2 - Formulation of Biologics Flashcards
Describe the 3 steps of formulation of biologics
- Sterilization – products; facilities
- Decontamination/ clearance – pyrogen removal
- Stability – physical, chemical, process (remove water)
Describe what sterilization techniques are used for biotechnological products
- *Can’t use heat to sterilize
- Filtration techniques for removal of mycobacterial contaminants
- Pre-filtration removes the bulk of bio burden and other particulate materials
- Sterilizing filtration is filtered through 0.2 or 0.22 um membrane filters
Describe sterilization of facilities to produce biotechnological products
- Think of 3E – equipment, excipient, environment
- Autoclave by dry heat (> 160 C for 30 min) – must be dry heat; sealed room w/ airflow into the room through a filter
- Chemical tx (use if can’t remove equipment)
- Gamma radiation (use if can’t remove equipment)
Define pyrogens. They have a high ___ charge, which helps them _____
- Any substance that can cause a fever (bacterial, viral, and yeast)
- High negative electrical charge
- Tendency to aggregate
- Form large units w/ MW of over 10^6 in water
- Tendency to absorb to surface
- These are properties we can play w/ to help remove them
Describe how endotoxin levels are measured. What level can cause sx in humans and what are the maximum levels for drugs?
- Endotoxin levels measured in “endotoxin units” EU
- 1 EU is ~ equivalent to 100 pg of E. coli lipopolysaccharide – the amount present in around 10^5 bacteria
- As little as 5 EU/kg body weight can cause human sx – fever, low BP, increased HR, low urine output
- Maximum permissible endotoxin levels for drugs distributed in US by FDA:
- Drug (injectable, intrathecal) = 0.2 EU/kg body weight
- Drug (injectable, non-intrathecal) = 0.5 EU/kg body weight
- Sterile water = 0.25-0.5 EU/mL depending on intended use
- Even small doses of endotoxin in the bloodstream are often fatal
Describe how a pyrogen-induced fever occurs
Bacteria/ viruses/ endotoxins -> phagocytic cells -> prostaglandin E2 (PGE2) -> hypothalamus -> elevated temp set-point -> vasoconstriction and shivering
What are the 2 tests for pyrogen detection?
- Rabbit test – rabbits have similar endotoxin tolerance (temperature) to humans but can’t quantitate
- Limulus amebocyte lysate (LAL) test
- Horseshoe crab blood forms clots when exposed to endotoxins so amoebocyte extract from horseshoe crab blood is mixed w/ samples to determine pyrogen levels
- Fast (~ 30 mins) and highly sensitive (up to 0.005 EU/mL sensitivity)
Pyrogen removal from biologics
- Aggregated endotoxins can be removed by activated charcoal
- Materials w/ large surfaces offering hydrophobic interactions (will bind to the charcoal, centrifuge the charcoal and we can separate the protein from the charcoal; this is then followed by an ion column in industry)
- Endotoxins can be removed by ion exchange chromatography (negative charge of pyrogen; won’t work if protein is also negatively charged)
Describe ion exchange chromatography for pyrogen removal
- Mix of protein and pyrogen flow through tube w/ immobilized cation surface
- Negatively charged pyrogens bind to immobilized cation surface
- Proteins flow through, separating proteins from pyrogens
Describe pyrogen removal from equipment
- Acid-base hydrolysis – can cleave Lipid A from the polysaccharide in the LPS molecule
- Oxidation – hydrogen peroxide is a low-cost pyrogen destroying solution and can be easily removed
- Heating – dry heating (250 C for 30 min) results in a 3log reduction of endotoxin levels
- Sodium hydroxide – used to clean ion exchange column after each batch
Examples of physical instability
- Denaturation
- Adsorption
- Aggregation
- Precipitation (makes crystals/ complexes, ex: long acting insulin; usually is a problem)
- Association w/ hydrophobic residues
What do proteins have a tendency to do? What can cause this?
- Non-glycosylated proteins have tendency to aggregate and precipitate
- Formation of aggregation can be due to:
- Hydrophobic and/or electrostatic interactions between molecules
- Formation of covalent bridge between molecules through disulfide bonds and ester amide linkages
What influences different aggregation states?
- pH
- Insulin concentration
- Ionic strength
- Specific excipients (Zn2+, phenol)
Describe and give examples of anti-adsorption and anti-aggregation agents
- Anti-adsorption agents reduce adsorption of the active protein to interfaces
- Hydrophobic sites of proteins (in the core of the native protein structure) bind when an interface is present => forms irreversible protein film on surfaces such as:
- Water/air
- Water/container wall
- Interfaces between the aqueous phase and utensils used to administer the drug (ex: catheter, needle)
- Albumin to prevent adsorption of insulin to the interface
- Lysine and arginine too small to affect protein adsorption, so we use albumin (large protein); works well
Native insulin in solution is in an equilibrium state between ____ forms
Monomeric, dimeric, tetrameric, and hexameric
How can fibrillar precipitates be prevented? Why is this needed?
- Low concentrations of phospholipids and surfactants, and proper pH inhibit fibrillar precipitates
- Needed b/c many proteins can form fibrillar precipitates
Give an example of irreversible aggregation
Adsorption to the hydrophobic interface = irreversible aggregation in the adsorbed protein films
What properties depend on pH? What makes up buffer systems in biotech formulations? What are some examples in which temporary pH changes can cause aggregation?
- Protein solubility, physical stability, and chemical stability depend on pH
- Buffer systems in biotech formulation = phosphate, citrate, acetate
- Temporary pH changes can cause aggregation, such as:
- Freezing step in a freeze-drying process
- If one of the buffer components is crystallized and the other is not, it will change the pH
- Na2HPO4 crystallizes faster than NaH2PO4 (drop in pH during the freezing step, which could cause aggregation)
What can be used to adjust tonicity of parenteral products?
- Saline solution
- Mono or disaccharide solution (glucose or dextrose)
- Sugars and polyhydric alcohols can stabilize protein structure
Give examples of additives to improve physical stability and why they help
- Salts – decrease denaturation and aggregation by ion binding to the protein
- Polyalcohol – stabilize the product by selective solvation
- Surfactants/ absorption – prevent adsorption of protein at interfaces
Caution of excipients
- Multivalent products – excipients aren’t always compatible w/ all the active ingredients
- Animal sourced excipients (ex: albumin)
What causes chemical instability? Which amino acids are readily oxidized?
- Due to the formation of a new chemical entity by cleavage or formation of new bond (ex: oxidation, deamidation, proteolysis, disulphide exchange, racemization)
- Methionine, cysteine, tryptophan, tyrosine, and histidine are amino acids that are readily oxidized, so proteins rich in these amino acids are liable to oxidative degradation
Ways to improve chemical stability
- Appropriate choice of conditions – pH, temp, ionic strength, addition of preservatives and antioxidants
- Genetic modification of proteins
- Site-directed mutagenesis: chemically reactive amino acids are replaced w/ ones that aren’t
- Chemical modification of proteins (coupling w/ PEG)
- Preservatives are needed in containers designed for multiple injection schemes
- Bacteriostatic rather than bactericidal in nature
- Mercury-containing preservatives
What can cause process instability?
- Lyoprotectants/ cake formers
- Storage conditions
- Typical excipients in a freeze-dried protein formulation, such as:
- Bulking agents (mannitol/ glycine for elegance/ blowout prevention; need to be added for freeze drying, mannitol also used as a preservative)
- Collapse temperature modifier (dextran, albumin/ gelatin to increase collapse temp)
- Lyoprotectant (sugars, albumin for protection of the physical structure of the protein)
What are the 3 stages of the freeze-drying process?
- Freezing step
- Primary drying step
- Secondary drying step
Advantages to freeze drying of proteins
- May provide the requested stability
- Water is removed through sublimation and not by evaporation
- Ice crystal doesn’t form at the thermodynamic or equilibrium freezing point but at supercooling
- Crystallization at -15 C or lower
What happens during the crystallization step of freeze drying?
- Temp may temporarily rise in the vial due to generation of crystallization heat
- pH and ionic-strength may change
- Protein denaturation
Describe the sizes of crystals during freeze drying?
- Small crystal w/ fast cooling
- Large crystal w/ lower cooling
- Small crystals are required for porous solids and fast sublimation rates
Is there any “free and fluid” water before the primary drying phase of freeze drying?
No
___ C is a typical freezing temp before sublimation is initiated through pressure reduction
-40 C
Describe the primary drying step of freeze drying
- Crystals and water not bound to protein/excipient are removed by sublimation
- Sublimation costs energy
- Temp in vials can drop
- Supply of heat from the shelf to the vial via direct shelf-vial contact, radiation, or gas conduction
Describe the secondary drying step of freeze drying
- Removal of water interacting w/ the protein and excipients
- Temp is slowly increased to remove “bound” water and the chamber pressure is still reduced
- Residual water content is a critical endpoint for the stability of freeze-dried products (1% or less residual water in the cake is recommended)
Describe a typical freeze-drying cycle
- Loading 4-5 h
- Freezing 2-6 h
- Primary drying 10-48 h
- Secondary drying 5-20 h
- Stopping, unloading 2-6 h
- Lower chamber pressure makes it easier for water to evaporate at lower temps; as temps go up, water will become free from the protein molecules
Shelf life of protein-based pharmaceuticals
- Proteins can be stored in an aqueous solution, a freeze-dried form, or in dried form in a compacted form (tablet)
- Stability of protein solutions depend on pH, ionic strength, temp, and presence of stabilizers
Components found in a parenteral formulation of biologic
- Active ingredient
- Solubility enhancers
- Anti-adsorption and anti-aggregation agents
- Buffer components
- Preservative and antioxidants
- Lyoprotectants/ cake formers
- Osmotic agents
- Carrier system
How are pyrogens produced?
Endotoxins are shed from gram-negative bacteria (lipopolysaccharides)
How is physical instability prevented in biologics? Give examples
Excipients (ex: solubility enhancers, anti-adsorption and anti-aggregation agents, buffer components, osmotic agents)
Approaches to enhance protein solubility
- Proper pH and ionic strength conditions
- Addition of amino acids (lysine or arginine) to solubilize tissue plasminogen activator (tPA)
- Surfactants (sodium dodecylsulfate, SDS) to solubilize non-glycosylated IL-2
What affect do surfactants have on adsorption?
Surfactants adsorb to hydrophobic interface w/ their own hydrophobic groups and render interface hydrophilic to the aqueous phase
How do sugars and polyhydric alcohols stabilize protein structure?
- Enhance the interaction of solvents w/ protein
- Excluded from protein surface layer
- Protein is hydrated
- Enhance the tendency of protein to self-associate
How can oxidative degradation of amino acids be prevented?
- Replacement of oxygen by inert gases
- Addition of antioxidants (ascorbic acid, acetylcysteine)
