Biologicals Flashcards
What are biologicals?
Medicinal products which are produced by, or derived from, a living system.
Can be from a whole organism or isolated cells. Also known as biopharmaceuticals.
What is the highest selling biologic?
Humira/adalimumab treatment for RA etc.
Production of therapeutic proteins relies largely on:
Recombinant DNA methods.
How does humanisation of antibodies occur?
Phage display
Transgenic mice
Immortalised memory B-cells
What are the benefits of using yeast cells to produce therapeutic proteins? [9]
- doubling time of ~4hrs
- High concentration production
- High MW proteins possible
- S-S bridges possible.
- Secretion sometimes possible.
- Singular/native aggregation state.
- Correct folding
- No pyrogens.
- Cheaper than mammalian cell use.
Why would we not want to use yeast cells to produce therapeutic proteins? [3]
- Retrovirus use not possible
- Secretion sometimes not possible
- More expensive than bacterial cells.
What are the benefits of using bacterial cells to produce therapeutic proteins? [3]
- 0.5hr doubling time.
- High concentration production possible.
- Cheaper than yeast and mammalian cell use.
What are the disadvantages to using bacterial cells for production? [6]
- Limited MW proteins possible.
- Limited S-S
- No secretion
- Misfolding can occur
- Retrovirus use not possible
- Pyrogens possible.
What are the advantages to using mammalian cells for production of therapeutic proteins? [7]
- High MW proteins
- No limits on S-S bridges
- Secretion possible
- Singular/native aggregation state.
- Correct folding
- Retrovirus use possible
- No pyrogens.
What are the disadvantages to using mammalian cells? [3]
- Long generation time: >24hrs.
- Expensive.
- Low concentrations/yield.
What two ways can protein inactivation occur?
- Conformational changes: formation of incorrect structures, aggregation.
- Chemical oxidation and other chemical changes see flashcards for biopharmaceuticals.
What are the possible consequences of aggregation/denaturation of protein product? [4]
- Altered formulation stability.
- Hyper or hypo-potency
- Off target binding, increase in side-effects, clearance.
- Patient may generate ATAs: neutralising antibodies: making drug ineffective, break in tolerance, cross-reactivity with endogenous proteins.
What types of aggregates exist?
Soluble and insoluble.
How do soluble aggregates differ to insoluble ones?
- Soluble are normally low in number and visually undetectable.
- They cannot be filtered out with 0.2micrometer filters.
- Soluble aggregation is reversible by dilution/pH.
How are insoluble aggregates different to soluble aggregates?
- Insoluble aggregates = more individual proteins, visually present as turbidity.
- Can generally be removed by filtration.
- Often irreversible, non-covalent can be dissociated by detergents.
How is the formation of soluble aggregates often reversed?
Dilution or changing the pH.
How can aggregation be precipitated? [8]
- Freezing and thawing.
- Agitation (interface formation)
- Sonication
- Contact with silicone oil.
- Low or High pH
- Low or High Salt
- Specific salts
- Heat
What are lyophilised formulations of drugs? [4]
- Freeze-dried protein with excipients.
- Shipped/stored at ambient temps.
- Requires reconstitution to take place and time allowed for full dissolution.
- Air entrapment can occur and agitation during and after dissolution.
Describe the liquid formulations of protein drugs:
- Shipped and stored at 2-8*C
- Volume required directly withdrawn, no need for reconstitution.
- Less manipulation needed.
- Agitation and inadvertent freezing can occur.
How can aggregate formation occur during the use of liquid formulations of protein drugs?
- agitation and inadvertent freezing and then thawing.
How are lyophilised formulations of drugs shipped/stored?
Ambient temps.
What IV diluents can be used with protein formulations?
Saline: normally ok, some small hydrophobic proteins are less soluble/insoluble at high salt conc.
Dextrose: normally not ok, can react with lysines on protein surface to form Schiff’s bases at ambient temperature.
Ringers solution: sometimes ok, check SPC.
Why can contamination occur with the use of lyophilised formulations?
They require a reconstitution stage. More stages required in the prep than with liquid alternatives.
Which IV diluents are normally not ok to use with protein formulations and why?
Dextrose is normally not ok to use as it can react with lysine residues on the protein surface to form schiff’s bases at ambient temperatures.
