IC7 & IC8 Flashcards
Usual body protein size
6-8 kDa
MW of proteins more than ____ may be eliminated via phagocytosis
200 kDa
Intestinal epithelium generally carries_____ charges
negative
Is convection affected by MW?
not affected by MW unless proteins are enormously large & gets trapped in ECM
Convection may be influenced by _______
steric hindrance & charge interactions
Larger proteins ____ move slowly across capillary membrane
> 16-20kDa
Are proteins metabolised by liver?
No, they are poor CYP substrates. Mostly metabolised via proteolysis.
Charge of glomerular basement membrane
Negative
Which protein sizes cannot be renally eliminated?
Proteins > 50 kDa not eliminated renally
Should vaccines be PEGlyated?
No, because we want immunogenicity
Ways to incr protein half-life
- Glycosylation of proteins
- PEGylation
- Incr size by means of fusion proteins
Two pathways in which proteins are degraded in mammalian cells
- Lysosomal pathway
- Proteasomal pathway
Normal levels of HIF-1alpha at normoxia
Low
Under normoxic conditions, HIF-1alpha is hydroxylated by oxygen-dependent _______
prolylhydroxylases
What happens to hydroxylated HIF-1alpha?
It gets recognized and targeted for ubiquitination
Role of HIF-1 alpha
A transcription factor responsible for oxygen homeostasis during hypoxic conditions; induces expression of genes involved in angiogenesis, cell migration and glycolytic pathway
Is lysosomal degradation a specific process?
No, as long as proteins are in lysosomes, regardless of identity, they are degraded.
(In higher eukaryotes, only membrane-associated proteins and alien proteins internalized by endocytosis are degraded in lysosomes.)
Is proteasomal degradation a specific process?
Yes, it takes place for ubiquitinated or some non-ubiquitinated proteins.
Is pinocytosis a specific process?
No
Role of 26 proteasome
responsible for specific degradation of regulatory proteins and removal of damaged proteins
Composition of 20s core structure of 26 proteasome
4 heptameric rings: 2 alpha outer subunits + 2 beta inner subunits
Structure of 26 Proteasome
20S core particle capped by a 19S regulatory particle at one or both ends
Inner rings of 26 proteasome house a central cavity containing ________
proteolytic active sites
Ubiquitin tag cleaved by _________ into monomers.
deubiquitinating enzymes
How many lysines do ubiquitin contain?
7 lysines
Ubiquitin is almost always attached to substrate protein through an isopeptide bond between ______ of Ub and the ______ Lys in the substrate.
C-terminal Gly; amino group of
Minimal signal necessary for proteasome targeting: a chain of ______ linked through _____
4 Ub monomers; Lys48
mW of traditional chemical-based drugs
< 1000Da
MW of biopharmaceutical products/ biologics
typically in kDa
All biopharmaceutical products are protein-based (T/F).
False. Not all. Majority (>95%) of biopharmaceuticals are protein products.
Definition of convection
Collective bulk movement of large mass of particles in a fluid (the flux is fluid- driven/driven by motion of the bulk fluid)
2 transport mechanisms for proteins upon SC administration in the ECM
- Diffusion
- Convection
Absorption for larger proteins > _____ kDa mostly occur through lymphatic system
16-20
Smaller proteins < 16-20 kDa, absorption can be via both _________
circulatory and lymphatic systems
Hypodermis is also known as ____
Subcutaneous tissue
Rate limiting factors that can cause changes to absorption rates of proteins drugs after SC/IM administration
- Interstitial fluid transport rate
- Lymphatic transport rate
Interstitial fluid refer to _____
Fluid embedding the ECM
Role of FcRn
responsible for transport of maternal IgG (1) across placenta to foetus, and (2) from mother’s milk across intestines of newborn babies.
FcRn name
Neonatal Fc receptor
At ______, FcRn binds to Fc domain of IgG or albumin to form FcRn-IgG or FcRn-albumin complex.
acidic pH 5-6
Charge of protein that allows it to have higher renal filtration
Positive charge (since glomerular basement mbm has neg charge)
Protein charge that gets reabsorbed by renal tubules
Positive charge
Factors affecting renal excretion of proteins
- Charge of protein
- MW of protein
- Shape & rigidity of protein
- Tubular reabsorption of protein
2 ways in which proteins can be eliminated
- Proteolytic degradation (extracellular and intracellular)
- Renal (glomerular) filtration (dominates renal excretion of protein drugs)
2 types of PEG
- PEG with free hydroxyl at both ends
- methoxylated PEG (mPEG) with hydroxyl at one or both ends methoxylated
Substances fused to proteins to incr circulation half-life
- Fc domain of antibody
- Albumin
Concern for fusion proteins with Fc domain of antibody
Fc domain may trigger unwanted effector functions -> trigger unwanted immune (inflammatory) responses
Can a biosimilar biologic be entirely identical to the innovator biologic? Why?
No, due to variability of the biological expression system and manufacturing process
sialylated glycans almost absent in Mabs produced in ______
CHO cells
