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OULU 2011

C 393

Narendar Kumar Khatri

OPTIMISATION OF RECOMBINANTPROTEIN PRODUCTIONIN PICHIA PASTORISSINGLE-CHAIN ANTIBODY FRAGMENT MODEL PROTEIN

UNIVERSITY OF OULU,FACULTY OF TECHNOLOGY,DEPARTMENT OF PROCESS AND ENVIRONMENTAL ENGINEERING

C 393

ACTA

Narendar K

umar K

hatri

C393etukansi.kesken.fm Page 1 Tuesday, October 11, 2011 11:19 AM

A C T A U N I V E R S I T A T I S O U L U E N S I SC Te c h n i c a 3 9 3

NARENDAR KUMAR KHATRI

OPTIMISATION OF RECOMBINANT PROTEIN PRODUCTION IN PICHIA PASTORISSingle-chain antibody fragment model protein

Academic dissertation to be presented with the assent ofthe Faculty of Technology of the University of Oulu forpublic defence in Kuusamonsali (Auditorium YB210),Linnanmaa, on 18 November 2011, at 12 noon

UNIVERSITY OF OULU, OULU 2011

Copyright © 2011Acta Univ. Oul. C 393, 2011

Supervised byDoctor Frank HoffmannProfessor Peter NeubauerProfessor Heikki Ojamo

Reviewed byDoctor Ursula RinasDoctor Juha-Pekka Pitkänen

ISBN 978-951-42-9584-3 (Paperback)ISBN 978-951-42-9585-0 (PDF)

ISSN 0355-3213 (Printed)ISSN 1796-2226 (Online)

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2011

Khatri, Narendar Kumar, Optimisation of recombinant protein production inPichia pastoris. Single-chain antibody fragment model proteinUniversity of Oulu, Faculty of Technology, Department of Process and EnvironmentalEngineering, P.O. Box 4300, FI-90014 University of Oulu, FinlandActa Univ. Oul. C 393, 2011Oulu, Finland

AbstractPotential lethal diarrhoea caused by enterotoxigenic Escherichia coli strains is one of the mostcommon diseases in young pigs. It can be cured by single-chain antibody fragments (scFv), whichcan be produced in recombinant microorganisms. Pichia pastoris, a methylotrophic yeast, isgenerally considered an interesting production system candidate, as it can secrete properly foldedproteins. These proteins accumulate in high concentrations during fermentation, reducing the costfor product recovery.

Strong inducible AOX1 promoter, widely used in P. pastoris for fast, inexpensive production,is typically induced by methanol. The high oxygen demand of methanol metabolism makesoxygen supply a major parameter in cultivations requiring special process design strategies. Instandard fed-batch cultivation, dissolved oxygen concentration inside a bioreactor is kept at acertain level by pumping air and pure oxygen into the reactor. There are safety concerns over thehandling of oxygen, especially at a large scale. Therefore, there is a need to develop a productionprocess under oxygen-limited conditions.

This dissertation studies the development of a cost-efficient production process of scFv in P.pastoris. Both methanol and oxygen parameters influence the production process and the objectivewas to find a robust production process. Fed-batch cultivations were performed in a 10 L scalebioreactor. The effects of lower oxygen level, methanol concentration, glycerol feeding durationand specific substrate-uptake rates on product formation were studied. A P. pastoris GS115 his4strain under an AOX1 promoter system expressing scFv was used in this study. The fed-batchfermentations were carried out in a bioreactor with basal salt media.

In this doctoral dissertation, a process was developed for a single-chain antibody fragment(scFv) production in P. pastoris. The product levels of 3.5 g L-1 scFv in culture supernatant wereachieved and a production process was designed without additional need of pure oxygen, thusrelieving safety requirements and lowering the amount of methanol. The process developed duringthis research may potentially be utilised by both academia and industry having interests inexpressing proteins in P. pastoris. The methanol-uptake control strategy is beneficial for thoseproducts that suffer from degradation or modification during limited feeding of methanol.

Keywords: fed-batch, fermentation, Pichia pastoris, recombinant protein production,scFv

Khatri, Narendar Kumar, Rekombinanttiproteiinin tuoton optimointi Pichiapastoris –hiivalla— scFv-vasta-ainefragmentti malliproteiinina. Oulun yliopisto, Teknillinen tiedekunta, Prosessi- ja ympäristötekniikan osasto, PL 4300, 90014Oulun yliopistoActa Univ. Oul. C 393, 2011Oulu

TiivistelmäEnterotoksigeenisten E.coli kantojen aiheuttama ripuli on porsaiden tavallisimpia tauteja, jokavoi johtaa jopa kuolemaan. Tautia voidaan hoitaa yhdistelmä-DNA-tekniikalla tuotetuilla vasta-ainefragmenteilla (scFv). Metylotrofista Pichia pastoris hiivaa pidetään kiinnostavana vasta-ainefragmenttien tuottoisäntänä, koska se pystyy erittämään oikealla tavalla laskostuneita prote-iineja. Näitä proteiineja kertyy fermentointiprosessissa solujen ulkopuolelle korkeina pitoisuuk-sina, mikä vähentää tuotteiden talteenottokustannuksia.

Vahva metanolilla indusoituva AOX1-promoottori on laajassa käytössä P. pastoris tuottosys-teemissä tuoton nopeuden ja alhaisten kustannusten ansiosta. Metanolin aineenvaihdunta vaatiipaljon happea, joten riittävän tehokas hapen liuottaminen on tärkeimpiä fermentointiparametre-ja ja vaatii erityisiä prosessin toteutusstrategioita. Perinteisessä fed-batch-fermentoinnissa liuen-neen hapen pitoisuus bioreaktorissa pidetään halutulla tasolla lisäämällä ilmaa ja puhdasta hap-pea reaktoriin. Koska hapen käsittelyyn liittyy turvallisuusriskejä erityisesti teollisuusmittakaa-vassa, happirajoitteisissa olosuhteissa toimiva tuotantoprosessi olisi hyödyllinen.

Tässä väitöstutkimuksessa kehitettiin kustannustehokasta prosessia scFv-:n tuottoon P. pasto-ris hiivalla. Metanoliin ja happeen liittyvät parametrit ovat olennaisia prosessiin vaikuttavia teki-jöitä. Tavoite oli kehittää yksinkertainen ja käytännöllinen prosessi. Työssä tutkittiin alhaisenhappitason, metanolin pitoisuuden, glyserolisyötön keston ja substraattien spesifisten kulutusno-peuksien vaikutuksia tuotteen muodostumiseen 10 litran bioreaktorissa. Isäntäkantana oli P. pas-toris GS115 his4, jossa scFv-ekspressiota säädeltiin AOX1 promoottorilla. Fed-batch fermen-tointien kasvatusalustana käytettiin Basal Salt Medium alustaa (BSM).

Väitöstyössä kehitettiin tavoitteiden mukainen vasta-ainefragmenttien tuottoprosessi P.pasto-ris hiivalle. Menetelmällä saavutettiin tuotepitoisuus 3,5 g L-1 kasvatusliemen supernatantissailman puhtaan hapen lisäystarvetta, ja siten metanolin kulutus väheni ja prosessiturvallisuusparani verrattuna perinteisiin prosesseihin. Kehitetty prosessi soveltuu käytettäväksi sekä akatee-misessa tutkimuksessa että teollisuudessa tuotettaessa erilaisia proteiineja P. pastoris hiivalla.Metanolin kulutuksen säätöstrategia on erityisen hyödyllinen tuotteille, joilla ongelmana on pro-teolyysi tai muokkautuminen metanolirajoitteisessa fermentoinnissa.

Asiasanat: fed-batch, fermentointi, Pichia pastoris, rekombinanttiproteiinin tuotto, scFv

To my grandparents, parents and family

8

9

Acknowledgements

This study was initially started at the Institute of Biochemistry and Biotechnology,

Martin Luther University Halle-Wittenberg, Germany and later it continued at the

Bioprocess Engineering Laboratory (BPEL), Department of Process and

Environmental Engineering at the University of Oulu, Finland. Direct and indirect

support for financing the study came from Novoplant GmbH Germany, Centre for

International Mobility (CIMO), Tauno Töningin Foundation, Biocenter Oulu,

Oskar Öflundin Säätiö and Oulun Yliopiston Apuraharahasto (Yliopiston

Apteekin Rahasto). I am grateful to all of them for supporting me during this

study.

I wish to express thanks to my supervisors, Dr Frank Hoffman at Halle, Prof.

Peter Neubauer, and Prof. Heikki Ojamo at Oulu, whose supervision and valuable

comments made this research work possible. I would like to thank the other co-

authors of my publications, Dr Oliver Trentmann and Dr Dörte Gocke for their

valuable contribution and suggestions. Special thanks to Dr Pekka Belt, Dr Janne

Harkonen, Dr Matti Mottonen and Prof. Riitta Keiski for their valuable comments

and support during the dissertation process. I appreciate technicians Uta Best and

Lilja Tuohimaa for their help in laboratory routines.

The reviewers, PD Dr Ursula Rinas and Dr Juha-Pekka Pitkänen are thanked

for their careful review of the thesis and providing many valuable comments. I

thank Edmund Ward, SfEP, for proofreading of the thesis.

I am thankful to all present and former staff of BPEL, Oulu for nice

discussions, translation help and providing good working atmosphere, not to

forget Liisa Myllykoski who took care of all financial and administration tasks,

Antti Vasala who provided generous help in settling us in this cold country. Many

thanks go to my dear friends Swapan Kumar Chatterjee, Naresh Paneru, Shashank

Shekhar, Tiina Ristikari and Martin Kögler for nice discussions and help in

everyday life.

I am grateful to my parents, grandparents, my uncle Dr Ashok Kumar Khatri

and other family members whose blessings and unlimited support through all

stages of my life are unforgettable. This thesis is dedicated to them.

Finally, my deepest gratitude goes to my wife, Neeta, and our children, Nitin

Kumar and Samiksha Rani, who endured my long working days. Their smiles

made me happy and motivated me to work hard.

Oulu, October 2011 Narendar Kumar Khatri

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11

List of abbreviations

µ specific growth rate (h−1)

µset set specific growth rate (h−1)

AOX Alcohol oxidase enzyme

AOX Alcohol oxidase promoter

BSM Basal salt medium

CDW Cell dry weight (g L−1)

CFW Cell fresh weight (g L−1)

DHAS Dihydroxyacetone synthase promoter

DO Dissolved oxygen concentration (%)

ELISA Enzyme-linked immunosorbent assay

exp exponential

FLD1 Formaldehyde dehydrogenase 1 promoter

GAP Glyceraldehyde 3-phosphate deydrogenase promoter

P. pastoris Pichia pastoris

PI control Proportional-integral control

PID control Proportional-integral-derivative control

qMeOH specific methanol-uptake rate (g g−1 h−1)

qp specific production rate or productivity (mg kg−1h−1)

scFv single-chain antibody fragment

tf time of feeding (h−1)

tind time of induction (h−1)

TLFB temperature limited fed-batch fermentation

YPD Yeast peptone dextrose

12

13

List of original publications

This dissertation is based on the following publications:

I Trentmann O, Khatri NK & Hoffmann F (2004) Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris. Biotechnol Prog 20: 1766–1775.

II Khatri NK & Hoffmann F (2006a) Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris. Biotechnol Bioeng 93: 871–879.

III Khatri NK, Gocke D, Trentmann O, Neubauer P & Hoffmann F (2011) Single-chain antibody fragment production in Pichia pastoris: Benefits of prolonged pre-induction glycerol feeding. Biotechnol J 6(4): 452–62.

IV Khatri NK & Hoffmann F (2006b) Oxygen-limited control of methanol uptake for improved production of a single-chain antibody fragment with recombinant Pichia pastoris. Appl Microbiol Biotechnol 72: 492–498.

The author of this dissertation has been the primary author in Articles II, III and

IV of the original publications and Number 2 in Article I. The researcher has been

responsible for formulating the research problems, collecting theoretical

background, formulating research questions, coordinating the collection of

empirical material, analysing the material, drawing conclusions, and finally being

either primary or secondary author for the articles. In Article I the author of this

dissertation was responsible for all fermentation-related experiments. In Articles

II, III and IV the role of co-authors has included reviewing and commenting on

the article manuscripts.

14

15

Contents

Abstract

Tiivistelmä

Acknowledgements 9 

List of abbreviations 11 

List of original publications 13 

Contents 15 

1  Introduction 17 

1.1  Background ............................................................................................. 17 

1.2  Objectives and scope ............................................................................... 18 

2  Literature review 19 

2.1  Pichia pastoris ........................................................................................ 19 

2.1.1  Methanol-utilising phenotypes ..................................................... 19 

2.1.2  Promoters for Pichia pastoris ....................................................... 20 

2.2  Fed-batch fermentations of Pichia pastoris ............................................ 21 

2.2.1  Fed-batch control strategies .......................................................... 22 

3  Materials and methods 27 

3.1  Strain and genetic constructs ................................................................... 27 

3.2  Medium ................................................................................................... 27 

3.3  Culture conditions ................................................................................... 27 

3.4  Purification of the scFv ........................................................................... 29 

3.5  Cell density analysis ................................................................................ 29 

3.6  scFv analysis ........................................................................................... 29 

4  Research contribution 31 

4.1  The effects of reduced oxygen supply on production process ................ 31 

4.1.1  Cultivation without addition of pure oxygen ................................ 31 

4.1.2  Cultivation with addition of pure oxygen ..................................... 33 

4.1.3  Discussion relating to Research Question 1 ................................. 34 

4.2  The impact of methanol concentration on scFv production under

oxygen-limited conditions ....................................................................... 34 

4.2.1  Discussion relating to Research Question 2 ................................. 36 

4.3  The effects of long glycerol feeding on scFv production under

oxygen-sufficient conditions ................................................................... 36 

4.3.1  Discussion relating to Research Question 3 ................................. 38 

4.4  Controlling methanol-uptake for improved production of a scFv ........... 38 

4.4.1  Discussion relating to Research Question 4 ................................. 43 

16

5  Conclusions 45 

References 47 

Original publications 55 

17

1 Introduction

1.1 Background

Potential lethal diarrhoea caused by enterotoxigenic Escherichia coli strains is

one of the most common diseases in young pigs. Bacterial adhesion to intestinal

surface is mediated by fimbriae and can be inhibited by appropriate monoclonal

antibodies (Sun et al. 2000). Production by hybridoma cells is well established; it

is very expensive for veterinary application as high doses are required. Therefore,

there is a need for a cost-efficient production process of antibodies. Cost-efficient

alternatives are single-chain antibody fragments (scFv), which, because of their

structural simplicity, can be produced in recombinant microorganisms.

The cheapest, easiest and quickest expression system of choice is Escherichia

coli, but it has many drawbacks. Proteins that are produced as inclusion bodies

are often inactive, insoluble, and require refolding (Demain & Vaishnav 2009).

Higher eukaryotic cells can produce functional proteins but the expression

systems are expensive. The yeast-based system P. pastoris is the choice in terms

of price and functionality.

Pichia pastoris, a methylotrophic yeast, is generally an interesting candidate

production system for protein production as it can secrete properly folded proteins,

which accumulate in high concentrations in the culture medium. More than 700

proteins have been expressed in P. pastoris (Zhang et al. 2009). In the controlled

environment of a bioreactor, it is possible to achieve high cell densities of about

400 g L−1 cell fresh weight or 130 g L−1 cell dry weight respectively with

P. pastoris (Jahic et al. 2002). The concentration of the product obtained from this

yeast is reported to be as high as 22 g L−1 for intracellular production (Hasslacher

et al. 1997) and 14.8 g L−1 of clarified broth for secreted proteins (Werten et al.

1999). Product yield of scFv is generally 10–50 mg L−1 (Shi et al. 2003, Panjideh

et al. 2008, Cai et al. 2009, Jafari et al. 2011, Sommaruga et al. 2011) or in some

cases up to 500 mg L−1 (Eldin et al. 1997, Fischer et al. 1999, Chang et al. 2008)

but by using a special sequence, up to 1.2 g L−1 product yield has been reported

(Freyre et al. 2000).

Crucial to the fast, inexpensive production in P. pastoris is the strong

inducible AOX1 promoter to drive recombinant protein expression, which is

induced in the presence of methanol. Other promoter systems are also available

but this promoter system is the most widely used.

18

The high oxygen demand of methanol metabolism makes oxygen supply a

major parameter in P. pastoris cultivations and therefore requires special process-

design strategies. In a standard P. pastoris fed-batch cultivation, dissolved oxygen

concentration inside the bioreactor is kept at a certain level by feeding air and/or

pure oxygen to the reactor. There are safety concerns over handling of oxygen,

especially at a large scale. Therefore, there is a need of a production process

under oxygen-limited (only aeration) conditions.

1.2 Objectives and scope

This dissertation studies the development of a cost-efficient production process of

a single chain antibody fragment in P. pastoris. In high cell density cultivations

during induction on methanol, P. pastoris cells respire fast and need more oxygen

to keep a constant dissolved oxygen concentration in a bioreactor. The main

objective has been to find a robust production process and study the effects of

methanol and oxygen conditions on the process.

This research problem is further divided into the following research questions:

RQ1 What are the effects of reduced oxygen supply on the production of single-

chain antibody fragment (scFv) by recombinant Pichia pastoris?

RQ2 What is the impact of methanol concentration on scFv production under

oxygen-limited conditions?

RQ3 What are the effects of a long glycerol feeding phase on scFv production in

Pichia pastoris under oxygen-sufficient conditions?

RQ4 How can the key process parameter “specific substrate uptake rate” be

controlled under substrate-sufficient conditions?

In this dissertation, each of the four research questions is answered by one

scientific journal article. This study has been carried out at a ten- litre scale. Other

magnitudes have been excluded.

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2 Literature review

2.1 Pichia pastoris

The methylotrophic yeast Pichia pastoris is currently one of the most effective

and versatile systems for the expression of heterologous proteins (Potvin et al.

2010). This expression system is popular for these reasons: (1) the molecular

biology techniques applied are the same as widely studied yeast Saccharoymyces

cerevisiae (2) its ability to express foreign proteins in high levels, either

intracellular or extracellular (3) the ability performing many posttranslational

modifications, such as glycosylation, disulphide bond formation and proteolytic

processing (4) the availability of the expression system as a commercial kit (5)

the efficient and tightly regulated promoter alcohol oxidase 1 (AOX1) that induces

up to 1000-fold when the carbon source is switched to methanol (6) its ability to

undergo high cell density cultivations in a bioreactor and the preference of a

respiratory rather than fermentative mode of growth (Cereghino & Cregg 2000,

Cereghino et al. 2002).

More than 700 proteins have been expressed in P. pastoris (Zhang et al.

2009). Both high cell density and high product levels are obtained in the

controlled environment of a bioreactor (Hasslacher et al. 1997, Werten et al. 1999,

Jahic et al. 2002, Gurramkonda et al. 2009, Gurramkonda et al. 2010).

Recently, P. pastoris genome sequence has been published (De Schutter et al.

2009, Mattanovich et al. 2009a, Mattanovich et al. 2009b) and a proteomic

analysis of its secretome in methanol induced cultures has been performed

(Huang et al. 2011). These developments open the door to the strain improvement

by systems biology approaches (Bollok et al. 2009).

2.1.1 Methanol-utilising phenotypes

There are three phenotypes of P. pastoris with regard to methanol utilisation

(Macauley-Patrick et al. 2005, Potvin et al. 2010). The Mut+, or methanol

utilisation plus phenotype has both AOX1 and AOX2 genes, grows on methanol at

wild-type rate and therefore requires a high dose of methanol in large-scale

cultivations (Cereghino & Cregg 2000). In the Muts, or methanol utilisation slow

phenotype, AOX1 gene is deleted and therefore the methanol-uptake rate is

slowed down due to weaker AOX2. In some cases, Muts strains have high

20

productivities compared to the wild-type strains (Cregg et al. 1987, Pla et al.

2006). The Mut-, or methanol-utilising minus phenotype, where both AOX genes

are deleted, is unable to grow on methanol and thus has low growth rate

(Macauley-Patrick et al. 2005). Recombinant protein expression is widely

reported with Mut+ phenotype strains (Macauley-Patrick et al. 2005). This study

is also based on this phenotype.

2.1.2 Promoters for Pichia pastoris

The most widely used and popular promoter system in Pichia pastoris for

recombinant protein production is AOX1 (Cereghino & Cregg 2000, Bushell et al.

2003, Lee et al. 2003a, Macauley-Patrick et al. 2005, Jahic et al. 2006, Potvin et

al. 2010). The promoter is repressed when P. pastoris cells are grown on glucose

or glycerol, but is induced strongly up to 1000-fold when the cells are shifted to

methanol medium (Cereghino et al. 2002). The ability of repressing expression of

a foreign protein is beneficial when the protein is toxic to the cells (Cereghino et

al. 2002). The Alcohol oxidase enzyme makes up to 33% of total cell proteins

when cells are grown on methanol (Couderec & Baretti 1980). The AOX1

promoter requires methanol for induction, which is highly combustive, toxic, and

requires special storage and handling issues. Therefore non-methanol-based

systems are desired.

The glyceraldehyde 3-phosphate deydrogenase promoter (GAP) is a strong

constitutive promoter in P. pastoris (Zhang et al. 2009). The common substrates

used are glucose, glycerol, and oleic acid but methanol can also be used (Zhang et

al. 2009). Mostly glycerol and glucose are used as carbon source because least

protein expression is reported when methanol is used (Waterham et al. 1997,

Zhang et al. 2007). The GAP promoter system is suitable for large-scale

production, as the need to change feeding solutions and fire and safety concerns

related to methanol are avoided. The expression levels obtained are comparable

with the AOX1 promoter (Waterham et al. 1997). However, it is not suitable if the

expressed protein is toxic to the cells (Cos et al. 2006a). Some studies report that

heterologous protein expression by GAP system is better than AOX1 (Waterham

et al. 1997, Döring et al. 1998, Delroisse et al. 2005), whereas others report that

AOX1 is better for protein expression (Sears et al. 1998, Boer et al. 2000,

Vassileva et al. 2001, Kim et al. 2009). Protein expression under the control of

both AOX1 and GAP promoters is an effective strategy to increase yields (Potvin

et al. 2010). Human granulocyte macrophage colony-stimulating factor and a

21

thermostable alkaline β-mannanase have been expressed successfully using this

strategy (Wu et al. 2003a, Wu et al. 2003b, He et al. 2008).

The FLD1 promoter based on glutathione-dependent enzyme formaldehyde

dehydrogenase regulates protein expression through induction with either

methanol as a sole carbon source (and ammonium sulphate as nitrogen source) or

methylamine as a sole nitrogen source (and glucose as a carbon source) (Shen et

al. 1998, Cereghino & Cregg 2000). The productivity of a Rhizopus oryzae lipase

in high cell density cultivation under the FLD1 promoter was similar to the AOX1

controlled system (Resina et al. 2005, Cos et al. 2005, Resina et al. 2009).

Some other promoter systems such as ICL1, TEF1, YPT1, DHAS for

recombinant protein expression with P. pastoris are reported but expression levels

are low and therefore AOX1 remains the most popular system (Cereghino &

Cregg 2000, Potvin et al. 2010). In this study, scFv expression is under AOX1

control.

2.2 Fed-batch fermentations of Pichia pastoris

With microbial cells, fed-batch fermentation is carried out to achieve high-density

cultivation. Typically, in a fed-batch process, after the depletion of substrate (such

as glucose) in the batch media, a growth limiting substrate is fed to the reactor

with a rate. The process is easy to control and very widely used to obtain high cell

density cultivations (Chiruvolu et al. 1997, Minning et al. 2001, Cos et al. 2005,

Sirén et al. 2006, Cos et al. 2006b, Yamawaki et al. 2007, Gurramkonda et al.

2009, Potvin et al. 2010, Gurramkonda et al. 2010, Park et al. 2011, Li et al. 2011,

Liu et al. 2011, Sommer et al. 2011, Yuan et al. 2011, Jung & Lee 2011, Abad et

al. 2011, Lopez-Cuellar et al. 2011, Vohra et al. 2011, Zhang et al. 2011, Parawira

& Tekere 2011).

The fed-batch cultivation of P. pastoris expressing a product under the

control of AOX1 consists of three, or in some cases, four phases: glycerol batch

phase, glycerol fed-batch phase, methanol fed-batch, and optional transition phase

(Zhang et al. 2000b, Cos et al. 2006a, Gurramkonda et al. 2009, Potvin et al.

2010). The aim of the first two phases is to produce enough biomass. The

maximum specific growth rate (µmax) of P. pastoris grown on glycerol is 0.18 h−1

(Cos et al. 2005, Potvin et al. 2010) and on methanol is 0.14 h−1 (Brierley et al.

1990, Potvin et al. 2010). Initially, in batch mode, basal salt media contains

40 g L−1 glycerol. When it is consumed, the dissolved oxygen concentration

increases in the bioreactor and at that time limited glycerol feeding is started. The

22

aim of this phase is to derepress the AOX1 promoter and make cells ready for

induction (Zhang et al. 2000b, Cereghino et al. 2002). In a standard protocol, this

intermediate glycerol feeding lasts for 2‒4 hours. After this phase, the methanol

feeding is started to induce the AOX1 promoter. The methanol feeding amount

and control strategy in a bioreactor influence residual methanol concentration,

specific growth rate of the culture, and heterologous protein expression levels

(Potvin et al. 2010).

In some cases, an optional transition phase is included between glycerol-

limited feeding and induction phase, so that cells adapt better for protein

expression. In this phase, a co-feeding of glycerol and methanol over a certain

time is performed whereby the glycerol feeding is decreased slowly and methanol

feeding is increased (Zhang et al. 2000a, Minning et al. 2001). A mixed feed of

glycerol and methanol allowed faster induction of alcohol oxidase and faster

adaptation of cellular metabolism than with a sole methanol feeding (Jungo et al.

2007).

Recently, a two-phase fed-batch protocol composed of a batch with high

glycerol (95 g L−1) and a feeding of methanol at constant rate is reported for the

successful production of Hepatitis B surface antigen (Gurramkonda et al. 2009)

and insulin precursor (Gurramkonda et al. 2010). The process scheme is easy to

implement and requires less instruments and control.

A high cell density fed-batch cultivation of P. pastoris under a GAP promoter

system consists of two phases: a batch phase and a fed-batch phase. The sole

carbon source is either glucose or glycerol. There are contrary reports claiming

which substrate is superior for recombinant protein production. Glucose is seen as

a preferred substrate over glycerol (Döring et al. 1998, Pal et al. 2006). On the

other hand, some authors report glycerol to be better than glucose (Zhang et al.

2007, Tang et al. 2009).

2.2.1 Fed-batch control strategies

Methanol is a carbon source and inducer for recombinant protein production in

AOX1-based Pichia cultivations. Its metabolism requires high amounts of oxygen

(Yurimoto et al. 2002). Methanol feeding rate has been identified as a major

parameter to optimise protein production with recombinant P. pastoris, and small

changes can have profound effects (Sinha et al. 2003). An unlimited methanol

supply can lead to oxygen depletion. Oxygen limitation can negatively affect the

23

protein expression (Cereghino & Cregg 2000). Dissolved oxygen concentration is

a critical parameter for high cell density cultivations (Cunha et al. 2004).

Therefore, fed-batch control strategies are reported with the aim to control

methanol feeding and achieve high product formation. The most common

methanol feeding strategies are 1) constant specific growth-rate feeding (μ-stat), 2)

constant methanol concentration feeding, 3) constant DO-based feeding (DO-stat)

and 4) temperature-limited fed-batch (TLFB).

μ-stat control

In this control method, methanol feeding rate is calculated on mass balance

equations to maintain a constant specific growth rate (µ) (Cos et al. 2006a). This

control method is easy to calculate and does not require any online monitoring of

system parameters, as long as the yield coefficient does not change (Potvin et al.

2010). The parameter µ is a good control strategy for process optimisation, as

many protein production processes are directly or indirectly related with cell

growth (Ren et al. 2003). Maintaining a constant μ enhances process-

reproducibility and facilitates study of growth-rate-related effects on recombinant

protein production (Potvin et al. 2010).

To keep a constant specific growth rate, models are devised using a methanol

feed profile (Potvin et al. 2010). A model based on a relation between the specific

growth rate, methanol concentration, and specific methanol consumption rate is

reported (Zhang et al. 2000a). According to them, the μmax calculated from this

model was 0.08 h−1 and the observed value was 0.0709 h−1. The highest level of

recombinant protein was achieved at about one third of μmax. A macrokinetic

model for P. pastoris expressing recombinant human serum albumin is reported

based on stoichiometric balances describing cell growth and protein production

(Ren et al. 2003). The μset was maintained by a combination of linear and

exponential feeding profiles. Also it was suggested that a μ set-point control

method is more efficient than maintaining a constant feed rate to maximising

production (Ren & Yuan 2005).

Sinha et al. designed a model based on cell growth on methanol with a

substrate-feed equation, and employed the model to control the process (Sinha et

al. 2003). They found that methanol feeding strategy is a very important factor

that controls the recombinant protein induction and protease production in

P. pastoris fermentation. An optimal value of 0.025 h−1 for specific growth rate (µ)

24

was reported, which could be controlled by methanol feeding profile (Sinha et al.

2003).

Trinh et al. reported three methanol feeding strategies for production of

mouse endostatin by P. pastoris (Trinh et al. 2003). First strategy was based on

methanol consumption; the second on dissolved oxygen concentration. In these

two control strategies, methanol supply was unlimited. In the last strategy, limited

methanol feeding was employed using an exponential feeding profile with set

growth rate (µset) of 0.02 h−1. Total recombinant protein produced was the same in

all cases (400 mg in 3L initial culture volume). However, specific product was

twice in limited methanol feeding compared to other strategies (Trinh et al. 2003).

Limited methanol feeding is widely used but not suited for those products

that get degraded or modified under these conditions (Zhou & Zhang 2002, Jahic

et al. 2003). To avoid methanol accumulation in the bioreactor, the specific

growth rate (μ) is maintained at a set value lower than μmax (Zhang et al. 2000a,

Ren et al. 2003).

Constant methanol concentration

Methanol concentration of P. pastoris culture broth is an important parameter to

control. The control strategy requires input from a methanol measurement system.

The most common methods for measuring methanol in offline mode are based on

chromatographic methods (GC and HPLC), and in online mode are based on

liquid gas equilibrium (Potvin et al. 2010). By controlling methanol concentration,

accumulation of methanol to high levels in the bioreactor is avoided.

Formaldehyde, the first product of methanol metabolism, is accumulated to toxic

levels when oxygen and methanol are in excess (Stratton et al. 1998). Stability of

the methanol concentration is important, as higher productivity is achieved at the

constant concentrations than with fluctuating concentrations (Guarna et al. 1997,

Minning et al. 2001).

Typical methanol control strategies are on-off control mode and Proportional-

integral (PI) or proportional-integral-derivative (PID) control. An on-off control

mode is a simple feedback control strategy and is used in P. pastoris cultivations

(Wagner et al. 1997, Guarna et al. 1997, Katakura et al. 1998, Zhang et al. 2000a).

The on-off control strategy is suitable for linear systems, but generally

recombinant protein production in P. pastoris is a more complex and highly non-

linear process, and therefore fluctuations around the set-point are reported (Zhang

et al. 2002, Cos et al. 2006a, Potvin et al. 2010).

25

In a PID control system, the system output depends on the controller gain, KC,

the integral time constant, τI, and derivative time constant, τD (Zhang et al. 2002).

Optimal controller settings are based on the specified desired biomass

concentration and the culture volume. Due to the nonlinearity and complexity of

fermentation process dynamics, the optimal PID controller settings are

determined by trial and error tuning, or by using other empirical methods (Zhang

et al. 2000a, Cos et al. 2006a, Potvin et al. 2010).

Cos et al. used a predictive control algorithm, together with a PI feedback

controller, to optimise the production of a Rhizopus oryzae lipase in P. pastoris

(Cos et al. 2006b). First-time derivative of methanol concentration was used as an

input; proportional gain and integral time constant of the PI controller were fixed

during the fermentation (Cos et al. 2006b).

Minning et al. showed that the methanol-control strategy based on an online

methanol measurement system was a better control strategy than the DO-stat for

the production of R. oryzae lipase in P. pastoris (Minning et al. 2001).

DO-stat control

The dissolved oxygen concentration (DO) in the medium is an important

parameter to control in P. pastoris cultivations. In cyclic fed-batch fermentation,

the specific product concentration depends linearly on the dissolved oxygen

concentration in quasi-steady state, with no product accumulation when DO is

below 15% (Bushell et al. 2003). Because of high demand of oxygen by Pichia

pastoris, generally DO is kept above 20% in the cultivations (Singh et al. 2008).

It is reported that for optimum protein production with P. pastoris, DO control is

very essential (Lim et al. 2003, Woo et al. 2005).

In DO-stat control strategy, the feeding of substrate is controlled to keep a

DO concentration at a constant level in the culture medium (Lee et al. 2003b, Hu

et al. 2008). High density cultures of P. pastoris exhibit oscillatory behaviour

with stirrer speed and aeration when methanol is fed into the bioreactor (Potvin et

al. 2010).

Although DO-stat control is used in several studies as a means to increase

process robustness and performance, the methanol concentration and specific

growth rate have not been held constant, making the influence of these parameters

on production difficult to determine (Inan & Meagher 2001, Lee et al. 2003b,

Oliveira et al. 2005, Cos et al. 2006a).

26

Temperature-limited fed-batch (TLFB)

In this fed-batch strategy, methanol limitation is replaced by temperature

limitation mainly to avoid oxygen deficiency at high cell densities (Jahic et al.

2003, Potvin et al. 2010). By this method, both oxygen and methanol

concentrations can be controlled. The methanol concentration in the broth is

maintained constant by DO-stat, µ-stat, or constant methanol concentration

methods and culture temperature is lowered to maintain DO at a set point (Potvin

et al. 2010). Cell growth is therefore limited by temperature (Jahic et al. 2003,

Potvin et al. 2010).

A recombinant fusion protein composed of a cellulose-binding module (CBM)

from Neocallimastix patriciarum cellulase 6A and lipase B from Candida

antarctica, was produced by P. pastoris fed-batch cultivations (Jahic et al. 2003).

Two fed-batch control strategies (a) methanol-limited fed-batch and (b)

temperature-limited fed-batch (TLFB) were applied and compared. The

production levels were twice in TLFB compared to methanol-limited fed-batch.

Other benefits include a low cell death rate, low protease activity in the culture

supernatant (Jahic et al. 2003) and increase in the activity of alcohol oxidase.

Suribas et al. reported that by using this control strategy, cell death during

cultivation is reduced and 1.3 times more final product was obtained compared to

methanol non-limited conditions (Surribas et al. 2007).

27

3 Materials and methods

3.1 Strain and genetic constructs

A single-chain antibody fragment (scFv) against fimbriae of enterotoxigenic

Escherichia coli F4 was obtained by phage display screening of a library

constructed from spleen cDNA of F4-challenged mice. From the phagemid vector

of a high-affinity binder, the scFv sequence called BA11 was amplified, joined

with sequences encoding the α-mating-factor secretion signal from

Saccharomyces cerevisiae in the plasmid pPic9 (Invitrogen, Carlsbad, CA) and a

His6 tag, and integrated in the genome of P. pastoris GS115 Mut+ strain to give

GS115:BA11. Successful transformants are selected for prototrophy after

complementation of the GS115 his4 strain by the HIS4 gene of pPic9; a high-

producing strain was obtained by screening for scFv secretion (Trentmann et al.

2004).

3.2 Medium

The growth medium was prepared according to the recommendation of the

Invitrogen Manual (EasySelect™ Pichia Expression Kit, Catalogue No. K1740-

01) : YPD (1% w/v yeast extract, 2% w/v peptone, 2% w/v dextrose) and BMGY

(1% w/v yeast extract, 2% w/v peptone, 100 mM KH2PO4 pH 6, 1.34% w/v yeast

nitrogen base with (NH4)2SO4 without amino acids, 4 10−5% biotin, 1% v/v

glycerol). To the basal salt medium BSM (26.7 mL L−1 phosphoric acid, 14.9

g L−1 MgSO4.7H2O, 0.93 g L−1 CaSO4

.2H2O, 18.2 g L−1 K2SO4, 4.13 g L−1 KOH,

40 g L−1 glycerol), 4 mL L−1 sterile-filtered trace element solution PTM1 (6 g L−1

CuSO4.5H2O, 0.08 g L−1 NaI, 3 g L−1 MnSO4

.H2O, 0.2 g L−1 Na2MoO4.2H2O, 0.02

g L−1 H3BO3, 0.5 g L−1 CoCl2, 20 g L−1 ZnCl2, 65 g L−1 FeSO4.7H2O, 0.2 g L−1

Biotin, 5 mL conc. H2SO4) was added after autoclaving (Zhang et al. 2000a). The

pH value was adjusted to pH 6 with 25% ammonium hydroxide solution.

3.3 Culture conditions

For pre-culture, 10 mL YPD were inoculated with a single colony of

GS115:BA11 from an agar plate and incubated 24 h at 30 °C and 160 rpm. Two

times 200 mL BMGY in 1000 mL shake flasks were inoculated with 1% (v/v) of

28

the first pre-culture and incubated for 24 h as above. The reactor was inoculated

with 5% (v/v) of the second pre-culture.

Cultivations were carried out in a Biostat C bioreactor with a working volume

of 10 L connected to a DCU3 digital control unit and the MFCS/win process

supervisory system (B. Braun Biotech International, Melsungen, Germany). The

initial culture volume was 6 L. The pH value was controlled at pH 6 by automatic

addition of 25% ammonium hydroxide solution. Dissolved oxygen (DO)

saturation was determined by a Clark electrode (Ingold, Steinbach, Germany),

and was maintained above 40% by increasing the stirrer speed from 300 to 1200

rpm, subsequently increasing the air flow rate from 2 to 16 L min−1, and—where

indicated—dosage of oxygen up to 10 L min−1, until the maximum oxygen

transfer capacity of the reactor was reached and the culture was harvested or the

DO concentration declined. The cultivation temperature was 30 °C. Antifoam was

added automatically on demand.

When the process control system detected the DO-controlled decrease in

stirrer speed and air flow rate upon depletion of glycerol from the BSM medium,

glycerol feeding was activated with a rate rglycerol given by Equation 1.

The parameter values used were µset = 0.07 h−1 (set growth rate), YX/S =

1 g g−1

setglycerol f setexp[ ( )]f

X S

r X t tY

(1)

(biomass yield coefficient on glycerol (experimental values are from 0.88 to 0.97

g g−1) and Xf = 38.5 g L−1 (expected cell density at the time of feeding start tf).

The initial rate was hence 2.7 g L−1 h−1.

For induction of scFv expression by methanol, the glycerol feeding was

stopped and the methanol concentration was increased in three steps to final

concentrations of 0.1%, 0.3%, and 0.5% (v/v) (for a set point concentration of

0.3%), additional steps of 1% and 2% (for a set point concentration of 1%), 3%

(for a set point concentration of 3%), while waiting for complete consumption of

previously added methanol between the additions. The recorded resistance values

were used to calibrate the methanol sensor (Alkosens electrode and Acetomat

controller, Heinrich Frings GmbH, Bonn, Germany). Afterwards, the methanol

concentration was controlled to the concentration indicated in the results section.

The methanol reservoir was placed on a balance to keep track of methanol

consumption.

29

3.4 Purification of the scFv

After cell removal by centrifugation, the pH value of the supernatant was adjusted

to 7.9 with 3 M K2HPO4. After overnight incubation with Ni-charged “His-Bind

resin” (Novagen, Madison, WI) the His-Bind resin was transferred to plastic

columns, washed with 5 vol. of 500 mM NaCl, 20 mM Tris-HCl pH 7.9, 5 mM

imidazol, and the scFv was eluted with 500 mM NaCl, 20 mM Tris-HCl pH 7.9,

500 mM imidazol. The eluate was extensively dialyzed against solutions of

decreasing NaCl concentrations (finally 1 mM NaCl) and lyophilised.

3.5 Cell density analysis

For cell density determination, 1.5 mL of culture was centrifuged in pre-weighed

Eppendorf tubes. Cell fresh weight (CFW) was determined after removal of

supernatant and cell dry weight (CDW) after drying at 60 °C for 48 hours. The

culture volume V was estimated from the initial volume V0 and mass of added

glycerol mglycerol and methanol mMeOH, neglecting density differences and volume

contraction (eq. 2)

0 glycerol methanolV V m m . (2)

3.6 scFv analysis

The concentration of scFv was quantified by ELISA in 96-well plates coated with

F4 fimbriae using an anti-His6 monoclonal antibody for detection. For ELISA

assay, fimbriae from E. coli F4 were purified following the thermal shock

procedure, re-suspended in PBS buffer (10 mM phosphate buffer, 150 mM NaCl)

and coated to 96-well microtitre plates (Nunc, Wiesbaden, Germany) overnight at

4 °C at 1 µg per well in carbonate buffer (pH 9.6). Wells were washed three times

with PBS buffer, blocked with 3% bovine serum albumin (BSA) in PBS buffer for

one hour, and washed again. Culture samples containing 3% BSA were added to

the wells and incubated for 2 h, followed by washing with PBS containing 0.05%

Tween®20 (Sigma) and washing three times with PBS. After incubation with anti-

polyhistidine the anti-mouse IgG horseradish-peroxidase conjugate (HRP) was

added to the wells. Bound antibody was detected with 1 mg mL−1 APTS (2,2’-

Azino-di-[3-ethylbenzthiazoline sulfonate] diammonium salt; Roche, Mannheim,

Germany) in 10 mM sodium acetate, 5 mM sodium hydrogen phosphate pH 4.2,

30

0.01% H2O2. The substrate turnover was measured at 405 nm in an ELISA-reader

(Tecan Spectra Image, Tecan, Crailsheim, Germany).

31

4 Research contribution

This chapter describes the results to all four research questions. Each research

question is answered through one article in a separate subchapter. The key results

of the original articles are summarised in these subchapters.

4.1 The effects of reduced oxygen supply on production process

Research Question 1 is answered through the results published in Article I

describing the effects of reduced oxygen supply on the production of single-chain

antibody fragment (scFv) by recombinant P. pastoris.

Two process conditions for the production of a scFv with P. pastoris were

compared. In the first experiment, dissolved oxygen (DO) concentration was

maintained by controlling stirrer speed and air flow, whereas additional pure

oxygen was fed in the second experiment.

4.1.1 Cultivation without addition of pure oxygen

A single-chain antibody fragment (scFv) was produced with recombinant Pichia

pastoris in a bioreactor having 6 L basal salt medium (BSM). When glycerol in

the medium is depleted, a limited glycerol feeding phase with a set specific

growth rate (µset) of 0.07 h−1 was started. This feeding lasted for about 2 hours.

The idea of this step was to de-repress the AOX1 promoter and make cells ready

for the induction. The cells were induced by feeding methanol into the reactor.

The concentration of methanol was raised stepwise to 2% (v/v) and afterwards

maintained at 1% with the help of an online methanol probe and control unit. The

dissolved oxygen concentration (DO) is set to 40% and achieved by increasing

first stirrer speed from 300 to 1200 rpm, followed by air flow rate from 2‒16

L min−1 (0.33 to 2.66 vvm). Only normal aeration is used as oxygen source and

additional pure oxygen were not employed. The process parameters of the

cultivation are shown in Fig. 1.

32

Table 1. Fig. 1. Growth and substrate uptake profiles without addition of pure oxygen.

(A) Growth profile monitored by CFW ( ) and CDW (O). (B) Specific growth rate. (C)

Gravimetric (thick line) and specific (thin line) feeding rates of methanol. (D) Dissolved

oxygen saturation (solid line), flow rate of air (dotted line), and oxygen uptake rate

(thick solid line). Time is given relative to induction. (I, published by permission of

John Wiley and Sons).

Fig. 1D shows that DO could not be maintained to 40% throughout the cultivation

as no additional pure oxygen is supplied. The methanol flow rate remained near

constant at about 55–65 g h−1 (Fig. 1C). Cell fresh weight (CFW) of 357 g L−1

corresponding to 116 g L−1 dry weight was reached at the end of cultivation with

a specific growth rate of 0.015 h−1 (Fig. 1A and B). The specific methanol-uptake

rate reached a maximum of 58 g g−1 h−1 at 10 h after induction but started

decreasing afterwards. The scFv was purified from culture supernatant by single

step His affinity chromatography and lyophilised afterwards. In this experiment, a

total of 1065 mg lyophilised scFv was obtained, thus yielding a productivity 27.3

mg h−1.

33

4.1.2 Cultivation with addition of pure oxygen

In this experiment pure oxygen was supplemented with inlet air to maintain the

dissolved oxygen concentration in the bioreactor. By this method, oxygen

limitation could be avoided and methanol-uptake rate reached a maximum of 200

g h−1 as shown in Fig. 2C.

Fig. 2. Growth and substrate uptake profiles with addition of pure oxygen. (A) Growth

profile monitored by CFW ( ) and CDW (O). (B) Specific growth rate. (C) Volumetric

(thick line) and specific (thin line) feeding rates of methanol. (D) Dissolved oxygen

saturation (solid line), flow rate of air (dotted line), and oxygen (dashed line). Time is

given relative to induction. (I, published by permission of John Wiley and Sons).

About 7 h after induction, the specific growth rate of 0.05 h−1 was observed and

maintained within ± 0.02 for 20 hours. The cell density at that time was 425 g L−1

fresh weight or 125 g L−1 dry weight (Fig. 2A and B). The specific methanol-

uptake rate reached maximum of 0.075 g g−1 h−1 at 10–20 h after induction, as the

biomass increased faster than the volumetric methanol consumption rate (Fig. 2C).

When the maximum oxygen transfer capacity was reached 15 h after induction,

the DO dropped to 10% saturation (Fig. 2D). About 22‒24 h after induction, DO

34

concentration increased steadily and ultimately oxygen and methanol

consumption deceased after 30 h induction (Fig. 2C and D). In this experiment, a

total 688 mg lyophilised scFv was obtained, thus yielding productivity of 21.1

mg h−1.

4.1.3 Discussion relating to Research Question 1

Two process conditions for the production of a single-chain antibody fragment

with P. pastoris were compared. In the first experiment, dissolved oxygen (DO)

was maintained by stirrer speed and air flow, whereas additional pure oxygen was

fed in the second experiment.

The product accumulation was fastest in the oxygen supplemented culture. It

is known that higher methanol consumption rates can accelerate production

(Curvers et al. 2001, Jahic et al. 2002). This is the case even with optimum

production with lower specific growth or methanol-uptake rates (Hong et al. 2002,

Cunha et al. 2004). But no impact of methanol-uptake rates on production has

been observed (Trinh et al. 2003). In this study, however, the final product yield

was not primarily determined by the synthesis rates. Rather, fast addition of

methanol compromised the stability of the oxygen-supplemented process, and a

small fluctuation in the feeding rate was followed by complete arrest of

respiration. The metabolic problems during the fast methanol addition prevented

growth and production already 54 h after induction, and the final purified scFv

was only 670 mg. Without oxygen supply, in contrast, production proceeded

smoothly, yielding about 1 g of lyophilised scFv 40 h after induction. Thus,

whereas oxygen limitation did not stop scFv accumulation, similar to the

production of other proteins (Hellwig et al. 2001), the process stability was

negatively affected by fast methanol consumption.

4.2 The impact of methanol concentration on scFv production under oxygen-limited conditions

Research Question 2 is answered through the results published in Article II

describing the impact of methanol concentration on scFv production in oxygen-

limited conditions.

A single-chain antibody fragment (scFv) was produced with recombinant

P. pastoris and secreted to the medium. After the batch phase, glycerol was fed at

35

limiting rate for 2–4 h to de-repress the AOX1 promoter. For induction of the scFv

production, the methanol concentration was increased stepwise to 0.1, 0.3, and

0.5% (v/v) (for a set point concentration of 0.3%), additional steps of 1% and 2%

(for a set point concentration of 1%), 3% (for a set point concentration of 3%),

and complete consumption was allowed between the pulses to adapt the cells to

methanol metabolism as illustrated in Fig. 3C.

Fig. 3. Growth and product formation at different methanol concentrations.

Recombinant gene expression was induced and the methanol concentration was

controlled to 0.3% (v/v) (circles, dotted lines), 1% (triangles, solid lines) or 3%

(squares, dashed lines). Time is given relative to the time of induction tind. (A) CFW

was measured offline (symbols) and estimated from online data (lines). (B) Product

concentration in the culture supernatant was determined by quantitative ELISA

(symbols). For the cultivation with 0.3%, a trendline is given reflecting the production

profiles for several similar cultivations. (C) The methanol concentration was measured

by methanol sensor in the culture broth. (D) Dissolved oxygen saturation was

measured by an Ingold sensor. (II, published by permission of John Wiley and Sons).

The cell densities during the production phase of cultivations with higher

methanol concentrations were low compared to the cultivation at 0.3% methanol

concentration (Fig. 3A). Product accumulation, however, was considerably

accelerated; especially, the production could be maintained at high rates until the

end of the cultivation as shown in Fig. 3B. The final product concentration

36

exceeded 150 mg L−1 with 1% methanol, and with 3% methanol reached 350

mg L−1 already 50 h after induction compared to 60 mg L−1 with 0.3% methanol

cultivations.

4.2.1 Discussion relating to Research Question 2

The impact of methanol concentration on the metabolism of recombinant

P. pastoris and on the production of a single-chain antibody fragment was

examined under oxygen limitation. Three and six times higher product

concentrations were found with methanol concentrations of 1 and 3% (v/v)

respectively, than with a low methanol concentration of 0.3%. Hence, not only the

specific production rate was improved at higher methanol concentrations, as

observed under oxygen-sufficient conditions (Katakura et al. 1998, Zhang et al.

2000a), but also the final product concentrations were enhanced. The biomass

yield, conversely, was lower at high methanol concentrations.

High methanol concentrations increase the selectivity of methanol conversion

to the recombinant protein by avoiding gratuitous biomass formation during the

production phase. Thereby, the specific methanol-uptake rate is maintained well

above the maintenance demand, and the active production phase prolonged. The

negative impact of low oxygen concentration can be compensated by higher

methanol concentration, improving the product quantity and quality. Thus,

efficient production of secreted proteins by recombinant P. pastoris under oxygen

limitation is possible, and as shown in Article II, high methanol concentrations

can mitigate the requirements for additional supply of pure oxygen.

4.3 The effects of long glycerol feeding on scFv production under oxygen-sufficient conditions

Research Question 3 is answered through the results published in Article III

describing the effects of long glycerol feeding on scFv production in Pichia

pastoris under oxygen-sufficient conditions.

Fed-batch cultivations were performed to study the impact of the duration of

the glycerol feeding under oxygen-sufficient conditions on the production process.

In all cultivations, after a batch phase on glycerol, additional glycerol was fed

exponentially at set growth rate of µset = 0.07 h−1. The duration of this feeding

was 2 h, 12 h, or 18 h as shown in Fig. 4C. The cell fresh weight at the end of this

phase reached 130, 216, and 270 g L−1 as shown in Fig. 4A. After induction by

37

switching to methanol, the volumetric methanol addition rate needed to maintain

a constant methanol concentration in the broth (0.3% v/v) was used as shown in

Fig. 4D.

At time point tf 30 hours (since glycerol feeding started), the maximum

methanol-uptake rate (rMeOH) of 235 g h−1 was observed in cultivation with a

shorter glycerol feeding phase whereas the cultivation with the longest glycerol

feeding phase reached only half as much, rMeOH = 115 g h−1. While more than

8000 L (0.5 vvm) pure oxygen was necessary to maintain a constant DO in the

early-induced culture, only 2200 L (0.2 vvm) was consumed with late induction.

Fig. 4. Impact of an extended glycerol feeding phase on the scFv production profiles.

After the batch phase, glycerol was fed for 2 h (triangles, dashed lines), 12 h (squares,

dotted lines), or 18 h (circles, solid lines) before induction of scFv production

(methanol induction points indicated by vertical lines). (A) Cell fresh weight CFW; (B)

scFv concentrations (functional product) as determined by quantitative ELISA;

(C) glycerol flow rates; (D) methanol flow rates. Time is given relative to the start time

of glycerol feeding, tf. The line indicates the trend of the experimental data. (III,

published by permission of John Wiley and Sons).

Quantitative analysis of the functional scFv by ELISA showed that the product

started accumulating 14 hours after induction with the 2 h glycerol feeding

experiment, whereas the product was found already 5 h after induction with 12 h

38

glycerol feeding, and immediately with 18 h glycerol feeding (Fig. 4B). The scFv

concentrations showed similar profiles in all three cultivation runs (Fig. 4B).

4.3.1 Discussion relating to Research Question 3

Limiting feeding of glycerol to de-repress the AOX1 gene prior to the addition of

methanol is known to be beneficial for an efficient uptake of methanol and

induction of the AOX1 promoter (Jahic et al. 2002), and therefore a short glycerol

feeding phase is mostly applied in P. pastoris fed-batch cultivations. Here, we

examined how the production of the scFv fragment under aerobic conditions is

affected by prolonged glycerol feeding followed by methanol feeding to maintain

constant methanol feeding inside the reactor.

In entirely aerobic cultivations, the accumulation of scFv in the culture

supernatant was delayed for 10–20 hours with the standard 2 hours glycerol

feeding procedure, whereas after prolonged glycerol feeding, immediate product

accumulation was achieved. The sum of duration of the glycerol feeding phase

and the product-lag phase after induction was about constant. The same final

product yield and similar high quality, i.e. low degradation, of secreted scFv

fragment were achieved after the same total length of a process. Advantages of

the prolonged glycerol feeding strategy include very significant savings of

methanol and pure oxygen of about 75%, and a 23% lower cell density which

facilitates the downstream purification process.

4.4 Controlling methanol-uptake for improved production of a scFv

Research Question 4 is answered through the results published in Article IV

describing the control of specific methanol-uptake rate for improved production

of a scFv in Pichia pastoris.

In previous cultivation conditions, it is observed that despite full aeration, the

dissolved oxygen saturation (DO) decreased to limiting levels after induction (Fig.

1D). To ensure efficient production under oxygen limitation, a high methanol

concentration was important. With a methanol concentration of 3% (v/v), a final

product concentration of 350 mg L−1 was obtained (Fig. 3B). The volumetric

methanol-uptake rate was constant, as it was determined by the oxygen transfer

capacity of the reactor. Therefore, with increasing cell densities, the specific

methanol-uptake rate qMeOH decreased from more than 0.05 g g−1 h−1 after the

adaptation phase to 0.02 g g−1 h−1 as shown in Fig. 5. The scFv production starts

39

only after a lag phase; the start of scFv accumulation coincided with a qMeOH of

0.02‒0.03 g g−1 h−1. Therefore, the methanol-uptake rate was lowered into the

optimum range from early after induction. Two approaches to control the specific

uptake rate were studied.

Fig. 5. Actual specific methanol-uptake rates. Specific methanol-uptake rates are

compared for cultivations with short glycerol feeding and full aeration throughout the

production phase with 3% methanol feeding (Khatri & Hoffmann 2006a) (thick lines,

Fig. 3), with prolonged glycerol feeding (thin solid lines, Fig. 6) and with restricted air

supply (thin dashed lines, Fig. 8). Time is given relative to the time of induction tind. (IV,

published by permission of Springer Science + Business Media).

Prolonged glycerol feeding

Initially, the specific methanol-uptake rate qMeOH can be controlled by the amount

of biomass present at the time of induction. The glycerol feeding phase before

induction was extended to 16 h (Fig. 6A), during which time glycerol was fed

with exponentially increasing rate, enabling growth with a constant specific

growth rate of 0.07 h−1. The cell density (CFW) reached 245 g L−1 at the time of

induction (Fig. 6C). After induction, the oxygen saturation fell to limiting levels

within 5 h despite full aeration (Fig. 6B). The specific methanol-uptake rate

showed a declining trend due to cell growth, but remained close to 0.02 g g−1 h−1

during the whole production phase (Fig. 5).

40

Fig. 6. Cultivation of Pichia pastoris with extended glycerol feeding phase. After batch

and 16 h fed-batch phase on glycerol, recombinant protein production was induced by

methanol. (A) The glycerol flow rate (thick lines) and methanol concentration (thin

lines). (B) The dissolved oxygen concentration (DO thin line), the airflow rate (thick

line). (C) The cell fresh weight (CFW). Time is given relative to the time of induction tind

(indicated by vertical lines). Solid and dashed lines, open and solid symbols, present

two independent cultivations. (IV, published by permission of Springer Science +

Business Media).

In contrast to cultivations with shorter glycerol feeding, product accumulation

started immediately after addition of methanol (Fig. 7A). The specific production

rate was constantly 80 mg kg−1 h−1 throughout the whole production phase (Fig.

7A). After three days of production, 2500 mg L−1 of scFv per culture supernatant

were obtained (Fig. 7A).

41

Fig. 7. Production of scFv under deliberate oxygen-limited control of the specific

methanol-uptake rate. The concentration of the recombinant single-chain antibody

fragment (scFv) in the culture supernatant was determined by quantitative ELISA

(circles). Specific production rates qP (triangles). (A) Production after extended

glycerol feeding phase in the cultivations of Fig. 6. (B) Production with restricted air

supply in the cultivations of Fig. 8. Lines are drawn to interpret the trend. Time is

given relative to the time of induction tind. Open and solid symbols present two

independent cultivations. (IV, published by permission of Springer Science + Business

Media).

Restricted air supply

In another approach to control the specific methanol-uptake rate during

production at low cell densities, the air flow rate was set to 0.8 vvm upon

induction, and was increased with an exponential profile proportional to exp

(0.03 h−1 * (t – tind)) (Fig. 8B). The DO consequently was below 10% during the

entire production phase (Fig. 8B). The cell density (CFW) increased from 135

g L−1 at induction to 300 g L−1 after three days (70 h) of production (Fig. 8C). The

specific methanol-uptake rate decreased from 0.04 g g−1 h−1 immediately after the

adaptation phase, to 0.02 g g−1 h−1 (Fig. 5).

42

Fig. 8. Cultivation of Pichia pastoris with restricted air supply. After batch and 4 h fed-

batch phase on glycerol, recombinant protein production was induced by methanol.

(A) The glycerol flow rate (thick lines), methanol concentration (thin lines), and (B)

dissolved oxygen saturation (DO thin line). The air flow rate (thick line) was controlled

to a predetermined profile. (C) The cell fresh weight (CFW). Time is given relative to

the time of induction tind (indicated by vertical lines). Solid and dashed lines, open and

solid symbols, present two independent cultivations. (IV, published by permission of

Springer Science + Business Media).

The product scFv accumulated in the culture supernatant only after a lag phase of

20 h after induction (Fig. 7B). The specific production rate was almost constant

during the next 50 h of production, averaging at 250 mg g−1 h−1 (Fig. 5B). This is

5‒8 times faster than in the culture with full aeration, in which the scFv

accumulated with an average specific production rate qP of 30–50 mg kg−1 h−1).

Likewise, qP was 2‒3 times faster as in the cultivations with long glycerol feeding

(Fig. 7A). After 70 h of production under restricted air supply, final product

concentrations of 3500 mg L−1 were reached (Fig. 7B).

43

4.4.1 Discussion relating to Research Question 4

Under oxygen limitation, the scFv accumulated in the culture supernatant only

after a lag phase of one day. During this time span, the specific methanol-uptake

rate declined from 0.06 g g−1 h−1 to 0.02 g g−1 h−1. The latter value optimises

recombinant protein production in methanol-limited fed-batch, chemostat, and

with methanol concentrations controlled to low levels (Zhang et al. 2000a,

d'Anjou & Daugulis 2001, Zhou & Zhang 2002, Trinh et al. 2003, Bushell et al.

2003, Sinha et al. 2003, Cunha et al. 2004). Therefore, the specific methanol-

uptake rate was controlled to lower values immediately after induction. Two

approaches were used, and both resulted in high product yields. Both approaches

differed, however, with respect to the starting time and rate of product

accumulation.

Three advantages stand out from the practical point of view: First, single-

chain antibody fragment titres in the gram-per-litre range were obtained, as in a

few other studies (Freyre et al. 2000, Damasceno et al. 2004), whereas scFv

product levels are usually in range of 50–250 mg L−1 (Fischer et al. 1999). The

specific production rate was increased to 250 mg kg−1 h−1, and final product

concentrations reached 3.5 g L−1. Second, the methanol-uptake can be controlled

even with products that suffer from degradation or modification during limiting

feeding of methanol (Zhou & Zhang 2002, Jahic et al. 2003, Trentmann et al.

2004). Third, problems related to insufficient oxygen transfer capacities in large

scale that cannot meet the high oxygen demand during protein production on

methanol (Curvers et al. 2001) can impede production (Cereghino et al. 2002,

Bushell et al. 2003, Lee et al. 2003a); the drawback of low oxygen transfer rate

can be circumvented by using deliberate oxygen limitation to control the

production process. Oxygen limitation may be superior to methanol limitation

with respect to release of host proteins, even if air is provided with maximum rate

(Charoenrat et al. 2005). Thus, oxygen-limited control of the methanol-uptake is a

versatile tool to optimise secreted protein production with recombinant P. pastoris.

44

45

5 Conclusions

In this doctoral dissertation, a process was developed for a single-chain antibody

fragment (scFv) production in methylotrophic yeast, Pichia pastoris. The product

levels of 3.5 g L−1 scFv in culture supernatant were achieved and production

process was designed without additional need of pure oxygen, thus relieving

safety requirements and lowering the amount of methanol fed into the bioreactor.

Key findings of the thesis were as follows:

1. Reduced oxygen supply by using only air for aeration contributed to the

robustness of the production process and resulted in the highest yield of

purified scFv. This is beneficial, as no additional oxygen is required and

therefore mixing issues and safety concerns regarding oxygen-handling, in

large-scale facilities, are relieved.

2. Efficient recombinant protein production under oxygen limitations requires

high methanol concentration. Both specific production rate and final product

level are enhanced under those process conditions.

3. Long-limiting glycerol feeding before induction under oxygen-sufficient

conditions not only increased volumetric productivity but also reduced the

need of methanol and oxygen by 75%. Additionally, final cell densities are

lower, thus reducing costs during the purification process.

4. Two strategies of (i) long glycerol feeding and (ii) limited aeration have been

demonstrated to control the specific substrate-uptake rate that has been found

optimal in substrate-limited processes. Both strategies resulted in a tenfold

increase in the final product concentration.

The process developed for scFv in this study can potentially be used for other

proteins expressed in P. pastoris. In other words, the process developed during

this doctoral research can prove beneficial for both academia and industry having

interests in expressing proteins in P. pastoris. The methanol-uptake control

strategy is beneficial for those products that suffer from degradation or

modification during limiting feeding of methanol.

46

47

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Original publications

I Trentmann O, Khatri NK & Hoffmann F (2004) Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris. Biotechnol Prog 20: 1766–1775.

II Khatri NK & Hoffmann F (2006a) Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris. Biotechnol Bioeng 93: 871–879.

III Khatri NK, Gocke D, Trentmann O, Neubauer P & Hoffmann F (2011) Single-chain antibody fragment production in Pichia pastoris: Benefits of prolonged pre-induction glycerol feeding. Biotechnol J 6(4): 452–62.

IV Khatri NK & Hoffmann F (2006b) Oxygen-limited control of methanol uptake for improved production of a single-chain antibody fragment with recombinant Pichia pastoris. Appl Microbiol Biotechnol 72: 492–498.

Reprinted with permission of John Wiley and Sons (I-III) and Springer Science +

Business media (IV).

Original publications are not included in the electronic version of the dissertation.

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Book orders:Granum: Virtual book storehttp://granum.uta.fi/granum/

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377. Malinen, Ilkka (2010) Improving the robustness with modified boundedhomotopies and problem-tailored solving procedures

378. Yang, Dayou (2011) Optimisation of product change process and demand-supplychain in high tech environment

379. Kalliokoski, Juha (2011) Models of filtration curve as a part of pulp drainageanalyzers

380. Myllylä, Markus (2011) Detection algorithms and architectures for wireless spatialmultiplexing in MIMO-OFDM systems

381. Muhos, Matti (2011) Early stages of technology intensive companies

382. Laitinen, Ossi (2011) Utilisation of tube flow fractionation in fibre and particleanalysis

383. Lasanen, Kimmo (2011) Integrated analogue CMOS circuits and structures forheart rate detectors and other low-voltage, low-power applications

384. Herrala, Maila (2011) Governance of infrastructure networks : developmentavenues for the Finnish water and sewage sector

385. Kortelainen, Jukka (2011) EEG-based depth of anesthesia measurement :separating the effects of propofol and remifentanil

386. Turunen, Helka (2011) CO2-balance in the athmosphere and CO2-utilisation : anengineering approach

387. Juha, Karjalainen (2011) Broadband single carrier multi-antenna communicationswith frequency domain turbo equalization

388. Martin, David Charles (2011) Selected heat conduction problems inthermomechanical treatment of steel

389. Nissinen, Jan (2011) Integrated CMOS circuits for laser radar transceivers

390. Nissinen, Ilkka (2011) CMOS time-to-digital converter structures for theintegrated receiver of a pulsed time-of-flight laser rangefinder

391. Kassinen, Otso (2011) Efficient middleware and resource management in mobilepeer-to-peer systems

392. Avellan, Kari (2011) Limit state design for strengthening foundations of historicbuildings using pretested drilled spiral piles with special reference to St. John’sChurch in Tartu

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Narendar Kumar Khatri

OPTIMISATION OF RECOMBINANTPROTEIN PRODUCTIONIN PICHIA PASTORISSINGLE-CHAIN ANTIBODY FRAGMENT MODEL PROTEIN

UNIVERSITY OF OULU,FACULTY OF TECHNOLOGY,DEPARTMENT OF PROCESS AND ENVIRONMENTAL ENGINEERING

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