Humanição glicosilação

8/11/2019 Humanição glicosilação

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Bösch Peter | 0240421 April 2005 | 1

Index

1. Introduction

2. Fundamentals

From DNA to protein

Secretory pathway

3. Glycosylation

Pichia pastoris

Glycosylation of therapeutic proteins

Differences between yeast and human N-glycosylation pathways

Humanizing glycosylation pathways in yeasts

A) Early approaches

B) Rebuilding the human glycosylation pathway

C) Galactosylation

D) Sialic acid transfer – the final step

Problems

4. Advantages and disadvantages of mammalian, yeast, fungal,

plant, and insect cells

Basis of evaluation

Mammalian cells

Yeast cellsFungal cells

Plant cells

Insect cells

5. Summary and perspectives

6. References



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Humanisation of posttranslational

modifications in yeasts

1. Introduction

Many proteins used for therapeutic reasons in medicine are not only a sequence of

amino acids determined by a specific gene, but require also trimming, editing of

amino acids, addition of sugars and many others. These alterations after the initial

translation of the proteins are called posttranslational modifications. Sometimes they

are crucial for the desired effect or enhance the effectivity of the substance ten fold or

render it useless.

Until now products for therapeutic use where expressed in mammalian cells, in

particular Chinese hamster ovary (CHO) cells. They can do all the necessary

alterations a human cell would do to produce a fully functional metabolite or enzyme.

But mammalian cells have a lot of drawbacks, so alternatives were searched for and

found in bacteria, especially Escherichia coli, yeasts like Saccharomyces cerevisiae

and Pichia pastoris, fungi, plants and insect cells. The most significant alteration

seems to be glycosylation, especially N-glycosylation.

Humanisation in this context means modification of a species; adaptation of skills not

to perform production of metabolites required in their natural habitat, but of

substances equivalent to those expressed in human cells including their

posttranslational modifications.

This text is focused on yeasts and their ability to glycosylate the proteins the human

way already or “learn” these processing steps and most important glycosylation by

the use of recombinant technology and selection so that they can be used for

production of substances for medical use.



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2. Fundamentals

The description given below is based on yeast, a simple eukaryote.

Eukaryotes in contrast to prokaryotes have got compartments enclosed by a bilayer

membrane made of phospholipids.

From DNA to protein

To construct any protein, the cell requires a plan which contains all the information to

build, modify and deliver it to the desired place. This plan is called gene, which is

embedded in a double stranded string along with other genes for other proteins.

Altogether they form the genome of a cell which is in eukaryotes surrounded by a

membrane to form the nucleus. Even in a multicellular organism the genome in

different cells is always exactly the same.

These genes are made of four nucleotides organised in a double helix where the

adenine nucleotide always pairs with the thymine nucleotide and the cytosine

nucleotide always pair with the guanine nucleotide. These sequences of nucleotides

which encode a gene are copied into mRNA, where the thymine nucleotides are

replaced by uracil nucleotides, capped, polyadenylated and spliced. This process is

called transcription. Transcription is highly regulated because of its direct

responsibility for the concentration of a certain protein. Several types of cancer occur

partly because one or more regulation mechanisms are knocked out.

The reason for copying the DNA to mRNA is simply to protect the genome and when

multiple copies are made a higher level of protein is achieved in a shorter period of

time.

After the transcription the mRNA is transferred from the nucleus to the cytosol.

In the cytosol, the medium that surrounds all the inner compartments of the cell, a

machinery called ribosome binds to the mRNA. The ribosome translates the plan

encoded by the mRNA strand into a sequence of 20 different amino acids which are

the units a protein is made of. After the complete sequence is read, the protein is

released and the mRNA is degraded.



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Depending on a signal sequence at the beginning of the mRNA the fate of a protein

is decided. It can either be released into the cytosol, peroxisomes, plastids,

endoplasmic reticulum (ER) or into the mitochondria. If the protein is expressed in the

cytosol it can stay there or it can be transferred into the nucleus via gated transport.

The translation/transfer of the protein into the ER is the starting point of the secretory

pathway.

Secretory pathway

The secretory pathway is mediated by vesicular transport, where membrane-

enclosed transport intermediates – which may be small, spherical transport vesicles

or larger, irregularly shaped organelle fragments – ferry proteins from one

compartment to another. The start point of this sequence of compartments is the ER,

followed by the Golgi, where it can either go to the late endosome and in progression

to the lysosomes where most of the substances are broken down or it buds from the

Golgi as a secretory vesicle targeted to the membrane of the cell with either

membrane proteins and other parts of the membrane or metabolites that are secreted

by the cell.

As mentioned before, a protein is expressed into the ER when it encodes a certain

signal sequence. This signal sequence is bound by a signal recognition protein which

attaches to a receptor in the ER membrane then translocation begins. When

translation and translocation are finished, the signal peptide is cleaved from the

protein. Most proteins synthesized in the rough ER are glycosylated by the addition of

an N-linked oligosaccharide. The sequence of the amino acids and the addition of the

oligosaccharides force the protein to fold into its native form, oligomerize and form

disulfide bonds. If not folded or oligomerized correctly, they are exported to the

cytosol, where they are deglycosylated, ubiquitylated, and degraded in proteasomes

[1].

The correctly folded and assembled proteins bud from the ER and reach the cis-

Golgi.

The Golgi finishes the glycosylation that has started in the ER by cleavage andaddition of sugars, phosphorylaton of oligosaccharides on lysosomal proteins and



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sulfation of tyrosines and carbohydrates. The proteins that leave the trans-Golgi are

sorted and transported to the lysosome, the plasma membrane or the secretory

vesicle.

The next chapter introduces the most important yeasts used for glycosylation

engineering so far and covers the glycosylation of proteins in more detail, the

necessary processing steps in yeast and in humans, their importance in

pharmaceutical products and the problems that occur by transforming the yeast

based glycosylation pathway into a humanlike pathway.



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3. Glycosylation

Pichia pastoris

The methylotrophic yeast Pichia pastoris is now one of the standard tools used in

molecular biology for the generation of recombinant protein. P. pastoris has

demonstrated its most powerful success as a large scale (fermentation) recombinant

protein production tool. What began more than 20 years ago as a program to convert

abundand methanol to a protein source for animal feed in the form of single cell

proteins has been developed into what are today two important biological tools: a

model eukaryote used in cell biology research and a recombinant protein production

system. In the controlled environment of a fermentor it is possible to grow the

organism to high cell densities (>100g/l dry cell weight), where the product in the

medium, the secreted proteins is roughly proportional to cell mass. Significant

advances in the development of new strains and vectors, improved techniques, and

the commercial availability of these tools coupled with a better understanding of the

biology of Pichia species have led to this microbe’s value and power in commercial

and research labs alike [2].

The Pichia pastoris system for expression of heterologous recombinant proteins is

being used increasingly because of the large yields of properly folded – processing of

signal sequences (both pre- and prepro-type), disulfide bridge formation - proteins

that result and the ease of scaling up pilot protocols into large-biomass fermentors.

Another advantage of this system centres on the type of glycosylation that result,

generally yielding protein-bound oligosaccharides of O- and N-linked type that are of

much shorter chain length than found in Saccharomyces cerevisiae [3].

With Pichia pastoris as expression system, yields of up to 15g/l (gelatin production)

were achieved, where production outcomes of >1g/l are termed as high protein titers.

For that reasons the text is focused on Pichia pastoris where major breakthroughs

have been reported in the last years and which is the best characterized system to do

humanization of yeast by the time of writing.



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Glycosylation of therapeutic proteins

As mentioned earlier, several hundred proteins have been expressed recombinanly

in the last couple of years in yeast. By now, the host system wasn’t able to do some

of the most critical modifications so a lot of highly interesting proteins such as some

antibodies, EPO, Serine protease inhibitor [4], hepatitis B [5] vaccine, Antithrombin III

[6] and many more couldn’t be fully considered for production in yeast systems so far.

This is mostly, because yeast does N-glycosylation of the high-mannose type or also

called hypermannosylation with up to 40 mannose residues. Human cells do not

hypermannosylate but use several additional types of sugars to synthesize an N-

glycan of the complex type. So if a human glycoprotein is expressed in yeast, the

sequence of the amino acids will be right, even the location of the glycosylation will

be right, but the glycan isn’t of the complex type but of the high-mannose type. The

best case scenario will lead to a fully functional glycoprotein which is not recognized

by the immune system of the patient it is applied on to. Most of the time the high-

mannose part is recognized by mannose receptors present on macrophages and

endothelial cells. The substance will show an extremely short half life and may not be

able to do any effect in its target tissue. If the glycosylation is skipped altogether the

protein will misfold (structural importance of glycosylation) and so is going to be

biologically inactive as well as cleared from circulation. Therefore no glycosylation at

all or the wrong type of glycosylation is not an option.

By now, the way to go was mammalian cells, especially Chinese hamster ovary

(CHO) cells as expression system. Although the glycosylation doesn’t match a

hundred percent, only minor differences have been found which can be eliminated by

modification to achieve maximum efficacy [7].But there are certain drawbacks with mammalian cell cultures. They grow slowly and

are very demanding. Even slight changes in system parameters can slash the

production or lead to other metabolites which require an additional step in down

stream processing after the fermentation. Mammalian cell cultures do not secrete

homologous glycoproteins but a heterogeneous, mixture of several glycoforms. Every

single glycoform has got its own kinetics and for approval after clinical phases these

proportions have to stay the same. The scale up from small cell cultures to bigfermentors is highly critical if sometimes simply not possible.



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To overcome these drawbacks one may attempt to further improve the mammalian

system, which is already on its limit or take another way.

This means, to change the production system radically and search for other options.

This will be discussed in Chapter 4, where the advantages and problems of other

systems are discussed in detail.

In the meantime the focus is on yeast as production system, the difference to

thehuman glycosylation pathway and the way it can be adapted.

Differences between yeast and human N-glycosylation pathways

The starting point either for human or yeast glycosylation is always the endoplasmic

reticulum. There, a preassembled core oligosaccharide (Glc3Man9GlcNAc2) bound

to the membrane component dolichol is transferred onto the nascent polypeptide

which has been imported into the ER because of its signal sequence (see Chapter 2:

the secretory pathway). This step is catalyzed by an enzyme complex named

oligosaccharyltransferase and has been conserved through all evolutionary steps

between yeast cells and a human cell. The transfer depends on the recognition of an

amino acid triplet which sequence is Asn-X-Ser/Thr (where X is any amino acid other

than proline). The core oligosaccharide is attached to the asparagine residue in this

sequence.

After these initial steps, the core oligosaccharide has to undergo several enzymatic

modification steps. First two enzymes called glucosidase I and glucosidase II cut

away the glucose sugars at the end of the branches which initiates a process called

glycan-mediated chaperoning. The last sugar that is removed in the ER is a

mannose, trimmed by a α-1,2-mannosidase. The glycoprotein leaving the ER has

always the same structure (Man8GlcNAc2). Incorrectly folded proteins are

reglucosylated transported into the cytosol for degradation. Therefore this process is

important for quality control, otherwise misfolded proteins might be secreted.

The resulting Man8GlcNAc2-containing glycoprotein is then transported to the Golgi

apparatus where the N-glycan takes different routes in yeast and humans. In

mammals the next step is cleavage of 3 mannose sugars by a α-1,2-mannosidase

which results in Man5GlcNAc2, the substrate for N-acetylglucosaminyltransferase I,



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which adds a single N-acetylglucosamine (GlcNAc) sugar onto the terminal of the

1,3-mannose.

Figure1 | Major N-glycosylation pathways in humans and yeast. Representative pathway

in humans (left) provides a template for humanizing N-glycosylation pathways in yeast (right).

ER, endoplasmic reticulum; GalT, galactosyltransferase; GlcNAc, N-acetylglucsoamine;

GnT I&II, N-acetylglucosaminyltransferase I & II; Man, mannose; Mns II, mannosidase II;

MnTs, mannosyltransferase; NANA, N-acetylneuraminic acid; ST, sialyltransferase. [8]

Mannosidase II removes the last two mannose sugar terminals which results in

GlcNAcMan3GlcNAc2. N-acetylglucosaminyl transferase II adds one GlcNAc sugar

and galactosyltransferase adds two galactose sugars. The final processing step is

catalysed by sialyltransferase where an N-acetylneuraminic acid (NANA) is

transferred to every terminal of the two branches. The product of the human

glycosylation pathway after ER and Golgi is NANA2Gal2GlcNAc2Man3GlcNAc2 also

known as complex type. In addition, there are other glycosyltransferases such as

GalNAc transferases, GlcNAc phosphotransferases and fucosyltransferases whichallow the spectrum of glycosylation to be much wider.



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Yeast alters the structure of the basic glycan exported from the ER with

mannosyltransferases which add mannose sugars as well as mannosylphosphate

transferases which leads to a hypermannosylation of the glycoprotein with varying

extents depending on the organism which gives a range of high-mannose type

oligosaccharides of Man9…100GlcNAc2.

In the end, the oligosaccharides are secreted either constitutively or regulated.

Constitutive secretion is done all the time and in balance with endocytosis. Regulated

secretion happens when a signal molecule triggers the release of the metabolite, for

example the release of the neurotransmitter acetylcholine.

Pichia pastoris’ glycan structures are typically smaller then those of the yeast

Saccharomyces cerevisiae and it has got a secretory pathway with distinct Golgi

stacks similar to those found in mammals [8].

Humanizing glycosylation pathways in yeasts

In the previous section the differences of the glycosylation pathways between yeast

and human cells were described. This section will cover all the steps required to

create a yeast cell enabled of doing complex N-glycosylation.

A) Early approaches

After the glycosylation pathways of both species were understood, or at least partially

unravelled strategies for achieving the transformation of a yeast where devised. The

most promising was knocking out the complete pathway in yeast that doesn’t match

the human one and substitute it with human enzymes. Because of the popularity and

well understood pathways in Saccharomyces cerevisiae early approaches targetedthat organism, although it has a much higher degree of mannosylation then Pichia

pastoris. The idea was to eliminate the α-1,6-mannosyltransferase Och1p which is

the core point of the outer chain elongation [8]. Jigami et al were the first who

attempted a knockout of both, the Och1 gene which encodes α-1,6-

mannosyltransferase and Mnn1 which encodes α-1,3-mannosyltransferase resulting

in mostly Man8GlcNAc2 type oligosaccharides [9]. Man8GlcNAc2 is the starting point

of the human glycosylation pathway. Chiba et al figured out in 1998 how to removethree mannose sugars after having the Man8GlcNAc2 substrate transported into the



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Golgi. They took a α-1,2-mannosidase from Aspergillus saitoi and placed it in the ER

of Saccharomyces cerevisiae. This led to a yield of 27% Man5GlcNAc2 the rest was

higher mannosylated [10]. By this time most of the attempts to humanize a yeast cell

failed because either a strain was chosen which doesn’t provide an appropriate

substrate or the elimination of the original N-glycosylation pathway in a way that it

doesn’t compete with the newly engineered on was difficult.

B) Rebuilding the human secretory pathway

Building a glycan is a step by step process that requires processing in a distinct

order. For example mannosidase II can only trim terminal α-1,6-mannose and α-1,3-

mannose if a terminal GlcNAc is present on the α-1,3 arm. The enzymes for N-

glycosylation have therefore to be arranged in a specific order along the ER / Golgi

pathway to be able to glycosylate efficiently and correctly.

So after overcoming the first obstacle by eliminating the old pathway and providing

the substrate for human glycosylation which is Man8GlcNAc2 the next milestone was

to arrange all the different mannosidases and glycosyltransferases along the way to a

glycan of the complex type in the right order on the right place.

The sequence of compartments a glycoprotein has to undergo is ER to cis-, medial –

and trans-Golgi compartment from where the product is transported to the membrane

where it is secreted. Most

glycosyltransferases and mannosidases

such as α-1,2-mannosidase,

mannosidase II and GlcNAcT I are

anchored to the membrane througha type II transmembrane anchor with an

N-terminal transmembrane domain.

The short N-terminus in the cytosol

and the nearby transmembrane region

encode the targeting information.

Figure2 | Type II membrane prot eins. The anchoring mechanism formost glycosyltransferases is through a type II transmembrane anchor,by which the C-terminal catalytic domain is anchored to the membrane

through an N-terminal transmembrane domain. [8]



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This finding was taken advantage of by the laboratory of Wildt and Gerngross [11].

Because it is not reliably predictable which sequence encodes targeting information

for a certain Golgi subcompartment and because the same sequence will lead to

different location of the enzyme in diverse species two libraries were built, one with

N-terminal fragments of ER and Golgi enzymes of Saccharomyces cerevisiae and

one with catalytic domains of several α-1,2-Mannosidases from Homo sapiens, Mus

musculus, Aspergillus nidulans, Caenorhabditis elegans, Drosophila melanogaster

and Penicillium citrinium, but without N-terminus.

Those two libraries were combined, every yeast N-terminus with each catalytical

domain of different species. That resulted in 608 so called chimeric fusion proteins.

As host was chosen a Pichia pastoris strain that lacks α-1,6-mannosyltransferase

activity but is able to secrete a hexahistidine-tagged fragment of human plasminogen

as a reporter protein. Only few strains were able to trim Man8GlcNAc2 to

Man5GlcNAc2.

Next step was the addition of an N-acetylglucosamine by N-acetylglucosaminyl

transferase I. The obstacles to overcome this problem were the requirement for

Man5GlcNAc2 which is the substrate, then the right localization of the enzyme and

the availability of UDP-GlcNAc in situ. Again a set of libraries was made with additionof a UDP-GlcNAc transporter from K. lactis for efficient UDP-GlcNAc transport. That

experiment yielded strains that where able to produce GlcNAcMan5GlcNAc2.

The GlcNAcMan5GlcNAc2 complex is substrate for the next enzyme, the

mannosidase II, which removes one terminal α-1,3- and one terminal α1,6-mannose.

That yields GlcNAcMan3GlcNAc2. The N-acetylglucosamine added in the step

before is essential; otherwise the reaction wouldn’t take place. This underlines the

importance of an approach that is similar to the order of the human glycosylationpathway.

Also N-acetylglucosaminyl transferase II was introduced into P. pastoris. This

enzyme extends the branch without N-acetylglucosamine so that the three mannose

sugar core is extended by an N-acetylglucosamine on each branch that is

GlcNAc2Man3GlcNAc2 [8].



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Figure3 | A working mod el for the cel lu lar dist ribution of glycosyl transferases

throughout the secretion pathway. Specific glycosyltransferases and glycosidases line the

luminal surface of the endoplasmic reticulum (ER) and Golgi, allowing the sequential processing

of glycoproteins as they are shuttled through the secretory pathway. GalT, galactosyltransferase;

GnT, N-acetylglucosaminyltransferase; ST, sialyltransferase; TGN, trans-Golgi network. [8]

C) Galactosylation

The previous step provided a paucimannose core (Man3GlcNAc2) plus an N-

acetylglucosamine on every branch. A galactosyl transferase should add one

galactose sugar onto every branch.

This part was rather tricky because of problems other groups also encountered

working with Saccharomyces cerevisiae [12].

The group around Wildt and Gerngross reported two approaches where only one is

described here. By expressing only galactosyl transferase the UDP-galactose would

be missing. So in addition, a UDP-galactose-4-epimerase was needed, that would

convert UDP-glucose from the cytosol into UDP-galactose. This enzyme was taken

out of S. pombe. Yet the UDP-galactose was in the cytosol, and the glycan and the



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galactosyl transferase are in the Golgi. From D. melanogaster a UDP-galactose

transporter was co-expressed in the Pichia pastoris strain.

In the Golgi all 4 components meet. The substrate (the GlcNAc2Man3GlcNAc2

synthesized earlier), the UDP-galactose made by UDP-galactose-4-epimerase out of

UDP-glucose present in the cytosol and transported from there to the Golgi by the

UDP-galactose transporter and the galactosyl transferase form a complex which

product is a Gal2GlcNAc2Man3GlcNAc2 biantennary glycan.

D) Sialic acid transfer – the final step

The last and probably most critical step is the transfer of sialic acid to each of the

antennarey of our glycoprotein. The method is the same as with earlier enzymes.

First a library with catalytic domains and a library with several N-terminal fragments

for the location are combined and screened. Several attempts for introducing

sialyltransferase have been successfull.

Second a pool of CMP-sialic acid has to be available. That is a major problem,

because there is no sialic acid available in yeast. Therefore a pathway to produce

CMP-sialic acid and a CMP-sialic acid transporter have to be introduced to the

system. Already there are some preliminary data available though not published yet[8]. Therefore it is difficult to judge the over all outcome and yield.

Problems

Although the first major breakthrough has been made by Wildt et al by reconstructing

the N-glycosylation pathway of humans in the yeast Pichia pastoris, there is a long

way to go. A problem is that also additional glycosyltransferases such as GalNAc transferases,

GlcNAc phosphotransferases and fucosyltransferases modify the final product.

So each glycoprotein has to be expressed within a specialized strain that can do the

specific alteration.

Further is not known, how effective this system works. James Cregg talks of a 50 to

75% probability of expressing any protein of interest in Pichia pastoris at a

reasonable level after having succeeded in generating an initial breakthrough suchas Gerngross et al [7]. This is concluded because the system then can be optimized



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by a couple of well known parameters which promise to yield more product. As

mentioned earlier up to 15g/l have been achieved so far, but a yield of >1g/l is

already excellent for an eukaryotic expression system [2].

Yeasts are well known to be robust and easy to scale up. But after having made so

many changes in the secretory pathway through recombinant protein technology it

might have lost a lot of it previous toughness. That is because the membrane is also

modified in this pathway and many of its proteins are glycosylated. But that has to be

proven yet.



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4. Advantages and disadvantages of mammalian,

yeast, fungal, plant, and insect cells

This text is primarily focused on yeast and its capacity to replace mammalian cells for

human protein expression. But there are other competitors in this sector with their

own advantages and drawbacks.

In this chapter these are summarised for a better overall view and to understand how

the current market for such systems has evolved or will. Escherichia coli and other

bacteria are not covered because of their total lack of compartments, folding

problems and the lack of higher glycosylation systems.

Basis of evaluation

If a novel protein has to be evaluated, there are five parameters [8] that have to be

considered before attempting:

• the cost of manufacturing and purification

A plant that can be grown anywhere and eaten without any purification step is much

cheaper than mammalian cells, which have to be passaged every other week and

require a high amount of purification steps to be below the tolerance for viral

contamination and other criteria of purity.

• the ability to control the final product including its posttranslational

processing.

Yeast are meant to do homogeneous glycosylation, mammalian cells don’t, yet

another important factor to slash costs on down stream processing.

• the time required from gene to purified protein.

That’s a big one with mammalian cells, because of the extremely long time a lot of

money is required and a rerun of the complete pipeline takes months.



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• the regulatory path to approve a drug produced on a given expression

platform

The approval of a drug is a time consuming and money robbing undertaking with

many hurdles. It accounts for the biggest junk of costs when developing a new

therapeutic agent. Proprietary expression systems are likely to encounter much

higher approval standards than for example a mammalian system that has been here

for decades.

• the overall royalties associated with the production of a recombinant

product in a given host.

Most of the time one or more patents have already been filed by other companies

while developing a new system and so can cause costs before the project has even

kicked of. This has to be considered because they can easily weigh out the merits.

These 5 points are considered for the evaluation of different expression systems [8].

Mammalian cells

Most of the critical points have been mentioned in the description of the key points.

Mammalian cells are expensive to maintain because they need a complex medium.

This is also a problem, because the growth factors required are added by calf serum

which can include contamination with viruses or prions. When the protein is secreted,

the downstream process is cheaper then with inclusion bodies that have to be

refolded. The product that is secreted is not homogeneous like that of yeast but

contains heterogeneous glycans.

The production cycle of a mammalian cell is extremely long and the yield is very

small compared to yeast.

Because a lot of drugs produced by mammalian cells have been approved, it’s rather

easy to push through a new substance based on the same systems, which has been

validated before. The idea of production with mammalian cells is not new and it’s

likely that a lot of patents have been developed on this field which directly leads to

royalty fees when considered.



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Yeast cells

Yields of up to 15g/l can be achieved with this system, which secrets its products.

Therefore the substance is found in the medium surrounding the cells which needn’t

to be destroyed for purification. In our case the glycoprotein that is secreted is

homogeneous. For that reason several critical and very expensive purification steps

can be avoided.

The time that an expression system with the right posttranslational modifications is

available to the time where a product is finally expressed is very short because of the

fast growth of yeast cells.

To obtain approval for glycoprotein made by yeast is another issue, because no

glycoprotein expressed by yeast has been yet approved; stringent qualification

criteria should be considered possible.

Glycoproteins made by yeast is a new idea, little work in this huge field has been

done by now; royalties are not a major concern right now.

Fungal cells

Fungal cells have pretty much the same properties as yeast cells, when it comes to

production costs. It should be mentioned that fungal cells have got much higher rates

of secretion then a yeast which is a big advantage because the concentration in the

medium is much higher which results in a cheaper down stream process.

Plant cells

Vaccines made by plants which can be taken in orally have the potential of solving a

lot of health issues in countries of the third world. They can be grown normal fields

like any other crop, stored for many months without the need for a fridge that keeps

the classical vaccines intact and taken in orally without the need of a doctor and

sterile conditions. Additionally plants can be cultivated in fermentors, but they grow

much slower then yeasts or fungi. The overall time from gene to product is a big

drawback because the cultivation takes place over many months.

A lot of transgenic plants have been released to the marked so there is enough

knowledge about getting a new hybrid seed approved. Many companies have

invested big money in researching transgenic plants.



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It’s unlikely that new products come without royalties.

Insect cells

The insect cell system is the closest system to the mammalian cells.

Recent reports have shown that insect cells can be grown without serum, so viruses

and prions are no longer an issue. But the medium is still very complex which makes

it more expensive then medium for yeasts.

Insect cells can secrete the desired product so down stream processing is

approximately the same as yeasts.

If the cells are grown in serum free medium, approval gets much easier and the down

stream process is much cheaper because of no additional steps to yield a higher

level of pureness.



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5. Summary and perspectives

For several years now, proteins for therapeutic reasons are expressed in yeast. That

is for example Insulin by NovoNordisk, Hepatitis B surface antigen by

GlaxoSmithKline, Merck and several others, Glucagon by NovoNordisk and many

more. They sum up to about 140 approved therapeutic proteins, with another 500 in

clinical trials [13]. Mammalian cells, the classic production system for recombinant

human glycoproteins, are able to do all the necessary posttranslational modifications

especially N-glycosylation of the complex type. N-glycosylation is of importance

because of its influence on half-life and hydrodynamic volume of the protein. If the

metabolite is glycosylated the wrong way or not at all, it may never develop a

complete level of impact.

But mammalian cells have a lot of drawbacks.

First they grow slowly, very slowly. Long generation times make minor alterations a

big issue. Their continuous need for passaging them take up a lot of manpower and

is a risk because of contamination.

Second the medium is very complex. They just won’t grow in any medium. They need

certain growth factors derived from animals such as calf serum, which can be a

problem because of the risk of introducing viruses or prions.

Third the scale up of mammalian cells can be a big issue because of the

heterogeneous mixtures of glycoforms that can be a consequence.

All these factors make this system very unattractive and expensive for expression of

recombinant proteins. But by now, it was the only way to go.

Because of this bottleneck, a lot of research on this particular field has been done tofind alternatives. One of the most promising is yeast. It is robust, the medium is well

defined and cheap, scale up is easy and genetic engineering has been done on

yeast for decades.

In the last couple of years groundbreaking discoveries have been made in the sector

of reconstructing the human N-glycosylation pathway in Pichia pastoris, an organism

used for the expression of several non-glycosylated recombinant therapeutic proteins

with outstanding features such as yields of up to 15g/l.



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This year, very promising results have been reported about having achieved a

complete reconstruction of the human N-glycosylation pathway in that yeast by Wildt

et al [8].

Experiences with yeast show, that once the major breakthrough has been made, the

optimisation and scale up of the system is quite easy.

This can directly lead to glycoproteins for therapeutic reasons in nearer future such

as human erythropoietin which is used for patients with anaemia or by athletes to

increase their performance. Other products are antibodies, such as Herceptin and

Rituxan, interferon-β for the treatment of multiple sclerosis and glucocerebrosidase.

While the production cycle from the start over fermentation and downstream

processing to the final product in yeast is much cheaper and shorter than in

mammalian cells, the clinical trial for approval of a drug is still the most expensive

step which has to be taken by either of the two systems. Therefore no big price drop

in therapeutic agents is anticipated because of conversion to yeast secreted proteins.

But unlike mammalian cells, yeasts secrete homogeneous glycoproteins which

characteristics can be researched in more detail than the mixture of glycoformsobtained in mammalian expression systems.

Therefore, yeast based expression systems for glycoproteins have a very good

chance to take over a big junk of the products made by mammalian cells right now.



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6. References

1 Bruce A., Johnson A., Lewis J., Raff M., Roberts K., Walter P., (eds.),

In Molecular Biology of the Cell (4th

ed.), Garland Science (2002),pp.665-709

The Cell, 665-709

2 Cregg J. M., Cereghino J. L., Shi J., Higgins D. R., ( 2000 )

Recombinant Protein Expression in Pichia pastoris

Molecular Biotechnology 16, 23-39

3 Bretthauer R. K., Castellino F. J., (1999),

Glycosylation of Pichia pastoris-derived proteins

Biotechnology and Applied Biochemistry 30, 193-200

4 Macauley-Patrick S., Fazenda M. L., McNeil B., Harvey L. M., (2005)

Heterologous protein production using the Pichia pastoris expression system

Yeast 2005, 22, 249-270

5 Martinet W., Maras M., Saelens X., Jou W. M., Contreras R., (1998)

Modification of the protein glycosylation pathway in the methylotrophic yeast

Pichia pastoris

Biotechnology Letters, 20, 1171-1177

6 Mochizuku S., Hamato N., Hirose M., Miyano K., Ohatani W., Kameyama S.,

Kuwae S., Tokuyama T., Ohi H., (2001)

Expression and Characterization of Recombinant Human Antithrombin III in

Pichia pastoris

Protein Expression and Purification, 23, 55-65

7 Tillman U Gerngross, (2004) Advances in the production of human therapeutic proteins in yeasts and

filamentous fungi

Nature Biotechnology, 22, 1409-1414

8 Wildt Stefan, Tillman U. Gerngross (2005)

The humanization of N-glycosylation Pathways in yeast

Nature Reviews in Microbiology, 3, 119-128



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9 Nakanishi-Shindo, Y., Nakayama, K., Tanaka, A., Toda, Y., Jigami Y., (1993)

Structure of the N-linked oligosaccharides that show the complete loss of α-

1,6-polymannose outer chain from Och1, Och1 Mnn1, and Och1 Mnn1 Alg3

mutants of Saccharomyces cerevisiae

Journal of Biological Chemistry 268, 26335-26345

10 Chiba, Y. et al. (1998)

Production of human compatible high mannose-type (Man8GlcNAc2) sugar

chains in Saccharomyces cerevisiae

Journal of Biological Chemistry 273, 26296-26304

11 Choi B., Bobrowicz P., Davidson R. C., Hamilton S. R., Kung D. H., Li H.,

Miele R. G., Nett J. H., Wildt S., Gerngross T. U. (2003)

Use of combinatorial genetic libraries to humanize N-linked glycosylation in the

yeast Pichia pastoris

Proceeding of the National Academy of SciencePNAS USA, 100, 5022-5027

12 Roy S. K., Yoko-o T., Ikenage H., Jigami Y. (1998)

Functional evidence for UDP-galactose transporter in Saccharomyces

cerevisiae through the in vivo galactosylation and in vitro transport assay

Journal of Biological Chemistry 273, 2583-259013 Walsh G., (2004)

Biopharmaceutical Benchmarks – 2003

Nature Biotechnology, 21, 865-870

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