Stability and Formulation of Biologics Flashcards
Why are Biologics so sensitive?
Because the structure gets disrupted by physical (handling, agitation) or chemical (pH, oxygen) changes
What are the chemical factors that cause protein instability?
-Proteolysis: proteases cutting peptide bonds
-Deamidation: pH and temperature-driven -> loss of -NH2 to aspartic acid or iso-aspartic acid
-Oxidation: O2-driven (catalyzed by metal ion)
-> Methionine to Metsulfoxide
-> Cysteine: -SH groups into -S-S- (more in extracellular space; inside the cell are more antioxidants neutralizing free radicals)
-Disulfide bridge scrambling: change of -S-S- position with an adjacent -SH group
-Isomerization: aging-driven: L to D-form -> change the 3D structure (stereoisomer: essential functional group won’t fit to the receptor anymore bc it is positioned in the opposite direction)
What are physical factors that cause protein instability?
-Disturbance of the tertiary structure by stress/shearing
-Stress: pH, temp., light, oxidation, mechanical, freeze/thaw, buffers
-f.e. shaking -> hydrophobic regions get exposed to water and will come closer together until they form aggregates
What are the physical challenges for biologics in syringes?
(Sources of Instability)
-Silicone oil (lubricant): after applying shear the hydrophobic regions get exposed to water -> now they bind to the hydrophobic silicon oil to hide from the water -> triggering aggregation
-Tungsten (metal in the needle) leaching out of the needle -> contaminates the drug
-Small needles: the smaller the radius, the greater the force that needs to be applied to move the liquid -> disrupt protein structure -> AGGREGATION
(Poisieulles equation: small diameter, more resistance, more force needed)
Why do misfolded proteins adsorb to the glass instead of water?
Because the surface tension between the glass and the protein is less than the surface tension between the protein and water
Why are biologics gently swirled rather than shaken?
-shearing exposes the core
-when exposed to water proteins are more stable on the glass interface or when they clump together (hydrophobic regions coming together)
-shaking triggers AGGREGATION
What are the consequences of Aggregation?
-Loss of potency -> Proteins are not able to bind to a substrate in the aggregated form
-Aggregates trigger anti-drug-antibodies (immune reaction)
-> ADAs can neutralize the drug, as well as the native secreted protein (insulin ADAs neutralizing native insulin)
What is the role of surfactants and inorganic salts in biological drugs?
SURFACTANTS
-surfactants have a hydrophilic and hydrophobic end
-they create a layer between the surface of the liquid and the glass of the vial -> reducing the surface tension and instability -> preventing aggregation
INORGANIC SALTS
-adjusting tonicity
Role of sugars and amino acids in a biological drug formulation
-Amino acids: Inhibit aggregation (L-arginine), buffer (L-histidine), antioxidant, specific interaction with proteins
-Sugars: increase interfacial tension in lipid bilayers -> providing protection (f.e. in lyophilization), increase solubility
Role of Proteins and Antioxidants
Proteins like Albumin or Gelatin prevent adsorption of the biological drug, bc they will adsorb to the plastic surface of the vial instead of the drug
Antioxidants: chelating agents (binding to metal ions) preventing oxidation
How does pH affect the Solubility of a protein drug?
-pH changes the ionization of specific functional groups of the protein drug
-> Change the Solubility
->may form interactions they are not supposed to
EXAM: Understand the graph
Why does the concentration of the growth hormone changes with pH?
Depending on the pKa and pH a functional group may or not be ionized
At which point is a protein drug the least ionized?
At its Isoelectric point, where the protein has no charge
Which pH would be chosen if the solubility is high at pH 3 and pH 6 if a soluble drug is preferred?
-pH 6 because it is closer to the physiological pH 7
-use a buffer that keeps the drug at the preferred pH!
Why do non-glycosylated proteins tend to aggregate, and how can drugs be formulated to counteract aggregations?
-Because glycosylation has OH groups (polar) allowing them to interact with water -> non-glycosylated proteins interact less with water -> so they are more prone to clump together and aggregate
-amino acids like Arginine (Alteplase) improve solubility (MOA is unknown)
