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\begin{document}

\title{Impact of next-generation high productivity perfusion cell culture
process on host cell protein profile and a comparison with fed-batch
cultures.}



\author[1]{Jiyoung Hong}%
\author[2]{Minni Aswath}%
\author[2]{Yuan Gao}%
\author[2]{Daisie Ogawa}%
\author[2]{Vaishali Sharma}%
\author[1]{Min Zhu}%
\affil[1]{Boehringer Ingelheim Fremont Inc}%
\affil[2]{Boehringer Ingelheim Corp USA}%


\vspace{-1em}



  
  \date{May 05, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Fed-batch culture currently represents the typical choice for the
production of monoclonal antibodies (mAbs) in the biopharmaceutical
industry. However, the implementation of perfusion culture process
combined with continuous manufacturing has gained attention due to
increased productivity and resource savings. In this paper, we compared
the host cell protein (HCP) production and profile of mAb1 between
fed-batch and perfusion culture processes. Our work demonstrated
differences in HCP production based on the type of cell culture process
for the first time. We focused on HCPs that get carried through the
purification process and are present in the final drug substance at
levels impacting antibody quality and stability. Perfusion process had
lower HCP levels and enabled higher clearance of problematic HCPs
compared to fed-batch suggesting a viable alternative process.
Furthermore, our work demonstrates proof of concept of the impact of
cell culture process on specific product quality and help to navigate
the process design when we move from traditional fed-batch to
next-generation perfusion cell culture.%
\end{abstract}%



\sloppy


\textbf{Impact of Next-generation High Productivity Perfusion Cell
Culture Process on Host Cell Protein Profile and a Comparison with
Fed-batch Cultures}

Jiyoung A. Hong, Minni Aswath\textsuperscript{1}, Yuan Gao, Daisie
Ogawa, Vaishali Sharma and Min Zhu

Protein Science, Process Science, Boehringer Ingelheim, Fremont, CA
04555

Correspondence\textsuperscript{1}:

Minni Aswath, Boehringer Ingelheim, Protein Science, Process Science,
Fremont, CA 94555.

Email: minni.aswath@boehringer-ingelheim.com

\textbf{Abstract}

Fed-batch cell culture processes is widely used in biopharmaceutical
industry for monoclonal antibodies (mAbs) manufacturing. As cell culture
platforms continue to evolve, perfusion process stand out an attractive
alternative to fed-batch process due to the high productivity,
manufacturing flexibility and low cost. In this study, the host cell
proteins (HCPs) that accumulated extracellularly in fed-batch and
perfusion process of the antibody (mAb1) were identified and quantified
using liquid chromatography-tandem mass spectrometry (LC-MS/MS),
followed by functional analysis. Due to the high viability and low cell
concentration, less HCPs were detected in steady state (SS) perfusion
process than fed-batch culture and non-steady state (NSS) perfusion. In
perfusion process of mAb1, altered distribution of charged variant
species may provide an opportunity to remove problematic HCPs, including
lipoprotein lipase (LPL). The HCP profile studies could provide guidance
on selecting the appropriate process to maintain good mAb quality for a
therapeutic antibody.

Keyword: perfusion, host cell protein, profiling, downstream process
development, polysorbate degradation



\textbf{Introduction}

Chinese hamster ovary (CHO) cells are the most common host organisms for
production of recombinant therapeutic proteins, including monoclonal
antibodies (mAbs). Fed-batch (FB) cell culture processes using CHO cells
have been established as a platform for most mAbs in the
biopharmaceutical industry (Zhu, 2012). As cell culture platforms
continue to evolve, perfusion process stands out as an attractive next
generation alternative to fed-batch process. High demand, competitive
landscape and fast-paced biopharmaceutical markets require higher
productivity, manufacturing flexibility and reduction in development
time (Rodrigues et al., 2010). In order to accept the current market
need, continuous downstream manufacturing combined with perfusion
upstream process offers great advantage on the intensification of the
volumetric productivity with limited investment. In the perfusion
process, media is fed on a regular basis and the product containing
media is harvested continuously. The continuous product removal and
short residence time reduce the variability in product modification and
lead higher product heterogeneity. Short residence time adds another
advantage for unstable molecules such as enzymes or bispecific
antibodies (Karst et al., 2018).

Perfusion cell culture have diverged to two mode of operations, steady
state (SS) and non-steady state (NSS) (Bielser et al., 2018). SS
perfusion mode maintains constant cell density over a lengthier culture
duration. The harvest stream is used to remove excess cells to keep a
steady concentration. NSS perfusion mode focuses on a higher intensified
cell density, which varies during the culture duration. Therefore,
perfusion rate must be adjusted dynamically to meet the cellular demand
and allow cells to grow at the maximum rate (Karst et al., 2017).
Changing the upstream processes can impact the host cell protein (HCP)
profile and the relative abundance of particular proteins in harvest
cell culture fluid (HCCF), as well throughout the downstream
purification (Hogwood et al., 2013; Park et al., 2017). Despite great
research interest in HCPs, there are no published investigations to
compare perfusion and fed-batch processes for differences in HCP
production and profiles, as well as the two modes of perfusion cell
culture process.

In this study, we collected HCCF from various bioreactor cultures (FB,
SS perfusion, NSS perfusion) at different growth phases of a
mAb1-producing CHO DG44 cell line. A complete analysis of HCPs will
establish the early proof of concept to understand the differences in
HCP production based on differences in cell culture processes. We
utilized liquid chromatography-mass spectrometry (LC-MS) based
proteomics throughout the study, which have recently shown great promise
for HCP characterization (Bracewell et al., 2015; Walker et al., 2017).
Discovery-based proteomics by data-dependent acquisition (DDA) is a
powerful technique for unbiased identification of proteins in a sample.
However, this method is not ideal for the consistent detection of low
abundance proteins. HCPs co-exist with highly concentrated mAb product
(dynamic range of concentrations [?] 5 orders) in the HCCF and
purification intermediates, which puts a great challenge for detection.
Here, to meet our needs, we developed a parallel reaction monitoring
(PRM) approach for the target proteins of interest. PRM uses targeted
tandem MS to simultaneously monitor product ions of a targeted peptide
with high mass resolution and accuracy (Kreimer et al., 2017).

In our HCP profiling studies, we focused on the comparison of one
particular group of HCPs that are known to affect product stability.
Particle formation in the polysorbate (PS)-formulated biotherapeutics
that has become a major quality concern and potential risk factor in the
industry. Recent published data and our own investigation have provided
clues for the leading cause of PS degradation by HCPs remaining in drug
product. Low levels of residual HCPs that have lipase activity, such as
lipoprotein lipase (LPL) (Chiu et al., 2017), hydrolyze polysorbates
cleaving them into fatty acids. The accumulation of free fatty acids
ultimately precipitates to form particles upon long or in some cases
short-term storage.

Particle formation in drug substances was observed in mAb1 and this
molecule was used in this study. Source materials were generated by
three different modes of cell culture processes, subsequently the
enzymes that potentially can hydrolyze polysorbates were identified,
quantified and compared. The aim of this body of work was to establish
proof of concept and methodology to compare FB and perfusion cell
culture processes. Perfusion process is shown to be a viable alternative
to utilize on the problematic molecules, such as mAb1 that have product
quality issues when cultured in traditional FB mode.

\textbf{Materials and Method}

\textbf{Cell line and cell culture}

A suspension-adapted recombinant CHO-DG44 cell line expressing mAb1 in
the dihydrofolate (dhfr) selection system was cultivated in FB, SS
perfusion and NSS perfusion in Boehringer Ingelheim's proprietary
in-house media. Fed-batch material was derived from a 12-kL stainless
steel bioreactor inoculated at a seeding density of 3.0 x
10\textsuperscript{5} cells/ml. Dissolved oxygen (DO) was maintained at
60\% and the temperature and agitation were 36.8\textsuperscript{o}C and
29 rpm, respectively. Nutrient feed media was continuously fed at 10\%
cell culture volume starting from day 3 and repeated every 48 hours with
a targeted final glucose concentration at 4 g/L. Culture supernatant
collected from the bioreactor was centrifuged at 5000 rpm for 55 L/min
and stored at -80\textsuperscript{o}C pending further analysis.

Perfusion cultures (SS, NSS) were established with a 2 L working volume
and subsequently seeded at 12.4 x 10\textsuperscript{6} cells/ml and
12.1 x 10\textsuperscript{6} cells/ml, respectively. In-house perfusion
growth medium (1x concentration) started perfusing immediately following
inoculation on day 0 at the rate of 1 vessel volume per day (1 vvd) and
increased gradually to 2 vvd on day 2. The SS reactor maintained a
constant 2 vvd perfusion rate from day 3-14 using a combination of three
concentrated feeds and diluent, the respective rates thereof adjusted
accordingly to maintain a residual culture osmolality of 310 \selectlanguage{ngerman}± 15 mOsm.
A culture bleed controlled by an Incyte permittivity probe (Hamilton,
Reno, NV) was used to maintain the viable cell density (VCD) target of
40 x 10\textsuperscript{6} cells/ml. The NSS reactor did not utilize a
cell bleed; cell culture proliferated to the maximum VCD attainable by
the system (feed, DO control, etc.). The addition rate of the three
concentrated feeds remained constant at a total of 0.5 vvd; the addition
rate of the diluent varied accordingly to maintain a residual culture
osmolality target of 330 ± 50 mOsm. Permeate was harvest by 0.2 \selectlanguage{greek}µ\selectlanguage{english}M
hollow fiber filtration throughout culture.

\textbf{Viable cell concentration and mAb assay}

Total cell counts and cell viability were measured daily by Trypan Blue
exclusion using a Vi-CELL cell counter (Beckman Coulter Life Sciences,
Indianapolis, IN). Daily permeate samples were collected for titer and
metabolite analysis on the Cedex BioHT (Roche Diagnostics GmbH,
Mannheim, Germany).

\textbf{Purification of mAb1}

Laboratory scale chromatographic separations were performed using an
\selectlanguage{ngerman}ÄKTA\selectlanguage{english} explorer chromatographic system (GE Healthcare, Uppsala, Sweden).
mAb1 is an IgG1 antibody with \emph{isoelectric point (} pI) value of
8.5. The antibody was captured from the HCCF by recombinant ProA
chromatography using MabSelect Sure (GE Healthcare, Uppsala, Sweden).

\textbf{Host cell protein quantification by ELISA}

HCPs were quantitatively determined by Gyrolab xP Workstation using
Gyrolab CHO-HCP E3G kit (Gyros Protein Technologies, Uppsala, Sweden).
The Gyrolab xP workstation is an automated analytical platform based on
microfluidic technology (Heo et al., 2014; Mora et al., 2010)\textbf{.}

\textbf{HCP profile analysis using mass spectrometry}

Protein (1.5 mg) from HCCF and ProA pool was diluted in 50 mM ammonium
bicarbonate (pH 8.0) to 1 mg/ml final concentration. Trypsin was added
into the solution at the ratio by weight of 50:1 (substrate to trypsin),
and incubated overnight at 37\textsuperscript{o}C. After adding 7.5 mM
dithiothreitol, the digested samples were boiled at
90\textsuperscript{o}C for 10 min. They were then cooled down rapidly
and precipitates were removed by centrifugation at 16000 x g for 5 min.
The remaining supernatants were applied to Pierce\selectlanguage{english} Peptide desalting
spin columns (Thermo Scientific, Rockford, USA).

Digested peptides were separated using a ACQUITY UPLC CSH C18 column
(130 A 1.7 \selectlanguage{greek}μ\selectlanguage{english}m,~2.1 x 150 mm) on an ACQUITY Arc high-pressure liquid
chromatography (HPLC) (Waters, Milford, USA). The column was held at
60\selectlanguage{ngerman}°C. A 200-min gradient from 98/2 to 55/45 water/acetonitrile (0.1\%
formic acid added) was used with a flow rate of 0.3 mL/min.~ The column
eluate was analyzed on a Thermo Q Exactive Plus mass spectrometer
(Thermo Scientific, Sunnyvale, CA) using DDA mode where the top 20 most
abundant peptide ions were subjected to MS/MS analysis (dynamic
exclusion 60 second, resolution 17500) after a survey scan (m/z range:
300-2000, resolution: 70000). Data analysis was performed by Proteome
Discoverer 2.2 (Thermo Scientific, Sunnyvale, CA) using SEQUEST
algorithm against CHO K1 proteome database (GCF\_000223135.1 CriGri\_1.0
from www.ncbi.nlm.nih.gov) by adding decoys and common contaminants. The
XCorr confidence thresholds were set as 1.2, 1.9, 2.3, and 2.6 for
charge state z = 1, 2, 3 and z \selectlanguage{english}[?] 4 individually. The delta
C\textsubscript{n} was set as 0.05, and the target false discovery rate
(FDR) was set as 0.01. The searched results were further filtered by
criteria such as unique pep \# \selectlanguage{english}[?] 2 and FDR Confidence=High. The semi
quantitative label-free quantification was performed using MS peptide
signals where the Minora algorithm performed untargeted feature
detection.

\textbf{Three lipases targeted using MS}

Three lipases were selected as HCP targets for PRM analysis. They are
lipoprotein lipase (LPL), putative phospholipase B-like 2 (PLBL-2), and
lysosomal phospholipase A2 isomer X1 (LPLA2). For each lipase, at least
three unique tryptic peptide sequences were selected for analysis. The
PRM LC-MS instrument setting was similar to the DDA analysis, but the LC
gradient was shortened to 80 min. The MS2 signal responses were analyzed
by Skyline 4.1 software (MacCoss Lab, University of Washington) where
four fragment ions were extracted for each peptide. The PRM
quantification was based on the signal intensities comparison to the
standard lipase proteins.

\textbf{GO and pathway analysis}

Gene enrichment and functional annotation analyses were performed using
the DAVID gene functional classification tool
(http://david.abcc.ncifcrf.gov.home.jsp) to elucidate the biological
functions of identified proteins (Huang da et al., 2009a, 2009b).

\textbf{Fluorescence micelle assay (FMA)}

FMA was used to determine the concentration of PS in the samples. The
FMA reagent contains 0.15~M sodium chloride, 0.05~M Tris, 5\% ACN,
5~\selectlanguage{greek}μ\selectlanguage{english}M~\emph{N} -phenyl-1-naphtylamine and 15~ppm (parts per million)
Brij35 with a pH 8.0. 10 \selectlanguage{greek}µ\selectlanguage{english}l of sample was mixed with 240 \selectlanguage{greek}µ\selectlanguage{english}l of FMA
reagent in a 96 well plate (Greiner Bio-One, Monroe, USA) and shaken for
60 sec at 35\textsuperscript{o}C. The fluorescence detector, Tecan Spark
(Tecan, Mannedorf, Switzerland) was set to an excitation wavelength of
350 nm and an emission wavelength of 420 nm. For quantitation, a
calibration curve ranging from 0.1mg/ml to 0.6 mg/ml PS was used as
standard.

\textbf{Lipase activity assay}

Lipase activity assay buffer was prepared with sodium phosphate, sodium
chloride, 3-{[}(3-cholamidopropyl) dimethylammonio{]}-1-propanesulfonate
hydrate (CHAPS) and 4-methylumbelliferyl decanoate (MUD4) at pH 7.4.
After the mixing, the plate was analyzed in the fluorescence detector,
Tecan Spark. The maximum excitation/emission wavelengths were determined
to be 340 nm and 450 nm. The kinetic measurement was reported over 1.5
hours with 45 seconds interval. The slope of fluorescence over time is
directly proportional to the hydrolytic activity of the sample used
within the linear measuring range. The hydrolytic activity allows
determination of lipase content in the sample.

\textbf{Results and Discussion}

\textbf{Cell culture performance}

The current industry standards for the production of stable mAbs is a
fed-batch process in stirred tank bioreactors. Perfusion methods offer
not only process advantages but also possibly low HCPs released into the
condition media due to high viability and less apoptosis in the culture.
Three culture processes, FB, SS perfusion and NSS perfusion, have been
compared from same host cell line, producing the same recombinant
product, mAb1. FB culture was harvested 11 days after the production
bioreactor was inoculated and perfusion supernatants were sampled daily.

Figure 1 shows the profiles of cell growth, viability and mAb1
production in culture supernatants during FB, SS and NSS perfusion
cultures. In FB cultures, a maximum viable cell concentration (MVCC) of
15.6 X 10\textsuperscript{6} cells/ml was achieved on day 7 (Fig1a). In
NSS perfusion, the trend for MVCC was very similar to fed-batch but
reached around 221.7 X 10\textsuperscript{6} cells/ml on day 7, which
was 14 times more cell density than FB culture. In SS perfusion, the
viable cell density was maintained at 40 X 10\textsuperscript{6}cells/ml
throughout the culture days with \textasciitilde{}95\% viability
(Fig1b). Fed-batch viability was reduced to \textasciitilde{}25\% and
NSS perfusion viability was also reduced to \textasciitilde{}37\% on day
11. Perfusion cultures were intended to have high productivity therefore
average productivity is 5.7 times greater in SS and 18.5 times greater
in NSS compared from FB (Fig1c).

\textbf{Identification and quantification of HCPs by LC-MS}

Total HCPs measurements of HCCF from different modes of cell culture
processes, FB, SS and NSS are listed in Table 1. Samples from both
unpurified (i.e. HCCF) and Protein A purified materials were analyzed.
For the perfusion systems, HCP counts across the harvest duration
provides insights to the HCP profile changes from early to late growth
stage.

During the early stages of cell culture growth, there is a significant
lag between central metabolism and post-translational modification. A
strategy was developed to exclude harvest product from the early days
(1-5 days) of production to ensure stable product quality. Hence, the
mAb1 quality and composition of HCPs were less varied depending on
culture days but maintained significant changes with the different
culture processes. The level of HCPs in HCCF were greater than
\textasciitilde{}10\textsuperscript{5} ppm for FB and NSS perfusion but
approximately 10\textsuperscript{4} ppm for SS perfusion. Low cell
viability for both FB and NSS perfusion culture processes can mean high
levels of HCPs released in the feed stream due to cell lysis.

General patterns of HCP distribution was visualized in 2D-PAGE shown
Figure 2. The loading of the gel is normalized by the amount of product.
Different culture processes show a comparable spot pattern and intensity
with mAb1 spots. For HCP comparison, SS shows less overall HCP
population. Day 5 perfusion product also shows less HCPs detected
compared to perfusion day 14. Interestingly, NSS perfusion demonstrates
additional HCPs in the HMW area (across pI range) while the FB shows
additional HCPs across the MW and PI ranges. There are several HCP
species (spots) and abundance (spot volume) varied among the culture
processes. These minor variations in HCP composition and abundance
between different processes suggest influence of cultivation process on
HCP in cell culture supernatants.

ELISA and 2D-PAGE have limited capacity to detect HCPs that are in low
abundance or poorly immunogenic. Orthogonal detection and monitoring
technique using MS offers the opportunity not only to arrive at an
accurate quantification of total HCPs but also provides the ability to
identify the individual protein species with high sensitivity (Levy et
al., 2014). Samples were analyzed by high-resolution LC-MS and HCPs were
identified by SEQUEST search against the CHO K1 proteome database (GCF
000223135.1 CriGri 1.0 from www.ncbi.nlm.nih.gov) through Thermo
Scientific Proteome Discoverer v2.2. Table 1 summarizes the group number
of HCPs in HCCF in the cultures with at least 2 identified unique
peptides. A total of 1310 HCPs were identified in FB culture
supernatants. Perfusion SS contained fewer detected HCPs
(\textasciitilde{}300) and NSS perfusion shows comparable numbers of
HCPs (\textasciitilde{}1700) as FB that were likely due to the high cell
concentration. Approximately 83\% of fed -batch, 70\% NSS perfusion,
92\% SS perfusion were commonly identified with other culture processes
using. HCP composition was significantly more diverse in the FB and NSS
perfusion culture than in the SS perfusion culture. These data confirm
similar trends in HCP population using ELISA.

The HCCF from the three types of cultures were later purified by ProA
chromatography and analyzed for HCP population. As indicated in Table 1,
results from both MS and ELISA method show drastic decrease on the HCP
level for both perfusions, but HCP levels remain relatively high for FB.
This result indicates that it might be easy to remove host cell protein
from perfusion process by ProA column. To investigate any significant
differences in product quality from FB, SS and NSS and their potential
impacts on methodologies used in the study, product quality indicators
such as aggregation, fragmentation, N-linked glycosylation patterns and
charge variants were evaluated after ProA purification (Supplementary
Table 1). Both aggregation and fragmentation were slightly reduced in
perfusion cultures compared to fed-batch. N-glycosylation showed similar
pattern for both types of culture. Perfusion NSS has been observed a
simpler glycosylation (G0F) increased as the runs progress and cell
densities go up due to the decrease in nutrient availability in all
culture processes. At the same time, high mannose (Man5) of NSS
perfusion increased and a similar correlation has also been reported in
other publications, which indicate that this could be a general
phenomenon (Pacis et al., 2011).

The charged variants analysis of mAb1 shows that FB has 24.4\% acidic
peaks, and 8.4\% basic peaks (Figure 3). With perfusion processes, the
abundance acidic species is significantly reduced to 13.6\% for SS and
15.8\% for NSS while more basic forms are produced to 14.6\% for SS and
13.4\% for NSS compared to FB. Other mAbs also exhibited similar trends
in charged variants with perfusion processes due to the increase
C-terminal lysine and/or less light chain N-terminal glutamine
cyclization (Gomez et al., 2020; Walther et al., 2019). The general
consensus according to a number of scientific publications and various
conventions is that C-terminal lysine and N-terminal glutamine
cyclization, are likely non-CQAs (critical quality attributes), which
may not have substantial effects on antigen binding affinity, efficacy
and safety of the antibody product (Du et al., 2012; Sing h et al.,
2016). However, charged variants can alter the composition of HCP
impurity profile if HCPs are binding to mAbs.

The relatively minor charge differences have been shown to have a large
impact on the total HCP association due to the single abundant charge
species that bind substantially more strongly to HCPs (Levy et al.,
2014). For ProA chromatography, post load wash is a critical step to
reduce HCPs.~In the mAb1 purification process, neutral pH (pH 7.4) was
employed for the capture and wash step. Since HCPs tend to have lower
isoelectric points (pI) (Lavoie et al., 2019), the majority of HCPs are
anionic whereas mAb1 (pI 8.5) is cationic at the neutral pH. Thus,
strong hydrophobic and electrostatic interactions can form between mAb1
and HCPs. Based on our proteomic analysis after ProA purification, FB
culture contains HCPs that have theoretical pI below neutral pH, pI
\textless{} 6 [?] 50\%, pI \textless{} 7 [?] 74\%, and pI \textless{} 8 [?]
82\%. This result showed that these low isoelectric points tend to bind
with acidic species of mAb1 in FB culture. Our work brings up the
awareness of potential differences of charged variants between fed-batch
and perfusion and the needs for further characterization of the product,
as well as HCPs. In general, the differences we have observed in all
product qualities tested, \emph{i.e.} aggregates, N-glycan, charged
variants, are not expected to have a noticeable impact on the
methodologies used in this work.

\textbf{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 (\textgreater{}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 O\textsubscript{2} 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
1.1x10\textsuperscript{5} pg/cell/day for NSS and
3.6x10\textsuperscript{3} 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.

\textbf{Problematic HCP}

The leading cause of particle formation following enzymatic degradation
pathway is from a group of host cell proteins, which exhibit polysorbate
degradation activity. Lipoprotein lipase (LPL) hydrolyze ester bonds
within triglycerides to form alcohols and fatty acid molecules. Knockout
mutants of LPL reduce polysorbate degradation by 50\% (Chiu et al.,
2017). In the development of mAb1, we observed elevated sub visible and
visible particles after long term storage at 2-8\textsuperscript{o}C and
also showed high PS20 degradation over 4 weeks storage at ambient
temperature. LPL was detected in the bulk drug substance in parts per
billion (ppb) levels (data not shown). In our proteomic studies, LPL,
was found in all three culture processes (FB, SS, NSS). After the ProA
purification, LPL was only detected in the fed-batch process, suggesting
fed-batch culture process may be harboring higher LPL levels (Table 2).

The DDA method was not adequately sensitive to detect the lipases in
purified samples, such as ProA pool. Instead, the PRM method was used to
further detect low ppm levels of lipases in mAb1 process sample. The
data-acquisition method for PRM contains a list of peptides, which are
exclusively present only in proteins that are of interest to the
researcher, and their elution time window. After the data is acquired
and analyzed, the information on the abundance of peptides observed by
PRM is then used to estimate the abundance level of their corresponding
proteins. PS degradation was also reported during the long-term storage
of CHO-derived purified mAbs with detectable quantities of endogenous
lysosomal phospholipase A2 isomer X1 (LPLA2) (Hall et al., 2016) and
PLBL2 (Dixit et al., 2016). We used PRM method for targeted quantitative
analysis of peptide pairs to identify LPL, LPLA2 and PLBL2 (Table 2).
Compared from the FB process, both perfusion processes shows less
overall lipases and later removed or greatly reduced the quantity after
the ProA purification.

Peroxiredoxin 6 (Prdx6) is the uniquely 1-Cys member of the
peroxiredoxin family with calcium-independent phospholipase A2
activities (Shanshan et al., 2017). Phospholipases such as LPLA2, Prdx6
and phospholipase A-2 activating protein (PLAA) were detected in
fed-batch and NSS perfusion processes but not in SS perfusion process.
LPLA2, Prdx6 and PLAA were also detected in fed-batch intermediate
purification pool after ProA purification but not in the both perfusion
processes.

For long-term storage of mAbs formulated with PS, difficult to remove
lipases that were identified need to be further cleared during the
downstream purification. In order to evaluate the mAb1 product
stability, PS was analyzed using two different methods after 4 weeks of
storage at 25\textsuperscript{o}C. Fluorescence micelle assay (FMA) was
utilized for PS quantification and 4-methylumbelliferyl decanoate
(4-Mud) assay for lipase activity. Table 2 summarized the PS
quantification, lipase activity and its relative abundance in different
processes samples. In HCCF, PS degraded from 400 ppm at day 0 to
approximately 20 ppm after 4 weeks of incubation. This result was
expected due to the high HCP concentration in the cell culture fluid.
After ProA purification, PS content was measured to above 200 ppm for
both perfusion processes but less than 100 ppm for the fed-batch
process. Lipase activity also shows great differences between perfusion
and fed-batch processes after the ProA purification. Combining all the
data collected in this study lead to the fact that fed-batch samples
contained more lipases involved in polysorbate degradation and particle
formation compared to both perfusion processes. These lipases were not
removed by chromatography methods.

\textbf{Conclusion}

In conclusion, clear differences in HCP profile were observed between
steady state and non-steady state cultures, as well perfusion and
fed-batch culture. Fewer HCPs were detected in SS perfusion process.
This is likely due to the high viability and lower cell counts in steady
state. Even though the level of HCPs in the final drug product would be
very low (1-100 ppm), those trace amounts of difficult or problematic
HCPs may effect on the quality attributes of mAb1. To characterize the
HCPs potentially affecting mAb quality, the detailed analysis of HCPs in
the culture supernatant of culture processing of mAb1 producing CHO
cells was performed in this study.

HCPs that were present in HCCF from fed-batch and perfusion processes of
mAb1 were identified and quantified using LC-MS approaches including
lipases that cause mAb1 stability. Different profiles and levels of
lipases were detected in the different cell culture processes. In
perfusion process of mAb1, altered distribution of charged variant
species may provide an opportunity to clear the lipases by downstream
process. Our work demonstrates that future bioprocessing can be shaped
by a deep understanding of the impact of cell culture process on
specific product quality when we move from traditional fed-batch to next
generation high productivity perfusion cell culture. A process specific
ELISA assay is likely needed for perfusion process. Combining advanced
downstream and analytical techniques in addition to tailored approaches
to perfusion cultures can offer greater control over product quality,
stability and ultimately the safety of biotherapeutics.

The authors would like to acknowledge contributions from the following:

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\textbf{Figure 1.} Profiles of (a) viable cell density (b) viability,
and (c) mAb1 productivity during fed-batch and perfusion processes.

\textbf{Figure 2.} 2D-gel image for HCP profile comparisons between
culture processes from mAb1-producing CHO HCCF. (a) fed-batch culture
process on day 11 (b) SS perfusion culture process on day 5 (c) SS
perfusion culture process on day 14 (c) NSS perfusion culture process on
day 5 (d) NSS perfusion culture process on day 14

\textbf{Figure 3.} Profiles of charge variation of mAb1 during fed-batch
(day11), SS perfusion (day12) and NSS perfusion (day12).

\textbf{Figure 4.} Heatmap showing the fold changes in the relative
abundance of HCPs. The y-axis reflects different individual proteins and
the x-axis represents the fold change in relative abundance of perfusion
processes respect from fed-batch process. The color scale illustrates
the relative abundance level of each protein; red and blue indicate high
and low abundance compared to the fed-batch culture process,
respectively. The color intensity indicates the degree of fold increase
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