UPTEC X 99 025 ISSN 1401-2138

MAY 1999

RICKARD JOHANNISSON

Production of
recombinant protein
by the methylotrophic
yeast Pichia pastoris

Master’s degree project

 Molecular Biotechnology Programme
Uppsala University School of Engineering

UPTEC X 99 025 Date of issue 1999-05

Author
Rickard Johannisson
Title (English)
Production of recombinant protein by the
methylotrophic yeast Pichia pastoris
Title (Swedish)
Abstract

The yeast Pichia pastoris is an efficient host for recombinant protein production. Pichia can
grow on methanol and has a strong promoter, tightly regulated by the presence of methanol in
the medium. In this study, Pichia has been grown in fermenters utilising a three-phase scheme
involving a batch and a fed-batch phase with growth on glycerol and a second fed-batch phase
with growth on methanol to induce production of the recombinant protein. The methanol
concentration in the outlet gas from the culture has been measured with a gas sensor and used
to control the methanol feeding rate, thereby avoiding starvation or intoxication of the culture.
A recombinant strain secreting human serum albumin (HSA) has been used as a model
system. The technology has been tested at different scales, ranging from 0.4 litres up to 100
litres. The HSA secreted into the medium has been analysed with gel filtration, gel
electrophoresis and Western blotting.
Keywords:
fed-batch fermentation, Pichia pastoris, human serum albumin, methanol control, scale-up

Supervisor
Andras Ballagi
Center for Surface Biotechnology, Uppsala University

Examiner
Gen Larsson, Dep. of Biotechnology, Royal Institute of Technology

Project name Sponsors

Language English Security

ISSN 1401-2138 Classification

Supplementary bibliographical information
Pages 36
Biology Education Centre Biomedical Center Husargatan 3 Uppsala
Box 592 S-75124 Uppsala tel +46 (0)18 174000 fax +46 (0)18 555217

1
Sammanfattning av examensarbetet
Albumin är ett protein (äggviteämne) som finns i blodet. Där transporterar det omkring
viktiga ämnen och hjälper till att upprätthålla vätskebalansen. Albumin ges bland annat vid
chock eller svåra blödningar, t.ex. i samband med operationer. Doserna som ges är på flera
tiotals gram, så det finns behov av stora mängder, totalt flera hundra ton per år i världen.
Hittills har albumin renats fram ur mänskligt blod, men rädsla för virus som t.ex. HIV gör att
man letar efter alternativa produktionssätt. Modern bioteknik gör så att man kan sätta det
mänskliga arvsanlaget för albumin i andra organismer, som får producera albuminet. Jag har
använt mig av en sorts jäst (Pichia pastoris) som kan växa på metanol (träsprit). För att jästen
skall få lagom mycket metanol har jag utvecklat en odlingsmetod, där en elektronisk apparat
mäter metanolhalten i odlingen och reglerar tillförseln av metanol. Denna teknologi har jag
testat på olika stora odlingar, från volymer på 0,4 liter upp till 100 liter. Olika biokemiska
tekniker har använts för att undersöka det producerade albuminet.

2
Table of Contents
1 INTRODUCTION AND AIMS OF THE PROJECT ........................................................................... 4

2 THEORY............................................................................................................................................... 4
2.1 FED-BATCH FERMENTATION ................................................................................................................ 4
2.2 PICHIA PASTORIS ................................................................................................................................. 5
2.2.1 Background ............................................................................................................................... 5
2.2.2 Genetics and Properties............................................................................................................. 5
2.2.3 Fermentation of Pichia pastoris ................................................................................................. 6
2.3 HUMAN SERUM ALBUMIN .................................................................................................................... 6
2.3.1 Structure and Function .............................................................................................................. 6
2.3.2 Synthesis.................................................................................................................................... 7
2.3.3 Use and Production ................................................................................................................... 7
2.4 METHANOL SENSOR ............................................................................................................................. 7
3 MATERIALS AND METHODS ........................................................................................................... 9
3.1 CHEMICALS ......................................................................................................................................... 9
3.2 FERMENTATIONS ................................................................................................................................. 9
3.2.1 Strains ....................................................................................................................................... 9
3.2.2 Equipment and Control .............................................................................................................. 9
3.2.2.1 1.5 litre Fermenter ............................................................................................................................... 9
3.2.2.2 0.4 litre Fermenter ..............................................................................................................................10
3.2.2.3 15 litre Fermenter ...............................................................................................................................11
3.2.2.4 100 litre Fermenter..............................................................................................................................12
3.2.2.5 Methanol Measurements and Regulation..............................................................................................12
3.2.3 Growth Media.......................................................................................................................... 15
3.2.3.1 Media Composition and Preparation ....................................................................................................15
3.2.3.2 Regulation of pH and foaming.............................................................................................................15
3.2.3.3 Precipitates .........................................................................................................................................15
3.2.4 Fermentation Protocol............................................................................................................. 16
3.2.4.1 Strain Propagation and Inoculum Culturing..........................................................................................16
3.2.4.2 Preparation of the Fermenter and Medium ...........................................................................................16
3.2.4.3 The Fermentation and its Three Phases ................................................................................................18
3.2.5 Sampling and Growth Monitoring ............................................................................................ 18
3.3 ANALYSIS OF PRODUCED PROTEIN ..................................................................................................... 19
3.3.1 Gel Filtration........................................................................................................................... 19
3.3.2 SDS-PAGE .............................................................................................................................. 19
3.3.3 Western Blot ............................................................................................................................ 20
4 RESULTS AND DISCUSSION........................................................................................................... 21
4.1 FERMENTATIONS ............................................................................................................................... 21
4.1.1 Fermentations 1–4 ................................................................................................................... 22
4.1.2 Fermentation 5 ........................................................................................................................ 23
4.1.3 Fermentation 6 ........................................................................................................................ 23
4.1.4 Fermentations 7 and 8 ............................................................................................................. 24
4.1.5 Fermentation 9 ........................................................................................................................ 25
4.1.6 Fermentations 10 and 11.......................................................................................................... 25
4.2 HSA ANALYSIS ................................................................................................................................. 27
4.2.1 Gel Filtration........................................................................................................................... 28
4.2.2 SDS-PAGE .............................................................................................................................. 28
4.2.3 Western Blot ............................................................................................................................ 29
4.3 DIFFERENT WAYS TO INCREASE THE YIELD AND TO AVOID PROTEOLYSIS............................................ 30
4.4 CONCLUSIONS ................................................................................................................................... 32
4.5 FURTHER STUDIES ............................................................................................................................. 32
5 ACKNOWLEDGEMENTS................................................................................................................. 33

6 REFERENCES .................................................................................................................................... 33
6.1 AUTHORED ARTICLES, BOOKS AND PATENTS...................................................................................... 33
6.2 OTHER REFERENCED DOCUMENTS AND WEB PAGES ........................................................................... 35

3
1 Introduction and Aims of the Project
This project was initiated for several reasons. One was to introduce to the Fermentation
Laboratory of the Centre for Surface Biotechnology at Uppsala University the know-how and
experience needed to culture Pichia pastoris as a host for recombinant protein production. In
doing this, it has been of great importance not only to apply the experience of other groups
working with P.pastoris, but also to try to improve the technology so that it requires less
supervision, while at the same time maximising both the cell mass production and the amount
of recombinant protein produced. A principal component of this improved fermentation
technology is the usage of a methanol measurement and control system to adjust the feed-rate
of the substrate to the metabolism of the yeast cells during the production phase. A second
reason for this study was to establish appropriate analytical methods to follow secretion of the
therapeutic protein human serum albumin (HSA) into the fermentation medium. A third was
to verify that the final technology was easily applicable at different fermentation scales. To
achieve these goals, a generally available model strain secreting HSA was used. All these
reasons have been considered during the course of the study, and though much remains do be
done, significant progress has been made towards achieving the goals mentioned. The report
does sometimes describe simple technical solutions. This is partly to make it possible to read
the report for those who do not have practical experience of fermentation, and partly to
facilitate the work for future participants in the project.

2 Theory

2.1 Fed-Batch Fermentation

The three most common modes of fermentation are batch, fed-batch and continuous culture.
In this study, fed-batch culture has been used. Batch and fed-batch are used today for most
industrial applications. In a batch fermentation, the inoculum is simply added to a fermenter
containing a certain amount of medium, and the micro-organisms grow until one of the
essential nutrients in the medium has been exhausted. A fed-batch fermentation (sometimes
referred to as ‘extended culture’ or ‘semi-batch’) consists of a batch phase, as described
above, followed by additions of either fresh nutrients or additives, such as precursors to
products. There are several advantages with this strategy as compared to the simple batch
culture. It allows utilisation of substrates which at high concentrations inhibit growth or are
toxic to the cells. It increases the production of biomass and it overcomes the problem of
catabolite repression. Compared to the continuous culture, in which the micro-organism is
grown for long periods at steady-state, all the time removing culture liquid and adding fresh
medium with the same flow rates, fed-batch culture reduces the problems of contamination,
mutation and plasmid instability. The only disadvantages with fed-batch fermentation, as
compared to a batch culture, are the need for expensive instrumentation for feed-back control
and the requirement for a substantial amount of operator skill (Brown 1990). When the
desired product is not growth-associated, i.e. production is not linked to cell growth, then
there is a need to have high concentrations of cells in the fermenter for long periods of time in
order to get a high concentration of the product. It can be shown that this is impossible with
the constant volume batch and the continuous fermenter. The solution is the fed-batch culture,
in which a high concentration of cells is built up, after which metabolism is switched to
induce production by altering the feed or by changing the growth conditions in another
suitable way (Sinclair and Cantero 1990). In this study, two fed-batch phases follow the batch
phase. In the first fed-batch phase, a substrate which promotes production of biomass is fed.
When a suitable cell concentration has been achieved, a second substrate which induces
production of recombinant protein is supplied in the feed.

4
2.2 Pichia pastoris

2.2.1 Background
In the production of recombinant proteins, yeasts offer advantages of both bacterial and
mammalian systems. Yeasts are easy to manipulate, have a relatively rapid growth rate, and
can be grown to higher cell densities than bacteria. On the other hand, as they are eukaryotic,
they offer the capacity to post-translationally modify the produced protein and to glycosylate
secreted proteins at a fraction of the cost of a mammalian expression system (Hagenson
1991). The first yeast selected for production of heterologous eucaryotic proteins was
Saccharomyces cerevisiae, due to the accumulated knowledge of its genetics and physiology.
It was also generally accepted as safe for human use through experience with the organism in
brewing and baking (Cregg et al. 1993). The first methylotrophic (i.e. capable of utilising
methanol as carbon and energy source) yeasts were discovered in 1969, although
methylotrophic bacteria had been known since the beginning of the century. The first
industrial application of a methylotrophic yeast was the production of single cell protein
(SCP) at Phillips Petroleum Company in the seventies. A strain of Pichia pastoris (sometimes
called Komagataella pastoris) was selected based on a very high cell mass yield, a high
protein content and stable fermentation characteristics (Hagenson 1991). Methylotrophic
yeasts offer considerable improvements of specific disadvantages often encountered in
S.cerevisiae-based production systems, e.g., mitotic instability of recombinant strains, a large
amount of undesirable overglycosylation and difficulties in adapting production schemes to
an industrial scale (Hollenberg and Gellissen 1997). One example is a study at Novo Nordisk
A/S (Denmark) where production of insulin by S.cerevisiae and P.pastoris has been examined
(Kjeldsen et al. 1999). The study showed that a fusion protein of an α-factor prepro leader
and an insulin precursor was more correctly processed and secreted from P.pastoris than from
S.cerevisiae, which secreted also hyperglycosylated and partly processed forms of the fusion
protein. Another advantage over production in S.cerevisiae is the existence of a strong and
tightly regulated promoter in P.pastoris, namely the AOX1 promoter (see 2.2.2 Genetics and
Properties).

2.2.2 Genetics and Properties

Being a methylotrophic yeast, P.pastoris can grow on methanol as a sole carbon and energy
source, and it has a highly inducible methanol utilisation pathway. The first enzyme of the
pathway, alcohol oxidase, can account for up to 35% of the total protein in cells grown on
methanol, whereas it is undetectable in cells grown on glucose, glycerol or ethanol
(Sreekrishna et al. 1997). A histidinol dehydrogenase mutant, GS115 (his4), is often used as
the host strain. This strain is incapable of synthesising histidine. The HIS4 gene from wild-
type P.pastoris is then incorporated into the cloning vector and used as a selectable marker for
transformation of this strain, grown in histidine-free media. Two types of vectors have been
developed, autonomous and integrative. Autonomous vectors can replicate outside of the
chromosome, but they are generally of low copy number, unstable, and do invariably integrate
at one ore more chromosomal loci, thereby necessitating a second screen for stable integrants.
Integrative vectors, which are generally preferred, must always integrate into the chromosome
in order to replicate. Integrations can be site-directed at the HIS4 gene or at the primary
alcohol oxidase (AOX1) locus. P.pastoris also contains a secondary alcohol oxidase gene
(AOX2), allowing growth on methanol even when the AOX1 gene is displaced by integration
of the vector (Wegner 1990). AOX1-deleted clones show a slower growth phenotype (MutS,

5
Methanol utilisation slow, sometimes referred to as Mut- in the literature) on minimal
methanol medium compared to AOX1 intact clones which have phenotypically normal
growth characteristics (Mut+) on minimal methanol medium (Sreekrishna et al. 1997).

2.2.3 Fermentation of Pichia pastoris

Pichia has been grown in fermenters in fed-batch and continuous culture. A high cell density
fermentation technology was developed for SCP production using a media of high salt
concentrations (Wegner 1983). Several proteins have been produced since. For a review, see
Cregg et al. 1993. Most commonly, Pichia is first grown to a high cell density on glycerol
during two phases, a batch and a fed-batch phase. Then the production of the recombinant
protein is switched on by feeding methanol. Invitrogen, who sell the Pichia transformation
and expression kits, recommend in their Fermentation Process Guidelines [5] that methanol
should be fed with a constant flow rate, which is increased a few times during the
fermentation. For Mut+ strains, the dissolved oxygen reading can be used to evaluate the state
of the culture. MutS strains, which metabolise methanol poorly, consume very little oxygen,
and hence the dissolved oxygen can not be used. Instead, the methanol feed is started with a
very low flow rate, and is increased in small increments over a rather long period of time. In
both cases (Mut+ and MutS), the process often requires a lot of manual intervention, also at
inconvenient hours. A control system for the methanol feed based on direct measurements of
the methanol concentration in the culture should reduce these problems and possibly increase
the yield of recombinant product. Other ways to increase the yield are discussed in section 4.3
Different Ways to Increase the Yield and to Avoid Proteolysis.

2.3 Human Serum Albumin

2.3.1 Structure and Function

Human serum albumin (HSA, Figure 1), a large protein with a molecular weight of 66500, is
the most abundant protein in human serum, with a concentration as high as 1 mM. HSA
contains 585 amino acids, all making up a single polypeptide chain, which is rich in cysteins,
the majority of which are involved in
disulphide bridges that cause the molecule to
fold into nine loops. The loops can be
grouped into three similar domains, with
different physiological ligands, e.g., metals
and long-chain fatty acids, binding to the
different domains. HSA is one of the few
plasma proteins that are essentially not
glycosylated (Putnam 1984). Except for
providing transportation of several ions and
molecules, including Ca2+, Na+, K+, fatty
acids, hormones, bilirubin, heavy metals and
drugs [4], HSA is also supposed to keep up
the colloidal osmotic pressure of blood, and
to function as a protein reserve (Andersson
Figure 1. Ribbon model of human serum albumin. 1976). Although no one seriously questions
Created with the software RasMol from the data in the PDB
database, entry no 1BJ5. the notion of albumin’s importance as an
osmotic agent, there is evidence suggesting
that ‘transport has always been the primary

6
raison d’être of albumin, and that the osmotic contribution… may simply be a fortuitous
fringe benefit’ (Doolittle 1984, p.239). In addition to the functions of HSA mentioned above,
there are other functions, which, though less understood, may be of great importance. One
example is leukocyte chemotaxis, which cannot occur in the absence of HSA (Czarnetzki and
Schulz 1980). Another is the discovery that HSA is a potent antioxidant, presumably
protecting the cells in the blood from reactive oxygen species, such as H2O2 (Cha and Kim
1996). Finally, HSA has been shown to inhibit the growth of certain breast cancer cell lines in
vitro, and there are experimental results indicating that this might be the case also in vivo
(Laursen et al. 1990).

2.3.2 Synthesis
HSA is synthesised as preproalbumin. The preprosequence consists of the first 24 amino
acids. Of these, the first 18 make up the signal peptide for secretion, and the last 6 are the so
called propeptide, which immediately precedes the polypeptide chain of mature albumin. The
signal sequence is cleaved off in the endoplasmic reticulum during the early stage of
secretion, leaving proalbumin. The function of the 6 amino acids in the propeptide is not
known, but they do not have to be removed before secretion (Putnam 1984). The prepro-
albumin is synthesised on membrane-bound ribosomes of liver cells. The formation of
disulphide bonds is believed to play an important role in the process of protein folding and for
tertiary structure stabilisation (Ikegaya et al. 1997).

2.3.3 Use and Production

Albumin is administered as a therapeutic agent in several cases, the two most common
probably being at shock and excessive bleeding (Putnam 1984). The usual clinical doses of
HSA are in excess of 10g/dose (Ohtani et al. 1998). An extracorporal application is the use of
albumin for perfusion of donated organs for transplantation (Claes 1976). HSA has
traditionally been produced from human plasma by fractionation according to the method of
Cohn et al. from the beginning of the forties (Anderson 1984). A varying supply of donor
plasma in combination with an increased awareness of the potential risks of contaminants,
e.g., hepatitis virus, HIV or prions, in products from blood, has lead to increasing efforts to
produce HSA recombinantly. In 1981, HSA was first cloned and expressed in Escherishia
coli (Lawn et al. 1981). It has since been expressed in several organisms, including Bacillus
subtilis (Saunders et al. 1987), S.cerevisiae (Sleep et al. 1990), tobacco and potato plants
(Sijmons et al. 1990) and the methylotrophic yeasts Kluyveromyces lactis (Fleer et al. 1991)
and Pichia pastoris (Sreekrishna et al. 1990, Barr et al. 1992, Ohi et al. 1994). The natural
structural complexity of HSA, together with its low unit price (a few US$ per gram) and its
large market volume (300t/year), make the conception of an industrial process for the
production of a pharmaceutical-grade recombinant serum albumin one of the most difficult
challenges of recombinant protein biotechnology (Fleer et al. 1991).

2.4 Methanol sensor

The methanol sensor used in this work is a MOS sensor, i.e. it consists of a metal oxide
semiconductor (in this case, it is SnO2 [3]) on a sintered alumina ceramic bead contained
within a flame arrestor [1]. When the sensor is heated to a high temperature (400°C) without
the presence of oxygen, free electrons flow easily through the grain boundaries of the tin
dioxide particles. In clean air, oxygen, which traps free electrons by its electron affinity, is
adsorbed onto the tin dioxide particle surface, forming a potential barrier in the grain

7
boundaries. This potential barrier restricts the flow of electrons, causing the electric resistance
to increase. When the sensor is exposed to a reducing gas, the tin dioxide surface adsorbs the
gas molecules and causes oxidation of the gas. This lowers the potential barrier, thereby
reducing the electrical resistance [2]. The reaction varies depending on the sensor
temperature, something which is used to create sensors with different specificities [1]. An
advantage with the MOS sensors is the ability to detect low concentrations of gases (50-500
ppm) over a wide range of temperatures. Limitations are the relative unspecificity and the
effect of humidity on sensor output. As humidity increases, sensor output increases and vice
versa [1,3].

8
3 Materials and Methods

3.1 Chemicals
The chemicals used were obtained from the following companies. All were pro analysi (p.a.)
unless otherwise stated. Antibodies were from DAKO, Glostrup, Denmark. Agarose (type
VII), histidine and HSA (fraction V, 96–99%) were from Sigma, St Louis, MO. β-
mercaptoethanol (puriss) and glycine were from Carl Roth, Karlsruhe, Germany. Acetic acid
(puriss), potassium sulphate (for fermentation 9), ammonia (puriss, for fermentation 9),
ferrous sulphate·7H2O (puriss, for fermentation 9), magnesium sulphate·7H2O (technical
quality, for fermentation 9) and methanol (purum, for all fermentations except no 11) were
from Kebo Lab, Spånga, Sweden. Ammonia, ferrous sulphate and potassium hydroxide were
also obtained from Merck, Darmstadt, Germany and used in all fermentations except no 9.
Methanol from Merck, Darmstadt, Germany, was used in fermentation 11 and for calibration.
Biotin and the molecular weight standard were from Pharmacia Biotech, Uppsala, Sweden.
Chlorhexidin was from Pharmacia & Upjohn, Stockholm, Sweden. Bromphenol blue was
from LKB, Bromma, Sweden. Glucose was from JT Baker, Deventer, Holland. Cobalt
chloride was from Fluka, Buchs, Switzerland. Hydrochloric acid was from Riedel-de-Haën,
Seeize, Germany. PPG (1025) was from BDH, Poole, UK. SDS was from Kebo, Stockholm,
Sweden. Silver nitrate was from CP Bakers, Phillipsburg, NJ. Yeast nitrogen base (without
amino acids) was from Difco, Detroit, MI. Sodium sulphite was from Labassco, Gothenburg,
Sweden. TRIS (Trizma base, reagent grade) was from Sigma-Aldrich, Steinheim, Germany.
Tween 20 (for synthesis) was from Merck, Hohenbrunn, Germany. Glycerol was from Merck,
Darmstadt, Germany, or from Struers, Albertslund, Denmark. All other chemicals were from
Merck, Darmstadt, Germany and were p.a. except gelatin (for microbiology),
glutardialdehyde (for synthesis), potassium hydroxide (extra pure, food grade) and sodium
iodide (suprapur).

3.2 Fermentations

3.2.1 Strains
Two strains of Pichia pastoris were used: GS115/MutS (see 2.2.2 Genetics and Properties)
HSA secreting strain from Invitrogen (San Diego, CA) and GS115 (his4) from NCPC
(China).

3.2.2 Equipment and Control

Four different fermenters were used in this study. They are described here in the order in
which they were employed.

3.2.2.1 1.5 litre Fermenter

In fermentations 1–4, a BioFlo C30 fermenter with a 1.5 litre cylindrical glass vessel (New
Brunswick Scientific, Edison, NJ) was used. This older fermenter had possibilities to
automatically control the temperature, pO2 (partial oxygen pressure, i.e. the amount of oxygen
dissolved in the liquid relative to the amount maximally dissolved), the pH and the stirrer
speed. Antifoams were added manually through a septum in the lid. The septum was also used
for taking samples and for injecting other additions, including the inoculum. Glycerol, acid,

9
base, and methanol were added through a three-way addition port in the lid. Glycerol was fed
with a 505U peristaltic pump (Watson-Marlow, Falmouth, UK) and methanol was fed with a
P1 peristaltic pump (Pharmacia, Uppsala, Sweden). This older fermenter did not function
properly due to several reasons, among which can be mentioned in particular that the air inlet
fittings were not tight, leading to that gas leaked out. There were also problems with the pO2
regulation and with foaming. Therefore a new 0.4 litre fermenter was installed as a
replacement.

3.2.2.2 0.4 litre Fermenter

Fermentations no 5–6 and 10–11 were performed in a 0.4 litre fermenter mounted on a SARA
control panel (Belach Bioteknik, Stockholm, Sweden). This fermenter was also made of glass,
but had a conical shape and was constructed with a number of threaded positions for
mounting different electrodes etc. The SARA control panel had built-in possibilities to control
temperature, pH, stirrer speed, pO2 and foam level by microprocessors and it also had built-in
pumps for addition of acid, base and antifoam. The control panel was connected to a PC with
the program Geasy 3.0 for logging of the five parameters mentioned above plus the methanol
signal from the methanol measurement device (see 3.2.2.5 Methanol Measurements and
Regulation). The temperature was controlled by heating the fermentation vessel through a
heating pad, on which the fermenter stood, and by cooling with cold water, which was turned
on and off by a magnetic valve and passed through a metal cooling finger. The pO2 was
controlled by the stirrer speed. The inlet air flow was controlled manually with a rotameter.
The outlet gas was bubbled through 3M NaOH and the cleaned gas was lead to a fume hood.

In fermentation no 5, it was noticed that the oxygen transfer rate (OTR) capacity of the
fermenter did not allow the pO2 to be kept at the desired level, even when the stirrer was
running at maximum speed (ca 1150 rpm). Therefore, pure oxygen was mixed with the inlet
air in fermentations 6, 10 and 11. The magnetic stirrer was made of teflon (with magnets
inside), and shaped as shown in Figure 2. Since the OTR of the vessel was not efficient
enough to keep up the pO2, efforts were put into improving the construction. The stirrer was
fitted with extra metal blades. The baffles of this fermenter were inward bends in the glass
wall, and therefore they were not sharp enough to stop the turbulence. To remedy this, an
extra metal baffle was constructed and mounted on the aeration tube/stirrer shaft of the vessel
(Figure 3). The extra stirrer blades and the baffle were used in fermentation no 11. Inoculation
and injection of trace salts and histidine as well as addition of acid, base and glycerol were
made through septa mounted on the threaded positions on top of the vessel. The septa were
also used for taking out samples with a sterile syringe. For addition of methanol, a longer
needle was used, reaching down through a septum and ending below the surface of the
fermentation liquid, see 4.1.1 Fermentations 1–4, for an explanation.

View from the side View from the top

Figure 2. The teflon stirrer of the 0.4 litre fermentation vessel

The foam level was measured as the relative conductivity between two electrodes, one of
which was connected to the fermentation liquid. The second electrode was an electrically

10
isolated steel tube mounted through a septum and reaching down into the vessel to the
maximum level of foam allowed. When foam built up and reached the second electrode, the
conductivity between the electrodes increased and antifoam was pumped in through the steel
tube. To allow time for the antifoam to take effect, the pump was active only for a short
period of time, after which there was a pause, the length of which could be set on the control
panel. To avoid problems with a moist layer forming on the inside of the vessel, connecting
the two foam level electrodes and thereby increasing the conductivity between them even
when there was no foam, the membrane housing around the second electrode had to be
heated. This was done by winding a laboratory heating tape (Thermolyne, Dubuque, IA)
around it. It was important that no ethanol was sprayed on the septum through which the
second electrode was inserted, since otherwise the conductivity might increase in spite of the
heating tape. It was, of course, also important to see that the second foam electrode did not
end too close to the surface, since increased stirrer speed as well as an increasing liquid
volume in the fermenter might cause the electrode to come into contact with the surface.

Inward bend baffle in

the glass vessel
Extra metal baffle
for decreasing the
turbulence Metal blades for
increased stirring effect
teflon magnetic stirrer
Figure 3. 0.4 litre fermentation vessel with our improved stirrer and baffle

In the glycerol fed-batch phase, glycerol was fed with a 101U/R peristaltic pump (Watson-
Marlow, Falmouth, UK), except in fermentation 5, when all glycerol was added at one time.
Methanol was fed with a P1 pump (Pharmacia, Uppsala, Sweden).

3.2.2.3 15 litre Fermenter

Fermentations 7 and 8 were performed in a 15 litre fermenter (Belach Bioteknik, Stockholm,
Sweden) made of stainless steel and coupled to an IBM PC with the software Geasy 3.0
(based on the Genesis Control Series, Iconics, Foxboro, MA) for control and logging of the
process parameters, namely temperature, pO2, pH, stirrer speed and addition of acid and base.
Heating and cooling of the fermenter was achieved by passing hot or cold water through the
mantle around the vessel. By filling the mantle with steam, the medium could be sterilised in
situ before the fermentations, and remaining cells could be killed after harvesting. pO2 was
measured by a polarographic oxygen electrode and was coupled to the stirrer speed. pH was
regulated by addition of acid and base with separate pumps. Airflow and pressure in the
fermenter were controlled manually, but the pressure was logged on the computer together
with the other parameters mentioned above. Inlet and outlet gas was filtered through a bonded
microfibre filter element (Headline Filters, Aylesford, UK). The stirrer was a Rushton turbine
with a magnet coupling to the motor below the fermenter. Air could be directed above the

11
surface of the liquid or below the turbine through a so called sparger, regulated by a manual
valve. Inoculation and addition of acid, base, glycerol, trace salts and histidine were made
through septa in the lid of the fermenter and samples were taken out through another septum
placed on the side of the fermenter and near the bottom. Methanol was added through a
specially designed addition valve mounted in the lid of the fermenter. The valve had a long
metal tube reaching down below the surface of the fermentation liquid. Glycerol was fed with
a 505U pump (Watson-Marlow, Falmouth, UK) and methanol with a P1 pump (Pharmacia,
Uppsala, Sweden). Foam control was carried out with a SARA control panel as described
above under 0.4 litre fermenter. To avoid problems with the foam control, the following
measures had to be taken: The membrane housing where the foam electrode was inserted was
heated with a heating tape (Thermolyne, Dubuque, IA) during fermentation 8. An electrical
short-circuiting was made between the fermentation vessel and the SARA control panel.
Furthermore, the power supply for the methanol control unit and the SARA control unit had
to be connected to different power outlets. For a discussion of the problems with foam control
in this fermenter, see 4.1.4 Fermentations 7 and 8.

3.2.2.4 100 litre Fermenter

The 100 litre fermenter (Chemoferm, Hägersten, Sweden) was used in fermentation 9. This
fermenter was also made of stainless steel with a mantle for sterilisation, heating and cooling
but it also had addition ports for acid and base and a sample port that could be sterilised by
clean steam. The fermenter was controlled by an Apple IIe computer (Apple Computer,
Cupertino, CA) with the control software Bioreactor Control System BRC 96, Pilot Plant
System Type PCP 300 (Chemoferm, Hägersten, Sweden) giving possibility to automatically
control and log temperature, pH, added amounts of acid and base, pO2 and stirrer speed, inlet
air flow and pressure. Inlet and outlet air was filtered through a teflon filter (Pall, East Hills,
NY). The sparger was controlled by a magnetic valve. pO2 was measured by a polarographic
oxygen electrode and was coupled to the stirrer speed. The stirrer was a Rushton turbine with
a magnetic coupling to the motor below the fermenter. Foam level control was performed
with a SARA control panel as for the 0.4 and 15 litre fermenters. No heating of the housing
with the membrane, through which the foam electrode was inserted, was necessary because
clean steam was passed around the joint between the fermentation vessel and the lid, thereby
heating the lid. As for the 15 litre fermenter, a short-circuit was introduced between the vessel
and the SARA control panel and the SARA and the methanol control unit were plugged into
different power outlets. Additions of trace salts, inoculation, feeding of glycerol and sampling
were made through the steam sterilisable septum located on the side of the vessel, near the
bottom. Both glycerol and methanol were fed with a 502S peristaltic pump (Watson-Marlow,
Falmouth, UK).

3.2.2.5 Methanol Measurements and Regulation

The methanol concentration in the fermentation liquid was determined indirectly by
measuring the concentration of methanol in the outlet gas flow, using a metal oxide
semiconductor (MOS) gas sensor TGS 822 from Figaro Engineering (Osaka, Japan), see
chapter 2.4 Methanol sensor. Part of the outlet gas was lead through a piston lab pump RP-
G150 (Fluid Metering Inc., Oyster Bay, NY) to the sensor. In the case of the 100 litre
fermenter, a 505U peristaltic pump (Watson-Marlow, Falmouth, UK) was used instead,
because the piston pump could not withstand the high pressure from the fermenter without
leaking. The pump was run at constant speed to guarantee a steady gas flow from the
fermenter to the sensor, regardless of the aeration rate of the fermenter and the dimensions of

12
the outlet gas tubings. In some of the experiments, it was necessary to dilute the outlet gas,
since it contained a too high concentration of methanol in relation to the sensitivity of the
MOS sensor. This was done with pressurised air, the flow rate of which was adjusted by a
reduction valve, and which was connected to the gas flow between the pump and the sensor.
In order to avoid the formation of droplets of condensed water in the outlet gas tubing before
the sensor, the outlet gas had to be heated. Otherwise methanol, dissolved in the droplets,
evaporated and disturbed the methanol sensor. Furthermore, accumulated condensed water
could be pumped into the sensor and cause malfunction. In the 1.5 and 0.4 litre fermentations,
a laboratory heating tape (Thermolyne, Dubuque, IA) was wound around the tubing and
connected to a power supply (EPS 500/400, Pharmacia Fine Chemicals, Uppsala, Sweden) set
to 150V and 200mA. The outlet gas from the 15-litre fermenter was heated by passing steam
through a condenser mounted on the lid of the fermenter, and in addition, the heating tape was
used as described above. As mentioned earlier, the lid of the 100-litre fermenter was heated
by injecting clean steam into the joint between the fermenter body and lid. This was enough
to heat the outlet gas and prevent formation of condense in the tubing. Due to the set-up with
the heating tape wound around the outlet gas tubing, it is not possible to say with certainty
that the temperature of the gas passing through the sensor lies within the temperature range
for which the sensitivity characteristics given can be obtained. According to the sensor’s
technical reference manual [3], the range is -10°C to about +40°C. It should also be
mentioned here that the sensor must be switched on at least a day before the experiments start,
in order to reach the required temperature and stabilise.

The methanol sensor was connected to the methanol control unit, which was developed and
constructed in collaboration with J. Kozma (Budapest, Hungary). In fermentations 1–10 only
an older model of control unit was used. It consisted of two devices, first one for
amplification and display of the measured sensor signal, and a second device for control of
the methanol feed. The control device had a knob for determining a set-point and compared
the methanol sensor signal from the first device with this set-point. If the signal was lower
than the set-point, a 220V outlet in the device was switched on, and vice versa. To this outlet,
the methanol feeding pump was connected. In fermentation 11, this older model was used
together with a new unit, containing all the functions of the two devices in one box. To test
the new unit, two sensors were positioned in series in the gas flow. A schematic picture of the
methanol control system is shown in Figure 4.

13
 Needle valve

Pressurised
air
MeOH controller

Inlet filter
MeOH pump

Electrical signal
Methanol
sensor
Liquid flow

Gas flow

MeOH

Out-gas
Piston pump

Fermentation
vessel

Needle
valve

Pressurised air

Flowhood
Magnetic
stirrer

NaOH

Figure 4. A schematic picture of the methanol control system. For further details, see the text.

14
Before each fermentation, the methanol unit was calibrated to establish a relationship between
the sensor output and the methanol concentration in the liquid of the fermenter. The fermenter
was filled with water, and the temperature regulation, aeration, stirrer etc. were started. Then
methanol was injected into the fermenter in amounts corresponding to the concentrations 0.5
g/l, 1.0 g/l, 2.5 g/l, 5.0 g/l and 7.5 g/l. The sensor signal was recorded, and this calibration
curve was used to determine the methanol set-point during the fermentation.

3.2.3 Growth Media

3.2.3.1 Media Composition and Preparation

The yeast cells were cultured on minimal dextrose (MD) plates, or, in the case of the his4-
strain (GS115), on MDH plates (MD + histidine). The inoculum cultures were grown in
minimal glycerol (MGY) medium, or, in the case of the his4-strain, in MGYH (MGY +
histidine). The plates and media were prepared and stored as described in the Pichia media
recipes [6]. MGY contains 1.34% Yeast Nitrogen Base (YNB) without amino acids, 1%
glycerol and 4x10-5% biotin. MGYH also contains 0.004% histidine. MD plates contain
1.34% YNB without amino acids, 4x10-5% biotin, 2% dextrose and 1.5% agar. MDH also
contains 0.004% histidine. The fermentations were performed in Fermentation Basal Salts
(FBS) medium, prepared as described in the Pichia fermentation process guidelines [5] and
sterilised as described in Preparation of the Fermenter and Medium below. One litre of FBS
contains 1.18g calcium sulphate·2H2O, 18.2g potassium sulphate, 14.9g magnesium
sulphate·7H2O, 4.13g potassium hydroxide, 26.7 ml 85% phosphoric acid and 40g glycerol.
To adjust the pH of the FBS medium before inoculation, 25% NH3-solution was added slowly
to the fermenter (except in fermentation 4, where 3M KOH was used instead). Before
inoculation, PTM1 Trace Salts solution (PTM1TS) was added. The PTM1TS was made with
the same ingredients as in [5] but to avoid a green precipitation, it had to be prepared in the
following way: For 1 litre, dissolve in the following amounts in water: 6.0g cupric
sulphate·5H2O in 50 ml, 0.08g sodium iodide in 1.0 ml, 3.0g manganese sulphate·H2O in 20
ml, 0.2g sodium molybdate·2H2O in 1.0 ml, 0.02g boric acid in 1.0 ml, 0.5g cobalt chloride in
5 ml, 20.0g zinc chloride in 100 ml, 65.0g ferrous sulphate·7H2O in 500 ml and 0.2g biotin in
100 ml. To dissolve the biotin, add 300 µl of 3M KOH to that aliquot. Add 5.0 ml of
concentrated sulphuric acid to dissolve the ferrous sulphate. Pour together all aliquots except
the one with biotin. Make sure that everything is mixed before adding the biotin solution.
Finally, add water to a final volume of 1.0 litre. Sterile filter (0.2µm) and store at room
temperature.

3.2.3.2 Regulation of pH and foaming

For regulation of pH, 3M phosphoric acid and 25% ammonia was used, except for the first
three fermentations, where 3M KOH was used instead of ammonia. The antifoam used was
polypropylene glycol (PPG). It is important that not too much PPG is added to the fermenter.
The two main reasons for this is that the solubility of oxygen goes down with increasing
concentration of PPG and that down-stream processing is made more difficult, since droplets
of PPG may interfere with the performance of chromatography columns etc.

3.2.3.3 Precipitates
Three different precipitates of unknown composition have appeared in this study. The first
was the green one appearing when PTM1TS is prepared according to the original recipe (see

15
3.2.3.1 Media Composition and Preparation above). The second one appears when the pH of
FBS is raised, either with NH3 or KOH. This precipitate is white and flaky, but seems to
dissolve after a while with stirring on. Finally, the third precipitate is the most difficult one to
eliminate. It appears when PTM1TS solution is added to the FBS medium before inoculation,
giving the medium a white, milky appearance. This precipitate has been noted already by
Siegel and Brierly (1989) but no further explanation was given. Experiments to elucidate the
nature of the precipitate have resulted in the following observations: The precipitate does not
appear if PTM1 is added to FBS without first raising the pH. Thus the reaction is pH-
dependent (or possibly, the NH4+ ion is involved in the precipitation). After the precipitate has
formed, the reaction cannot be reversed by lowering the pH of the medium with H3PO4. When
FBS is prepared with H2SO4 instead of H3PO4 (and adjusted to the same pH), no precipitation
is seen either when the pH is raised with NH3 or when PTM1TS is added. This indicates that
the phosphate ion is involved in both the precipitate that appears when the pH of FBS is
raised and the one that appears upon addition of PTM1TS to FBS. Since the former precipitate
appears whether pH is raised with NH3 or KOH, this last finding indicates that the NH4+ ion is
not involved in the precipitation at all. More likely, a metal ion from FBS forms the
precipitate with the phosphate ion when the pH is raised, or when PTM1TS is added. In order
to find out the nature of the formed precipitate upon addition of PTM1TS to FBS, the solution
was filtered and the precipitate was dried on the filter paper and collected. The powder was
then examined with powder x-ray crystallography, using an Expectron XDC-1000 Guinier-
Hägg camera with the radiation of the Cu Kα1 line with a wavelength of 1,54Å. If the
precipitate had been crystalline, then diffraction lines would have appeared on a film, and the
compound would have been identified in a computer database, containing more than 60,000
inorganic salts. Unfortunately, no lines from the sample were visible on the film, and
therefore the conclusion was drawn that the precipitate was amorphous.

3.2.4 Fermentation Protocol

The fermentation protocol was originally taken from the Invitrogen Pichia Fermentation
Process Guidelines [5], but several details were altered during the course of this study. An
outline of the adapted protocol will be given here. Several technical details are mentioned in
order to facilitate recreation of the process by future workers in the project.

3.2.4.1 Strain Propagation and Inoculum Culturing

The yeast was grown on MD (for the transformed strain) or MDH (for the his4 strain GS115)
plates at 30°C. Inoculum cultures were grown in MGY (for the transformed strain) or in
MGYH (for GS115) in Erlenmeyer flasks at 30°C and shaken at 200-300 rpm until the OD600
was between 2–6. The inocula for fermentations 1–3 were grown in baffled shake flasks and
the rest of the inocula were grown in normal flasks. The volume of medium in the shake flask
was not more than maximum 20% of the shake flask volume, but most often not more than
10% of the volume to assure adequate aeration. In some of the fermentations, the inoculum
culture was diluted in fresh medium to delay the time when the desired OD600 was reached.
This procedure was, if necessary, repeated.

3.2.4.2 Preparation of the Fermenter and Medium

Before the fermentation, the methanol sensor was calibrated as described in 3.2.2.5 Methanol
Measurements and Regulation, and the inoculum was cultured as described above. Before
sterilisation of the fermentation vessel, the pH and pO2 electrodes were calibrated and if

16
necessary, electrical contacts were covered and (for galvanic pO2 electrodes) short-circuited.
Rubber septa for additions and sampling, as well as air inlet and outlet filters were checked
and if necessary changed. For the 1.5 and 0.4 litre fermenters, the FBS medium was prepared
as described in 3.2.3.1 Media Composition and Preparation and poured into the fermentation
vessel before autoclaving. In the case of the 15 and 100 litre fermenters, the FBS medium was
prepared in separate vessels (another 15 litre fermenter or a stainless steel medium tank,
respectively) and sterilised in situ by steam injection into the mantle of the vessel. It was then
transferred to the actual fermentation vessel to be used by connecting the two vessels by
Teflon tubing which was sterilised by clean steam, and by applying an overpressure to the
vessel containing the FBS medium. Before the transfer, the fermentation vessel was also
sterilised in situ by steam injection into the mantle. During this step, the fermenter was filled
with phosphate buffer of pH 7 to protect the pH electrode. Simultaneously, clean steam was
passed through the methanol feeding device to sterilise the valve.

The pO2 electrode of the 1.5 litre fermenter seemed to show a better resistance to the harsh
conditions during the sterilisation (high temperature and pressure, in combination with the
low pH of the FBS medium, ca 1.6–1.8) than the pO2 electrodes of the other fermenters, even
though the membrane of the electrode was broken during fermentation 3. The electrodes of
the 0.4 and 15 litre fermenters were destroyed by sterilisation in FBS, and the electrode of the
0.4 litre fermenter did not seem to stand autoclaving at all, even in neutral salt buffer. This is
why the FBS medium was sterilised separately for the 15 and 100 litre fermenters. In the case
of the 0.4 litre fermenter, the pO2 electrode had to be inserted into the vessel after autoclaving
and thus, the FBS medium could be sterilised in the fermenter without harming the electrode.
To assure sterility, the electrode was sterilised for a minimum of 10 minutes in each of the
following solutions before being inserted into the fermenter: chlorhexidin (klorhexidinsprit) 5
mg/ml, 70% ethanol, and sterile distilled water.

The two small fermenters were autoclaved for 20 min at 121°C and 1,1 bar overpressure in a
Vapoklav 500E autoclave (Dr Hoiß+Partner, Munich, Germany). Since no foam control was
possible with the 1.5 litre fermenter, antifoam (polypropylene glycol, PPG) was added before
autoclaving. This resulted in a greyish colour of the medium after autoclaving. The larger
fermenters were sterilised in situ. The conditions were the same as for the small fermenters,
and the sterilisation process was controlled by the computers coupled to the fermenters.
Before the start of a fermentation, tubing had to be prepared and autoclaved. For the 1.5, 0.4
and 15 litre fermenters, tubings for acid, base, antifoam (not for the 1.5 litre fermenter),
glycerol and methanol were needed. The 15 litre fermenter also required a tubing for
inoculation. The 100-litre fermenter used steam sterilisable teflon tubings for addition of acid
and base, so only tubings for inoculation, glycerol, antifoam and methanol needed to be
prepared. All tubings were made of silicone rubber and had dimensions suited for the
respective additions. Before autoclaving, the ends of the tubings were wrapped in aluminium
foil to protect sterility until use. Into each piece of tubing, a small amount of water was added
to facilitate heat transfer during sterilisation.

After sterilisation of the vessel and FBS medium (and in the case of the 0.4 litre fermenter,
mounting of the pO2 electrode) all tubings and cables were connected, and aeration, agitation
and temperature regulation were started. pH of the medium was then raised to 4.5–4.9 by
addition of 25% NH3 solution (except for fermentation 4, when 3M KOH was used instead).
On addition of the base, a white precipitate appeared, but this dissolved again. To minimise
problems with the precipitate, pH was raised slowly over a period of minimum 15 minutes.

17
Inoculum Phase 1 Phase 2 Phase 3
culture Glycerol Batch Phase Glycerol Fed-Batch Phase Methanol Fed-Batch Phase
1–2 days ca 24h 4–24h Up to one week

Figure 5. The different phases of a fermentation and the time of each phase.

When the medium had become clear again, PTM1TS was added, 4.35 ml/l of FBS. Upon this
addition, a milky white precipitate appeared, see Precipitates. When the correct temperature
(30°C) was reached, the pO2 was set to 100% for the small fermenters, and 100*(1+x)%
where x is the overpressure in the fermenter in bars, for the larger fermenters. The last item to
be done before inoculation of the fermenter was to start the heating of the outlet gas tubing,
see 3.2.2.5 Methanol Measurements and Regulation, and to check that all the set-points were
set to the correct values, i.e., temperature 30°C, pO2 30%, pH 4.9, stirrer speed controlled by
pO2 and foam level 15% during fermentations 5–7 and 75% for fermentations 8–11.

3.2.4.3 The Fermentation and its Three Phases

The volume of the inoculum (with an OD600 of 2–6) was 5–10% of the volume of the FBS in
the fermenter. Immediately after inoculation, a sample was taken out with a sterile syringe.
Later during the fermentation, samples were taken out at irregular intervals, but most often at
the end of each phase and at least once a day, see 3.2.5 Sampling and Growth Monitoring.

The fermentation consisted of three phases: the glycerol batch phase, the glycerol fed-batch
phase and the methanol fed-batch phase. In the glycerol batch phase, the cells consumed the
amount of glycerol that was present in the FBS from the beginning (4%). The phase was
ended when all glycerol was finished, which could be seen as a sharp increase in the pO2
signal to about 100%, or, when pO2 was coupled to the stirrer speed, by a decrease in the
stirrer speed, because less agitation was needed to keep up the pO2 when the substrate was
consumed. The second phase, the glycerol fed-batch phase, involved feeding the culture with
a 50% glycerol solution, containing 1.2% PTM1TS solution. The original procedure involved
a feeding for four hours at 18.15 ml/h/l of initial culture, but this was varied between the
fermentations, see 4.1 Fermentations. When the intended amount of glycerol had been fed, or
the culture had reached a particular density, the second phase was ended, and the third phase
was commenced when all remaining glycerol had been consumed. In the methanol fed-batch
phase, a concentrated methanol feed containing 1.2% PTM1TS solution was pumped into the
culture, at a couple of occasions at a constant rate, but more often the methanol feed was
controlled by a specially designed methanol control equipment, which is described in the
section 3.2.2.5 Methanol Measurements and Regulation. A summary of the fermentation
process, together with approximate times for each phase, is shown in Figure 5.

3.2.5 Sampling and Growth Monitoring

To monitor the growth of the cells, samples were taken out from the fermentation liquid
through rubber septa with sterile syringes, which were either single-use or stored in 70%
ethanol between the samplings. Sterility of the culture was ensured by placing a pad of cotton
wool, soaked in ethanol, over the septum and covering the sample port with aluminium foil.
Before sampling, the cotton wool was removed using a pair of tweezers stored in ethanol and
the sample port was flushed with ethanol to remove any contaminants before inserting the
sampling needle. The sample port of the 100-litre fermenter could not be treated in this way.

18
Instead, during the batch phase, the port was sterilised by clean steam before each sampling.
During the fed-batch phases, the septum was thoroughly rinsed with ethanol before insertion
of the needle.

The samples were analysed for cell mass by optical density (OD) and/or wet weight (ww).
Some samples were also analysed for pH or examined visually under a light microscope (Carl
Zeiss, Stockholm, Sweden) to discover possible contaminants and to evaluate the fraction of
the cells which were budding. The OD measurement was carried out at 600nm (OD600) using
a Spectronic 601 (Milton Ray, Rochester, NY). Zeroing was done with phosphate-buffered
saline (PBS, 0.07M NaCl, 0.075M Na-phosphate, pH 7.3) and the samples were diluted in
PBS until the reading was below 0.3 OD units, since the photometer was not linear above this
range. Wet weight determination was done by pipetting an exact amount of the sample into
pre-weighed Eppendorf tubes, which were subsequently spun in a microfuge until the cells
were pelleted, not less than 5 min at 12200 rpm, but sometimes twice that time. The
supernatant was removed, the tubes were weighed again, and the weight of the packed cell
pellet could be calculated. Some of the samples were stored at –20°C and others at –70°C and
sometimes the supernatant was also stored in the freezers until analysis of produced HSA (see
3.3 Analysis of Produced Protein below).

3.3 Analysis of Produced Protein

3.3.1 Gel Filtration

For analysis of the produced HSA, culture supernatant was subjected to gel filtration using an
ÄKTA Explorer with the control and evaluation software Unicorn Version 3.00.10
(Amersham Pharmacia Biotech, Uppsala, Sweden). The column was a Pharmacia Superdex
200 HR 10/30 (inner diameter 10 mm, height of packed bed 30 cm, bed volume 24 ml).
Standard curves for quantification of the HSA were made with an albumin standard from the
Finnish Red Cross (FRK Blodtjänst, Finland). A series of dilutions was made and the
concentration of HSA was determined by measuring the A280 in a Lambda 11 Spectrometer
(Perkin Elmer, Norwalk, CT) or in a Spectronic 601 (Milton Ray, Rochester, NY). The
concentration was calculated as A280/0.53. The running buffer was 0.5M NaCl, 50mM
Sodium Phosphate, pH 7.0. The flow rate was 0.5 ml/min and 0.5 ml of each sample was
applied. The samples from fermentations 6 and 9 were diluted 2–4 times. Detection was made
at 215nm, 230 nm and 280nm. The area under the UV absorption curve was proportional to
the amount of HSA, as was seen in the obtained standard curves (data not shown).

3.3.2 SDS-PAGE
Analysis of the HSA secreted from the cells into the fermentation broth was made with
sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on a PhastSystem
electrophoresis unit (Pharmacia Biotech, Uppsala, Sweden). The gel used was a PhastGel
gradient 8–25 and as the buffer PhastGel SDS Bufferstrips for discontinuous buffer
separations were used. Each sample was prepared by mixing 5µl of β-mercaptoethanol, 10µl
of 25% SDS solution, 50 or 75 µl of the supernatant, TE buffer (10 mM TRIS/HCl, 1 mM
EDTA, pH 8.0) up to 98µl and 2µl of sample loading dye containing bromophenol blue. Then
the sample was boiled in a water bath for 5 minutes and centrifuged for 3 minutes at 14000
rpm. The electrophoresis was run according to Separation Technique File no 110 [7], with the
change that the separation was carried out for 75 Vh (accumulated volt hours) instead of 65
Vh. 1 or 4µl of the prepared sample was applied to each lane. The gel was developed with

19
silver stain according to Development Technique File no 210 [7] with the change that the
preserving solution used in development step 11 contained 13% acetic acid and 10% glycerol.
The gel was scanned with a GS700 Imaging Densitometer (Biorad Laboratories, Hercules,
CA) and analysed using the software Biorad molecular analyst 1.3 (same company) and
AlphaEase 3.3e (Alpha Innotech, San Leandro, CA).

3.3.3 Western Blot

SDS-PAGE was performed as described above. After the separation, the proteins were
transferred to a nitrocellulose membrane (pore size 0.2µm, Pharmacia Biotech, Uppsala,
Sweden) as described in Development Technique File no 221 [7]. For transfer, three filters
were used on each side of the gel and membrane (PhastTransfer filterpapers, Pharmacia
Biotech, Uppsala, Sweden). The membrane and filters were soaked in transfer buffer prior to
use (25mM Tris, 192mM glycine, pH 8.3, containing 20% methanol). The transfer was
carried out for 5 Vh. The membrane was then incubated in blocking solution, wash solution,
in a primary antibody solution (rabbit anti human serum albumin), in wash solution again and
then with a secondary antibody (goat anti rabbit IgG with alkaline phosphatase). Finally, the
membrane was washed again and incubated in TBS as described in [7], before being
developed with Sigma FAST BCIP/NBT tablets according to the descriptions (Sigma
Biosciences, St.Louis, MO). The tablets contained a substrate for the enzyme alkaline
phosphatase which was coupled to the secondary antibodies. Thereby the bands which
contained HSA became coloured. The following incubation times were used: blocking
solution 1 hour, primary antibody (diluted 500 times) 2 hours. The other times were as
described. The membrane was scanned and analysed as described for the gels above.

20
4 Results and Discussion

4.1 Fermentations
During the course of this study, eleven fermentations have been started. Out of these
fermentations, named fermentation 1–11, some have been more successful than others, but
even so, something has been learned from each of them. Many technical details that have been
found out about how the equipment should be configured in order to avoid problems, or to
increase the performance of the system, are described in section 3 Materials and Methods.
The fermentations have been carried out using two different strains, one without the HSA
gene, requiring addition of histidine for growth, and one with the HSA gene. The latter strain
was obtained from two different sources. As one of the aims of the project was to scale-up the
process, fermentation has been carried out at four different scales (0.4, 1.5, 15 and 100 litres).
A summary of the fermentations with respect to the strain used, the scale, the duration of the
experiment and the method of HSA analysis used, if any, is shown in Table 1. In the
following sections, the main features of all fermentations are described in short. Table 2
shows the lengths of the three phases for all fermentations and the optical density and cell wet
weight reached at the end of the phases.

Table 1. The fermentations performed in the study.

Fermentation Strain Scale Duration Albumin analysisa
number (days)
1 GS115 Alb secr UK 1.5 litres 2 -
2 - “- 1.5 litres 2 -
3 - “- 1.5 litres 1 -
4 - “- 1.5 litres 2 -
5 - “- 0.4 litres 4 -
6 - “- 0.4 litres 6 GF + SDS-PAGE
7 GS115 (his4) 15 litres 3 -
8 GS115 (his4) 15 litres 4 -
9 GS115 Alb secr UK 100 litres 8 GF + SDS-PAGE
10 - “- 0.4 litres 7 SDS-PAGE
11 GS115 Alb secr Hungary 0.4 litres 7 SDS-PAGE + WB
a
GF – gel filtration, SDS-PAGE – sodium dodecylsulphate polyacrylamide gel
electrophoresis, WB – Western Blotting

21
Table 2. Data of the lengths of the three fermentation phases and the cell concentration reached at the end of the
phases. Gl.B – glycerol batch phase, Gl.FB – glycerol fed-batch phase, MeOH FB – methanol fed-batch phase,
N.D. – not determined. The values for OD and wet weight given in parantheses are not measured at the exact
time of the end of the phase but a few hours before or after.
Ferm. Gl.B ODa wwb Gl.FB OD ww MeOH OD ww
number (h) (h) FB (h)
1 24 70 N.D. -c - - 21 140 N.D.
2 28 65 108 4 184 186 18.5 200 212
3 25.5 N.D. N.D. - - - - - -
4 34 70 80 11d 136 141 7.5 48? 148
5 34 (32) (42) 16.5e (192) (206) 46.5 N.D. 155
6 22 N.D. 117 4 N.D. N.D. 114 N.D. 320
7 23 56 105 4 108 172 42.75 107 186
8 21.5 118 111 4 150 190 68.5 N.D. 256
9 27.5 N.D. N.D. 5 132 168 154 N.D. 163
10 22.5 70 107 24f 450 396 128 N.D. 395
11 22.5 32 N.D. 25.5f 444 460 119.5 560 382
a
OD600
b
wet weight in g/l
c
In fermentation 1, the glycerol fed-batch phase was omitted.
d
The 50% glycerol solution was diluted three times and fed to the fermenter at a lower rate, but the amount of
glycerol fed was the same as was fed in 4 hours in fermentation 2.
e
All glycerol in the fed-batch phase was added at one occasion.
f
Much more glycerol than usual was fed to examine the possibility of obtaining a higher cell concentration.

4.1.1 Fermentations 1–4

As is shown in Table 1, the first 4 fermentations were performed in the 1.5 litre fermenter.
After the fourth fermentation, which revealed many technical problems (among other things,
the lid was worn out and therefore not tight), it was obvious that this fermenter was not
functional any more, and therefore the 0.4 litre fermenter was obtained to the laboratory. In
the first two fermentations, the methanol fed-batch phase failed due to technical problems, but
the glycerol phases worked as expected. The third fermentation was not successful due to a
broken pO2 electrode membrane, leading to that the fermentation liquid leaked out. In
fermentation 4, KOH was used instead of NH3 to raise the pH of the fermentation basal salts
(FBS) medium prior to inoculation. This made me aware of that the only source of nitrogen
for the yeast was the NH3, and that lack of nitrogen was the reason for the slow growth during
this fermentation (see Figure 6). This was proven by addition of (NH4)2SO4 to the culture,
which lead to a rapid decrease in the pO2, indicating an increase in the respiration. Since KOH
was used for pH regulation in the first four fermentations, the cells could grow during the first
two phases of fermentations 1 and 2, only because NH3 had been used to raise the pH of the
FBS before the fermentations were started. The major difference between fermentations 1 and
2 is that in fermentation 1, the glycerol fed-batch phase was omitted due to shortage of time.
Fermentation 4 was the first time that the methanol regulation was tried, and it was then
discovered that the methanol level detected by the sensor did not correctly represent the
methanol level of the fermentation liquid. This was due to droplets of concentrated methanol
hanging from the methanol feeding tube. In all subsequent fermentations in the other
fermenters, methanol was fed through a tube ending below the surface of the liquid, thereby
eliminating this problem.

22
 wet weight (g/l) 1000

100
ferm e n t a t io n 2
ferm e n t a t io n 4
10

1
0 10 20 30 40
tim e s i n c e i n o c u l a t i o n / h

Figure 6. A comparison between the growth rates in the glycerol batch phase of fermentations 2 and 4. The low
slope of the curve for fermentation 4 lead to the discovery that the only source of nitrogen is the NH3 used for
pH regulation. The high value in the beginning of this fermentation is probably due to large amounts of
precipitate in the medium. Note that the scale on the y-axis is logarithmic.

4.1.2 Fermentation 5
This was the first fermentation in the 0.4 litre fermenter, and also the first fermentation of the
strain GS115 (his4), leading to a need for addition of histidine to the medium. Because no
pump with low-enough flow rate was available, all glycerol of the fed-batch phase was added
at one single time. At the start of the methanol fed-batch phase, the fermenter was coupled to
a computer for registration of the fermentation parameters pH, temperature, pO2, stirrer speed,
foam level and methanol concentration in the program Geasy. During this fermentation,
problems with the foam control lead to addition of a large amount of PPG, and the high cell
density in combination with the high PPG concentration resulted in an inability of the stirrer
to keep the pO2 above the set-point (30%). Despite these problems, the fermentation was run
for 2 days on methanol. During this time, two different methanol set-point levels were tried,
first a lower level to allow the cells to adapt to methanol metabolism, and later a higher level.
The control seemed to function properly, with quite small deviations from the set-point value,
see Figure 7, which shows a screen-dump from the program Geasy.

4.1.3 Fermentation 6
In this fermentation, the HSA secreting strain was used in the 0.4 litre fermenter for the first
time. No problems with the foam control appeared, and by mixing pure oxygen with the inlet
air when necessary, the pO2 could be kept above 30% at all times. A new pump allowed
glycerol addition at a very low flow rate during the second phase. The methanol phase was
run for more than 4 days, and the regulation appeared to work well. Samples from this
fermentation were subjected to gel filtration and sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE), to determine if any HSA was produced, see HSA Analysis.

23
Figure 7. A screen-dump of the main control screen from fermentation 5. Only the methanol fed-batch phase is
shown. Methanol (red line) is regulated around two different set-points. The blue line shows the pO2 which could
not be held up. The stirrer speed is at its top value almost the whole time.

4.1.4 Fermentations 7 and 8

Fermentations 7 and 8 were the first attempts to scale up the process. They were run in a 15
litre fermenter with the strain GS115 (his4), requiring addition of histidine for growth. The
produced cell mass was meant to be sent to Hungary to immunise rabbits for production of
antibodies, for use in an enzyme-linked immunosorbent assay (ELISA). The ELISA was
meant for the detection of unwanted residual P.pastoris components in a purified recombinant
product. Only the cell mass from fermentation 8 was used for this purpose, due to reasons
which will be described below. In fermentations 7 and 8, the exhaust gas was heated by
passing steam through a condenser device, fitted on the lid of the fermenter, as well as
winding the heating tape around the silicone tubing, leading outlet air from the fermenter to
the methanol sensor, as described in section 3.2.2.5 Methanol Measurements and Regulation.
An important observation from fermentation 7 was that the heating of the outlet gas must be
switched on from the start of the fermentation. Otherwise, condense water accumulated in the
silicone tubing, and when the pump leading to the sensor was switched on, water was pumped
through the sensor, and in the process destroying it. A new sensor was used to replace the old
one, destroyed by the water, but since the new one was not calibrated, it was thought that this
resulted in an undesirably high methanol concentration in the culture. Later use revealed that
this new sensor was much more sensitive than the old one.

Another problem with fermentation 7 was the foam control. This lead to that large amounts of
antifoam (PPG) were added to the fermenter, resulting in that the cell mass could not be used.
The probable explanation for this problem was that there was a voltage between the fermenter
and the SARA control unit used for foam control, or that the foam electrode was too close to
the surface of the fermentation liquid, something which when the stirrer speed as well as the
volume increased, lead to contact between the electrode and the surface. Another possible
explanation could be that the foam level reading increased suddenly each time the methanol

24
pump was switched on or off by the methanol control device. The first of these possible
reasons was overcome by connecting the fermenter to the cabinet of the SARA control unit,
the second by raising the electrode, and the third by plugging the methanol device and the
control panel into different power points. In addition to these measures, the set point of the
foam level was also increased (from 15% to 75%) since it was noted that at foam formation,
the foam level reading increased to over 100% conductivity. After all these precautions were
made, there was still the possibility that a moist film was generated and thereby connecting
the two foam electrodes, resulting in the increase in conductivity and signalling foam, when,
in fact, there was none. The problem was solved before fermentation 8 in the same way as for
the small fermenter, namely by winding a heating tape around the membrane housing through
which the foam electrode was inserted.

Fermentation 8 was successful and the cell mass was sent to Hungary and used for
development of an ELISA as mentioned above. In order to release intracellular proteins ,
attempts were made to disrupt the cells with 4-mm diameter glass beads, using a bead beater
(Edmund Bühler, Tübingen, Germany), but the beads were too large, as examination by light
microscopy revealed many intact cells (data not shown). Furthermore, attempts were made to
concentrate the proteins in the supernatant using ultrafiltration (Ultrasette Tangential Flow
Device, MW cut-off 3K, Filtron Technology, Northborough, MA), but the filter cut-off was
too small and thus the filter was very quickly clogged with proteins(data not shown).

4.1.5 Fermentation 9
This was the only fermentation performed in the 100-litre fermenter. It was observed that a
too high aeration created too much foam, leading to addition of relatively large amounts of
PPG. During the methanol fed-batch phase, methanol could not be added below the surface of
the liquid, due to technical problems.

The methanol phase worked well for about three days, but then the methanol sensor signal
went up for an unknown reason and stayed at the elevated level. The culture was left for a day
with the methanol pump switched off, to see if the signal would go down again (when the
methanol was consumed), but it did so only to a small extent. When the outlet gas from the
fermenter to the sensor was stopped, the signal decreased slowly over a period of 16 hours,
but the signal increased when the outlet gas was started again, indicating that the problem
might lie before the sensor. Just to be sure, the sensor was changed for a new one on the sixth
day on methanol, but the signal did not decrease. On the seventh day of the methanol fed-
batch phase, the cells did not breathe any longer, and the fermentation was terminated. One
thing that could possibly explain why the methanol signal decreased slowly when the gas flow
to the sensor was stopped, is that the piston pump used in the other fermentations was
changed for a peristaltic pump in this fermentation, because the piston pump could not
withstand the pressure from the fermenter. This change meant that a piece of silicone tubing
was in the peristaltic pump, where it could not be heated by the heating tape. Therefore it
might have accumulated a larger amount of methanol in the walls than in other fermentations.
Supernatant from this fermentation was examined by gel filtration and SDS-PAGE, see 4.2
HSA Analysis.

4.1.6 Fermentations 10 and 11

These runs were made in the 0.4 litre fermenter. The HSA secreting strain was used, but in
fermentation 10, it was the strain obtained from England (the same as was used in earlier

25
fermentations) and in fermentation 11, the same strain was obtained from Hungary. The
reason for this was that we wanted to test whether the low production of HSA was due to any
mutation or if it was a true characteristic of the strain. In both these fermentations, a
prolonged glycerol fed-batch phase was tried, in order to increase the cell mass before
induction with methanol. Instead of feeding glycerol for 4 hours as described [5], glycerol
was added for 24 hours at a lower speed, resulting in that a total amount of 110 ml 50%
glycerol was fed, instead of only 14.5 ml as in fermentations 5 and 6. Cell growth as a
function of the fed amount of glycerol is shown in Figure 8. It shows that both OD600 and wet
weight in g/l reach values between 400 and 450 with the new feeding strategy as compared to
values around 200 for the old strategy. With such a high cell concentration, a rather high
proportion of pure oxygen had to be mixed with the inlet air to keep the pO2 above 30% as
desired. The oxygen needed could be reduced by providing the fermenter with an improved
stirrer (see Figure 3), used in fermentation 11. In fermentation 10, approximately half of the
culture volume was removed from the vessel after 1.5 days on methanol. After five days on
methanol, the yeast cells did not seem to bud any longer, and did not appear to consume any
methanol, as indicated by the fact that the methanol pump was never switched on by the
control unit. The reason for the ended consumption is not known.

500
OD600 f5
400 Wet weight f5

300 Wet weight f6

OD600 f10
200
Wet weight f10

100 OD600 f11

Wet weight f11
0
0 10 20 30 40 50
time since inoculation/h

Figure 8. Growth of the fermentation culture as measured by OD600 and wet weight (g/l), during the first two
phases of fermentations 5, 6, 10 and 11. It is clearly seen the large difference between the obtained cell
concentration for the two groups of fermentations. In fermentation 5 and 6, only 14.5 ml of 50% glycerol was
fed, whereas in fermentations 10 and 11, 110 ml was fed. In the figure legend, the fermentations are identified by
the letter f followed by the fermentation number. OD600 measurements are shown as dashed lines and wet
weights as solid lines.

Fermentation 11 was used to evaluate a new generation of methanol control units, see 3.2.2.5
Methanol Measurements and Regulation. The methanol fed-batch phase ran smoothly for
three days, after which the lab voltage was shut down for electrical work. During that time
(about 22 hours), no outlet gas was pumped to the methanol sensor, and thus methanol was
continuously pumped into the fermenter. In spite of this, the pO2 was still around 30% all the
time, indicating that the cells were breathing. When the problem was detected, and the outlet
gas pump was switched on again, the methanol signal immediately went up to its maximum.
As a test, the piston pump to the sensor was switched off again. The methanol sensor signal
was still high. Due to that the recorder and methanol unit were disarranged during a

26
reorganisation of the equipment (during the time that the pump was off for electrical work),
the settings were not reliable any more. During the time before the power cut, when the
methanol regulation was working, one problem was detected. Since the methanol pump was
set at a low speed and since it was turned off by the methanol control device for rather long
periods, the methanol was diffusing out through the silicone tubing that was used while air
diffused in instead. This lead to the formation of air bubbles in the tubing. Thus, when the
pump was switched on again, it often took some time before methanol reached the fermenter
and increased the concentration again. All this resulted in larger deviations from the set-point
value for methanol. The problem might have been caused by an aged methanol tubing. Later
experiments with new tubing have not shown the same behaviour. Figure 9 shows logged data
of methanol concentration, stirrer speed and pO2 from fermentation 11. These three
parameters covariate because of the poor methanol regulation, due to the formation of air
bubbles in the tubing for methanol feeding. The same large deviations from the set-point
values were not seen in fermentation 10.

1000 100
900 90
800 80
stirrer speed/rpm

700 70
600 60

pO2/%
500 50
400 40
300 30
200 20
100 10
0 0
2881
1 3595
715 4309
1429 5023
2143 5761
2857
Time since inoculation/min

methanol stirrer pO2

Figure 9. Logged data from fermentation 11. Only the methanol concentration (arbitrary units), stirrer speed and
pO2 are shown. Only part of the fermentation is shown, starting in the beginning of the methanol fed-batch
phase.

Samples from fermentations 10 and 11 were run on SDS-PAGE and samples from
fermentation 11 were subjected to Western blot analysis, see 4.2 HSA Analysis.

4.2 HSA Analysis

In fermentations 1–4 the HSA secreting strain was used, but these fermentations were not
successful due to different technical problems. In fermentations 5, 7 and 8 the strain GS115
(his4), which did not contain the HSA gene, was used. Thus HSA analysis was tried only on
samples from fermentations 6, 9, 10 and 11.

27
4.2.1 Gel Filtration
Samples from fermentations 6 and 9 were subjected to gel filtration in order to quantify the
amount of HSA secreted into the culture medium. In the sample chromatograms, no albumin
peaks were visible. Controls of pure HSA showed peaks at the correct eluted volume. This
would indicate either that there is no albumin secreted or that the amount is too low to be
detected by this method. Therefore, the more sensitive method of SDS-PAGE with silver
staining of the bands was tried.

4.2.2 SDS-PAGE
To test whether any HSA at all was secreted into the culture supernatant, SDS-PAGE
followed by silver staining was performed on one sample from each of the two fermentations
6 and 9, which had been examined by gel filtration with negative results. It turned out that
albumin was secreted from this strain but the production level was low. The sample from
fermentation 6, taken after ca 2.5 days on methanol, contained only about 5-10 mg per litre of
HSA and the sample from fermentation 9, taken after 3 days on methanol, contained ca 70
mg/l. Both these fermentations had been carried out with the same strain (GS115/MutS HSA
secreting), which was also used in fermentation 10. Samples from fermentation 10, taken after
a half day, one and a half day, four days and five and a half days on methanol were run on
SDS-PAGE, see Figure 10A. On each gel, a standard series of dilutions of pure HSA was also
run. In order to quantify the amount of HSA in the samples, the gels were scanned and
densitometry was used to evaluate the bands, both with respect to the absolute amount of
protein in the HSA band, and with respect to the relative amount of HSA as compared to the
total amount of protein in all the bands in a lane. This time the amount of HSA in the broth
seemed to increase during the whole course of the fermentation, reaching a highest level of ca
300 mg/l on the last day of the methanol fed-batch phase, which lasted for five days. The
reason for the higher level of HSA in the samples from fermentation 10 compared to
fermentation 6 and 9 might be the higher biomass produced during the longer glycerol fed-
batch phase.

To make certain that the low production level (at least 10-fold higher was hoped for) was not
due to any mutation, the strain was obtained again from another source, and used in
fermentation 11. Samples from four consecutive days of fermentation 11 (after one, two, three
and four days on methanol) were subjected to SDS-PAGE. Figure 10B shows a silver-stained
gel with samples from fermentation 11. Densitometry gives that the maximal amount of HSA
in the culture supernatant of this fermentation can be found in the sample taken after two days
on methanol, shown in lane 6. This amount is also approximately 300 mg/l and it lies in the
range of the produced amount of the same strain in fermentation 10. Thus, this production
level is probably characteristic for this strain grown under these conditions. It must be noted
here that the strain is not designed for high level production but rather as a demonstration of a
MutS protein-secreting strain. Although the production in fermentations 10 and 11 lies within
the same order of magnitude, a significant difference is that the amount of protein in the HSA
band in fermentation 11 reaches its maximum after only two days on methanol, but for
fermentation 10 the amount seems to increase during the whole methanol fed-batch phase,
resulting in the same amount of HSA in the full length form after more than five days as is
obtained after only two days in fermentation 11.

28
 A B
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

Figure 10. Silver stained SDS-PAGE gels. A. Fermentation 10. Lanes 1 contains a molecular weight standard,
lanes 2–4 contain pure HSA (a standard series of dilutions) and lanes 5–8 contain samples from fermentation 10
taken after 0.5, 1.5, 4 and 5.5 days on methanol. B. Fermentation 11. Lanes 1–4 contain pure HSA (a standard
series of dilutions) and lanes 5–8 contain samples from fermentation 11 taken after 1, 2, 3 and 4 days on
methanol.

It can be seen in Figure 10B (lanes 5–8) that besides the band with HSA in its full-length
form, other bands are prominent. This indicates the proteolytic degradation of the albumin
peptide chain into shorter fragments, a phenomenon also known from other P.pastoris strains
(Ferrarese et al. 1998, J-C. Janson, Personal communication). In order to verify the identity of
the other bands on the gel, Western blotting of the supernatant from fermentation 11 was
used. In the samples from fermentation 10, the amount of protein in other bands increases
with time. The relative amount of protein in other bands compared to the amount in the band
with HSA in its full length form is, however, not as large in the samples from fermentation 10
as in those from fermentation 11. It is not known why there is a difference between the results
from fermentations 10 and 11, but clearly the growth conditions were not identical. As
mentioned above (4.1.6 Fermentations 10 and 11), the methanol regulation did not keep the
methanol concentration at a constant level, especially during fermentation 11. This may have
affected the metabolism of the cells in some way, leading to the observed differences in the
amount of secreted HSA in different forms. Possibly, since the total amount of protein in each
lane in Figure 10B decreases towards the end of the methanol fed-batch phase, the secreted
protein was degraded and used as a substrate. In the samples from fermentation 10 on the
other hand, this phenomenon is not seen, but instead the total amount of secreted proteins
increased during the whole methanol fed-batch phase.

4.2.3 Western Blot

Western blotting of the product from fermentation 11 showed that all the bands that were
visible on the SDS-PAGE silver stained gel reacted with anti-HSA antibodies (data not
shown), indicating that they contained either the full-length form or proteolytic cleavage
products of HSA.

29
4.3 Different Ways to Increase the Yield and to Avoid Proteolysis
As was shown by the chromatography and electrophoresis experiments performed, the
obtained amount of HSA did not reach a satisfactory level. In this section I will review some
of the literature concerning possible changes to the production system, i.e. Pichia pastoris. If
the produced level of protein is not high enough with respect to the intended use and the
production economics, several different options exist to increase the yield. One thing is to
look at the strain. Autonomously replicating or integrating cloning vector, site of integration,
and type of methanol utilisation are only a few of the things to consider. As an integration of
the construct results in a higher expression cassette stability and in addition allows creation of
multi-copy strains and control of the site of integration (HIS4 or AOX loci), it is normally
preferred over an autonomous vector. When it comes to the site of integration, both loci have
been used successfully. However, if the HIS4 locus is used, there is a risk of gene conversion
between the chromosomal mutant copy of the his4 and the good HIS4 gene of the expression
cassette, resulting in a loss of the ability to produce histidine. As regards type of methanol
utilisation (Mut+ or MutS), the best choice differs from case to case. For intracellular
expression, MutS is generally preferred because of the lower levels of alcohol oxidase which
make the expressed protein easier to purify (Sreekrishna et al. 1997). This is due to that in
Mut+ strains, alcohol oxidase and the desired protein are expressed at about the same level,
but in MutS strains the level of alcohol oxidase (produced from the AOX2 locus) is about one
tenth compared to that of the wanted protein. For secretion of HSA, no significant difference
has been seen between Mut+ and MutS phenotypes (Barr et al. 1992) but for other proteins,
differences have been noticed. In a study by Brierly et al. (1993), the volumetric productivity
(secreted amount of bovine lysosyme/volume and time) of the induction phase could be
increased 6.5-fold by exchanging a MutS strain for a Mut+ strain.

The gene dosage, i.e. the number of copies of the recombinant gene introduced, is also highly
important for the production level. In general, multi-copy integrants produce the largest
amounts of recombinant protein (see for example Clare et al. 1991, where recombinant
tetanus toxin fragment C was expressed intracellularly to a level of 12 g/l in a strain
containing fourteen copies of the gene). In some rare instances, an increase in copy number
has a negative effect on the production level. It is also important to look at the design of the
expression cassette and the recombinant gene in particular. The 5’-UTR of the gene should be
as similar as possible to the native P.pastoris AOX1 leader sequence, and the translation start
codon AUG context should be relatively free of secondary structure. Furthermore, it has been
noticed that genes with high A+T content are not transcribed efficiently due to premature
termination (Sreekrishna et al. 1997). In redesigning the gene to contain 30–55% A+T, use of
P.pastoris preferred codons is recommended (Sreekrishna 1993). Also important on the gene
level is the secretion signal used, if any. Many proteins have been produced at high levels
intracellularly. The highest production obtained is probably that of hydroxynitrile lyase from
the tropical rubber tree Hevea brasiliensis, reaching a level of 22g/l. In this case, the protein
was found in the cytosol, and attempts to produce the protein extracellularly by applying
different leader peptides were not successful (Hasslacher et al. 1997). If possible, it is often
more convenient to have the product secreted because it significantly simplifies the
downstream processing. A few secretion signals have been tried. The most common is
probably the secretion signal (prepro-leader) of the α-mating factor from S.cerevisiae.
Increased secretion has been observed when using a hybrid prepro-leader consisting of the
P.pastoris acid phosphatase secretion signal fused to a synthetic pro sequence (Laroche et al.
1994). Examples of highly secreted proteins are dextranase from Penicillium minioluteum,
reaching a level of more than five grams of protein per litre of broth (Rodríguez-Jiménez et
al. 1997) and invertase from S.cerevisiae, which has been secreted at a level of 2.5 g/l

30
(Hagenson 1991). HSA has been reported to have been secreted at a level of 4 g/l (Cregg et
al. 1993).

The fermentation conditions can be optimised to obtain the maximal amount of functional
product. An important parameter is the length of the induction phase. In this study induction
has not been tried for more than ca 150h (in fermentation 9) and in several fermentations
around 120–130h (see Table 2). Hasslacher et al. (1997) found that the highest specific
activity of hydroxynitrile lyase was obtained after 200h of induction. In this context, there is
of course the question of product stability in the fermentation broth. As was shown by SDS-
PAGE and Western blotting, the secreted HSA was proteolytically degraded and the
proportion of degraded forms of HSA increased over time. In other studies, it has been found
that additions of yeast extract, peptone, Gibco histidine assay medium or casaminoacids
reduce the degradation of secreted proteins from P.pastoris (Barr et al. 1992). The reason is
probably that short peptides present in these additives bind to proteases in the medium and
thereby inhibit cleavage of the product. The reason for choosing a simple medium in this
study is that complex media are undefined, more expensive and introduce difficulties into the
down-stream processing of the product. One could also imagine that a simple sugar-free
medium decreases the risk of unwanted glycosylation. The use of protease deficient strains
has been reported to double the amount of produced ghilanten (a leech anticoagulant protein)
(Brankamp et al. 1995). When it comes to HSA, however, it appears that the addition of
peptone and yeast extract, even with a protease deficient strain, was essential for secretion
(Sreekrishna 1993).

The methanol feeding strategy can affect the production significantly. Different feeding
strategies have been examined by Minning et al. (1998) and by Rodríguez-Jiménez et al.
(1997). In these studies, strategies based on the pO2 of the culture were the most promising
and lead to the highest yield of recombinant protein, both with respect to the amount of
methanol fed and to the biomass formed. The results show that both a too low and a too high
methanol feed rate affect the production rate negatively, and that the feed rate should be
adjusted to the metabolic rate and to the current biomass concentration in the fermenter. This
is what the present study has tried to accomplish. As the cell mass increases and the cells
adapt to methanol metabolism, the feeding pump can be switched on more and more
frequently, and if the methanol concentration cannot be kept at the selected level, then the
feed pump rate can be increased manually. On the other hand, if the speed with which the
culture consumes methanol decreases, an intoxication of the cells, as would result with a
constant methanol feeding rate, does not occur, since the feeding pump can in such a case be
switched on more rarely.

Except for optimising the feeding strategy by controlling the methanol feed in a fed-batch
mode of fermentation (as in this study), two other possibilities can be tried: mixed glycerol-
methanol feed or continuous culture. Brierly et al. (1993) have been able to increase the
secretion of bovine lysosyme more than 4-fold by feeding a mixture of glycerol and methanol
in the proportions 2:1 (w:w), using a MutS host. They also tried continuous culture of a Mut+
strain. The total production of bovine lysosyme after 200 hours of continuous culture was
between 17 and 19 g, as compared to 3 g produced in the original MutS fed-batch culture of
125 hours.

Several other fermentation parameters can be of great importance for the yield of recombinant
protein. These include temperature, medium composition and pH. The use of a well defined
synthetic medium instead of a complex medium has in some cases caused a lower production

31
level (Minning et al. 1998). The recommended pH range is between 2.8 and 6.5. Secretion of
HSA has been positively affected by a high pH, but other proteins are proteolytically
stabilised by lowering the pH (Sreekrishna et al. 1997).

4.4 Conclusions
Since the first industrial process involving high cell density fermentation of the methylo-
trophic yeast Pichia pastoris for production of single cell protein (Wegner 1983),
considerable research has been done on the development of this system, in particular the
production of recombinant proteins. As a consequence of the advantages and properties of this
yeast, it has been and is investigated for production of several therapeutic proteins. HSA
secreted from P.pastoris has been shown to have the same primary and secondary structure
and binding properties as HSA from plasma. It has also after purification been tested in pre-
clinical studies and in phase I clinical trials, showing no toxic effects (Sumi et al. 1993,
Yokoyama and Ohmura 1995). Therefore, production of recombinant HSA from P.pastoris is
a feasible way to obtain large quantities for therapeutic use. The levels of HSA obtained in
this study (ca 300 mg of HSA per litre of culture supernatant) compared to the amount needed
for economical production (around 5-10 g/l) indicate that alterations of the strain, or the use of
another strain may be required. Nevertheless, the fermentation technology involving on-line
measurement and control of the methanol feed can be of great use for further optimisation and
production of recombinant proteins in general, and of HSA in particular.

4.5 Further Studies

A further step as regards the methanol regulation could be to couple the methanol sensor
device to a computer via an analogue/digital (A/D) converter. The computer could be supplied
with a program for control of the methanol feed using better control algorithms than the
simple on-off regulation of the pump used in this study. Also, to better control the level of
methanol to which the set-point is adjusted, the methanol in the culture medium should be
quantified by another method. For this purpose, a gas chromatograph has been acquired by
our laboratory and is now being installed and tested. Furthermore, the problems with the
sensitivity of the methanol sensor to temperature and humidity have led to initial trials with
alternative ways to convey the atmosphere in the fermenter to the gas sensor without using the
outlet gas stream. So far, the results are promising. To examine the possibility to increase the
proteolytic stability of the secreted HSA in the fermenter, the suggestion has been made by
J-C. Janson (Personal communication) to add a protease inhibitor to the fermentation, in a
form that does not affect the well-being of the cells. A serine protease has been identified in
the culture supernatant of P.pastoris, and its N-terminal amino acid sequence shows close
homology to that of S.cerevisiae carboxypeptidase Y (Ohi et al. 1996). If this protease is
responsible for the proteolysis of HSA, then a possible solution would be to add to the
fermenter a suitable protease inhibitor of serine proteases. Experiments to this end have
already been initiated. Furthermore, additional experiments to overcome the problems with
the precipitates have been started.

32
5 Acknowledgements
I would like to express my gratitude towards my supervisor Andras Ballagi for his
enthousiasm and guidance during the study. I also thank Jan Zabielski and Erika Karlsson for
help with practical things and for making the fermentation laboratory a nice place to work,
Bing-Ze Xu for help with the electrophoresis and Western blotting, Feng-Hui Fan for help
with the chromatography, Bo Ersson for practical modifications and repair of the equipment,
Jan-Christer Janson for providing chemicals, equipment and information, J. Kozma for
construction of the methanol devices, Paul Lowings (CVL, Weybridge, UK) and Ferenc
Felföldi (Institute of Enzymology, Budapest, Hungary) for providing the HSA secreting
strains. Emma Nyström, Tomas Sandberg, Åsa Rosengren and Johan Engström for interesting
discussions and Karin Caldwell, Ulla Widenberg, Anita Vitina and all other people at the
Center for Surface Biotechnology for creating a pleasant atmosphere. Finally, I thank my
girlfriend Maria for support and for critically reading the manuscript.

6 References

6.1 Authored Articles, Books and Patents

Andersson, L-O. 1976. “Transportproteiner; Serumalbumin; Biokemi”. In: Blombäck, B. and Hanson, L.Å. Eds.,
Plasmaproteiner, 53–66, Bohuslänningens AB, Uddevalla, Sweden

Barr, K.A., Hopkins, S.A. and Sreekrishna, K. 1992. “Protocol for efficient secretion of HSA developed from
Pichia pastoris”. Pharm Eng 12, 48–51

Brankamp, Robert G., Sreekrishna, Koti, Smith, Philip L., Blankenship, Dale T. and Cardin, Alan D. 1995.
“Expression of a synthetic gene encoding the anticoagulant-antimetastatic protein ghilanten by the
methylotrophic yeast Pichia pastoris”. Protein Expr Purif 6, 813–820

Brierly, R.A., Bussineau, C., Kosson, R., Melton, A. and Siegel, R.S. 1990. “Fermentation development of
recombinant Pichia pastoris expressing the heterologous gene: bovine lysozyme”. Ann NY Acad Sci 589, 350–
362

Brown, A. 1990. “Fed-batch and continuous culture”. In: McNeil, B. and Harvey, L.M. Eds., Fermentation – a
practical approach, 113–130, Oxford University Press, Oxford, UK

Cha, M-K. and Kim, I-H. 1996. “Glutathione-linked thiol peroxidase activity of human serum albumin: A
possible antioxidant role of serum albumin in blood plasma”. Biochem Biophys Res Comm 222, 619–625

Claes, G. 1976. “Transportproteiner; Serumalbumin; Extrakorporeal organförvaring med kontinuerlig

albuminperfusion”. In: Blombäck, B. and Hanson, L.Å. Eds., Plasmaproteiner, 83–84, Bohuslänningens AB,
Uddevalla, Sweden

Clare, J.J., Rayment, F.B., Ballentine, S.P., Sreekrishna, K. and Romanos, M.A. 1991. “High-level expression of
tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene”.
Biotechnology 9, 455–460

Cregg, J.M., Vedvick, T.S. and Raschke, W.C. 1993. “Recent advances in the expression of foreign genes in
Pichia pastoris”. Biotechnology 11, 905–910

Czarnetzki, B.M. and Schulz, W. 1980. “Role of purified serum components in polymorphonuclear leukocyte
chemotaxis”. Int Archs Allergy Appl Immun 61, 424–430

Doolittle, R.F. 1984. “Evolution of the vertebrate plasma proteins”. In: Putnam, F.W. Ed., The Plasma Proteins
– Structure, Function, and Genetic Control, Volume IV. 2nd ed., 317–360, Academic Press, Orlando, Florida

33
Ferrarese, L., Trainotti, L., Gattolin, S. and Casadoro, G. 1998. “Secretion, purification and activity of two
recombinant pepper endo-β-1,4-glucanases expressed in the yeast Pichia pastoris”. FEBS lett 422, 23–26

Fleer, R., Yeh, P., Amellal, N., Maury, I., Fournier, A., Bacchetta, F., Baduel, P., Jung, G., L’Hôte, H., Becquart,
J., Fukuhara, H. and Mayaux, J.F. 1991. “Stable multicopy vectors for high-level secretion of recombinant
human serum albumin by Kluyveromyces yeasts”. Biotechnology 9, 968–975

Hagenson, M.J. Skogen. 1991. “Production of recombinant proteins in the methylotrophic yeast Pichia
pastoris”. Bioprocess Technol 12, 193–212

Hasslacher, M., Schall, M., Hayn, M., Bona, R., Rumbold,K., Lückl, J., Griengl, H., Kohlwein, S.D. and
Schwab, H. 1997. “High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree
Hevea brasiliensis in microbial hosts”. Protein Expr Purif 11, 61–71

Hollenberg, C. P. and Gellissen, G. 1997. “Production of recombinant proteins by methylotrophic yeasts”. Curr
Opin Biotechnol 8, 554–560

Ikegaya, K., Hirose, M., Ohmura, T. and Nokihara, K. 1997. “Complete determination of disulfide forms of
purified recombinant human serum albumin, secreted by the yeast Pichia pastoris”. Anal Chem 69, 1986–1991

Kjeldsen, T., Pettersson, A.F. and Hach, M. 1999. “Secretory expression and characterization of insulin in Pichia
pastoris”. Biotechnol Appl Biochem 29 (Pt 1), 79–86

Laroche, Yves, Storme, Véronique, Meutter, Jan De, Messens, Joris and Lauwereys, Marc. 1994. “High-level
secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic
yeast, Pichia pastoris”. Biotechnology 12, 1119–1124

Laursen, I., Briand, P. and Lykkesfeldt, A.E. 1990. “Serum albumin as a modulator on growth of the human
breast cancer cell line, MCF-7”. Anticancer Res 10 (2A), 343–351

Lawn, R.M., Adelman, J., Bock, S.C., Franke, A.E., Houck, C.M., Najarian, R.C., Seeburg, P.H. and Wion, K.L.
1981. “The sequence of human serum albumin cDNA and its expression in E.coli”. Nucl Acids Res 9 (22),
6103–6114

Minning, S., Schmidt-Dannert, C. and Schmid, R.D. 1998. “Functional expression of Rhizopus oryzae lipase in
Pichia pastoris: high-level production and some properties”. J Biotechnol 66, 147–156

Ohi, H., Miura, M., Hiramatsu, R. and Ohmura, T. 1994. “The positive and negative cis-acting elements for
methanol regulation in the Pichia pastoris AOX2 gene”. Mol Gen Genet 243 (5), 489–499

Ohi, H., Ohtani, W., Okazaki, N., Furuhata, N. and Ohmura, T. 1996. “Cloning and characterization of the
Pichia pastoris PRC1 gene encoding carboxypeptidase Y”. Yeast 12, 31–40

Ohtani, W., Ohda, T., Sumi, A., Kobayashi, K. and Ohmura, T. 1998. “Analysis of Pichia pastoris components
in recombinant human serum albumin by immunological assays and by HPLC with pulsed amperometric
detection”. Anal Chem 70, 425–429

Putnam, F.W. 1984. “Alpha, Beta, Gamma, Omega– The structure of the plasma proteins”. In: Putnam, F.W.
Ed., The Plasma Proteins – Structure, Function, and Genetic Control, Volume IV. 2nd ed., 45–166, Academic
Press, Orlando, Florida

Rodríguez-Jiménez, E., Sánchez, K., Roca, H. and Delgado, J.M. 1997. “Different methanol feeding strategies to
recombinant Pichia pastoris cultures producing high level of dextranase”. Biotechnol. Techniques 11 (7), 461–
466

Saunders, C.W., Schmidt, B.J., Mallonee, R.L. and Guyer, M.S. 1987. “Secretion of human serum albumin from
Bacillus subtilis”. J Bacteriol 169 (7), 2917–2925

34
Siegel. R.S. and Brierley, R.A. 1989. “Methylotrophic yeast Pichia pastoris produced in high-cell-density
fermentations with high cell yields as vehicle for recombinant protein production”. Biotechnol Bioeng 34, 403–
404

Sijmons, P.C., Dekker, B.M.M., Schrammeijer, B., Verwoerd, T.C., van den Elzen, P.J.M. and Hoekema, A.
1990. “Production of correctly processed human serum albumin in transgenic plants”. Biotechnology 8, 217–221

Sinclair, C.G. and Cantero, D. 1990. “Fermentation modelling”. In: McNeil, B. and Harvey, L.M. Eds.,
Fermentation – a practical approach, 65–112, Oxford University Press, Oxford, UK

Sleep, D., Belfield, G.P. and Goodey, A.R. 1990. “The secretion of human serum albumin from the yeast
Saccharomyces cerevisiae using five different leader sequences”. Biotechnology 8, 42–46

Sreekrishna, K. 1993. “Strategies for optimizing protein expression and secretion in the methylotrophic yeast
Pichia pastoris”. In: Baltz, R.H., Hegeman, G.D. and Skatrud, P.L. Eds., Industrial Microorganisms: Basic and
Applied Molecular Genetics. American Society for Microbiology, Washington, DC, 119–126

Sreekrishna, K., Potenz, Rica H.B., Cruze, John, A., McCombie, William R., Parker, Kathryn A., Nelles, Lynn,
Mazzaferro, Paul A., Holden, Kathy A., Harrison, Roger G., Wood, Phyllis D., Phelps, Doris A., Hubbard, Cindi
E. and Fuke, Motohiro. 1988. “High level expression of heterologous proteins in methylotrophic yeast Pichia
pastoris”. J Basic Microbiol 28, 265–278

Sreekrishna, K., Nelles, L., Potenz, R., Cruze, J., Mazzaferro, P., Fish, W., Fuke, M., Holden, K., Phelps, D.,
Wood, P. and Parker, K. 1989. “High-level expression, purification, and characterization of recombinant human
tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris”. Biochemistry 28, 4117–4125

Sreekrishna, K., Barr, K.A., Hoard, S.A., Prevatt, W.D., Torregrossa, R.E., Livingston, R.E., Cruze, J.A. and
Wegner, G.H. 1990. “Expression of human serum albumin in Pichia pastoris”. In: Oliver, S.G. and Wickner, R.
Eds.,. 15th Int. Congr. Yeast Genetics and Molecular Biology, 1990, The Hague, The Netherlands. Yeast 6
(Special Issue). Wiley. New York, NY, page S447

Sreekrishna, K., Brankamp, R.G., Kropp, K.E., Blankenship, D.T., Tsay, J-T., Smith, P.L., Wierschke, J.D.,
Subramaniam, A. and Birkenberger, L.A. 1997. “Strategies for optimal synthesis and secretion of heterologous
proteins in the methylotrophic yeast Pichia pastoris”. Gene 190, 55–62

Sumi, A., Ohtani, W., Kobayashi, K., Ohmura, T., Yokoyama, K., Nishida, M. and Suyama, T. 1993.
“Purification and physicochemical properties of recombinant human serum albumin”. In: Rivat, C. and Stoltz, J.-
F. Eds., Colloque INSERM 227 (Biotechnology of Blood Proteins), John Libbey Eurotext Ltd., 293–298

Wegner, E.H. 1990. “Emerging applications of the methylotrophic yeasts”. FEMS Microbiol Rev 87, 279–284

Wegner, E.H. 1983. “Biochemical conversions by yeast fermentation at high cell densities”. U.S. Patent no
4,414,329

Yokoyama, K. and Ohmura, T. 1995. “Production of human serum albumin by biotechnology”. Jpn J Apheresis
14, 19–20

6.2 Other Referenced Documents and Web Pages

1. Biosystems 1998-07-15. How oxygen, electrochemical toxic, and metal oxide semiconductor sensors work.
http://www.biosystems.com/appnotes/howoxyge.htm

2. Figaro gas sensors, Figaro Engineering, Osaka, Japan

3. TGS 822 Technical reference manual, Figaro Engineering, Osaka, Japan

4. Swiss-Prot. Q13140. http://expasy.hcuge.ch/cgi-bin/get-sprot-entry?Q13140. Accessed on 16 July 1998

5. Pichia fermentation process guidelines , Invitrogen, San Diego, CA

35
6. Pichia media recipes, Invitrogen, San Diego, CA

7. PhastSystem Technical reference manual, Pharmacia Biotech, Uppsala, Sweden

36