FIGURE LEGENDS
Figure 1. Cell culture profiles of a mAb-expressing CHO cell
line cultured at various pH set points. (A) Viable cell density, (B)
viability, (C) cumulative viable cells, (D) titer, (E) specific growth
rate, and (F) specific productivity (qP) at pH 6.7 (low), 6.9 (medium),
7.1 (high). Error bars denote mean ± SD (n=3).
Figure 2. Groups of correlated product quality attributes
identified through hierarchical clustering. Heatmap of hierarchical
clustering of product quality attributes (rows) across cell culture
samples (columns). The product quality attributes include N-glycoforms,
charge variants (Total Acidic, Main, Total Basic) and aggregation (Total
LMW, Monomer, Total HMW). The samples are grouped by a horizontal bar
with increasingly darker shades of red (low pH), green (medium pH) and
blue (high pH) at increasing time-points.
Figure 3. Global analysis of multi-omics data. (A) PCA of
individual omics datasets. Each data point is a single replicate sampled
on a particular day of a pH set point. A polynomial spline trend line is
added for each pH set point. The numbers in each plot denote culture day
for the sample. (B) Overview of data analysis steps. Overlapping
pathways across significantly enriched Reactome pathways are identified
by individual omics analyses. The numbers of enriched pathways are shown
in the Venn diagrams.
Figure 4. Localization of glycan variant profiles and associated
differential omics features in the N-glycosylation pathway among the
three different pH set-points. The process of N-glycosylation takes
place in the ER and Golgi complex along a luminal pH gradient of
increasing acidity. Accumulation of group 1 “early glycans” at the
more basic cis- to medial-Golgi were observed with increased expression
of features associated with precursor glycan assembly (at the ER),
demannosylation and GlcNAc extension in the low-pH cultures. Culture pH
had minimal effect on distribution of group 2 medial-Golgi
“intermediate glycans” which include the dominant G0F variant. Group 3
“complex glycans” at the more acidic trans-Golgi mainly decreased with
time but with a slightly higher proportion observed in high-pH cultures;
associated omics features reflected the same trend over time but were
not differentially expressed by culture pH. Omics features are
differentially expressed at FDR < 0.01 (except for those
marked with ‘*’) between cultures of different pH.1,2,3Glycan: Group 1, 2 or 3 of mAb quality attributes
in the samples that were clustered according to their distributions over
pH and time. ER: endoplasmic reticulum; TGN: trans-Golgi network; T:
transcriptomics, P: proteomics, M: metabolomics.