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Year 3 - Biotechnology > 2 - Formulation of Biologics > Flashcards

2 - Formulation of Biologics Flashcards

1
Q

Describe the 3 steps of formulation of biologics

A
  1. Sterilization – products; facilities
  2. Decontamination/ clearance – pyrogen removal
  3. Stability – physical, chemical, process (remove water)
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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
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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)
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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
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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
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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

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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)
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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)
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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
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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
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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
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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
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13
Q

What influences different aggregation states?

A
  • pH
  • Insulin concentration
  • Ionic strength
  • Specific excipients (Zn2+, phenol)
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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
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15
Q

Native insulin in solution is in an equilibrium state between ____ forms

A

Monomeric, dimeric, tetrameric, and hexameric

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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
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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
Q

What are the 3 stages of the freeze-drying process?

A
  • Freezing step
  • Primary drying step
  • Secondary drying step
26
Q

Advantages to freeze drying of proteins

A
  • 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
Q

What happens during the crystallization step of freeze drying?

A
  • Temp may temporarily rise in the vial due to generation of crystallization heat
  • pH and ionic-strength may change
  • Protein denaturation
28
Q

Describe the sizes of crystals during freeze drying?

A
  • Small crystal w/ fast cooling
  • Large crystal w/ lower cooling
  • Small crystals are required for porous solids and fast sublimation rates
29
Q

Is there any “free and fluid” water before the primary drying phase of freeze drying?

A

No

30
Q

___ C is a typical freezing temp before sublimation is initiated through pressure reduction

A

-40 C

31
Q

Describe the primary drying step of freeze drying

A
  • 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
Q

Describe the secondary drying step of freeze drying

A
  • 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
Q

Describe a typical freeze-drying cycle

A
  • 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
Q

Shelf life of protein-based pharmaceuticals

A
  • 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
Q

Components found in a parenteral formulation of biologic

A
  • Active ingredient
  • Solubility enhancers
  • Anti-adsorption and anti-aggregation agents
  • Buffer components
  • Preservative and antioxidants
  • Lyoprotectants/ cake formers
  • Osmotic agents
  • Carrier system
36
Q

How are pyrogens produced?

A

Endotoxins are shed from gram-negative bacteria (lipopolysaccharides)

37
Q

How is physical instability prevented in biologics? Give examples

A

Excipients (ex: solubility enhancers, anti-adsorption and anti-aggregation agents, buffer components, osmotic agents)

38
Q

Approaches to enhance protein solubility

A
  • 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
Q

What affect do surfactants have on adsorption?

A

Surfactants adsorb to hydrophobic interface w/ their own hydrophobic groups and render interface hydrophilic to the aqueous phase

40
Q

How do sugars and polyhydric alcohols stabilize protein structure?

A
  • Enhance the interaction of solvents w/ protein
  • Excluded from protein surface layer
  • Protein is hydrated
  • Enhance the tendency of protein to self-associate
41
Q

How can oxidative degradation of amino acids be prevented?

A
  • Replacement of oxygen by inert gases

- Addition of antioxidants (ascorbic acid, acetylcysteine)

Year 3 - Biotechnology (11 decks)

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