Lec 3- Biotechnology-based pharmaceutics Flashcards
1
Q
Pharmaceutical biotechnology
A
- Pharmaceuticals manufactured by industrial processes that use biological systems
- i.e. inherently biological in nature
2
Q
Drug discovery
NME
A
- NME (New Molecular Entity)
- New drug products containing as their active ingredient a chemical substance marketed for the first time in the USA (FDA) =>
- Made by reactions between organics and/or inorganic chemicals =>
- Conventional drugs including aspirin or statins
3
Q
Drug discovery
NBE
A
- New Biological Entities
- Defined as biological products used to prevent or treat diseases and include proteins, nucleic acids, cells or tissue =>
- Can be made from humans, animals, micro-organisms or produced by recombinant DNA technology =>
- Vaccines, gene therapy, mAb
4
Q
Types of biotechnology products
A
- Recombinant blood factors
- Recombinant hormones
- Hematopoietic growth factors
- Recombinant interferons
- Interleukins
- Vaccines
- mAb-based products
- DNA and anti-sense based products
5
Q
Biotech pharmaceuticals
A
- There are generally two categories of biotechnology pharmaceuticals
- Those that have previously only been available from natural sources
- Isolated from human tissue, blood or urine
- Insulin used to be bovine- problems with immunogenicity
- Those that can’t be effectively isolated from their natural source
- Insufficient quantities
6
Q
Biotech-based pharmaceutical production
A
- Initially, proteins and peptides were produced by direct extraction from the native source material
- Associated problems
- Quantity of drug extracted
- Quality of drug extracted
7
Q
Advantages of pharmaceutical production by recombinant means
A
8
Q
Technologies which facilitate biotech drug production
A
- Recombinant DNA: hybrid DNA is produced by joining pieces of DNA from different sources
- Genetic engineering: Facilitates the large-scale production of a protein once its amino acid sequence has been determined
- Hybridoma technology: Facilitates the large scale production of mono-specific Ab raised against virtually any Ag/receptor
9
Q
Recombinant DNA production
A
- Insert gene into a plasmid (Normally E.coli)
- Use restriction enzyme to cut DNA (leaving sticky end)
- Add fragement of DNA of favourable gene
- Allow new peices of DNA to be inserted
- Combined by DNAlygase
10
Q
Genetic engineering
A
*
11
Q
Background
A
- Recombinant DNA technology has made commercial production of proteins and peptides possible
- The first chemical synthesis of a therapeutic peptide as oxytocin (1953)
- First approved drug produced by rDNA technology was human insulin in 1982 (Also a peptide)
- HUMULIN (Lilly)
12
Q
Biotech. protein therapeutics
A
- These can substitute, enhance or block physiological reactions of human metabolism
- For the successful development of biotech proteins & peptides, you need to know
- The mode of action of the native human protein (how the protein acts in a healthy body)
- It’s interaction in the pathophysiological status of the patient (how the protein acts in an ill body)
13
Q
Functional classification of protein therapeutics
Group I
A
- Group I: protein therapeutics with enzymatic or regulatory activity
- A) replacing a protein that is deficient or abnormal (Table 1&2)
- Generally endocrine and metabolic disorders with defined aetiology
- Insulin (humulin- can say glargine- insulin analogue giving slower, longer onset of action)- regulate blood glucose and cause shift of K into cells
- B) Augmenting an existing pathway (Table 3&4)
- Mainly therapies that augment haematological endocrine pathways and immune responses
- Erythropoietin- erythropoisis- anaemia (chemo, renal failure)
- C) Providing a novel function or activity (Table 5)
- Botox- botulinum toxin- cervical dystonia, correct squint, cosmetics- cleaves SNAP25 at NMJ to disrupt SNARE complex and prevent ACh release- flaccid paralysis
14
Q
Functional classification of protein therapies
Group II
A
- Group II: protein therapeutics with special targeting activity
- A) Interfering with a molecule or organism
- B) Delivering other compounds or proteins
- These proteins use their specific targeting to blocking the function of molecules or organisms, targeting them for destruction, or stimulating a signalling pathway
- mAb are the main examples
15
Q
Functional classification of protein therapeutics
A
- Group III- protein vaccines
- A) Protecting against a deleterious foreign agent
- B) Treating an autoimmune disease
- C) Treating cancer
- Group IV- protein diagnostics (Table 10)
- These are diagnostic used in clinical decision-making
- Glucagon- slow GI motility, reversal of hypoglycaemia
16
Q
Recombinant protein production
NB- look at tables on slides pick an example from each one
A
- The major host for production of recombinant proteins are
- Escherichia Coli (E.Coli)
- Saccharomyces cerevisiae
- P.pastoris
- Chinese hamster ovarian cells
- Baby hamster kidney cell lines
17
Q
E.coli: the workhorse of biotech
Problems
A
- They don’t excrete proteins- must break cell to access protein
- Foreign proteins are degraded easily- immunogenicity
18
Q
Mammalian cell lines
A
- Good protein expression occurs
- Often they export proteins
- Products are not likely to be immunogenic to humans
19
Q
Fermentation of mammalian cells
A
- Cultivation: The protein is either
- Secreted into the culture medium
- Or accumulates in the cell
- Purification: the first step is to separate cells and cell debris
- By centrifugation or microfiltration techniques
- Finally, the product is purified by chromatographic techniques
20
Q
Disadvantages of using mammalian cell cultures
A
- Manufacturing costs are high
- Due to culture requirements
- Long process times
- Mammalian cell cultures have a relatively slow growth rate
- They are sensitive to shear rates
- There are relatively low levels of product titres- a slower growth rate
- Additional purification steps needed (To remove potential viral contaminants of raw materials of biological origin)
21
Q
Biotech manufacturing issues
A
- Biotech production is burdened by the potential risk of introducing foreign agents or contamination
- Source includes
- Host cell lines, raw materials or by inappropriate process conditions, personel
22
Q
Sources of microbial of viral contamination
A
- Sterility is the most critical factor in determining the success rate of mammalian cell culture fermentation
- Contamination sources
- Media supply
- Aeration
- Sampling systems
- Therefore, special equipment and skilled operators are required
23
Q
Categories of components that should not be present in the final bio-drugs
A
-
Components present due to process conditions
- Host cell derived components
- DNA + proteins
- Process-derived components
- Lipids, proteins, antifoam agents, antibiotics, substances used for production isolation, cleansing agents
- Host cell derived components
-
Components present due to contaminations
- Viruses, virus-like particles, bacteria, fungi and tramsissible pathogens
24
Q
Methods used to evaluate protein pharmaceuticals
A
- Liquid chromatography- Used to assess the purity and degradation profiles of proteins
- The most commonly used methods: Reverse phase HPLC; Ion exchange; Size-exclusion
- Optical spectroscopy- Quantitation of the way protein absorb, emit and scatter light provides valuable info about the amount of protein, its confirmation and its tendency to aggregate
- Techniques used: UV and visible absorption spec;
- Electrophoresis- Based on the difference in the electrically induced migration of a protein in a sieve-like gel
- Migration depends on molecules size and their charge
- Immunoassays and biological activity assays
- The ultimate test of the stability of the native protein conformation is its maintenance of biological activity- these are included instability monitoring of protein formulations
