Therapeutic Protein From Plant

7/31/2019 Therapeutic Protein From Plant

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So far, pharmaceuticals used for the treatment of

diseases have been based largely on the natural or

synthetic organic molecules produced by microbes or

synthesised by organic chemists. However, the post-

genomic-era scenario projects a remarkable change

in the therapeutics market, with the realisation that

proteins have many potential therapeutic advantagesfor preventing and curing diseases and disorders.

Proteins are the ultimate players of cellular

function. Information stored in DNA directs the

protein-synthesising machinery of the cell to

produce specific proteins required for its structure,

function and regulation. There are many proteins

that are essential for good health that some people

cannot produce because of genetic defects. The

advent of sophisticated genomics and proteomics-

based functional identification has unravelled large

numbers of candidate proteins with a potential for therapeutic intervention. Several market research

groups have predicted that the growth of the

biopharmaceutical market might reach as high as

15% annually and result in a critical shortage in

protein-manufacturing capacity in the near future.

Short peptide chains consisting of fewer than 30

amino acids can be synthesised chemically. Proteins

larger than that are best produced by living cells.

These ‘natural bioreactors’ support the sustained

yield of target protein. The protein-encoding DNA

is inserted into cells. As the cells grow, theysynthesise the protein, which is subsequently

harvested and purified.

Production of proteins by micro-organisms is well

entrenched in the industry. More recent knowledge

of the molecular and cellular mechanism of diverse

hosts has led to envisioning the art of expression of

foreign proteins in mammalian cell culture and

transgenic animals and plants. However, several key

issues, namely complexity of the protein, quantity

requirement and reliability of the expression system,

need to be assessed before choosing a host system for

a particular therapeutic protein. Microbial and plant

hosts can be utilised for a moderate and large

quantity of low-complexity proteins. Complex

proteins can be produced in relatively small and large

quantities in animal cell culture and transgenic

animals, respectively.

Since 1982, more than 100 therapeutic proteins and

peptides have been licensed for production using

bacterial, fungal and mammalian cells, and many

therapeutic proteins are currently being developed

and tested in a variety of host cells.1 An overview of

the technologies available and technical hurdles that

are encountered in these processes is presented here.

Produ c t i on o f L ow - c omp l e x i t y

P ro t e i n s

M i c r o b i a l C e l l F a c t o r i e s

Humans have exploited microbial transformations

for centuries – initially only in the food industry,

but, lately, they have provided immense benefits to

the pharmaceutical industry as bioreactors for

antibiotic production. The birth of genetic

engineering in the late 1970s, and subsequent

technological expansion, has provided molecular

tools for producing heterologous proteins in a wide

range of micro-organisms including bacteria, yeast

and filamentous fungi, thus largely extending their manufacturing capabilities.2

Following transformation of a best protein-

expressing host (for example Escherichia coli ,

Saccharomyces cerevisiae and Pichia pastoris), best

protein-expressing clones are identified and further

optimised for protein expression. Because of

scalability and well-characterised genetics, microbial

host systems remain the best option for low-

complexity, moderate-volume protein production at

relatively low cost.3 A major hurdle in microbial

1. R Andersson and R Mynahan, “The Protein Production Challenge”, http://www.windhover.com

2. “Old bugs for new tasks; the microbial offer in the proteomics era”, http://www.microbialcellfactories.com/content/1/1/4

3. R Andersson and R Manahan , “The Protein Production Challenge”, In Vivo, Windhover Information Inc., May 2001,

pp. i–5.

Dr Nilanjan Roy

Shruti Agarwal

Dr Nilanjan Roy is Head of theProteomics Laboratory of the

National Institute of Pharmaceutical

Education and Research (NIPER).

In addition to research, he is

actively engaged in teaching in

the areas of advanced techniques

in biotechnology, genomics,

proteomics, bio and pharma

informatics. As well as his research

articles, he is the author of several

reports and popular articles. Dr

Roy holds a PhD from Bose

Institute, Kolkata, India, and has

seven years of research experience

from one of the premier hospitals

of the US, The Cleveland Clinic

Foundation, Cleveland, OH.

Shruti Agarwal is an active

researcher at NIPER’s Proteomics

Laboratory. She has been involved

in deciphering the role of

oxidative stress regulators in

the ageing process.

a report by

Dr N i l a n j a n Roy and Sh ru t i A g a rwa l

Head and Researcher, Proteomics Laboratory, National Institute of Pharmaceutical Education and Research

(NIPER)

Therapeut i c Prote in Product ion – An Overv iew

B U S I N E S S B R I E F I N G : F U T U R E D R U G D I S C O V E R Y 2 0 0 3

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Technology PROTEOMES & PROTEOMICS


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production is in the post-production recovery of

proteins. Examples of strategies for improved

bioprocesses are as follows.

• Use of a secretory signal peptide fused to the

N-terminus of the target protein can ensure

efficient secretion of the protein. This eliminates

the need for cell disruption and reduces

interaction with host proteins. However, dilution

of protein can be a shortcoming. This can be

overcome by tagging a hydrophobic tail to the

target protein, which can alter the partitioning of

the protein in two-phase systems for efficient

purification and concentration. Otherwise,

tagging of an affinity tail (for example His-tag) can

be an option for low-cost purification.

• The addition of charged amino acid residues or

mutation of amino acids of a protein to alter theisoelectric point can improve the purification by ion

exchange chromatography followed by production.

However, proteins with complicated folding

requirements and post-translational modification

steps cannot be produced with this system.

T r a n s g e n i c P l a n t s

The use of genetically engineered plants to produce

valuable proteins is increasing slowly. Vaccines,

mammalian blood constituents, enzymes, antibodies

and low-calorie sweeteners are being produced intobacco leaves. In order for a plant to be used to

produce specific molecules, a novel gene is inserted

into its chromosomes. A regulatory code inserted

with that gene dictates to the plant where to produce

the desired protein – in its leaves, roots or seeds.

Since the plant proteins are not modified post-

translationally, transgenic plants are likely to be a

viable alternative for less complex proteins.4 With

well-established transgenic technology and easier

expansion by means of clonal propagation, transgenic

plants are specially suited to high-volume production

of proteins that otherwise cannot be produced cost-effectively with conventional microbial systems.5

Protein extraction and purification are required

much the same way, as are the requirements in the

case of cell culture methods. However, recent

chloroplast transformation techniques have been

perfected and have achieved high-level protein

production in chloroplasts. Because of protein

accumulation, the size of transgenic chloroplasts

increases and this facilitates purification.

The issues surrounding objections and concerns to

transgenic crops are as follows.

• There may be damage to human health due to

allerginicity of contaminating plant proteins.

• Crop-to-crop and crop-to-weed transfer of

foreign protein genes and negative effects on

herbivorous animals are a risk. Thus, it is of

utmost importance to ensure that the gene pool is

not contaminated. Adequate quarantine measures

can therefore increase production cost.6

• There are also some possibilities of disturbances

in traditional farming practices and economies,

especially in developing countries, wheremolecular farming can be a lucrative alternative

to traditional processes.5

Thus, cost-efficient production of transgenic plants

can alter the economics of recombinant protein

synthesis. To make best use of this promising

opportunity, it is important that government

regulatory agencies take active interest in developing

the agronomic and manufacturing regulations

needed to ensure safety, consistency and potency of

plant-made pharmaceuticals.

Produ c t i o n o f C omp l e x P ro t e i n s

A n i m a l C e l l C u l t u r e s

Animal cell lines have been utilised increasingly as

hosts for the production of recombinant proteins and

currently dominate some product families such as

antibodies. Considerable research effort has been

directed towards the development and optimisation

of the cell culture process, with the objective of

delivering the quantity and quality of protein

required by the healthcare industries. Many potentialproducts are already in the clinical trial pipeline.

However, many of the mammalian cell lines that are

used for industrial-scale recombinant protein

production undergo apoptotic death due to

deprivation of key nutrients (such as amino acids,

glucose and serum), oxygen, the use of virus-based

protein expression systems and cytostatic agents in

the bioreactor environment.7 This limits culture

4. R Fischer, et al., “Towards molecular farming in the future: transient protein expression in plants”, Biotechnol. Appl.

Biochem., 30 (1999), pp. 113–116.

5. B R Thomas, A V Deynze and K J Bradford, “Production of Therapeutic Proteins in Plants”, AgriculturalBiotechnology in California Series, Pub-8078.

6. R Fischer and N Emans, “Molecular farming of Pharmaceutical proteins”, Transg. Res., 9 (2000), pp. 279–229.

7. B D Kelley, “Biochemical engineering: Bioprocessing of therapeutic proteins”, Curr. Opin. Biotechnol., 12 (2001),

pp. 173–174.



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duration and productivity. Thus, though the

technique is fairly well characterised, the capacity

constraints call for accurate manufacturing strategy,

therefore limiting use to low-volume requirements

of high-complexity proteins.

Again, the set-up costs are prohibitively expensive.

Building or expanding a manufacturing facility to

include 10,000-litre bioreactors – the minimum size

for truly large-scale commercial production – takes

three to five years and costs US$250–500 million.8

Process control equipment, assay development and

quality control systems are major expenses.

Moreover, the time required to construct large-

scale facilities has imposed significant business and

financial risks on pharmaceutical companies. For

the facility to be able to work by the time US Food

and Drug Administration (FDA) approval is

sanctioned, the company needs to start investingwhen the drug is somewhere in the Phase III stage

of clinical trials.9 Given the uncertainty related to

the outcome of clinical trials, pharmaceutical

companies face the risk of loss due to under-

building or over-building capacities. Thus, an

appropriate decision needs to be taken before

manufacturing proteins with limited markets.

Before the successful commercialisation of this

technology, significant research inputs are required

in the areas of cell line selection, bioreactor

configuration, online bioreactor controls andproduction modes: suspension culture in serum or

protein-free media, fed batch or perfusion

technology and protein purification techniques.

Also, the production of biologics must be managed

carefully to avoid unintentional transmission of viral

diseases that could infect humans.

T r a n s g e n i c A n i m a l s

The term ‘transgenic animals’ describes animals with

chromosomes that contain stably integrated copies of

genes or gene constructs derived from other speciesor not normally found in the host animal. The use

of transgenics as a technology for producing

recombinant proteins has made remarkable strides in

the past few years. The production of the first

transgenic farm animals was reported in 1985, and

biopharmaceutical production by these animals

followed shortly thereafter. ‘Pharming’ is the

production of human pharmaceuticals in farm

animals, a process still in the developmental stages,

with good prospects of large-scale commercialisation

in the near future.

The process involves microinjection of the DNA

solution into the pronucleus of the embryo using a

very fine glass needle. The injected zygote is then

transferred into a hormonally prepared recipient and

brought to term. Finally, positive transgenic animals

are matured and the level of expression of the

transgene is determined. However, transgenic

animals are costly to produce. The cost of making

one transgenic animal ranges from US$20,000 to

US$300,000 and only a small portion of the attempts

succeeds in producing a transgenic animal. The rate

of transgenesis is 5% to 25% of live births,10 but the

unit cost per protein should be significantly less

when animals are used as bioreactors to produce

human proteins.

Different transgenic species of cow, sheep, goat, pig

and rabbit are in use. Although gene expression and

heterologous protein production is possible in many

different tissues and fluids of the animal, transgenic

milk production is currently the method that is most

feasible and furthest along in development and the

regulatory process, making ruminants the best choice

in this regard. Secretion in milk is made possible by

coupling a mammary gland-targeting signal sequence

with the gene for making protein.11 The transgenic

protein can be harvested and purified from milk. The

advantages of this method are:

• a high expression level and volume output;

• the ability to express complex proteins;

• low initial capital investment is required;

• low operational costs; and

• the production ‘facility’ is reproducible –

inbreeding could pass an animal’s ability to

produce transgenic protein to its offspring.

However, as with any other developing technology,

a transgenic animal also has its own constraints and

shortcomings that need to be resolved. The major issue is regulatory approval. For animal-based

transgenic companies, the FDA is expected to be the

primary regulatory body, along with regulations that

govern moral animal treatment, protein purification

and extensive testing.12 Stringent rules are still in the

process of development and implementation.

Currently, there is a high degree of uncertainty

related to long-term policy associated with FDA

approval of a transgenically produced protein. s

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8. A Ingley, J Pavlik and T Smith, “Transgenic Protein Production as an alternative Manufacturing Technology for

Pharmaceutical Companies”.9. “Biopharmaceutical outsourcing: will a supply crunch hit its growing market?”, http://www.biotech.about.com

10. W G Gavin, “The future of Transgenics”, Regulatory Affairs Focus, May 2001

11. M Herper, “Milking Genetically Modified Cows”, http://www.forbes.com

12. FDA Directive 75/318/EEC, “Use of Transgenic Animals in the Manufacture of Biological Medicinals”.



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