Host cells for the production of biopharma-ceuticals

Many of biopharmaceuticals, especially proteins : produced by recombinant DNA technology using var-ious expression systems

Expression systems : E. coli, Bacillus, Yeast(Saccharomyces cerevisiae) , Fungi(Aspergillus), animal cells (CHO), plant cells, in-sect cells

E. coli and mammalian cells : most widely used

Typical biopharmaceuticals produced by recombinant DNA technology : Cytokines, therapeutic proteins, etc.

Use of appropriate expression system for specific biopharmaceuticals :

- Each expression system displays its own unique set of advantages and disadvantages - Expression level (soluble form), Glycosylation, Easy purification, cultivation process, cell density Cost effectiveness feasibility Production system for therapeutic proteins - Cultured in large quantity, inexpensively and in a short time by standard cultivation methods

Eschericia coil

Most common microbial species to produce het-erologous proteins of therapeutic interest

- Heterologous protein : protein that does not occur in host cells ex) The first therapeutic protein produced by E. coli : Human in-

sulin (Humulin) in 1982, tPA (tissue plasminogen activator) in 1996

Major advantages of E. coli - Served as the model system for prokaryotic genetics Its molecular biology is well characterized - High level expression of heterologous proteins : - High expression promoters (~30 % of total cellular

protein - Easy and simple process : Rapid growth, simple and in-

expensive media, appropriate fermentation technol-ogy, large scale cultivation

Intracellular accumulation of proteins in the cy-toplasm

Complicate downstream processing compared to ex-tracellular production

Additional primary processing steps : cellular ho-mogenization, subsequent removal of cell debris by filtration or centrifugation

Extensive purification steps to separate the protein of interest

Inclusion body - Insoluble aggregates of partially folded protein - Formation via intermolecular hydrophobic interac-

tions

Draw-backs

High level expression of heterologous proteins over-loads the normal cellular protein-folding mecha-nisms

Hydrophobic patch is exposed, promoting aggre-gate formation via intermolecular hydrophobic interac-tions

Inclusion body displays one processing advantage - Easy and simple isolation by single step centrifu-gation - Denaturation using 6 M urea - Refolding via dialysis or diafiltration

Prevention of inclusion body formation - Growth at lower temperature (20 oC) - Expression with fusion partner : GST, Thioredoxin, GFP, - High level co-expression of molecular chaperones

Inability to undertake post-translational modifica-tion, especially glycosylation : limitation to the pro-duction of glycoproteins

Cf) Unglycosylated form of glycoprotein : little effect on the biological activity (ex : IL-2 E. coli can be used as a good host system)

The presence of lipopolysaccharide (LPS) on its sur-

face : pyrogenic nature More complicated purification procedure

Yeast Saccharomyces cerevisiae, Pichia pastoris

Major advantages Their molecular biology is well characterized, facilitat-

ing their genetic manipulation Regarded as GRAS-listed organisms (generally re-

garded as safe) with a long history of industrial appli-cations (e.g., brewing and baking)

Fast growth in relatively inexpensive media, outer cell wall

protects them from physical damage Suitable industrial scale fermentation equipment/

technology is already available Post-translational modifications of proteins, especially

glycosylation : Highly mannosylated form

Drawbacks Glycosylation pattern usually differs from the pattern

observed in the native glycoprotein : highly mannosy-lation pattern

Trigger the rapid clearance from the blood stream

Low expression level of heterologous proteins : < 5 %

Major therapeutic proteins produced in yeast for gen-eral medical use:

ex) Insulin, colony stimulating factor(GM-CSF) for bone marrow transplantation, Hirudin for anticoagu-lation,

Fungal production systems Aspergillus niger

Mainly used for production of industrial enzymes : a-amylase, glucoamylase, cellulase, lipase, protease

etc..

Advantages High level expression of heterologous proteins (~ 30

g/L) Secretion of proteins into extracellular media easy and simple separation procedure Post-translational modifications : glycosylation - Different glycosylation pattern compared to that in human

Disadvantage Produces significant quantities of extracellular pro-

teases Degradation of heterologous proteins Use of mutant strain with reduced level of pro-teases

Animal cells Major advantage : Suitable for production of glycopro-

tein especially glycosylation Chinese Hamster Ovary (CHO) and Baby Hamster

Kidney (BHK) cells Typical proteins produced in animal cells : EPO, tPA,

Interferons, Immunoglobulin antibodies, Blood factors etc.

Drawbacks Very complex nutritional requirements : growth fac-

tors expensive complicate the purification procedure Slow growth rate: long cultivation time Far more susceptible to physical damage Increased production cost

CHO cells

Transgenic animals

Transgenic animals : live bioreactor

Generation of transgenic animals : Direct microinjection of exogenous DNA into an egg cell Stable integration of the target DNA into the genetic complement of the cell After fertilization, the ova are implanted into a surro-

gate mother Transgenic animal harbors a copy of the transferred

DNA

In order for the transgenic animal system to be practi-cally useful, the target protein must be easily and simply separable from the animal without any injury

: Simple way : to produce a target protein in a mam-mary gland Easy recovery of a target protein from milk

Mammary-specific expression : Fusion of a target gene with the promoter-containing regulatory sequence of a gene coding for a milk-specific protein

ex) Regulatory sequences of the whey acid protein (WAP, the most abundant protein in mouse milk), β-casein, α- and β-lactoglobulin genes

ex) Production of tPA in the milk of transgenic mice - Fusion of the tPA gene to the upstream regulatory sequence of the mouse whey acid protein More practical approach : production of tPA in the

milk of transgenic goats

Production of proteins in the milk of transgenic ani-mals : ex) tPA (goat) : 6 g/L,

Growth hormone (Rabbit) : 50 mg/L

Goats and sheep : Most attractive host system High milk production capacities : 700-800 L/year for

goat Ease of handling and breeding Ease of harvesting of crude product : simply requires

the animal to be milked

Pre-availability of commercial milking systems with maximum process hygiene

Low capital investment : relatively low-cost animals replace high-cost traditional cultivation equipment, and low running costs

High expression levels of proteins are potentially at-tained :

> 1 g protein/L milk

On-going supply of product is guaranteed by breeding Ease downstream processing due to well-character-

ized properties of major native milk proteins

Issues to be addressed for practical use Variability of expression levels (1 mg /L ~ 1 g/L) Different post-translational modifications, especially

glycosylation, from that in human Significant time lag between the generation of a

transgenic embryo and commencement of routine product recovery:

- Gestation period ranging from 1 month to 9 months - Requires successful breeding before beginning to lactate - Overall time lag : 3 years in the case of cows, 7 months in the case of rabbits

Inefficient and time-consuming in the use of the mi-cro-injection technique to introduce the desired gene into the egg

Other approaches than microinjection Use of replication-defective retroviral vectors : consis-

tent delivery of a gene into cells and chromosomal integration

Use of nuclear transfer technology Manipulation of donor cell nucleus so as to harbor a gene coding for a target protein Substitution of genetic information in un unfertilized egg with donor genetic information Transgenic sheep, Polly and Molly, producing human blood factor IX, in 1990s

No therapeutic proteins produced in the milk of transgenic animals had been approved for general medical use

Alternative approach : production of therapeutic pro-teins in the blood of transgenic pigs and rabbits

Drawbacks - Relatively low volumes of blood can be harvested - Complicate downstream processing because of complex serum - Low stability of proteins in serum

Transgenic plants

Expression of heterologous proteins in plant : Introduction of foreign genes into the plant

species : Agrobacterium-based vector-mediated gene transfer

- Agarobacterium tumefaciens A. rhizogenes : soil-based plant pathogens

When infected, a proportion of Agarobacterium Ti plasmid is trans-located to the plant cell and inte-

grated into the plant cell genome

Expression of therapeutic proteins in plant tissue : Table 3.16

Potentially attractive recombinant protein pro-ducer

Low cost of plant cultivation Harvest equipment/methodologies are inexpensive and well established Ease of scale-up Proteins expressed in seeds are generally stable Plant-based systems are free of human

pathogens(eg., HIV)

Disadvantages Variable/low expression levels of proteins Potential occurrence of post-translational gene silenc-

ing (a sequence specific mRNA degradation mechanism) Different glycosylation pattern from that in human Seasonal/geographical nature of plant growth

Most likely focus of future transgenic plants : Production of oral vaccines in edible plants or fruit,

such as tomatoes and bananas - Ingestion of transgenic plant tissue expressing re-

combinant sub-unit vaccines induces the production of antigen-specific antibody responses

Direct consumption of plant material provides an inexpensive, efficient and technically straightfor-ward mode of large-scale vaccine delivery

Several hurdles Immunogenicity of orally administered vaccines vary widely Stability of antigens in the digestive tract varies widely Genetics of many potential systems remain poorly character-

ized Inefficient transformation systems and low expression levels

Insect cell-based system Laboratory scale production of proteins Infection of cultured insect cells with an engineered

baculovirus (a viral family that naturally infects in-sects) carrying the gene coding for a target protein

Most commonly used systems Silkworm virus Bombyx mori nuclear

polyhedrovirus(BmNPV) in conjunction with cultured silkworm cells

Virus Autographa californica nuclear polyhedrovirus(AcNPV) in conjunction with cultured armyworm cells

Advantages High level intracellular protein expression - Use of strong promoter derived from the viral polyhedrin : ~30-50 % of total intracellular protein - Cultivation at high growth rate and less expensive

media than animal cell lines - No infection of human pathogens, e.g., HIV

Drawbacks - Low level of extracellular secreted target protein -Glycosylation patterns : incomplete and different

No therapeutic protein approved for human use

Alternative insect cell-based sys-tem

Use of live insects - Live caterpillars or silkworms Infection with the engineered baculovirus vector Ex) Veterinary pharmaceutical company, Vibragen

Owega - Use of silkworm for the production of feline interferon ω

Plant cell system