Therapeutic Proteins Flashcards
Sola and Griebenow (2010) Glycosylation of Therapeutic Proteins: An Effective Strategy to Optimize Efficacy. Biodrugs, 24, 9-21
Bacterial Restriction Endonucleases
cut DNA at specific 4-8bp recognition sequence, EcoRI (palindromic) GAATTC, leaving sticky ends can be rejoined to form recombinant, insert into vector (using DNA ligase and ATP)
Bacteria mRNA used to make cDNA
No introns
Bacteria has no introns
Use mRNA to make cDNA and poly thymidated promoter for polyadenylated tail of mRNA
Vectors
Plasmid
virus
Control expression by
artificial chromosomes
Making protein - key points (3)
Naturally produced in species? all machinery present
soluble? require folding
post-translational modification (N + O glycosylation only in eukaryotes)
codon in species? frequency - number tRNAs –> mistakes
product toxic?
Advantages of using prokaryotes (5)
easy culture rapid growth expression easy to induce easy retrieval no ethics
Disadvantages of using prokaryotes (5)
low expression rapid degradation no post-trans modification can't glycosylate doesn't secrete product
Advantages of using simple eukaryotes
established tech
high product level
cost effective
easy to adapt for industry
Pichia Pastoris
well defined growth and high density
simple glycosylation
expression controlled by AOX1 gene - change methanol conc - methylotropic
saccharomyces cerevisae
make viral fragments for vaccine - HOV
disadvantage - addition of mannose at N-glycosylation sites
Filamentous fungi - aspergillus
- trichoderma
- produce pectinases for food industry - more juice from fruit
- produce antibody fragments for medicine
Eukaryotic algae - chlamydomonas reinhardtii
motile single cell - translated proteins folded and assembled
Insects
express cDNA well
transcriptional processing of introns
folding and post-trans mod
protein secreted
Mammalian cell lines advantages
full post-trans mod
some secrete protein
easy recovery and purification
Mammalian cell line disadvantages
complex conditions
purification may be required
slow growth
expensive
Transgenic animals
production of recombinant therapeutic proteins in milk
Transgenic Plants
Use plant virus as vector - vaccine production
advan - easy to grow, cheap, easy drug delivery
disadvan - change taste, stable?, choice, where will gene go
Glycosylation
oligosaccharides added to the protein during synthesis and processing through ER and golgi
Glycan
carbohydrate structure
affects bioactivity, pharmacokinetics, secretion, in vivo clearance, solubility, recognition, antigenicity
Main glycosidic links
N- –> bound to ASN residue Asn-X-Ser/Thr
Complex/Hybrid (both golgi)/High Mannose (ER)
O- –> bound to hydroxyl of Ser/Thr, hydroxylysine, hydroxyproline
short, but longer on ABO antigens
N-linked glycan assembly
precursor - dolichol (lipid) linked to glycan by pyrophosphate
Oligosaccharyltransferase - enzyme attaches asn in ER
glucosidases on ER membrane
gycosidases and glycotransferases in golgi
addition sialic acid, fucose, poly-N-acetyl lactosamine
O-linked glycan assembly
added post translationally to folded portein
on ser/thr in golgi
GalNAc transferase adds N-acetylygalactosamine for mucin type
Glycosylation under control of
metabolism and cell growth
Glycans Biological Activtiy
enhance thermal stability
protection from proteolysis
improve solubility
inhibit aggregation
Glycans - folding
carbohydrate negatively charged - interact AA side chain
Erythropoietin
sialoglycoprotein - secreted by kidneys - hypoxia/low red cell mass - stimulate erythrocyte production from bone marrow
Erythropoietin - Glycan
ensure adequate residence in bloodstream
Recombinant Erythropoietin (Dongmei et al 2010)
Increase sialylation —> increase half-life - not removed by asialoglycoprotein receptor
Glycan Antibody function
IgG Fc has 2 N-linked complex type
High fucose - effectively recruit polymorphonuclear cells
low fucose - enhance NK cell activity
Glycan Pharmacokinetics - Circulating Residence Time
Function of pharmacokinetic (PK) behaviour including clearance rate, serum half-life and max and min concs
glycosylation affects PK
FSH short half life
Glycosylation increases this
Mannose and asiaglycoprotein receptors remove certain glycoforms
Lenercept - terminal GlcNAc
Growth Hormone
191 AA Single chain, non-glycosylated
recombinant used in HGH deficiency and wasting from AIDS
Insulin
51 AA (30+21) Ultra short analogue - not form hexamers/dimers so absorbed quicker Lente - intermediate Levemir - long-acting - myristyric acid attached to albumin so remains in circ longer
Blood clotting proteins Haemophilia A
impairment of gene synthesise factor VIII - this has 25 N-glycosylation sites
unglycosylated = immunogenic and cleared quickly
Haemophilia B
Impair gene synthesises Factor IX - so is transfused in treatment
post-trans modifications required - peptide removal,vitamin K dependent carboxylation, ser- phosporylation, tyr sulfation, glycosylation
Interferons
140-170 AA - alpha (B lymphocytes) , beta (fibroblasts) and gamma (T lymphocytes)
antiviral activity and retard cancer cells
Interferon Alpha
Interfere cancer cells growth and mulptilication
increase antigen presentation on cancer cells
activate NK cells
given for renal malignant/multiple myeloma + leukaemia
Recombinant Alpha
treat hepatitis C - has human albumin attached, in blood for 4 weeks, no SE
Interferon Beta (recombinant)
3 types - treat multiple sclerosis
Betarson - non-glycosylated
Avonex and rebif are glycosylated - higher specific activity and lower production of neutralising antibodies
