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


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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The Causes of Common Types of Cell Line Instability and Their Impact

Figure 2. Cell line instability manifests itself in various forms. Plotted is the specific productivity (Qp, picograms/cell/day) for 24 clonal cell lines for a Phase 1 antibody project during 90 days of continuously culturing the cells. Cell lines showing a stable Qp profile are marked by black symbols, cell lines showing gradual instability are marked by blue symbols, and cell lines showing acute instability are marked by red symbols.
Figure 2 shows the stability profiles for the 24 highest performing clones for a Phase 1 antibody project. These 24 clones were selected from hundreds of clones that were initially subjected to two rounds of high throughput clone screening. The figure illustrates not only the types of instability we observe (acute and gradual), but also shows how the performance of high-performing clones may change in even a very short amount of time. This is clearly seen for the nine clones, designated with red lines and symbols, where a 50% drop in expression occurred within two weeks, and product expression was completely lost within 50 days of continuous culturing. The ramifications of this lack of predictability in clone performance can affect the entire upstream and downstream process development teams during the critical period when they are striving to bring together the clone, the cell culture process, and the purification process to achieve the desired performance and product quality.

Variability in Instability

Table 1. Frequency of acute and gradual instability in Phase 1 antibody projects. This table contains information on the number of clones demonstrating acute and gradual cell line instability in seven different Phase 1 antibody projects. For each project, 24 adapted clones were monitored over a similar length of time.
To further emphasize this point regarding unpredictability, Table 1 shows the frequency of clones exhibiting acute and gradual instability for seven different antibody projects for which the same platform procedures were used. For each project, a total of 24 clones were assessed. The numbers illustrate that the level of instability is quite variable from project to project. This variability in instability emphasizes our inability to consistently predict how our system will perform. We therefore attempted to determine the causes of instability we observe, and to see if this information might enable us to take corrective action to reduce or eliminate instability from our cell line development process.

Figure 3. Rearranged DHFR in an unstable Phase 1 antibody cell line. A) The small red triangles mark the specific productivity (Qp, picograms/cell/day) for a Phase 1 antibody expressing cell line over 150 days in continuous culture. The large black triangles denote the time points (34, 62, 102, 119, and 147 days in culture) at which RNA was prepared for the northern blot shown in Panel B. B) Each lane was loaded with 3 μg of isolated 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.
The results presented in Figure 3 are representative of one type of instability that was observed in several Phase 1 antibody projects. Platform procedures, as outlined in Figure 1, were followed for all activities from transfection through cell line adaptation to cell banking. As shown in Figure 3A, a high producing clone from this project demonstrated a dramatic and eventually complete loss of antibody expression (as marked by the red line and triangles in Figure 3A). RNA was prepared at the time points marked by the black triangles in Figure 3A, and was run on the northern blot shown in Figure 3B, which was hybridized with a probe specific for the selectable marker dihydrofolate reductase (DHFR). At the earlier time points, the expected heavy chain (HC)-DHFR bicistronic transcript (marked by an arrow, labeled "HC-DHFR") was seen. Over time, however, there was a detectable loss of this HC-DHFR bicistronic transcript with the appearance of a smaller transcript (also marked by an arrow, labeled "rearranged-DHFR"). To show that each lane contained equivalent amounts of RNA, the blot was subsequently hybridized with a probe encoding the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and similar signal intensities were observed across all the lanes. Cloning and sequencing the smaller transcript demonstrated that it lacked any HC sequence, but it still contained an intact DHFR gene. These results indicated that DHFR and HC had become uncoupled: although this clone continued to synthesize DHFR and to grow in the presence of methotrexate, it was no longer producing HC protein or intact antibody.

In referring back to the timeline depicted in Figure 1, this clone had already made it through clone screening and small-scale productivity assessments, and its performance was in the process of being evaluated in benchtop bioreactors. It was considered to be a top performer and a favorite for being selected as the production clone for this project. However, because of its instability, this clone was immediately eliminated as a contender, and both the upstream and downstream groups began to focus their efforts on an alternative clone whose performance was adequate, but relatively inferior, compared to the unstable clone before it became unstable. Although deadlines and material needs were successfully met for this project, some additional bench-scale bioreactor work had to be performed at a late stage on the timeline to sufficiently assess the performance of the alternative clone.

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