Recombinant protein and pharmaceuticals production in microbes

1. RECOMBINANT PROTEIN AND PHARMACEUTICALS
PRODUCTION IN MICROBES
By
Assist.. Professor
Dr. Berciyal Golda. P
VICAS

3. 1982 Human insulin, created using recombinant DNA
technology
Interferon alfa and muromonab-CD3 approved
1986
1993 CBER's Office of Therapeutics Research and
1997
2002
2006
Review (OTRR) formed
First whole chimeric antibody, rituximab, and
first humanized antibody, daclizumab, approved
$30 billion share of biotechnological drugs of
$400 billion in yearly worldwide pharmaceutical
sales
An inhaled form of insulin (Exubera) approved

5. 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 manufacture Bioreactor process Downstream
Medium developmentdevelopment & scale-upurification
Analytical characterization

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

7. • 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

8. 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).

10. INSECT CELLS
• High level of expression
• Correct folding
• More difficult to work with
• Expensive
• Slow generation time
• Not suitable for proteins with repetitive sequences
PLANTS
• competitive cost
• the availability of established practices for their
efficient harvesting, transporting,
• sorting and processing

11. • 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

12. 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.

15. Some recombinant
proteins approved for
human use
Company
Baxter, Bayer
Genetics Institute
Genetech
Protein
Factor VIII
Factor IX
Tissue plasminogen
activator (TPA)
Insulin
Human growth
hormone
Erythropoietin
DNase I
Various interferons
(IFN)
Eli Lilly, Novo Nordisk
Eli Lilly, Genetech, Upjohn,
Novo Nordisk
Amgen, Ortho Biotech
Genetech
Schering, Biogen, Chiron,
Genetech
Disorder
Hemophilia A
Hemophilia B
Acute myocardial
infarction
Diabetes mellitus
GH deficiency in
children (dwarfism)
Anemia
Cystic fibrosis
Hepatitis B and C,
multiple sclerosis

17. 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

20. Schematic representation of the process used to
purify ATryn from the milk of transgenic goats.

21. 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

23. • to respond to a human influenza pandemic.
•
to respond to a human influenza pandemic.
Vaccine Production at industry level

24. • 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

25. rhDNase I

26. CANCER VACCINE

27. Bio therapeutics are delicate drugs
• Much
complex than
larger and more
traditional
pharmaceuticals
with
of unstable
a precise
• Composed
proteins
structure
• Easily damaged by
unfavorable temperature
history during storage