An Alternative Vector Strategy
Following these results and additional work, alterations were made to our vector strategy. A head-to-head comparison of clones
resulting from transfections with both the newly engineered vectors and the previously used ("old") vectors, with the same
antibody molecule, showed no differences in recombinant protein expression or cell growth performance during clone screening
before cell line adaptation (data not shown). High-producing clones from this head-to-head comparison were grown in continuous
culture, and RNA was prepared at various time points and was run on a northern blot hybridized with probes encoding DHFR and GAPDH.
The results are shown in Figure 4A. The lane labeled "old vector clone" contained RNA prepared at a single time point from
a single clone derived using the previous vector system, and showed that there was rearranged DHFR transcript present. The other lanes (labeled 1, 2, 3, 4, 5) contained RNA from five clones arising from transfection with
the newly engineered vectors at several time points (these time points ranged from 11 to 113 generations). None of these clones
were found to have rearranged DHFR. Information contained in Figure 4B shows that of the 12 high-producing clones derived using the new vectors, none exhibited
rearranged DHFR transcripts, whereas 9 of the 12 high producing clones derived using the previous ("old") vectors demonstrated a loss of
product expression during the same time period, and these latter clones were all found to have rearranged DHFR.
Figure 4. Alterations in antibody vector constructs result in improved cell line stability. A) RNA was prepared at indicated
time points (in generations) from five clones derived from a transfection using newly engineered vector constructs, and from
a single clone derived from a transfection using previous "old" vector constructs. Each lane was loaded with 3 μg of RNA,
the gel was transferred to nitrocellulose, and the nitrocellulose was hybridized with probes encoding DHFR and GAPDH. Migration
of HC-DHFR, rearranged DHFR, and GAPDH (control) are as indicated. B) Refer to text for explanation of table.
Although the alternative vector strategy significantly reduced the frequency of DHFR rearrangement, it did not completely eliminate the ability of cells to uncouple DHFR and HC. This is represented by the data shown in Figure 5. Using the new vector strategy, platform procedures were followed
for all activities from transfection through cell line adaptation to cell banking. Figure 5A shows the growth and productivity
profiles for a clone that demonstrated a cell-specific productivity (Qp) of >30 picograms per cell per day out to 42 generations.
From 42 generations onward, the growth rate (marked by the red line and triangles) of the clone appeared relatively stable,
however, a precipitous drop in antibody expression (Qp shown in the black line and triangles) was observed starting at about
77 generations, resulting in a total loss of product expression by 107 generations. RNA was prepared at several time points
(marked by blue generation numbers on Figure 5A) during the time that this clone was continuously cultured, and presented
in Figure 5B is the northern blot of these RNA samples hybridized with a DHFR probe.
Figure 5. DNA rearrangement in an unstable Phase 1 antibody cell line. A) A clone from a Phase 1 antibody project was continuously
cultured for 107 generations. Plotted are the specific productivity (Qp, picograms/cell/day) designated by black triangles,
and the growth rate (hr-1) designated by red triangles. RNA was isolated at the time points (19, 42, 63 and 100 generations)
indicated on the graph. B) Each lane was loaded with 3 μg of isolated RNA, the gel was transferred to nitrocellulose, and
the nitrocellulose was hybridized with a probe encoding DHFR. The migration of rearranged DHFR is indicated.
As shown, there was little change in the level of expected HC-DHFR bicistronic transcript from 19 to 63 generations. By 100 generations, however, there was a detectable loss of the expected
HC-DHFR transcript concomitant with the appearance of an even smaller, rearranged DHFR transcript than was previously observed (see Figures 3B and 4A). The cloning and sequencing of this smaller transcript demonstrated
that it contained an intact DHFR-coding sequence and no HC sequence. Thus, the cells were able to grow in the presence of methotrexate by continuing to synthesize
DHFR. By 107 generations, however, they were clearly expressing no HC protein and therefore, no intact antibody.