IC7 & 8: Chew Eng Hui Flashcards
What is the definition of biosimilar?
A biologic that is almost an identical (or ideally identical) alternative version of the original biologic (called innovator biologic/reference biologic) that is manufactured by a different company -> term is reserved ONLY for biologics
Can a biosimilar be entirely identical to innovator biologic? Explain your answer.
No. Each biological product displays variability even between different batches of the same product due to the variability of the biological expression system and manufacturing process
State the 2 pathways for protein degradation
Lysosomal Degradation and Proteasomal Degradation
Describe the components of the 26S Proteasome
1) Composed of a 20S core particle capped by a 19S regulatory particle at one or both ends.
2) 20S core particle made up of 4 heptameric rings assembled to form cylindrical structure
o 2 outer rings (closer to upper lid) = 2 α subunits
o 2 inner rings (touching each other) = 2 β subunits
o Inner rings house a central cavity containing proteolytic active sites at the walls; has protease activity
o Gate to 20S core particle is usually closed.
3) Degradation chamber reached through a channel running along the long axis of core particle
4) Narrow entrance to channel (13 Å) -> folded proteins partially unfolded (into more primary structure) before they can be translocated into the 20S core particle (non-ubiquitinated proteins cannot enter)
o Those in native conformation cannot enter -> not meant to be degraded
o Upon entering the channel, protein unfolds stretches along channel hydrolyzed to short peptides of 3-25 amino acids released from opposite end of channel
5) 19S regulatory particle arranged into lid and base.
o Contains ATPase subunits (energy dependent reaction), gates entrance to degradation channel and plays a role in substrate recognition, unfolding and translocation into the 20S core particle
o Some only present in lid and not base but either way, short peptides get released from the base.
How are substrates delivered to proteasome? (3 routes)
a. Substrates bind directly to proteasome by interacting with the 19S regulatory particle subunits
b. Substrates brought to proteasome by adaptor proteins that bind both proteasome and polyubiquitin chain on the substrate to deliver it for degradation.
c. Some protein substrates are degraded by proteasome without being ubiquitinated (minor path)
State why proteins have poor oral bioavailability
- Poor protein stability
o Complex pH environments e.g. acidity of gastric fluids
o Presence of digestive enzymes - Poor permeability
o Viscous mucus layer lining entire GIT -> high viscosity impedes speed that molecules can travel through (affects chemical drugs less as they are small)
o Negatively charged intestinal epithelium (phospholipid bilayer) and tight junctions exist between epithelial cells to restrict absorption of hydrophilic peptides/proteins) - Immune cells present in mucosal epithelia may recognize the administered peptide/proteins as foreign particles (the larger the protein, the more likely) and degraded.
List the strategies available to improve PK profile of protein therapeutics
1) N-linked Glycosylation
2) PEGylation
3) increase size by means of Fusion protein
Explain how glycosylation improves PK profile of protein therapeutics and state its potential disadvantages as well.
- Increases circulation half life by increasing size of protein or modifying binding to glycoprotein receptors.
Potential Disadvantage:
1) Glycosylation may lead to decrease in efficacy of binding to receptors
o e.g Fucosylated anitbodies e.g human IgGs (contain N-linked glycans at Asn297) yet removal of Fucose (a type of glycan) improves binding of Fc domain of IgG to Fc receptor which is desired
2) Certain glycans are rapidly eliminated which may decrease circulation half-life of protein drugs.
o Engineering antibodies containing high mannose glycans found to be rapidly eliminated compared to other glycosylated antibodies. Mannose and asialoglycoprotein receptors believed to be responsible for rapid removal of these antibodies (May be good or bad depending on when you want the antibodies to be removed.)
Which form of PEGylyation (branched or linear) is more effective in extending half life?
Branched PEG is more effective than linear PEG in extending half life (but enhancement of half life is more significant for small molecules rather than big molecules)
Explain the ways that PEGylation improves circulation half life.
1) Increase in the size of conjugated protein
o Glomerular filtration of small proteins is retarded if PEG molecules with MW 40-50 kDa is conjugated to the proteins (cut off MW)
2) Decrease elimination by proteolysis (form protective layer on surface, decreasing chance for proteolytic enzymes to interact with and break down proteins.)
3) Decrease elimination by action of antibodies and activated immune cells
o PEG molecules form a protective layer on surface of protein molecules, decreasing recognition by antibodies or activated immune cells (e.g. macrophages, dendritic cells, NK cells, etc) -> escape tagging by antibodies
o This is also the property that make PEGylated proteins have reduced immunogenicity/antigenicity -> hence vaccines are usually not PEGylated as high immunogenicity is desired to create immune response to antigen.
Explain the ways that fusion proteins improves circulation half life and state potential drawbacks
1) Larger molecule, slower clearance
2) Fc domain of antibody or albumin fused to a therapeutic protein allows the protein to undergo FcRn mediated recycling
Drawback:
Fc domain may trigger unwanted effector functions -> trigger unwanted immune response.
State how proteins are metabolised
Via proteolysis by proteolytic enzymes (e.g. activated proteases; if protein drug not taken orally)
Are proteins substrates of CYP enzymes?
No
State where proteolysis may occur
1) In the interstitial fluid (extracellular fluid) present in tissues/organs.
o In the ECF, there are proteases released by activated immune cells and other cell types. Involved in proteolysis.
o There are immune cells lying in ambush in ECF take part in phagocytosis and proteolysis
2) On cell surfaces -> proteases either bound to or secreted by cells
3) Intracellularly once protein drugs are taken up into cells (see protein degradation for more info)
o Proteases are not the ones found inside the lysosomes but are just present in the cytoplasm
State the routes whereby protein drugs may be eliminated
Protein drugs can be eliminated via:
* 1. Proteolytic degradation (extracellular and intracellular)
* 2. Renal (glomerular) filtration
