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

ADVERTISEMENT

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.


BioPharm International Supplements


Methylated DNA Immunoprecipitation (MeDIP)


Figure 8. Schematic of the methylated DNA immunoprecipitation (MeDIP) procedure. For a more detailed explanation, refer to the text.
As an alternative, we developed and now use a methylated DNA immunoprecipitation (MeDIP) procedure.4 This procedure is illustrated in Figure 8: DNA is denatured and digested, and is incubated with an antibody that specifically recognizes 5-methylcytidine. The resultant immune complexes are incubated with protein A/G beads, and following washing and centrifugation steps, the captured material is eluted and can be subjected to endpoint PCR, or, if quantitation is desired, to qPCR. The advantages of this procedure compared to a methylation-sensitive Southern blot are that it is high throughput, requires no radioactivity, allows for quantitative results using qPCR, and additionally, through the use of specific PCR primers, can enable a determination of which region(s) of DNA are methylated.


Figure 9. MeDIP assay results of 16-6F and subclone 16-6F A5. Genomic DNA from the 211 generation time point of the unstable cell line 16-6F described by Figure 7 and associated text was subjected to the MeDIP procedure illustrated in Figure 8. Forward and reverse PCR primers were specific for 5' and 3' sequences just upstream and downstream of the promoter region of the transfected vector construct. The lane labeled "+ control" is in vitro methylated plasmid also subjected to the MeDIP procedure. 16-6F was later re-cloned, and a resultant stable subclone, A5, was also subjected to the MeDIP procedure. The arrow marks the migration of the methylated PCR product.
Figure 9 shows the results of a MeDIP experiment on the same 16-6F genomic DNA from the Southern blot experiment shown in Figure 7. Endpoint PCR made use of primers directed to the promoter region of our transfected vector construct, and as expected, the results showed no detectable PCR product from untransfected CHO cells. Also as expected, the lane labeled "+control" showed a very strong signal for in vitro methylated DNA plasmid. The lane labeled "16-6F," which contained the MeDIP PCR product from the genomic DNA sample from the phenotypically unstable 16-6F cell line prepared at 211 generations (recall from Figure 7 that this DNA was not fully digested with the methylation sensitive enzyme AatII), showed a strong signal as well. Although the size of DNA fragments generated during the digestion step of the MeDIP procedure needs to be considered, these latter results suggested that at least a portion of the methylation is in the vicinity of the promoter region of our integrated transfected vector construct. The 16-6F cell line was later re-cloned, and genomic DNA from a resultant high-expressing subclone, 16-6F A5, whose protein expression profile was stable, was subjected to the same MeDIP and PCR assay conditions. Figure 9 shows that in the lane labeled "16-6F A5," there is a much reduced methylation signal. Taken together, these data indicated that recloning resulted in the isolation of a clone having reduced levels of expression vector methylation, and that the markedly reduced methylation in this clone correlated with its increased protein expression and increased stability.

The MeDIP assay allows us to increase our understanding of the methylation status of our integrated expression constructs. By applying it broadly to our Phase 1 antibody projects, the MeDIP assay will enable us to monitor the extent to which epigenetic changes of this nature occur in our cell lines and negatively affect the outcome of our cell line development efforts.


blog comments powered by Disqus

ADVERTISEMENT

ADVERTISEMENT

AbbVie to Acquire Shire for $54.7 Billion
July 18, 2014
AstraZeneca Reveals Design for New Global R&D Center and Corporate Headquarters
July 18, 2014
Particulate Matter Prompts Baxter's Recall of IV Solutions
July 17, 2014
Mylan to Acquire Abbott's Non-US Businesses in $5.3 Billion Stock Deal
July 14, 2014
Shire and AbbVie Discuss Possible Deal
July 14, 2014
Author Guidelines
Source: BioPharm International Supplements,
Click here