IC7 Protein Degradation and ADME of Biologics

Question 1

What are the 2 pathways protein are degraded in mammalian cells?

Answer 1

1. Lysosomal degradation
2. Proteosomal degradation

Question 2

What is lysosomal degradation? what are the 3 types of cell uptake?

Answer 2

Lysosomal degradation

• pH 4-5 (acidic)
• Non-specific, the material must enter the lysosome to be degraded by enzymes in it
• Usually, must be endocytosed such that it is placed in an endosome that fuses with the lysosome

1. Phagocytosis
    “cell eating”
    Damaged, unwanted materials, cell debris, protein aggregates, foreign particles etc. are taken up by the cell into phagosomes, fuse with lysosome and degraded by enzymes
    Non-specific
2. Pinocytosis
    Cell “drinks” extracellular fluids and in the meantime take up solutes
    Fuse with lysosome and degraded by enzymes
    Non-specific
3. Receptor-mediated endocytosis
    Hormones, metabolites, proteins and viruses bind to the receptors found on cell surface
    Taken up by endocytosis
    Placed in coated vesicles and fuse with endosomes
    Fuse with lysosome and degraded by enzymes
    Specific

Question 3

What is proteosomal degradation? What are the subunits involved? Describe them and their functions?

Answer 3

Proteosomal Degradation (more common way of protein degradation)

• Specific
• Needs to be tagged (Ubiquitin tag)
• 26S Proteosome (most common proteosome)
  o Found in all cells
  o Central cavity, proteolytic active sites

1. 19s regulatory particles (as the lid and cap at the bottom)
    Has ATPase to break down ATP to release energy for the following activities
    Recognize the polyubiquitin chain
    Ubiquitin tags cleaved by deubiquitinating enzymes (DUBs) into monomers, which then escapes from proteosome and recycled
    Unfold the protein
    Translocate the protein into the core particle
2. 20s core particle
    2 outer alpha rings
    2 inner beta rings
    Fully unfold the protein
    Breakdown the protein
    short peptides are released out from the bottom

Question 4

What are ubiquitin tags? How are they attached to the protein substrate?

Answer 4

• 76 a.a polymer (have 7 Lys)
• C-terminal of Glycine of ubiquitin tag attaches to amino-group of Lysine of protein by isopeptide bond

Question 5

What is the Minimal no. of tags needed to be recognized/targeted by proteosome?

Answer 5

4 tags

Question 6

When is monoubiquitination used?

Answer 6

Monoubiquitination:

• For histones and transcription factors (won’t be degraded but maybe inactivated)
• Of surface cell receptors for endocytosis and degradation in lysosomes

Question 7

What are the ways for protein to be degraded by proteosome? (3 ways)

Answer 7

Ways for protein to be degraded by proteosome:

1. Directly attach to 19s particle of the proteosome (have polyubiquitin chain)
2. Adaptor proteins that bind polyubiquitin tag on protein substrate to proteosome
3. Proteins degraded by proteosome without being ubiquitinated

Question 8

What happens when a patient has von-hippel lindau disease?

Answer 8

Short-lived protein + Disease:

• HIF-1α
  o transcription factor that upregulates transcription of genes involved in angiogenesis, cell migration and glycolysis during hypoxia
• Von-Hippel Lindau (VHL) Disease
  o autosomal dominant mutation to allele of genes VHL
• During normoxia, HIF-1α level is low
• Without the disease, protein VHL (an enzyme, part of the ubiquitin proteosome system) and HIF-1α the substrate –> pVHL will tag HIF-1α to be degraded by proteosome
• In VHL disease, HIF-1α is not degraded
  o  accumulation leads to constant upregulation of angiogenesis, cell migration and glycolysis during normoxia
  o  increase invasion and metastasis
  o  higher predisposition of developing tumor

Question 9

What are the 2 types of biopharmaceutical products?

Answer 9

2 Types of biopharmaceutical products:

1. Biopharmaceuticals e.g. biologics, recombinant proteins
2. Clinical diagnostics and devices

Question 10

What are the challenges of using biopharmaceutical products?

Answer 10

Challenges using biopharmaceutical products:

1. Immunogenicity
2. Proteins susceptible to denaturation and protease degradation in extracellular fluids (phagocytes) –> especially when MW of proteins > 200kDa, phagocytosis will be involved
3. Proteins susceptible to degradation in intracellular fluids (lysosomes, proteosomes)
4. Distribution of proteins to tissues limited by porosity of vasculatures

Question 11

What are the usual ROA for biopharmaceutical products?

Answer 11

Usual ROA: IV, IM, SC

Question 12

What are the reasons for PO being rarely used as a ROA for biopharmaceuticals?

Answer 12

Rarely PO, poor oral bioavailability:
1. acidic stomach secretions (protein degradation)
2. enzymes in gut (protein degradation)
3. intestinal wall barrier (too big to pass through)
4. negatively charged mucous lining layer (interaction prevents it from passing through the wall, interaction NOT good)
5. Immune cells phagocytose protein (degradation)

Question 13

What are the ways biopharmacueticals are absorbed when administered via SC/IM?

Answer 13

SC/IM absorption (move through ECM via):

1. Diffusion (into blood capillaries)
   o From high (SC tissues) to low (blood) concentration
   o Usually, small molecules <16-20kDa e.g. insulin
   o Less likely to be trapped in ECM
   o Rate limiting factor:
    perfusion rate
    interstitial fluid transport rate
2. Convection (into lymphatic system)
   o Convection force is due to hydrostatic pressure and oncotic pressure that pushes fluids out from plasma at arteriole end
   o Move bulk fluids
   o Advantageous to large molecules e.g. MABs
   o Steric hindrance & charge interactions have influence
   o Small molecules could possibly move through this method as well
   o Excess fluids are drained into lymphatic capillaries
   o Rate limiting factor:
    interstitial fluid transport rate
    Lymphatic transport rate
3. Negatively charged proteins
   o ECM is negatively charged
   o Negatively charged proteins will repel and move faster
   o Interaction NOT good
   - Some are intentionally huge and positively charged
   o Remains longer in ECM
   o Slower release
   o E.g. protamine insulin (intermediate acting)

Question 14

How are the biopharm products distributed in the blood and into the targeted tissues? What are the factors in the 2 pore model?

Answer 14

Once in blood capillaries –> into interstitial fluid –> tissues (can be reversed since dynamic)

1. Protein binding e.g. albumin
   o Increase circulation half-life
   o Reduce degradation by enzymes (since it is huge)
   o Provides greater efficiency in travelling to targeted site
2. Passive movement of protein drugs from plasma into interstitial fluids –> diffusion and convection
   o Both across and between endothelial cells

Two pore model:
Factors:

1. Pore size of membranes (small and large)
2. MW of protein
   o Larger proteins: more limited and slower distribution

Question 15

How and where are biopharmaceutical products degraded?

Answer 15

Proteins are NOT degraded in Liver (different from chemicals!)

1. Proteolysis (proteases-released immune cells, lysosomal degradation, proteasomal degradation)
   o Interstitial fluids (extracellular)
   o Cell surfaces
   o Intracellularly
2. Phagocytosis and proteolysis in immune cells

Question 16

How are biopharmaceutical products excreted? What are the factors affecting excretion of these products?

Answer 16

Renal excretion

• Most proteins are excreted out via glomerular filtration

Factors:

1. MW
   o >50kDA CANNOT be filtered out, not renally eliminated
2. Charge of protein
   o Positively charged proteins excreted quicker
   o Interaction GOOD
   o Negatively charged proteins will repel the glomerular basement membrane
3. Shape and rigidity
   o Must fit the pore shape
4. Tubular reabsorption
   o Tubular epithelium have net negative charge
   o Positively charged proteins get reabsorbed more
   o Interaction NOT good (if want to be eliminated)

Question 17

State the common strategies to enhance circulation half-life of protein therapeutics? (no need to describe)

Answer 17

1. Glycosylation
2. PEGylation
3. Fusion proteins to increase MW

Question 18

How does glycosylation enhance circulation half-life of protein therapeutics?

Answer 18

Glycosylation

• Requires enzyme
• Adding N-linked glycan
   Poorer substrate to proteolysis
   Increase size / MW, limiting glomerular filtration
   Modify binding to glycoprotein receptors
   prevents it from being degraded by other enzymes
   –> longer half-life
• Removal of fucose
   Improves affinity of de-fucosylated IgG Fc domain to Fc receptors (good!)
• Mannose glycan
   mannose glycan causes the protein to bind to Mannose and asialoglycoprotein receptor on phagocytes
   will increase degradation
   –> shorter half-life

Question 19

How does PEGylation enhance circulation half-life of protein therapeutics?

Answer 19

PEGylation

• Polyethylene Glycol is an inert polymer made of ethylene oxide monomers (chemical)
• Free OH at both ends OR methoxyl at 1 end or both ends
• Amphiphilic (mainly hydrophilic –> compatible with water)
• Bulky, 40-50kDa
• More for small protein drugs
• Increase circulation half-life

1. Prevent antibodies and immune cell from recognizing it as foreign
   –> prevent degradation, increase stability, reduce immunogenicity
2. Prevents degradation by proteolytic enzymes or proteosomes –> act as protective layer
3. Increase MW / size –> retards glomerular filtration of small molecules
4. Increase solubility of drug –> increase stability

• Cons:
  –> Takes longer to be excreted since MW > 50kDa (could be a pro too)

Question 20

How does Fusion proteins enhance circulation half-life of protein therapeutics?

Answer 20

Fusion protein with albumin or Fc domain*****

• Neonatal Fc Receptor (FcRn)
   IgG Fc domain or albumin can bind to FcRn on cell surfaces and be intracellularly trafficked to be recycled
   Escapes degradation by lysosomes

1. Cellular recycling (increase t1/2 of IgG and albumin)
   a. Pinocytosis
   b. pH 5-6, Bind to FcRn in endosome
   c. recycled by binding to cell surface
   d. in neutral pH, release IgG and albumin
2. Transcytosis (allow transport of IgG and albumin)
   a. At apical side of mucosal epithelial cell, acidic pH, binds to FcRn
   b. Transcytosed and form endosome
   c. Endosome fuse with basolateral side of epithelial cell
   d. At neutral pH, release IgG and albumin

• Attach proteins to Fc domains or albumin (utilize FcRn-mediated recycling)
  –> increase size (MW)
  –> increase circulation half-life
• Cons:
   May lead to unwanted triggering of immune system –> unwanted inflammation / responses
   Albumin has 3 pockets, usually 2 pockets required for binding to FcRn, if the pockets are occupied by smaller molecules/fatty acids –> compromise FcRn binding and FcRn-mediated recycling