclonal antibody, it illustrated how a single ACE
can be used to audition cell lines for a desired trait without having to separately engineer both lines from the beginning
of an engineering process.