Protein ADME Flashcards
What are the 2 different degradation pathways in mammalian cells?
Which is the main pathway? Which pathway is specific/ non-specific?
- Lysosomal degradation (non-specific)
- Proteasomal degradation (main) (specific)
How are therapeutic proteins taken up by cells degraded?
By both lysosomal and proteasomal degradation
3 types of endocytosis?
- Phagocytosis
- Pinocytosis
- Receptor-mediated endocytosis
Describe the structure of the proteasome
- 20S core particle capped by 19S regulatory particle at one or both ends
- 20S core particle made up of 4 heptameric rings → outer rings = α subunits; inner rings = β subunit
- Inner rings have a central cavity containing proteolytic active sites
Role of the 19S regulatory particle?
- Contains ATPase subunits
- Energy needed for substrate recognition, unfolding and translocation into 20S core particle
What happens to the polyubiquitin tag after it brings the substrate to the proteasome?
- Polyubiquitin tag cleaved by deubiquitinating enzymes into monomers
- Recycled to label other protein substrates
What is the minimal signal necessary for proteasome targeting?
Chain of 4 Ub monomers linked through Lys48
What are the 3 routes where substrates are delivered to proteasome?
- Substrates bind directly to 19S regulatory particle subunit
- Substrates brought to proteasome by adaptor proteins that bind to both proteasome and polyubiquitin chain on substrate
- Some protein substrates degraded without being ubiquinated (minority)
Upon SC administration, what are the 2 ways which small and large proteins move through the ECM?
- Diffusion (small)
- Convection (large)
- Collective bulk movement of large mass of particles in fluid (driven by motion of bulk fluid)
How are large and small proteins absorbed?
- Larger proteins (> 16-20 kDa): lymphatic system → circulatory system
- Smaller proteins (< 16-20 kDa): both lymphatic and circulatory system (perfusion is limiting factor of absorption)
What are the rate-limiting factors that affect absorption of proteins?
- Interstitial fluid transport rate
- Lymphatic transport rate
What does the 2 pore model show?
Proteins can also move out of tissues into interstitial fluid then drained into lymphatic flow and recycled back into systemic circulation
How are protein drugs metabolised?
NOT metabolised by liver but by proteolytic enzymes
What are the 2 pathways in which FcRn recycles lgG and serum albumin?
- Cellular recycling of lgG and albumin (increase t1/2 of lgG and albumin)
- Transcytosis of lgG and albumin (allows transport of lgG and albumin)
How are protein drugs eliminated?
What is the cut-off MW of proteins that cannot be renally excreted?*
Do positively charged proteins have lower or higher renal filtration/ tubular reabsorption than negatively charged proteins?
- Proteolytic degradation
- Renal filtration (not for proteins > 50 kDa)
Positively charged proteins: higher renal filtration and tubular reabsorption
What are the 3 common strategies to improve PK profile of protein therapeutics?
- Glycosylation of proteins (N-linked glycosylation)
- PEGylation of proteins
- Increase in size (MW) by means of fusion proteins
What are the 2 different types of glycosylation?
- Removal of fucose improves affinity of binding of Fc domain in lgG to Fc receptor
- High mannose glycans eliminated rapidly compared to other glycosylated Ab
What are the 3 ways in which PEGylation works to increase circulation t/12 of drugs?
- Increase in size of conjugated protein (glomerular filtrated slowed with conjugation of PEG molecules MW 40-50 kDa)
- Decrease elimination by proteolysis (PEG molecules as protective layer)
- Decrease elimination by action of Ab and activated immune cells (PEG molecules as protective layer hence reduced immunogenicity)
How does increase in size (MW) by means of fusion proteins improve their PK profile?
Larger proteins → slower clearance
- Includes FcRn-mediated recycling
What are 2 major challenges that protein pharmaceuticals face?
- Antigenicity
- Stability
How can protein stability be affected physically (1) and chemically (4)?
Physical:
- Aggregation
Chemical:
- Deamidation
- Oxidation
- Proteolysis
- Disulfide bond breakage and formation
- Hydrolysis
What can cause protein aggregation? (5)
- Temperature (slow unfolding at elevated temp)
- pH
- Adsorption
- Shaking and shearing
- Non-aqueous solvents
- Repeated freeze-thaw
- Photodegradation
Name an amino acid which is susceptible to photodegradation
Tryptophan: side chain cleavage of Trp upon photodegradation
Deamidation is the most common degradation pathway.
Which are the amino acids susceptible to deamidation?
Asn (asparagine) and Gln (glutamine)
Which amino acids are susceptible to hydrolysis?
Asp-Gly and Asp-Pro
Which amino acids are most susceptible to hydrolysis?
Which enzyme catalyses oxidation?
Thiol groups of Cys (C) and Met (M)
Transitional metal ions
Which amino acids is susceptible to disulfide bond formation?
Cys
How can we improve the stability of protein pharmaceuticals via chemical modification?
- Amino acid substitution/ modification
- Introduction of disulfide bond
- PEGylation
- Acylation (chemical attachment of fatty acids)
How can we change the properties of solvent to improve the stability of protein pharmaceuticals?
- Stabilizers: sugars, polyols
- Solubility enhancers (Lys, Arg, surfactants)
- Anti-adsorption and anti-aggregation agents (albumin, surfactants)
- Buffer components (phosphate salts)
- Preservatives and anti-oxidants (inert gas, thimerosal, phenol, benzyl alcohol)
Disadvantages of using E. coli as recombinant protein host cells? (3)
- Recombinant protein accumulates intracellularly (need additional steps to purify)
- Lack ability to perform post-translational modifications
- Presence of lipopolysaccharides (LPS) on surface that act as pyrogens
Between E. coli and CHO cells, which do we choose if we want to make:
1) Large proteins (>100 kDa)
2) Small proteins (<30 kDa)
3) Require post-translational modification
4) Production of protein where solubility and native folding essential?
5) High yields, low cost
1) Large proteins: CHO
2) Small proteins: E. coli
3) Require post-translational modification: CHO
4) Production of protein where solubility and native folding essential?: CHO
5) High yields, low cost: E. coli
How easy/ difficult is it to generate biosimilars for:
1) Chemical drugs
2) Biologics or relatively low MW
3) Mabs which are larger proteins with post-translational modifications
How easy/ difficult is it to generate biosimilars for:
1) Chemical drugs: easy
2) Biologics or relatively low MW: easier compared to Mab
3) Mabs which are larger proteins with post-translational modifications: impossible to engineer a 100% identical biosimilar
