Lec 3- Biotechnology-based pharmaceutics

Question 1

Pharmaceutical biotechnology

Answer 1

• Pharmaceuticals manufactured by industrial processes that use biological systems
• i.e. inherently biological in nature

Question 2

Drug discovery

NME

Answer 2

• 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

Question 3

Drug discovery

NBE

Answer 3

• 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

Question 4

Types of biotechnology products

Answer 4

• Recombinant blood factors
• Recombinant hormones
• Hematopoietic growth factors
• Recombinant interferons
• Interleukins
• Vaccines
• mAb-based products
• DNA and anti-sense based products

Question 5

Biotech pharmaceuticals

Answer 5

• 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

Question 6

Biotech-based pharmaceutical production

Answer 6

• Initially, proteins and peptides were produced by direct extraction from the native source material
• Associated problems
  
  • Quantity of drug extracted
  • Quality of drug extracted

Question 7

Advantages of pharmaceutical production by recombinant means

Answer 7

Question 8

Technologies which facilitate biotech drug production

Answer 8

1. Recombinant DNA: hybrid DNA is produced by joining pieces of DNA from different sources
2. Genetic engineering: Facilitates the large-scale production of a protein once its amino acid sequence has been determined
3. Hybridoma technology: Facilitates the large scale production of mono-specific Ab raised against virtually any Ag/receptor

Question 9

Recombinant DNA production

Answer 9

• 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

Question 10

Genetic engineering

Answer 10

*

Question 11

Background

Answer 11

• 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)

Question 12

Biotech. protein therapeutics

Answer 12

• 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)

Question 13

Functional classification of protein therapeutics

Group I

Answer 13

• 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

Question 14

Functional classification of protein therapies

Group II

Answer 14

• 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

Question 15

Functional classification of protein therapeutics

Answer 15

• 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

Question 16

Recombinant protein production

NB- look at tables on slides pick an example from each one

Answer 16

• 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

Question 17

E.coli: the workhorse of biotech

Problems

Answer 17

• They don’t excrete proteins- must break cell to access protein
• Foreign proteins are degraded easily- immunogenicity

Question 18

Mammalian cell lines

Answer 18

• Good protein expression occurs
• Often they export proteins
• Products are not likely to be immunogenic to humans

Question 19

Fermentation of mammalian cells

Answer 19

• 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

Question 20

Disadvantages of using mammalian cell cultures

Answer 20

1. Manufacturing costs are high
   
   • Due to culture requirements
2. Long process times
   
   • Mammalian cell cultures have a relatively slow growth rate
3. They are sensitive to shear rates
4. There are relatively low levels of product titres- a slower growth rate
5. Additional purification steps needed (To remove potential viral contaminants of raw materials of biological origin)

Question 21

Biotech manufacturing issues

Answer 21

• 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

Question 22

Sources of microbial of viral contamination

Answer 22

• 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

Question 23

Categories of components that should not be present in the final bio-drugs

Answer 23

• 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
• Components present due to contaminations
  
  • Viruses, virus-like particles, bacteria, fungi and tramsissible pathogens

Question 24

Methods used to evaluate protein pharmaceuticals

Answer 24

• 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