What are Biopharmaceuticals?

Biopharmaceuticals are defined as

pharmaceuticals (protein, peptides (amino
acids-based )or nucleic acid ) manufactured
by biotechnology methods, with the products
having biological sources, usually involving
live organisms or their active components,
transgenic animals, recombinant DNA.
Basic Protein (Peptide) Structure
 Protein Definitions

Peptide: Short chain of residues (< 10 amino acids )with a defined

sequence but with no fixed three-dimensional conformation (e.g.
calcitonin-leuprolide). Could be synthetically obtained.

Polypeptide: Longer chain, usually of defined sequence and length

(more than 10 amino acids).

Polyamino acids: Random sequences of varying lengths generally

resulting from nonspecific polymerization of one or more amino acids.

Protein: Polypeptides that occur naturally and have a definite 3-D

structure under physiological conditions(> 50 amino acids ).
Very difficult to obtain by synthetic means.
Structure

Primary structure:
Order of the individual amino acids
Secondary structures: (Regular structure)

Coiled α-helix and β-pleated sheets and β-turns

characterized by hydrogen bond(s) between NH and C=O

groups
Tertiary structure: (3-D structure)

Chain folding, arising from crosslinking through hydrogen

bonding or disulfide bridges forming “Domains”
Domain

A protein domain is a part of protein

sequence and structure that can:

- Evolve,
- Function, and
- Exist

independently of the rest of the

protein chain
Quaternary structure (the protein):

Arrangement of multiple folded protein molecules in a multi-

subunit complex

Aggregation of tertiary structural units.

Protein stability assessment: Implies knowledge of 1ry, 2ry, 3ry
and 4ry should be known
 Classification of Proteins

• According to their biological roles

- Enzymes – Catalyses virtually all chemical reactions i.e. 6GDH
- Transport proteins i.e. Haemoglobin of erythrocytes
- Contractile or Motile proteins i.e. Actin and Myosin
- Structural proteins i.e.Collagen
- Defense proteins i.e. Immunoglobulins and Antibodies
- Regulatory proteins i.e. insulin
- Nutrient and storage proteins i.e. albumin
Solution Properties of peptides and
proteins

1-Hydrophobicity.
2- Solubility.
Hydrophobicity
- Amphiphilic nature.
- Regular configuration of synthetic peptides:
 Hydrophobic core
 Regular distance between hydrophilic and hydrophobic aa

-Expression of hydrophobicity:
- Log P (heptane/ethylene glycol): -1.72 / -3.38
-Hydrophobic character relative to glycine (hydrophobic character=0)
(high positive values indicate very hydrophobic aa and negative values
indicate aa with hydrophilic characters.).

-Indices of hydrophobicity of individual a.a.

-Actual hydrophobic character is also determined by 2ry and 3ry structures
Solubility of peptides and proteins
-Vary from very soluble to the virtually insoluble proteins.
Variable solubility in physiological conditions
- solubility occurs due to hydration of hydrophilic a.a. side chains

Factors affecting protein solubility

• pH: I.P

• Temperature.CST (cataract)

• Salts (e.g. hemoglobin)

• Organic solvents ( during encapsulation).

 Solubility
Effect of pH
- pH relative to the IP
- Near IP: Increased tendency for self-association
- Away from IP: Greater electrostatic repulsion
- Protein unfolding at extreme pHs and exposure of hydrophobic core
 Isoelectric point:
It is the point at which the protein has no net charge and
therefore has agreat tendency to associate.

At a solution pH that is above the IP, the surface of the protein is

predominantly negatively charged and therefore like-charged
molecules will exhibit repulsive forces.

Likewise, at a solution pH that is below the IP, the surface of the

protein is predominantly positively charged and repulsion between
proteins occurs.

However, at the IP the negative and positive charges cancel, repulsive

electrostatic forces are reduced and the attraction forces predominate.
The attraction forces will cause aggregation and precipitation. The IP
of most proteins is in the pH range of 4-6.
As the net charge increases, the affinity of the protein
for the aqueous environment increases and the protein
molecules exert a net repulsion.

Extremes of pH cause protein unfolding which exposes

the hydrophobic non-polar groups.

The greatest disadvantage to isoelectric point

precipitation is the irreversible denaturation caused by the
mineral acids.
2- Effect of Ionic strength:
Significance
-e.g: effect of salts on hemoglobin.

-Affects choice of buffer system used to prepare proteins

Relation between protein solubility and ionic strength of solution.

- Effect of Ionic strength:
.1- Optimum ionic strength
2- At very low ionic strength: Increased solubility
 Peptization (Charge sheilding)

3- At high ionic strength: Decreased solubility

Interaction of salts with bulk water
salting-out
Increased water Surface Tension
3- Effect of Temperature
• CST for protein-water solution
 Important in manufacture and formulation
 Pathophysiological implications Phase transition
e.g. Lens opacification

4- Effect of Organic solvents

• Lowering D.E. constant and so decrease stability.

5- Effect of Polymers
• Decreased protein solubility as they attract water molecules from
the solvation layer of proteins: Liquid phase separation
 Volume exclusion
 Unfavorable interactions between polymer (PEG-high
mW) and charged protein surface
 Protein Formulation and delivery
In protein formulation, the following challenges should
be considered:
Challenges:

- Instability.

- Diffusion ( TRANSPORT)

- Rapid clearance.

- Spatial and temporal delivery

22
 1-STABILITY of PROTEINS
&
PEPTIDES
Physical instability Chemical instability

Changes in the higher- Modification of the protein

order structure via bond formation or
(secondary and above) cleavage, yielding a new
chemical entity
Denaturation
Protein deamination
Protein oxidation
Racemization
Aggregation
Proteolysis
Surface adsorption
β-elimination
Precipitation
Disulfide formation
 Stabilty Problem with Proteins
(in vitro – in the bottle)

Noncovalent Covalent

- Denaturation - Deamidation
- Aggregation - Oxidation
- Precipitation - Disulfide exchange
- Adsorption - Proteolysis
- - Racemization.
24
 Protein instability/inactivation
 Proteins/peptides inactivate by two separate pathways
◦ Conformational changes (folding) (Non-covalent)
◦ Chemical alteration (covalent changes)

For globular proteins

Native = folded 3D structure.
Minimum energy.
Biological function
 Physical instability (Non-covalent)
1- Denaturation

- Loss of the unique biologically active 3-D structure

- Reversible and irreversible denaturation

Causes of denaturation
Exposure of Increased Ionic strength and
hydrophobic temperature pH near IP
residues Organic solvents

Aggregated molecule
 How to revert denaturation

•By dialysis to remove the denaturant.

•Addition of SAA +/-dilution with water

 Noncovalent Processes
WHY:
1- due to the presence of a native 3-D conformation.
2-Amphiphile: hydrophobic inside and hydrophilic outside.

Denaturation Adsorption Aggregation Precipitation

28
3- Surface adsorption and precipitation
Adsorption on container walls leading to dosage error.

- Insulin preparations:
 Frosting, then denaturation and precipitation with
subsequent blocking of delivery port.
 Effect of large headspace.

Comment: Large head space should be avoided in insulin

preparations.
The blocking of delivery port during insulin iv admin is a
common problem.
The use of narrow and tall containers are preferred for
protein solution
2- Aggregation:

Aggregation:
• Irreversible process: interaction between denaturated
proteins.

• It is the formation of intermediates of low solubility upon

addition of denaturant (e.g.: addition of acid or salts).

E.g.: Deactivation of bovine growth hormone and γ-interferon by

addition of denaturants as acids or salts
2- Aggregation: Aggregation versus association

Compare

-Self-association in aqueous solutions oligomers

• Reversible process in appropriate solvent/add surfactants.

E.g.: zinc hexamer of Insulin is a complex of insulin and Zn which

dissolves slowly to give dimers, then monomers and so used as
Long acting.
Formulation and protein physical stabilization:

1- Prevention of adsorption
- Containers with hydrophilic surfaces (glass silylation)
- Additives to compete for binding sites on the glass surface.
• Serum albumin
• SAA (safety): poloxamers or polysorbates: SAA align
themselvesat the liquid/air interface excluding he protein
from surface.

Comment: BSA is added to insulin

during IV infusion?
Formulation and protein physical stabilization:

2- Minimization of exposure to air

- Air/solution interface is hydrophobic
- Effect of shear forces during manufacture (filtration-pump…)
- Addition of SAA e.g poloxamers can reduce denaturation)
- Stability testing (stress conditions) and assessment of
protein configuration involve shaking of protein soln for several
hours and then assessment of protein configuration.

Explain the role of poloxamer (pluronics)

during the encapsulation of growth
hormone in PLGA microparticles
3- Addition of cosolvents: (PEG- Glycerol)

Minimize denaturation by solvation

Cosolvent
Preferential Binding to
protein hydration protein surface

Cosolvent exclusion Shift the protein to a more

compact structure inhibiting
protein aggregation (as glycerol)
• Steric effect (PEG) Cosolvent
• Surface tension (sugars) Water
• Charge effects
 Thermodynamic interpretation

Addition of cosolvents causing preferential hydration

Cosolvent Increase SFE Decreased SA

Compact conformation
Rx
rh Dnase I 2 mg
Glycerol :water 50:50 4 mL???
Tris buffer pH 7 Q.s

Glycerol shift the protein to a more compact state inhibiting

aggregation
4- Optimization of pH
• ± 0.5 pH units IP
• pH 5 – 7
To avoid stability problems arising from charge
neutralization and to ensure adequate stability

Rx
rh Dnase I 2 mg
Glycerol :water 50:50 4 mL
Tris buffer pH 7 Q.s ???
Characterization of degradation
If the formulation fails to protect the protein physical
instability, therefore, we should:
- Study the pharmacology, immunogenicity, and toxicology
of the denatured or aggregated protein (Safety and efficacy)

- Soluble aggregate may affect the pharmacokinetics and

immunogenicity of the protein

- Insoluble aggregates are generally unacceptable

 INSTABILITY
Because of their instability in solution, protein
solutions are usually freeze dried.

HOW?
Procedure (Steps):
 Freeze liquid sample in container
 Place under strong vacuum
 Solvent
sublimates leaving only solid or
nonvolatile compounds
 Reduces moisture content to <0.1%
38
 Storage (Freeze Drying)
Problem of freeze drying:
1-Many proteins in lyophilized form can undergo moisture-
induced aggregation leading to reduced activity, stability and
diffusion.
2- Stress encountered during freeze drying may affect protein
stability

Solution:
The use of excipients (sugars, organic acids, as mannitol-
sorbitol, trehalose…) called cryoprotectants .
Owing to their ability to preferentially absorb water.

39
 Lyophilized proteins
- Instability in solution
- In lyophilised form many protein moisture-induced aggregation
- Lyoprotectants and cryoprotectants: low & high mW sugars which
can preferentially absorb water.

Time dependent change in solubility of

rhAco lyophilized with various conc
with sorbitol (dashed line represent
protein without sorbitol.

40
rhA solubility after a one-day incubation as a
function of sorbitol-to-rhA weight ratio

41
• Cryoprotectant:

•Example: Mannitol-sorbitol-trehalose.

•Action: Added before freeze drying to protect the protein against

moisture induced aggregation and stress encountered during freeze
drying.

•Mechanism of action: Preferential absorption of moisture.

42
 Water adsorption isotherm
• Proteins are very sensitive to moisture.
•It is then important to determine moisture sorption ability in
preformulation studies

• Effect of water content on loss of rHA

• 43
 Tutorial:
I- Comment on the following:
1- Protein secondary structure is important in determinig protein
hydrophilicity.
2- Protein solution cannot be prepared at its isoelectric point.
3- Glycerol is added to solution of protein formulation.
4-Large head space should be avoided in insulin preparations.
5- The blocking of delivery port during insulin iv admin is a
common problem.
6-The use of narrow and tall containers are preferred for protein
solution.
7- BSA is added to insulin during IV infusion.
8-PEG is frequently added to protein solutions.

44
II- Compare between protein aggregation and
association.

III- Explain the effect of Poloxamer addition

during the formulation of protein in
microparticles.

IV-Discuss the following and illustrate ur answers

with graphs:
1- Effect of salts on the solubility of proteins.
2- Effect of pH on the solubilty of proteins.

45
 Tutorial:
V-True or false:
1- Glycine is the least hydrophobic amino acid.
2- Aspartic acid (log P=-3.38) is more hydrophobic than valine
(log P= -2.08).
3- hydrophilic surfaces are preferred for protein containers rather
than hydrophobic ones.

VI- Discuss, Accelerated stability testing of proteins.

46
Protein and peptide transport

Significance (Importance): 2 main issues should be regarded

- 1-Permeability through cell membrane
hydrophilic and large cannot pass by passive diffusion or
paracellular through junctions and pores.

- 2- Diffusion coefficient (passive diffusion) from hydrogel matrix

devices and other delivery vehicles and membrane transport.

o o o o o o
o o o o o
o o o
o

47
Factors affecting protein transport:
- Partition coefficient: Positive correlation between permeability
coefficient “P” and partition coefficient “K” between heptane and
ethylene glycol.

- Hydrophilicity and breaking of hydrogen bonds.

- Molecular size, shape and interaction with solvent molecules:

Large molecules cannot diffuse and hence needs (Permeation
enhancers.)

- Change in shape during membrane transport in lipid environment:

Change in shape can affect protein stability and activity

48
 Administration
Hence, They are mostly administered via Parentral route.

- For non-Parentral administration

- Major challenge: Poor permeability of water-soluble and hygroscopic

macromolecules in non parenteral dosage forms.

Solution:
- Permeation enhancers to provide self-administered formulations with
improved safety and potentially reduced cost (non-parentral)
- Mixed micelles with peptides and proteins

49
 General Classification of Permeation Enhancers

Class Effectiveness Safety and Examples

Recovery
I Strong and rapid Relatively safe due to fast Fatty acids such as oleic, capric, linoleic
action recovery rate acids, and their monoglycerides

II Moderate and Relatively safe due to fast Bile salts such as cholate, taurocholate
fast activity recovery rate and derivatives,

III Strong to May exhibit tissue Surfactants such as sodium dodecyl

moderate disturbance because sulphate (SDS), Triton X-100,
activity these molecules tend to Chelating agents such as EDTA.
remain associated with
cells
IV Moderate Comparatively safe but Ethanol, dimethyl sulfoxide (DMSO),
activity and may produce systemic dimethyl formamide (DMF).
remained as side effects at high
water miscible concentration
solvents

50
 Novel Protein Delivery Strategies
Permeation Enhancement

- Mixed micelles with peptides and proteins: aggregates formed from

amphiphiles (SAA) in presence of proteins.
- Protection against metabolic processes and protein denaturation besides
enhancing permeation via the following mechanism:

51
 Mixed micelles
- In aqueous environment, at high conc of enhancers (amphoteric
cpds), they form micelles that momentarily disrupt and permeate
the cell membrane allowing the passage of proteins.
- At low conc of permeation enhancer, they form soluble monomers
instead of micelles, these are membrane inactive and allow the cell
membrane to return to its normal state and resume its role as a
physiological barrier.
 Examples:
1-Interferon

• Rectal administration of interferon with permeation enhancers is

distributed directly to lymph nodes compared to IV providing a
unique opportunity for interferon use in cancer therapies.

•Intravenously administered interferon cannot preferentially distribute

in the lymph and blood concentrations are always higher than lymph
concentrations.

53
 Example 2

Cyclosporine

Anticancer polypeptide
Formulation: oral emulsion (Neoral) with permeation
enhancers (cremophore & polysorbate)
Inhibit p gp thereby improving BAV (2 times).

54