Proteins and peptides have received increased interest in the current drug therapies Recently, approved recombinant protein therapeutics have been developed to treat a wide variety of clinical indications, including cancers, exposure to infectious agents, autoimmunity/ inflammation and genetic disorders. Their high potency and selectivity. Their low accumulation in tissues. They have potentially lower toxicity than the small drug molecules. Provide abroad range of targets, which could represent a basis for personalized medication.

1. PHARMACEUTICAL
PROTEINS
PREPARED BY:
Dalia Hijjo

2. P R O T E I N B A S E D P H A R M A C E U T I C A L S
• Proteins and peptides have received increased interest in the
current drug therapies
• Recently, approved recombinant protein therapeutics have been
developed to treat a wide variety of clinical indications,
including cancers, exposure to infectious agents, autoimmunity/
inflammation and genetic disorders.

3. ADVANTAGES OF PHARMACEUTICAL PROTEINS
Their high potency and selectivity.
Their low accumulation in tissues.
They have potentially lower toxicity than the small drug
molecules.
Provide abroad range of targets, which could represent a basis
for personalized medication.

4. LIMITATIONS OF PHARMACEUTICAL PROTEINS
The manufacturing and production of therapeutic proteins are
highly complex process.
Protein therapeutics can not be completely synthesized by
chemical processes and have to be manufactured in living cells or
organisms which my affect the final product characteristics.
As the products are synthesized by cells or organisms complex
purification processes are involved.
Viral clearance processes such as removal of virus particles by
using filters or detergents, are important to prevent the serious
safety issue of viral contamination of protein drug substances.
Therapeutic proteins are larger in size than small molecule drug,
having molecular weights exceeding 100KDa

6. Exhibit complex secondary structures that must be maintained.
All of these limitations may affect the stability of protein based
drugs and studying the shelf life of protein based pharmaceuticals
is highly desirable to ensure their safety and efficacy

7. S H E L F L I F E
• shelf life is typically expressed in
units of months, i.e: 24 months.
• Shelf life/ expiry date reflect the time
where a product will work both safety
and effectively this is why “shelf life
testing” is also referred to as “
stability testing”
• The amount of time a product can stay
stable under certain environmental
conditions equals to its shelf life.

8. STABILITY OF PHARMACEUTICAL PROTEINS
• It may be defined as the capability of a particular formation in
a specific container to remain within its physical, chemical,
microbiological, toxicological protective and informational
specifications.
• Stability testing thus evaluates the effect of environment
factors on the quality of the a drug substance or formulated
product which is utilized for prediction of its shelf life,
determine proper storage conditions and suggest labelling
instructions

9. Storage
Protein can be stored as:
1. An aqueous solution
2. Freeze –dried form
3. Dried from in a compacted state
Proteins in solution often don’t meet the preferred stability
requirement.
The presence of water promotes chemical and physical degradation
process.
So…. How to
solve this
problem?

10. FREEZE – DRYING OF PROTEINS
• Also knowns as lyophilization or cryodesiccation , is a low
temperature dehydration process which involves freezing the
protein, lowering pressure, then removing the ice by sublimation
.
• This is in contrast to dehydration by most conventional methods
that evaporate water using heat .

11. BASIC PROCESS OD PROTEIN FREEZING - DRYING
The technical procedures of freeze-drying consist of:
1) Preparation and freezing.
2) Primary drying (sublimation drying)
3) Secondary drying (desorption drying)
4) Package
The freeze –dried drugs (or proteins) can be stores at room
tempreture or in refrigerator for a long time

12. 1. PREPARATION AND FREEZING OF
DRUGS.
 The concentration of protein must be a specified.
 Excipients should be added to reinforce the structure of freeze-dried
products.
 Lyoprotectant should be added into the proteins to protect them from
denaturation.
During the freezing stage, the material is cooled below its triple point
(the lowest temperature at which the solid liquid and gas phases of the
material can coexist). This ensures that sublimation rather than melting
will occur.

14. 2. PRIMARY DRYING (SUBLIMATION
DRYING)
 Performed at low temperature and vacuum.
 The pores or channels formed by the sublimation ice become the ways of
vapor to escape.
 The boundary between drying layer and frozen layer is known as the
sublimation interface.
 90-95% water in protein is removed after primary drying.

15. 3. SECONDARY DRYING (DESORPTION
DRYING)
• Purpose: to remove a portion of the bound water.
• The moisture content of protein is lower than 3% after secondary
drying.
• Because of large absorption energy, the product temperature is
secondary drying must be increased high enough to remove the
bound water, and on the other hand, this temperature cannot
induce denaturation of proteins.

16. PACKAGE (ENCAPSULATION
PROCESS)
• WHEN THE SECONDARY DRYING
PROCESS IS COMPLETE, DIRECTLY
PLUG THE VIALS OF PROTEIN IN
ORDER TO PREVENT THE FREEZE-
DRIED DRUGS FROM OXIDATION AND
WATER ABSORPTION.

17. C o m m o n c o n d i t i o n s f o r p r o t e i n
s t a g e s

18. GENERAL CONSIDERATIONS FOR PROTEIN STORAGE
 TEMPERATURE
1. Generally, proteins should be stored at <4C in clean, autoclaved glass
or polypropylene tubes.
2. Storage at room temperature often leads to protein degradation and/or
inactivity, commonly as a result of microbial growth.
3. For short term storage of 1 day to a few weeks, many proteins may be
stored at 4 c.
4. For long term storage (from 1 month to 1 year), bead single-use aliquots
of the protein in liquid nitrogen and store it in clean plastic
containers under liquid nitrogen.
5. This method involves adding the protein solution dropwise (about 100ml
each) into a pool of liquid nitrogen, then collecting the drop-sized

20. 1.Freezing at -20c or -80c is the more common form of frozen
protein storage.
2.Avoid repeated freeze-thaw cycles, which decrease protein
stability. Instead, prepare small working aliquots so that, once
thawed, the protein solution will not have to be refrozen.
3.Adding 50% glycerol or ethylene glycol will prevent solutions
from freezing at -20c, by helping in stabilizing proteins and
preventing the formation of ice crystals enabling repeated use
from a single stock.

21.  PROTEIN CONCENTRATION
1. Dilute protein solutions of less than 1 mg per ml are more prone to
inactivation and loss as a result of low-level binding to the storage
vessel. Therefor, it is best to store proteins in more concentrated
form.
2. However, the addition of a carrier protein, such as purified bsa (final
concentration of 10-15 mg/ml), to dilute protein solutions helps to
protect against such degradation and loss.

22.  ADDITIVES
Many compounds may be added to protein solutions to lengthen shelf
life:
1. Cryoprotectants such as glycerol or ethylene glycol to final
concentration of 25-50% help to stabilize proteins by preventing the
formation of ice crystals at -20c that destroy protein structure.
2. Protease inhibitors prevent proteolytic cleavage of protein.
3. Anti-microbial agents such as sodium azide (nan3) at a final
concentration of 0.02 – 0.05% (w/v) or thimerosal at a final
concentration of 0.01% (w/v) inhibit microbial growth.

23.  PH AND IONIC STRENGTH
1. The pH value of the solvent in which the proteins are dissolved, have
been shown to drastically alter their stability.
2. The effect, the pH value has on the stability of the protein is a
testament to the importance of the ionizable groups of the protein.
3. The ph value of the solvent induces a change in the overall charge of
the protein, as the titration groups or sites of the protein are
ionized, which ultimately affects the electrostatic interactions
comprising protein stability.

24. THANK YOU