DISCUSSION
A multi-omics data-driven systems biotechnology approach is presented
here, characterizing the effect of pH on titer and various quality
profiles of mAbs produced in CHO cells. This approach is necessary for
the dissection of underlying molecular mechanisms as the changes in
specific rates of by-product production alone could not explain the
variations in observed quality profiles. Specifically, our work clearly
demonstrated that N-glycan structures are influenced by transcript and
protein expression changes in various pathways including N-glycan
biosynthesis, ER and Golgi vesicular transport, fatty acid β-oxidation,
and responses to unfolded proteins (UPR), ER stress and oxidative
stress. Similarly, temporal variations in gene expression profiles of
UPR and response to ER stress were associated with high molecular weight
(HMW) species as the culture progressed. The pH-dependent changes in
charge variants were also positively correlated with the expression
differences of relevant post-translational modification enzymes.
The relative abundance of early glycans in low-pH cultures may be
explained by the pH specificity of intracellular organelles where the
resting pH of the Golgi ranges from ~pH 6.7 in the
cis-Golgi to ~pH 6.0 in the trans-Golgi (Figure
4 ). The maintenance of ER and Golgi pH homeostasis is important for ER
and Golgi functions such as protein glycosylation, membrane trafficking
and protein sorting (Kellokumpu, 2019; Paroutis et al., 2004). We
postulate that the influx of H+ into the cell in
low-pH cultures decreases the pH of the cytoplasm and the luminal pH of
the ER and Golgi via increased expression of v-ATPases
(Supplementary Figure 7 ) and PAM (Supplementary Figure
8A ), the latter a proposed v-ATPase associated pH sensor within the
secretory pathway (Rao et al., 2019) (Figure 5, left panel ).
The resultant disruption of luminal pH gradient through acidification
may constitute a significant perturbation in the more basic compartments
of the early secretory pathway and is associated with an increased
expression of the corresponding localized N-glycosylation enzymes.
Coupled with higher expression of members of ER and Golgi vesicular
trafficking pathways in low-pH cultures (Supplementary Figure
6 ), the implied increase in general secretory pathway trafficking and
thus a shorter residence time for cargo proteins in ER and Golgi will
result in higher abundance of early mAb glycoforms.
Cellular stress resulting from prolonged high production of mAb may
account for the decreased relative abundance of late glycans, with
culture time having a much more pronounced effect in comparison to
culture pH. As the cell culture progresses, we hypothesize there is a
disruption of ER homeostasis due to ER stress associated with increased
levels of unfolded proteins (Gerlach et al., 2012) as well as oxidative
stress associated with oxidative protein folding in the ER and increased
fatty acid β-oxidation that generate reactive oxygen species (ROS)
(Malhotra and Kaufman, 2007) (Figure 5, right panel ); the
increase in the bioenergetic fatty acid β-oxidation is possibly a
response to higher energy demand for protein productivity by the cells.
ER stress and oxidative stress are also known to exacerbate each other
(Nakka et al., 2016), and their resultant disruption of ER homeostasis
promotes accumulation of unfolded proteins which can lead to aggregation
of mAb into HMW species (Li et al., 2016; Zhou et al., 2018). The
decrease in complex (late) mAb glycoforms, resulting in
hypoglycosylation and reduced glycodiversity, may arise from accumulated
irreversible oxidative damage to glycosylation enzymes that causes
increasing loss of functionality over time (Korovila et al., 2017). Such
damage will naturally have a larger cumulative effect further down in a
sequential chain of enzymatic reactions.
While previous studies have investigated the effects of pH on quality
attributes such as N-glycosylation (Aghamohseni et al., 2014; Ivarsson
et al., 2014; Jiang et al., 2018), aggregation (Paul et al., 2018) and
charge variants (Xie et al., 2016), to the best of our knowledge, no
report has investigated the underlying causes of these pH-specific
effects. In this regard, this study systematically characterizes the
effects of pH on quality attributes at various levels of the cellular
hierarchy, i.e. transcripts, proteins and metabolites. Such integrative
analyses allow us to understand that some cellular processes emerge at
transcriptional level whereas others emerge only at the
post-transcriptional or metabolite levels. The increased expression of
vesicular transport genes in low-pH cultures, and its potential
contribution to higher levels of early glycans, was evidenced by
transport, protein and metabolite datasets. Such concordant observations
across multiple omics datasets strongly suggest the activation or
suppression of certain pathways and its consequences at downstream
layer, thereby allowing us to derive relevant hypotheses at the global
level which may not be possible with only one of the omics datasets.
However, certain observed changes in product quality were found to be
linked to a specific omics dataset – for example, the variations in mAb
aggregation levels were found to be associated with differential
expression of several protein disulfide isomerases only at the protein,
i.e. post-transcriptional, level. We also noted a decrease in
glycodiversity as the cell culture progressed, irrespective of pH with
concomitant increased expression of multiple ER stress and oxidative
stress markers in all three omics datasets (Supplementary
Figures 3, 4 ).
Overall, the integration of multi-omics data allowed us to unravel
mechanisms involving multiple pathways and enzymes that are associated
with variations in product quality, and also highlighted the complex and
dynamic interactions between these quality profiles and pathways. Taken
together, the systematic approach adopted in the current study augments
our understanding of factors affecting mAb quality attributes, and we
believe that the insights gained from this study can contribute towards
the development of targeted approaches that result in more effective
product quality control strategies.