207

CHO Cell Engineering for Improved Process Performance

and Product Quality
Simon Fischer 1 and Kerstin Otte 2
1 Boehringer Ingelheim Pharma GmbH & Co.KG, Cell Line Development CMB, Bioprocess & Analytical

Development, Birkendorfer Straße 65, 88397 Biberach an der Riss, Germany

2
University of Applied Sciences Biberach, Institute of Applied Biotechnology, Hubertus-Liebrecht-Strasse 35,
88400 Biberach, Germany

9.1 CHO Cell Engineering

Although Chinese hamster ovary (CHO) cells have been successfully employed as
a manufacturing host cell system for more than 30 years, these cell lines still suffer
from naturally occurring limitations with regard to growth rates and recombi-
nant protein production capacity [1]. The molecular basis for these limitations
may be based on the fact that this cell type has not evolved to exhibit superior
growth and recombinant protein production properties in large-scale stirred tank
bioreactors, but rather to accomplish its task as a fibroblast cell in an ovary tis-
sue. Nonetheless, today, an entire industry is primarily relying on CHO cells as a
manufacturing host system for therapeutic protein production [2, 3].
Historically, the most important modified CHO cell lines, which eventually
paved the way for an economical utilization of CHO cells for biopharmaceuti-
cal manufacturing, were the different dihydrofolate reductase (DHFR)-deficient
CHO sublines named DXB11 and DG44, respectively [4, 5]. They mark the start-
ing point of the commercial exploitation of CHO cells in biotechnology in the
mid-1980s [6]. Several years later, a more effective metabolic selection system was
introduced, which was based on the glutamine synthetase (GS) enzyme that can
be inhibited by methionine sulfoximine (MSX), enabling a more stringent selec-
tion and thus generation of high-expressing recombinant CHO cell lines [7, 8].
In the meanwhile, the number of GS-deficient CHO host cell lines, besides the
first CHO-K1SV [9], has expanded considerably and several biopharmaceutical
companies are already using these cell lines for commercial manufacturing. Both
DHFR- and GS-deficient CHO cell lines can be selected for stable transfectants
in growth media lacking hypoxanthine/thymidine and l-glutamine, respectively,
if the cells were previously transfected with an expression plasmid encoding a
transgene in combination with a functional DHFR or GS gene copy.

Cell Culture Engineering: Recombinant Protein Production,

First Edition. Edited by Gyun Min Lee and Helene Faustrup Kildegaard.
© 2020 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2020 by Wiley-VCH Verlag GmbH & Co. KGaA.
208 9 CHO Cell Engineering for Improved Process Performance and Product Quality

Monoclonal antibodies (mAbs) are still the most frequently developed class of
biologics [10]. However, the number of multispecific formats as well as highly
potent fusion proteins (in the following sections, referred to as “multispecifics”)
have also increased dramatically over the past few years [11, 12]. Certainly, these
molecules have never undergone an evolutionary process and thus represent new
territories for production cells in terms of translation, intracellular processing,
folding, and secretion. Thus, it seems little surprising that these novel and com-
plex therapeutics often turn out to be difficult-to-express for CHO cells [13–15].
Although the state-of-the-art industrial cell line development (CLD) workflows
frequently deliver clonal cell lines exceeding product yields of 5 g/l, also stan-
dard IgGs were found to be challenging to be expressed by CHO cells [16, 17],
e.g. if the molecule design is suboptimal. For instance, if the primary amino acid
sequence of an IgG exhibits aggregation-prone regions on the surface or very
unusual amino acid residues at certain positions, compared to a wide range of
known IgG sequences, it can also have a dramatic influence productivity [18–20].
Consequently, there is an urgent demand to steadily improve industrial CHO host
cell lines in order to be prepared for future bioprocessing challenges. In addi-
tion, health care systems are already facing tremendous costs associated with
the increasing demand for therapeutic proteins to address unmet medical needs
[21]. Hence, industrial manufacturing processes of biopharmaceuticals are highly
dependent on nonexpensive and high-yielding production platforms in order to
maximize production yields but also to reduce associated costs. Therefore, host
cell engineering of CHO cells represents a valuable strategy to overcome limita-
tions in the production of biologics.
There are different opportunities to counteract limitations of mammalian cell
factories. In the following chapters, we have summarized the most relevant
cell line and state-of-the-art engineering techniques currently applied for CHO
cell engineering including overexpression or knockout of genes, as well as the
usage of noncoding RNAs. Furthermore, we provide an overview on applications
of cell line engineering approaches in CHO cells to enhance recombinant
protein production, repress cell death and accelerate growth, and modulate
posttranslational modifications (PTMs).

9.2 Methods in Cell Line Engineering

9.2.1 Overexpression of Engineering Genes
After the identification of beneficial genes for the production of biopharma-
ceutical proteins, the overexpression of these genes is one of the promising
strategies to improve the performance of mammalian production cell lines. This
technique has frequently been exploited during the past 25 years using both
transient and stable overexpression strategies. To achieve overexpression of
beneficial genes, the usually codon-optimized complementary DNA (cDNA)
lacking any intronic sequences is isolated and cloned into a mammalian expres-
sion vector [22]. The plasmid DNA (pDNA) is subsequently delivered into the
cells by transfection preferentially via electroporation or lipofection. Transfected
 9.2 Methods in Cell Line Engineering 209

cells are then subjected to antibiotic selection pressure to generate cell pools
with the plasmid DNA stably integrated into their genome. The expression of
the gene of interest (GOI) is often driven by strong viral or cellular promoter
and enhancer sequences to ensure high expression levels [23], while the selective
gene is normally controlled by weak promoters to increase the overall expression
level [24]. After the selection process, the resulting cell culture represents
a heterogeneously mixed pool of cells showing various extent of transgene
overexpressions. This procedure, however, results in phenotypic differences
between individual cells, and thus, cell lines derived from a single progenitor
cell subsequently have to be established (process is called “single cell cloning”)
to obtain a homogenous host cell line exhibiting a strong and stable engineered
phenotype.

9.2.2 Gene Knockout

Instead of overexpressing beneficial GOIs to improve production characteristics
of CHO cells, there is the possibility to knockout disadvantageous genes to engi-
neer host cell lines [25]. For the stable deletion of genes from the genome, several
methods are available including chemical- or radiation-induced random muta-
genesis in addition to a variety of precise genome editing techniques. However,
targeted genome engineering with high specificity has become a preferentially
used methodology to random mutagenesis, especially from a regulatory point of
view. In this conjunction, the current state-of-the-art technologies mainly com-
prise the use of zinc-finger nucleases (ZFNs), transcription activator-like effector
nucleases (TALENs), meganucleases, or the recently introduced clustered regu-
larly interspaced short palindromic repeats (CRISPRs/Cas9) system [26–29]. The
CRISPR/Cas9 technology has recently entered the field of CHO cell engineer-
ing because of advantages such as the ease of use of the methodology as well
as less time-consuming, and much more cost-effective procedures compared to
the more sophisticated and expensive alternatives. Hence, in the context of ratio-
nal design of mammalian cell factories, this novel methodology doubtlessly has
the potential to revolutionize current cell line optimization strategies [30] and
is of particular interest for multiplexed CHO cell engineering approaches where
several genes can be rapidly knocked out.

9.2.3 Noncoding RNA-mediated Gene Silencing

Instead of deleting a gene from the genome of a production cell, disadvantageous
genes may be silenced. This can be achieved by RNA interference (RNAi), and as
the gene is silenced, this method is also known as knockdown of gene expression.
RNAi was originally discovered in in Caenorhabditis elegans (C. elegans) [31],
and since then, gene silencing using small double-stranded RNAs (dsRNAs),
which are also termed small-interfering RNAs (siRNAs), has become a frequently
applied technology in cell engineering. SiRNAs are small double-stranded RNA
molecules of 20–25 base pairs in length and exhibit complete sequence com-
plementarity to their target messenger RNA (mRNA) [32]. After exogenous
delivery of siRNAs by directly introducing small dsRNA into the cytoplasm of
210 9 CHO Cell Engineering for Improved Process Performance and Product Quality

a cell or by expression from small hairpin RNA (shRNA) containing vectors,

the RNA molecules are cleaved by the RNase-III enzyme DICER and loaded
onto an Argonaute-2 (AGO2) protein [33, 34], which is the only member of
the AGO family exhibiting slicer activity [34]. The resulting RNA-induced
silencing complex (RISC) is established in the cytoplasm [35] and binding to the
target mRNA leads to immediate mRNA degradation [36]. The thermodynamic
stability at the 5′ -terminus of the dsRNA determines which strand will be favored
as the guide strand for binding to the mRNA target [37]. Although siRNAs are
considered artificially designed molecules for targeted gene silencing, recent
studies demonstrated the presence of naturally occurring siRNAs in eukaryotes.
Endogenous siRNAs can be derived from transposons, repetitive sequences,
long stem loop structures, or sense–antisense transcripts [38–41]. Notably, the
high specificity of an siRNA limits its application for multiplexed knockdown of
a larger number of engineering genes and thus for modulating several cellular
pathways in parallel.
A second species of noncoding RNA molecules are microRNAs (miRNAs),
which have recently entered the field of CHO cell engineering. MiRNAs are
19–25 nucleotides in length and bind to the 3′ untranslated region (3′ UTR) of a
target gene by imperfect base pairing. Although the seed sequence, usually com-
posed of nucleotides 2–8, binds as a perfect match, the remaining nucleotides
only partially bind by imperfect base pairing and thereby allow for the targeting
of multiple genes [42, 43]. Therefore, these endogenous small RNAs are capable
of regulating entire cellular pathways [44, 45], and their use as engineering tools
may enable the modulation of entire signaling pathways. This may improve
phenotypic outcome because of the fact that changes in cellular phenotypes
are most likely not the result of altering the expression of an individual gene
but rather of a plethora of genes involved in the same or different pathways. To
substantiate this hypothesis, recent studies discovered that large numbers of
miRNAs can actually regulate multiple different cellular pathways concomitantly
in order to keep the cell in homeostasis [46]. These properties in addition to the
fact that overexpression of a noncoding miRNA does not add any additional
translational burden to the host cell make miRNAs attractive molecular tools for
next-generation host cell engineering. In order to functionally analyze a large
number of miRNAs in CHO cells, high-content functional miRNA screening
approaches [47], as well as miRNome profiling studies, helped to unravel novel
target molecules to be used for CHO cell engineering [48–50].
First identified in 1993 as a critical regulator of development in the nema-
tode C. elegans [51], miRNAs have been demonstrated to play essential roles
in the regulation of virtually all cellular process in metazoans as a fine-tuner of
gene expression [42]. Their important role in cellular regulation is emphasized
by the exceptionally high evolutionary conservation of sequences among species
[52, 53]. Of note, about 50% of all miRNA loci in mammals are in close proxim-
ity to other miRNAs [34], enabling the generation of miRNA clusters, which are
transcribed from a single polycistronic transcription unit [54].
The fine-tuning of the expression of many different target genes is achieved by
the imperfect nature of target recognition of miRNAs and thereby lowering target
specificity of an individual miRNA to its mRNA targets [36, 55, 56]. MiRNAs with
 9.3 Applications of Cell Line Engineering Approaches in CHO Cells 211

identical seed sequences are grouped into families [43]; however, miRNAs from
the same seed family can have surprisingly different roles in vivo. The function of
miRNAs highly depends on the composition of the cellular transcriptome, which
impedes a clear functional classification for individual miRNAs [47, 57].
The lack of genomic sequence information before 2011 substantially hindered
miRNA research in CHO cells [58], and different strategies for functional miRNA
analysis had been pursued such as transient transfections of either miRNA mim-
ics or artificial expression vectors encoding chimeric miRNA precursor hairpins
[59, 60]. However, chimeric miRNA expression vectors were shown to be infe-
rior to vectors coding for the endogenous Cricetulus griseus miRNA sequence
[47, 61]. Taking advantage of next-generation RNA sequencing technology, 307
mature miRNAs and 200 pre-miR sequences were initially found in total RNA
samples of different CHO cell lines cultivated under various culture conditions
[62, 63]. These miRNA sequences were subsequently annotated as cgr-miRNAs
in miRBase [64, 65]. Recently, thorough in silico re-analysis of the CHO genome
revealed the presence of an additional 71 mature miRNAs in CHO cells [66].

9.3 Applications of Cell Line Engineering Approaches

in CHO Cells
9.3.1 Enhancing Recombinant Protein Production
The challenge of improving recombinant protein expression and thereby overall
yield from culturing of production cells can be met through various molecular
approaches of cell line engineering and as well by targeting a variety of cellular
processes or signal transduction pathways. These may include gene transcription,
protein translation/modification, unfolded protein response (UPR), ubiquitina-
tion/proteasomal degradation, metabolism, intracellular trafficking, cytoskeleton
dynamics, or secretion/exocytosis. In the following chapter, we will discuss a
number of examples demonstrating the breadth of used approaches. A compre-
hensive list of cell engineering studies aiming at improving recombinant protein
production in CHO cells is provided in Table 9.1.
Cellular metabolism is one of the most interesting processes to be addressed
while aiming to improve culture performance of CHO production cells. Tailor-
ing the metabolic activity of CHO production cells by overexpressing specific
genes, which affected cellular metabolism, was performed about two decades
ago to increase culture longevity and product yields. The forced expression of
vitreoscilla hemoglobin (VHb) in CHO cells reported by Pendse and Bailey led
to a 40–100% increase in human tissue plasminogen activator (tPA) productivity
[85]. Several years later, nutrient consumption and accumulation of toxic byprod-
ucts in the cell culture medium were addressed by other groups to contribute to
expand the list of metabolic engineering genes [86, 87, 89–94]. In addition, most
of the mentioned studies also demonstrated increased product yields as a result
of improved media utilization apart from optimizing the metabolic activity of
engineered CHO cells. Recently, overexpressing glutamate-cysteine ligase mod-
ifier subunit (GCLM) was shown to improve specific productivity, titer, and the
frequency of generating high-producing CHO clones by 70% [76].
Table 9.1 CHO cell engineering approaches for enhanced recombinant protein production in CHO cell lines.

Cellular Origin of
pathway engineering gene Target gene Engineered phenotype References

Protein Not indicated Protein disulfide isomerase (PDI) Increased productivity of monoclonal Borth et al. [67]
synthesis (overexpression) antibodies
CHO (overexpression) ERp57 (an isoform of PDI) 2.1-Fold increase in specific thrombopoietin Hwang et al. [68]
(TPO) productivity without decreasing cell
growth
CHO (overexpression) Calnexin (CNX) and calreticulin (CRT) 1.9-Fold increase in specific TPO productivity Chung et al. [69]
without negatively influencing cell growth and
biological activity of the recombinant TPO
Bovine Nonphosphorylatable version of the Enhanced transient expression of recombinant Underhill et al. [70]
(overexpression) eukaryotic translation initiation factor proteins
2 α (eIF2α)
Artificial Artificial zinc finger protein 10-Fold increase in IgG titer Kwon et al. [71]
(overexpression) transcription factor (ZFP-TF)
CHO (overexpression) Activating transcription factor 4 (ATF4) Increase in human antithrombin III (AT-III) Ohya et al. [72]
titer
CHO (overexpression) Growth arrest and DNA 40% increase in human AT-III titer Omasa et al. [73]
damage-inducible protein 34 (GADD34)
Human Mammalian target of rapamycin Increased cell growth, viability, apoptosis Dreesen and
(overexpression) (mTOR) resistance, and specific productivity of Fussenegger [74]
glycoproteins
Human Heat-shock 70 kDa protein 5 (BIP), Increased expression of difficult-to-express Pybus et al. [17]
(overexpression) activating transcription factor 6C monoclonal antibodies and reduced cell growth
(ATF6C), and X-box binding protein 1
(XBP1)
CHO (overexpression) Ying Yang 1 (YY1) Increased production of several product genes Tastanova et al. [75]
(SEAP, VEGF165, and IgG including rituximab)
CHO (overexpression) Glutamate-cysteine ligase modifier Increased specific productivity, mAb titer, and Orellana et al. [76]
subunit (GCLM) frequency of high producer clones by 70%
CHO (knockout) Dihydrofolate reductase (DHFR) Transgene amplification for increased protein Urlaub et al. [5]
production Page and Sydenham
[77]
CHO (knockout) Glutamine synthetase (GS) Transgene amplification for increased protein Sanders et al. [8]
production Cockett et al. [7]
CHO (knockout) DHFR Rapid establishment of DHFR−/− cells within Santiago et al. [29]
one month
CHO (knockout) GS Increased selection efficiency of Fan et al. [9]
high-producing CHO cells
CHO (knockdown) DHFR >100% increase in specific IgG productivity Wu et al. [78]
and 30% improved stability of transgene
expression
CHO (knockout) Insulin-like growth factor 1 receptor Increased production of a difficult-to-express Romand et al. [79]
(IGF1R) protein (IGF1)
CHO (knockout) FAM60A Increased expression stability of mAb Ritter et al. [80]
production clones
CHO (knockout) C12orf35 Several fold increased mAb productivity of Ritter et al. [81]
stable pools and clones
CHO (knockout) Activating transcription factor 6 β Improved mAb productivity Pieper et al. [82]
(ATF6β)
CHO Ceramide synthase 2 (CerS2) and Rab1 Improved mAb productivity Pieper et al. [83]
(knockout) GAP Tbc domain family member 20
(Tbc1D20)
CHO Breast cancer 1 (BRCA1) Increased mAb productivity of up to 5.3-fold Matsuyama et al.
(knockdown) [84]
Table 9.1 (Continued)

Cellular Origin of
pathway engineering gene Target gene Engineered phenotype References

Metabolism Vitreoscilla Vitreoscilla hemoglobin (VHb) 40–100% increase in specific human tPA Pendse and Bailey
(overexpression) productivity [85]
Rat (overexpression) Carbamoyl phosphate synthetase I (CPS 25–33% Decreased accumulation of Park et al. [86]
I) and ornithine transcarbamoylase ammonium and 15–30% increased cell growth
(OTC)
Yeast (overexpression) Pyruvate carboxylase 2 (PYC2) 35% Decrease in lactate production and Fogolin et al. [87]
twofold increase in product titer
Human Pyruvate carboxylase (PC) Increased viability because of 21–39% Kim and Lee [88]
(overexpression) decreased lactate production
Mouse Glucose transporter protein 5 (GLUT5) Less lactate production and higher cell Wlaschin and Hu
(overexpression) densities in fructose fed-batch processes [89]
Mouse GLUT5 Less lactate production, increased growth rate, Le et al. [90]
(overexpression) prolonged culture duration, and higher product
titer
CHO (overexpression) Malate dehydrogenase II (MDH2) Increased intracellular levels of ATP and Chong et al. [91]
NADH led to an 1.9-fold improvement in
integral viable cell number
CHO (overexpression) Taurine transporter (TAUT) Improved viability and increased IgG titer Tabuchi et al. [92]
CHO (overexpression) TAUT and alanine aminotransferase 1 Higher IgG yield in shorter cultivation time Tabuchi and
(ALT1) Sugiyama [93]
CHO (knockout) Lactate dehydrogenase A (LDHA) 45–79% Reduced lactate concentrations and Kim and Lee [94]
diminished glucose consumption
CHO (knockout) LDHA Diminished medium acidification because of Jeong et al. [95]
decreased lactate production leading to less
apoptosis
 CHO (knockout) Enolase 1 (ENO1) Increase in viable cell density Doolan et al. [96]
CHO (knockout) LDHA and pyruvate dehydrogenase 68–90% Increase in IgG titer Zhou et al. [97]
kinase (PDHK)
CHO (knockdown) Knockdown of LDHA combined with Improved culture longevity because of Jeong et al. [98]
BCL2 overexpression decreased lactate production and increased
apoptosis resistance
Secretion Human X-box binding protein 1 (XBP1) Higher endoplasmic reticulum content and Tigges and
(overexpression) increase in product titer Fussenegger [99]
Becker et al. [100]
Human Spliced form of XBP-1 (XBP1s) Fourfold increase in specific IgG productivity Ku et al. [101]
(overexpression) Gulis et al. [102]
Human Suppressor of loss of YPT1 protein 1 15-Fold increase in IgG production Peng and
(overexpression) (SLY1) and syntaxin binding protein 3 Fussenegger [103]
(MUNC18C)
Human Tricystronic expression of SLY1, 20-Fold increase in IgG production Peng and
(overexpression) MUNC18C, and XBP1 Fussenegger [103]
Human Ceramide transfer protein (CERT) Increase in specific productivity of human Florin et al. [104]
(overexpression) serum albumin (HSA) and monoclonal
antibodies
Human Mutant form of CERT (S132A) 35% Increase in specific t-PA productivity Rahimpour et al.
(overexpression) [105]
Human Synaptosome-associated protein of Increase in SEAP productivity by enhanced Peng et al. [106]
(overexpression) 23 kDa (SNAP-23) and secretory capacity
vesicle-associated membrane protein 8
(VAMP8)
Human Human signaling receptor protein 14 Improved secretion and production of Le Fourn et al. [107]
(overexpression) (SRP14) difficult-to-express proteins
Table 9.1 (Continued)

Cellular Origin of
pathway engineering gene Target gene Engineered phenotype References

Cell cycle Human Cyclin-dependent kinase inhibitor 1A Growth arrest and 10- to 15-fold increase in Fussenegger et al.
(overexpression) (p21CIP1 ) and CCAAT/enhancer-binding specific SEAP productivity [108]
protein 𝛼 (C/EBP-𝛼)
Human Tricystronic expression of p21CIP1 , Growth arrest and 30-fold increase in specific Fussenegger et al.
(overexpression) C/EBP-𝛼, and BCL-xL SEAP productivity [108]
Astley et al. [109]
Human Cyclin-dependent kinase inhibitor 1B Increased specific SEAP productivity Mazur et al. [110]
(overexpression) (p27KIP1 )
Human p21CIP1 Fourfold increase in IgG production Bi et al. [111]
(overexpression)
Human Myelocytomatosis oncogene (C-MYC) >70% Increase in maximal cell density without Kuystermans and
(overexpression) additional supply of nutrients Al-Rubeai [112]
CHO (knockout) Ataxia telangiectasia and Rad3 related Fourfold increase in specific IgG productivity Lee et al. [113]
(ATR) and threefold improved IgG titer
microRNA CHO (inhibition) miR-7a-5p (inhibition) Reduced growth and enhanced SEAP Barron et al. [102]
productivity Meleady et al. [56]
Sanchez et al. [114]
CHO (overexpression) miR-30a, c, d, e Enhanced mAb and SEAP productivity Fischer et al. [47]
CHO (overexpression) miR-2861 Enhanced mAb and SEAP productivity Fischer et al. [115]
CHO (overexpression) miR-17-5p Enhanced growth and EPO-Fc productivity Jadhav et al. [59]
Clarke et al. [116]
Jadhav et al. [117]
Loh et al. [118]
CHO (overexpression) miR-19b Enhanced mAb productivity Loh et al. [118]
Clarke et al. [116]
CHO (overexpression) miR-20a Enhanced mAb productivity Loh et al. [118]
Clarke et al. [116]
CHO (overexpression) miR-17-92a Enhanced mAb and EPO-Fc productivity Jadhav et al. [117]
Loh et al. [118]
CHO (overexpression) miR-92a Enhanced mAb productivity Loh et al. [119]
Human miR-1287 Enhanced mAb productivity Strotbek et al. [60]
(overexpression)
Human mitosRNA-1978 Enhanced mAb productivity Strotbek et al. [60]
(overexpression)
CHO (inhibition) miR-34a Enhanced SEAP productivity Kelly et al. [120]
CHO (overexpression) miR-483-3p Enhanced mAb and rAAV productivity Emmerling et al.
[121]
CHO (inhibition) miR-23 Enhanced SEAP productivity Kelly et al. [122]
CHO (overexpression) miR-143-3p Enhanced productivity of difficult-to-express Schoellhorn et al.
proteins [123]
Human miR-557 Twofold increase in difficult-to-express mAb in Strotbek et al. [60]
(overexpression) fed-batch cultivation Fischer et al. [124]
218 9 CHO Cell Engineering for Improved Process Performance and Product Quality

Moreover, the cellular protein synthesis machinery has been exploited by

stable genomic introduction of genes promoting protein production to increase
the yield of recombinant proteins (r-proteins) using CHO cell cultures. Over-
expression of specific transcription factors such as ZFP-TF, ATF4, GADD34,
or more recently YY1 was shown to substantially boost volumetric yields of
various r-proteins of up to 10-fold compared to parental cells [71–73, 75].
In addition, forced ectopic expression of mammalian target of rapamycin
(mTOR), which is another key protein critically involved in protein synthesis,
substantially increased overall culture performance of recombinant CHO cells
leading to increased cell growth, viability, apoptosis resistance, and specific
productivity [74].
Pybus et al. reported that co-overexpression of heat shock 70 kDa protein
5 (BIP), activating transcription factor 6C (ATF6C), and X-box binding
protein 1 (XBP1) could increase the productivity of difficult-to-express mAbs in
CHO cells [17]. This approach is especially of interest, as the demand for com-
plex and difficult-to-express therapeutic proteins increases. Hence, strategies
have to be developed to provide sufficient amount of clinical-grade material for
clinical studies and market supply. Recently, Pieper et al. found that the stable
knockdown of activating transcription factor 6 β (ATF6β), a repressor of the
prosurvival and UPR promoting factor ATF6α, significantly improved antibody
titer and viable cell density (VCD) in CHO-IgG cells under fed-batch condi-
tions [82]. This was associated with an elevated expression of the UPR genes
glucose-regulated protein 78 (GRP78), homocysteine-inducible ER protein with
ubiquitin-like domain 1 (HERPUD1), and CCAAT/enhancer-binding protein
homologous protein (CHOP) [82]. Another study on overcoming challenges
of difficult-to-express proteins reported that the production of insulin-like
growth factor 1 (IGF1) using CHO cells turned out to be difficult because of an
activation of the endogenous IGF-1 receptor (IGF1R) on the production cell line
by IGF1 itself. Elevated production of IGF1 resulted in growth retardation and
low IGF1 product titers [79]. In an elegant approach, Romand et al. knocked out
cgr-IGF1R in the CHO production cell line using ZFN technology to block the
activated inhibitory signaling induced by the recombinantly expressed IGF1 and
hence could increase IGF1 productivity by sevenfold [79].
Another promising strategy to increase productivity is the engineering of the
secretory capacity of the CHO production cell. Le Fourn et al. overexpressed the
human signaling receptor protein 14 (SRP14) to engineer the secretory capacity
of CHO cells, which successfully improved product yields [107]. Further genetic
engineering efforts to increase secretion yielded several exciting concepts
describing the exploitation of various genes involved in the secretory pathway.
Overexpression of protein disulfide isomerase (PDI), suppressor of loss of
YPT1 protein 1 (SLY1), syntaxin binding protein 3 (MUNC18C), X-box binding
protein 1 (XBP1), ceramide transfer protein (CERT), synaptosome-associated
protein of 23 kDa (SNAP23), vesicle-associated membrane protein 8 (VAMP8),
or combinations thereof could be demonstrated to increase product titer of
recombinant proteins [67, 99, 101, 103–106]. A different approach to engineer
the secretory pathway was reported by Pieper et al., where ectopic expression
of a human mitochondrial genome-encoded small RNA (mitosRNA-1978)
 9.3 Applications of Cell Line Engineering Approaches in CHO Cells 219

in an IgG expressing CHO cell line strongly improved specific productivity

and the combined stable knockdown of two mitosRNA-1978 target genes,
ceramide synthase 2 (CerS2) and the Rab1 GAP Tbc domain family member 20
(Tbc1D20), resulted in dramatically increased antibody production in CHO-IgG
cells accompanied by enhanced cell growth [83].
A dominant issue in bioprocessing using mammalian expression systems is
a continuous acidification of the culture medium because of the generation
of lactic acid as a result of pyruvate conversion by the lactate dehydrogenase
(LDH) [125]. As a result of this, lactate-mediated decrease in culture pH cell
growth is impeded. Strategies to avoid oxidation of pyruvate to lactate comprise
the repression of LDH and different strategies have been applied to achieve this
goal. Using siRNAs directed against LDHA resulted in a decrease of LDHA lev-
els below 11–25% of residual enzyme activity and a decline of lactate levels below
21% without impairing cell proliferation and productivity [88]. The simultane-
ous siRNA-mediated knockdown of LDH and pyruvate dehydrogenase kinase
(PDHK) activity resulted in reduced lactate concentrations and increased vol-
umetric mAb productivities [97]. Jeong et al. took advantage of using antisense
mRNA for specific gene knockdown and showed that in CHO cells constitutively
expressing LDH antisense mRNA erased LDHA activity successfully and dimin-
ished acidosis mediated apoptosis in CHO cells [95]. Notably, a recent study
revealed that a complete knockout of LDH is lethal in CHO cells [126], a fact
that always has to be taken into account if complete gene knockout strategies are
envisioned to induce particular cell phenotypes using precise genome editing.
Influencing cytoskeleton dynamics can lead to improved phenotypes of phar-
maceutical production cells since cell division, intracellular trafficking, cell sta-
bility, and secretion might be optimized by genetic engineering [127]. A number
of studies investigated differences between high- and low-producing mammalian
cell factories and have identified cytoskeleton genes such as vimentin and annexin
to be downregulated in high-producing cell lines, while other cytoskeleton genes
were found to be upregulated [128–132]. Another key regulator protein of the
actin cytoskeleton, cofilin-1 (CFL1) was identified to be highly downregulated
when cell-specific secreted alkaline phosphatase (SEAP) productivity increased
[133], its transient siRNA-mediated knockdown in CHO cells led to enhanced
recombinant protein productivity by up to 80%, and stable downregulation
led to a 65% and 47% increase in cellular SEAP and tPA productivity, respec-
tively [134]. Although the modulation of cytoskeleton dynamics appears to be
auspicious for improving CHO cell behavior, it still remains a neglected field for
genetic engineering of mammalian production cell factories.
Recently, it was reported that the lack of a telomeric region of chromosome 8
correlates with increased productivities and higher production stabilities of mAb
producing CHO cell lines [81]. Further studies indicated that the knockout of the
gene Fam60A, which is located within this telomeric region on Chr8 in CHO-K1a
cells, leads to the isolation of significantly more clones exhibiting higher protein
production stabilities of mAbs during long-term cultivation [80], and disruption
of a second gene within this region, C12orf35, leads to increased productivities
in recombinant CHO cell lines [135].
220 9 CHO Cell Engineering for Improved Process Performance and Product Quality

The extraordinarily high potential of miRNAs as a powerful cell engineering

tool has recently been highlighted by various experimental setups to improve
productivity of CHO cell lines. Barron et al. discovered that introduction of
miR-7a-5p mimics in CHO cells decelerated cellular proliferation rates but
increased cell-specific SEAP productivity [136]. Conversely, stable inhibition
of miR-7a resulted by a different group in an increase in culture longevity and
therefore elevated volumetric SEAP expression levels [114]. In addition, stable
overexpression of members of the miR-17-92a cluster enhanced cell-specific
mAb productivity, while enforced expression of the entire miR-17-92a cluster did
not improve cellular performance [118]. These results are in line with findings by
Jadhav et al., who discovered that stable overexpression of miR-17-5p increases
recombinant protein expression in CHO cells by approximately threefold [117].
A recently published follow-up study by Loh et al. unraveled that overexpression
of miR-92a resulted in enhanced recombinant protein production in CHO
cells primarily because of modulation of cholesterol biosynthesis and increased
Golgi volume, which in turn increased protein secretion [119]. Targeting the
well-characterized tumor suppressor miRNA, miR-34a, by stable depletion
using a miRNA sponge decoy vector, the overall product yield in both fed-batch
and small-scale clonal batch CHO production cultures was enhanced by
approximately twofold, despite having a negative impact on cell growth [120].
A transient screening performed by Strotbek et al. identified nine miRNAs
to increase mAb titer. Two of these were human encoded, hsa-miR-557 and
hsa-miR-1287, which could be demonstrated to positively affect growth-
and cell-specific productivity after stable overexpression in CHO cells [60].
A demonstration of the huge potential of the miRNA technology, especially
with regard to difficult-to-express biologics, was recently provided by Fischer
et al., who engineered CHO host cells by stable introduction of miR-557 [124].
MiRNA-engineered CHO cells were tested against control host cells (lacking
the proproductive miR-557), and compared in two independent CLD campaigns
using an easy as well as a difficult-to-express antibody [124]. Constitutive
overexpression of miR-557 increased overall CLD performance and clonally
derived final production cell lines exhibited an average increase in mAb titer of
twofold in representative fed-batch cultivation processes, without having any
detectable effects on product quality [124].
An unexpectedly huge number of miRNAs influencing diverse biotechno-
logically relevant cellular functions such as protein production, cell growth,
apoptosis, and necrosis were identified in an even more comprehensive study
[47]. Out of these, the entire miR-30 family contributed to enhanced recom-
binant protein expression in CHO cells, which could eventually be ascribed
to an increase in cell-specific productivity (miR-30c, miR-30e) or cell growth
(miR-30a) by stable miR-30 overexpression [47]. It later turned out that the
proproductive effects of the miR-30 family in CHO cells was partly mediated by
the regulation of genes involved in the ubiquitin pathway such as the S-phase
kinase associated protein 2 (SKP2) [137]. In addition, miR-2861 was identified
in the large-scale miRNA screen as a novel miRNA in CHO cells and could be
presented as an enhancer of protein production by modulating HDAC5 without
negatively influencing product quality attributes [115]. Furthermore, modulation
 9.3 Applications of Cell Line Engineering Approaches in CHO Cells 221

of oxidative metabolism and mitochondrial activity by stable sequestration of

miR-23 expression in CHO cells resulted in an average threefold increase in spe-
cific SEAP productivity by modulating LETM1 and IDH1 [122]. Emmerling et al.
presented miR-483 as a species and product-independent enhancer of cellular
productivity in mammalian cells, which enhanced recombinant mAb as well as
adeno-associated virus production in CHO and HeLa cells, respectively [121].
Recently, miR-143 was reported to improve production of difficult-to-express
proteins by targeting MAPK7 [123]. Given the substantial increase in CHO cell
engineering studies using miRNAs, the authors believe that all these examples
are just the beginning of a larger number of miRNA species, which will soon
become available for next-generation CHO cell engineering.

9.3.2 Repression of Cell Death and Acceleration of Growth

A typical observation toward the end of a manufacturing process is that the via-
bility of the grown cell culture decreases because of an increasing percentage of
dying cells in the bioreactor. A dying cell culture population usually consists of
a mixture of cells suffering from various forms of cell death including apopto-
sis, necrosis, and autophagy [138, 139]. A comprehensive list of cell engineering
studies aiming at the modulation of cell death or cell proliferation in CHO cells
is provided in Table 9.2.
The programmed cell death, which is also called apoptosis, represents a
well-characterized cellular pathway because of its fundamental importance in
the development of cancer where this form of cell death has been lost by tumor
cells [164]. Apoptosis is an essential cellular process and can be modulated by
changing the expression of key proteins of the different apoptotic pathways, the
intrinsic and extrinsic apoptotic pathways. Apoptosis is induced by nutrient
depleted (spent) media or by the accumulation of toxic metabolites toward the
end of a cultivation process [140, 141, 143, 144, 146, 154, 156].
In this context, a major problem is that dying cell cultures directly contribute to
a loss of product quality attributes such as mAb fragmentation or decreased mat-
uration of N-glycans at the Fc part of the antibody. This loss of product integrity
occurs as proteases and other enzymes are released from nonviable cells and
accumulate in the bioreactor at the end of the bioprocess [165]. Another issue
connected with increased cell death is a dramatic loss of cell culture harvest per-
formance because of markedly reduced filterability properties of the cell culture
supernatant. When it comes to the development of a commercial production
process, harvest performance is of critical importance for a successful implemen-
tation and scale-up of a bioprocess. Hence, a delayed onset of cell death during
bioprocessing would be highly desirable to increase harvest viabilities, culture
longevity, and ultimately product yields [166, 167].
Ectopic expression of antiapoptotic genes has become one of the mostly favored
strategies to overcome apoptosis in CHO manufacturing cell lines. Overex-
pression of antiapoptotic genes such as B-cell lymphoma XL (BCL-XL), B-cell
lymphoma 2 (BCL2), apoptosis and caspase activation inhibitor (AVEN), Fas
apoptotic inhibitory molecule (FAIM), X-linked inhibitor of apoptosis (XIAP),
or myeloid cell leukemia 1 (MCL1) could be demonstrated to prolong cultivation
Table 9.2 CHO cell engineering approaches for enhanced apoptosis resistance and cell growth in CHO cell lines.

Cellular Origin of
pathway engineering gene Target gene Engineered phenotype References

Apoptosis Human (overexpression) B-cell lymphoma 2 (BCL2) 75% Increase in maximum viable cell density Tey et al. [140]
Human (overexpression) BCL2 and BCL2-like 1 (BCL-xL) Improved viability and enhanced apoptosis Mastrangelo et al.
resistance [141]
Meents et al. [142]
Human (overexpression) X-linked inhibitor of apoptosis Improved apoptosis resistance Sauerwald et al.
(XIAP) [143]
Human (overexpression) Apoptosis, caspase inhibitor (AVEN) Improved apoptosis resistance Figueroa et al. [144]
and BCL-xL
Human (overexpression) BCL-xL 88% Increase in viability and enhances IgG Chiang and Sisk
titer by >2-fold [145]
Human (overexpression) Myelocytomatosis oncogene (c-myc) Improved apoptosis resistance and increased Infandi and
and BCL2 viable cell density Al-Rubeai (2005)
CHO (overexpression) Fas apoptotic inhibitory molecule Improved apoptosis resistance leading to 80% Wong et al. [146]
(FAIM) increased VCD and 2.5-fold enhanced
interferon 𝛾 (IFN𝛾) titer
Bombyx mori silkworm Apoptosis-inhibiting 30K protein Increased viable cell density and enhanced Choi et al. [147]
hemolymph (30Kc6) erythropoietin (EPO) titer of up to 10-fold
(overexpression)
Human (overexpression) Telomerase reverse transcriptase Increased apoptosis resistance resulting in Crea et al. [148]
(TERT) higher viable cell density
Human and adenoviral AVEN and control protein E1B 19K Increased viable cell density and viability; 50% Figueroa et al. [149]
(overexpression) (E1B-19K) enhanced IgG titer
Mouse (overexpression) Murine double-mutant 2 (MDM2) Increased apoptosis resistance Arden et al. [150]
Human (overexpression) E2F transcription factor 1 (E2F1) 20% increased viable cell density and Majors et al. [151]
improved proliferation
Human and adenoviral AVEN, E1B-19K and a mutant form 60% Increase in viable cell density and 80% Dorai et al. [152]
(overexpression) of XIAP (EAX197) enhanced IgG titer
CHO (overexpression) Heat-shock proteins 27 and 70 Extended culture longevity and 2.5-fold Lee et al. [153]
(HSP27 and HSP70) increase in IFN𝛾 titer
Human (overexpression) Myeliod cell leukemia 1 (MCL1) Improved viability and 20–35% increased IgG Majors et al. [154]
titer
Human (overexpression) RAC-𝛼 serine/threonine-protein Delayed onset of apoptosis and autophagy Hwang and Lee
kinase (AKT1) during batch culture [155]
Human (overexpression) Mutated form of BCL-xL (Asp29Asn Improved apoptosis resistance Majors et al. [156]
variant)
CHO (overexpression) Chinese hamster heat-shock protein 2.2-Fold higher peak VCD, sustained viability, Tan et al. [157]
27 (HSP27) and 2.3-fold increase in mAb titer
CHO (knockout) BCL2-associated X protein (BAX) Increased apoptosis resistance because of Cost et al. [158]
and BCL2-antagonist/killer (BAK) inhibition of caspase activation leading up to
fivefold increase in IgG titer
CHO (knockout) BAX, BAK, and FUT8 Prolonged culture longevity because of Grav et al. [159]
diminished apoptosis; engineered clones
produced afucosylated IgGs
CHO (knockdown) Caspase 3 Extended culture longevity by more than two Kim and Lee (2002)
days in batch cultures
CHO (knockdown) Caspase 3 and 7 Enhanced cell viability and 55% increase in Sung et al. [160]
hTPO titer
CHO (knockdown) Caspase 8 and 9 Enhanced viability in batch and fed-batch Yun et al. (2007)
cultivations
CHO (knockdown) Alpha-1,3/1,6-mannosyltransferase Elevated cell density and culture longevity; Wong et al. [146]
(ALG2), Requiem (REQ), Fas 1.2- to 2.5-fold increase in IFN𝛾 titer
(TNFRSF6)-associated via death
domain (FADD), and Fas apoptotic
inhibitory molecule (FAIM)
Table 9.2 (Continued)

Cellular Origin of
pathway engineering gene Target gene Engineered phenotype References

CHO (knockdown) Bax and Bak Enhanced cell viability and 35% increase in Lim et al. [161]
IFN𝛾 titer
Autophagy and CHO (overexpression) BCL-xL Delayed onset of autophagy and apoptosis Kim et al. [34]
apoptosis
Human (BCL-2) and CHO BCL-2 and Beclin-1 Extended culture longevity and higher viable Lee et al. [162]
(Beclin-1) (overexpression) cell density because of decreased apoptosis
and autophagy
Proliferation Human (overexpression) Cyclin-dependent kinase like 3 Increased maximum viable cell density Jaluria et al. [163]
(CDKL3) and cytochrome c oxidase
subunit (COX15)
CHO (overexpression) Valosin-containing protein (VCP) Increased cell proliferation and viability Doolan et al. [96]
CHO (knockdown) Cofilin (CFL1) 65% (SEAP) and 47% (tPA) increase in Hammond and Lee
specific productivity [134]
miRNA Human (overexpression) miR-557 Enhanced cell growth Strotbek et al. [60]
Fischer et al. [124]
CHO (overexpression) miR-30a Enhanced cell growth Fischer et al.
[47, 115]
 9.3 Applications of Cell Line Engineering Approaches in CHO Cells 225

processes by inhibiting apoptosis [140–144, 146]. Of note, a recent study

demonstrated that overexpression of heat-shock protein 27 (HSP27) in CHO
cells delayed the activation of caspases and hence apoptosis, which finally led to
increased monoclonal antibody yields in fed-batch cultivation process [157].
Inhibition of proapoptotic genes can also be exploited to constitutively increase
cell line performance of CHO manufacturing cell lines. Permanent genomic
knockout of BCL2-associated X protein (BAX) and BCL2-antagonist/killer
(BAK) expression in CHO production cells via ZFN improved resistance
to apoptosis by decreasing the activation of caspases and thus enhanced
therapeutic protein production by up to fivefold compared to controls
[158]. In order to achieve an efficient knockdown of target gene siRNAs
are often applied to specifically repress gene function in order to achieve
improved apoptosis resistance, modified glycosylation pattern, increased
metabolic efficiency, or simply enhanced recombinant protein production
[88, 97, 125, 134, 146, 160, 161, 168–182]. Stable siRNA-mediated knockdown
of BAX and BAK had been reported to increase culture longevity of CHO
cell processes at nutrient-depleted or high osmolytic cultivation conditions,
which finally led to improved cell growth and final interferon 𝛾 (IFN𝛾) titer
[161]. Another antiapoptotic cell engineering strategy comprised repression of
caspase-3 (CASP3) and -7 (CASP7), which were both silenced in parallel result-
ing in an increased performance of siRNA-engineered CHO cells during sodium
butyrate (NaBu) treatment [160]. Sodium butyrate is an unspecific inhibitor
of histone deacetylases (HDACs) and treatment of CHO cells with NaBu has
shown to increase cell-specific productivity but also to induce apoptosis. In
the presence of 1 mM sodium butyrate, knockdown of CASP3 and CASP7
increased cell viability and prolonged the cultivation process finally resulting in
a substantial increase in human thrombopoietin (hTPO) yield.
In another study, Wong et al. performed transcriptomics studies in CHO cells,
which identified four early apoptosis signaling genes to be differentially expressed
at the end of the cultivation process [146]. In this study, two proapoptotic genes
(REQUIEM and ALG2) were stably inhibited by siRNAs, and higher apoptosis
resistance, increased cell growth, and thus enhanced final IFNγ product yield
were observed in engineered cell lines [146]. A recent study showed that sta-
ble knockdown of breast cancer 1 (BRCA1) gene in CHO cells using shRNAs
increased mAb productivity up to 5.3-fold as well as production stability com-
pared to control cells [183]. Although mock control cells exhibited decreased
H3K4 methylation levels after long-term cultivation, H3K4 methylation levels
remained unaffected in stable BRCA1 downregulated cell lines [183].
Besides apoptosis, autophagy and necrosis contribute to cell death in bio-
processes. Autophagy is triggered under starvation conditions when nutrient
supply is limited at the end of a CHO production process [184]. Autophagy
is a process that enables mammalian cells to generate energy by degradation
of their own organelles and other cellular constituents [185]. Necrosis (or
necroptosis) is defined as a form of cell death where the cell loses its mem-
brane integrity because of mechanical or osmotic stress. CHO cell engineering
approaches employing overexpression of the serine–threonine protein kinase
1 (AKT1) or the combination of BCL2 and Beclin-1 (BECN1), a component
226 9 CHO Cell Engineering for Improved Process Performance and Product Quality

of the phosphatidylinositol-3-kinase (PI3K) complex, have successfully been

applied to simultaneously target both autophagy and apoptosis pathways in
order to keep cell viability at a high level and to prolong cultivation times
[155, 162]. Interestingly, forced expression of a core autophagy pathway genes
called autophagy-related protein 9A (ATG9A) in CHO cells could not improve
susceptibility to autophagy [186]. However, as overexpression of ATG9A has
only been investigated in a single CHO cell line, it might be possible that this
particular cell line did not react but other CHO cell lines may be improved.
Given that autophagy plays an essential role in CHO cells during bioprocessing,
it seems surprising that this cellular process has been rather neglected by CHO
cell researchers in the past. In the opinion of the authors, autophagy represents
a promising cellular mechanism to target in CHO cells in order to enhance
bioprocess performance in fed-batch cultivation processes. However, we still
need to understand autophagy in more detail and how it exactly works in CHO
cells cultivated in serum-free media.
Improving cell division rates and proliferation to accelerate growth phases
in bioprocesses has also been frequently addressed by cell line engineering
studies. As cell cultures usually show decreased cell-specific productivity during
exponential growth, shortening the period for biomass production would add
considerable value. As a result, production phases would be extended and prod-
uct yields likely will increase. Doolan et al. demonstrated that overexpression
of the valosin-containing protein (VCP) results in enhanced cell proliferation
and viability of recombinant CHO cell lines [96]. Other cell line engineering
approaches were targeting genes involved in cell cycle progression to improve
growth rates of CHO production cells. For instance, co-overexpression of the
cyclin-dependent kinase like 3 (CDKL3) and cytochrome-c oxidase subunit 15
(COX15) proteins resulted in an increased maximum VCD [163]. Kuystermans
and Al-Rubeai stably introduced the myelocytomatosis oncogene (c-MYC) into
CHO cells and increased maximum VCD by 70% without additional nutrient
supply [112]. Surprisingly, even a blockage of cell cycle progression might
be useful to enhance bioprocess performance, as overexpression of cell cycle
inhibitors such as p21CIP1 or p27KIP1 increased cell-specific productivity in
CHO cells at the expense of accompanying cell cycle arrest [108, 110, 111, 187].
To specifically accelerate cell growth and proliferation, cell cycle checkpoint
kinases constitute interesting targets to be addressed by host cell engineering
as these regulatory proteins control cell cycle progression and proliferation
[188]. Knockdown of the cell cycle checkpoint kinase ataxia telangiectasia and
Rad3 related (ATR) in mAb-producing CHO cells facilitated a more rapid estab-
lishment of high-producing cell clones during methotrexate (MTX)-mediated
gene amplification compared to control cells. Cell cycle checkpoint regulators
such as ATR induce cell cycle arrest as a consequence of MTX-mediated DNA
replication stress in treated CHO cells [189]. In this conjunction, the observed
accelerated cellular recovery was explained by the fact that ATR inhibition
abrogated MTX-mediated blockage of the cell cycle.
Apoptosis engineering also presents a suitable topic to be addressed using the
novel miRNA technology in order to enhance process robustness because of
increased harvest viabilities and resistance to stressful cultivation conditions in
 9.3 Applications of Cell Line Engineering Approaches in CHO Cells 227

a bioreactor [190]. The first functional investigation of the potential of miRNAs

to protect CHO cells from apoptotic cell death as presented by Druz et al.
using, miR-466h. The authors discovered miR-466h to induce apoptosis in
CHO cells during starvation in nutrient-depleted culture medium [191, 192].
ShRNA-mediated repression of miR-466h in CHO-SEAP cells increased the
integral of VCD and culture longevity because of inhibition of apoptosis [193].
In addition, Druz et al. could further demonstrate that knockdown of miR-466h
led to increased expression of the five antiapoptotic genes Bcl-2-like protein
2 (BCL2L2), Dolichyl-diphosphooligosaccharide—protein glycosyltransferase
subunit (DAD1), baculoviral IAP repeat-containing protein 6 (BIRC6), signal
transducer and activator of transcription 5A (STAT5A), and smoothened
(SMO). All of these genes actually harbor partial complementary binding sites
for miR-466h within the 3′ UTR of their transcripts, suggesting that these genes
might serve as direct targets of miR-466 in CHO cells [193]. Conserved gene
and microRNA function might be exploited to develop novel cell engineering
strategies for CHO cells. For instance, miR-34a has previously been demon-
strated to induce apoptosis in human and murine cells [194, 195]. MiR-34
actually represents the first miRNA to be investigated for its potential as a
cancer therapeutic in clinical trials. Based on the proapoptotic function of
miR-34, Kelly et al. stably sequestered miR-34a in CHO-SEAP cells in order to
protect cells from apoptosis. Unexpectedly, engineered cells lacking miR-34a
exhibited enhanced cell-specific productivity (see also Section 9.3.1), but no
increased resistance to apoptosis [120]. These results show that constitutive
repression of miRNAs in CHO cells might not always result in the expected
phenotype. Furthermore, because of a functional redundancy among miRNAs
in mammalian cells [46], it is very likely that other proapoptotic miRNAs present
on the CHO genome might balance out a loss of a single miRNA in a specific
cellular pathway. Consequently, gain-of-function approaches using miRNA
overexpression potentially represents a better strategy toward improved CHO
cell phenotypes compared to a miRNA knockout or knockdown.

9.3.3 Modulation of Posttranslational Modifications to Improve

Protein Quality
Although therapeutic proteins produced by CHO cells are predominantly used
for application in humans and are generally considered to be safe, they only
exhibit human-like but not human-identical PTMs [196–198]. Numerous cell
line engineering approaches have aimed at modulating PTMs and product
quality of recombinant proteins expressed in CHO cells and a comprehensive
list of engineering studies is provided in Table 9.3.
Among PTMs, glycosylation is of particular relevance because the glyco-
sylation state critically affects the properties of recombinant proteins such
as serum half-life, stability, immunogenicity, and functionality in the human
body [177, 224, 225]. Control and optimization of protein N-glycosylation
present on many recombinant glycoproteins are therefore crucial and alteration
in the glycan composition can be used to modulate the production quality
of a recombinant biotherapeutic. In addition, improper N-glycosylation of
Table 9.3 CHO cell engineering approaches for improved product quality in CHO cell lines.

Origin of
Cellular pathway engineering gene Target gene Engineered phenotype References

Glycosylation Human (overexpression) Alpha 2,6 sialyltransferase (ST6GAL) Expression of partly 𝛼2,6-sialylated Lee et al. [199]
recombinant proteins Zhang et al. [200]
Bragonzi et al. [201]
Rat (overexpression) ST6GAL Expression of 𝛼2,6-sialylated recombinant Minch et al. [202]
human tissue plasminogen activator (tPA)
Bovine (GnT-IV ) and 𝛼-1,3-d-mannoside 𝛽 1,4 56.2% Increase in tetra-antennary sugar Fukuta et al. [203]
human (GnT-V ) N-acetylglucosaminyltransferase chains on recombinant IFN𝛾
(overexpression) (GnT-IV) and 𝛼-1,6 d-mannoside 𝛽-1,6
N-acetylglucosaminyltransferase
(GnT-V)
Mouse (ST3GAL), rat ST3GAL, ST6GAL, and GnT-V Increase in the extent of sialylation of Fukuta et al. [204]
(ST6GAL) and human human recombinant IFN𝛾 of up to 80%
(GnT-V )
(overexpression)
Rat (overexpression) ST6GAL Improved sialylation and therapeutic Jassal et al. [205]
activity of recombinant IgG3
Human (overexpression) 𝛼-2,3 sialyltransferase (ST3GAL) and 𝛽 Expression of homogeneously distributed Weikert et al. [206]
1,4 galactosyltransferase (GalT) and >90% sialylated glycoproteins
Human (overexpression) ST6GAL and GalT Increased sialylation level of recombinant Jeong et al. [98]
human EPO
CHO (overexpression) CMP-sialic acid transporter (CMP-SAT) 4–16% Increased sialylation of human IFN𝛾 Wong et al. [207]
Human (overexpression) CMP-sialic acid synthetase (CMP-SAS), Further enhanced sialylation of Jeong et al. [208]
CMP-SAT, and ST3GAL recombinant human EPO
CHO (CMP-SAT) and Mutant uridine diphosphate-N-acetyl >10-Fold increase in CMP-sialic acid Son et al. [209]
human (ST3GAL) glucosamine 2-epimerase (GNE), concentration leading to a 32% increase in
(overexpression) CMP-SAT, and ST3GAL sialylation of human recombinant EPO
Rat (overexpression) 𝛽-1,4 N-acetylglucosaminyltransferase Expression of antibodies with increased Davies et al. [210]
III (GnT-III) bisecting glycan chains that resulted in a
20-fold lower antibody dosage with high
ADCC
Rat (GnT-III) and human GnT-III and Golgi 𝛼-mannosidase II Expression of nonfucosylated antibodies Ferrara et al. [211]
(ManII) (overexpression) (ManII) possessing N-glycans of the complex type
Human (overexpression) 𝛽-1,6 N-acetylglucosaminyl-transferase Redirection of the O-glycosylation pathway Prati et al. [212]
(C2GnT) in CHO cells
CHO (knockout) 𝛼-1,6-fucosyltransferase (FUT8) Production of completely nonfucosylated Yamane-Ohnuki
antibodies resulting in 100-fold enhanced et al. [213]
antibody-dependent cellular cytotoxicity
(ADCC)
CHO (knockout) FUT8 Production of completely nonfucosylated Malphettes et al.
antibodies with enhanced ADCC [214]
CHO (knockout) FUT8 Genomic knockout of FUT8 with Cristea et al. [215]
simultaneous integration of an antibody
expression cassette
CHO (knockout) N-acetylglucosaminyl-transferase 1 Production of recombinant proteins with Zhong et al. [216]
(MGAT1) Man5 as the predominant N-linked Sealover et al. [217]
glycosylation species
CHO (knockout) GDP-fucose transporter (SLC35C1) and Production of recombinant antibodies Zhang et al. [218]
CMP-sialic acid transporter (SLC35A1) lacking both fucose and sialic acid to
increase ADCC
CHO (knockout) MGAT4A, 4B, and 5 Expression of EPO with almost Yang et al. [219]
homogeneous biantennary N-glycans with a
minor amount of
poly-N-acetyl-lactosamine (poly-LacNAc)
CHO (knockout) MGAT4A/4B/5 and 𝛽-1,4 >90% Reduction in galactosylation of Yang et al. [219]
galactosyltransferase 1 (B4GALT1) recombinant EPO
CHO (knockout) 𝛽-1,3 N-acetylglucosaminyltransferase 2 Expression of recombinant EPO lacking Yang et al. [219]
(B3GNT2) poly-LacNAc
Table 9.3 (Continued)

Origin of
Cellular pathway engineering gene Target gene Engineered phenotype References

CHO (knockout) ST3GAL4/6 and MGAT4A/4B/5 Expression of recombinant EPO with Yang et al. [219]
heterogeneous tetra-antennary N-glycans
without sialylation
CHO (knockout) FUT8 and B4GALT1 Expression of an IgG1 with homogenous Yang et al. [219]
biantennary N-glycans without fucose and
almost no galactose
CHO (knockdown) Sialidase (NEU2) 60% Decrease in sialidase activity led to Ferrari et al. (1998)
increased sialic acid content in IFN𝛾 Ngantung et al.
[176]
CHO (knockdown) Sialidases NEU1 and 3 98% Decrease in sialidase activity led to Zhang et al. [182]
26–33% increase in sialic acid content of
IFN𝛾
CHO (knockdown) 𝛼-1,6-fucosyltransferase (FUT8) Reduction in core fucose by 60–88% Mori et al. [175]
resulted in 100-fold improved ADCC of the Beuger et al. [168]
produced IgG
CHO (knockdown) GDP-fucose 4,6-dehydratase (GMD) Production of 100% defucosylated Kanda et al. [172]
recombinant antibodies if culture medium
lacks l-fucose
CHO (knockdown) FUT8 and GMD Production of fully nonfucosylated Imai-Nishiya et al.
antibodies with improved ADCC [220]
CHO (knockdown) GDP-fucose transporter (GFT) 10–40% Increase in defucosylated AT-III Omasa et al. [73]
Posttranslational CHO (knockout) Peptidylglycine 𝛼-amidating Reduction in C-terminal amidated species Skulj et al. [221]
modifications monooxygenase (PAM) of recombinant monoclonal antibodies
Drug product CHO (knockout) Lipoprotein lipase (LPL) Removal of LPL host cell protein decreased Chiu et al. [222]
stability polysorbate-20 and -80 degradation and
increased drug product stability
Viral resistance CHO (knockout) CMP sialic acid transporter (SLC35A1) Increased resistance to MVM virus Mascarenhas et al.
infection [223]
 9.3 Applications of Cell Line Engineering Approaches in CHO Cells 231

pattern of recombinant antibodies can lead to severe immunological reactions

[169]. Therefore, interfering with genes involved within the glycosylation
pathways might be a promising strategy to generate CHO cells that will express
recombinant proteins with tailored glycosylation patterns. Genomic knockout
of N-acetylglucosaminyltransferase (MGAT1) in CHO cells could be shown to
enable production of glycoproteins with Man5 as the predominant N-linked
glycosylation species [216, 217]. An impressive example toward the development
of novel designer CHO cells capable of producing glycoproteins with predefined
glycopatterns was a detailed ZFN-assisted knockout screening of 19 different
glycosylation genes in recombinant CHO cells [219]. The authors nicely demon-
strated a variety of knockout combinations to achieve a desired glycosylation
profile on recombinant proteins by combinatorial genome editing approaches,
thereby highlighting future potential of this genetic engineering technology
[219]. Further engineering efforts comprised the combinatorial introduction
of genes involved in different parts of the N-glycosylation pathway to steadily
improve N-glycan structures of recombinant proteins for therapeutic use.
These included different N-acetylglucosaminyltransferases (GnT-III, IV, and V),
uridine diphosphate-N-acetyl glucosamine 2-epimerase (GNE), CMP-sialic acid
synthetase (CMP-SAS), and Golgi α-mannosidase II (ManII) [98, 203, 206–211].
It has been reported that monoclonal antibodies lacking core fucose induce
stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and at lower
antibody doses [181]. As monoclonal antibodies represent by far the most
important class of therapeutic glycoproteins being manufactured, research
toward development of strategies to produce low or nonfucosylated Mab glycans
has been intensified. Modulation of α-1,6-fucosyltransferase (FUT8), an enzyme
that catalyzes the transfer of the core fucose to the Fc part of an antibody, has
been addressed by numerous groups. Homologous recombination-mediated
stable genomic knockout of FUT8 completely erased FUT8 activity in CHO
cells [213]. Similarly, ZFN-based genome editing leads to stable excision of the
FUT8 gene from the genome [214] and complete abolishment of fucosylation
on the Fc domain of IgG. To demonstrate the functionality of a combined
knockout/knockin strategy in CHO cells, a FUT8 knockout was performed
simultaneously with the introduction of an antibody expression cassette [215].
Other methods to reduce the fucosylation level of antibody glycans were also
examined. Mori et al. reported that an 80% knockdown of FUT8 mRNA by
anti-FUT8 siRNA resulted in >60% reduction of mAb fucosylation, leading
to a 100-fold improved ADCC [175]. Furthermore, constitutive knockdown
of FUT8 resulted in 88% defucosylated anti-IGF-1 receptor antibody without
negative effect on productivity [168]. Loss-of-function studies involving other
genes of the fucosylation pathway revealed that attenuation of GDP-mannose
4,6-dehydratase (GMD) expression was superior to FUT8 knockdown and
yielded entirely nonfucosylated mAbs [172]. Finally, a combined knockdown
of both FUT8 and GMD also resulted in fully defucosylated mAbs exhibiting
enhanced ADCC [220]. The first report that employed the novel CRISPR/Cas9
tool for precise genome editing technique in CHO cells described the genomic
disruption of two genes at a time involved in the O- and N-glycosylation
pathways, core 1 β3GalT specific molecular chaperone (COSMC) and FUT8
232 9 CHO Cell Engineering for Improved Process Performance and Product Quality

[226]. Subsequently, Grav et al. demonstrated that CRISPR/Cas9 can be used to

rapidly generate multiple knockout phenotypes in a single approach, enabling
the simultaneous disruption of FUT8, BAX, and BAK without noticeable
off-target effects [159]. An alternative approach used ZFNs, TALENs, and the
CRISPR-Cas9 to inactivate the GDP-fucose transporter (SLC35C1) in CHO cells
resulting antibodies lacking core fucose [227].
Another important PTM is sialylation. Desialylated serum glycoproteins
have significantly lower circulatory half-lives as compared to their sialylated
counterparts [169]. Thus, improving sialylation is an important factor for the
manufacturing of therapeutic proteins. In contrast to human and mouse cells,
CHO cells were shown to lack expression of α-2,6-sialyltransferase and only
express 𝛼-2,3-sialyltransferase [228]. Consequently, CHO cells inherently cannot
produce glycoproteins with similar terminal sialic acid content as compared to
human cell lines [229, 230]. Already in 1989, a CHO cell line that stably overex-
pressed 𝛽 galactoside 𝛼-2,6-sialyltransferase (ST6GAL) was generated, which was
able to secrete recombinant proteins additionally harboring 𝛼2,6-sialylated gly-
can residues [199]. Other groups later confirmed that overexpression of ST6GAL
of various species (human, mouse, and rat) could indeed increase sialylation con-
tent on N-glycans of recombinant proteins produced in genetically engineered
CHO cells [200–202, 204, 205]. Recently, a combinatorial approach of overex-
pression of 𝛼-2,6 sialyltransferase and disruption of ST3GAL4 and ST3GAL6
genes by CRISPR/Cas9 was performed to minimize the 𝛼-2,3 sialylation. This
leads indeed to increased 𝛼-2,6 sialylation level relative to 𝛼-2,3 sialylation [231].
Also, transient approaches have been tested where the expression of ST6Gal1 in
CHO and HEK293 513 cells increased the 𝛼-2,6 sialylation of an antibody [232]
and transient coexpression of ST6Gal1 and β4GalT1 significantly enhanced anti-
body sialylation [232]. In a combinatorial approach, an engineered CHO cell line
stably expressing the human 𝛼2,3-sialyltransferase (ST3Gal3), the rat-mutated
GNE/MNK-R263L-R266Q glycosylation enzymes, and the Chinese hamster
CMP-sialic acid transporter was successful in increasing tetra-sialylation of
recombinant human erythropoietin (EPO) [209]. The combinatorial knockout
of two different key carbohydrate transporters for GDP-fucose (SLC35C1) and
CMP-sialic acid (SLC35A1) using ZFN technology yielded an engineered CHO
cell line that produces afucosylated and asialylated glycoproteins [218, 233].
Applying the opposite rationale, glycoprotein sialylation levels could be
increased by inhibition of sialidases. The knockdown of different CHO sial-
idases by shRNA or siRNA improved the sialylation level of recombinant
proteins, particularly for the sialidases NEU2 and NEU3 [176, 182]. Recently,
the co-overexpression of the N-acetylglucosaminyltransferases I (MGAT1) and
IV (MGAT4) was reported to improve sialylation of albumin-EPO. The sialic
acid content of the recombinant protein was highest in cells with excess MGAT4
gene, and these cells showed a higher tri- and tetra-antennary structure than
control cells [234].
Finally, although the modulation of N-linked glycosylation pattern on recom-
binant proteins has gained major attention during the last decades, modulation
of O-glycosylation has also been studied in CHO cells [212]. In addition, a
recent study has found some interesting new features of the O-glycoproteome
in liver cells [235]. However, it still needs to be elucidated if the findings made
 9.4 Conclusions 233

by Schjoldager et al. can be confirmed in CHO cells as well. Furthermore, as

alterations in N-glycosylation still seem to have a more dramatic impact on the
therapeutic potential and safety profile of recombinant proteins, further efforts
regarding modulation of O-glycosylation will be less expected.
The presence of C-terminal amidated species of mAbs produced by CHO
cells is another PTM of amino acid residues influencing the variability of
monoclonal antibodies and thereby their quality [236–238]. The ZFN-mediated
genomic knockout of the peptidylglycine 𝛼-amidating monooxygenase in CHO
cells led to a significant reduction in C-terminal amidated mAbs, resulting in a
more homogeneous product [221]. Heterogeneity of C-terminal lysine levels is
often observed in therapeutic monoclonal antibodies. A recent study showed
that the CRISPR/Cas9 knockout of carboxypeptidase D (CpD) completely
abolished C-terminal lysine cleavage [239].
In the past, the influence of the production cell line and its cellular entities on
drug product stability had rather been a neglected field of investigation. A sur-
prising influence of host cell protein (HCP) content on polysorbate degradation
was recently presented by Chiu et al. [222]. In this study, the authors demon-
strated that an individual HCP, lipoprotein lipase (LPL), which was completely
copurified with the therapeutic protein, led to product instability in the final drug
product formulation because of polysorbate degradation [222]. Genomic knock-
out of LPL in CHO cells using CRISPR and TALENs, however, could markedly
improve drug product stability because of the absence of polysorbate degradation
of material produced by engineered CHO cell lines [222].
Viral safety is another very important factor for biopharmaceutical process
development. In the past, it was shown that viruses such as the parvovirus
minute virus of mice (MVM) can successfully infect and replicate in CHO
cell cultures and thus represents a vital financial and regulatory threat for
CHO-based production processes, as occurrence of any viral contamination can
oblige the manufacturer to shut down an entire production facility for months
[240]. Consequently, efforts are currently aiming at increasing regulatory safety
of CHO manufacturing cell lines by host cell engineering in order to reduce the
risk of viral contaminations. As the presence of sialic acids on cell surface recep-
tors play a critical role in viral entry mechanisms of MVM viruses [241, 242],
Mascarenhas et al. knocked out a sialic acid transporter (SLC35A1) using ZFN
in CHO host cells [223]. Knockout cell lines showed almost entirely asialylated
cell surface proteins [223]. As a result, SLC35A1 knockout cells were completely
resistant to MVM infection [223]. Of note, however, if sialylation is an important
PTM of a therapeutic protein such as EPO, these engineered CHO host cell lines
lacking the ability to sialylate were of course not the vital option to be employed
for commercial production.

9.4 Conclusions
Isolated more than 60 years ago, various sublines of the original CHO cell line
have found a solid place both within the biopharmaceutical industry and in
academic research laboratories all over the world. There are certainly numerous
key factors that have eventually contributed to the great success of CHO cells
234 9 CHO Cell Engineering for Improved Process Performance and Product Quality

to become the most frequently employed mammalian expression system for

therapeutic proteins. However, the biopharmaceutical industry is still facing
tremendous challenges, e.g. (i) an increasing number of complex biological
formats that need to be produced at similar amounts as classical monoclonal
antibodies, (ii) the requirement for tailored product characteristics (biosimilars),
(iii) reductions in health care system budgets, and (iv) an overall elevated number
of drug candidates that puts enormous pressure on the global development and
manufacturing capacity for biologics. These and additional factors eventually
require regular and systematic “updates” of CHO host cell lines. Although we
are still far away from holistically understanding the complexity of CHO cells,
the abovementioned cell line engineering approaches have valuably contributed
to substantial improvements of CHO manufacturing cell lines over the past
decades. However, there is still room for improvement! As current CHO cell
lines express a plethora of endogenous genes that are presumably not required
or even disadvantageous to advanced recombinant protein production and
growth in chemically defined media in stirred tank bioreactors, new cell line
engineering technologies such as precise genome editing or the use of noncoding
RNA-mediated pathway engineering will pave the way for generation of even
more advanced CHO cell lines. Advanced host cell lines in combination with
other critical cornerstones such as a high-performing vector system, a rapid cell
line generation process, an advanced and stable cell culture media platform, as
well as a scalable process finally forms the basis for successful and cost-effective
biopharmaceutical drug developments. Therefore, consideration of all these
elements will be the key toward successful and cost-effective biological drug
development in the future.

References
1 Kunert, R. and Reinhart, D. (2016). Advances in recombinant antibody man-
ufacturing. Appl. Microbiol. Biotechnol. 100: 3451–3461.
2 Lalonde, M.E. and Durocher, Y. (2017). Therapeutic glycoprotein production
in mammalian cells. J. Biotechnol. 251: 128–140.
3 Wells, E. and Robinson, A.S. (2017). Cellular engineering for therapeu-
tic protein production: product quality, host modification, and process
improvement. Biotechnol. J. 12 (1): https://doi.org/10.1002/biot.201600105.
4 Urlaub, G. and Chasin, L.A. (1980). Isolation of Chinese hamster cell
mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci.
U.S.A. 77: 4216–4220.
5 Urlaub, G., Kas, E., Carothers, A.M., and Chasin, L.A. (1983). Deletion of
the diploid dihydrofolate reductase locus from cultured mammalian cells.
Cell 33: 405–412.
6 Wurm, F.M. and Hacker, D. (2011). First CHO genome. Nat. Biotechnol. 29:
718–720.
7 Cockett, M.I., Bebbington, C.R., and Yarranton, G.T. (1990). High level
expression of tissue inhibitor of metalloproteinases in Chinese hamster
 References 235

ovary cells using glutamine synthetase gene amplification. Biotechnology 8:

662–667.
8 Sanders, P.G., Hussein, A., Coggins, L., and Wilson, R. (1987). Gene amplifi-
cation: the Chinese hamster glutamine synthetase gene. Dev. Biol. Stand. 66:
55–63.
9 Fan, L., Kadura, I., Krebs, L.E. et al. (2012). Improving the efficiency of CHO
cell line generation using glutamine synthetase gene knockout cells. Biotech-
nol. Bioeng. 109: 1007–1015.
10 Shukla, A.A., Wolfe, L.S., Mostafa, S.S., and Norman, C. (2017). Evolving
trends in mAb production processes. Bioeng. Transl. Med. 2: 58–69.
11 Jost, C. and Pluckthun, A. (2014). Engineered proteins with desired speci-
ficity: DARPins, other alternative scaffolds and bispecific IgGs. Curr. Opin.
Struct. Biol. 27: 102–112.
12 Spiess, C., Zhai, Q., and Carter, P.J. (2015). Alternative molecular formats
and therapeutic applications for bispecific antibodies. Mol. Immunol. 67:
95–106.
13 Johari, Y.B., Estes, S.D., Alves, C.S. et al. (2015). Integrated cell and process
engineering for improved transient production of a “difficult-to-express”
fusion protein by CHO cells. Biotechnol. Bioeng. 112: 2527–2542.
14 Lee, G.W., Fecko, J.K., Yen, A. et al. (2007). Improving the expression of a
soluble receptor:Fc fusion protein in CHO cells by coexpression with the
receptor ligand. Cell Technol. Cell Prod. 29–39.
15 Mathias, S., Fischer, S., Handrick, R. et al. (2018). Visualisation of intracel-
lular production bottlenecks in suspension-adapted CHO cells producing
complex biopharmaceuticals using fluorescence microscopy. J. Biotechnol.
271: 47–55.
16 Bentley, K.J., Gewert, R., and Harris, W.J. (1998). Differential efficiency of
expression of humanized antibodies in transient transfected mammalian
cells. Hybridoma 17: 559–567.
17 Pybus, L.P., Dean, G., West, N.R. et al. (2014). Model-directed engineering
of “difficult-to-express” monoclonal antibody production by Chinese hamster
ovary cells. Biotechnol. Bioeng. 111: 372–385.
18 Mason, M., Sweeney, B., Cain, K. et al. (2012). Identifying bottlenecks in
transient and stable production of recombinant monoclonal-antibody
sequence variants in Chinese hamster ovary cells. Biotechnol. Progr. 28:
846–855.
19 Seeliger, D., Schulz, P., Litzenburger, T. et al. (2015). Boosting antibody
developability through rational sequence optimization. mAbs 7: 505–515.
20 van der Kant, R., Karow-Zwick, A.R., Van Durme, J. et al. (2017). Prediction
and reduction of the aggregation of monoclonal antibodies. J. Mol. Biol. 429:
1244–1261.
21 Tartari, F., Santoni, M., Pistelli, M., and Berardi, R. (2017). Healthcare cost of
HER2-positive and negative breast tumors in the United States (2012–2035).
Cancer Treat. Rev. 60: 12–17.
22 Makrides, S.C. (1999). Components of vectors for gene transfer and expres-
sion in mammalian cells. Protein Expression Purif. 17: 183–202.
236 9 CHO Cell Engineering for Improved Process Performance and Product Quality

23 Gopalkrishnan, R.V., Christiansen, K.A., Goldstein, N.I. et al. (1999). Use

of the human EF-1alpha promoter for expression can significantly increase
success in establishing stable cell lines with consistent expression: a study
using the tetracycline-inducible system in human cancer cells. Nucleic Acids
Res. 27: 4775–4782.
24 Wurm, F.M. (2004). Production of recombinant protein therapeutics in culti-
vated mammalian cells. Nat. Biotechnol. 22: 1393–1398.
25 Kramer, O., Klausing, S., and Noll, T. (2010). Methods in mammalian cell
line engineering: from random mutagenesis to sequence-specific approaches.
Appl. Microbiol. Biotechnol. 88: 425–436.
26 Cho, S.W., Kim, S., Kim, J.M., and Kim, J.S. (2013). Targeted genome engi-
neering in human cells with the Cas9 RNA-guided endonuclease. Nat.
Biotechnol. 31: 230–232.
27 Galetto, R., Duchateau, P., and Paques, F. (2009). Targeted approaches for
gene therapy and the emergence of engineered meganucleases. Expert Opin.
Biol. Ther. 9: 1289–1303.
28 Miller, J.C., Tan, S., Qiao, G. et al. (2011). A TALE nuclease architecture for
efficient genome editing. Nat. Biotechnol. 29: 143–148.
29 Santiago, Y., Chan, E., Liu, P.Q. et al. (2008). Targeted gene knockout in
mammalian cells by using engineered zinc-finger nucleases. Proc. Natl. Acad.
Sci. U.S.A. 105: 5809–5814.
30 Lee, J.S., Grav, L.M., Lewis, N.E., and Faustrup Kildegaard, H. (2015).
CRISPR/Cas9-mediated genome engineering of CHO cell factories: appli-
cation and perspectives. Biotechnol. J. 10: 979–994.
31 Fire, A., Xu, S., Montgomery, M.K. et al. (1998). Potent and specific genetic
interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:
806–811.
32 Hamilton, A.J. and Baulcombe, D.C. (1999). A species of small antisense
RNA in posttranscriptional gene silencing in plants. Science 286: 950–952.
33 Bagasra, O. and Prilliman, K.R. (2004). RNA interference: the molecular
immune system. J. Mol. Histol. 35: 545–553.
34 Kim, V.N., Han, J., and Siomi, M.C. (2009). Biogenesis of small RNAs in ani-
mals. Nat. Rev. Mol. Cell Biol. 10: 126–139.
35 Forstemann, K., Horwich, M.D., Wee, L. et al. (2007). Drosophila microR-
NAs are sorted into functionally distinct argonaute complexes after
production by DICER-1. Cell 130: 287–297.
36 John, B., Enright, A.J., Aravin, A. et al. (2004). Human MicroRNA targets.
PLoS Biol. 2: e363.
37 Tomari, Y. and Zamore, P.D. (2005). Perspective: machines for RNAi. Genes
Dev. 19: 517–529.
38 Golden, D.E., Gerbasi, V.R., and Sontheimer, E.J. (2008). An inside job for
siRNAs. Mol. Cell 31: 309–312.
39 Lippman, Z. and Martienssen, R. (2004). The role of RNA interference in
heterochromatic silencing. Nature 431: 364–370.
40 Okamura, K., Balla, S., Martin, R. et al. (2008). Two distinct mechanisms
generate endogenous siRNAs from bidirectional transcription in Drosophila
melanogaster. Nat. Struct. Mol. Biol. 15: 581–590.
 References 237

41 Werner, A., Cockell, S., Falconer, J. et al. (2014). Contribution of natural

antisense transcription to an endogenous siRNA signature in human cells.
BMC Genomics 15: 19.
42 Bartel, D.P. (2009). MicroRNAs: target recognition and regulatory functions.
Cell 136: 215–233.
43 Gurtan, A.M. and Sharp, P.A. (2013). The role of miRNAs in regulating gene
expression networks. J. Mol. Biol. 425 (19): 3582–3600.
44 Barron, N., Sanchez, N., Kelly, P., and Clynes, M. (2011). MicroRNAs: tiny
targets for engineering CHO cell phenotypes? Biotechnol. Lett. 33: 11–21.
45 Hackl, M., Borth, N., and Grillari, J. (2012). miRNAs–pathway engineering of
CHO cell factories that avoids translational burdening. Trends Biotechnol. 30:
405–406.
46 Fischer, S., Handrick, R., Aschrafi, A., and Otte, K. (2015). Unveiling the
principle of microRNA-mediated redundancy in cellular pathway regulation.
RNA Biol. 12: 238–247.
47 Fischer, S., Buck, T., Wagner, A. et al. (2014). A functional high-content
miRNA screen identifies miR-30 family to boost recombinant protein pro-
duction in CHO cells. Biotechnol. J. 9: 1279–1292.
48 Gammell, P., Barron, N., Kumar, N., and Clynes, M. (2007). Initial identifica-
tion of low temperature and culture stage induction of miRNA expression in
suspension CHO-K1 cells. J. Biotechnol. 130: 213–218.
49 Hammond, S., Swanberg, J.C., Polson, S.W., and Lee, K.H. (2012). Profil-
ing conserved microRNA expression in recombinant CHO cell lines using
Illumina sequencing. Biotechnol. Bioeng. 109: 1371–1375.
50 Maccani, A., Hackl, M., Leitner, C. et al. (2014). Identification of microR-
NAs specific for high producer CHO cell lines using steady-state cultivation.
Appl. Microbiol. Biotechnol. 98 (17): 7535–7548.
51 Lee, R.C., Feinbaum, R.L., and Ambros, V. (1993). The C. elegans hete-
rochronic gene lin-4 encodes small RNAs with antisense complementarity to
lin-14. Cell 75: 843–854.
52 Ibanez-Ventoso, C., Vora, M., and Driscoll, M. (2008). Sequence relationships
among C. elegans, D. melanogaster and human microRNAs highlight the
extensive conservation of microRNAs in biology. PLoS One 3: e2818.
53 Pasquinelli, A.E., Reinhart, B.J., Slack, F. et al. (2000). Conservation of the
sequence and temporal expression of let-7 heterochronic regulatory RNA.
Nature 408: 86–89.
54 Lee, Y., Jeon, K., Lee, J.T. et al. (2002). MicroRNA maturation: stepwise
processing and subcellular localization. EMBO J. 21: 4663–4670.
55 Arvey, A., Larsson, E., Sander, C. et al. (2010). Target mRNA abundance
dilutes microRNA and siRNA activity. Mol. Syst. Biol. 6: 363.
56 Meleady, P., Gallagher, M., Clarke, C. et al. (2012). Impact of miR-7
over-expression on the proteome of Chinese hamster ovary cells. J. Biotech-
nol. 160: 251–262.
57 Ventura, A., Young, A.G., Winslow, M.M. et al. (2008). Targeted deletion
reveals essential and overlapping functions of the miR-17 through 92 family
of miRNA clusters. Cell 132: 875–886.
238 9 CHO Cell Engineering for Improved Process Performance and Product Quality

58 Bratkovic, T., Glavan, G., Strukelj, B. et al. (2012). Exploiting microRNAs for
cell engineering and therapy. Biotechnol. Adv. 30: 753–765.
59 Jadhav, V., Hackl, M., Bort, J.A. et al. (2012). A screening method to assess
biological effects of microRNA overexpression in Chinese hamster ovary
cells. Biotechnol. Bioeng. 109: 1376–1385.
60 Strotbek, M., Florin, L., Koenitzer, J. et al. (2013). Stable microRNA expres-
sion enhances therapeutic antibody productivity of Chinese hamster ovary
cells. Metab. Eng. 20: 157–166.
61 Klanert, G., Jadhav, V., Chanoumidou, K. et al. (2014). Endogenous
microRNA clusters outperform chimeric sequence clusters in Chinese
hamster ovary cells. Biotechnol. J. 9: 538–544.
62 Hackl, M., Jakobi, T., Blom, J. et al. (2011). Next-generation sequencing
of the Chinese hamster ovary microRNA transcriptome: identification,
annotation and profiling of microRNAs as targets for cellular engineering.
J. Biotechnol. 153: 62–75.
63 Johnson, K.C., Jacob, N.M., Nissom, P.M. et al. (2011). Conserved microR-
NAs in Chinese hamster ovary cell lines. Biotechnol. Bioeng. 108: 475–480.
64 Griffiths-Jones, S., Grocock, R.J., van Dongen, S. et al. (2006). miRBase:
microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 34:
D140–D144.
65 Kozomara, A. and Griffiths-Jones, S. (2014). miRBase: annotating high con-
fidence microRNAs using deep sequencing data. Nucleic Acids Res. 42:
D68–D73.
66 Diendorfer, A.B., Hackl, M., Klanert, G. et al. (2015). Annotation of addi-
tional evolutionary conserved microRNAs in CHO cells from updated
genomic data. Biotechnol. Bioeng. 112 (7): 1488–1493.
67 Borth, N., Mattanovich, D., Kunert, R., and Katinger, H. (2005). Effect of
increased expression of protein disulfide isomerase and heavy chain binding
protein on antibody secretion in a recombinant CHO cell line. Biotechnol.
Progr. 21: 106–111.
68 Hwang, S.O., Chung, J.Y., and Lee, G.M. (2003). Effect of
doxycycline-regulated ERp57 expression on specific thrombopoietin pro-
ductivity of recombinant CHO cells. Biotechnol Prog. 19 (1): 179–184.
69 Chung, J.Y., Lim, S.W., Hong, Y.J. et al. (2004). Effect of
doxycycline-regulated calnexin and calreticulin expression on specific
thrombopoietin productivity of recombinant Chinese hamster ovary cells.
Biotechnol Bioeng. 85 (5): 539–546.
70 Underhill, M.F., Coley, C., Birch, J.R. et al. (2003). Engineering mRNA trans-
lation initiation to enhance transient gene expression in chinese hamster
ovary cells. Biotechnol Prog. 19 (1): 121–129.
71 Kwon, R.J., Kim, S.K., Lee, S.I. et al. (2006). Artificial transcription factors
increase production of recombinant antibodies in Chinese hamster ovary
cells. Biotechnol. Lett. 28: 9–15.
72 Ohya, T., Hayashi, T., Kiyama, E. et al. (2008). Improved production of
recombinant human antithrombin III in Chinese hamster ovary cells by
ATF4 overexpression. Biotechnol. Bioeng. 100: 317–324.
 References 239

73 Omasa, T., Takami, T., Ohya, T. et al. (2008). Overexpression of GADD34

enhances production of recombinant human antithrombin III in Chinese
hamster ovary cells. J. Biosci. Bioeng. 106: 568–573.
74 Dreesen, I.A. and Fussenegger, M. (2011). Ectopic expression of human
mTOR increases viability, robustness, cell size, proliferation, and antibody
production of chinese hamster ovary cells. Biotechnol. Bioeng. 108: 853–866.
75 Tastanova, A., Schulz, A., Folcher, M. et al. (2016). Overexpression of YY1
increases the protein production in mammalian cells. J. Biotechnol. 219:
72–85.
76 Orellana, C.A., Marcellin, E., Gray, P.P., and Nielsen, L.K. (2017). Overex-
pression of the regulatory subunit of glutamate-cysteine ligase enhances
monoclonal antibody production in CHO cells. Biotechnol. Bioeng. 114:
1825–1836.
77 Page, M.J. and Sydenham, M.A. (1991). High level expression of the human-
ized monoclonal antibody Campath-1H in Chinese hamster ovary cells.
Biotechnology (N Y). 9 (1): 64–68.
78 Wu, S.C., Hong, W.W., and Liu, J.H. (2008). Short hairpin RNA targeted
to dihydrofolate reductase enhances the immunoglobulin G expression
in gene-amplified stable Chinese hamster ovary cells. Vaccine. 26 (38):
4969–4974. https://doi.org/10.1016/j.vaccine.2008.06.081.
79 Romand, S., Jostock, T., Fornaro, M. et al. (2016). Improving expression of
recombinant human IGF-1 using IGF-1R knockout CHO cell lines. Biotech-
nol. Bioeng. 113: 1094–1101.
80 Ritter, A., Nuciforo, S., Schulze, A. et al. (2017). Fam60A plays a role for
production stabilities of recombinant CHO cell lines. Biotechnol. Bioeng. 114:
701–704.
81 Ritter, A., Voedisch, B., Wienberg, J. et al. (2016). Deletion of a telomeric
region on chromosome 8 correlates with higher productivity and stability of
CHO cell lines. Biotechnol. Bioeng. 113: 1084–1093.
82 Pieper, L.A., Strotbek, M., Wenger, T. et al. (2017). ATF6beta-based
fine-tuning of the unfolded protein response enhances therapeutic anti-
body productivity of Chinese hamster ovary cells. Biotechnol. Bioeng. 114:
1310–1318.
83 Pieper, L.A., Strotbek, M., Wenger, T. et al. (2017). Secretory pathway opti-
mization of CHO producer cells by co-engineering of the mitosRNA-1978
target genes CerS2 and Tbc1D20. Metab. Eng. 40: 69–79.
84 Matsuyama, R., Yamano, N., Kawamura, N., and Omasa, T. (2017). Length-
ening of high-yield production levels of monoclonal antibody-producing
Chinese hamster ovary cells by downregulation of breast cancer 1. J Biosci
Bioeng. 123 (3): 382–389. https://doi.org/10.1016/j.jbiosc.2016.09.006.
85 Pendse, G.J. and Bailey, J.E. (1994). Effect of Vitreoscilla hemoglobin expres-
sion on growth and specific tissue plasminogen activator productivity in
recombinant Chinese hamster ovary cells. Biotechnol. Bioeng. 44: 1367–1370.
86 Park, H., Kim, I.H., Kim, I.Y. et al. (2000). Expression of carbamoyl phos-
phate synthetase I and ornithine transcarbamoylase genes in Chinese
hamster ovary DHFR-cells decreases accumulation of ammonium ion in
culture media. J. Biotechnol. 81: 129–140.
240 9 CHO Cell Engineering for Improved Process Performance and Product Quality

87 Fogolin, M.B., Wagner, R., Etcheverrigaray, M., and Kratje, R. (2004). Impact
of temperature reduction and expression of yeast pyruvate carboxylase on
hGM-CSF-producing CHO cells. J. Biotechnol. 109: 179–191.
88 Kim, S.H. and Lee, G.M. (2007). Down-regulation of lactate
dehydrogenase-A by siRNAs for reduced lactic acid formation of Chinese
hamster ovary cells producing thrombopoietin. Appl. Microbiol. Biotechnol.
74: 152–159.
89 Wlaschin, K.F. and Hu, W.S. (2007). Engineering cell metabolism for
high-density cell culture via manipulation of sugar transport. J. Biotechnol.
131: 168–176.
90 Le, H., Vishwanathan, N., Kantardjieff, A. et al. (2013). Dynamic gene
expression for metabolic engineering of mammalian cells in culture. Metab.
Eng. 20: 212–220.
91 Chong, W.P., Reddy, S.G., Yusufi, F.N. et al. (2010). Metabolomics-driven
approach for the improvement of Chinese hamster ovary cell growth: over-
expression of malate dehydrogenase II. J. Biotechnol. 147: 116–121.
92 Tabuchi, H., Sugiyama, T., Tanaka, S., and Tainaka, S. (2010). Overexpression
of taurine transporter in Chinese hamster ovary cells can enhance cell viabil-
ity and product yield, while promoting glutamine consumption. Biotechnol.
Bioeng. 107: 998–1003.
93 Tabuchi, H. and Sugiyama, T. (2013). Cooverexpression of alanine amino-
transferase 1 in Chinese hamster ovary cells overexpressing taurine trans-
porter further stimulates metabolism and enhances product yield. Biotechnol.
Bioeng. 110: 2208–2215.
94 Kim, S.H. and Lee, G.M. (2007). Functional expression of human pyruvate
carboxylase for reduced lactic acid formation of Chinese hamster ovary cells
(DG44). Appl. Microbiol. Biotechnol. 76: 659–665.
95 Jeong, D., Kim, T.S., Lee, J.W. et al. (2001). Blocking of acidosis-mediated
apoptosis by a reduction of lactate dehydrogenase activity through antisense
mRNA expression. Biochem. Biophys. Res. Commun. 289: 1141–1149.
96 Doolan, P., Meleady, P., Barron, N. et al. (2010). Microarray and pro-
teomics expression profiling identifies several candidates, including the
valosin-containing protein (VCP), involved in regulating high cellular growth
rate in production CHO cell lines. Biotechnol. Bioeng. 106: 42–56.
97 Zhou, M., Crawford, Y., Ng, D. et al. (2011). Decreasing lactate level and
increasing antibody production in Chinese Hamster Ovary cells (CHO) by
reducing the expression of lactate dehydrogenase and pyruvate dehydroge-
nase kinases. J. Biotechnol. 153: 27–34.
98 Jeong, Y.T., Choi, O., Lim, H.R. et al. (2008). Enhanced sialylation of recom-
binant erythropoietin in CHO cells by human glycosyltransferase expression.
J. Microbiol. Biotechnol. 18: 1945–1952.
99 Tigges, M. and Fussenegger, M. (2006). Xbp1-based engineering of secretory
capacity enhances the productivity of Chinese hamster ovary cells. Metab.
Eng. 8: 264–272.
 References 241

100 Becker, E., Florin, L., Pfizenmaier, K., and Kaufmann, H. (2008). An XBP-1
dependent bottle-neck in production of IgG subtype antibodies in chemi-
cally defined serum-free Chinese hamster ovary (CHO) fed-batch processes.
J Biotechnol. 135 (2): 217–223. https://doi.org/10.1016/j.jbiotec.2008.03.008.
101 Ku, S.C., Ng, D.T., Yap, M.G., and Chao, S.H. (2008). Effects of overex-
pression of X-box binding protein 1 on recombinant protein production
in Chinese hamster ovary and NS0 myeloma cells. Biotechnol. Bioeng. 99:
155–164.
102 Gulis, G., Simi, K.C., de Toledo, R.R. et al. (2014). Optimization of heterol-
ogous protein production in Chinese hamster ovary cells under overexpres-
sion of spliced form of human X-box binding protein. BMC Biotechnol. 14:
26. https://doi.org/10.1186/1472-6750-14-26.
103 Peng, R.W. and Fussenegger, M. (2009). Molecular engineering of exocytic
vesicle traffic enhances the productivity of Chinese hamster ovary cells.
Biotechnol. Bioeng. 102: 1170–1181.
104 Florin, L., Pegel, A., Becker, E. et al. (2009). Heterologous expression of the
lipid transfer protein CERT increases therapeutic protein productivity of
mammalian cells. J. Biotechnol. 141: 84–90.
105 Rahimpour, A., Vaziri, B., Moazzami, R. et al. (2013). Engineering the cel-
lular protein secretory pathway for enhancement of recombinant tissue
plasminogen activator expression in Chinese hamster ovary cells: effects of
CERT and XBP1s genes. J. Microbiol. Biotechnol. 23: 1116–1122.
106 Peng, R.W., Abellan, E., and Fussenegger, M. (2011). Differential effect of
exocytic SNAREs on the production of recombinant proteins in mammalian
cells. Biotechnol. Bioeng. 108: 611–620.
107 Le Fourn, V., Girod, P.A., Buceta, M. et al. (2014). CHO cell engineering to
prevent polypeptide aggregation and improve therapeutic protein secretion.
Metab. Eng. 21: 91–102.
108 Fussenegger, M., Schlatter, S., Datwyler, D. et al. (1998). Controlled prolif-
eration by multigene metabolic engineering enhances the productivity of
Chinese hamster ovary cells. Nat. Biotechnol. 16: 468–472.
109 Astley, K., Naciri, M., Racher, A., and Al-Rubeai, M. (2007). The role of
p21cip1 in adaptation of CHO cells to suspension and protein-free culture.
J Biotechnol. 130 (3): 282–290.
110 Mazur, X., Fussenegger, M., Renner, W.A., and Bailey, J.E. (1998). Higher
productivity of growth-arrested Chinese hamster ovary cells expressing the
cyclin-dependent kinase inhibitor p27. Biotechnol. Progr. 14: 705–713.
111 Bi, J.X., Shuttleworth, J., and Al-Rubeai, M. (2004). Uncoupling of cell
growth and proliferation results in enhancement of productivity in
p21CIP1 -arrested CHO cells. Biotechnol. Bioeng. 85: 741–749.
112 Kuystermans, D. and Al-Rubeai, M. (2009). cMyc increases cell number
through uncoupling of cell division from cell size in CHO cells. BMC
Biotechnol. 9: 76.
113 Lee, J.S., Ha, T.K., Park, J.H., and Lee, G.M. (2013). Anti-cell death engi-
neering of CHO cells: co-overexpression of Bcl-2 for apoptosis inhibition,
Beclin-1 for autophagy induction. Biotechnol Bioeng. 110 (8): 2195–2207.
https://doi.org/10.1002/bit.24879.
242 9 CHO Cell Engineering for Improved Process Performance and Product Quality

114 Sanchez, N., Kelly, P., Gallagher, C. et al. (2013). CHO cell culture longevity
and recombinant protein yield are enhanced by depletion of miR-7 activity
via sponge decoy vectors. Biotechnol. J. 9 (3): 396–404.
115 Fischer, S., Paul, A., Wagner, A. et al. (2015). miR-2861 as novel HDAC5
inhibitor in CHO cells enhances productivity while maintaining product
quality. Biotechnol. Bioeng. 112 (10): 2142–2153.
116 Clarke, C., Henry, M., Doolan, P., et al. (2012). Integrated miRNA, mRNA
and proteinexpression analysis reveals the role of post-transcriptional reg-
ulation incontrolling CHO cell growth rate. BMCGenomics. 13:656. doi:10
.1186/1471-2164-13--656.
117 Jadhav, V., Hackl, M., Klanert, G. et al. (2014). Stable overexpression of
miR-17 enhances recombinant protein production of CHO cells. J. Biotech-
nol. 175: 38–44.
118 Loh, W.P., Loo, B., Zhou, L. et al. (2014). Overexpression of microRNAs
enhances recombinant protein production in Chinese hamster ovary cells.
Biotechnol. J. 9 (9): 1140–1151.
119 Loh, W.P., Yang, Y., and Lam, K.P. (2017). miR-92a enhances recombinant
protein productivity in CHO cells by increasing intracellular cholesterol
levels. Biotechnol. J. 12 (4): https://doi.org/10.1002/biot.201600488.
120 Kelly, P.S., Gallagher, C., Clynes, M., and Barron, N. (2015). Conserved
microRNA function as a basis for Chinese hamster ovary cell engineering.
Biotechnol. Lett. 37 (4): 787–798.
121 Emmerling, V.V., Fischer, S., Stiefel, F. et al. (2016). Temperature-sensitive
miR-483 is a conserved regulator of recombinant protein and viral vector
production in mammalian cells. Biotechnol. Bioeng. 113 (4): 830–841.
122 Kelly, P.S., Breen, L., Gallagher, C. et al. (2015). Re-programming CHO
cell metabolism using miR-23 tips the balance towards a highly productive
phenotype. Biotechnol. J. 10: 1029–1040.
123 Schoellhorn, M., Fischer, S., Wagner, A. et al. (2017). miR-143 targets
MAPK7 in CHO cells and induces a hyperproductive phenotype to
enhance production of difficult-to-express proteins. Biotechnol. Progr. 33
(4): 1046–1058.
124 Schoellhorn, M., Fischer, S., Wagner, A. et al. (2017). miR-143 targets
MAPK7 in CHO cells and induces a hyperproductive phenotype to
enhance production of difficult-to-express proteins. Biotechnol Prog. 33
(4): 1046–1058. https://doi.org/10.1002/btpr.2475.
125 Neermann, J. and Wagner, R. (1996). Comparative analysis of glucose and
glutamine metabolism in transformed mammalian cell lines, insect and
primary liver cells. J. Cell. Physiol. 166: 152–169.
126 Yip, S.S., Zhou, M., Joly, J. et al. (2014). Complete knockout of the lactate
dehydrogenase A gene is lethal in pyruvate dehydrogenase kinase 1, 2, 3
down-regulated CHO cells. Mol. Biotechnol. 56 (9): 833–838.
127 Kim, J.Y., Kim, Y.G., Han, Y.K. et al. (2011). Proteomic understanding of
intracellular responses of recombinant Chinese hamster ovary cells cul-
tivated in serum-free medium supplemented with hydrolysates. Appl.
Microbiol. Biotechnol. 89: 1917–1928.
 References 243

128 Carlage, T., Hincapie, M., Zang, L. et al. (2009). Proteomic profiling of
a high-producing Chinese hamster ovary cell culture. Anal. Chem. 81:
7357–7362.
129 Charaniya, S., Karypis, G., and Hu, W.S. (2009). Mining transcriptome data
for function-trait relationship of hyper productivity of recombinant antibody.
Biotechnol. Bioeng. 102: 1654–1669.
130 Fomina-Yadlin, D., Mujacic, M., Maggiora, K. et al. (2015). Transcriptome
analysis of a CHO cell line expressing a recombinant therapeutic protein
treated with inducers of protein expression. J. Biotechnol. 212: 106–115.
131 Kantardjieff, A., Jacob, N.M., Yee, J.C. et al. (2010). Transcriptome and pro-
teome analysis of Chinese hamster ovary cells under low temperature and
butyrate treatment. J. Biotechnol. 145: 143–159.
132 Yee, J.C., Gerdtzen, Z.P., and Hu, W.S. (2009). Comparative transcriptome
analysis to unveil genes affecting recombinant protein productivity in mam-
malian cells. Biotechnol. Bioeng. 102: 246–263.
133 Hayduk, E.J. and Lee, K.H. (2005). Cytochalasin D can improve heterologous
protein productivity in adherent Chinese hamster ovary cells. Biotechnol.
Bioeng. 90: 354–364.
134 Hammond, S. and Lee, K.H. (2012). RNA interference of cofilin in Chinese
hamster ovary cells improves recombinant protein productivity. Biotechnol.
Bioeng. 109: 528–535.
135 Ritter, A., Rauschert, T., Oertli, M. et al. (2016). Disruption of the gene
C12orf35 leads to increased productivities in recombinant CHO cell lines.
Biotechnol. Bioeng. 113: 2433–2442.
136 Barron, N., Kumar, N., Sanchez, N. et al. (2011). Engineering CHO cell
growth and recombinant protein productivity by overexpression of miR-7.
J. Biotechnol. 151: 204–211.
137 Fischer, S., Handrick, R., and Otte, K. (2015). The art of CHO cell engineer-
ing: a comprehensive retrospect and future perspectives. Biotechnol. Adv. 33:
1878–1896.
138 Al-Rubeai, M., Singh, R.P., Goldman, M.H., and Emery, A.N. (1995). Death
mechanisms of animal cells in conditions of intensive agitation. Biotechnol.
Bioeng. 45: 463–472.
139 Rangan, S., Kamal, S., Konorov, S.O. et al. (2018). Types of cell death and
apoptotic stages in Chinese Hamster Ovary cells distinguished by Raman
spectroscopy. Biotechnol. Bioeng. 115: 401–412.
140 Tey, B.T., Singh, R.P., Piredda, L. et al. (2000). Influence of BCL-2 on cell
death during the cultivation of a Chinese hamster ovary cell line expressing
a chimeric antibody. Biotechnol. Bioeng. 68: 31–43.
141 Mastrangelo, A.J., Hardwick, J.M., Bex, F., and Betenbaugh, M.J. (2000).
Part I. BCL-2 and BCL-XL limit apoptosis upon infection with alphavirus
vectors. Biotechnol. Bioeng. 67: 544–554.
142 Meents, H., Enenkel, B., Eppenberger, H.M. et al. (2002). Impact of coex-
pression and coamplification of sICAM and antiapoptosis determinants
bcl-2/bcl-x(L) on productivity, cell survival, and mitochondria number in
CHO-DG44 grown in suspension and serum-free media. Biotechnol Bioeng.
80 (6): 706–716.
244 9 CHO Cell Engineering for Improved Process Performance and Product Quality

143 Sauerwald, T.M., Betenbaugh, M.J., and Oyler, G.A. (2002). Inhibiting
apoptosis in mammalian cell culture using the caspase inhibitor XIAP and
deletion mutants. Biotechnol. Bioeng. 77: 704–716.
144 Figueroa, B. Jr., Chen, S., Oyler, G.A. et al. (2004). AVEN and BCL-XL
enhance protection against apoptosis for mammalian cells exposed to vari-
ous culture conditions. Biotechnol. Bioeng. 85: 589–600.
145 Chiang, G.G. and Sisk, W.P. (2005). Bcl-x(L) mediates increased produc-
tion of humanized monoclonal antibodies in Chinese hamster ovary cells.
Biotechnol Bioeng. 91 (7): 779–792.
146 Wong, D.C., Wong, K.T., Nissom, P.M. et al. (2006). Targeting early apop-
totic genes in batch and fed-batch CHO cell cultures. Biotechnol. Bioeng. 95:
350–361.
147 Choi, S.S., Rhee, W.J., Kim, E.J., and Park, T.H. (2006). Enhancement of
recombinant protein production in Chinese hamster ovary cells through
anti-apoptosis engineering using 30Kc6 gene. Biotechnol Bioeng. 95 (3):
459–467.
148 Crea, F., Sarti, D., Falciani, F., and Al-Rubeai, M. (2006). Over-expression
of hTERT in CHO K1 results in decreased apoptosis and reduced serum
dependency. J Biotechnol. 121 (2): 109–123.
149 Figueroa, B. Jr., Ailor, E., Osborne, D. et al. (2007). Enhanced cell culture
performance using inducible anti-apoptotic genes E1B-19K and Aven in
the production of a monoclonal antibody with Chinese hamster ovary cells.
Biotechnol Bioeng. 97 (4): 877–892.
150 Arden, N., Majors, B.S., Ahn, S.H. et al. (2007). Inhibiting the apoptosis
pathway using MDM2 in mammalian cell cultures. Biotechnol Bioeng. 97 (3):
601–614.
151 Majors, B.S., Arden, N., Oyler, G.A. et al. (2008). E2F-1 overexpression
increases viable cell density in batch cultures of Chinese hamster ovary cells.
J Biotechnol. 138 (3–4): 103–106.
152 Dorai, H., Kyung, Y.S., Ellis, D. et al. (2009). Expression of anti-apoptosis
genes alters lactate metabolism of Chinese Hamster Ovary cells in culture.
Biotechnol Bioeng. 103 (3): 592–608. https://doi.org/10.1002/bit.22269.
153 Lee, Y.Y., Wong, K.T., Tan, J. et al. (2009). Overexpression of heat shock
proteins (HSPs) in CHO cells for extended culture viability and improved
recombinant protein production. J Biotechnol. 143 (1): 34–43. https://doi.org/
10.1016/j.jbiotec.2009.05.013.
154 Majors, B.S., Betenbaugh, M.J., Pederson, N.E., and Chiang, G.G. (2009).
Mcl-1 overexpression leads to higher viabilities and increased production of
humanized monoclonal antibody in Chinese hamster ovary cells. Biotechnol.
Progr. 25: 1161–1168.
155 Hwang, S.O. and Lee, G.M. (2009). Effect of Akt overexpression on pro-
grammed cell death in antibody-producing Chinese hamster ovary cells.
J. Biotechnol. 139: 89–94.
156 Majors, B.S., Chiang, G.G., Pederson, N.E., and Betenbaugh, M.J. (2012).
Directed evolution of mammalian anti-apoptosis proteins by somatic hyper-
mutation. Protein Eng. Des. Sel. 25: 27–38.
 References 245

157 Tan, J.G., Lee, Y.Y., Wang, T. et al. (2015). Heat shock protein 27 overex-
pression in CHO cells modulates apoptosis pathways and delays activation
of caspases to improve recombinant monoclonal antibody titre in fed-batch
bioreactors. Biotechnol. J. 10: 790–800.
158 Cost, G.J., Freyvert, Y., Vafiadis, A. et al. (2010). BAK and BAX deletion
using zinc-finger nucleases yields apoptosis-resistant CHO cells. Biotechnol.
Bioeng. 105: 330–340.
159 Grav, L.M., Lee, J.S., Gerling, S. et al. (2015). One-step generation of triple
knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment.
Biotechnol. J. 10: 1446–1456.
160 Sung, Y.H., Lee, J.S., Park, S.H. et al. (2007). Influence of co-down-regulation
of caspase-3 and caspase-7 by siRNAs on sodium butyrate-induced apoptotic
cell death of Chinese hamster ovary cells producing thrombopoietin. Metab.
Eng. 9: 452–464.
161 Lim, S.F., Chuan, K.H., Liu, S. et al. (2006). RNAi suppression of Bax and
Bak enhances viability in fed-batch cultures of CHO cells. Metab. Eng. 8:
509–522.
162 Lee, J.S., Ha, T.K., Park, J.H., and Lee, G.M. (2013). Anti-cell death engi-
neering of CHO cells: co-overexpression of BCL-2 for apoptosis inhibition,
Beclin-1 for autophagy induction. Biotechnol. Bioeng. 110: 2195–2207.
163 Jaluria, P., Betenbaugh, M., Konstantopoulos, K., and Shiloach, J. (2007).
Enhancement of cell proliferation in various mammalian cell lines by gene
insertion of a cyclin-dependent kinase homolog. BMC Biotechnol. 7: 71.
164 Blagosklonny, M.V. (2005). Molecular theory of cancer. Cancer Biol. Ther. 4:
621–627.
165 Kaneko, Y., Sato, R., and Aoyagi, H. (2010). Changes in the quality of anti-
bodies produced by Chinese hamster ovary cells during the death phase of
cell culture. J. Biosci. Bioeng. 109: 281–287.
166 Arden, N. and Betenbaugh, M.J. (2004). Life and death in mammalian cell
culture: strategies for apoptosis inhibition. Trends Biotechnol. 22: 174–180.
167 Simon, L. and Karim, M.N. (2002). Control of starvation-induced apoptosis
in Chinese hamster ovary cell cultures. Biotechnol. Bioeng. 78: 645–657.
168 Beuger, V., Kunkele, K.P., Koll, H. et al. (2009). Short-hairpin-RNA-mediated
silencing of fucosyltransferase 8 in Chinese-hamster ovary cells for the pro-
duction of antibodies with enhanced antibody immune effector function.
Biotechnol. Appl. Biochem. 53: 31–37.
169 Ghaderi, D., Zhang, M., Hurtado-Ziola, N., and Varki, A. (2012). Production
platforms for biotherapeutic glycoproteins. Occurrence, impact, and chal-
lenges of non-human sialylation. Biotechnol. Genet. Eng. Rev. 28: 147–175.
170 Hong, W.W. and Wu, S.C. (2007). A novel RNA silencing vector to improve
antigen expression and stability in Chinese hamster ovary cells. Vaccine 25:
4103–4111.
171 Irani, N., Wirth, M., van Den Heuvel, J., and Wagner, R. (1999). Improve-
ment of the primary metabolism of cell cultures by introducing a new
cytoplasmic pyruvate carboxylase reaction. Biotechnol. Bioeng. 66: 238–246.
172 Kanda, Y., Imai-Nishiya, H., Kuni-Kamochi, R. et al. (2007). Establishment
of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: a new
246 9 CHO Cell Engineering for Improved Process Performance and Product Quality

strategy for generating completely non-fucosylated recombinant therapeutics.

J. Biotechnol. 130: 300–310.
173 Kellems, R.E. (1991). Gene amplification in mammalian cells: strategies for
protein production. Curr. Opin. Biotechnol. 2: 723–729.
174 Koterba, K.L., Borgschulte, T., and Laird, M.W. (2012). Thioredoxin 1 is
responsible for antibody disulfide reduction in CHO cell culture. J. Biotech-
nol. 157: 261–267.
175 Mori, K., Kuni-Kamochi, R., Yamane-Ohnuki, N. et al. (2004). Engineer-
ing Chinese hamster ovary cells to maximize effector function of produced
antibodies using FUT8 siRNA. Biotechnol. Bioeng. 88: 901–908.
176 Ngantung, F.A., Miller, P.G., Brushett, F.R. et al. (2006). RNA interference of
sialidase improves glycoprotein sialic acid content consistency. Biotechnol.
Bioeng. 95: 106–119.
177 Ohtsubo, K. and Marth, J.D. (2006). Glycosylation in cellular mechanisms of
health and disease. Cell 126: 855–867.
178 Pallavicini, M.G., DeTeresa, P.S., Rosette, C. et al. (1990). Effects of
methotrexate on transfected DNA stability in mammalian cells. Mol. Cell.
Biol. 10: 401–404.
179 Patel, M.S. and Korotchkina, L.G. (2001). Regulation of mammalian pyru-
vate dehydrogenase complex by phosphorylation: complexity of multiple
phosphorylation sites and kinases. Exp. Mol. Med. 33: 191–197.
180 Wu, S.-C. (2009). RNA interference technology to improve recombinant
protein production in Chinese hamster ovary cells. Biotechnol. Adv. 27:
417–422.
181 Yamane-Ohnuki, N. and Satoh, M. (2009). Production of therapeutic anti-
bodies with controlled fucosylation. MAbs 1: 230–236.
182 Zhang, M., Koskie, K., Ross, J.S. et al. (2010). Enhancing glycoprotein sialyla-
tion by targeted gene silencing in mammalian cells. Biotechnol. Bioeng. 105:
1094–1105.
183 Matsuyama, R., Yamano, N., Kawamura, N., and Omasa, T. (2017). Length-
ening of high-yield production levels of monoclonal antibody-producing
Chinese hamster ovary cells by downregulation of breast cancer 1. J. Biosci.
Bioeng. 123: 382–389.
184 Kim, J.Y., Kim, Y.G., and Lee, G.M. (2012). CHO cells in biotechnology for
production of recombinant proteins: current state and further potential.
Appl. Microbiol. Biotechnol. 93: 917–930.
185 Levine, B. (2005). Eating oneself and uninvited guests: autophagy-related
pathways in cellular defense. Cell. 120 (2): 159–162.
186 Lee, J.S. and Lee, G.M. (2012). Estimation of autophagy pathway genes for
autophagy induction: overexpression of Atg9A does not induce autophagy in
recombinant Chinese hamster ovary cells. Biochem. Eng. J. 68: 221–226.
187 Astley, K., Naciri, M., Racher, A., and Al-Rubeai, M. (2007). The role of
p21CIP1 in adaptation of CHO cells to suspension and protein-free culture.
J. Biotechnol. 130: 282–290.
188 Walworth, N.C. (2000). Cell-cycle checkpoint kinases: checking in on the cell
cycle. Curr. Opin. Cell Biol. 12: 697–704.
 References 247

189 Lee, K.H., Onitsuka, M., Honda, K. et al. (2013). Rapid construction of
transgene-amplified CHO cell lines by cell cycle checkpoint engineering.
Appl. Microbiol. Biotechnol. 97: 5731–5741.
190 Muller, D., Katinger, H., and Grillari, J. (2008). MicroRNAs as targets for
engineering of CHO cell factories. Trends Biotechnol. 26: 359–365.
191 Druz, A., Betenbaugh, M., and Shiloach, J. (2012). Glucose depletion acti-
vates mmu-miR-466h-5p expression through oxidative stress and inhibition
of histone deacetylation. Nucleic Acids Res. 40: 7291–7302.
192 Druz, A., Chu, C., Majors, B. et al. (2011). A novel microRNA
mmu-miR-466h affects apoptosis regulation in mammalian cells. Biotechnol.
Bioeng. 108: 1651–1661.
193 Druz, A., Son, Y.J., Betenbaugh, M., and Shiloach, J. (2013). Stable inhibition
of mmu-miR-466h-5p improves apoptosis resistance and protein production
in CHO cells. Metab. Eng. 16: 87–94.
194 Cho, W.C. (2007). OncomiRs: the discovery and progress of microRNAs in
cancers. Mol Cancer. 6: 60.
195 Raver-Shapira, N., Marciano, E., Meiri, E. et al. (2007). Transcriptional acti-
vation of miR-34a contributes to p53-mediated apoptosis. Mol Cell. 26 (5):
731–743.
196 Butler, M. and Spearman, M. (2014). The choice of mammalian cell host
and possibilities for glycosylation engineering. Curr. Opin. Biotechnol. 30:
107–112.
197 Hossler, P., Khattak, S.F., and Li, Z.J. (2009). Optimal and consistent protein
glycosylation in mammalian cell culture. Glycobiology 19: 936–949.
198 Wang, Q., Stuczynski, M., Gao, Y., and Betenbaugh, M.J. (2015). Strate-
gies for engineering protein N-glycosylation pathways in mammalian cells.
Methods Mol. Biol. 1321: 287–305.
199 Lee, E.U., Roth, J., and Paulson, J.C. (1989). Alteration of terminal glycosyla-
tion sequences on N-linked oligosaccharides of Chinese hamster ovary cells
by expression of beta-galactoside alpha 2,6-sialyltransferase. J. Biol. Chem.
264: 13848–13855.
200 Zhang, X., Lok, S.H., and Kon, O.L. (1998). Stable expression of human
alpha-2,6-sialyltransferase in Chinese hamster ovary cells: functional con-
sequences for human erythropoietin expression and bioactivity. Biochim.
Biophys. Acta 1425: 441–452.
201 Bragonzi, A., Distefano, G., Buckberry, L.D. et al. (2000). A new Chinese
hamster ovary cell line expressing alpha2,6-sialyltransferase used as universal
host for the production of human-like sialylated recombinant glycoproteins.
Biochim. Biophys. Acta 1474: 273–282.
202 Minch, S.L., Kallio, P.T., and Bailey, J.E. (1995). Tissue plasminogen activator
coexpressed in Chinese hamster ovary cells with alpha(2,6)-sialyltransferase
contains NeuAc alpha(2,6)Gal beta(1,4)Glc-N-AcR linkages. Biotechnol.
Progr. 11: 348–351.
203 Fukuta, K., Abe, R., Yokomatsu, T. et al. (2000). Remodeling of sugar chain
structures of human interferon-gamma. Glycobiology 10: 421–430.
248 9 CHO Cell Engineering for Improved Process Performance and Product Quality

204 Fukuta, K., Yokomatsu, T., Abe, R. et al. (2000). Genetic engineering of CHO
cells producing human interferon-gamma by transfection of sialyltrans-
ferases. Glycoconjugate J. 17: 895–904.
205 Jassal, R., Jenkins, N., Charlwood, J. et al. (2001). Sialylation of human
IgG-Fc carbohydrate by transfected rat alpha2,6-sialyltransferase. Biochem.
Biophys. Res. Commun. 286: 243–249.
206 Weikert, S., Papac, D., Briggs, J. et al. (1999). Engineering Chinese hamster
ovary cells to maximize sialic acid content of recombinant glycoproteins.
Nat. Biotechnol. 17: 1116–1121.
207 Wong, N.S., Yap, M.G., and Wang, D.I. (2006). Enhancing recombinant gly-
coprotein sialylation through CMP-sialic acid transporter over expression in
Chinese hamster ovary cells. Biotechnol. Bioeng. 93: 1005–1016.
208 Jeong, Y.T., Choi, O., Son, Y.D. et al. (2009). Enhanced sialylation of recom-
binant erythropoietin in genetically engineered Chinese-hamster ovary cells.
Biotechnol. Appl. Biochem. 52: 283–291.
209 Son, Y.D., Jeong, Y.T., Park, S.Y., and Kim, J.H. (2011). Enhanced sialylation
of recombinant human erythropoietin in Chinese hamster ovary cells by
combinatorial engineering of selected genes. Glycobiology 21: 1019–1028.
210 Davies, J., Jiang, L., Pan, L.Z. et al. (2001). Expression of GnTIII in a recom-
binant anti-CD20 CHO production cell line: expression of antibodies with
altered glycoforms leads to an increase in ADCC through higher affinity for
FC gamma RIII. Biotechnol. Bioeng. 74: 288–294.
211 Ferrara, C., Brunker, P., Suter, T. et al. (2006). Modulation of therapeu-
tic antibody effector functions by glycosylation engineering: influence of
Golgi enzyme localization domain and co-expression of heterologous beta1,
4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II.
Biotechnol. Bioeng. 93: 851–861.
212 Prati, E.G., Matasci, M., Suter, T.B. et al. (2000). Engineering of coordinated
up- and down-regulation of two glycosyltransferases of the O-glycosylation
pathway in Chinese hamster ovary (CHO) cells. Biotechnol. Bioeng. 68:
239–244.
213 Yamane-Ohnuki, N., Kinoshita, S., Inoue-Urakubo, M. et al. (2004). Estab-
lishment of FUT8 knockout Chinese hamster ovary cells: an ideal host
cell line for producing completely defucosylated antibodies with enhanced
antibody-dependent cellular cytotoxicity. Biotechnol. Bioeng. 87: 614–622.
214 Malphettes, L., Freyvert, Y., Chang, J. et al. (2010). Highly efficient dele-
tion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that
produce completely nonfucosylated antibodies. Biotechnol. Bioeng. 106:
774–783.
215 Cristea, S., Freyvert, Y., Santiago, Y. et al. (2013). In vivo cleavage of trans-
gene donors promotes nuclease-mediated targeted integration. Biotechnol.
Bioeng. 110: 871–880.
216 Zhong, X., Cooley, C., Seth, N. et al. (2012). Engineering novel Lec1 gly-
cosylation mutants in CHO-DUKX cells: molecular insights and effector
modulation of N-acetylglucosaminyltransferase I. Biotechnol. Bioeng. 109:
1723–1734.
 References 249

217 Sealover, N.R., Davis, A.M., Brooks, J.K. et al. (2013). Engineering Chinese
hamster ovary (CHO) cells for producing recombinant proteins with sim-
ple glycoforms by zinc-finger nuclease (ZFN)-mediated gene knockout of
mannosyl (alpha-1,3-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase
(Mgat1). J. Biotechnol. 167: 24–32.
218 Zhang, P., Haryadi, R., Chan, K.F. et al. (2012). Identification of functional
elements of the GDP-fucose transporter SLC35C1 using a novel Chinese
hamster ovary mutant. Glycobiology 22: 897–911.
219 Yang, Z., Wang, S., Halim, A. et al. (2015). Engineered CHO cells for
production of diverse, homogeneous glycoproteins. Nat. Biotechnol. 33:
842–844.
220 Imai-Nishiya, H., Mori, K., Inoue, M. et al. (2007). Double knockdown of
alpha1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase
(GMD) in antibody-producing cells: a new strategy for generating fully
non-fucosylated therapeutic antibodies with enhanced ADCC. BMC Biotech-
nol. 7: 84.
221 Skulj, M., Pezdirec, D., Gaser, D. et al. (2014). Reduction in C-terminal ami-
dated species of recombinant monoclonal antibodies by genetic modification
of CHO cells. BMC Biotechnol. 14: 76.
222 Chiu, J., Valente, K.N., Levy, N.E. et al. (2017). Knockout of a
difficult-to-remove CHO host cell protein, lipoprotein lipase, for improved
polysorbate stability in monoclonal antibody formulations. Biotechnol. Bio-
eng. 114: 1006–1015.
223 Mascarenhas, J.X., Korokhov, N., Burger, L. et al. (2017). Genetic engineer-
ing of CHO cells for viral resistance to minute virus of mice. Biotechnol.
Bioeng. 114: 576–588.
224 Elliott, S., Lorenzini, T., Asher, S. et al. (2003). Enhancement of therapeu-
tic protein in vivo activities through glycoengineering. Nat. Biotechnol. 21:
414–421.
225 Sinclair, A.M. and Elliott, S. (2005). Glycoengineering: the effect of gly-
cosylation on the properties of therapeutic proteins. J. Pharm. Sci. 94:
1626–1635.
226 Ronda, C., Pedersen, L.E., Hansen, H.G. et al. (2014). Accelerating genome
editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target
finding tool. Biotechnol. Bioeng. 111: 1604–1616.
227 Chan, K.F., Shahreel, W., Wan, C. et al. (2016). Inactivation of GDP-fucose
transporter gene (SLC35C1) in CHO cells by ZFNs, TALENs and
CRISPR-Cas9 for production of fucose-free antibodies. Biotechnol. J. 11:
399–414.
228 Jenkins, N., Parekh, R.B., and James, D.C. (1996). Getting the glycosyla-
tion right: implications for the biotechnology industry. Nat. Biotechnol.
14: 975–981.
229 Bork, K., Horstkorte, R., and Weidemann, W. (2009). Increasing the sialyla-
tion of therapeutic glycoproteins: the potential of the sialic acid biosynthetic
pathway. J. Pharm. Sci. 98: 3499–3508.
250 9 CHO Cell Engineering for Improved Process Performance and Product Quality

230 Butler, M. (2005). Animal cell cultures: recent achievements and perspectives
in the production of biopharmaceuticals. Appl. Microbiol. Biotechnol. 68:
283–291.
231 Chung, C.Y., Wang, Q., Yang, S. et al. (2017). Integrated genome and protein
editing swaps alpha-2,6 sialylation for alpha-2,3 sialic acid on recombinant
antibodies from CHO. Biotechnol. J. 12 (2): https://doi.org/10.1002/biot
.201600502.
232 Raymond, C., Robotham, A., Spearman, M. et al. (2015). Production of
alpha2,6-sialylated IgG1 in CHO cells. mAbs 7: 571–583.
233 Haryadi, R., Zhang, P., Chan, K.F., and Song, Z. (2013). CHO-gmt5, a novel
CHO glycosylation mutant for producing afucosylated and asialylated
recombinant antibodies. Bioengineered 4: 90–94.
234 Cha, H.M., Lim, J.H., Yeon, J.H. et al. (2017). Co-overexpression of
MGAT1 and MGAT4 in CHO cells for production of highly sialylated
albumin-erythropoietin. Enzyme Microb. Technol. 103: 53–58.
235 Schjoldager, K.T., Joshi, H.J., Kong, Y. et al. (2015). Deconstruction
of O-glycosylation–GalNAc-T isoforms direct distinct subsets of the
O-glycoproteome. EMBO Rep. 16: 1713–1722.
236 Gramer, M.J. (2014). Product quality considerations for mammalian cell cul-
ture process development and manufacturing. Adv. Biochem. Eng. Biotechnol.
139: 123–166.
237 Johnson, K.A., Paisley-Flango, K., Tangarone, B.S. et al. (2007). Cation
exchange-HPLC and mass spectrometry reveal C-terminal amidation of an
IgG1 heavy chain. Anal. Biochem. 360: 75–83.
238 Tsubaki, M., Terashima, I., Kamata, K., and Koga, A. (2013). C-terminal
modification of monoclonal antibody drugs: amidated species as a general
product-related substance. Int. J. Biol. Macromol. 52: 139–147.
239 Hu, Z., Zhang, H., Haley, B. et al. (2016). Carboxypeptidase D is the only
enzyme responsible for antibody C-terminal lysine cleavage in Chinese
hamster ovary (CHO) cells. Biotechnol. Bioeng. 113: 2100–2106.
240 Moody, M., Alves, W., Varghese, J., and Khan, F. (2011). Mouse minute virus
(MMV) contamination–a case study: detection, root cause determination,
and corrective actions. PDA J. Pharm. Sci. Technol. 65: 580–588.
241 Cotmore, S.F. and Tattersall, P. (2007). Parvoviral host range and cell entry
mechanisms. Adv. Virus Res. 70: 183–232.
242 Lopez-Bueno, A., Rubio, M.P., Bryant, N. et al. (2006). Host-selected amino
acid changes at the sialic acid binding pocket of the parvovirus capsid mod-
ulate cell binding affinity and determine virulence. J. Virol. 80: 1563–1573.