Dynamic analysis of GS‐NS0 cells producing a recombinant monoclonal antibody during fed‐batch culture

Abstract

In this study we have analyzed the dynamic covariation of the mammalian cell proteome with respect to functional phenotype during fed-batch culture of NS0 murine myeloma cells producing a recombinant IgG(4) monoclonal antibody. GS-NS0 cells were cultured in duplicate 10 L bioreactors (36.5 degrees C, 15% DOT, pH 7.0) for 335 h and supplemented with a continuous feed stream after 120 h. Cell-specific growth rate declined continuously after 72 h of culture. Cell-specific recombinant monoclonal antibody production rate (qP) varied sixfold through culture. Whilst qP correlated with relative recombinant heavy chain mRNA abundance up to 216 h, qP subsequently declined, independent of recombinant heavy chain or light chain mRNA abundance. GS-NS0 cultures were sampled at 48 h intervals between 24 and 264 h of culture for proteomic analyses. Total protein abundance and nascent polypeptide synthesis was determined by 2D PAGE of unlabeled proteins visualized by SYPRO Ruby and autoradiography of (35)S-labeled polypeptides, respectively. Covariation of nascent polypeptide synthesis and abundance with biomass-specific cell growth, glucose and glutamate consumption, lactate and Mab production rates were then examined using two partial least squares regression models. Most changes in polypeptide synthesis or abundance for proteins previously identified by mass spectrometry were positively correlated with biomass-specific growth rate. We conclude that the substantial transitions in cell physiology and qP that occur during culture utilize a relatively constant complement of the most abundant host cell machines that vary primarily with respect to induced changes in cell growth rate.

Dynamics of unfolded protein response in recombinant CHO cells

“…It increases until day 4 and decreases subsequently. A similar profile for productivity has been reported earlier in a study with GS-NSO cells (Stansfield et al 2007), where productivity of MaB increased up to day 8, and declined thereafter in a fed-batch culture. Other studies have also shown a similar productivity profile (Kaufmann et al 1999;Hendrick et al 2001;Yoon et al 2006a, b).…”

“…Note that this promoter has been reported to possess sites for many different transcription factors (Sambucetti et al 1989;Yurochko et al 1997). It is interesting to note that IgG HC and LC were also under the control of the same promoter in the GS-NSO cells used in the study mentioned above (Stansfield et al 2007). The expression from this promoter seems to be inversely related to growth.…”

Media photo‐degradation in pharmaceutical biotechnology – impact of ambient light on media quality, cell physiology, and IgG production in CHO cultures

“…Productivity data suggested that the overall reduction in cell concentration is the main limiting factor in photo‐damaged medium, as was reported elsewhere 28. However, it was found that additionally the IgG processing machinery seems to be altered in light‐irradiated medium, and the ‘intracellular titer’ is somewhat decoupled from the extracellular concentration.…”

Mammalian cells as biopharmaceutical production hosts in the age of omics

“…Hence, it is very difficult to distinguish between cellular changes resulting from the decrease in growth rate and increase in q. A substantial effect of changes in growth rate on the proteome of GS-NS0 cells during fed-batch cultures was confirmed by Stansfield and coworkers [19]. They observed that both q and growth rate varied during culture, each following a distinct pattern.…”

“…Changes in growth rate have a significant impact on the proteome [19], possibly explaining why structural and growth-related proteins accounted for the largest differences between the cell lines [18].…”