Optimization of Key Factors in Serum Free Medium for Production of Human Recombinant GM-CSF Using Response Surface Methodology

Document Type : Research article


1 Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

2 Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.


Researchers add serum to a classical medium at concentrations of 5 to 10% (v/v) to grow cells
in-vitro culture media. Unfortunately, serum is a poorly defined culture medium component
as its composition can vary considerably while serum-free cell culture media are an excellent
alternative to standard serum-containing media and offer several major advantages. Advantages
of using serum-free media include a lower risk of infectious agents, lower risk of interfering
components, less contaminant, avoids ethical issues. According to previous studies insulin,
selenium, transferrin and glucose are important component of serum that affect cell growth. In
the present study, we optimized amount of these factors in order to serum free culture medium
fabrication. Response surface methodology (RSM) was employed for optimization of key factors
in serum free medium to enhance recombinant human GM-CSF (rhGM-CSF) production in CHO
cell line. Four important process parameters including insulin concentration (0-2 g/L), transferrin
concentration (0-1 g/L), selenium concentration (0-0.001 g/L) and glucose concentration (0-5
g/L) were optimized to obtain the best response of rhGM-CSF production using the statistical
Box–Behnken design. The experimental data obtained were analyzed by analysis of variance
(ANOVA) and fitted to a second-order polynomial equation using multiple regression analysis.
Numerical optimization applying desirability function was used to identify the optimum
conditions for maximum production of rhGM-CSF. The optimum conditions were found to be
insulin concentration = 1.1 g/L, transferrin concentration = 0.545 g/L, selenium concentration =
0.000724 g/L and glucose = 1. 4 g/L. Maximum rhGM-CSF production was found to be 3.5 g/L.


Main Subjects

(1) Andersen DC and Krummen L. Recombinant protein
expression for therapeutic applications. Curr. Opin.
Biotechnol. (2002) 13: 117-23.
(2) Khan KH. Gene expression in mammalian cells and
its applications. Adv. Pharm. Bull. (2013) 3: 257-63.
(3) Babaeipour V, Vahidi H, Alikhani S, Ranjbari
J, Alibakhshi A and Tabarzad M. Effect of acyl
homoserine lactone on recombinant production of
human insulin-like growth factor-1 in batch culture
of Escherichia coli. Protein Pept. Lett. (2018) 25:
(4) Baik JY, Dahodwala H, Oduah E, Talman L, Gemmill
TR and Gasimli L. Optimization of bioprocess
conditions improves production of a CHO cell‐
derived, bioengineered heparin. Biotechnol. J.
(2015) 10: 1067-81.
(5) Ranjbari J, Babaeipour V, Vahidi H, Moghimi
H, Mofid MR and Namvaran MM. Enhanced
production of insulin-like growth factor I protein in
Escherichia coli by optimization of five key factors.
Iran. J. Pharm. Res. (2015) 14: 907-17.
(6) Xing Z, Kenty B, Koyrakh I, Borys M, Pan SH and
Li ZJ. Optimizing amino acid composition of CHO
cell culture media for a fusion protein production.
Process Biochem. (2011) 46: 1423-9.
(7) Altamirano C, Paredes C, Cairo J and Godia
F. Improvement of CHO cell culture medium
formulation: simultaneous substitution of glucose
and glutamine. Biotechnol. Prog. (2000) 16: 69-75.
(8) Garcia-Kirchner O, Segura-Granados M and
Rodriguez-Pascual P. Effect of media composition
and growth conditions on production of β-glucosidase
by Aspergillus niger C-6. Twenty-Sixth Symposium
on Biotechnology for Fuels and Chemicals. Springer
(9) Even MS, Sandusky CB and Barnard ND. Serumfree hybridoma culture: ethical, scientific and safety
considerations. Trends Biotechnol. (2006) 24: 105-8.
(10) Shah G. Why do we still use serum in the production
of biopharmaceuticals? Dev. Biol. Stand. (1999)
99: 17-22.
(11) Ritacco FV, Wu Y and Khetan A. Cell culture
media for recombinant protein expression in
Chinese hamster ovary (CHO) cells: History,
key components, and optimization strategies.
Biotechnol. Prog. (2018) 34: 1407-26.
(12) Gstraunthaler G. Alternatives to the use of fetal
bovine serum: serum-free cell culture. ALTEX
(2003) 20: 275-81.
(13) Gu C, Li P, Liu W, Zhou Y and Tan WS. The
role of insulin in transdifferentiated hepatocyte
proliferation and function in serum‐free medium.
J. Cell. Mol. Med. (2019) 23: 4165-78.
(14) Bayat H, Hossienzadeh S, Pourmaleki E, Ahani R
and Rahimpour A. Evaluation of different vector
design strategies for the expression of recombinant
monoclonal antibody in CHO cells. Prep. Biochem.
Biotechnol. (2018) 48: 160-4.
(15) Saia-Cereda VM, Aquino A, Guest PC and Martinsde-Souza D. Two-dimensional gel electrophoresis:
A reference protocol. Adv. Exp. Med. Biol. (2017)
974: 175-82.
(16) Solanki AB, Parikh JR and Parikh RH. Formulation
and optimization of piroxicam proniosomes by
3-factor, 3-level Box-Behnken design. AAPS
PharmSciTech. (2007) 8: 43.
(17) Miki H and Takagi M. Design of serum-free
medium for suspension culture of CHO cells on the
basis of general commercial media. Cytotechnology
(2015) 67: 689-97.
(18) Xu J, Yan FR, Li ZH, Wang D, Sheng HL and Liu
Y. Serum-free medium optimization based on trial
design and support vector regression. Biomed. Res.
Int. (2014) 2014: 269305.
(19) Kim DY, Lee JC, Chang HN and Oh DJ. Development of serum-free media for a recombinant CHO
cell line producing recombinant antibody. Enzyme
Microb. Technol. (2006) 39: 426-33.