ADME of Recombinant Proteins

Question 1

Why do protein drugs generally have poor oral systemic absorption?

Answer 1

Poor protein stability due to pH and digestive enzymes

Poor permeability

Innate cellular system laying ambush ready to attack

Question 2

List some of the challenges protein drugs require to go through resulting in its poor permeability

Answer 2

Mucus layer lining the entire GIT

Intestinal epithelium has overall negative charge

Tight junction between epithelial cells make it tougher for absorption

Question 3

How are protein molecules absorbed SC? Describe their mechanism

Answer 3

Diffusion: movement of single particles from high to low concentration

Convection: collective bulk movement of large mass of particles into a fluid

Question 4

What is the relationship between absorption and MW size of particles?

Answer 4

Diffusion is inversely related to MW size of particles

Convection has no limit unless particles are excessively big

Question 5

How do larger proteins get absorbed into the body?

Answer 5

Slow movement across the capillary membrane

Proceeds into the lymphatic system to be drained into lymph nodes and larger vessels

Enters circulatory system

Question 6

How do smaller proteins get absorbed into the body?

Answer 6

Both circulatory and lymphatic system at the same time

Question 7

What are some factors to consider about rate of absorption?

Answer 7

Perfusion: varies with site of injection or patient’s poor peripheral perfusion

Immune cells in hypodermis
- can cause protein degradation

Basement membrane of lymph vessels
- can be porous

Question 8

Define the rate limiting factors of absorption

Answer 8

Interstitial fluid transport rate

Lymphatic transport rate

Question 9

What is interstitial fluid transport rate dependent on?

Answer 9

Charge
Hydrostatic pressure
Oncotic pressure
Presence of fibrous tissue

Anatomical issues influencing drug transport

Question 10

What influences lymphatic transport rate?

Answer 10

Depends on patient’s lymphatic system and whether he has cardiac problems

Question 11

Define the two way model

Answer 11

Movement of protein from circulation into interstitial fluid and then tissues

Question 12

Define what the two pore model means

Answer 12

The two pore model is used to describe trans vascular movement of protein drugs of various size.

Movement is typically reversible where proteins move out of into tissue into interstitial fluid; drained into lymphatic flow and recycled back to systemic circulation.

Question 13

What are the key characteristics of the two pore model?

Answer 13

Endosomal space: Porous tissue microvascular endothelium

Big pore and small pore represent how fluid can pass through both pores and recirculate from plasma space to interstitial space.

This is done by diffusion and fluid phase convection

Question 14

Does metabolism occur for recombinant proteins? Why?

Answer 14

No.

Proteolysis occurs with the help of proteolysis enzymes

Question 15

Where do proteolysis occur?

Answer 15

Interstitial fluid in tissue and organs

Cell surfaces

Intracellularly

Question 16

How is neonatal FcRn transported to the baby?

Answer 16

Transport mother’s IgG through placenta to fetus and mother’s milk

Question 17

What are the key roles neonatal FcRn plays?

Answer 17

IgG homeostasis mediating antibody recycling

Recycling of serum albumin

Question 18

How does FcRn increase half life of IgG and albumin?

Answer 18

IgG and albumin dissolved in blood and taken up by endothelial cells by pinocytosis forming early endosomes

Acidic endosomes containing internalized FcRn fuse with endosomes containing IgG and albumin; forming a complex

Complex formed is recycled to cell surface to undergo exocytosis of IgG and albumin

Neutral pH of blood then disfavours FcRn binding to IgG and hence, allow IgG and albumin to dissociate from complex into blood

Question 19

How does FcRn allow transport of IgG and albumin?

Answer 19

At apical site of mucosal epithelial cells, acidic pH allow binding of FcRn to ligands

Binding between FcRn and ligand occur within acidified endosomes and is further processed to complexes

Endosomes then fuse into basolateral site of epithelial cells and expose complex to neutral pH in interstitium, thus releasing IgG and and albumin

Question 20

How can elimination of recombinant proteins be done?

Answer 20

Proteolytic degradation

Renal filtration

Question 21

What are the factors affecting renal excretion?

Answer 21

Cut off MW

Charge: negative charged basement membrane

Shape rigidity

Tubular reabsorption:

Negatively charged hence those positively charged are reabsorbed

Question 22

What are the 3 strategies to improve PK profile of recombinant proteins?

Answer 22

Glycosylation

PEGylation

Increasing size by fusing proteins

Question 23

Define glycosylation

Answer 23

Addition of glycans to specific amino acids in proteins

Question 24

How does glycosylation help with improving PK profile of recombinant proteins?

Answer 24

Enhances receptor binding

Increase half life of proteins

Question 25

What are some example of traditional glycosylation and glycoengineering?

Answer 25

Traditional: N-linked glycosylation

Glycoengineering:
- Removal of fucose
- Addition of high mannose glycans

Question 26

Describe what PEG is

Answer 26

Addition of amphiphilic, chemically inert polymers made of repeating units of ethylene oxide

Question 27

How does PEG help to increase half lives of recombinant proteins?

Answer 27

Increase size of conjugated proteins help retard the glomerular filtration

Decreases elimination by proteolysis by forming a protective layer

Decreases elimination by action of antibodies and activated immune cells also by forming a protective layer

Question 28

How is an increase in protein size done by fusion?

Answer 28

Done by fusing proteins with a Fc domain of antibody or albumin

Question 29

How does albumin help to increase protein size?

Answer 29

Albumin naturally has 3 domains with each domain containing binding pockets

Altering the Fc domain through fusion with albumin helps by allowing the protein to attract binding of smaller molecules and making it bigger

This compromises FcRn binding and FcRn mediated recycling