Biologics Flashcards
Define biopharmaceutics.
Biopharmaceutics are drugs that have biological active ingredient that have a therapeutic effect.
What are some examples of biopharmaceutics in market today (5 examples)?
Carbohydrates (low molecular weight heparin)
Protein and peptide hormones (Insulin)
Monoclonal antibodies (Trastuzumab)
Vaccines (Diphtheria-Pertussis-Tetanus vaccine)
Cytokines (Interferons)
What are some of the issues that remain today regarding biopharmaceuticals in relation to the route of administration?
Biopharmaceutics have high molecular weights (>5000 Da) and suffer with stability problems upon storage and after administration meaning that they remain unavailable in non-parenteral formulation.
The active structure of biologics make them more likely to induce an immune response.
What are some of the benefits of using biologics?
They have a much larger surface area in comparison to smaller molecules and they are able to bind much more specific to their drug target (they are evolved to their drug target).
Ultimately more selective binding results in less induced side effects.
What are some of the drawbacks of using biologics?
More complex synthesis
Storage instability
Parenteral formulations only
More likely to be antigenic and evoke an immune response compared to smaller molecules
What are some of the benefits of using small molecules?
Small molecules are synthesised using medicinal chemistry techniques which is relatively straight forward and does not have the added complexity of growing the molecules in cell lines.
The manufacturing approach to synthesising small molecules is done at a large production scale with refined techniques in attempt to drive manufacturing costs down.
Small molecules tend to be membrane permeable - even if the drug has increased hydrophilicity- usually small enough to still be uptaken into the cell, or by an alternative route.
What are some of the key differences between small molecules and Biologics?
In addition to biologics being much more complex, there are some key differences between the two:
Small molecules are synthesised through a predictable chemical process and form identical copies whereas biologics are synthesised through a cell line; similar but not identical copies are made.
Biologics are much more difficult to characterise than small molecules
Biologics are much more sensitive to degradation so greater consideration is required during handling and storage
Immunogenicity with small molecules is relatively unexpected however with biologics there is a much higher potential for an immune response (many biologics are immune-modulating)
Describe the structure of insulin.
Insulin is a peptide consisting of 51 amino acids and has an alpha (21 residues) and beta (30 residues) chain linked by di-sulphide bridges.
Describe how insulin was first manufactured.
First insulin was extracted from the pancreas of a cow but the purification process was complex.
Then insulin began to be mass produced by a strategy known as isoelectric precipitation.
However it became apparent that use of animal insulin was inconsistent and causing some adverse reactions within patients.
In the 1970s recombinant DNA technology where cells are re-programmed to produce insulin (synthetic insulin) reducing the need for animal insulin.
Describe in detail the recombinant production of therapeutic proteins in application of insulin.
The gene that is response for coding for insulin identified, isolated and amplified before being cloned into complementary DNA, becomes expressed on a plasmid and then transferred into the host cell. The protein becomes expressed following choice of an appropriate cell line has been selected. This protein is then expanded in a fermentation medium (bioreactor) by growth of cells following normal replication.
The protein product in this case insulin is then isolated by centrifugation/filtration and the protein itself is purified by chromatographic techniques.
The protein then needs to undergo post-translational modifications responsible for it’s folding.
Is the recombinant production technique a homologous or heterologous expression?
Heterologous expression as the gene has to be inserted into the cell for its expression, the cell doesn’t naturally produce the protein.
What are some examples of host organisms used in the process of recombinant DNA?
E coli
Chinese Hamster Ovary cell lines
Yeast cells
What are the advantages of using E coli?
Molecular biology is well characterised
High expression of heterologous proteins are possible
Quick and cheap
Possible to scale up large fermentation culture
What are the disadvantages of using E coli?
Formation of LPS on the surface of E coli (pyrogenic) provoking an immune response
Heterologous proteins accumulate intracellularly not extracellularly and therefore have to lysis the cell before extracting them
Inability of the cell to undergo post translational modifications but is critical for desired activity of many proteins
Formation of inclusion bodies (insoluble aggregates of partially soluble heterologous proteins)
What are some examples of post-translational modifications?
Glycosylation
Phosphorylation
Sulfation
What are post-translational modifications?
Any covalent modification of the peptide chain after synthesis, can be critical for the therapeutic activity (may not fold or bind correctly) this is outside of the template of DNA, RNA, mRNA sequence.
What are the steps in the process of creating a functional protein?
DNA codes for RNA which codes for mRNA which then codes for the protein. There is a translation of the mRNA sequence on to the ribosome. The protein is then folded and bound to co-factors which provides the molecule with shape and potential sites for binding interactions.
Which type of bonds provide the folding process in the molecule?
Normally hydrogen bonds in addition to other non-covalent bonds. These are not the same as the post-translational modifications of the molecule which impact its therapeutic effect which are caused by covalent bonds.
What function does the post-translational modification of glycosylation have to the function of the peptide?
Increases the solubility
Alters the biological half life and activity of the molecule
What function does the post-translational modification of phosphorylation have to the function of the peptide?
Regulates the activity of many polypeptide hormones
What function does the post-translational modification of sulfation have to the function of the peptide?
Regulates activity of some neuropeptides and processing of other polypeptides
Do all proteins rely on glycosylation for its desired activity and therapeutic effect?
No, some proteins can be relatively unaffected by the removal of a glycosylation group however some proteins rely on glycosylation for their activity. An example of this is IgG which strongly relies on one glycosylation site for its activity.
What are the two types of glycosylation?
N-glycosylation which is more common and O-glycosylation
Where does N-glycosylation occur?
It occurs in the rough endoplasmic reticulum
The fully synthesised glycosyl chain is bound into the membrane is transferred to the newly synthesised peptide chain by the enzyme oligosaccharyl transferase.
