Early prediction of instability of chinese hamster ovary cell lines expressing recombinant antibodies and antibody‐fusion proteins

Abstract

One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian cell line that maintains stability of production. Problems with process yield, lack of effective use of costly resources, and a possible delay in obtaining regulatory approval of the product may ensue otherwise. Therefore the stability of expression in a number of Chinese hamster ovary (CHO) derived production cell lines that were isolated using the glutamine synthetase (GS) selection system was investigated by defining a culture as unstable if the titer (which is a measure of productivity) of a cell line expressing an antibody or antibody-fusion protein declined by 20-30% or more as it underwent 55 population doublings. Using this criterion, a significant proportion of the GS-selected CHO production cell lines were observed to be unstable. Reduced antibody titers correlated with the gradual appearance of a secondary, less productive population of cells as detected with flow cytometric analysis of intracellular antibody content. Where tested, it was observed that the secondary population arose spontaneously from the parental population following multiple passages, which suggested inherent clonal instability. Moreover, the frequency of unstable clones decreased significantly if the host cell line from which the candidate production cell lines were derived was apoptotic-resistant. This data suggested that unstable cell lines were more prone to apoptosis, which was confirmed by the fact that unstable cell lines had higher levels of Annexin V and caspase 3 activities. This knowledge has been used to develop screening protocols that identify unstable CHO production cell lines at an early stage of the cell line development process, potentially reducing the cost of biotherapeutic development.

Building a Cell Culture Process with Stable Foundations: Searching for Certainty in an Uncertain World

“…Older generation cells had an altered metabolism together with an elevated level of the transcription factor GADD153 mRNA, indicative of increased cellular and specifically ER stress (Bailey et al 2012). Related to these observations, Dorai et al (2012) observed that unstable CHO clones were more prone to apoptosis, indicated by higher levels of the apoptotic markers annexin V and caspase 3.…”

“…This threshold level is thought to be the point at which the downstream biosynthetic reactions utilising recombinant mRNAs are saturated, and therefore only when the transcript copy number declines further will there be any phenotypic consequence. Cell line instability can be observed both in the presence and absence of selective pressure, although it may be both more likely and more precipitous once the selective pressure has been removed (Dorai et al 2012;Kim et al 2011;Osterlehner et al 2011;Yang et al 2010;Chusainow et al 2009). Where a decline in recombinant Mab mRNA has been observed, it can affect both the HC and LC genes in cell lines created using both DHFR and GS expression systems, as well as in low and high productivity clones (Kim et al 2011;Yang et al 2010;Chusainow et al 2009).…”

“…A study by Dorai and colleagues (2012) uncovered a correlation between the cell culture population IAA profiles (measured by flow cytometry) and incipient instability, where unstable cell lines displayed a heterogenous IAA profile with a minority secondary population of cells with much lower IAA even at early passage. With increasing number of population doublings the population of GS-CHO cells with low IAA increased as final culture titre declined (Dorai et al 2012).…”

Proteomic Analysis of Bioreactor Cultures of an Antibody Expressing CHOGS Cell Line that Promotes High Productivity

“…In order to reduce cost and time in biopharmaceutical development, emphasis is on scale-down models and predictive technologies. However, the shake-flask culture, which is the widely used scale-down model of fedbatch bioreactor, does not mimic the environmental conditions of a stirred-tank, fed-batch bioreactor well [16,39]. Consequently, in our laboratory, the final step in the cell line screening process is routinely carried out in bioreactors.…”

“…Additionally, robust and highly productive host cell lines can be deployed by increasing the efficiency of gene expression that regulates proliferation [11] survival and longevity [12][13][14]. Finally, methods that allow early prediction of unstable and stable cell lines by measuring intra-cellular Ab content for example, by flow cytometry [15,16] are highly desirable.…”