PROTEIN FORMULATION AND

STABILIZATION

SCHOOL OF PHARMACY ITB

 PROTEIN
STRUCTURE &
FUNCTION
Solubility:
- pH
- electrolyte
 PROTEIN INSTABILITY
PHYSICAL INSTABILITY (a change in the
secondary, tertiary, or quaternary structure):
 Denaturation  Aggregation *)
 Precipitation  Adsorption to surfaces

CHEMICAL INSTABILITY (covalent modification

of the protein via bond formation or cleavage):
 Hydrolysis Deamidation *)
 Oxidation *) Racemization
 Disulfide formation/exchange
 β-elimination
PROTEIN INSTABILITY
 PHYSICAL INSTABILITY
 Unfolding of stable forms can lead to adsorption,
aggregation or further chemical reactivity.
 Aggregation can lead to precipitation.
 Loss of the unique biologically active three-
dimensional structure (i.e. denaturation), can be
caused by:
 heating
cooling or freezing,
 extremes of pH,
 contact with organic chemicals and denaturants
 1. DENATURATION: reversible, irreversible

Denaturation refers to a disruption of the

tertiary and secondary structure of the protein
molecule.

Denaturation must first occur before the other

processes such as aggregation, adsorption and
precipitation can proceed
 1. DENATURATION: reversible, irreversible
Unfolding : a sharp transition in structure from native
to an unfolded state at the melting temperature.
The melting temperature Tm : the temperature at
the midpoint of unfolding at which 50% of the
molecules are completely unfolded.

U* = unfolding intermediate;
Liquid crystalline state

X = a collection of inactive molecules (e.g. aggregated

protein)  blocked from changing to N or U
 1. DENATURATION: reversible, irreversible
To recover the native state of a misfolded
protein  add Denaturants such as
GuanidineHCl/urea, followed by Dialysis

Reversible unfolding protein:

 typically small (<30 kDa),
 dilute (<1 mglml)
 highly charged to inhibit aggregation
 2. AGGREGATION
Soluble & insoluble aggregates; from originally 30-50 A
coagulate into 0.1-0.2μm primary particles then
floculate to form 10-20 μm :
 Loss biological activity
 Blockage of infusion set
 Increase in effective MW  more immunogenic

Two steps of protein aggregation:

1. Protein unfolding: exposing the buried
hydrophobic amino acid
2. Association of unfolded protein
 2. AGGREGATION
Factors influence aggregation:
1. Energy input
2. The change of pH, salt concentration,
solvent composition

Sources of energy input in protein unfolding:

 Temperature increase
 Radiation
 Ultrasound
 2. AGGREGATION
INCLUSION BODY (IB): during over production
Reducing condition in E.coli cytoplasm is unsuitable for
disulfide bond formation
 aggregation of partially folded/misfolded.
urea/GdHCl+dithiothreitol IB dissociation&
solubilization
refolded to the correct native structure

Aggregation must be avoided during refolding=

PEG = bind the unfolded form & be preferentially

excluded from a folded native
Protein stabilization: cosolvent PEG, glycerol
 3. ADSORPTION
At unfold form, the hydrofobic part interact with the
surface  adsorption  can be followed by
aggregation

Adsorption is determined by:

 primary sequence of protein
 Size : higher MW tends to be adsorp at multiple
contact
 A disulfide proteins are less likely to unfold  less
surface active
 Charge interaction of polyelectrolite depends on
pH, ionic strength
 3. ADSORPTION
Competition of proteins (BSA/hIgG) and surfactants
(Tween 80 or lechitin) on the surface

Adsorption higher at air-water interface as compared

to solid-water interface  shaking allows large scale
denaturation/aggregation.

Adsorption at oil-water interface i.e polymixin to lyse

bacterial membrane.
 3. ADSORPTION
Adsorption losses are very significant for dilute
solutions but become less of a concern at
concentrations higher than 5 IU/ml (-0.2 μg/ml, -0.03
mM).

Insulin adsorption may be minimized by the addition of

0.1 to 1% albumin

The adsorption rate of Lysozyne increase as the pH

close to pI. Addition of salts, the adsorption of
lysozymes at the air-water interface increased when
the electrostatic interactions decrease.
 PROTEIN LYOPHILIZATION

Removal of the water shell

influence thermodynamic of protein
residual water content for proteins should
be optimized
 to maintain the correct folded structure of
protein
 PROTEIN LYOPHILIZATION
3 stages of freeze drying process:
1. FREEZING : local concentrate of excluded excipient
salts  the change in ionic strength; the components
of mixed buffers may selectively crystalized that shift
the pH  lead to exposure of buried hydrophobic
residues  increase in aggregation (may be
irreversible on reconstitution)
2. PRIMARY DRYING at lower temperatures  to
remove most of water
3. SECONDARY DRYING at ambient or higher
temperatures to minimize the final unbound water
content
First, water immobilized in the sample as ice crystals or
bound with amorphous components of the sample at a
glass state.

Cryoprotectant = to increase galss transition (Tg)

e.g Saccharides
 PROTEIN FORMULATION/STABILIZER
1. pH/Buffers
Buffers should be kept to a minimum
concentration to maintain the pH
Mixed buffer systems, such as phosphate,
should be used cautiously as pH shifts can occur
on freezing
Histidine, citrate, or Tris buffers being
preferable.
 PROTEIN FORMULATION/STABILIZER
2. Salts
Inorganic salts reduce the glass transition
temperature (Tg′) of the formulation
limit the temperature at which primary drying can
be performed
salt concentrations should be reduced to a
minimum temperature at which primary drying can
be performed.
Alternative osmotic modulators can be selected that
have less impact on the Tg′ should be preferred.
 PROTEIN FORMULATION/STABILIZER
3. Stabilizers
Hydrogen bond-forming sugars, such as trehalose or
sucrose as excellent stabilizers for freeze-drying of
proteins.
Other stabilizers may be more specific in their effects
(such as metal ions, e.g., zinc)

mannitol or glycine, are valuable as matrix

formers without offering the stabilizing benefits
of sucrose/trehalose
 PROTEIN FORMULATION/STABILIZER
4. Protein concentration
Proteins themselves are often common choices as
stabilizers for use in formulating for freeze-drying for
low concentrations of recombinant proteins
i.e Albumin:
 It has a high Tg′, is a good cake-former
 Blocks nonspecific binding of proteins to glass
surfaces,
 Protein stabilization

Improvement of thermal stability, cause self-association

and modulate solubility by the addition of:
1. sugars (trehalose, sucrose, maltose or glucose)
2. salts (potassium phosphate, sodium citrate or
ammonium sulphate),
3. heparin

 Chelating agents (EDTA)  form complexes with metal

dependent proteases/peptidases  reduce catalytic
degradation processes
 Protein stabilization

Cyclodextrins, in particular hydroxypropylcyclodextrins

with unclear stabilization mechanism

 Non-ionic surfactants (Pluronic F 68) stabilizes against

self-aggregation

 Cationic (cetrimide) and anionic (SDS) surfactants

facilitate absorption of peptides and proteins across
biological membranes
 OXIDATION
Amino acids that may undergo oxidation:
 Methionine  cysteine  triptophan
 Histidine  tyrosine.

Sources of oxidation:
 peroxide: excipient impurities in
polysorbates surfactan, PEG
 Metal ion
 light
 base
 free-radical initiator
 OXIDATION

The solving:
 Antioxidant  reducing agent may
induce degradation in long run
 Packed under N2
 EDTA
DISULPHIDE FORMATION /EXCHANGE
OXIDATION –β ELIMINATION
 HYDROLYSIS

Peptide cleavage at acidid solution:

 Aspartame  proline
HYDROLYSIS
 DEAMIDATION

The amide groups of asparaginyl or glutaminyl

(Asn and Gln) residues are labile
at extremes of pH  may be hydrolyzed easily
to a free carboxylic acid.

Deamidation of As residue > Gln residue

Deamidation occurs at neutral – alkaline pH

DEAMIDATION
 DEAMIDATION

The rate of deamidation is determined by:

 primary, secondary, tertiary structure
 Temp
 pH
 Buffer species
 Ionic strength
 Special intermolecular interaction
THANK YOU