Biologics Flashcards
What are biologics?
These are larger molecules, that are synthesised by cells & organisms. These molecules are highly specific.
What are some of the drawbacks of using biologics?
Not accessible by chemical synthesis.
Not membrane permeable
Antigenic.
They are also harder to characterise because they are larger molecules.
Are Biologics stable?
More sensitive than small molecules to handiliing and storage.
How are biologics made?
Recombinant production of therapeutic proteins.
What is Recomibnant production of therapeutic proteins?
Heterologous expression of recomibnant proteins -> Introduction of gene or cDNA coding for a protein into a suitbale producer organism.
What does heterologous mean?
A protein that does not occur naturallty within the cell.
What are the man steps in protein production by recombinant DNA techonology?
Identification - Amplification and isolation of the target gene.
Integration - Of target gene into a cloning vector (Plasmid)
Growth - Of the cell in-vitro (Done in a bioreactor).
Identification - Of the cells contatining the target protein.
Isolation of purification of the target protein.
What are the advantages of E.coli as a host?
Molecular biology is well characterised.
High expression lvls of heterologous proteins are possible.
Quick and cheap - Rapid growth on simple and inexpensive media.
Possible to scale up to large fermentation culture.
What are the disadvantages of E.coli as a host?
Heterologous proteins accumulate intracellularly.
Inability to undertake post-translational modifications.
Presence of LPS on E.coli surface.
Formation of inclusion bodies (Insoluble aggregates of partially soluble heterologous proteins)
What can happen to biologics once they’ve been formed?
Post-translational modifications.
What are the step sin the creation of a functional protein?
Translation of mRNA sequence into amino acids sequence on the ribosome.
Completed polypeptide chain must (Fold correctly into its 3D conformation, bind any necessary cofactors, assemble with its partner protein)
These are driven by non-covalent bond formation.
Post translational modifications are covalent modifications of selected AA.
What are the types of Post Translational Modifications?
Proteolytic processing - Many proteins become active after proteolysis.
Glycosylation - Glycosyation can increase solubility, alter t1/2 & activity.
Phosphrylation - Regulates acti ity of many polypeptide hormones.
Acetylation - Fuction unclear.
Acylation - May increase anchoring of proteins in membranes.
Amidation - Regulates activity of some polypeptides.
Sulfation - Regulates activity of some neuropeptides and processing of their polypeptides.
Hydroxylation - Important in strucural assembly blood proteins to bind to Ca2+
What is N-linked glycosylation?
Occurs co-translationally at sites with consensus sequence:
- Asn-Xxx-Ser / Thr OR Asn-Xxx-Ser where Xxx-is any AA except Pro.
Common oligosaccharide trimming:
- 3 glucose and 1 mannose after later removed in the ER by glucosidases.
- Further saccharides removed and added accordingly but a core motif is always retained.
What is O-linked Glycosylation
Occurs post-transitionally in ER/Golgi
Ser/Thr residues.
Up to eight different core structures.
What are the challaneges of producing biologics - Glycoforms?
Variations in glycosylation patterns give different glycoforms.
Different glycoforms of ne protein can give different:
- Stability
- Solubility
- Serum t1/2
- Biological activity
- Immunogenicity.
