2 - Formulation of Biologics Flashcards
(41 cards)
1
Q
Describe the 3 steps of formulation of biologics
A
- Sterilization – products; facilities
- Decontamination/ clearance – pyrogen removal
- Stability – physical, chemical, process (remove water)
2
Q
Describe what sterilization techniques are used for biotechnological products
A
- *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
3
Q
Describe sterilization of facilities to produce biotechnological products
A
- 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)
4
Q
Define pyrogens. They have a high ___ charge, which helps them _____
A
- 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
5
Q
Describe how endotoxin levels are measured. What level can cause sx in humans and what are the maximum levels for drugs?
A
- 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
6
Q
Describe how a pyrogen-induced fever occurs
A
Bacteria/ viruses/ endotoxins -> phagocytic cells -> prostaglandin E2 (PGE2) -> hypothalamus -> elevated temp set-point -> vasoconstriction and shivering
7
Q
What are the 2 tests for pyrogen detection?
A
- 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)
8
Q
Pyrogen removal from biologics
A
- 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)
9
Q
Describe ion exchange chromatography for pyrogen removal
A
- 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
10
Q
Describe pyrogen removal from equipment
A
- 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
11
Q
Examples of physical instability
A
- Denaturation
- Adsorption
- Aggregation
- Precipitation (makes crystals/ complexes, ex: long acting insulin; usually is a problem)
- Association w/ hydrophobic residues
12
Q
What do proteins have a tendency to do? What can cause this?
A
- 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
13
Q
What influences different aggregation states?
A
- pH
- Insulin concentration
- Ionic strength
- Specific excipients (Zn2+, phenol)
14
Q
Describe and give examples of anti-adsorption and anti-aggregation agents
A
- 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
15
Q
Native insulin in solution is in an equilibrium state between ____ forms
A
Monomeric, dimeric, tetrameric, and hexameric
16
Q
How can fibrillar precipitates be prevented? Why is this needed?
A
- Low concentrations of phospholipids and surfactants, and proper pH inhibit fibrillar precipitates
- Needed b/c many proteins can form fibrillar precipitates
17
Q
Give an example of irreversible aggregation
A
Adsorption to the hydrophobic interface = irreversible aggregation in the adsorbed protein films
18
Q
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?
A
- 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)
19
Q
What can be used to adjust tonicity of parenteral products?
A
- Saline solution
- Mono or disaccharide solution (glucose or dextrose)
- Sugars and polyhydric alcohols can stabilize protein structure
20
Q
Give examples of additives to improve physical stability and why they help
A
- 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
21
Q
Caution of excipients
A
- Multivalent products – excipients aren’t always compatible w/ all the active ingredients
- Animal sourced excipients (ex: albumin)
22
Q
What causes chemical instability? Which amino acids are readily oxidized?
A
- 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
23
Q
Ways to improve chemical stability
A
- 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
24
Q
What can cause process instability?
A
- 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)
25
What are the 3 stages of the freeze-drying process?
- Freezing step
- Primary drying step
- Secondary drying step
26
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
27
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
28
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
29
Is there any "free and fluid" water before the primary drying phase of freeze drying?
No
30
___ C is a typical freezing temp before sublimation is initiated through pressure reduction
-40 C
31
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
32
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)
33
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
34
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
35
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
36
How are pyrogens produced?
Endotoxins are shed from gram-negative bacteria (lipopolysaccharides)
37
How is physical instability prevented in biologics? Give examples
Excipients (ex: solubility enhancers, anti-adsorption and anti-aggregation agents, buffer components, osmotic agents)
38
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
39
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
40
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
41
How can oxidative degradation of amino acids be prevented?
- Replacement of oxygen by inert gases
| - Addition of antioxidants (ascorbic acid, acetylcysteine)
