RESULTS
Impact of culture pH and time on cell performance and mAb
quality attributes
14-day fed-batch 2 L bioreactor cultures of a mAb-producing GS-CHO cell
line were performed at three defined pH set points of 6.7 (low), 6.9
(medium) and 7.1 (high) in biological triplicates. Growth profiles of
cultures at medium and high pH were shown to be comparable achieving
peak viable cell densities (VCD) at 8.68 × 106 – 1.10
× 107 cells/mL (Figure 1A ) and cumulative
viable cells (CVC) at 8.20 × 107 – 9.71 ×
107 cells.days/mL (Figure 1C ). The first 5
days of culture was considered as exponential growth phase with all
cultures achieving highest growth rates at their respective culture pH
conditions. Stationary phase occurred between days 5 – 10 during which
the cultures maintained steady growth and cell viability. Death phase
was assigned to cultures after day 10 when both viable cell density and
viability began to decline.
Overall, cultures controlled at low pH exhibited lower VCD ranging from
5.40 – 6.85 × 106 cells/mL, suggesting that
maintenance of low pH hindered cell growth as reflected in the specific
growth rates during the first 6 days (Figure 1E ). In agreement
with the lower peak VCD, bioreactor cultures at low pH showed a
significantly lower mAb titer compared to cultures controlled at medium
pH and high pH (Figure 1D ; repeated measures two-way ANOVA with
Tukey’s post hoc tests, p <0.05) although specific
productivity (qP) was slightly higher in low pH in later phases of the
cell culture (Figure 1F ). Daily glucose, lactate, glutamine,
glutamate and ammonium concentrations were obtained throughout the
14-day bioreactor cultures (Supplementary Figure 1A-E ) and used
to calculate consumption or production rates across culture growth
phases (Supplementary Figure 1F-J ). Glutamine and glutamate
concentrations (Supplementary Figure 1C and 1D ) and specific
consumption rates (Supplementary Figure 1H and 1I ) were
comparable regardless of culture pH set points. However, glucose
consumption and lactate production rates under high-pH culture set point
trended higher than those of their lower pH set points
(Supplementary Figure 1F and 1G ). Notably, such trend was also
similar to a previous study where increased glucose consumption and
lactate production were observed in mAb-producing GS-CHO cells under
high pH cultures (Jiang et al., 2018). In addition, we noted that the
low-pH cultures accumulated ~3× higher concentrations of
ammonium than their medium- and high-pH counterparts from day 8
(Supplementary Figure 1E ) with the most dramatic differences in
ammonium production rate occurring between days 2 – 6
(Supplementary Figure 1J ). This time period coincided with
slower growth (Figure 1A, 1C ) and lower titer (Figure
1D ) and lower glucose consumption (Supplementary Figure1F ) in the low-pH cultures.
Due to the observed changes in extracellular biochemical profiling, we
investigated whether the variation in mAb quality profiles are affected
across different pH conditions. To do so, cell culture supernatant
samples were obtained from the three pH set points over the 14-day runs
and analyzed for N-glycosylation, charge variants and aggregation levels
(quality attributes). Using hierarchical clustering (seeMethods ), we analyzed how the quality attributes vary with
respect to pH and time and observed the clustering of the quality
attributes into three groups (Figure 2; Supplementary Figure
2 ). Group 1 attributes were elevated in low-pH – high-mannose
structures, G0, G0-GlcNAc, G0F-GlcNAc (“early glycans”) and Total
Basic; group 2 attributes increased with time – agalactosylated
structures G0F, G0F+GlcNAc (“intermediate glycans”), Total Acidic and
Total HMW; and group 3 attributes decreased with time – galactosylated
structures G1’, G1F/G1F’, G2F and sialylated structures A1G1F and A1G2F
(“complex glycans”), Main, Total LMW and Monomer. Similar to earlier
reports (Ivarsson et al., 2014; Jiang et al., 2018; Xie et al., 2016),
we observed N-glycosylation and charge variant differences arising from
cell culture pH. Among the three pH conditions, low pH culture exhibited
the most dramatic effects on group 1 attributes compared to their medium
and high pH counterparts (Figure 2 ). Common observations for
groups 2 and 3 product quality attributes for the three pH conditions
are that the changes are culture time dependent. Although the overall
degree of changes varies, the directionality of change is consistent
across the pH conditions (Figure 2 ).
We noticed that mAb N-glycosylation varied with respect to both culture
pH set points and time. Early glycans (group 1) such as high-mannose
structures and G0 were consistently elevated in low pH across most of
the days in cell culture. On the other hand, intermediate glycans (group
2) tended to increase with time, with greater effect at medium and high
pH than low pH. Complex glycans (group 3) generally decrease with time
suggesting that none of the culture conditions were able to maintain the
same levels of complex N-glycosylation over the entire cell culture.
Previously, G1F+G2F was reported to increase with pH in the range of 6.9
– 7.1 but remained relatively unchanged outside of this range (Jiang et
al., 2018). In this regard, while we found galactosylated structures to
be most dramatically influenced by time, they decreased slightly as pH
set point decreased (16.2%, 15.0%, 10.7% in high-, medium-, low-pH
cultures respectively on day 14). We also observed increasing amounts of
high-mannose (1.17%, 1.26%, 1.79% respectively) and agalactosylated
structures (82.7%, 83.7%, 86.6% respectively). Similar to
N-glycosylation, charge variants also varied with culture pH and time.
Total basic variants (group 1) was higher in low-pH culture and
conversely, total acidic variants (group 2) was higher in high-pH
culture, while both increased with time (particularly in low-pH and
high-pH respectively). The decrease of acidic variants in pH-downshift
culture conditions (pH 6.95 to 6.75) and increase over time regardless
of pH were similarly reported in 14-day fed-batch CHO cell cultures (Xie
et al., 2016). Unlike N-glycosylation and charge variants, product
aggregation levels do not appear to be affected by pH set points,
similar to previous studies that found no significant impact of pH on
protein aggregation in batch and fed-batch CHO cell cultures (Paul et
al., 2018; Xie et al., 2016). It could be observed that HMW (high
molecular weight) species (group 2) increases with culture time while
LMW (low molecular weight) species and monomer levels (group 3) decrease
with time, irrespective of pH set points.
Intracellular multi-omics profiling of key biological
pathways impacted by culture
pH
To understand how culture pH could alter the observed cell culture
performance and product quality, multi-omics profiling including
transcriptomic, proteomic and metabolomic (polar and lipid metabolites)
approaches, was carried out on cell pellet samples obtained from the
inoculum and at specific time-points (days 1, 5, 8, 10, 12, 14)
throughout the bioreactor runs. The raw omics files were preprocessed
and normalized for principal component analysis (PCA) and correlations
with mAb quality profiles (Figure 3 ). PCA of the individual
omics datasets (Figure 3A ) showed that culture time (PC1) was
the largest contributor of variation among the samples, followed by
culture pH (PC2) which had more pronounced effects on the transcriptome
and proteome than the metabolome. The metabolomic data showed a biphasic
effect as a function of culture time (PC1) and this may indicate that
the general cellular metabolic changes are governed by differences
between exponential (up to day 5) and stationary (days 5 - 10) phases.
The differences observed for transcriptomic and proteomic findings
indicating that transcription and translation activities are impacted by
culture pH conditions, are consistent with our hierarchical clustering
analysis (Figure 2 ) of the product quality phenotypes.
In order to further elucidate the specific cellular transcripts and
proteins that are responsible for these observed phenotypes, we first
identified the differentially expressed features (FDR-adjustedp -value < 0.01) in all three omics datasets across the
different pH conditions (Figure 3B ). This analysis uncovered
3,246 transcripts, 212 proteins and 364 metabolites that were
differentially expressed upon pH variations. Enrichment analysis of the
differentially expressed features further revealed 231, 126 and 40
biochemical pathways to be differentially regulated at transcript,
protein and metabolite levels respectively (Figure 3B ). Among
the cellular pathways significantly enriched (p -value <
0.05) in at least two of the omics datasets (Supplementary Table
1 ), pathways directly involved in the process of secretory protein
expression, including generic transcription and translation processes as
well as glycosylation, nucleotide sugar transport and vesicular
trafficking in the endoplasmic reticulum (ER) and Golgi cisternae, are
identified and subsequently found to correlate well with observed
product quality differences in glycosylation and charge variant species.
Additionally, differential expression analysis also indicated
pH-specific effects on cell cycle and apoptosis in agreement with cell
culture profiles (Figure 1 ), with potential involvement of the
PI3K-Akt signaling pathway accounting for significantly lower viability
observed at later stages of culture in high pH condition (seeSupplementary Results for details). Culture pH also has a
differential effect on the metabolism of various classes of biomolecules
and the cellular response to culture stress, such as hypoxia, heat
stress, cellular senescence and reactive oxygen species
(Supplementary Table 1 ).
To gain further insights into specific biological pathways associated
with variation in mAb quality attributes, we ranked the omics features
by their average Pearson’s correlation to each group of quality
attributes with similar trends (Figure 2 ), and carried out gene
set enrichment analyses using GSEA Preranked (Subramanian et al., 2005).
Various biological pathways, many of which were already identified in
the enrichment analysis of differentially expressed features, correlated
with these three groups of quality attributes (Figure 3B ;Supplementary Tables 2 - 4 ). For example, ER-to-Golgi
anterograde transport, intra-Golgi transport and Golgi-to-ER retrograde
transport protein expression are positively correlated with early
glycans (group 1), indicating that proteins involved in vesicle-mediated
transport between ER and Golgi tend to be more highly expressed in
low-pH conditions. In addition, proteins present in the pathways “IRE1α
activates chaperones” and “detoxification of reactive oxygen species”
are positively correlated with intermediate glycans (group 2) which tend
to increase with time, indicating that there is an upregulation of ER
stress and oxidative stress response genes as the culture progresses.
Culture time regulates ER and oxidative stress
responses
Biosynthesis of all secretory proteins occurs through the intracellular
secretory pathway that begins with co-translational translocation from
the cytosol into the endoplasmic reticulum (ER) (Figure 4 ).
N-linked glycosylation of secretory proteins is initiated in the ER
where the glycan is added onto proteins at the consensus sequence
Asn-X-Ser. The ER luminal pH (~pH 7.2) is slightly more
acidic than the cytosolic pH (~pH 7.4), thus ensuring
oligosaccharyltransferase efficiency in transferring precursor glycans
to the co-translational translocated protein acceptors. In addition to
being the site of glycosylation initiation, the ER is also the quality
check center that monitors cellular stresses and triggers proper stress
responses. As the glycoproteins move through the Golgi cisternae and
secretory vesicles as they exit into the extracellular milieu, the
luminal pH of different organelles in the secretory pathway maintain a
gradient of increasing acidity (Figure 4 ) which is essential
for proper post-translational processing and trafficking of secretory
proteins (Paroutis et al., 2004).
Since each organelle has an optimal pH, we speculate that pH culture set
point may alter the efficiency of each organelle and in turn affect mAb
product quality profiles. GSEA Preranked analysis using the Pearson’s
correlation values of omics features with product quality attributes,
revealed that intermediate glycans (group 2 attributes) were correlated
with gene expression related to unfolded protein response and ER stress
response (“IRE1α activates chaperones”, proteomics p< 0.001, transcriptomics p = 0.0026)(Supplementary Figure 3A; Supplementary Tables 2C and 3C ) and
oxidative stress response (“detoxification of reactive oxygen
species”, proteomics p = 0.022) (Supplementary Figure
3B ). These pathways included known markers of ER stress (e.g. HSPA5,
HYOU1) and oxidative stress (e.g. SOD2, CAT) which showed increased
expression as the culture progressed, indicating that CHO cells
experienced increasing levels of ER and oxidative stress independently
of culture pH. In addition, as levels of intermediate glycans are
positively correlated with expression of proteins involved in fatty acid
metabolism (Supplementary Figure 4A; Supplementary
Table 3C; proteomics p = 0.061), we further inspected a key
component of fatty acid metabolism - fatty acid β-oxidation, a catabolic
process that is associated with formation of reactive oxygen species
such as superoxide and hydrogen peroxide (Quijano et al., 2016). We
found that the expression of several genes (e.g. HADH, SLC25A20 at
protein level; CD36, CRAT, MLYCD at transcript level)
(Supplementary Figure 4B ) and abundance of short-chain
acylcarnitines statistically increased as the culture progressed
(Supplementary Figure 4C; FDR-adjusted p <
0.01). Overall, during exponential growth phase, stress responses were
low, but were elevated as the cultures entered stationary phase and into
death phase, and these correlated with increase in intermediate glycans
(concomitantly a decrease in complex glycans) towards the end of
culture.
Total high molecular weight (HMW) species increased with culture time,
similarly to intermediate glycans (Figure 2 ;Supplementary Figure 2 ). The increase of HMW species at later
stages of culture corresponded strongly with unfolded protein response
(UPR), ER stress and oxidative stress, all of which suggest an
accumulation of unfolded proteins in the ER and disruption to ER
homeostasis. Aggregation of mAb can be induced by partial protein
unfolding, leading to structural changes that expose hydrophobic
stretches which can constitute aggregation nuclei (Li et al., 2016), and
the protein unfolding and aggregation can be mediated by protein
adsorption to bulk interfaces, chemical degradation (e.g. deamidation,
oxidation) or fragmentation of the mAb (Roberts, 2014). Protein
disulfide isomerases (PDIs) act as chaperone proteins to induce the
refolding of misfolded or unfolded proteins, and the protein expression
profiles of four PDIs – P4HB, PDIA3, PDIA4 and PDIA6 – corroborated
the presence of unfolded proteins in our cell cultures; they were found
to be highly correlated with HMW levels and significantly upregulated
over culture time (FDR-adjusted p < 0.01;Supplementary Figure 5 ). Aggregation at late stage culture is
also previously observed to arise from the lack of N-glycosylation (as a
result of deglycosylation or aglycosylation), which promoted the
formation of mAb dimers likewise through exposure of hydrophobic
residues upon absence of glycosylation (Onitsuka et al., 2014). The
dimer subsequently acts as an aggregation nucleus and promotes
polymerization of the antibody into large aggregates. The cause of
absence of glycosylation is not known but given that ER is the site of
glycan precursor synthesis and attachment, disruption of ER homeostasis
may potentially contribute to protein aglycosylation.
Culture pH- and time affect protein trafficking and
glycosylation
When we further examined correlation analyses of mAb quality profiles,
we found that protein and transcript expression of ER-to-Golgi
anterograde transport genes (proteomics GSEA Preranked p=0.0037,
transcriptomics p<0.001), intra-Golgi and Golgi-to-ER
retrograde transport genes (proteomics and transcriptomics GSEA
Preranked p<0.001) were influenced by culture pH set point
(Supplementary Figure 6; Supplementary Tables 2A and 3A ).
Particularly, evaluation of the genes involved in protein trafficking in
the secretory pathway revealed higher expression of certain ER and Golgi
vesicular transport proteins in low-pH cultures, with a similar
distribution to that of early glycans in these cultures
(Supplementary Figure 6 ). These proteins include RAB1A and
RAB1B, Rab GTPases which are localized to ER-Golgi intermediate
compartment and Golgi, and are required for targeting and fusion of
ER-derived vesicles to Golgi; YKT6 which is a component of v-SNARE
(vesicular soluble NSF attachment protein receptor) complex that
mediates vesicle docking to cis-Golgi; COPI coatomer complex subunits
COPA and COPB1 which are involved in intra-Golgi transport as well as
retrograde vesicle transport from the Golgi to the ER; and finally,
ADP-ribosylation factor (Arf) ARF5 and guanine nucleotide exchange
factor GBF1 which activates Arfs such as ARF5, in turn mediating COPI
coatomer recruitment and vesicle formation at the ER-Golgi interface.
Moreover, increased levels of lyso-phosphatidylcholine (LysoPC) suggest
vesicle fusion with ER and Golgi membranes occurred more frequently in
low-pH cultures (McIntyre and Sleight, 1994), while the trend of higher
qP in low-pH cultures observed throughout culture duration
(Figure 1F ) further supports increased protein trafficking
through these organelles, diminished residence time within these two
compartments and reduced glycan processing in low-pH cultures. This also
helps to account for the higher proportion of complex glycans observed
in high-pH cultures, in which products have a relatively longer
residence time within the ER and Golgi. As ER and Golgi pH homeostasis
are important for the functions of these organelles such as protein
glycosylation, membrane trafficking and protein sorting (Kellokumpu,
2019; Paroutis et al., 2004), we searched for pH-specific differences in
the expression levels of vacuolar-type H+-ATPases (v-ATPases), which
pump protons into intracellular organelles to reduce the luminal pH. We
found that the expression levels of v-ATPases tend to be higher in
low-pH cultures than medium and high-pH (Supplementary Figure
7 ), suggesting that there is a reduction of luminal pH in the ER and
Golgi in low-pH cultures.
In order to better understand the pH-specific N-glycosylation
variations, differentially expressed features of the glycosylation
pathway from all three omics datasets were further examined
(Figure 4 ). The distribution of mAb N-glycan variants with
corresponding differential expression of the N-glycosylation pathway
omics features revealed possible perturbation to the pathway in a
pH-dependent manner with a higher proportion of glycans derived from the
more basic cis- to medial-Golgi compartments in the low-pH cultures, and
from the more acidic trans-Golgi compartment in the high-pH cultures
(Figures 2 and 4 ). Higher expression of omics features
associated with GDP-mannose synthesis (MPI, GMPPB, PPM2), lipid-linked
oligosaccharide (LLO) precursor synthesis (UDP-GlcNAc transferase ALG13)
and mannose trimming (mannosidases MAN1A1, MAN2A1, MAN2A2) was
associated with higher accumulation of high-mannose glycans in low-pH
cultures. ALG13 not only catalyzes the beginning of the precursor glycan
assembly but is also a possible regulator for flux control of the LLO
pathway (Averbeck et al., 2008). Higher expression of UDP-GlcNAc
synthesis enzyme (UAP1) and transporters (SLC35A3, SLC35A4, SLC35D2),
and GlcNAc transferases (MGAT5) were also observed with increased
G0-GlcNAc, G0F-GlcNAc and G0 in the low-pH cultures. While percentage
composition of complex glycans with terminal galactose and sialic acid
added was higher in high-pH cultures, no corresponding pH-dependent
differential omics feature was found. However, the decrease of complex
glycans over time co-occurred with decreasing levels of direct glycan
precursors, UDP-Gal and CMP-Neu5Ac (sialic acid). These observations
implied that there are cellular processes besides glycosyltransferase
levels likely to be involved in the pH-dependent distribution of complex
glycans among the cultures.
Culture pH- and time regulates genes associated with mAb
charge variant
distributions
Charge variants were significantly affected by both pH and time
(Figure 2B ). More basic species were observed in low-pH
cultures and conversely, more acidic species in high-pH cultures. Acidic
variants increased over time regardless of pH, with low-pH showing the
least pronounced increase.
Chemical degradation pathways are known to contribute to the formation
of either acidic or basic species (Hintersteiner et al., 2016; Khawli et
al., 2010). For example, C-terminal α-amidation gives rise to basic
species, while pyroglutamate formation through cyclization of N-terminal
glutamine residues and C-terminal lysine clipping lead to acidic
species. The peptidylglycine α-amidating monooxygenase enzyme PAM
catalyzing the occurrence of C-terminal α-amidation on the mAb heavy
chain (Hu et al., 2017) was found to be more highly expressed at the
transcript level in low-pH cultures particularly on day 14
(Supplementary Figure 8A ). PAM has an acidic optimum pH, and is
active in the lumen of secretory granules (Vishwanatha et al., 2014) and
extracellular region, suggesting that the activity of PAM increases with
decreasing culture pH, in turn increasing basic species. In contrast to
basic variants, acidic variants were most highly represented in high-pH
cultures, and additionally at late stages of culture regardless of pH
(Figure 2B ). Pyroglutamate formation occurs through the
cyclization of N-terminal glutamine and glutamic acid residues, which
respectively lead to loss of an amine group (resulting in acidic
variants) and simultaneous loss of amine and carboxylic acid groups
(demonstrated to result in basic variants) (Liu et al., 2019).
Pyroglutamate formation is catalyzed by the glutaminyl cyclase
iso-enzymes QPCT and QPCTL (Perez-Garmendia and Gevorkian, 2013), and
QPCTL transcript expression was found to increase with pH
(Supplementary Figure 8B ). Pyroglutamate formation can also
occur through nonenzymatic processes by modifying the products in the
culture media. The efficiency of nonenzymatic N-terminal glutamine
cyclization is reported to be higher at pH 7.2 than 6.2, as a
deprotonated N-terminal amino group will favor the nucleophilic reaction
(Dick Jr. et al., 2007; Gazme et al., 2019). This may have contributed
to the increased acidic species in high-pH cultures as well as its
accumulation in late-stage cultures. Another possible cause for acidic
species accumulation over time was the corresponding increased
transcript expression of the carboxypeptidase D enzyme (CPD) that
catalyzes C-terminal lysine clipping (Hu et al., 2016)
(Supplementary Figure 8C ).
When we compared the levels of charge variants with N-glycans, the total
percentage of basic variants trended similarly with early glycans, while
total acidic variants were higher in high-pH than low-pH cultures
similar to complex glycans (Figure 2A ). High-mannose structures
may contribute to charge differences by conferring subtle conformational
differences that affect charge distribution (Du et al., 2012), and might
have partially contributed to the increase in basic variants as they
were demonstrated previously to be enriched in basic fractions of a mAb
produced in CHO cells (Hintersteiner et al., 2016). Sialic acid is known
to contribute to the formation of acidic charge variants (Khawli et al.,
2010) and some increase in sialylated glycans was observed in high-pH
cultures.