Managing Cell Line Instability and Its Impact During Cell Line Development - By considering stability as part of the cell line selection and cell banking paradigm, we can ensure that instability probl

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Managing Cell Line Instability and Its Impact During Cell Line Development
By considering stability as part of the cell line selection and cell banking paradigm, we can ensure that instability problems are not observed during clinical or commercial manufacturing.


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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.

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.
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 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.
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.

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.


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