Comparison of HCP distribution and Cluster analysis of
quantified HCP
The relative fold changes of quantified HCPs were calculated by
comparing the protein abundance between the perfusion processes to
fed-batch and visualized through a heat map shown in Figure 4. Each band
represents one HCP species as identified by mass-spec based proteomics.
Blue corresponds to fold decrease and red to fold increase in abundance
comparing FB to perfusion processes. HCPs were further classified in
three clusters according to their concentration profiles during the
cultures. Cluster 1 and cluster 3 represents HCPs that have a large fold
increase and decrease (>2.5 fold) respectively from the
fed-batch cultures. Cluster 2 contains HCPs that show inconsistent fold
changes throughout the culture days. Although there were many HCPs that
did not show large fold changes from FB to perfusion processes, distinct
red and blue represent the HCPs that show a significant increase and
decrease in protein abundance regarding the specific culture processes.
SS perfusion culture shows large portions of HCPs with reduced
abundances. Depending on the manufacturing processes, these prevalent
differences in HCP population suggest to develop “process-specific”
HCP assays. Commercially available ELISA kits, typically used the
reagents derived from FB, can be limited in their utility by poor
sensitivity and coverage.
A cutoff of 2.5 fold was used for the absolute fold changes based on the
%CV of the dataset. HCPs that have more than 2.5 fold changes were
accounted for functional annotation and biological interpretation of
proteomics data. To understand the biological roles of the identified
HCPs, GO analysis was performed based on relative quantification from
fed-batch culture using DAVID (Supplementary figure 1). Most upregulated
HCPs in cluster 1 (Figure 4) were associated with transcription
(spliceosome) and translation (RNA transport, ribosome, lysosome).
Increased level of transcription and translation correlate to increase
in the final titer produced in CHO cells. For example, transcription
factor BTF3, cellular nucleic acid binding protein (CNBP) and 60s
ribosomal protein L36a (RPL36A) were highly upregulated (more than 10
fold) in both perfusion processes. In perfusion culture (cluster 3),
HCPs that associated with metabolic pathway were downregulated with a
response to changes in concentrations of key metabolites in the cell.
NSS perfusion shows downregulation of phagosome and proteasome, which
are responses found in starved and growth arrested cells due to the high
production of mAb (Vergara et al., 2018). A recent study reported that
the primary cellular function of HCPs is an important factor to be
considered when preparing feedstocks for ProA purification (Lintern et
al., 2016). Cells with a high productivity such as perfusion process
expected to produce more mAb per unit of O2 and nutrient
consumed (Pan et al., 2017).
Cellular component analysis shows that compared to the fed-batch,
proteins located in both the cytoplasm and nucleus are greatly impacted
in both perfusion cultures (Supplementary figure 1). For SS perfusion,
the percentage of HCPs associated with the extracellular region,
extracellular space was significantly higher due to the high viability.
However, in the NSS perfusion, HCPs that are associated with cytosol and
nucleoplasm emerged as the most impacted proteins. This result suggests
that HCPs in the culture supernatants in SS perfusion culture process
include a high percentage of extracellular proteins secreted from viable
cells, whereas HCPs in NSS perfusion culture process involve a
significant portion of intracellular proteins resulting from cell lysis.
Those intracellular HCPs included proteins that were not naturally
secreted and could only be found if cells lysed. HCPs that are
associated with membrane proteins are upregulated in the early days but
downregulated in later days of the culture. In earlier days, a previous
study suggested that membrane proteins are secreted by exocytosis after
being sorted in the trans-Golgi network into transport vehicles(Le
Borgne and Hoflack, 1998). In our case, we believe in both perfusion
processes, the active secretion is decreased due to cell lysis as the
number of culture days increase. Furthermore, the concentration of
lactate dehydrogenase (LDH), which is most widely used to quantify the
degree of cell lysis, increased toward the end of the cultures. For the
NSS, LDH level is significantly increased that demonstrates shear
sensitivity on cell. At the end of the culture, LDH level was
1.1x105 pg/cell/day for NSS and
3.6x103 pg/cell/day for SS. Naturally secreted HCPs
include proteins present in extracellular matrix and proteins excreted
through exosomes. Overall, the numbers of secreted proteins were
upregulated in SS perfusion process while intracellular HCPs are the
main contributor to the HCP compositions of NSS perfusion process.