Reduction in Cell Line Development Timelines
Expression System I with CHO dhfr– host cell lines. Standard transfection and amplification of a CHO dhfr– cell line are performed by plating cells in serum containing commercially available media after electroporation and subsequently
increasing the methotrexate (MTX) concentration to 500 nM in the same 96-well plates. These cell lines may be further amplified
to 2 μM MTX in batch cultures. The process has included two rounds of subcloning, occurring before and after adaptation to
the serum-free commercially available medium, followed by adaptation to MTCM medium. This entire process takes approximately
12 months (Figure 2A).
Figure 2. Effect of using a single medium throughout cell line development for Expression Systems I and II
Conditions were then developed to accelerate the process in the commercial medium without serum right from the initiation
of cell line development, a process that included optimizing transfection efficiency. This shortened the cell line development
timeline by up to four months and eliminated the need for a lengthy serum-weaning process and one round of subcloning (Figure
2A). However, using only MTCM media during the entire process of cell line development would eliminate media transitions and
potentially reduce the timeline to seven months for a 2 μM MTX amplified cell line (Figure 2A).
Expression System II with CHO dhfr+ host cell lines. A similar process could be carried out with Expression System II, which does not require amplification. Initial protocols
used serum in a commercially available medium at the transfection stage, which was then removed during adaptation. In a manner
similar to Expression System I above, the conditions were optimized using the same commercially available medium without serum
throughout transfection and subcloning. As Figure 2B indicates, by using only MTCM media, we could accelerate the cell line
development timeline to five months. In addition, further reduction in the timeline by another month could be achieved using
bulk transfection and selection methods.
In both cases, pre-adapted host cell banks in MTCM media were generated in a serum-free freezing mixture. Transfection with
these well-characterized host cell banks eliminated media transitions and reduced the timelines for cell line development,
as illustrated in Figure 2.
Amplification Using MTCM Media
In a controlled experiment for Expression System I (Figure 3), CHO colonies in 96-well plates were gradually amplified to
1 μM MTX in a commercial medium (parental 1 μM) and subcloned (clone 1 μM). The parental line was heterogeneous, as indicated
by the increase in gene copy number from 18 to 40 upon subcloning. On the other hand, the efficiency of MTCM media for amplification
was monitored by amplifying up to 500 nm MTX and subcloned (Figure 3). This process yielded a cell line (clone 0.5 μM) with
23 gene copies and better volumetric productivity compared to the parental cell line (parental 0.5 μM). Several MTCM-derived
clones were pooled and amplified to 2 μM MTX. The pooled culture was increased from an average of 29 gene copies per cell
at 500 nm MTX (pool 0.5 μM) to 34 copies at 2 μM MTX (pool 2 μM). Subcloning of the bulk amplified pool resulted in cell lines
(clone 2 μM) producing over 500 mg/L in batch culture with 47 and 54 gene copies per cell. Copy number analysis clearly demonstrated
the increase in gene copies and productivities in correlation with increasing resistance to higher MTX concentration in MTCM
medium. In addition, bulk amplification was also successful with MTCM media, as monitored by qPCR copy number analysis.
Figure 3. Productivity and gene copy number analysis during amplification and subcloning for a HuMAb cell line derived from
Expression System I