Biologics Flashcards
(39 cards)
What are biologics?
biologics are large, strucuturally complex molecules that have therapeutic effect. Usually drugs are small.
What are some advantages and disadvantages of biologics as drugs?
+ Have high specificity for drug targets
- They aren’t accessible by chemical synthesis
- They are not membrane permeable unlike previous small drugs
- They are more likely to be antigenic- provoke an immune response
- More sensitive to handling and storage conditions
Can biologics be chemically synthesised?
NO- We have to convince certain cells to make the desired proteins e.g. insulin
What is the average size of a biologic (+ compare it to a ‘normal’ small drug)?
The average biologic molecular weight is >2 KD
e.g For a certain monoclonal antibody drug that is a biologic e.g. rituximab, its MW = 144,000 daltons but for a small molecule medicine e.g. Acetylsalicylic acid (Aspirin)= 180 daltons
OR
the anticancer agent paclitaxel has a size of 854 Daltons (Da), while the commercially available G-CSF (i.e. filgrastim), has a size of 18 000 Da.
What are some examples of current biologics in use?
The Ebola vaccine
Monoclonal antibodies e.g. Rituximab & trastuzumab
Insulin
Erythropoietin
What is recombinant production of therapeutic proteins?
The heterologous expression (heterologous as the protein of interest doesn’t occur naturally in the cell) of recombinant proteins =
‘Involves the introduction of a gene or cDNA coding for the protein of interest into a suitable producer organism’
What are the steps for recombinant production of therapeutic proteins?
- Identify the target gene, then isolate and amplify it
- Integrate the target gene into a suitable cloning vector such as plasmid DNA
- Then introduce this vector into the host cell. Host cells e.g. E.coli, yeast, animal cell lines
- Grow this host cell in vitro (in the lab)
- Assess all the cells grown to identify which contain the target protein
- Once identified, Isolate e.g. by centrifugation or filtration and purify e.g. by chromatography the target protein for use
What are the commonly used host organisms for producing recombinant proteins?
- Yeast, especially s.cerevisiae
- Microorganisms e.g. E.coli
- Animal cell lines
- Insect cells
- Plants
What are the advantages and disadvantages of using E.coli as a host?
+ It is well understood as a micro-organism
+ Produces a high yield of heterologous proteins
+ Is quick and cheap- allows for rapid growth of proteins in inexpensive media
+ Is possibility to scale up production
- Heterologous proteins accumulate intracellulaly- meaning they have to lyse the cells and extract the proteins
- There is the presence of LPS on the surface of E.Coli which is pyrogenic and so would induce a fever
- There is inability to undertake post-translational modifications (MAIN DISADVANTAGE)
What is a post-translational modification?
These are any changes via covalent modifications of the peptide sequence (of chosen amino acids) once the peptide chain has been synthesised.
e.g These can be glycosylation, phosphorylation, sulphation, glycation, deamidation, and deimination to amino acids
Using E.Coli as a host has many advantages, so why isn’t is always used?
Because E.coli is unable to undertake any post-translational modifications as it is a prokaryotic cell
How are proteins made?
- mRNA sequence is translated into an amino acid sequence in a ribosome
- The completed polypeptide chain must then be folded correctly into its 3D conformation. This is NOT covalent
- But any post-translational modifications (PTM) are covalent e.g. Glycosylation, phosphorylation, acylation, hydroxylation, Acetylation
What are some examples of post-translational modifications?
Glycosylation- Attachment of carbohydrates
phosphorylation- Attachment of a phosphate group
acylation- Attachment of an acyl group-
hydroxylation- Attachment of a hydroxyl group
Acetylation - attatchment of an acetic acid group
What is glycosylation?
The enzymatic process that attaches polysaccharides to proteins/lipids/other organic molecules. There are 2 types- N-linked (most common) and O-linked glycosylation.
What is an ogliosaccharide?
An oligosaccharide is a saccharide polymer containing a small number (typically three to ten) of monosaccharides (simple sugars)
What is the difference between N-linked and O-linked glycosylation?
In N-linked glycosylation, a protein can exhibit multiple glycan linkages ranging from one to many. In contrast, O-linked glycosylation typically includes only a single sugar residue added to a serine or threonine residue.
Good explanation from online:
Glycosylation is an important modification to eukaryotic proteins because the added sugar residues are often used as molecular flags or recognition signals to other cells than come in contact with them.
- There are two types of protein glycosylation, both of which require import of the target polypeptide into the ER.
- N-linked glycosylation actually begins in the endoplasmic reticulum, but O-linked glycosylation does not occur until the polypeptide has been transported into the Golgi apparatus. Therefore, it is also the case that N-linked glycosylation can (and is) usually beginning as a co-translational mechanism, whereas O-linked glycosylation must be occurring post-translationally.
- Other major differences in the two types of glycosylation are (1) N-linked glycosylation occurs on asparagine (N) residues within an N-X-S or N-X-T sequence (X is any amino acid other than P or D) while O-linked glycosylation occurs on the side chain hydroxyl oxygen of either serine or threonine residues determined not by surrounding sequence, but by secondary and tertiary structure
- N-linked glycosylation begins with a “tree” of 14 specific sugar residues that is then pruned and remodeled, but remains fairly large, while O-linked glycosylation is based on sequential addition of individual sugars, and does not usually extend beyond a few residues
What is a glycoform?
These are different forms of one protein caused by variation in glycosylation patterns.
They have the same protein sequence, but different sites of glycosylation
Can alter:
- Stability
- Solubility
- Serum half-life (bioavailability)
- Biological activity
- Immunogenecity
Can prokaryotes perform post-translational modifications?
No because they don’t have the required machinery- as they don’t have membrane-bound organelles e.g. nuclei as they are prokaryotic cells
- This is where the use of E.coli becomes a limiting factor as it doesn’t have the necessary glycosylation, machinery to lead to these post-translational modifications.
What cells are recommended for producing recombinant proteins?
Eukaryotic cells
Especially:
- Chinese hamster ovary cells
- Human embryonic kidney cells
What is the HAMA response when trying to produce monoclonal antibodies?
HAMA = Human anti-mouse antibody response
This is a problem that occurs when using mouse cells to grow the monoclonal antibodies as when they are then injected into a human, it provokes an immune response leading to self-immunogenicity.
How were the monoclonal antibodies produced using mouse cells made?
Via Hybridoma technology
What is the process of Hybridoma technology to grow monoclonal antibodies in mouse cells?
- The mouse is immunised with specific antigens
- The immune system of the mouse will then produce antibodies to these antigens
- These antibodies are fused with immortal cell lines (long lasting) such as myeloma cells and fused with PEG and so they continue to replicate
- The cells are grown in a medium in large numbers = high yield
- The cells are then analysed and the most potent cells that produced the largest number of antibodies are selected.
- Then isolation and purification is needed- the process depends on whether the cell has secreted the desired protein or if it is still intracellular
Hybridoma = desired = mouse plasma cell + myeloma cell
What factor determines which isolation and purification techniques are used and what are the 2 methods?
Depends on whether the cell has secreted the desired protein or if it is still intracellular.
- Recovery of protein from producing organism-
- Treat with chemicals e.g. detergents or alkaline conditions
- sonification (Sonication is the act of applying sound energy to agitate particles in a sample) or homogenisation
- Agitation in the presence of abrasives e.g. glass beads - Chromatographic purification
What is a biosimilar?
Biosimilars are agents that are biologically and clinically comparable to the innovator product but not exactly the same
When a patent of a biologic expires (usually 20 years from submission), then different companies can try and make the drug- but it won’t be the exact same and wouldnt pass trials like small drugs would. The new drug may have the same protein sequence but has different properties due to post-translational modifications and so needs to be completely re-tested.
