Protein ADME

Question 1

What are the 2 different degradation pathways in mammalian cells?

Which is the main pathway? Which pathway is specific/ non-specific?

Answer 1

1. Lysosomal degradation (non-specific)
2. Proteasomal degradation (main) (specific)

Question 2

How are therapeutic proteins taken up by cells degraded?

Answer 2

By both lysosomal and proteasomal degradation

Question 3

3 types of endocytosis?

Answer 3

1. Phagocytosis
2. Pinocytosis
3. Receptor-mediated endocytosis

Question 4

Describe the structure of the proteasome

Answer 4

• 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

Question 5

Role of the 19S regulatory particle?

Answer 5

• Contains ATPase subunits
• Energy needed for substrate recognition, unfolding and translocation into 20S core particle

Question 6

What happens to the polyubiquitin tag after it brings the substrate to the proteasome?

Answer 6

• Polyubiquitin tag cleaved by deubiquitinating enzymes into monomers
• Recycled to label other protein substrates

Question 7

What is the minimal signal necessary for proteasome targeting?

Answer 7

Chain of 4 Ub monomers linked through Lys48

Question 8

What are the 3 routes where substrates are delivered to proteasome?

Answer 8

1. Substrates bind directly to 19S regulatory particle subunit
2. Substrates brought to proteasome by adaptor proteins that bind to both proteasome and polyubiquitin chain on substrate
3. Some protein substrates degraded without being ubiquinated (minority)

Question 9

Upon SC administration, what are the 2 ways which small and large proteins move through the ECM?

Answer 9

1. Diffusion (small)
2. Convection (large)
   - Collective bulk movement of large mass of particles in fluid (driven by motion of bulk fluid)

Question 10

How are large and small proteins absorbed?

Answer 10

• 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)

Question 11

What are the rate-limiting factors that affect absorption of proteins?

Answer 11

1. Interstitial fluid transport rate
2. Lymphatic transport rate

Question 12

What does the 2 pore model show?

Answer 12

Proteins can also move out of tissues into interstitial fluid then drained into lymphatic flow and recycled back into systemic circulation

Question 13

How are protein drugs metabolised?

Answer 13

NOT metabolised by liver but by proteolytic enzymes

Question 14

What are the 2 pathways in which FcRn recycles lgG and serum albumin?

Answer 14

1. Cellular recycling of lgG and albumin (increase t1/2 of lgG and albumin)
2. Transcytosis of lgG and albumin (allows transport of lgG and albumin)

Question 15

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?

Answer 15

1. Proteolytic degradation
2. Renal filtration (not for proteins > 50 kDa)

Positively charged proteins: higher renal filtration and tubular reabsorption

Question 16

What are the 3 common strategies to improve PK profile of protein therapeutics?

Answer 16

1. Glycosylation of proteins (N-linked glycosylation)
2. PEGylation of proteins
3. Increase in size (MW) by means of fusion proteins

Question 17

What are the 2 different types of glycosylation?

Answer 17

1. Removal of fucose improves affinity of binding of Fc domain in lgG to Fc receptor
2. High mannose glycans eliminated rapidly compared to other glycosylated Ab

Question 18

What are the 3 ways in which PEGylation works to increase circulation t/12 of drugs?

Answer 18

1. Increase in size of conjugated protein (glomerular filtrated slowed with conjugation of PEG molecules MW 40-50 kDa)
2. Decrease elimination by proteolysis (PEG molecules as protective layer)
3. Decrease elimination by action of Ab and activated immune cells (PEG molecules as protective layer hence reduced immunogenicity)

Question 19

How does increase in size (MW) by means of fusion proteins improve their PK profile?

Answer 19

Larger proteins → slower clearance
- Includes FcRn-mediated recycling

Question 20

What are 2 major challenges that protein pharmaceuticals face?

Answer 20

1. Antigenicity
2. Stability

Question 21

How can protein stability be affected physically (1) and chemically (4)?

Answer 21

Physical:
- Aggregation

Chemical:
- Deamidation
- Oxidation
- Proteolysis
- Disulfide bond breakage and formation
- Hydrolysis

Question 22

What can cause protein aggregation? (5)

Answer 22

1. Temperature (slow unfolding at elevated temp)
2. pH
3. Adsorption
4. Shaking and shearing
5. Non-aqueous solvents
6. Repeated freeze-thaw
7. Photodegradation

Question 23

Name an amino acid which is susceptible to photodegradation

Answer 23

Tryptophan: side chain cleavage of Trp upon photodegradation

Question 24

Deamidation is the most common degradation pathway.

Which are the amino acids susceptible to deamidation?

Answer 24

Asn (asparagine) and Gln (glutamine)

Question 25

Which amino acids are susceptible to hydrolysis?

Answer 25

Asp-Gly and Asp-Pro

Question 26

Which amino acids are most susceptible to hydrolysis? Which enzyme catalyses oxidation?

Answer 26

Thiol groups of Cys (C) and Met (M) Transitional metal ions

Question 27

Which amino acids is susceptible to disulfide bond formation?

Answer 27

Cys

Question 28

How can we improve the stability of protein pharmaceuticals via chemical modification?

Answer 28

1. Amino acid substitution/ modification 2. Introduction of disulfide bond 3. PEGylation 4. Acylation (chemical attachment of fatty acids)

Question 29

How can we change the properties of solvent to improve the stability of protein pharmaceuticals?

Answer 29

- 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)

Question 30

Disadvantages of using E. coli as recombinant protein host cells? (3)

Answer 30

1. Recombinant protein accumulates intracellularly (need additional steps to purify) 2. Lack ability to perform post-translational modifications 3. Presence of lipopolysaccharides (LPS) on surface that act as pyrogens

Question 31

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

Answer 31

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

Question 32

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

Answer 32

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