\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[greek,ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{Autophagy-inducing Peptide Increases CHO Cell Monoclonal Antibody
Production in Batch and Fed-batch Cultures}



\author[1]{Katrin Braasch}%
\author[2]{Marko Kryworuchko}%
\author[1]{James M. Piret}%
\affil[1]{University of British Columbia}%
\affil[2]{BC Centre for Disease Control}%


\vspace{-1em}



  
  \date{May 05, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
The development of generic biopharmaceuticals is increasing the
pressures for enhanced bioprocess productivity and yields. Autophagy
(``self-eating'') is a cellular process that allows cells to mitigate
stresses such as nutrient deprivation. Reputed autophagy inhibitors have
also been shown to increase autophagic flux under certain conditions,
and enhance recombinant protein productivity in Chinese Hamster Ovary
(CHO) cultures. Since peptides are commonly added to bioprocess culture
media in hydrolysates, we evaluated the impact on productivity of an
autophagy-inducing peptide (AIP), derived from the cellular autophagy
protein Beclin 1. This was analyzed in CHO cell batch and fed-batch
serum-free cultures producing a human IgG1. Interestingly, the addition
of 1 to 4 \selectlanguage{greek}µ\selectlanguage{english}M AIP enhanced productivity in a concentration-dependent
manner. Cell-specific productivity increased up to 1.8-fold in batch
cultures, while in fed-batch cultures a maximum 2-fold increase in
volumetric productivity was observed. An initial drop in cell viability
also occurred before cultures recovered normal growth. Overall, these
findings strongly support the value of investigating the effects of
autophagy pathway modulation, and in particular, the use of this AIP
medium additive to increase CHO cell biotherapeutic protein production
and yields.%
\end{abstract}%



\sloppy




\textbf{Introduction}

Mammalian cell cultures are widely used for the production of protein
biopharmaceuticals because of their capacity for complex and
physiologically relevant post-translational modifications, often
required for therapeutic efficacy (Varki et al., 2009). Chinese Hamster
Ovary (CHO) cells have become the most used cell line for industrial
production of recombinant glycoproteins due to their in-depth
characterization and capacity to produce human-like glycosylation
(Hamaker and Lee, 2018; Hong et al., 2018).

In the past decades, there has been an increasing demand for large-scale
production of biopharmaceuticals, especially antibodies, from mammalian
cell culture processes (Lalonde and Durocher, 2017). More than 90\% of
all antibodies on the market are produced in mammalian cells (Ecker et
al., 2014). Over the years, cell culture engineering has made
significant advances with improved growth media, culture condition
control and cell lines (Butler and Meneses-Acosta, 2012; Kelly et al.,
2018). In media development, formulations based on cellular metabolic
needs (Ghaffari et al., 2020), as well as the addition of small
molecules such as butyrate (Mimura et al., 2001) or butyrated ManNAc
(Yin et al., 2018), have increased recombinant protein production.
Improvements in cell line engineering include the development of
superior mammalian expression systems (Bebbington et al., 1992; Lucas et
al., 1996; Hamaker and Lee, 2018; Amann et al., 2019; Bult\selectlanguage{ngerman}é et al.,
2020) and the inclusion of anti-apoptotic genes to improve cell survival
(Kim and Lee, 2002; Mastrangelo et al., 2000; Tey et al., 2000; Figueroa
et al., 2007) with the goal of enhancing specific and volumetric
productivity.

While apoptosis caused by nutrient limitations is the most well-known
cell death mechanism impacting bioprocesses (Kim et al., 2012),
autophagy (``self-eating'') is being studied in this context as well,
often functioning as a pivotal tipping point between cell survival and
death (Doherty and Baehrecke, 2018). Indeed, through vacuolar
engulfment, degradation, and recycling of large protein complexes to
entire organelles, autophagy is a critical mitigating response to
metabolic stressors including nutrient depravation, thereby maintaining
the cell's energetic needs and homeostasis (Hwang and Lee, 2008; Zustiak
et al., 2008; Mizushima and Klionsky, 2007; Mizushima et al., 2008;
Doherty and Baehrecke, 2018). We previously reported that the addition
of 3-Methyl Adenine (3-MA), a reputed autophagy inhibitor, increased
recombinant protein productivity in CHO cell fed-batch cultures (Jardon
et al., 2012). This phenomenon was further studied by Baek et al.
(2016), testing 3-MA and eight other chemical inhibitors of autophagy,
and their respective effects on cell culture productivity. An increased
specific productivity was confirmed for 3-MA, and also observed in
response to two other reputed autophagy inhibitors including
dorsomorphin (AMP-activated protein kinase inhibitor), and SP600125
(c-Jun NH2-terminal kinase inhibitor). However, Baek et al. (2016)
recognized that these agents did not inhibit autophagy but rather
appeared to induce it, thereby linking 3-MA-enhanced autophagic flux
rather than inhibition with increased bioprocess productivity. This
induction of autophagy in response to 3-MA, particularly under
nutrient-rich culture conditions, is consistent with previous studies by
Wu et al. (2010).

In this study, a novel autophagy-inducing peptide was tested to further
investigate the influence of this cellular process on secreted protein
production. While the addition of chemicals to induce autophagy in cell
culture is possible, their use comes with the risks of confounding
effects (e.g. 3-MA (Klionsky et al., 2016, Wu et al., 2010)). The use of
an alternative, more specific agent that could be more readily
incorporated into an industrial bioprocess and at lower concentrations
is preferable. Indeed, current media formulations often contain peptides
as part of hydrolysates added to the medium to increase growth and
productivity (Spearman et al., 2014). This made the use of a unique and
specific cell-permeable, autophagy-inducing peptide (AIP), derived from
the autophagy protein Beclin 1 (Shoji-Kawata et al., 2013), suitable for
this purpose. Therefore, we evaluated and developed the capacity of this
AIP to enhance therapeutic protein productivity in CHO cells maintained
in batch culture as well as in the more industrially relevant fed-batch
culture. In addition to therapeutic protein productivity, we determined
the impact of the AIP on cell culture growth and viability.

\textbf{Materials and Methods}

\textbf{Cell line and culture maintenance}

The Chinese Hamster Ovary (CHO) cell line expressing human monoclonal
anti-Interleukin-1\selectlanguage{greek}β \selectlanguage{english}IgG1 (Kennard et al., 2009) ChK2 437.89.34 was
provided by Pfizer (St. Louis, MO). The cell line was maintained in
shake tubes (Corning or Nalgene) in a humidified incubator (Kuhner
shaker, Basel, Switzerland) at 37@\selectlanguage{english}C, 5\% CO\textsubscript{2} and 225
rpm. The cells were passaged every 3 days and grown in chemically
defined CD CHO medium (Gibco-Invitrogen, Grand Island, NY), supplemented
with 100 \selectlanguage{greek}µ\selectlanguage{english}g/mL hygromycin B (Gibco), 4.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL bleocin (Calbiochem, La
Jolla, CA) and 4 mM glutamine (Gibco).

\textbf{Autophagy-Inducing Peptide (AIP)}

The autophagy-inducing peptide (AIP) and negative control peptide were
provided by Dr. Beth Levine (Howard Hughes Medical Institute and
University of Texas Southwestern Medical Center, Dallas, TX), and based
on her group's previous studies (Shoji-Kawata et al., 2013). The
cell-permeable AIP was comprised of a portion of the Beclin 1 autophagy
protein spanned by amino acids 267-284, joined via a diglycine linker to
the HIV-1 Tat protein transduction domain (PTD) (Shoji-Kawata et al.,
2013, van den Berg and Dowdy, 2011). The control peptide consisted of an
identical Tat PTD domain linked to a scrambled Beclin 1 amino acid
sequence that does not induce autophagy (Shoji-Kawata et al., 2013).
Both peptides were reported to be well-tolerated in mice when
administered \emph{in vivo} (Shoji-Kawata et al., 2013).

\textbf{Batch Induction Experiments}

The batch induction experiments were run in 24-well deep well plates
(Axygen Scientific, Union City, CA) using Duetz covers (Kuhner shaker).
The cultures were seeded at 2 x 10\textsuperscript{5} cells/mL in 3.5 mL
CD CHO medium supplemented with 100 \selectlanguage{greek}µ\selectlanguage{english}g/mL hygromycin B, 4.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL
bleocin and 4 mM glutamine. Cultures were incubated at 37\selectlanguage{ngerman}°C, 5\%
CO\textsubscript{2} and 225 rpm in a humidified incubator (Kuhner
Shaker). The experiment was initiated by adding autophagy-inducing
peptide (AIP) stock solution (1.65 mM) to the cultures to obtain the
medium concentrations of 0, 1, 2, 3 and 4 \selectlanguage{greek}µ\selectlanguage{english}M. Every two days, a 300 \selectlanguage{greek}µ\selectlanguage{english}L
sample was taken for monitoring of cell density, cell viability, cell
diameter, and cell aggregation (Cedex analysis) using a Cedex automated
cell counting instrument (Innovatis, Bielefeld, Germany), according to
the manufacturer's instructions. Once cultures reached a cell density
above 2 x 10\textsuperscript{6} cells/mL, 100 \selectlanguage{greek}µ\selectlanguage{english}L of the culture sample
were mixed with 200 \selectlanguage{greek}µ\selectlanguage{english}L of 1X Dulbecco's phosphate-buffered saline (DPBS)
(Gibco) and used for cell count and viability measurements at a 1:3
dilution. The remainder of the culture sample was centrifuged to remove
the cells and approximately 150 \selectlanguage{greek}µ\selectlanguage{english}L of the supernatant were retained to
determine the IgG production by ELISA. Each AIP medium concentration was
evaluated in duplicate cultures and the entire batch induction
experiment was repeated two additional times (for a total of 3 times).
In addition, a batch induction experiment was performed using the
negative control peptide.



\textbf{Fed-Batch Induction Experiments}

The fed-batch induction experiments were performed in 24-well deep well
plates (Axygen Scientific) using Duetz covers. The cultures were seeded
at 2 x 10\textsuperscript{5} cells/mL in 3.5 mL CD CHO medium
supplemented with 100 \selectlanguage{greek}µ\selectlanguage{english}g/mL hygromycin B, 4.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL bleocin, and 4 mM
glutamine, and incubated at 37\selectlanguage{ngerman}°C, 5\% CO\textsubscript{2} and 225 rpm in
a humidified incubator (Kuhner shaker). The experiment was initiated by
adding the AIP stock solution (1.65 mM) to obtain medium concentrations
of 0, 1, 2, 3 and 4 \selectlanguage{greek}µ\selectlanguage{english}M. Starting on day 1, the fed-batch regime was
initiated with a 280 \selectlanguage{greek}µ\selectlanguage{english}L sample taken daily from the culture. This volume
was then replaced with 140 \selectlanguage{greek}µ\selectlanguage{english}L CD CHO Efficient Feed A (Gibco), 70 \selectlanguage{greek}µ\selectlanguage{english}L
amino acid mixture A {[}10 mM L-cystine disodium salt hydrate (MP
Biomedicals, Illkirch, France), 15 mM L-tyrosine disodium salt (Sigma,
St. Louis, MO) and 10 mM aspartic acid (Gibco) dissolved in 0.1 N HCl{]}
and 70 \selectlanguage{greek}µ\selectlanguage{english}L amino acid mixture B {[}10 mM L-glutamic acid (Sigma) and 75
mM L-asparagine (Sigma) in 0.1 N NaOH{]}. Every two days, 150 \selectlanguage{greek}µ\selectlanguage{english}L of the
removed culture volume was mixed with 150 \selectlanguage{greek}µ\selectlanguage{english}L of DPBS and used for Cedex
analysis at a 1:2 dilution. As above, when cultures reached a
concentration of greater than 2 x 10\textsuperscript{6} cells/mL, a 100
\selectlanguage{greek}µ\selectlanguage{english}L aliquot was taken to determine cell count and viability at a 1:3
dilution with DPBS. Similar to the batch cultures, the remainder of the
culture sample was centrifuged and the supernatants were retained for
IgG quantitation by ELISA. Each AIP medium concentration was evaluated
in duplicate cultures during a run and the entire fed-batch induction
experiment was repeated (for a total of 3 times).

\textbf{Cell count and viability determination}

Viable cell concentration was measured by trypan blue exclusion using a
Cedex automated cell counting instrument, according to the
manufacturer's instructions (Innovatis). Measurements were performed
every two days during the culture period using 0.4\% (w/v) trypan blue
(Gibco).

\textbf{Recombinant protein quantification}

Levels of IgG1 in culture supernatants were evaluated by ELISA. Standard
ELISA protocols were followed using 96-well Maxisorb plates (Thermo
Fisher Scientific) and signals measured using a plate reader (Molecular
Devices, Sunnyvale, CA) at 405 nm (reference at 492 nm). The primary
(coating) antibody used was a goat anti-human IgG (Fc\selectlanguage{greek}γ
\selectlanguage{english}fragment-specific) (Jackson ImmunoResearch \#109-005-098). The secondary
(detection) antibody used was a goat anti-human IgG (H+L
specific)-alkaline phosphatase antibody (Jackson ImmunoResearch
\#109-055-088). Quantification was achieved by interpolation from a
standard curve obtained from 1.9 - 125 ng/mL dilutions of purified IgG.

The volumetric productivity was determined by the overall production of
IgG for each culture volume, while the specific productivity for each
culture was calculated based on the product concentration over the
integral viable cell density (IVCD) (Renard et al., 1988).

\textbf{Statistical analysis}

Differences between measurements was evaluated by one-way ANOVA followed
by multiple comparison tests (Tukey-Kramer HSD Post-Hoc test). P values
of \textless{} 0.05 were considered statistically significant.



\textbf{Results and Discussion Autophagy enhances recombinant antibody
production in batch cultures}

Our initial investigations evaluated whether addition of the
autophagy-inducing peptide (AIP), derived from the autophagy protein
Beclin 1 (Shoji-Kawata et al., 2013), could enhance monoclonal antibody
production in batch cultures. CHO cells expressing a human anti-IL1\selectlanguage{greek}β
\selectlanguage{english}IgG1 (CHO-aIL1\selectlanguage{greek}β\selectlanguage{english}) were cultured in medium supplemented with AIP at 0, 1,
2, 3 and 4 \selectlanguage{greek}µ\selectlanguage{english}M concentrations, and IgG production was evaluated by ELISA,
as described in Materials and Methods. Figure 1A compares the specific
and volumetric productivities in the control- and AIP- treated cultures
from a representative batch induction experiment. The specific
productivity for the AIP-treated cultures increased from 11.0 \selectlanguage{ngerman}± 0.9 to
19.2 ± 0.7 \selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6} cells/day at 1 and 4 \selectlanguage{greek}µ\selectlanguage{english}M
concentrations, respectively. The specific productivities observed for
the 2, 3, and 4 \selectlanguage{greek}µ\selectlanguage{english}M AIP-containing cultures were significantly increased
(ANOVA, P \textless{} 0.05) relative to the control culture (10.6 \selectlanguage{ngerman}± 0.6
\selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6}cells/day). Similarly, the volumetric
productivity increased from 404.9 \selectlanguage{ngerman}± 28.3 at 1 \selectlanguage{greek}µ\selectlanguage{english}M AIP to 565.5 \selectlanguage{ngerman}± 39.6
\selectlanguage{greek}µ\selectlanguage{english}g/mL at 4 \selectlanguage{greek}µ\selectlanguage{english}M AIP. However, the increase in volumetric productivity was
statistically significant only for the 4 \selectlanguage{greek}µ\selectlanguage{english}M AIP concentration, as
compared to the control culture (ANOVA, P \textless{} 0.05). Thus,
substantial increases were observed, up to 82\% and 46\% in specific and
volumetric productivities, respectively.

Peptide hydrolysates have been added to cell culture manufacturing media
to increase cell growth and productivity (Franek et al., 2003; Franek
and Fussenegger, 2008). To determine whether the impact of the AIP was
indeed due to its capacity to induce autophagy, a negative control
peptide containing the same amino acids, but in a scrambled arrangement,
was also tested. Figure 1B shows that 3 or 4 \selectlanguage{greek}µ\selectlanguage{english}M concentrations of the
negative control peptide had little if any influence on the protein
productivity compared to the control culture containing 0 \selectlanguage{greek}µ\selectlanguage{english}M peptide.
Furthermore, it was confirmed that the addition of the AIP to the
culture medium induced cellular autophagy in CHO-aIL-1\selectlanguage{greek}β \selectlanguage{english}cells, while the
control peptide did not, as evaluated by cellular p62 protein
degradation in Western blot analyses (Shoji-Kawata et al., 2013 and data
not shown). Therefore, the increase in productivity observed was linked
to the induction of autophagic flux in the cell culture, and this is
consistent with our previous findings (Jardon et al., 2012) as
interpreted and confirmed by Baek et al. (2016). Shoji-Kawata et al.
(2013) have shown that the AIP used in these experiments specifically
triggers the autophagy cascade by promoting the release of Beclin 1 from
one of its inhibitors, the Golgi-associated plant pathogenesis-related
protein 1 (GAPR-1). However, the molecular mechanism by which autophagic
flux, in response to the AIP, leads to enhanced protein productivity in
culture supernatants is unclear at present. It is interesting to note
that the increased autophagy and CHO cell productivity in response to
3-MA was associated with the induction of the unfolded protein response
(UPR) at the transcriptional level (Baek et al. 2018). These authors
further suggested that UPR induction may enhance endoplasmic reticulum
(ER) capacity and thereby the cellular productivity, a possibility that
requires further investigation.

\textbf{The effect of the autophagy-inducing peptide on cell growth and
viability in batch culture}

Given the encouraging productivity results, it was important to consider
the effects of the AIP on the growth and viability of the CHO cells in
culture. The results obtained with the AIP were compared to two control
cultures containing either no peptide or the scrambled negative control
peptide that did not induce autophagy (Figure 1B). Compared to the two
control cultures, with the addition of increasing AIP concentrations,
there was an increasing lag in growth over the first 4-8 days of culture
(Figure 2A). Subsequently, the AIP-containing cultures had similar rapid
growth phases, resulting in a \textasciitilde{}5 day extended batch
culture duration. These cultures reached maximum cell densities from 8.3
\selectlanguage{ngerman}± 0.1 to 9.9 ± 0.6 x 10\textsuperscript{6} cells/mL, with Integral
Viable Cell Density (IVCD) values from 26.2 ± 0.3 to 40.9 ± 0.3 x
10\textsuperscript{6} cells-day/mL. After reaching the maximum, the
viable cell densities consistently declined rapidly over the ensuing 2
days. Notably, the addition of 2-4 \selectlanguage{greek}µ\selectlanguage{english}M AIP to the cultures resulted in
significantly lower (ANOVA, P \textless{} 0.05) maximum cell densities
and IVCDs compared to the control and 1 \selectlanguage{greek}µ\selectlanguage{english}M AIP-containing cultures.
These differences, along with the more pronounced lag phases at higher
AIP concentrations, are likely the result of the complex control exerted
over cell survival and death by the autophagy pathway (Doherty and
Baehrecke, 2018).

The batch culture viability profiles are shown in Figure 2B. The control
cultures maintained a \textgreater{}90\% viability until day 6, before a
rapid viability decline. For all cultures treated with AIP, the lag in
growth was accompanied by an initial drop in viability observed on day
2. Similarly, the extent of the drop was AIP concentration dependent,
with 4 \selectlanguage{greek}µ\selectlanguage{english}M of AIP exerting the largest, \textgreater{}90\% decrease in
cell viability. This decrease in viability was not observed in the
control peptide culture, nor would it be expected from peptide
hydrolysate medium supplementation. Also, the decrease in viability was
not attributable to the protein transduction domain of the tat motif
present in both peptides to facilitate their uptake into the cell (Herce
and Garcia, 2007). The day 2 decreased viability likely resulted from
the induction of autophagic cell death or ``autosis'', which can be a
consequence of autophagic flux in response to the AIP, as previously
reported (Liu et al., 2013). Interestingly however, the viability in all
AIP-treated cultures recovered rapidly after day 2, reaching over 90\%
by day 6. After day 8, another rapid decrease in viability was observed
as these batch cultures entered the decline phase. It appears that the
autophagic flux in response to the AIP, the ensuing cell death, and
rapid subsequent expansion of surviving cells, creates conditions and/or
selects for cells that have superior protein production capabilities.
This merits further investigation, especially in the context of the
fed-batch processes most commonly used for monoclonal antibody
manufacturing.

\textbf{Induction of the autophagy pathway enhances antibody
productivity in fed-batch cultures}

Since the most common culture systems found in industry are fed-batch
cultures, the ability of the AIP to enhance protein production was also
evaluated on this culture platform. CHO-aIL-1\selectlanguage{greek}β \selectlanguage{english}cells were cultured in
the presence or absence of AIP using fed-batch and the production of IgG
was evaluated by ELISA. Figure 3 shows the specific and volumetric
productivity in AIP-treated and control (0 \selectlanguage{greek}µ\selectlanguage{english}M peptide) cultures. The
specific productivity increased from 9.6 \selectlanguage{ngerman}± 1.5
\selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6}cells/day in control cultures up to 13.7 \selectlanguage{ngerman}± 0.6
\selectlanguage{greek}µ\selectlanguage{english}g/10\textsuperscript{6}cells/day in AIP-treated cultures. The increase
in specific productivity above that of controls was statistically
significant in 2 and 4 \selectlanguage{greek}µ\selectlanguage{english}M AIP-containing cultures (ANOVA, P \textless{}
0.05). The volumetric productivity also increased significantly (ANOVA,
P \textless{} 0.05) to 1260.1 \selectlanguage{ngerman}± 87.1 and 1229.7 ± 90.0 \selectlanguage{greek}µ\selectlanguage{english}g/mL in cultures
with 3 and 4 \selectlanguage{greek}µ\selectlanguage{english}M AIP concentrations, so by over 2-fold compared to the
control cultures. These findings are consistent with those obtained in
batch cultures, but with the volumetric productivity increase over twice
as much as the 46\% increase observed in batch culture.

\textbf{Growth and viability of fed-batch cultures in response to AIP}

As in the batch cultures, we investigated the effects of AIP on the
growth and viability profiles of CHO-aIL-1\selectlanguage{greek}β \selectlanguage{english}cells in the fed-batch
cultures. AIP-treated cells were compared to the 0 \selectlanguage{greek}µ\selectlanguage{english}M AIP control
(Figure 4). Both the control cultures and those stimulated with 1 \selectlanguage{greek}µ\selectlanguage{english}M AIP
grew relatively rapidly with similar kinetics before slowing and
reaching a maximum by day 10, and then rapidly dropping off thereafter.
As in the Figure 2A batch cultures, correlated with higher additions of
AIP, an increasing lag in growth was observed (Figure 4A). After this
initial lag phase, the 2, 3 and 4 \selectlanguage{greek}µ\selectlanguage{english}M AIP-containing cultures grew
rapidly for an additional 6 days before reaching a maximum, with 3 and 4
\selectlanguage{greek}µ\selectlanguage{english}M AIP cultures exhibiting a stationary phase before declining. Compared
to the control (20.2 +/- 1.7 x 10\textsuperscript{6} cells/mL),
significantly lower maximum cell densities (ANOVA, P \textless{} 0.05)
were observed at AIP concentrations of 3 \selectlanguage{greek}µΜ \selectlanguage{english}(13.4 +/- 0.9 x
10\textsuperscript{6} cells/mL) and 4 \selectlanguage{greek}µΜ \selectlanguage{english}(10.3 +/- 0.7 x
10\textsuperscript{6} cells/mL). Concomitantly, for the 3 and 4 \selectlanguage{greek}µ\selectlanguage{english}M AIP
cultures, the IVCD values were significantly increased (ANOVA, P
\textless{} 0.05) compared to the control, 1 \selectlanguage{greek}µ\selectlanguage{english}M, and 2 \selectlanguage{greek}µ\selectlanguage{english}M cultures,
indicating lower maximum growth but increased culture longevity.

The viability profiles for all cultures are shown in Figure 4B. Control
and 1 \selectlanguage{greek}µ\selectlanguage{english}M AIP-containing cultures maintained viabilities
\textgreater{}90\%, until they decreased after day 10. Cultures
containing 2 - 4 \selectlanguage{greek}µ\selectlanguage{english}M AIP exhibited an initial and AIP dose-related drop
in viability by day 2. In particular, both the 3 and 4 \selectlanguage{greek}µ\selectlanguage{english}M AIP cultures
had viabilities of less than 50\% by culture day 2. Similar to the batch
cultures, a rapid recovery in viability for all AIP cultures was
observed on day 4. The cell viability remained above 90\% after day 6
until cultures reached their maximum cell concentration, before
declining again. After day 10, a rapid decline in viability was observed
for control, 1 and 2 \selectlanguage{greek}µ\selectlanguage{english}M cultures, while 3 and 4 \selectlanguage{greek}µ\selectlanguage{english}M cultures had a more
prolonged decline phase. Overall, as for the batch cultures, the initial
decline in cell viability could be due to autophagic death, and then the
autophagy induction may promote the subsequent rapid recovery and
altered growth characteristics (prolonged stationary phase), either
selecting cells or providing the conditions that enhance recombinant
protein productivity.



\textbf{Conclusion}

Overall, this study provides strong support for modulating cellular
autophagy to increase monoclonal antibody production in the CHO cell
culture system, both in batch and fed-batch cultures. Despite the
negative impacts on cell growth and viability (that could be reduced),
the positive impacts on both cell specific and volumetric antibody
production were substantial. This unique peptide and its capacity to
specifically activate the autophagy cascade, merits further
consideration as a media additive for bioprocess refinement, and is thus
of particular relevance to industrial biotechnology.

\textbf{Acknowledgements}

The authors acknowledge research funding in support of this work from
the University of British Columbia, the Saskatchewan Health Research
Foundation and the Natural Sciences and Engineering Research Council of
Canada. Dr. Beth Levine is kindly acknowledged for providing the
autophagy-inducing and negative control peptides, as well as Pfizer for
providing the CHO cell line. All authors approved the final version of
the manuscript and declare that they have no conflicts of interest.

\textbf{References}

Amaan, T., Hansen A. H., Kol, S., Hansen, H. G., Arnsdorf, J.,
Nallapareddy, S., et al. Glyco-engineered CHO cell lines producing
alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized
N-glycosylation profiles. \emph{Metabolic Engineering} , \emph{52} ,
143-152. doi: 10.1016/j.ymben.2018.11.014

Baek, E., Kim, C. L., Kim, M. G., Lee, J. S., \& Lee, G. M. (2016).
Chemical inhibition of

autophagy: examining its potential to increase the specific productivity
of recombinant CHO cell lines.~\emph{Biotechnology and
Bioengineering},~\emph{113}(9), 1953-1961. doi: 10.1002/bit.25962

Baek, E., Lee, J.S. \& Lee, G.M. (2018). Untangling the mechanism of
3-methyladenine in enhancing the specific productivity: transcriptome
analysis of recombinant Chinese Hamster Ovary cells treated with
3-methyladenine. \emph{Biotechnology and Bioengineering} ,\emph{115}
,2243--2254. doi: 10.1002/bit.26777

Bult\selectlanguage{ngerman}é, D. B., Palomares, L. A., Gómez Para, C., Martínez, J. A.,
Contreras, M. A, Noriega, L. G., \& Ramirez, O. T. (2020).
Overexpression of the mitochondrial pyruvate carrier reduces lactate
production and increases recombinant protein productivity in CHO
cells.\emph{Biotechnology and Bioengineering} ,

Bebbington, C. R., Renner, G., Thomson, S., King, D., Abrams, D., \&
Yarranton, G. T. (1992).

High-Level Expression of a recombinant antibody from myeloma cells using
a glutamine synthetase gene as an amplifiable selectable
marker.~\emph{Nature Biotechnology},~\emph{10}(2), 169-175. doi:
10.1038/nbt0292-169

Butler, M., \& Meneses-Acosta, A. (2012). Recent advances in technology
supporting

biopharmaceutical production from mammalian cells.~\emph{Applied
Microbiology and Biotechnology},~\emph{96}(4), 885-894. doi:
10.1007/s00253-012-4451-z

Doherty, J., \& Baehrecke, E. H. (2018). Life, death and
autophagy.~\emph{Nature Cell Biology} , \emph{20} (10),

1110-1117. doi: 10.1038/s41556-018-0201-5

Ecker, D. M., Jones, S. D., \& Levine, H. L. (2014). The therapeutic
monoclonal antibody

market.~\emph{mAbs} ,~\emph{7} (1), 9-14. doi:
10.4161/19420862.2015.989042

Figueroa Jr., B., Ailor, E., Osborne, D., Hardwick, J. M., Reff, M., \&
Betenbaugh, M. J. (2007).

Enhanced cell culture performance using inducible anti-apoptotic
genes\emph{E1B-19K} and \emph{Aven} in the production of a monoclonal
antibody with Chinese Hamster Ovary cells. \emph{Biotechnology and
Bioengineering}. 97, 877-892. doi: 10.1002/bit.21222

Franek, F., \& Fussenegger, M. (2008). Survival factor-like activity of
small peptides in

hybridoma and CHO cells cultures.~\emph{Biotechnology
Progress},~\emph{21}(1), 96-98. doi: 10.1021/bp0400184

Franek, F., Eckschlager, T., \& Katinger, H. (2003). Enhancement of
monoclonal antibody

production by lysine-containing peptides.~\emph{Biotechnology
Progress},~\emph{19}(1), 169-174. doi: 10.1021/bp020077m

Ghaffari N., Jardon M.A., Krahn N., Butler M., Kennard M., Turner
R.F.B., Gopaluni B., Piret

J.M. (2020). Effects of cysteine, asparagine or glutamine limitations in
CHO cell batch and fed-batch cultures. \emph{Biotechnology Progress},
36(2), e2946. doi: 10.1002/btpr.2946

Hamaker, N. K., \& Lee, K. H. (2018). Site-specific integration ushers
in a new era of precise

CHO cell line engineering. \emph{Current Opinion in Chemical
Engineering}, 22, 152-150. doi: 10.1015/j.coche.2018.09.011

Herce, H. D., \& Garcia, A. E. (2007). Molecular dynamics simulations
suggest a mechanism for

translocation of the HIV-1 TAT peptide across lipid
membranes.~\emph{Proceedings of the National Academy of
Sciences},~\emph{104}(52), 20805-20810. doi: 10.1073/pnas.0706574105

Hong, J. K., Lakshmanan, M., Goudar, C., \& Lee, D.-Y. (2018). Towards
next generation CHO

cell line development and engineering by systems
approaches.\emph{Current Opinion in Chemical Engineering}, 22, 1-10.
doi: 10.1016/j.coche.2018.08.002

Hwang, S. O., \& Lee, G. M. (2008). Nutrient deprivation induces
autophagy as well as apoptosis

in Chinese hamster ovary cell culture.~\emph{Biotechnology and
Bioengineering},~\emph{99}(3), 678-685. doi: 10.1002/bit.21589

Jardon, M. A., Sattha, B., Braasch, K., Leung, A. O., Côté, H. C. F.,
Butler, M., Gorski, S.M., \& Piret, J. (2012). Inhibition of
glutamine-dependent autophagy increases t-PA production in CHO cell
fed-batch processes.~\emph{Biotechnology and Bioengineering}
,~\emph{109} (5), 1228-1238. doi: 10.1002/bit.24393

Kelly, P. S., Miguez, A. A., Alves, C., \& Barron, Niall. (2018). From
media to mitochondria-

rewiring cellular energy metabolism of Chinese Hamster Ovary cells for
the enhanced production of biopharmaceuticals. \emph{Current Opinion in
Chemical Engineering}, 22, 71-80. doi: 10.1016/j.cohce.2018.08.009

Kennard, M. L., Goosney, D. L., Monteith, D., Zhang, L., Moffat, M.,
Fischer, D., \& Mott, J.

(2009). The generation of stable, high mAb expressing CHO cell lines
based on the artificial chromosome expression (ACE)
technology.~\emph{Biotechnology and Bioengineering},~\emph{104}(3),
540-553. doi: 10.1002/bit.22406

Kim, N. S., \& Lee, G. M. (2002). Response of recombinant Chinese
Hamster Ovary cells to

hyperosmotic pressure: effect of Bcl-2 overexpression.~\emph{Journal of
Biotechnology},~\emph{95}(3), 237-248. doi:
10.1016/s0168-1656(02)00011-1

Kim, J. Y., Kim, Y.-G., \& Lee, G. M. (2012). CHO cells in biotechnology
for production of

recombinant proteins: current state and further potential. \emph{Applied
Microbiology and Biotechnology}, 93, 917-930. doi:
10.1007/s00253-011-3758-5

Klionsky, D. J., Abdelmohsen, K., Abe, A., Abedin, M. J., Abeliovich,
H., Acevedo Arozena,

A. et al. (2016). Guidelines for the use and interpretation of assays
for monitoring autophagy (3rd edition).~\emph{Autophagy},~\emph{12}(1),
1-222. doi: 10.1080/15548627.2015.1100356

Lalonde, M.-E., \& Durocher, Y. (2017). Therapeutic glycoprotein
production in mammalian

cells. \emph{Journal of Biotechnology} , 251, 128-140. doi:
10.1016/j.jbiotec.2017.04.028

Liu, Y., Shoji-Kawata, S., Sumpter, R. M., Wei, Y., Ginet, V., Zhang, L.
et al. (2013). Autosis

is a Na+,K+-ATPase-regulated form of cell death triggered by
autophagy-inducing peptides, starvation, and
hypoxia-ischemia.~\emph{Proceedings of the National Academy of
Sciences},~\emph{110}(51), 20364-20371. doi: 10.1073/pnas.1319661110

Lucas, B. K., Giere, L. M., DeMarco, R. A., Shen, A., Chisholm, V., \&
Crowley, C. W. (1996).

High-level production of recombinant proteins in CHO cells using a
dicistronic DHFR intron expression vector.~\emph{Nucleic Acids
Research},~\emph{24}(9), 1774-1779. doi: 10.1093/nar/24.9.1774

Mastrangelo, A. J., Hardwick, J. M., Zou, S., \& Betenbaugh, M. J.
(2000). Part II.

Overexpression of bcl-2 family members enhances survival of mammalian
cells in response to various culture insults.~\emph{Biotechnology and
Bioengineering},~\emph{67}(5), 555-564. doi:
10.1002/(sici)1097-0290(20000305)67:5\textless{}555::aid-bit6\textgreater{}3.0.co;2-t

Mimura, Y., Lund, J., Church, S., Dong, S., Li, J., Goodall, M., \&
Jefferis, R. (2001). Butyrate

increases production of human chimeric IgG in CHO-K1 cells whilst
maintaining function and glycoform profile.~\emph{Journal of
Immunological Methods},~\emph{247}(1-2), 205-216. doi:
10.1016/s0022-1759(00)00308-2

Mizushima, N., \& Klionsky, D. J. (2007). Protein turnover via
autophagy: implications for

metabolism.~\emph{Annual Review of Nutrition},~\emph{27}(1), 19-40. doi:
10.1146/annurev.nutr.27.061406.093749

Mizushima, N., Levine, B., Cuervo, A. M., \& Klionsky, D. J. (2008).
Autophagy fights disease

through cellular self-digestion.~\emph{Nature} ,~\emph{451} (7182),
1069-1075. doi: 10.1038/nature06639

Renard, J. M., Spagnoli, R., Mazier, C., Salles, M. F., \& Mandine, E.
(1988). Evidence that

monoclonal antibody production kinetics is related to the integral of
the viable cells curve in batch systems.~\emph{Biotechnology
Letters},~\emph{10}(2), 91-96. doi: 10.1007/bf01024632

Shoji-Kawata, S., Sumpter, R., Leveno, M., Campbell, G. R., Zou, Z.,
Kinch, L. et al. (2013).

Identification of a candidate therapeutic autophagy-inducing
peptide.~\emph{Nature},~\emph{494}(7436), 201-206. doi:
10.1038/nature11866

Spearman, M., Lodewyks, C., Richmond, M., \& Butler, M. (2014). The
bioactivity and

fractionation of peptide hydrolysates in cultures of CHO
cells.~\emph{Biotechnology Progress},~\emph{30}(3), 584-593. doi:
10.1002/btpr.1930

Tey, B. T., Singh, R. P., Piredda, L., Piacentini, M., \& Al-Rubeai, M.
(2000). Influence of Bcl-2

on cell death during the cultivation of a Chinese hamster ovary cell
line expressing a chimeric antibody.~\emph{Biotechnology and
Bioengineering},~\emph{68}(1), 31-43. doi:
10.1002/(sici)1097-0290(20000405)68:1\textless{}31::aid-bit4\textgreater{}3.0.co;2-l

van den Berg, A., \& Dowdy, S. F. (2011). Protein transduction domain
delivery of therapeutic

macromolecules.~\emph{Current Opinion in Biotechnology},~\emph{22}(6),
888-893. doi: 10.1016/j.copbio.2011.03.008

Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P.,
Bertozzi, C. R., Hart, G. W.,

\& Etzler, M. W. (2009).~\emph{Essentials of Glycobiology}~(2nd ed.).
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Walsh, G. (2003). Biopharmaceutical benchmarks - 2003.~\emph{Nature
Biotechnology} , \emph{21} (8), 865-870. doi: 10.1038/nbt0803-865

Wu, Y., Tan, H., Shui, G., Bauvy, C., Huang, Q., Wenk, M. R. et al.
(2010). Dual role of 3-

methyladenine in modulation of autophagy via different temporal patterns
of inhibition on Class I and III Phosphoinositide
3-Kinase.~\emph{Journal of Biological Chemistry},~\emph{285}(14),
10850-10861. doi: 10.1074/jbc.m109.080796

Yin, B., Wang, Q., Chung, C.-Y., Ren, X., Bhattacharya, R., Yarema, K.
J., \& Betenbaugh, M. J.

(2018). Butyrated ManNAc analog improves protein expression in Chinese
Hamster Ovary cells. \emph{Biotechnology and Bioengineering}, 115,
1531-1541. doi: 10.1002/bit.26560

Zustiak, M. P., Pollack, J. K., Marten M. R., \& Betenbaugh, M. J.
(2008). Feast or famine:

autophagy control and engineering in eukaryotic cell culture. Current
Opinion in Biotechnology, 19, 518-526. doi: 10.1016/j.copbio.2008.07.007



\textbf{Figure Legends}

\textbf{Figure 1.} A) The effect of AIP on the specific and volumetric
productivity of aIL-1\selectlanguage{greek}β-\selectlanguage{english}producing CHO cells in a batch culture compared
to control cultures containing no added peptide. Error bars indicate the
standard deviation for two technical replicate cultures in one
experiment, assuming that the data is normally distributed. These data
are from one representative run out of three exhibiting the same trend.
B) The effect of a negative control peptide (NCP) on the specific and
volumetric productivity in CHO-aIL-1\selectlanguage{greek}β \selectlanguage{english}cells batch culture with 3 \selectlanguage{greek}µ\selectlanguage{english}M and
4 \selectlanguage{greek}µ\selectlanguage{english}M NCP added to the basal cell culture medium on day 0.

\textbf{Figure 2.} The effect of autophagy on the A) growth profiles and
B) viability for aIL-1\selectlanguage{greek}β-\selectlanguage{english}producing CHO cells in batch cultures over time.
Cultures with no added peptide (Control) or with the negative control
peptide (NCP 3 \selectlanguage{greek}µ\selectlanguage{english}M) were experimental controls. The AIP concentrations
tested were 1 \selectlanguage{greek}µ\selectlanguage{english}M, 2 \selectlanguage{greek}µ\selectlanguage{english}M, 3 \selectlanguage{greek}µ\selectlanguage{english}M, and 4 \selectlanguage{greek}µ\selectlanguage{english}M. Error bars indicate the standard
deviation for two technical replicate cultures within one run, assuming
that the data is normally distributed. These data are from one
representative run out of three exhibiting the same trend.

\textbf{Figure 3.} The effect of AIP (1-4 \selectlanguage{greek}µΜ\selectlanguage{english}) on the specific and
volumetric productivity of aIL-1\selectlanguage{greek}β-\selectlanguage{english}producing CHO cells in fed-batch
cultures compared to the control cultures containing no additional
peptide. Error bars indicate the standard deviation for two technical
replicate cultures within one run, assuming that the data is normally
distributed. These data are from one representative run out of three
exhibiting the same trend.

\textbf{Figure 4.} The effect of AIP on the A) growth profiles and B)
viability of aIL-1\selectlanguage{greek}β-\selectlanguage{english}producing CHO cells in fed-batch cultures. Cultures
without AIP were used as experimental controls. Error bars indicate the
standard deviation for two technical replicate cultures within one run,
assuming that the data is normally distributed. These data are from one
representative out of three exhibiting the same trend.

\par\null\par\null

\selectlanguage{english}
\FloatBarrier
\end{document}

