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How to obtain Pharmaceutical Proteins?

1. Isolation from different sources

2. Recombinant DNA (cloning) ~ rDNA

Protein as a Drug

1.Insulin and analogue 2.Growth hormone 3.Factor VIII and IX 4.Cytokines : - Interferron alpha for Hep C - INF beta for multiple sclerosis 5.Erythropoietin 6.Granulocyte colony stimulating factor

Expression system
Which ? Why? How? Commercial aspect?

Expression systems
1. Prokaryotic :

a. E.coli b. B.subtilis

2. Eukaryotic : a. yeast : S.cerevisiae, H. polymorpha, Pichi pastoris b. Mammalian systems: CHO, BHK c. plant cell
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Prokaryotic system Advantages: - high level of recombinant protein expression

- rapid growth of cell - simple media requirement

Disadvantages: - intracellular accumulation of heterologous protein

- possibilities of protease degradation - bacterial endotoxin

Eukaryot ic system Advantages: - grow relatively quickly and are highly adaptable to large-scale
production - capable of glycosilating proteins up to a certain extent like mammalian cells - do not produce endotoxins

Disadvantages: - cost of production is high because expensive medium cells

Why we have to use eukaryotic cells to produce pharmaceutical protein?

1. Unstable product when expressed in prokaryotic cell
2. Lack of biological activities due to incorrect folding Prion disease CJD (Creutzfield Jacob Disease) 3. Proteolytic cleavage of a precursor form 4. Some of bacterial proteins are toxic to human (pyrogenic) 5. Post-translational process

Posttranslational Modifications
Correct disulfide bond formation Protein disulfide isomerase Amino acid removal from initial polypeptide O-linked or N-linked glycosylation
About 30% of eukaryotic proteins are glycosylated
No single eukaryotic host can satisfy post-translational modifications ~ select other

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Production and Purification of rRDNA produts

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Layout of recombinant DNA

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Factors to be considered in rDNA production

1. 2. Plasmid instability Compatibility of rDNA product in different host systems Stability of cell bank containing rDNA Process validation: - fermentation and production

3. 4.

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Intracellular

FERMENTATION

Extracellular

CELL RECOVERY
Process

CELL REMOVAL

REMOVAL CELL DEBRIS Concentration of Product Isolation and Purification

Sterile Filtration

Product
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PROTEIN FARMASETIK

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