TD: Introduction to Biotechnology Flashcards
What is biotechnology?
Biotechnology refers to use of biological systems (cells, tissues, or enzymes) for the manufacture of biomolecular drugs.
What are biotechnology products like? Difference to traditional small molecule pharmaceuteicals?
- Termed biopharmaceuticals, these drugs are usually large polypeptides or proteins >1 kDa.
- Different from traditional small molecule pharmaceuticals (100’s atomic mass units) i.e. not chemically synthesised.
- Biotechnology products are not exclusively protein but very often are protein or peptide in nature. Proteins >25 amino acids cannot be chemically synthesised, and human/animal tissue extracts had been used.
What does biotechonology products overcome?
What can this technology permit?
- Production overcomes ethical issues and safety associated with using extracts e.g. Creutzfeldt-Jakob disease.
- Transferring e.g. skin post mortum can pass on infection so synthesis can avoid this
- Also synthesis of e.g. hormones can help overcome ehtical/ religous beliefs
- Some of this technology can permit amino acid alterations designed to improve action e.g.
- faster or delayed onset,
- extended duration of action.
Biopharmaceuticals are able to be manufactured on large scales due to the advent of recombinant DNA technology. This allows for the use of bacterial plasmids to have genes inserted not their sequences which causes them to produce peptides/proteins which they wouldn’t normally manufacture.
This removes the problem of having to isolate and purify proteins from human/animal sources which does not produce high yields and also can result in infection passing from donor to recipient (e.g. Creutzfeldt-Jakob disease.). The use of plasmids also allows for the alteration of proteins to suit a particular purpose i.e. fast/slow acting proteins or extended duration of action.
Current bipharmaceuticals
- GNRH/LHRH treat breast or prostate cancer.
- Somatostatin analogues used in treatment of thyroid cancer.
- Immunopeptides (mAb) used to treat both multiple sclerosis and hepatitis C.
- Calcitonins treat calcium deficiency e.g. osteoporosis.
- Antiplatelet peptides reduce risk of infarction.
- Vasopressins treat diabetes insipidus.
Describe the production of biopharmaceuticals
- Production can be divided into upstream and downstream processing.
- Upstream processing involves the initial fermentation process resulting in product manufacture, requiring:
−selection of cell culture system ie bacterial, fungal, animal,
−construction of suitable molecular clones,
−optimal cell growth conditions to maximise production.
Describe the basics of the cloning proceedure
- Identidy gene of intrest - this is a piece of DNA that contains a gene which can be transcribed and translated into a desired protein
- Insert gene of intrest into cloning vector - plasmid
- Recombinant DNA/ plasmid taken up by cellular expression system (e.g bacteria)
- sequence in plasmid is copied and transcribed to make multiple copies.
What are transgenic animals?
Transgenic animals are animals (most commonly mice) that have had a foreign gene deliberately inserted into their genome. Such animals are most commonly created by the microinjection of DNA into the pronuclei of a fertilised egg which is subsequently implanted into the oviduct of a pseudopregnant surrogate mother.
Designed to e.g. produce a protein, an altered protein or an absence of a protein
What cell culture systems can be used?
- Cellular expression systems include bacteria, yeast/fungi, animal cells (CHO/BHK), insect cells, and transgenic animals or plants.
- Most biopharmaceuticals are produced by bacteria eg special strains of E. coli (K12) expressing a cloned foreign gene.
Why is E.coli typically used for the manufacture of biopharmaceuticals?
- E. coli can be rapidly and cheaply cultured in large quantity by standard fermentation.
- Gene cloning in E. coli allows production of biopharmaceutical to exceed 30% total cell protein giving excellent yield.
Manufacture of biopharmaceuticals is predominantly carried out using E.coli as it has a number of advantages.
- It’s molecular biology is well characterised and so is easy to manipulate to produce a wide range of proteins
- E.coli grow rapidly in cheap, easily obtained conditions and so high yields of proteins can be easily achieved. It can grow rapidly in a wide variety of conditions and is robust so is unlikely to be killed during manufacture i.e. it is a reliable method. Can be cultures in large quantities using standard fermentation – allows for biopharmaceutical production to exceed 30% if totally cell protein.
Limitations of E.coli for manufacture of biopharmaceutics
- Can be difficult to extract and purify proteins from E.coli as they accumulate inside bacteria cells i.e. not excreted from cells on production. Therefore certain extraction techniques must be performed to harvest the protein before it can then be purified and formulated.
- Products can be contaminated with lipopolysaccharide from the surface of E.coli (Lipopolysaccharides are pyrogenic and so will cause fever in the patient)
- Difficult to manipulate proteins produced. It is difficult to manipulate their structure on harvesting and purification
- •Drawbacks include lack of secretion by E. coli, inclusion body formation, and no capacity for post-translational modification.
- As product is intracellular its recovery is more complex, also complicated by need to remove all LPS endotoxin.
- Use of the yeast S. cerevisiae is favoured for certain products eg the short acting insulin NovoLog.
What is the only other significant method for culture if prokaryotes cannot be used?
Advantages?
Limitations?
Uses?
Animal cells are potentially more useful than bacterial cells as the proteins they produce can have the structures more readily modified after harvesting – are able to undergo post-translational modification e.g. correct glycosylation unlike bacterial cells.
However animal cells are far more fragile than bacterial cells and require more costly and complex conditions in order to grow. They are also far more likely to be damaged during manufacture and so result in the failure of the manufacturing process or produce lower than expected yields.
Currently animal cell culture is the only other significant method for production.
- Major advantage is the ability to post-translationally modify product eg correct glycosylation.
- However, animal cells have complex nutritional requirements, grow very slowly in limited quantity (small yield), and are easily contaminated or damaged.
- Favoured for production of most interleukins and interferons, EPO, Factor VIII and mAbs.
What is an example of trasngenic animals?
What are benefits/limitations of transgenic animals?
- Transgenic animals that secrete biotherapeutic protein in their milk have been developed.
- Production yield is potentially huge (~ 6 g L-1 tPA), correct post-translational modification undertaken, and purification more simple, but costs are huge.
Transgenic animals have their genetic code altered to produce the desired protein. This is normally done with mammals which produce milk e.g. cows.
Advantages include:
- Mammals produce large amount of milk regularly and already farmed which means that the high yield of protein from low cost outlay can be achieved.
- Continuation of transgenic variety guaranteed through normal breeding practices. Therefore although initial costs may be high the transgenic variety can be cheaply propagated thereafter.
Limitations:
- Takes much longer to produce transgenic animals then to grow bacteria as you must first impregnate a cow with its transgenic fertilised egg, then wait for the calf to be born and then mature before it can start to regularly produce milk and preed (i.e months/years compared to days/weeks for bacteria)
- Fraught with ethical considerations as changing an animal genetic code is highly controversial whereas doing the same with bacteria is a minor consideration
- As looking after a herd of animals can be expensive
How are cells grown during the upstream process?
How long does this process take
- Large scale fermentation vessels are used for growth of bacteria, yeast and some animal cells.
- Typical fermenters include the following features (see picture)
- Growth medium (minimal/complex) is prepared and sterilised in situ within the vessel.
- After inoculation with seed culture, the fermentation takes several days and biomass accumulates.
- Facilities must operate to exacting standards of GPMP, with similar QC and QA requirements for working in clean rooms.
When does the downstream processing occur?
What does this involve
What are the conditions like?
So once the cell growths have peaked then downstream processing occurs:
- DS processing permits
- –recovery and purification of product.
- –formulation and packaging of final product.
- Occurs under conditions equivalent to clean rooms to prevent contamination.
Describe the downstream process
- Proteins are fragile so processes must not cause denaturation of the molecule.
- Initially for intracellular products the cells are collected by centrifugation.
- Cell destruction then follows using either chemicals or physical disruption, not heat.
- Following rupture several stages involving ultrafiltration (UF) or column chromatography are used.
Downstream processing is tailored to permit maximum biopharmaceutical recovery
