Production of Recombinant Therapeutic

Proteins

Outline

1. Introduction2. Methods used to produce recombinant

proteins3. How are transgenic animals produced? 4. Advantage of transgenic systems5. Limitations of the transgenic

expression systems6. List of therapeutic proteins

1.INTRODUCTION Production of Recombinant Therapeutic Proteins

Recombinant DNA technology is widely used in the production of therapeutic agents such as;

hormones, cytokines, growth factors, antibiotics, vaccines, blood products like albumin, thrombolytics, fibrynolytics, clotting factors such as factor VII, factor IX, tissue plasminogen activator and many more.

• All these therapeutic agents can be produced in a large quantity and also more economically by using rDNA technology.

• Many proteins which may be used for medical treatment or for research are normally expressed at very low concentrations.

• Through rDNA technology, a large quantity of proteins can be produced. This involves inserting the desired protein gene into an "expression vector" which must contain a promoter so that the protein can be expressed.

2. METHODS USED TO PRODUCE RECOMBINANT PROTEINS

(i) Production of recombinant proteins in microbial bioreactors Examples E.coli expression system Saccharomyces cerevisiae

(ii) Mammalian cell derived bioreactors E.g. Chinese Hamster Ovary cell (CHO) bioreactors.

(iii) Animal Bioreactors “Pharming” Production of Recombinant Therapeutic Proteins in the Milk of

Transgenic Animals Eg, Cows, sheep, pigs etc.

(i) Microbial bioreactors• The first microbial bioreactors, in particular

Escherichia coli (bacterial) and Saccharomyces cerevisiae & Pichia pastoris (yeasts) were found to be satisfactory for the production of simple polypeptides such as insulin and human growth hormone

• However, microbial bioreactors were found to be unsuitable for proteins with complex post-translational modifications or intricate folding requirements, such as the coagulation factors, or monoclonal antibodies.

• This led to the development of large-scale mammalian cell culture, for example, the use of Chinese Hamster Ovary (CHO) cell cuture bioreactors.

Limitations of microbial bioreactors• Bacteria often improperly fold complex proteins,

leading to involved and expensive refolding processes and ;

• Both bacteria and yeast lack adequate post-translational modification machinery for mammalian-specific N- and O-linked glycosylation, γ-carboxylation, and proteolytic processing

CODING SEQUENCEINTRON poly A signalPROMOTER

Building the Transgenes

Transgene

bacterial genes•antibiotic marker•replication origin

Selectable Marker Gene

Plasmid DNA Construct

ON/OFF Switch Makes Protein stop sign

An overview of the recombination process in Escherichia coli bioreactor

(ii) Cell culture bioreactors • These technologies permitted the development of

numerous monoclonal antibodies, cytokines, and other complex bioactive biomolecules.

• However, there are proteins that, due to a combination of complex structure and large therapeutic dosing have until now eluded (fail to be attained) recombinant production using traditional bacterial and cell culture bioreactors

• E.g Commercial recombinant production of complex molecules, such as antithrombin and alpha1-antitrypsin, has not yet been achieved in microbial or mammalian cell derived bioreactors

• Cell culture systems require high initial capital expenditures, lack scale-up (or down) flexibility and use large volumes of culture media

• This led to development of transgenics technology i.e Production of Recombinant Therapeutic Proteins in the Milk of Transgenic Animals

(iii) Production of Recombinant Therapeutic Proteins in the Milk of Transgenic Animals

What is a transgenic animal?• A transgenic animal is one which has been genetically

altered to have specific characteristics (genes) it otherwise would not have.

• Different types of transgenic animals have been invented to carter to specific societal needs.

• It includes; transgenic disease models, transgenic pharmers, xenotransplanters and transgenic food source.

3. How are transgenic animals produced?

The foreign DNA can be inserted in three ways:

(i) DNA microinjection Fusing an expression vector, comprising a gene

that is encoded for the human or humanized target protein with mammary gland-specific regulatory sequences, and then inserting into the germline of the selected production species

.

• When integrated, the milk-specific expression construct becomes a dominant genetic characteristic that is inherited by the progeny of the founder animal (Figure 1).

• This general strategy makes it possible to harness the ability of dairy animal mammary glands to produce large quantities of complex proteins.

Fig 1.

Electrofusion: Fusion induced by electric pulse

Cells brought close together

fusion pulse

Heterokaryon phase: nuclei distinct

fusion product

Cont………(ii) Retrovirus-Mediated Gene Transfer• A retrovirus is a virus that carries its genetic

material in the form of RNA rather than DNA

• retroviruses used as vectors to transfer genetic material into the host cell, resulting in a chimera

• chimeras are inbred for as many as 20 generations until homozygous transgenic offspring are born

(iii) Embryonic Stem Cell-Mediated Gene Transfer• This method involves isolation of totipotent stem

cells from embryos

• The desired gene is inserted into these cells

• Cells containing the desired DNA are incorporated into the host’s embryo, resulting in a chimeric animal

Advantage of transgenic systems

• Transgenic livestock can be maintained and scaled-up in relatively inexpensive facilities

• Use animal feed as raw material

• Can achieve impressive yields of recombinant proteins.

Limitations of the transgenic expression systems

• Limitations are related to potential adverse effects of bioactive heterologous proteins on the health of the production animals and to a lesser extent, to initial timelines. E.g. 12-18 months in goats

• Although transgenic expression systems are able to perform complex post-translational modifications, such as γ-carboxylation, β-hydroxylation or N- and O-linked glycosylation,

-there are species-specific and tissue-specific characteristics for these modifications that may affect the appropriateness of a given system for the expression of specific proteins.

This is also a challenge found with mammalian cell culture, microbial expression systems or transgenic plants.

•Transgenic goats Producing anti-thrombin (rhAT) therapeutic protein. • It is used to treat clotting defects as in Haemophilia and is used to prevent DIC and DVT also used before surgery

Webster and Peter

Nexia Biotechnologies transferred the silk gene from Orb spiders into goats

The resulting male goats were used to sire silk-producing female goats

Each goat produces several grams of silk protein in her milk

The silk is extracted, dried to a white powder and spun into fibers

The fibers are stronger and more flexible than steel

Transgenic young male goats carrying silk gene

Tracy the sheep (1997).

The first transgenic animal to produce a recombinant protein drug in her milk alpha-1-antitrypsin (AAT) treatment for emphysema & cystic fibrosis. Created by PPL Therapeutics & The Roslin Institute

Herman the bull

-15/12/1990: first transgenic bull carrying transgene for human lactoferrin (Gene Pharming, The Netherlands)

-Dec 1992: permission to generate 50 offsprings by AI

-Oct 1993: first calf born

-March 1996: 5 cows producing human lactoferrin in their milk

•Some Biotherapeutic companies has received approval to sell human anti-thrombin (hAT) purified from goat’s milk in Europe

•Technology is not restricted to cows,

goats, & sheep, there is interest in

using rabbits since housing costs are

significantly less & generation time is faster

•Chickens which produce recombinant drugs in their eggs have been produced by The Roslin Institute.

= Is it good to make transgenics from Mr. D.O.G??

THE END