Applied Microbiology and Biotechnology (2023) 107:1063–1075

https://doi.org/10.1007/s00253-022-12342-x

MINI-REVIEW

Progress in fed‑batch culture for recombinant protein production

in CHO cells
Wen‑Jing Xu1,2 · Yan Lin1,3 · Chun‑Liu Mi1 · Jing‑Ying Pang4 · Tian‑Yun Wang1,5

Received: 13 September 2022 / Revised: 13 December 2022 / Accepted: 15 December 2022 / Published online: 17 January 2023
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023

Abstract
Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve
better cell growth and high-yield recombinant protein, fed-batch culture is typically used for recombinant protein produc-
tion in CHO cells. According to the demand of nutrients consumption, feed medium containing multiple components in
cell culture can affect the characteristics of cell growth and improve the yield and quality of recombinant protein. Fed-batch
optimization should have a connection with comprehensive factors such as culture environmental parameters, feed com-
position, and feeding strategy. At present, process intensification (PI) is explored to maintain production flexible and meet
forthcoming demands of biotherapeutics process. Here, CHO cell culture, feed composition in fed-batch culture, fed-batch
culture environmental parameters, feeding strategies, metabolic byproducts in fed-batch culture, chemostat cultivation, and
the intensified fed-batch are reviewed.
Key points
• Fed-batch culture in CHO cells is reviewed.
• Fed-batch has become a common technology for recombinant protein production.
• Fed batch culture promotes recombinant protein production in CHO cells.

Keywords CHO cell · Process development · Recombinant therapeutic proteins · Fed-batch culture

Introduction (EMA) and the U.S Food and Drug Administration (FDA)
with thousands of billion dollars in market value (Kaplon
In recent years, with the growing demand for recombinant et al. 2020). A report has estimated that market value about
therapeutic proteins (RTPs) in the pharmaceutical market, monoclonal antibodies (mAbs) will has reached over US$
including recombinant antibody, the production of RTPs has 300 billion by 2025 (Lu et al. 2020). Among the recombinant
developed rapidly. More than 120 recombinant antibodies protein expression system, Chinese hamster ovary (CHO)
have been approved by the European Medicines Agency cells are the preferred cells for RTP production (Ritacco
et al. 2018; Gupta et al. 2021) due to several advantages:
* Tian‑Yun Wang ① not susceptible to human virus infection with high safety
[email protected] (Lalonde and Durocher 2017); ② RTPs with post- transla-
1
International Joint Research Laboratory for Recombinant tional modifications similar to those of human cells (Stach
Pharmaceutical Protein Expression System of Henan, Xinxiang et al. 2019); ③ high-density CHO cells can accommodate
Medical University, Xinxiang 453003, Henan, China suspension culture with the chemically defined serum-free
2
School of Pharmacy, Xinxiang Medical University, medium (CD-SFM) and produce proteins that secrete into
Xinxiang 453003, Henan, China the culture medium (Ritacco et al. 2018; Dahodwala and
3
School of Nursing, Xinxiang Medical University, Lee 2019).
Xinxiang 453003, Henan, China Nowadays, mAbs production in mammalian cell culture
4
School of the First Clinical College, Xinxiang Medical is mainly performed with fed-batch processes in large-scale
University, Xinxiang 453000, Henan, China bioreactors (Schellenberg et al. 2022). CHO cells are usu-
5
School of medicine, Xinxiang University, Xinxiang 453003, ally cultivated in a fed-batch mode to achieve maximum cell
Henan, China density and product titer, with either continuous or bolus

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1064 Applied Microbiology and Biotechnology (2023) 107:1063–1075

feed addition (Romanova et al. 2022). The initial cellular fed-batch process has become an important production
growth is supported by the basal medium. Concentrated process for the large-scale production of RTPs. However,
feed medium is added in fed-batch culture to replenish maintaining high productivity of cells and quality attributes
nutrients and prolong the culture time (Ritacco et al. 2018; of protein is still a challenge. Therefore, fed-batch culture
Chee Furng Wong et al. 2005). Feed medium can avoid the has shifted from the high-yield recombinant protein merely
nutrients limitation, and it can promote higher production of to the dual goals of high-yield and high-quality production
recombinant proteins (Rish et al. 2022; Bibila and Robinson (Ha et al. 2022).
1995; Lu et al. 2013). The culture time of cells in stationary
phase after fed-batch treatment is longer than that of batch
culture (Sellick et al. 2015). Fed-batch culture requires an Feed composition in fed‑batch culture
understanding of the characteristics of cell growth and cell
metabolism, and the key process parameters that affect the Optimizing the critical feed composition and the concentra-
quality and yield of RTPs (Ghaffari et al. 2020). CHO cell tions of components are crucial to process development (Zou
grows at a high speed in exponential phase, with an acceler- et al. 2020). In fed-batch industry, Raman spectroscopy with
ated consumption of glucose and amino acids, resulting in partial least squares regression (PLSR) or multiple linear
accumulation of lactate and ammonium (Horvat et al. 2020; regression (MLR) are applied to monitor and control the
Zagari et al. 2013). In fed-batch culture, temperature, pH, concentrations of glucose and amino acids (Kozma et al.
and other process parameters also have an impact on anti- 2018). Besides Raman spectroscopy, liquid chromatography-
body expression and quality attributes (Kuwae et al. 2018; mass spectrometry (LC–MS) has been considered to analyze
Pan et al. 2017; Graham et al. 2019). Fed-batch culture can amino acids and inorganic salt (Hoang et al. 2021). For the
improve the expression and production of RTPs in CHO first time, lipidomics analysis has been performed for lipid
cells, but traditional bolus feed delivery also causes accumu- identification and lipid quantitation in fed-batch culture (Ali
lation of metabolic byproducts (Xiao et al. 2021). Therefore, et al. 2018). In addition, feed composition has been linked
a reasonable feeding strategy is quite important (Baik et al. with cell metabolism regulation to demonstrate fed-batch
2015; Zhang et al. 2013; Mellahi et al. 2019). Fed-batch culture (Braasch et al. 2021) (Fig. 1).
culture has focused on reducing the cost of RTP produc- The components of feed medium are similar to SFM,
tion, which has become the research focus in pharmaceutical including amino acids, carbon sources, inorganic salts, trace
industry (Ghaffari et al. 2020). elements, vitamins, lipids, and putrescine and yeast hydro-
lysate etc. (Table 1).

CHO cell culture

Amino acid
Adherent CHO cell grows under DMEM/F12 medium con-
taining 10% fetal bovine serum (FBS) at 37℃, 5% ­CO2 (Li During upstream process optimization, amino acid is one of
et al. 2021). SFM is chosen to suspension culture, support- the major concerns (Fan et al. 2015). Amino acids deplete
ing a transition from adherent culture to suspension culture in batch culture, which is detrimental to RTP production
mode. The basal medium is used for the adherent culture (Ghaffari et al. 2020). Amino acids’ feeding is related with
including serum components from fetal bovine. Serum tricarboxylic acid cycle (TCA cycle) (Rish et al. 2022). The
components are complex, and it is prone to contamination TCA cycle depends on alternative metabolites to comple-
(Lalonde and Durocher 2017). SFM is divided into ordi- ment intermediates (Hong et al. 2018; Ghorbaniaghdam
nary serum-free medium, xeno-free medium, animal-free et al. 2014). Amino acids consumed from feed medium can
medium, protein-free medium, and chemically defined be directed to the TCA cycle by conversion between the
serum-free medium (Gélinas et al. 2019; Yao and Asayama cytosol and the mitochondria. Furthermore, the catabolism
2017). When it comes to cell suspension culture, SFM can of amino acids is supply for the TCA cycle. Amino acids in
effectively avoid disadvantages caused by serum culture, feed medium are limited. AMP that leads to boosting cell
such as contamination and difficulty in purification. health is associated with the purine nucleotide cycle. Malate
Batch culture refers to a culture method that can harvest and glycine accumulation are from the TCA cycle. Cell pro-
recombinant proteins at one time after adding SFM once. liferation has a negative impact with ammonia concentra-
Due to short culture time and unsatisfactory cell density at tions above 5 mM (Yang and Butler 2000), and ammonia
the time of sample collection, RTP production is unideal. is derived from the hydrolysis of amino acids (Rish et al.
Fed-batch mode improves RTP production. It is reported that 2022). Thus, controlling feeding of amino acids may reduce
mAb titer in fed-batch culture has reached more than 10 g/L byproducts accumulation (Rish et al. 2022). Although cells
(Handlogten et al. 2018; Kelley 2009). In recent years, can synthesize non-essential amino acids, cell growth, and

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Applied Microbiology and Biotechnology (2023) 107:1063–1075 1065

According to metabolic flux analysis (MFA), Xing et al.

optimized feed medium to produce antibody fusion protein
(B1) in CHO cells. The concentration of alanine, arginine,
glutamine and glycine were reduced, while the concentra-
tion of methionine, tryptophan, asparagine and serine were
increased. Finally, B1 protein titer was ameliorated by 27%
in fed-batch culture (Xing et al. 2011). Fouladiha et al. used
the computational modeling method to design a feeding
strategy in CHO cells. To study the effect of the produc-
tion of mAb in CHO cells, 15 metabolites were analyzed
by Plackett – Burman (PB) design. The results showed that
the production of mAbs could be significantly improved by
2 times after threonine treatment (Fouladiha et al. 2020).
Hecklau et al. reported that S-sulfo-L cysteine (SSC)
replaced cysteine and was highly stable in fed-batch culture;
feed medium supplemented with SSC prolonged cell viabil-
ity and enhanced product titers by 78%. Meanwhile, this
strategy reduced ammonium (­ NH4+) accumulation (Hecklau
et al. 2016). Zhang et al. took advantage of high concentra-
tion tyrosine and low concentration tyrosine experimental
groups, and they cultured CHO cells expressing anti-CD20
IgG1 in bioreactor. Apart from feed medium containing
tyrosine, 88 mM tyrosine solution was added in the high
concentration tyrosine group. High concentration tyrosine
promoted the cell growth and reduced the osmolality of bio-
reactor culture environment and increased the production of
mAbs (Zhang et al. 2020a).
Amino acids are key components of feed medium, and
their concentration and properties can affect yield and qual-
ity of RTPs in fed-batch culture. Amino acids involved in
fed-batch culture require a strict design. However, some high
concentration amino acids are easy to precipitate due to the
limitation of the solubility. Modified amino acids are based
on the original structure. We need to comprehensively con-
sider the characteristics of CHO cells and establish the best
addition strategy.
Fig. 1  Fed-batch culture in CHO cell. Gene of interest (GOI) is intro-
duced in CHO cell via transfection. Positive CHO cells are used for
shake flask culture. Then, CHO cells and feed medium are added in
bioreactor. In fed-batch culture mode, nutrients are monitored and Carbon source
controlled to achieve the yield and quality of RTPs. The production of
RTPs is indicated by fed-batch system. Glucose is the primary carbon source of the TCA cycle in
exponential phase, and glucose consumes fast at the later
the expression of recombinant proteins still rely on amino stage of the exponential phase (Kirsch et al. 2022). How-
acids in feed medium (Horvat et al. 2020). ever, glucose consumption causes accumulation of pyru-
N-lactoyl-leucine (Lac-Leu) and N-lactoyl-isoleucine vate and lactate, which leads to inhibition of cell growth.
(Lac-Ile) can replace unmodified leucine and isoleucine, Wilkens et al. found that 3.6 g/L glucose concentration
respectively. Lac-Leu and Lac-Ile improved the solubility in shortened CHO cell culture time and lactate concentra-
culture medium. Adding 2 × concentrated feed medium with tion attained 2.7 g/L after 120 h. Although both 1.08 g/L
a 70% mixture of two above-mentioned N-Lactoyl amino glucose and 2.52 g/L galactose are applied to diminish
acids (Lac-AA) increased the yield of recombinant protein accumulation of lactate, and tissue plasminogen activator
by 58%, compared with the addition of 1 × concentrated feed (t-PA) production was increased to 100% when 1.08 g/L
medium. Schmidt et al. found that 2 × concentrated medium glucose and 2.52 g/L galactose was added to CHO cell
reduced the volume of feed medium (Schmidt et al. 2021). culture, final concentration of glucose approached zero

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Table 1  The components in fed- Category Additives Effect Reference

batch culture
Amino acid Lac-Ile Improved yield of protein Schmidt et al. (2021)
Lac-leu Improved yield of protein Schmidt et al. (2021)
Alanine Inhibited the TCA pathway Xing et al. (2011)
Arginine Increased cytotoxic level Xing et al. (2011)
Glutamine Increased ammonium production Xing et al. (2011)
Glycine Increased ammonium production Xing et al. (2011)
Methionine Prevented amino acid limitation Xing et al. (2011)
Tryptophan Prevented amino acid limitation Xing et al. (2011)
Asparagine Decreased ammonium production Xing et al. (2011)
Serine Provided a carbon donor Xing et al. (2011)
Threonine Increased antibody production Fouladiha et al. (2020)
SSC Promoted protein productivity Hecklau et al. (2016)
Tyrosine Enhanced antibody production Zhang et al. (2020a)
Carbon resource Glucose Increased byproduct accumulation Wilkens et al. (2011)
Glutamine Accumulated lactic acid Sun et al. (2021)
Galactose Improved protein expression and Xiao et al. (2019)
Fructose sialic acid content Wlaschin and Hu (2007)
Promoted cell growth
Inorganic salt Na+ Regulated osmolality Zhu et al. (2005)
Trace element Selenite Increased antibody production Zhang et al. (2006)
Fe ­(Fe2+) Enhanced acidic charge variants Chung et al. (2019)
Zn ­(Zn2+) Promoted protein production Graham et al. (2020)
Cu ­(Cu2+) Improved antibody titer Xu et al. (2016)
Vitamin Vitamin C Decreased phosphorylation level Hou et al. (2019)
Nicotinamide Decreased phosphorylation level Hou et al. (2019)
B vitamins Improved antibody titer Vijayasankaran et al. (2013)
Vitamin K Decreased Gla level Lee et al. (2017a)
Lipid Ethanolamine Promoted antibody titer Zhang et al. (2013)
Lipid mixture Promoted antibody titer Zhang et al. (2013)
Other additives Putrescine Increased antibody production Zhang et al. (2013)
3-MA Increased protein production Jardon et al. (2012)
AIP Improved antibody titer Braasch et al. (2021)
YE Increased protein production Hu et al. (2018)
deoxyuridine Increased antibody production Takagi et al. (2017)
thymidine Increased antibody production Takagi et al. (2017)
deoxycytidine Increased antibody production Takagi et al. (2017)
MPPB Increased antibody production Aki et al. (2021)

after 160 h and cell viability was decreased (Wilkens et al. According to the glucose content in the culture medium,
2011). During the cell culture, the minimum glucose con- the high concentration glucose solution can be added to the
centration needs to be controlled at 2 ~ 3 g/L (Sun et al. CHO cell culture medium. Because the high concentration
2021). of carbon source also leads to metabolic byproducts accu-
Glutamine is the secondary carbon source in the early mulation, so the concentration of carbon source should be
exponential phase of cells, but it increases lactate levels strictly controlled. Proper alternative carbon sources can
(Kirsch et al. 2022). It was found that the expression of avoid inhibition of cell growth by byproducts after glucose
Fc-fusion protein in CHO cells was increased by 43% and consumption.
the total sialic acid content of cells was increased by 37%
when galactose was supplemented with 40% replacement
ratio and glucose carbon source was replaced (Xiao et al. Inorganic salts and trace elements
2019). CHO cells transfected with GLUT5 fructose trans-
porter were cultivated in fed-batch culture system, and feed Fed-batch culture also requires inorganic salts to regulate
medium containing 14.5 g/L fructose was added. Fructose pH and osmolality in cell culture environment. Zhu et al.
improved cell growth, and it decreased accumulation of lac- used 2L bioreactor for fed-batch culture, and they added ­Na+
tate. Wlaschin et al. indicated that CHO cells had an ability to control osmolality and partial pressure of ­CO2 ­(pCO2).
to replace carbon source through the expression of GLUT5 Both 140 ~ 160 mmHg ­pCO2 and 400 mOsm/kg osmolality
fructose transporter (Wlaschin and Hu 2007). decreased the viable cell density (VCD) of CHO cells by

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18%, but these factors clearly augmented the production of mL and the antibody titer of 378 mg/L, which was threefold
protein B1 (Zhu et al. 2005). In cell culture, selenium (Se) higher than batch culture (Zhang et al. 2013). Therefore, lipid
in the form of selenite can protect cells from oxidative dam- can appropriately supplement in feed medium.
age. Zhang et al. found that the VCD exceeded 1 × ­107 cells/
mL and product titer of approximately 3 g/L was achieved Other components
in 14-day fed-batch cultures with the addition of selenite
(Zhang et al. 2006). Putrescine is essential for cell growth (Jänne et al. 2004).
Gangwar et al. found that Zn, Cu, Fe, and Mn impact Zhang et al. confirmed that putrescine was designed by PB
charge heterogeneity, and reduce accumulation of meta- design and it sharply increased mAb production. A signifi-
bolic byproducts in cell culture. Fe, Mn, and Ni enhance cant increase in TNFR-Fc production was observed after
the acidic variants while Cu inhibits it. Zn reduces the basic adding feed medium with the concentration of 41.67 mg/L
variants (Gangwar et al. 2021). Chung et al. found that Fer- of putrescine (Zhang et al. 2013). Jardon et al. found that
rous sulfate (­ Fe2+) in feed medium stored for 7 weeks can autophagy negatively affected recombinant proteins pro-
reduce the charge heterogeneity, but it improved the yield of duction. The autophagy inhibitor 3-methyl adenine (3-MA)
mAb expressed by CHO cells (Chung et al. 2019). Graham in fed-batch culture resulted in a 2.8-fold increase in t-PA
et al. found that both 50 and 100 μM zinc ions (­ Zn2+) added production compared with control group without 3-MA
in bioreactor promoted the production of β-Glucuronidase (Jardon et al. 2012). Autophagy-inducing peptide (AIP),
(GUS) in CHO cells and harvested 2 times increase in spe- a derivative of the autophagy protein Beclin 1, facilitated
cific activity of GUS (Graham et al. 2020). 5 μM copper the production of mAbs in CHO cells by regulating cel-
sulfate ­(Cu2+) in feed medium obtained the final mAb titer lular autophagy. The addition of 3 µM of AIP in fed-batch
of 11.9 ± 0.6 g/L to express IgG1 in CHO cells, which was culture ameliorated the expression level of human IgG1,
4.8 times higher than the final mAb titer of conventional and IgG1 concentration exceeded 1200 µg/mL (Braasch
fed-batch culture (Xu et al. 2016). et al. 2021). The yield of Fc-fusion protein (Fc) in fed-
batch culture raised twofold by Yeast hydrolysate (YE),
and YE also increased CHO cells size and intracellular
Vitamin nucleotide content (Hu et al. 2018). CHO cells expressing
human mAb and Fab fragments were cultivated in 14-day
Vitamin enhances productivity in CHO cells, and impacts fed-batch culture, and the concentration of 25 mg/L deox-
charge heterogeneity. Gangwar et al. also indicated that yuridine was added at day 2. VCD and protein produc-
vitamins can inhibit the acidic variants while it enhances tion were increased. Takagi et al. harvested 9.2 g/L human
the basic variants (Gangwar et al. 2021). Hou et al. found mAb and over 4 g/L Fab fragment antibody with deoxyu-
that increasing rate of vitamin C and nicotinamide feeding ridine, thymidine, and deoxycytidine (Takagi et al. 2017).
can reduce the phosphorylation level in CHO cells to 3% Since deoxyuridine, thymine, and deoxycytidine are all
and attenuated the hydroxylation level to 9.4% (Hou et al. pyrimidine nucleotides, exogenous nucleoside may give
2019). Adding B vitamins including 0.005 g/L vitamin B2, a platform for efficient antibody production by providing
0.0035 g/L pyridoxine (vitamin B6), 0.03 g/L pyridoxal nucleosides precursors through the remedial synthesis
(vitamin B6), 0.0985 g/L folic acid and 0.024 g/L vitamin pathway of pyrimidine nucleotides. Aki et al. screened
B12 can improve packed cell volume (PCV) and antibody 4-(2,5-dimethyl-1H-pyrrol-1-yl) -N- (2,5-dioxopyrroli-
titer (Vijayasankaran et al. 2013). Lee et al. added 10 g/L din-1-yl) benzamide (MPPB). The mAb concentration
vitamin K solution prepared with 13% polysorbate 20 (PS20) of MPPB treatment was increased to 1098 mg/L, which
in 4-day fed-batch culture for cultured CHO cells expressing was 1.5-fold higher than the mAb concentration of control
recombinant human factor II (rhFII). When the final concen- group (Aki et al. 2021). Further studies in fed-batch culture
tration of vitamin K was 0.1 mg/L, gamma-carboxygluta- facilitate the development of more additives, besides these
mate (Gla) level was decreased (Lee et al. 2017a). above components.

Lipid Fed‑batch culture environmental

parameters
Ethanolamine is one of the components of the phospho-
lipid structure in mammalian cell membranes (Spens and Culture parameters can affect CHO cell growth, recombinant
Häggström 2007). As for TNFR-Fc production in CHO cells, protein expression, protein glycosylation, cell metabolites,
the addition of the feed medium supplemented with ethanola- and other factors (Butler 2005). Bioreactors are designed
mine and lipids resulted in a maximum VCD of 4.3 × ­106 cells/ for the cultivation of suspension culture for RTP production

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1068 Applied Microbiology and Biotechnology (2023) 107:1063–1075

in CHO cells (Schmid et al. 2022). Cells are expanded by galactosylation, and increased the proportion of high-
the cryovial or shake flask, and then grows in bioreactor mannose glycosylation (man5) (Qin et al. 2019). Xiao
(Hernández Rodríguez and Frahm 2020; Schmid et al. et al. found that continuous feeding resulting in osmo-
2022). For fed-batch culture, culture environmental param- lality below 450 mOsm/kg reduced the high-mannose
eters in bioreactor are usually optimized through design-of- glycosylation to less than 5% and increase the yield of
experiments (DoE) or one-factor-at-a-time (OFAT) studies, mAb to 10 g/L (Xiao et al. 2021). Lee et al. investigated
like temperature, dissolved oxygen (DO), and pH (Weng the effect of high osmolality on protein glycosylation in
et al. 2020; Schneider et al. 2019). CHO cells. Osmolality reached over 400 mOsm /kg after
adding betaine, and Fc-fusion protein sialylation in CHO
cells decreased (Lee et al. 2017b). Inorganic salt and high
Culture temperature feed volume can lead to high osmolality in fed-batch cul-
ture. Although high osmolality increased the cell-spe-
Culture temperature downshifting is generally performed cific productivity (qp) of CHO cells, but it increased the
in exponential growth phase. The culture temperature is proportion of high mannose and inhibited the growth of
switched from 37 ℃ to a lower temperature ranged from CHO cells. Romanova et al. launched proteome study of
29 ℃ to 35 ℃ for the sake of decreasing cell apoptosis and hyperosmolality-exposed CHO cells together with control
promoting antibody synthesis (Moore et al. 1997; Kou et al. cells, and harvested cell samples at days 2, 6, and 8 in
2011). Low temperature has a positive impact on integral fed-batch culture, respectively. Whole protein lysates of
cell density, cell viability, and cell-specific productivity hyperosmolality-exposed cell group and control cell group
(McHugh et al. 2020). sampled on days 2, 6, and 8 were analyzed by western
Torres et al. cultured CHO cell expressing human eryth- blot. Septins protein was obviously up-regulated by about
ropoietin (hEPO) using a fed-batch culture process. They 2-threefold at day 6 after treated with hyperosmolality,
observed that low temperature not only had the advantage and up-regulated about 1-threefold at day 8 (Romanova
of improving the specific productivity of CHO cell express- et al. 2022).
ing recombinant proteins, but also significantly increased Exposure of CHO cell to highly concentrated feed
the gene encoding the specific transcription activator of medium causes an increasing osmolality over 300 mOsm/
unfolded protein response, reducing the possibility of protein kg in respect of cell physiology, morphology, and proteome
degradation; Hypothermia also enhanced the expression of (Romanova et al. 2022). In fed-batch culture, appropri-
hEPO by regulating the cell cycle (Torres et al. 2021). Cell ate additives can obtain feasible osmolality. Furthermore,
culture temperature can significantly affect the VCD and an available feeding strategy is also used for avoiding
protein expression in CHO cells. When CHO cells express- hyperosmolality.
ing anti-CD20 mAb were cultured at 35 °C, 33 °C, and 31 °C
with the addition of feed medium at days 3, 6, and 9, 35 °C
can increase cell density and expression of anti-CD20 mAb
(Kong et al. 2020). Stiefel et al. that the effect of shifting pH
temperature from 37 °C to 30 °C on miRNA expression in
CHO cells was investigated. Low temperature condition The pH of cell culture requires optimization to produce
increased IgG production through upregulating the expres- high-quality recombinant protein. The pH of CHO cell
sion of miRNAs related with inhibition of apoptosis, pro- growth ranges from 7.2 to 7.6 (Michl et al. 2019). CHO cells
motion of cell growth and protein production in CHO cells expressing mAb against vascular endothelial growth factor
(Stiefel et al. 2016). Thus, low temperature is one of the (VEGF-MA) were cultured in 3 L bioreactor and the culture
most normal factors of cell culture. conditions were optimized. The pH was adjusted by adding
sodium bicarbonate. When the pH value was set to 7.10,
the yield of VEGF-MA increased to 4.1 g/L, and the charge
Osmolality heterogeneity, glycosylation level and protein purity were
26.1%, 59.1%, and 95.1% respectively (Feng and Shi 2020).
Nutrient supplementation and metabolic byproducts accu- It is necessary to follow the standard pH range of CHO cells
mulation are able to increase extracellular osmolality at when it comes to designing the pH in fed-batch culture. If
the late stages of culture in fed-batch culture (Alhuthali the pH of cell culture maintains below 6.8 or above 7.6,
et al. 2021). Both nutrient and alkaline supplemented CHO cell growth is negatively affected. The pH of cell cul-
can increase the osmolality of the culture environment ture is usually interactively regulated by C
­ O2 and inorganic
(Ha et al. 2022). In fed-batch process, NaCl maintained salts to improve CHO cell growth, which can achieve large-
high osmolality. It increased the titer of mAb, reduced scale recombinant proteins production.

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Dissolved oxygen 437 mg/L (Yuan et al. 2020). In order to improve the
expression of human mouse chimeric anti epidermal
DO is one of the key environmental parameters in large-scale growth factor receptor variant III (EGFR vIII) antibody
process, which affects cell growth and recombinant protein C12 in CHO cells, continuous feeding strategy was
production (Chotigeat et al. 1994; Restelli et al. 2006). Most adopted and feed medium was added to the bioreactor
of DO values in CHO cells are usually in the range of 20 to from day 3. Compared with bolus feeding methods, the
50% (Fleischaker and Sinskey 1981). Ventini et al. reported continuous feeding method reduced the ratio of cell meta-
that the final recombinant thyroid-stimulating hormone bolic byproducts, culture osmolality and high mannose
(rTSH) titer was 1.6 mg/L when DO value was 20% in fed- type, improved the VCD and viability of CHO cells, and
batch culture mode (Ventini et al. 2011). To improve the the antibody production exceeds 10 g/L (Xiao et al. 2021).
yield and quality of human IgG1 mAb, Mahé et al. adjusted Horvat et al. used continuous feeding to optimize fed-batch
the DO value to 40%, and CHO cells expressing human IgG1 culture process to culture CHO cells. From day 4, glucose
mAb were cultured in fed-batch culture supplied with Cop- solution with a concentration higher than 400 g/L was sup-
per Acetate and Ferric Citrate. The yield of mAb reached plemented to keep final glucose concentration less than
more than 10 g/L in 14 days fed-batch culture, Copper Ace- 5.55 mmol/L. From day 5, feed medium containing serine
tate and Ferric Citrate diminished man5 content and acidic was continuously added (Horvat et al. 2020). Continuous
charge variants of mAb and increased the proportion of G1F feeding can reduce the osmolality and mannosylation ratio
(Mahé et al. 2022). of the cell culture, but it increased the mAb yield. If the
VCD and cell viability of CHO cells show a downtrend in
the late exponential phase or in the early stationary phase,
feed medium should be added in cell culture. Great feed
Feeding strategy strategy has a positive impact on RTP production.
Monitoring and control system is introduced in fed-batch
In addition to adjusting the composition of feed medium culture, which maintains key cellular nutrients at an ideal
and setting reasonable process parameters, it is neces- level. Bolus feed delivery strategy is widely used for fed-
sary to design and develop available feeding strategy to batch culture, but it cannot adjust the medium concentration
produce the high-yield and high-quality of recombinant to the dynamic nutrient consumption rate of cells at different
proteins (Horvat et al. 2020). The control criterion and the metabolic stages (Xie and Wang 1994a, b).
mode of feeding should be taken into consideration in the Raman spectroscopy is used for monitoring nutrient and
aspect of feeding strategy. Keeping the concentration of byproducts (glucose, lactate, and amino acid) (Domján et al.
one or two main nutrients at an ideal level is completed 2022). Dynamic feeding strategies are used to control the deliv-
by control criterion. According to the stoichiometric feed ery of feed medium via Raman spectroscopy. In an early phase
composition, the rest of nutrients are added to culture of the development, Domján et al. established a partial least
system (Costa et al. 2014). Feeding strategy is usually squares (PLS) calibration model that took the cell metabolic
divided into two addition methods: continuous feeding behavior and nutritional consumption into account. Subse-
and bolus feeding. Bolus feeding has the advantages of quently, the nutrients are maintained at the desired level (Dom-
simple operation, low facility requirements and low pro- ján et al. 2022). For mAb production in CHO cells, Eyster et al.
duction costs. Bolus feed delivery strategy increases the developed Raman spectroscopy to control lactate and glucose
expression of mAbs in CHO cells and is the most widely in fed-batch culture, and established PLS model for predict-
used fed-batch culture method for large-scale mammalian ing glucose and lactate concentrations. Three feeding strate-
cell culture. However, the addition of a large amount of gies were evaluated in terms of cell metabolism, productiv-
feed medium leads to accumulation of metabolic byprod- ity, and product quality. Ammonium levels were reduced by
ucts and the increase of osmolality. Continuous feeding 68% through controlling lactate at 2 g/L, while mAb galacto-
can deal with two problems above, and it can adjust the sylation levels were increased by approximately 50% (Eyster
feeding rate to maintain the concentration of nutrients, et al. 2021) .
which is based on the nutrients consumption. Therefore,
continuous feeding has more advantages in the aspect of
cell culture (Xiao et al. 2021; Kamachi and Omasa 2018). Metabolic byproducts in fed‑batch culture
To promote the high expression of anti-HER2 mAb in
CHO cells, Feed 4 was added at 5% of the initial culture In the late stages of cell culture, fed-batch culture usually
volume at days 3, 5, 7, and 9. Peak VCD was 17.1 × ­106 stores byproducts such as lactate and ammonium (Brun-
cells/mL in fed-batch culture and mAb yield reached ner et al. 2017; Fan et al. 2015). High concentrations of

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1070 Applied Microbiology and Biotechnology (2023) 107:1063–1075

metabolic byproducts are detrimental to cell growth and The intensified fed‑batch
antibody expression (Torres et al. 2018; Zhang et al. 2019;
Konakovsky et al. 2016; Karengera et al. 2018). Glucose is In the biomanufacturing industry, intensified processes
transferred to the cytoplasm by transport protein GLUT1 require more time to develop compared with conventional
in CHO cells, and a large amount of pyruvate is generated fed-batch.
through glycolysis. Part of pyruvate using lactate dehydro- Schulze et al. performed standard fed-batch (sFB) and
genase is further converted to lactate (Li et al. 2012). To intensified fed-batch (iFB) processes in 15 mL and 250-
culture CHO cells expressing Fc-fusion protein and reduce mL bioreactors, starting with 4% (vol / vol) feed medium
lactate accumulation in fed-batch culture, galactose is used A and 4% (vol / vol) feed medium B. The final glucose
as a carbon source to reduce accumulation of lactate (Xiao concentration was 5 g/L. Cell concentrations inoculated by
et al. 2019). CHO-K1 cells expressing anti-HIV antibody N-1 perfusion reached 1 × ­108 cells/mL, which are used for
VRC01 were cultured in fed-batch culture. Feed medium the iFB. In 15 mL bioreactors, two iFBs inoculated at 2.5
was added daily starting from day 3. After 12 h of cell inocu- and 5 × ­106 cells/mL were from N-1 perfusion. Compared
lation, 10 mM and 30 mM ­NH4Cl were added to the CHO with sFB process, similar titers were reached in shorter
cell culture medium. Compared with the control group, VCD process times, but respectively, the space–time yield
and culture time of group treated with 30 mM ­NH4Cl were (STY) was dramatically increased by 16% and 36% for the
significantly reduced, and byproducts including lactate, glu- 2.5 and 5 × ­106 cells/mL methods. In 250-mL bioreactors,
cose, ammonium, and glutamine were accumulated. Particu- iFB inoculated at 5 × ­106 cells/mL added 2.5 mM butyric
larly, ammonium had a negative impact on antibody titer and acid (BA) after 48 h. Compared with iFB inoculated at
cell-specific productivity (Chitwood et al. 2021). 5 × ­106 cells/mL without BA, KPI was found by raising
The addition of TCA cycle intermediates including alpha- the STY by 50% after BA treatment. The average cell-
ketoglutarate (α-KG), malate, and succinate to CHO cells specific productivity was increased from 25 to 37 pg/cell/
can reduce accumulation of lactate and ammonium in fed- day through BA supplementation (Schulze et al. 2022a, b).
batch culture and improve cell-specific productivity and
antibody titer (Zhang et al. 2020b). Xiao et al. designed
continuous feeding strategy in fed-batch culture. They found
that continuous feeding reduced accumulation of lactate and Discussion
ammonium because of massive nutritional feed medium in a
7-L bioreactor. Finally, the production of C12 antibody was RTPs are fast developing. About 40 novel antibodies are
promoted (Xiao et al. 2021). In fed-batch culture, byproducts introduced each year. How to implement high-level expres-
should be avoided. It is advisable for fed-batch culture to sion and production of RTPs is a challenge which limits
choose available feeding strategies to reduce the probability RTP drugs development. These are state-of-the-art strat-
and concentration of metabolic byproducts accumulation. egies for boosting RTP production and quality, such as
optimizing gene sequence, expression vector, and glyco-
sylation (Zhang et al. 2022).
Chemostat cultivation A report has shown that the biosimilar development
of pembrolizumab can be directed by Quality by design
For improving cell-specific productivity of RTPs, success- (QbD) (Jaffar-Aghaei et al. 2022). QbD is an efficient but
ful strategies require to manipulate culture temperature and challenging approach for the development of biosimilar
glucose concentration in media. Unfortunately, changes in due to the complex relationship among process, quality,
cell culture affect the specific growth rate, especially in both and efficacy (Zhang et al. 2020c). To ensure critical qual-
operational variables. To produce recombinant human tissue ity attributes (CQAs) about the production of mAbs, an
plasminogen activator (rh-tPA) in CHO cell, Vergara et al. uti- understanding of the production process is highlighted by
lized chemostat cultivation and set three groups about feeding QbD (Gibbons et al. 2022). Jaffar-Aghaei et al. found a
media with 20 mM, 30 mM, and 40 mM glucose concentra- pharmaceutical measurement guided by QbD to under-
tions. A total of 40 mM glucose limited cell growth, but it take the Pembrolizumab biosimilar candidate PSG-024
increased cell-specific productivity of rh-tPA and cells in the (Keytruda®) in fed-batch culture. Currently, Keytruda®
G2/M phase. Furthermore, glucose consumption and lactate has become the ace of therapeutic mAbs (Jaffar-Aghaei
production rates were reduced when temperature condition et al. 2022).
was 33 °C. Vergara et al. indicated that a reduced specific Despite the fact that glucose concentration above 1 g/L
growth rate together with high feed glucose rapidly improves had brought 10 g/L mAb titer, Handlogten et al. still
protein productivity in CHO cell (Vergara et al. 2018). pointed out that bolus feeding was defective (Handlogten

13
Applied Microbiology and Biotechnology (2023) 107:1063–1075 1071

et al. 2018). Recently, the final mAb titer of 4 g/L was manuscript; TYW edited the review content. TYW and YL funded
reached by the glucose concentration of 12 g/L in fed- this manuscript.
batch platform process (Schellenberg et al. 2022); mean- Funding This work was funded by National Natural Science Founda-
while, the final glucose concentration was 5 g/L. This glu- tion of China (No.32101232), and Basic Research Foundation of Key
cose feeding strategy also promoted cell growth efficiently. Scientific Research of Universities in Henan Province (No. 20zx013).
Cysteine greater than 2.5 mM with low cell densities
Data Availability All data included in this study are available upon
resulted in counteracted oxidative stress, but Komuczki request by contact with the corresponding author.
et al. presented that high cysteine concentration with a
higher cell concentration can counteract oxidative stress Declarations
(Komuczki et al. 2022). Wang et al. reported that an upturn
in terms of the product titer was influenced by tempera- Human and animal rights and informed consent This paper does not
contain any studies with human participants or animals performed by
ture shifted to 33℃ in 14-day fed-batch culture, and 100% any of the authors.
normalized titer was set as the highest titer on day 14.
Fed-batch optimization is available and nevertheless, opti- Conflict of interest The authors declare no competing interests.
mizing all process parameters is impossible on account of
limited resources and timeline (Wang et al. 2022).
Biosimilars will enter the markets in the context of
COVID-19 (Schulze et al. 2022a, b; Tichy et al. 2022), References
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