RECOMBINANT THERAPEUTIC PROTEINS

ATRACTIVE FEATURES

• Therapeutic proteins are used for the treatment of abnormal health conditions.

• Replacing chemically synthesized drugs• Highly specific function• less interference with normal biological processes • faster clinical development and FDA approval time • less likely to elicit immune responses.

Evolution of Protein Therapeutic

1982 Human insulin, created using recombinant DNA technology

1986 Interferon alfa and muromonab-CD3 approved 1993 CBER's Office of Therapeutics Research and

Review (OTRR) formed 1997 First whole chimeric antibody, rituximab, and

first humanized antibody, daclizumab, approved

2002 $30 billion share of biotechnological drugs of $400 billion in yearly worldwide pharmaceutical sales

2006 An inhaled form of insulin (Exubera) approved

Classification of Protein therapeutics

• Group I: protein therapeutics with enzymatic or regulatory activity

• Group II : protein therapeutics with special targeting activity

• Group III : protein vaccines• Group IV : protein diagnostics

5

Process of Drug Production

Cells and plasmid

+

Cell line

Transfection Cell culture Purification

Drug substance (crude)

Drug substance (pure)

Drug product -(sterile)

Formulation/Filling

Cell line manufactureMedium development

Bioreactor process development & scale-up

Downstream purification

Analytical characterization

World market

- EPO alone : ~ $ 11

Billion per year

- - $ 50 Billion

(2007) $ 190

Billion (2015)

- Antibodies > 50

%

- Intensive

investment in

monoclonal

antibodies:

Therapeutic proteins will form the back-born of future medicinal therapy

PRODUCTION SYSTEMS FOR PRODUCING THERAPEUTIC PROTEINS:

• simple physiology • short generation times, as bacteria grow and multiply

rapidly • large yields of product - up to 10 % of mass (low cost)• The expressed proteins often do not fold properly and

so are biologically inactive.• The synthesised protein is often toxic to bacteria

preventing the cell cultures from reaching high densities

BACTERIAL CELLS

Yeast cells

• Yeast is a simple eukaryote and performs many of the post-translational modifications required for human proteins

• Presence of active proteases that degrade foreign (expressed) proteins, thereby reducing their yield

• (a solution to this problem is the construction of yeast strains from which the protease genes have been deleted).

Bacillus Sp.

Actinomycetes Sp.

Eschericia coli

(Yeast)

MICROBES USED FOR PROTEIN PRODUCTION

• High level of expression • Correct folding

• More difficult to work with • Expensive

• Slow generation time • Not suitable for proteins with repetitive sequences

INSECT CELLS

• competitive cost • the availability of established practices for their

efficient harvesting, transporting, • sorting and processing

PLANTS

Transgenic animals

• cheap method • produce the desired proteins in vast quantities when

using larger animals like cows.• long lead time to generate a herd of transgenic

animals • concerns about the health of the animal. • cause serious negative health effects

In vitro systems• E. coli extract; plant wheatgerm extract; and mammalian sources, rabbit reticulocyte lysate.

• lack both the genomic material and the cellular boundary system

• contain the cellular components required for transcription and/or translation of genes.

Comparison of Recombinant Protein Expression

PURIFICATION STEPS

Some recombinant proteins approved for human use

Protein Company Disorder

Factor VIII Baxter, Bayer Hemophilia A

Factor IX Genetics Institute Hemophilia B

Tissue plasminogen activator (TPA)

Genetech Acute myocardial infarction

Insulin Eli Lilly, Novo Nordisk Diabetes mellitus

Human growth hormone

Eli Lilly, Genetech, Upjohn, Novo Nordisk

GH deficiency in children (dwarfism)

Erythropoietin Amgen, Ortho Biotech Anemia

DNase I Genetech Cystic fibrosis

Various interferons (IFN)

Schering, Biogen, Chiron,Genetech

Hepatitis B and C, multiple sclerosis

PRODUCTION OF RTPs

DRUG ANIMAL USED GENETIC MODIFICIATION

WHO/WHERE PRODUCED

Anti-Cancer Drugs (currently in the

process of making).

Chickens The anti-cancer drug is produced in the chickens eggs.

Roslin Institute in the United Kingdom is

LACTOFERRIN (Breast Milk Supplement)

COWS Recombinant DNA targets lysosome from the breast

milk and modifies it in the cow which

gives a more nutritional boost

for infants

Hermitech, Inc. in Sioux Falls, South

Dakota.& China

Drugs in transgenic animals

http://www.pharmaceutical-technology.com/projects/eli_lilly/index.html

Production of Recombinant Therapeutic Proteins in the Milk of

Transgenic Animals

Schematic representation of the process used to

purify ATryn from the milk of transgenic goats.

Recombinant Hepatitis vaccine

• The hepatitis B virus (HBV) vaccine– surface antigen purified from the

blood of infected individuals. – Due to safety concerns, the HBV

vaccine became the first to be produced using recombinant DNA technology (1986)

– Produced in bakers’ yeast (Saccharomyces cerevisiae

• to respond to a human influenza pandemic.•

to respond to a human influenza pandemic.

Vaccine Production at industry level

FOLLICLE STIMULATING HORMONE (FSH)

• single vector containing the coding sequences for both subunit genes.

• After transfection, a genetically stable transformant producing biologically active recombinant FSH was isolated

• 150–450 gene copies were present in mammalian cells.

• FSH products from cell culture supernatants.

• collected from a perfusion-type bioreactor

• downstream purification

rhDNase I

CANCER VACCINE

Bio therapeutics are delicate drugs

• Much larger and more complex than traditional pharmaceuticals

• Composed of unstable proteins with a precise structure

• Easily damaged by unfavorable temperature history during storage