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FEEDING STRATEGIES IN RECOMBINANT HUMAN ERYTHROPOIETIN PRODUCTION
BY Pichia pastoris FERMENTATION IN STIRRED-TANK BIOREACTOR
by
SHAHKILLAHWATI BINTI MOHD RIDHWAN
Thesis submitted in fulfillment of the requirements for the degree of
Master of Science
August 2014
ii
ACKNOWLEDGEMENTS
First and foremost, I wish to express my sincere gratitude to my supervisor
Assoc. Prof. Dr. Ahmad Ramli Mohd Yahya, for his continuous support, guidance
understanding, valuable advices, brilliant ideas, criticism and patience, in all the
possible way, throughout my research work.
I am thankful to my co-supervisor Assoc. Prof. Dr. Yahya Mat Arip for his
constructive comments and for his important support throughout this work. Not
forgetting, to Assoc. Prof. Dr Amirul Al-Ashraf for his guidance and for giving me
the opportunity to use the equipments for this research and also for his valuable
advices and criticisms.
I warmly thank Rennuka, Adila, Shantini, Nur Ashiffa Kai Hee, Zainatul
Asyikin for all their encouragement and interest throughout my graduate program. I
would also like to thank all my friends from Lab 318, lab 218 and lab 414 for their
co-operations for the use of laboratories facilities
I am also thankful to all academic, administrative and technical staffs in
School of Biological Sciences, for their assistance throughout my education.
Above all, I would like to deeply thank to my uncle, Dato’ Mohd Shafiq Bin
Abdullah for his continued support and help through out my education and to my
family for loving and encouraging me all through my life.
iii
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
TABLE OF CONTENTS iii
LIST OF TABLES ix
LIST OF FIGURES x
LIST OF PHOTOGRAPHS xvi
LIST OF ABBREVIATIONS xvii
LIST OF APPENDICES xx
LIST OF PUBLICATIONS AND SEMINARS xxi
ABSTRAK xxii
ABSTRACT xxiii
CHAPTER 1.0: INTRODUCTION 1
1.1 Motivation and scope of the project 2
1.2 Objectives of the study 3
CHAPTER 2.0: LITERATURE REVIEW 4
2.1 Human erythropoietin (EPO) 4
2.1.1 Primary role in red blood cell production 4
2.1.2 Secondary roles 5
2.1.3 EPO gene structure 6
2.2 Pichia pastoris 7
iv
2.2.1 Metabolism of Pichia pastoris 8
2.2.2 Expression system of Pichia pastoris 12
2.2.3 Methanol utilization phenotypes of Pichia pastoris 14
2.2.5 Proteolytic degradation in Pichia Pastoris 15
2.3 Medium design and bioreactor operation parameters 16
2.3.1 Medium design 16
2.3.2 Carbon source 17
2.3.3 pH 18
2.3.4 Temperature 20
2.4 Fed-batch fermentation of Pichia pastoris 21
2.4.1 High cell density cultivation 21
2.4.2 Fed-batch process control strategies 23
2.4.2.1 μ-stat control 23
2.4.2.2 Constant methanol concentration 25
2.4.2.3 DO-stat control 26
2.4.3 Fed-batch feeding strategy 26
2.4.3.1 Constant volume fed-batch 27
2.5 Concluding remark 28
v
CHAPTER 3.0: MATERIALS AND METHODS 30
3.1 Chemicals 30
3.2 General methodologies 30
3.2.1 Microorganism 30
3.2.2 Sterilization 30
3.2.3 Centrifugation 30
3.2.4 Measurement of pH 31
3.2.5 Cell concentration 31
3.2.6 Material weighing 31
3.2.7 Culture incubation 31
3. 3 Buffers and stock solutions 32
3.3.1 Breaking buffer 32
3.3.2 SDS-PAGE buffer and solutions 33
3.3.3 Coomassie blue staining 34
3.4 Growth medium and storage of microorganism 34
3.4.1 Solid medium 34
3.4.2 Precultivation medium and glycerol stock solution 35
3.4.3 Production medium and trace salt 36
3.4.4 Inocula preparation 37
3.5 Production in a shake flask system 38
3.5.1 Identification of rhuEPO 38
vi
3.5.2 Cultivation medium formulation 39
3.5.3 Effect of carbon sources 40
3.5.5 Effect of methanol concentration 40
3.5.6 Effect of induction temperature 41
3.6 RhuEPO production in 3.6L bench-top bioreactor 42
3.6.1 Batch cultivation 42
3.6.2 Fed-batch cultivation with constant low rate of
glycerol 43
3.6.3 Methanol fed-batch phase (MFB) 45
3.6.3.1 Methanol induction every 24 hours 45
3.6.3.2 Constant flow rate of methanol 45
3.6.3.3 Effect of different methanol feeds 45
3.6.3.4 Stepwise increase of methanol 47
3.6.3.5 DO-stat control 48
3.7 Analyses 49
3.7.1 Protein Analyses 49
3.7.1.1Total protein 49
3.7.1.2 EPO concentration 50
3.7.1.3 SDS-Polyacrylamide Gel Electrophoresis
(SDS-PAGE) 50
3.7.1.4 Staining the SDS-PAGE gels 52
vii
3.7.2 Yeast lysate 52
3.7.3 Protease analysis 53
CHAPTER 4.0: RESULTS AND DISCUSSION 54
4.1 Production in a shake flask system 54
4.1.1 Identification of rhuEPO 54
4.1.2 Cultivation medium formulation 57
4.1.3 Effect of carbon sources 60
4.1.4 Effect of methanol concentration 65
4.1.5 Effect of induction temperature 67
4.2 Batch fermentation in 3.6 L bioreactor 69
4.3 Fed batch cultivation 72
4.3.1 Fed-batch cultivation with constant flow rate of
glycerol 72
4.3.2 Methanol fed-batch phase (MFB) 75
4.3.2.1 Methanol induction every 24 hours 75
4.3.2.2 Constant flow rate of methanol 78
4.3.2.3 Effect of different methanol feeds 80
4.3.2.4 Stepwise increase flow rate of methanol 82
4.3.2.5 DO-stat control 86
CHAPTER 5.0: CONCLUSSION 93
5.1 Recommendation for future research 95
viii
REFERENCES 97
APPENDICES
ix
LIST OF TABLES
Page
Table 2.1: Advantages and disadvantages of P. pastoris 13
Table 3.1: The compositions of SDS-PAGE buffer and solution 32
Table 3.2: Staining recipes for SDS-PAGE gel using Coomassie
Blue. 33
Table 3.3: Induction of different methanol concentration in
different flasks. 40
Table 3.4: Different methanol flow rate modified from
Invitrogen, (2002). 45
Table 4.3.2.5: Effects of methanol flow rate on EPO production. 91
x
LIST OF FIGURES
Page
Figure 2.1: The structure of human erythropoietin 6
Figure 2.2: The glycerol catabolic pathway in P. pastoris 10
Figure 2.3: The methanol pathway in P. pastoris. 11
Figure 3.1: Scale-up steps and the bench-top scale bioreactor system. 42
Figure 3.2: The simulation setup using IRIS V 5.3 for constant
flow rate of glycerol. 43
Figure 3.3: The simulation setup using IRIS V 5.3 for stepwise
increase flow rate. 46
Figure 3.4: The simulation setup using IRIS V 5.3 for DO-stat
control. 47
Figure 4.1.1: Analysis on 1% (w/v) agarose gel of direct
yeast colony by PCR. 54
Figure 4.1.2 (a): Growth kinetic in PBM and BMGY 57
Figure 4.1.2 (b): pH changes of P. pastoris in PBM and BMGY culture
media. 57
Figure 4.1.3 (a): Variation in P. pastoris cell concentration with time
in PBM medium containing sorbitol and glycerol. 57
Figure 4.1.3 (b): Coomassie Blue staining on SDS-PAGE showed
xi
extracellular proteins produced in laboratory scale
shake flask in P pastoris. 57
Figure 4.1.3 (c): EPO concentration in production medium
contained sorbitol with induction every 24 hours
of 1% (v/v) methanol. 60
Figure 4.1.3 (d): Coomassie Blue staining on SDS-PAGE gel of
extracellular protein produced in laboratory scale
baffle shake flask in P pastoris. 62
Figure 4.1.3 (e): EPO concentration in production medium containing
sorbitol with addition of 1% (v/v) methanol every
24 hours interval time. 62
Figure 4.1.4 (a): Growth curve of Pichia pastoris GS115 at different
methanol concentration. 64
Figure 4.1.4 (b): EPO concentration in different methanol concentration
at 84 hours of cultivation. 64
Figure 4.1.5 (a): Growth kinetic of Pichia pastoris GS115 at different
temperature. 66
Figure 4.1.5 (b): EPO concentration in different temperature; 25 oC
xii
and 30 oC. The methanol was induced at every
24 hours time interval. 66
Figure 4.2 (a): Graph showing the growth kinetic of P. pastoris in batch
cultivation system and graph. 69
Figure 4.2 (b): Graph showing the dissolve oxygen (blue line),
stirrer speed (red line), pH (green line) and temperature
(black line) in batch cultivation system.
69
Figure 4.3.1(a): The growth kinetic of Pichia pastoris in glycerol
fed-batch cultivation. Fed-batch was initiated
(from 24 to 48hr) by feeding 50% (v/v) of glycerol
containing PTM at 7 mL/h constant flow rate. 72
Figure 4.3.1(b): Dissolved Oxygen profile (blue line) and stirrer
speed (red line) in glycerol fed-batch with constant
flow rate of 50% glycerol. 72
Figure 4.3.2.1 (a): Growth kinetic of P. pastoris in glycerol batch (GB),
glycerol fed-batch (GFB) and methanol fed-batch (MFB). 75
Figure 4.3.2.1(b): Dissolved Oxygen profile (blue line) for 50% glycerol
with 1% (v/v) methanol (every 24 hours). 75
Figure 4.3.2.1 (c): EPO concentration and biomass
xiii
in MFB with 1% methanol. 75
Figure 4.3.2.2 (a): Growth kinetic of P. pastoris in constant flow rate
of methanol. 77
Figure 4.3.2.2 (b): showing the dissolved oxygen profile (blue line) and
stirrer speed (red line). 77
Figure 4.3.2.2 (c): Graph showed EPO concentration in MFB with 1% (v/v)
methanol added every 24 hours. 77
Figure 4.3.2.3 (a): Variation of EPO concentration for different feeding
strategies. 79
Figure 4.3.2.3 (b): Variation of tyrosine concentration for different
methanol feeds strategies with different flow rate. 79
Figure 4.3.2.4 (a): Graph showing growth kinetic of P. pastoris in constant
flow rate of glycerol in glycerol fed-batch (GFB) and
stepwise increase feed volume in methanol fed-batch
(MFB). 82
Figure 4.3.2.4 (b): Graph profile for dissolved oxygen of constant
glycerol feed in glycerol fed-batch (GFB) and
step wise increase in methanol fed-batch (MFB). 82
xiv
Figure 4.3.2.4 (c): Graph showing the extracellular EPO concentration 83
Figure 4.3.2.4 (d): Tyrosine concentration for stepwise increase feeding
in methanol fed-batch phase. 83
Figure 4.3.2.4 (e): Intracellular EPO concentration for stepwise increase
feeding in methanol fed-batch phase. 83
Figure 4.3.2.5 (a): Graph profile for dissolved oxygen (blue line) and stirrer
speed (red line) for constant flow rate of 50% (v/v) glycerol
and DO-stat control 87
Figure 4.3.2.2 (b): graph showing a growth kinetic of constant glycerol
flow rate in glycerol fed-batch (GFB) and DO-stat control
in methanol fed-batch (MFB) with flow rate 0.1mL/min.
The orange arrows indicated the start (72 hours) and end
time (84 hours) of methanol feed. 87
Figure 4.3.2.5 (c): Feeding profile for DO-stat control in MFB phase. 90
Figure 4.3.2.5 (d): The EPO concentration in MFB for DO-stat control
feeding in methanol fed-batch phase. 88
Figure 4.3.2.5(e): Tyrosine concentration for DO-stat control
of methanol feed in methanol fed-batch phase. 90
Figure 4.3.2.5 (f): The intracellular EPO concentration for
xv
DO-stat control of methanol in methanol
fed-batch phase. 90
xvi
LIST OF PLATES
Plate 4.1.1 (a): Pichia pastoris GS115 colonies on minimal
methanol agar. 53
Plate 4.1.1 (b): Pichia pastoris under light microscope at
1000X magnification. 53
Plate 4.1.2: Growth of P. pastoris in Phosphate buffer medium
(PBM) and Buffer minimal glycerol complex medium
(BMGY). 58
Plate 4.3.2.2: Pichia pastoris cells before MFB; A Cells
at 60 hours and during MFB; B are the cells at
72 hours which are bigger than the cells at
60 hours, C are the cells at 84 hours. 89
xvii
LIST OF ABBREVIATIONS
Abbreviations
AOX Alcohol oxidase
BMGY Buffered glycerol-complex medium
BMMY Buffered methanol complex
BFU-E Burst forming unit erythroid cells
C Concentration in the medium g L-1 or mol m-3
CBB G- 250 Coomassie brilliant blue G-250
CDW Cell dry weight
CFU-E Colony forming unit-erythroid
DMSO Dimethyl sulpoxide
DNA Deoxyribonucleic acid
DO Dissolved oxygen
EDTA Ethylene diaminetetraacetic acid
EPO Human erythropoietin hormone
g Relative centrifugal force (centrifugation)
hGH Human growth hormone
HPLC High pressure liquid chromatography
PCR Polymerization chain reaction
Mut+ Methanol utilization plus
Mut- Methanol utilization slow
N Agitation rate min–1
OD Oxygen demand mol m-3 sec-1
OD600 Optical density at 600 nm
xviii
PAGE Polyacrylamide gel electrophoresis
PBM Phosphate buffer medium
PBS Phosphate buffer saline
PMSF Phenylmethylsulfonyl fluoride
rhuEPO Recombinant human erythropoietin
SDS Sodium dodecyl sulphate
SDS-PAGE Sodium Dodecyl sulphate polyacrylamide gel electrophoresis
TCA Tricarboxylic acid
TEMED N,N,N’,N’-tetramethylethylenediamine
Tris Tris [hydroxymethyl] aminomethane
TweenR 20 Polyoxyethylene-sorbitan mono-laurate
YNB Yeast nitrogen base
YPD Yeast peptone dextrose
F Flow rate mL h-1
t Cultivation time h
T Temperature, °C
U One unit of an enzyme
V Volume of the bioreactor L
Y Yield (overall) g g-1
Greek Letters
ρ Density g L-1
μset Desired specific growth rate h-1
μs,max Maximum specific growth rate on sorbitol h-1
xix
Subscripts
0 Refers to initial condition
AOX Refers to alcohol oxidase
G Glycerol
M Refers to methanol
O Refers to oxygen
S Refers to sorbitol or substrate
X Refers to cell
xx
LIST OF APPENDICES
1.1: Standard curve for Pichia pastoris GS115 2 (5)
1.2: Standard curve for Bradford assay, total protein.
1.3: Standard curve of Erythropoietin
1.4: Standard curve of tyrosine
1.5: Example of feeding strategies setting with IRIS software
xxi
LIST OF PUBLICATION AND SEMINARS
Seminars
1. Production of recombinant human erythropoietin in Pichia pastoris fermentation.
2. Effect of feeding modes on biomass and rhuEPO production.
xxii
STRATEGI SUAPAN DALAM PENGHASILAN REKOMBINAN
ERYTHROPOIETIN MANUSIA MELALUI Pichia pastoris FERMENTASI
DALAM REAKTOR TANGKI TERANDUK
ABSTRAK
Pengunaan erythropietin atau EPO sebagai terapeutik dapat meningkatkan
kualiti hidup di kalangan pesakit kanser, buah pinggang dan anemia. Mengetahui
kepentingan EPO dan permintaan yang tinggi di pasaran biofarmaseutikal,
rekombinan erythropoietin manusia (rhuEPO) telah dihasilkan secara genetik
menggunakan Pichia pastoris strain GS115 2 (5) sebagai sel perumah melalui proses
pengkulturan tiga-peringkat. Kultur pertama kali dikulturkan dalam medium
penghasilan yang mengandungi gliserol sebagai sumber karbon (fasa I) sebelum
memperolehi sel berkepadatan tinggi (fasa II) dan diaruhkan dengan metanol untuk
penghasilan protein (fasa III). Formulasi medium dan peningkatan kultur
sekelompok diselidiki sebelum fermentasi suap-kelompok. Pelbagai strategi
pemakanan dan keadaan aruhan metanol yang mempengaruhi proses penghasilan
protin juga dikaji. Sebuah proses penghasilan sel berkepekatan tinggi telah dihasilkan
mengambil masa 60 jam untuk mencapai tujuh kali ganda sel dengan jumlah sel
sebanyak 132.10 g/L menggunakan suap malar. Keadaan aruhan juga telah
diperbaiki mengunakan suapan menaik secara berperingkat. Ia hanya mengambil
masa 19 jam dari fasa aruhan untuk menghasilkan sejumlah 104.8 ng/L EPO dengan
hasil produk per biojisim (Y p/x) 1.19 ng g-1 berbanding dengan cara pengkulturan
biasa 36 jam.
xxiii
FEEDING STRATEGIES IN RECOMBINANT HUMAN ERYTHROPOIETIN
PRODUCTION BY Pichia pastoris FERMENTATION
IN STIRRED-TANK BIOREACTOR
ABSTRACT
Administration of erythropoietin or EPO as a therapeutic improves the quality
of life in patients with cancer, renal and anemia. Knowing the importance of EPO
and the high demand in the biopharmaceutical market, Pichia pastoris strains GS115
2 (5) has been genetically engineered to produce recombinant human erythropoietin
(rhuEPO) from the synthetic gene using three-stage cultivation process. The culture
was first grown in production medium containing glycerol as a carbon sources (phase
I) prior to obtained high cell density culture (phase II) and was induced with
methanol for protein production (phase III). Medium formulation and enhanced batch
cultivation was investigated prior to fed-batch fermentation. Various feeding
strategies and methanol induction conditions which highly influenced the production
of protein were also investigated. A process of high cell density cultivation was
developed which took 60 hours for the culture to reach seven fold of cells 132.10 g/L
using constant flow rate. An improved induction condition was also developed using
step wise increase feeding strategy. It took only 19 hours from the induction phase to
produce a total of 104.8 ng/L of EPO with the yield of product over biomass (Y p/x)
1.19 ng g-1 compared to the preliminary 36 hours.
1
1
CHAPTER 1.0: INTRODUCTION
Pichia pastoris is methylotrophic yeast capable of utilizing methanol as a sole
carbon and energy source; it is a promising foreign protein expression host, offering
numerous advantages. It requires simple molecular genetic manipulations. The high
cell densities culture can be achieve on simple, inexpensive media (Thorpe et al.,
1999). In the controlled environment of a bioreactor, it is possible to achieve high
cell densities of about 400 g/L cell fresh weight or 130 g/L cell dry weight,
respectively with P. pastoris (Jahic et al., 2002). Moreover, it has good secretion
ability for proteins (Jahic et al., 2006). More than 700 proteins have been expressed
in P. pastoris (Zhang et al., 2009). The most essential to the fast, inexpensive
production in P. pastoris is the strong inducible AOX1 promoter which drives the
expression of recombinant protein and is inducible in the presence of methanol.
Erythropoietin (EPO) is a glycoprotein hormone function as a red blood cell
formation stimulator in bone marrows and also initiates hemoglobin production. It
synthesized by interstitial fibroblasts in kidney during adult and liver by per
sinusoidal cells during fetal and predominantly during adulthood (Ahmed et al.,
2002; Buemi et al., 2005; Siren et al., 2009). Administration of EPO as a therapeutic
improved the quality of life in patients with cancer, renal and anemia.
Knowing the importance of EPO and the high demand in the
biopharmaceutical market, Pichia pastoris strains GS115 2 (5) has been genetically
engineered (Bustami et al., 2009) to produce recombinant human erythropoietin
(rhuEPO) from the synthetic gene. This transformed produce a strain with the
phenotype of hEPO-Mut+ for an extracellular recombinant human erythropoietin.
2
Achieving high cell density has always been a vital step since the production
of heterologous protein expression is frequently dictated by the cell density. In this
research, the development of fermentation process for high cell density culture of
recombinant Pichia pastoris has been carried out in shake flask and bench-top scale
bioreactor system. Growth and production parameters such as selection of cultivation
medium, carbon sources, methanol concentration, induction temperature as well as
methanol feeding strategies were investigated in batch and fed-batch cultivation for
high cell density culture and EPO production. An improved fermentation process for
biomass and EPO production was developed from this research.
1.1 Motivation and scope of the project
Several studies of recombinant human erythropoietin production from cDNA
using Pichia pastoris as a host has been reported and conducted by other researchers
in other parts of the world such as Turkey and Indonesia (Bayraktar et al., 2009;
Calik et al., 2009; Celik et al., 2007; Celik et al., 2010; Soyaslan and Calik, 2011)
and a few has been conducted in Malaysia (Teh et al., 2011). However none of
synthetic recombinant human erythropoietin production has been reported in large
scale fermentation.
The main focus of this project was to produce synthetic recombinant human
erythropoietin from genetically engineered Pichia pastoris having a methanol
utilization phenotypes hEPO-Mut+ in a high cell density culture and to investigated
the effect of feeding strategies on EPO production. The research began with shake
flask experiments for the selection of cultivation medium, carbon sources and
methanol induction concentration as well as induction temperature for enhanced EPO
3
production. In bench-top bioreactor studies, in order to get high cell density cultures,
three phase fed-batch cultivation was performed which contained glycerol batch
(GB), glycerol fed-batch (GFB), and methanol fed-batch phase (MFB). During the
GFB phase, a predetermined limiting substrate was fed at a constant flow rate. In
MFB phase, the effect of using different methanol feeding strategies on cell growth
and rhuEPO production was investigated. Quantification of extracellular and
intracellular EPO was done by using an accurate determination of cytokine protein
concentration enzyme-linked immunosorbent assays (ELISA).
1.2 Objectives of the study
This work studies the development of a fermentation process for the
production of human erythropoietin in P. pastoris. This is included in all of the
objectives below:
1. To investigate the effect of glycerol and sorbitol as a carbon sources and
methanol induction on biomass and EPO production as well as to obtain a
high cell density culture in glycerol fed-batch phase.
2. To access different methanol feeding strategies in methanol fed-batch phase
toward biomass and EPO production.
4
CHAPTER 2.0: LITERATURE REVIEW
2.1 Human erythropoietin (EPO)
The synthesis of red blood cells in our body is controlled by a glycoprotein
hormone named erythropoietin, also known as erythropoetin or erthropoyetin (EPO).
Erythropoietin is a protein signaling molecule or a cytokine for erythrocyte
precursors in the bone marrow (Ahmet et al., 2011; Siren et al., 2009).
Hematopoietin or human EPO is manufactured by interstitial fibroblasts in kidney.
The molecular weight of EPO is 34 kDa (Atkins et al., 1991; Lu et al., 2006).
Furthermore it is also produced in the liver by perisinusoidal cells during fetal period
and predominant during adulthood. Besides regulating the making of red blood cell,
EPO was also known to have association in brain response especially in neuronal
injury and engaged in healing wound (Clavio et al., 2004; Dordal and Goldwasser,
1982; Kao et al., 2011; Nissenson, 1989; Youssoufian et al., 1990).
2.1.1 Primary role in red blood cell production
The erythropoiesis process will never arise in the absence of EPO (Osterborg,
2000). In order to increase the production of the red blood cell, the kidney will
directed toward CFU-E pro-erythroblast and basophilic erythroblast, subsets in the
differentiation before manufacturing and generating erythropoietin
under hypoxic conditions (Coleman and Brines, 2004). The survival of red blood cell
progenitors in human bone marrow can be prolonged by erythropoietin which will
defend these cells from programmed cells death process (Ferri and Grassi, 2010).
5
In addition, the development of erythroid linage from multipotent progenitors
required erythropoietin which act as a primary erthropoietic factor to cooperate with
multiple growth factor such as IL-3, IL-6, glucocorticoids, and SCF (Benz et al.,
1992; Soubasi et al., 1999).
Erythropoietin receptor expression is initiated by the burst forming unit
erythroid (BFU-E) cells, however the colony forming unit-erythroid (CFU-E), is
entirely reliant on erythropoietin for further differentiation and is able to expresses in
maximal density erythropoietin receptor (Teh et al., 2011; Youssoufian et al., 1993).
2.1.2 Secondary roles
Besides performing many primary important deeds, erythropoietin hormones
also performed secondary action such as vasoconstriction-dependent hypertension,
stimulating angiogenesis, and proliferation of smooth muscle fibers. In addition, it
also can increase the adsorption of iron in blood by suppressing the hepcidin
hormone (Haroon et al., 2003). Furthermore, Chen et al. (2006a) has successfully
detected about 100x of EPO in brain with hypoxic damage as a natural response. Due
to this finding, the researchers believed that EPO can also affect neuronal protection
during hypoxic conditions such as stroke (Chen et al., 2006b; Lin et al., 2008).
Nevertheless, no studies on human subjects are reported at the present.
6
Figure 2.1: The structure of human erythropoietin adapted from European Molecular
Biology Laboratory (http://www.ebi ac.uk/pdbe-srv/view/entry/1eer/taxonomy).
2.1.3 EPO gene structure
Erythropoietin is a glycoprotein hormone which comprised of 165 amino
acids. The molecular mass is made up with 40% of carbohydrate (Rebollo et al.,
2003). The amino acid group of EPO is able to migrate from 18,398 to 30,000
Dalton. This process will merely occur in fully glycosylated form of EPO. The
glycosylation of EPO does not affect the biological activity (Macdougall, 1999).
Furthermore, the biological half-life of EPO molecule is only 8.5 hours. There are
fours of the carbohydrate hydrate side chains bound with sialic acid residues. The
hydrophobic polypeptide of EPO contains one O- and three N-linked sites (Wilson
and Jolliffe, 1999). The glycosylation site of three complex types N-glycans is sited
at positions 24, 38 and 83 asparagine residues and a mucin- type-O glycan located at
7
ser-126. In addition, the oligosaccharaide side chain is the site for processing and
transportation of EPO from its cell (Mayeux et al., 1994).
2.2 Pichia pastoris
Pichia pastoris is mesophilic yeast which is unicellular, having an oval shape
approximately 1-5 μm wide and 5-30 μm long. Pichia pastoris are classified under
the Kingdom Fungi, Division Eumycota, Subdivision Ascomycotina, Class
Hemoascomycetes, Order Endomycetales, Family Saccharomycetaceae and Genus
Pichia (Bayraktar et al., 2009). The eukaryotic cell structure is generally possessed a
thick polysaccharide cell wall. The yeast is classified as a facultative anaerobe based
on its oxygen requirement (Tanaka et al., 1986).
Yeast, capable of utilizing methanol as a sole carbon and energy source, is
known as methylotrophic yeast (Tanaka et al., 1986). Amongst many, only four
genera were identified up to now; namely, Hansenula, Pichia, Candida and
Torulopsis (Faber et al., 1994). Phillips Petroleum was the first company who
developed the protocols and cultivation media for P. pastoris in 1970s. The interest
on P. pastoris has increased dramatically and at present, almost more than 700 of
recombinant proteins have been produced by P. pastoris (Cregg et al., 2000).
Pichia pastoris, as a promising foreign protein expression host, offers
numerous advantages. It required simple molecular genetic manipulations. Pichia
pastoris can be growth on simple, inexpensive media (Thorpe et al., 1999).
Moreover, it has good secretion ability for proteins (Jahic et al., 2006). A detailed list
showing advantages and disadvantages of P. pastoris is given in Table 2.1(Cregg et
al., 1999; Daly and Hearn, 2005; Macauley-Patrick et al., 2005).
8
2.2.1 Metabolism of Pichia pastoris
According to the Invitrogen fermentation procedure Invitrogen, (2002)
P. pastoris can be grown first on glycerol as a substrate rather then methanol for
higher biomass yield and maximum specific growth rate. On the other hand, the
protein expression is repressed during growth on glycerol (Cregg and Higgins, 1995).
In glycerol utilization pathways (Figure 2.2), the glycerol is phosphorylated
to glycerol-3-phosphate through a glycerol kinase. The FAD-dependent glycerol-3-
phosphate dehydrogenase is further oxidized the product to dihydroxyacetone
phosphate, which will then entering the glycolytic pathway (Nevoigt and Stahl
1997). According to Nevoigt and Stahl, (1997) alternative pathways which use
NAD-dependent glycerol dehydrogenase kinase and a dihydroxyacetone for glycerol
dissimilation are performed by certain yeast only. The product of glycolysis is
pyruvate. The pyruvate dehydrogenase will then oxidize the pyruvate to acetyl–CoA.
Afterward, acetyl–CoA will enter the tricarboxylic acid (TCA) cycle, to form various
metabolites which are crucial for the synthesis of cellular constituents such as amino
acids, nucleic acids as well as cell wall components (Ren et al., 2003). It is crucial to
have better understanding Pichia metabolism for better control of growth and
product formation.
Methanol metabolism emerged as an important pathway for the P. pastoris
expression system (Dehoop et al., 1991; Johnson et al., 2001). The initial utilization
reaction occurs in the peroxisomes and later in the cytoplasm (Macauley-Patrick et
al., 2005).The first step of methanol utilization pathway is catalyzes by the alcohol
oxidase enzyme (AOX), which is responsible for the oxidation of methanol to
formaldehyde and hydrogen peroxide (Figure 2.3). The catalase enzyme was then
degraded the hydrogen peroxide formed in the first reaction to oxygen and water.
9
The formaldehyde which leaved the peroxisome was oxidized further to formate and
carbon dioxide and was used as an energy source and for growth. Formaldehyde
which still remain will be assimilated to form cell constituents in cyclic pathway and
begin the reaction with formaldehyde and xylulose 5-monophosphate. The products
of this reaction departed the peroxisome and enter the cytoplasmic pathway
reproduced xylulose 5-monophosphate and other cell supplies (Charoenrat et al.,
2006; Cregg et al., 2000).
10
Glycerol
Substrate-Level ATP
Phosphorylation ADP
Glycolysis Glycerol 3-phosphate Biomass
2 NAD 2ADP
2 NADH 2ATP CO2 NADH NAD
Pyruvate Acetaldehyde Ethanol
CO2 NAD NAD NADH
NADH NADH
0.5 O2 Acetyl-CoA Acetate
ADP NADH ATP 3NAD
ATP NAD ADP 3NADH
H2O 2CO2
Respiratory Chain Tricarboxylic Acid (TCA) Cycle
Figure 2.2: The glycerol catabolic pathway in P. pastoris (adapted from Ren et al.
(2003).
11
Peroxisome Cytosol
CH3OH CH3OH
O2 GSH
H2O2
HCHO HCHO GS-CH2OH HCOOH CO2
½ O2+H2O NAD NADH NAD NADH
GAP DHA X05P
Rearrangement reactions 1/3GAP
Cell
Constituents
DHA DHAP
ATP ADP FBP F6P
GAP Pi
Figure 2.3: The methanol pathway in P. pastoris. Adapted from; (Bayraktar et al.,
2009; Cregg et al., 1999; Ren et al., 2003).
12
2.2.2 Expression system of Pichia pastoris
Pichia pastoris with AOX1 promoter should be grown on methanol for
recombinant protein expression. In order to utilized methanol, several enzymes are
required and these enzyme are only expressed when cells are grown on methanol
(Cereghino et al., 2002; Jahic et al., 2006). When the cells are grown on other carbon
sources such as glucose and glycerol, the AOX1 promoter is partially suppressed.
However, the promoter can be strongly induced by methanol (Cereghino et al., 2002;
Jahic et al., 2006). There are two types of alcohol oxidase genes producing alcohol
oxidase which is AOX1 gene and AOX2 gene. Almost 85% of alcohol oxidase
activity in the cell is modulated by AOX1 gene (Zhang et al., 2002). On the other
hand, the AOX2 gene is only capable generated 10–20 times less AOX activity than
the AOX1 gene (Macauley-Patrick et al., 2005). The total concentration of AOX
promoter, in a methanol-limited fed-batch culture, reaches over 30% of total soluble
protein.
13
Table 2.1: Advantages and disadvantages of P. pastoris adapted from Acik et al. (2009).
Advantages Disadvantages
• High yield and productivity
• Non pathogenic
• Strong promoter (AOX1)
• Chemically defined media-simple,
inexpensive formulation
• Broad pH range: 3- 7
• Ability of utilizing methanol
• Preference for respiratory growth
rather than fermentative, a major
advantage relative S. cerevisiae
• Stable production strains
• Low purification cost
• No endotoxin problem
• Crabtree-negative
• Hyper-glycosylation is not as much
as in S. cerevisiae.
• High levels of expression of
intracellular and secreted proteins
• Eukaryotic post-translational
• Potential of proteolysis, non-native
glycosylation.
• Long time for cell cultivation
compared to bacteria
• Monitoring methanol during a
Process is difficult in order to induce
AOX1 promoter.
• Since methanol is a petrochemical
substance, it may be unsuitable for
use in the food industry and also
storing of this in industrial scale is
undesirable because it is a fire
hazard.
14
Despite the fact that AOX1 is the most efficient promoter for expressing numerous
foreign genes, the usage of methanol inducing gene expression may not be
appropriate in some conditions, especially in food production, since methane, a
petroleum-related compound is not suitable for human consumption (Boze et al.,
2001). Thus, the expressions of certain genes that do not rely on methanol or an
alternative to the AOX1 promoter are more attractive such as GAP, FLD1, PEX8,
and YPT1 promoters (Cereghino and Cregg, 2000).
2.2.3 Methanol utilization phenotypes of Pichia pastoris
The ability of P. pastoris on utilizing methanol can be divided into three
phenotypes; P. pastoris plus phenotype, Mut+, which comprised AOX1 and AOX2
genes and is able to growth on methanol at the wild-type rate. Jungo reported the
maximum specific growth rate of Mut+ on methanol is 0.14 h-1 (Jungo et al., 2006).
The second type is methanol utilization slow phenotype, Muts, which has a
dislocated AOX1 gene and only depends on the weaker AOX2 gene to metabolized
methanol. The disruptions in the AOX1 gene take place during transformation of the
expression vector. Deletion and substitution of AOX1 gene which is conducted by
the expression cassette and marker gene acquired only 10 to 20 % of transformation
actions. Therefore Muts merely relied on the weaker AOX2 gene for growth on
methanol since its AOX1 gene is disrupted (Cregg et al., 1987). The maximum
specific growth rate of Muts on methanol is 0.04 h-1(Bayraktar et al., 2009). The
large difference of specific growth rates for both phenotypes can be used to
differentiate between the two transformed simply by cultivating these two
phenotypes on methanol minimal medium (MM), whereby the Muts grew slower and
only formed a small colony compared with the Mut+. The last phenotype is methanol
15
utilization minus, Mut-, which has both AOX1 and AOX2 genes dislocated, which
makes it incapable grew on methanol. Yet, methanol is still required for the induction
of recombinant protein expression (Macauley-Patrick et al., 2005).
2.2.4 Proteolytic degradation in Pichia Pastoris
Pichia pastoris is one of the most desirable expression systems for producing
foreign proteins. However, in high cell density cultures, proteolytic degradation is a
concern (Kobayashi et al., 2000b; Sinha et al., 2003; Van den Ende et al., 2005).
Proteolysis in the production of recombinant protein can cause reduction of product
yield, product degradation, loss of biological activities and product contamination
due to intermediate degradation in downstream process (Macauley-Patrick et al.,
2005; Zhang et al., 2007).
Proteases in P. pastoris are well characterized. It was reported that the most
common type of protease released from lysed cell in the medium are extracellular
proteases, cell-bound proteases and intracellular proteases (Jahic et al., 2002;
Macauley-Patrick et al., 2005). The level of protease in yeast vacuoles can vary and
highly depended on their nutrition (Hansen and Stougaard, 1997; Sinha et al., 2005).
In addition, according to Jahic et al. (2006) vacuolar protease is probably the major
type of the proteases secreted by P. pastoris since the extracellular proteases was
founded only at low level in the medium (Jahic et al., 2006). Moreover, the vacuolar
proteases do not necessitate ATP and have low substrate specificity so they can
executed on any peptide bond in secreted protein structure, which result in protein
degradation.
Most of the proteases presented in the medium are probably released from
lysed cells (Jahic et al., 2006). There are several factors which can lead to cell lysis
16
such as change of heat and pH, starvation or toxic chemicals since these factors stress
the cells. In addition, proteins impairment due to oxidative stress and heat-shock
responses were also elicited proteolytic response (Sweeney and Holoman, 2001;
Zhang et al., 2007).
2.3 Medium design and bioreactor operation parameters
The yield and the production of recombinant proteins are affected by various
parameters. Medium composition and bioreactor operation parameters such as pH,
temperature and oxygen transfer are the most vital since they can influence metabolic
pathways and changing metabolic fluxes consequently generating miscellaneous
effects on product formation especially in aerobic fermentation processes (Celik et
al., 2009).
2.3.1 Medium design
In bioprocess development, cultivation medium is the most crucial factor as
cell growth and product formation is highly influenced by the uptake and conversion
of nutrients component in the medium. According to Nielsen and Villadsen, (1994)
the cultivation medium should contain carbon, nitrogen and energy sources.
Furthermore, it should also contain all essential mineral required for growth and
contain all necessary growth factors to ensure rapid growth and high yield of the
desired product. In addition, the cultivation medium should always be readily
available and stable. Finally, it should not pose any difficulty in downstream
processing (Wang et al., 2001).
17
The essential nutrients for cell growth can be divided into two groups, which
can be classified based on the required concentrations of the nutrients in the medium.
The first group is the macronutrients. Carbon, oxygen, nitrogen, hydrogen, sulfur,
phosphorus and magnesium are the main macronutrients which are generally
required at concentrations higher than 10-4 M. The second group is the
micronutrients, with the required concentrations less than 10-4 M. Trace elements
such as Ca, Cu, Fe, Na, Mn, Mo, Zn, and vitamins are some micronutrients and they
are usually added to the culture medium as mineral salts (Fiecher et al., 1984).
2.3.2 Carbon source
Carbon source play essential roles in cell growth and recombinant protein
production. Most commonly used carbon sources for Pichia pastoris are methanol,
glycerol, sorbitol, glucose, mannitol, trehalose, etc. (Brierley et al., 1990; Inan and
Meagher, 2001b; Sreekrishna et al., 1997; Thorpe et al., 1999). Methanol is not only
used as a sole carbon source, but also used as an inducer of AOX1 to trigger the
expression of recombinant protein (Celik et al., 2008). Beside carbon sources,
nitrogen is also required as an essential macronutrient. In P. pastoris fermentation
protocol, the ammonium hydroxide is typically used as a nitrogen sources as well as
pH controller to maintain the pH at the preferred level (Cos et al., 2006a). According
to Brankamp et al., (1995) the use of ammonium ions as a nitrogen source
throughout recombinant protein production is likely the most frequently used method
for reducing proteolytic degradation due to nitrogen-limitation. In addition,
ammonium ions can probably act as a substitute and competing substrate for
proteases with lower substrate specificity (Brankamp et al., 1995). On the contrary,
18
extension of lag phase was observed when a high ammonium concentration was used
in the medium and thus inhibited the growth of the cells and protein production (Xie
et al., 2003; Yang et al., 2004).
The major types of growth media can be divided into three categories;
defined medium, which contain specific amounts of pure chemical compounds with
known chemical compositions, semi-defined medium, and complex medium,
containing natural compounds whose chemical composition is not exactly known
(Shuler et al., 1994). Complex media frequently contains an organic nitrogen source,
essential minerals and different growth factors with difference in the composition
(Nielsen and Villadsen, 1994).
In industrial applications, defined medium is more preferred in bioprocess
due to the lower number of steps in subsequent downstream processing and effortless
process validation (Macauley-Patrick et al., 2005). Basalt salt medium (BSM) along
with the trace salts (PTM1) is the most common medium used in high cell density
fermentation for methylotrophic yeast. Even though it may not be the optimum
medium for growth but it can still be considered as a standard medium due to its
popularity. Nevertheless, it may possess some important problems, such as,
precipitates and high ionic strength (Cereghino et al., 2002; Cos et al., 2006b).
2.3.3 pH
Microbial cells have a distinguished capability to sustain their intracellular
pH at constant level, although with large fluctuation of pH in the medium, provided
that, the energy demands for maintenance is only increase at the outflow, since Gibbs
19
free energy has to be used for maintaining the proton gradient across the cell
membrane (Nielsen and Villadsen, 1994).
Microbial growth rate, the activity of enzymes, transport mechanisms and
other extracellular and intracellular actions are mainly regulated by hydrogen ion
(H+) concentration which is also one of the most imperative bioreactor operation
parameters.
Pichia pastoris can tolerate a broad pH, range between 3.0 and 7.0 and more
preferable at pH around 3.5 to 5.5 as this range gave minimum effect on the growth
rate and provide substantial alternative in adjusting the pH and can minimize
proteases activity in the medium (Macauley-Patrick et al., 2005; Sreekrishna et al.,
1997). The expression of recombinant protein and its stability robustly depends on
the optimum pH value. Hence, different pH values were found to be favorable for
different proteins (Macauley-Patrick et al., 2005); for instance, the production of
recombinant human erythropoietin was optimum at pH 6.0 when the protein was
produce in Buffer minimal glycerol medium (BMGY) and pH 3.0 was found to be
optimal in production of insulin-like growth factor-I and cytokine growth-blocking
peptide (Brierley et al., 1990; Calik et al., 2010; Koganesawa et al., 2002). In
addition the production of recombinant ovine interferon was higher at pH 6.0 due to
the substantial reduction of proteolytic activity and protein degradation however the
proteolytic response increased at pH 5.0 with an average of 2836 U/mL protease or
1243.9 U of protease/mg protein in fermentation supernatant Kobayashi et al., (2000)
and Sinha et al., (2003) observed that, during recombinant human serine albumin
production, protease activity rapidly increased when the pH was set below pH 5.0
and was decrease at pH 5.6 and they has successfully minimizing the degradation
20
rate of human albumin serum or HAS at pH 5.6 (Kobayashi et al., 2000b; Sinha et
al., 2003).
2.3.4 Temperature
Microorganisms do not have the capability to regulate their inner
temperature. Hence, their internal temperature is always equal to their external
temperature and all cellular biochemical reactions occurred directly depend on the
external temperature. Beside the reaction rates, the metabolic regulation, nutritional
requirements, biomass composition and product formation can also influenced by
temperature. In order to inquire the optimum temperature, consideration should be
taken for optimum growth temperature and product formation since both can be
diverge. In addition, when temperature is elevated higher than the optimum
temperature, the maintenance necessities of cells begin to amplify (Nielsen and
Villadsen, 1994). Moreover, secondary and tertiary structures of the protein are also
highly contingent on temperature since lower temperature facilitated to diminishing
protein misfolding and able to generate more correctly folded proteins (Georgiou et
al., 1996).
The upshot of temperature on the maximum specific growth rate of a
microorganism is equivalent to enzyme activity. Moreover, protein denaturation will
rapidly increase when temperature is abruptly increases to a high level. In addition,
the maximum specific growth rate is greatly increases the same way as in normal
chemical rate constant whilst temperatures are lower (Nielsen and McCammon,
2003). According to Shay et al. (1983), the optimum temperature for growth of P.
pastoris is 30 °C and temperatures higher than 32 °C can be detrimental to protein
expression and may lead to cell death (Invitrogen, 2002). Temperatures above 30 °C
are conducive to cell lysis and will elicit protease activity in fermentation media and
21
are not appropriate for the production of recombinant protein because high
temperature will result in cell death (Inan et al., 1999). Li et al. (2001) observed that,
during the production of herring antifreeze proteins, decreasing the temperature from
30 oC to 23 oC improved the yield from 5.3 mg/L to 18.0 mg/L, and increased cell
viability (Li et al., 2001). Moreover, Hong et al. (2002) were successfully acquiring
more active laccase when decreasing the production temperature from 30 oC to 20
oC. In addition, it is observed that, when the cultivation temperature is lowered, the
rates of proteases activity were also decreased (Hong et al., 2002; Li et al., 2001;
Macauley-Patrick et al., 2005).
Zhang et al. (2007) was observed that, the stability of intracellular protein
was ameliorate when the production temperature was around 25-27 °C as compared
with 30°C. Thus, optimization of production temperature may also minimize
degradation (Zhang et al., 2007). However, it has to be remark that, many other cases
where the production of recombinant proteins expressed by P. pastoris strains does
not extensively influence by lowering production temperature below 30 °C (Curvers
et al., 2001; Hong et al., 2002; Inan et al., 1999; Kupcsulik and Sevella, 2005). In
this study, temperature was kept at 30 °C during the entire process.
2.4 Fed-batch fermentations of Pichia pastoris
2.4.1 High cell density cultivation
In fermentation, fed-batch is the most preferred mode of cultivation as such
system proficient for achieving high cell densities and easier to control (Chiruvolu et
al., 1998; Cos et al., 2005a; Huy et al., 2011; Li et al., 2011b; Liu et al., 2011a; Tang
22
et al., 2010; Zhang et al., 2011). Typical fed-batch fermentation for Pichia pastoris
consist of growth and production phase. In growth phase glycerol was commonly
used to support the growth of the cell whereas in production phase methanol was
used to induce protein expression. P. pastoris with AOX1 can be cultivated to three
phases; glycerol batch phase (GB), glycerol fed-batch phase (GFB), methanol fed-
batch (MFB) and in some cases additional methanol transition phase (MT) (Cos et
al., 2006b; Gurramkonda et al., 2009; Potvin et al., 2012; Tang et al., 2010; Zhang et
al., 2001b).
In glycerol batch, glycerol will be included in define medium to culture the
cell. Subsequently, when the substrate is depleted the dissolved oxygen (DO) will
suddenly increase, the substrate will be feed at limited feed rate to further increase
cell concentration. The end time of glycerol fed-batch is depends on the desired
biomass. The maximum specific growth rate (μmax) of P. pastoris grew on glycerol
is 0.18 h−1 (Cos et al., 2005b; Liu et al., 2011b). An optional methanol transition or
transition phase can also be included between glycerol limited feeding (GFB) and
induction phase (MFB), allowing the cells to adapt to methanol and synthesizing
some of alcohol oxidase (AOX) enzyme to trigger the first step of methanol
catabolism before utilize methanol. In addition, according to Zhang et al. (2001), the
length of transition phase can be shorten by simultaneously feeding a limited amount
of glycerol with methanol over 3 hour period and the feed rate of glycerol is set to
ramp down linearly and at the same time the methanol will be continually added for
additional 2 hours to accelerate the synthesis of AOX enzyme. The transition phase
can be terminated when the dissolve oxygen start to decrease abruptly after adding
several drop of methanol. The cell density in this phase should be around 5-10 g/L
23
due to the addition of glycerol and the length of the transition phase is between 5-6
hours (Minning et al., 2001; Zhang et al., 2001b). Finally, after the transition phase,
the production phase can be initiated to produce the recombinant protein. The end
time of MFB will be based on the desire biomass. The maximum specific growth rate
(μmax) of P. pastoris, Mut+ grew on methanol is 0.14 h−1 (Siegel and Brierley, 1989;
Tang et al., 2010).
2.4.2 Fed-batch process control strategies
In fed-batch fermentation, the main factor to control under AOX1 regulated
system is methanol addition in production phase as the feed rate, the residual and the
methanol concentration will directly affecting the rate of expression, proteolytic
degradation, cell growth, cell lysis and oxygen transfer (Potvin et al., 2012; Tang et
al., 2010). Numerous strategies has been proposed and implemented to control the
addition of methanol to enhance protein production. The most frequent methanol
control strategies are include constant specific growth-rate feeding (μ-stat), constant
methanol concentration feeding, constant DO-based feeding (DO-stat), oxygen
limited fed-batch (OLFB) and temperature-limited fed-batch (TLFB) (Khatri et al.,
2011; Tang et al., 2010).
2.4.2.1 μ-stat control
In μ-stat control strategy, methanol feeding rate can be determined base on
mass balance equations for maintaining a constant specific growth rate (μ) (Cos et
al., 2006b). Since most of the protein production processes are directly or indirectly
related with cell growth, μ-stat is a good control strategy for process optimization,
24
does not required any on-line monitoring for parameters system (Ren et al., 2003). A
constant specific growth rate can be achieved by using methanol feed profile models
devised (Tang et al., 2010). Zhang et al. (2001b) was successfully developed a model
which involved the combination of specific growth rate, methanol concentration, and
specific methanol consumption rate. The μmax calculated from their model was 0.08
h−1 however the highest level of protein production was attained at 0.03 h-1.
Furthermore, Ren et al. (2003) has reported a macrokinetic model for P. pastoris
expressing recombinant human serum albumin based on stoichiometric balances to
describe the cell growth and protein production. They have successfully maintained
the μset by combining the linear and exponential feeding profiles (Khatri et al.,
2011).
Three methanol feeding strategies for production of mouse endostatin by
P. pastoris were reported by Trinh et al. (2003) which involved comparison between
methanol consumption, dissolved oxygen concentration and exponential feeding
(Trinh et al., 2003). Unlimited methanol was supplied in first two strategies and
limited methanol was employed in exponential feeding with (μset) of 0.02 h−1. The
specific productivity was two times higher in limited methanol feeding compared to
other strategies (Trinh et al., 2003). Despite the fact that the limited methanol
feeding is widely used but this strategy is not suited for recombinant protein which
will easily get dissipated under these conditions (Jahic et al., 2003; Khatri et al.,
2011; Trinh et al., 2003; Zhou and Zhang, 2002).
