Stability of Drug Products

Shelf-Life and Stability of Drug Products

Learning Outcomes

• Explain the meaning of a product's shelf-life.
• Discuss stability factors affecting shelf-life.
• Describe common chemical degradation reactions.
• Identify types of drugs likely to undergo different degradation pathways.
• Discuss the stability of protein formulations.

Shelf-Life

• Products have expiry dates.
• After compounding or manufacturing, products change over time.
• Shelf-life dictates the expiry date and is the period after manufacture during which the product is expected to perform as intended, within specified limits, when stored under recommended conditions.
• The expiry date specifies the exact date after which a particular batch cannot be guaranteed to be safe and effective.

Stability

• Ideally, drug products should have a long shelf-life, indicating good stability.
• Three types of instability:
  • Chemical degradation of the drug or excipients (chemical stability).
  • Microbiological contamination (microbiological stability).
  • Physical changes (physical stability).
• The shelf-life of a product may be limited due to chemical, microbiological, or physical instability reasons.

Microbiological Stability

• Measures resistance to microbial (bacterial or fungal) contamination during storage and use.
• Even a few microbes introduced may grow and multiply, compromising safety.
• Likely in products with high water content.
• Certain products, such as injections and eye drops, must be sterile throughout their shelf-life.
  • Single-dose units: no preservative.
  • Multiple doses: contain preservative.

Physical Stability

• Examples of physical instability in various dosage forms:
  • Coated tablets: Cracks, mottling, tackiness in coating.
  • Uncoated tablets: Cracks, mottling, swelling, discoloration.
  • Dry powders: Caking into a hard mass.
  • Solutions: Precipitation or formation of gases, or evidence of microbial growth.
  • Creams: Emulsion breakage, crystal growth, shrinking due to evaporation.
  • Ointments: Change in consistency, formation of granules, presence of liquid.
  • Suppositories: Excessive softening, dryness, or hardening.
  • Hard or soft gelatin capsules: Hardening or softening of the shell.
  • Suspensions: Caked solid phase, presence of large crystals.

Chemical Stability

• Chemical degradation of the active drug:
  • Leads to loss of dose.
  • May produce toxic products.
• Chemical degradation of excipients can also occur.
• Can lead to changes in appearance (e.g., color) and therapeutic effect.
• Shelf-life is typically defined as the time during which the drug concentration remains at 90-95% of its original concentration.

Chemical Decomposition

• Common chemical decomposition pathways include:
  • Hydrolysis
  • Oxidation
  • Isomerization
  • Photochemical degradation
  • Polymerization

Hydrolysis

• Very common degradation pathway.
• Chemical groups susceptible to hydrolysis are derivatives of carboxylic acids, including:
  • Esters
  • Amides
  • Lactones
  • Lactams
  • Imides
  • Carbamates

Controlling Hydrolysis

• Hydrolysis often involves general acid or base catalysis, so maintaining an appropriate pH is crucial.
• Altering the dielectric constant of the solution by adding alcohol, glycerol, or propylene glycol can help.
• Since only the portion of the drug in solution hydrolyzes, suppressing degradation by making the drug less soluble (e.g., using a suspension) can be effective.
• Forming a complex (e.g., caffeine and benzocaine).
• Solubilization of the drug by surfactants.
• Modification of the chemical structure (as long as the desired effect is retained).

Oxidation

• After hydrolysis, oxidation is the next most common degradation pathway.
• Oxidative degradation can occur by autoxidation (involving oxygen).
• May involve chain processes consisting of three concurrent reactions:
  • Initiation: Free radicals form from organic molecules due to heat, light, or transition metals (e.g., copper, iron) present in some buffers.
  • Propagation: Molecular oxygen combines with the free radical.
  • Termination: The reaction proceeds until all free radicals are destroyed.

Oxidation - Functional Groups

• Susceptible functional groups:
  • Unsaturated carbon-carbon bonds (e.g., alkenes).
  • Phenols (e.g., phenols in steroids).
  • Catechols (e.g., catecholamines like dopamine, isoproterenol).
  • Ethers (R-O-R', e.g. diethylether).
  • Thiols (RCH₂SH, e.g. dimercaprol (BAL)).
  • Thioethers (R-S-R', e.g., phenothiazines like chlorpromazine).
  • Carboxylic acids (RCOOH, e.g., fatty acids).
  • Nitrites (RNO₂, e.g., amyl nitrite).
  • Aldehydes (RCHO, e.g., paraldehyde).

Stabilization Against Oxidation

• Replacing oxygen in the pharmaceutical container with N2 or CO2.
• Storage at reduced temperatures.
• Use of antioxidants:
  • Agents that interrupt propagation by interfering with free radicals (e.g., ascorbic acid).
  • Agents that preferentially oxidize (e.g., sodium bisulfite).
  • Chelating agents (e.g., EDTA), which form complexes with heavy metal ions required to initiate oxidation reactions.

Isomerization

• Conversion into optical or geometrical isomers.
  • Adrenaline at low pH changes from the active form (L isomer) to the less active (D-isomer).
  • Tetracycline undergoes epimerization to 4-epi-tetracycline.
• Some isomers do NOT have the same therapeutic effect.

Photochemical Degradation

• The drug can absorb UV light in the range of incident light and degrade.
• Excipients in the formulation may absorb light (photosensitizers) and transfer the absorbed energy to the drug, causing it to degrade.
• Leads to loss of effect and change in appearance.
• Can occur during storage and use.
• Need to assess photostability in the final product.
• Stabilization against photochemical degradation:
  • Use colored glass and store in the dark.
  • Coat tablets with a polymer film containing UV absorbers.

Polymerization

• Example:
  • Dimerization and hydrolysis of ampicillin involving the opening of the lactam ring and the formation of amide links.
  • Refer to Scheme 3.12 for a detailed illustration.

Formulation of Protein Drugs

• Very challenging due to the delicate nature, large molecular weight, and numerous functional groups of proteins.
• Proteins exhibit both physical and chemical instability.
• Need to preserve the protein’s native conformation during processing and storage.
• Changes in the 3D structure caused by physical or chemical degradation render the protein therapeutically inactive.

Overview of Protein Stability

• Chemical Instability:
  • Deamidation
  • Oxidation
  • Hydrolysis
  • Racemization
  • Disulfide exchange
  • Beta Elimination
• Physical Instability:
  • Denaturation
  • Surface Adsorption
  • Aggregation
  • Precipitation

Chemical Structure of Proteins

• Proteins are composed of amino acids.
• Primary structure refers to the amino acid sequence.
• 20 normally occurring amino acids are found in proteins.
• All amino acids have an amino group and a carboxyl group.
• Amino acids are classified as acidic/basic or polar/nonpolar based on their side chains.
• Peptides and proteins are formed by peptide bonds between the NH_2 and COOH groups on amino acids.
• Molecules with >50 amino acids are called proteins; <50 are called peptides.

Chemical Instability of Proteins

• Proteolysis
• Oxidation
• Deamidation:
  • Asparagine and glutamine can be deamidated.
• Racemization:
  • Can occur for all chiral amino acids; can change bioactivity.
• Disulfide formation:
  • Interchange of disulfide bonds can result in altered 3D structure.
• Beta elimination:
  • Thermal stress can destroy disulfide bonds.

Protein Structure and Stability

• The presence and position of an amino acid in the sequence determine whether it occupies the surface or core of the protein.
• Influences the overall structure of the protein.
• Ionizable and polar amino acids tend to occupy the surface.
• Nonpolar side chains cluster to form a hydrophobic core.

Physical Instability of Proteins

• A potential problem for peptides and proteins.
• Caused by changes in the protein’s conformation, referred to as denaturation.
  • Alteration of the tertiary and frequently secondary structure of globular proteins from their native conformation.
• Possible outcomes of protein and peptide instability are:
  • Surface adsorption
  • Aggregation
  • Precipitation

Denaturation of Proteins

• How easily a protein denatures depends on the forces keeping the protein in its native conformation.
• Hydrophobic residue interactions play a critical role.
• Changes in temperature, pH, and the presence of organic solvents or denaturants can interfere with bonding forces and push the protein towards an unfolded, denatured state.
• Denaturation can be reversible or irreversible.
• As a protein denatures, internal hydrophobic residues are often exposed to the solvent.
• These residues can interact with nonpolar surfaces such as container surfaces, leading to surface adsorption.

Surface Adsorption of Proteins

• The physical and chemical nature of both the molecule and the surface govern the type and extent of adsorption.
• Altering the ionic strength and pH of the media can enhance or reduce the tendency to adsorb.
• Adsorption is usually greatest near the protein’s isoelectric point.
• The extent and reversibility of protein-surface interactions are dependent on time, temperature, and agitation.
• Often, prolonged exposure to surfaces, high temperatures, and agitation cause irreversible loss of proteins.

Surface Adsorption and Protein Concentration

• Surface adsorption is also determined by the available surface area.
• Once a closely packed monolayer is formed on the surface, the adsorption process is saturated.
• Further loss occurs only by surface-induced denaturation in the bulk of the solution.
• Product loss is negligible when there is a high protein concentration.
• At low concentrations (e.g., insulin), a significant proportion can be lost due to adsorption.

Reducing Surface Adsorption

• Once an adsorbed protein dissociates from the surface, its denatured conformation could result in aggregation.
• Reduce this by:
  • Incorporating serum albumin in the formulation to compete for surface-active sites.
  • Using surfactants such as copolymers of ethylene oxide (Pluronics) and polysorbates (Tweens, Triton X 100), which can be effective in preventing adsorption.

Aggregation and Precipitation of Proteins

• Often the end product of protein instability and denaturation.
• Resulting aggregates often lose bioactivity.
• Also have other undesirable effects such as increased immunogenicity, altered pharmacokinetics, pharmacology, and toxicology.
• When aggregation occurs on a macroscopic scale, it can cause blockage of tubing and pumps delivering the drug.
• Can occur during ordinary product handling.
• Contact with hydrophobic air interfaces, shearing, shaking during shipment, and passage through needles can result in denaturation and self-association.

Formulation and Stabilization of Proteins

• Optimize the pH:
  • Select a pH at least 0.5 units above or below the isoelectric point to ensure adequate solubility and avoid charge neutralization.
  • Difficult to achieve as a pH range of 5-7 is usually required to minimize chemical breakdown, and this frequently coincides with the isoelectric point.
• Adding a cosolvent:
  • PEGs, glycerol:
  • Either cause preferential hydration of the protein or bind to the protein surface.

Temperature, Agitation, and Additives Effect on Protein Stability

• Temperature and agitation:
  • Denaturation is accelerated by heat (thermal denaturation) and agitation (mechanical denaturation).
  • Susceptibility is influenced by temperature, presence of water, and other additives.
• Refrigerate and use appropriate additives such as salts, sugars, and glycerol.
• Do not freeze, as the physical environment changes, and stresses involved impact protein stability.
• Can lyophilize (freeze-drying) to afford a solid product:
  • Use cryoprotectants (e.g., glycine or mannitol) to protect proteins.