Formulation approaches for Protein stabilization : Formulation approaches for Protein stabilization Slide 2: 1. Protein stabilization in soln using additive.
Most stable in solution that mimic their natural environment.
e.g. 1.Stability of B-galactosidase-enhanced in presence of MILK protein.
2. Many protein like caseins that bind to ca2+ require small amt of ion to maintain their native structure.
1.PH– Buffer salt selection-guided by pH range of interest acceptability of buffer for use in medicinal pdt.
e.g. Human growth hormone, a sodium phosphate buffer concn of abt 5 mM produces less aggregation of the protein compound with solution of 2.5,10,20 mM.
e.g. Tissue plasminogen activator-solubility of protein at optimally stable pH was insufficient for therapeutic application.
So a +vely charged A.A arginine included to ↑ the solubility at desired pH range. Slide 3: 2. Oxidation : Presence of NaCl in monoclonal antibody rhu HER2
caused an ↑ in oxidation at high temp.after contect wid S.S.
Anti Oxidants :Methionine & sodium thiosulphate.
3. Non-ionic surfactants(polysorbate 80)-included in several marketed formulation & same to inhibit protein aggregation
Mechanism -Surfactant molecule align themselves at the liquid/air
Excluding protein from interface
Inhibiting surface denaturation. Slide 4: 4. Solvent Additives: co solvents
-may inhibit aggregation
Mechanism –Their preferential exclusion from protein surface.
With the cosolvent preferentially excluded
Protein surface preferentially hydrated
Key structural elements remains in their native confirmation. Slide 5: Mechanism of exclusion-divided into 2 classes.
The First class- Interactions determined by proportion of solvent.
Exclusion of the larger additive molecule compared with the smallest
water molecule. [Eg. PEG]
Preferential hydration of protein.
Additive such as sugar, A.A,many salts may act by increasing the
surface tension of water.
Leads to preferential hydration at protein solvent interface Slide 6: 2nd Class of Cosolvents- Solvophobicity and
Replusion from charges on protein surface
Additive’s preferential exclusion.
e.g. glycerol & other polyols (sorbitol & Mannitol)
Net effect of the stabilizing/destabilizing agent, depends on balance
b/w exclusion from the protein surface(stabilizing).
Propensity of additive to bind to the protein by electrostatic or
5.) Globular Proteins- ability to adsorb to the surface & to inhibit adsorption of a low concentration of therapeutic protein e.g.Albumin Slide 8: B. Protein stabilization in dried solid state
Solution formulation are not chemically & physically stable for 1 year.
Retarding degradation using low temp drying.
Lyophilization & spray drying.
Use to dehydrate heat sensitive molecules & inhibit degradative reaction that may be observed when proteins are formulated in solution.
LYOPHILIZATION & PROTEIN FORMULATION DEVELOPMENT
Lyophilization-Water is removed from a product after it is frozen & placed under vacuum allowing the ice to change directly from solid to vapor without going there a liquid phase. Slide 9: 3 Steps-
1.) freezing of the solution.
2.) primary drying where sublimation of ice water takes place.
3.)Secondary drying where tightly bound water is removed(desorption)
Tissue type plasminogen activator can withstand repeated freeze there cycles-no less in activity.
Loss in activity for lactice dehydroganase.
aggration of recombinant-45.
1. Recovery of protein activity after freezing by reducing the unfolded state can be influenced by addition of certain protective additive-sugars,polyols,aminoacid,certain salts. Slide 10: Cryoprotective agents-stabilize solution by preferential exclusion from protein surface, making it thermodynamically unfavorable for protein to unfold.
E.g. certain carbohydrates(disaccharides Such as sucrose & trehalose)-preserve phosphofructokinase during freeze drying/ air drying.
2. Formation of an amorphous glass opposed to crystaline matrix.
Additive used- Carbohydrates,polyols,polymers or mixtures thereof. Slide 11: 3. By increasing glass transition temperature of protein formulation.
4. Stabilizing protein by binding to the dried protein to act as water substitute after removal of hydration shell.
e.g. Trehalose,lactose(satisfy hydrogen bonding requirement of polar groups in dried protein.
5. Residual moisture in final products must be relatively low. Excipients – For Stabilization : Excipients – For Stabilization Sucrose
Mannitol Bulking Agent : Bulking Agent Used in lyophilized formulation.
To enhance appearance
To reduce residual moisture
Provide physical support to the cake to prevent collapse
e.g. Mannitol Spray Drying : Spray Drying Techniques- Spray drying
Spray freeze drying
Milling of lyophilized powders
Super critical fluid precipitation
Co-precipitation with polymers Slide 15: Advantages:
1.Good flow properties
2.Narrow size distribution
3.Reproducible size & shape
4. Produce particles of a range of sizes (dependent of application) Slide 16: e.g.
Trehalose stabilize protein during spray drying
Disaccharides (sucrose,trehalose)-preserve protein structure & activity in solution state.
Surfactants (Polysorbate 20; Polysorbate 80) protect proteins from harmful hydrophobic interfacial interactions.
E.g.—Addition of 0.1%w/v Polysorbate 20 to hGH reduce spray drying by 70-85%.