L29 - Biologicals Flashcards
What are biologicals?
Medicinal products which are produced/derived from a living system
- whole organism, iolated cells
- aka biopharmaceuticals
What are types of biologicals?
- blood, blood fractionation products
- vaccines
- therapeutic proteins
- nucleic acids & gene therapies
- cell therapies
Proteins vs small molecules drug (proteins): (8)
- BIG (~5,000 >150,000 Da)
- Synthesis (biochemical rxns (mostly), within cells)
- Usually, very water soluble
- No oral absorption
- Not membrane permeable
- No active catabolites
- Variable circulating half-life
- Success in the clinic (higher – can be highly specific)
Protein vs small molecules drugs (small molecule drugs): (8)
• Small (<1000 Da)
• Synthesis (organic rxns, within vessels)
• May have limited water solubility
• Variable oral absorption
• Membrane permeable
• Potentially active catabolites
• Variable circulating half-life
• Success in the clinic (lower)
Therapeutic effect: activation of receptor signaling:
What are antibodies? Produced by? What are they like?
Proteins produced by B cells in blood in response to foreign substances
- B cells derived from single ancestral B cell
- highly specific to their target antigen
What is the therapeutic effect: inhibition of receptor signaling? Eg. VEGF signaling, anti-TNF-a therapies
- ligand binds to antibody
- ligand binds to protein
- antibody binds to receptor
= prevent harmful physiological effect
What is the therapeutic effect: inhibition of receptor signaling? E.g. human epidermal growth factor receptor
Herceptin (trastuzumab) is a mAb binds to HER2
- prevents ligand from binding
= no cell growth, as not activated
What are other therapeutic effects?
E.g. Rituximab
- used to treat certain autoimmune disorders and blood cancers
What are the steps in the production of therapeutic proteins?
Relies largely on recombinant DNA methods
- get mRNA from gene of interest
- reverse transcriptase to get DNA RNA hybrid
- DNA pol for cDNA
What are the steps in the production of therapeutic mAbs: hybridomas?
- immunisation of BALB/c mice with antigen to stimulate antibody prod
- isolation of antibody producing B cells
- fusion and generation of hybridomas
- selection of specific hybridomas
- expansion of selected hybridoma to produce mAbs
What are the different types of mAbs that can be produced?
- mouse
- chimeric (half human, half mouse)
- humanised (with complementarity determining regions
- human
How do you scale up therapeutic protein production of bacteria?
Bigger vat
How do you scale up therapeutic protein production of mammalian cells?
- more difficult and expensive
- cell lines are adherent - microcarriers can be used
- cells that grow in suspension are easier
E.g. chinese hamster ovary (CHO) cells
What are the characteristics/properties of protein folding & stabilisation?
• Hydrophobic interactions (80% internal)
• Electrostatic (repulsions, ion pairing)
• H-bonding (– Inter- and intramolecular)
• VDW forces
• Steric effects
• Hydration
• Disulphide bridges
What are the 2 different irreversible protein inactivations?
- conformational change
- chemical changes
What is conformational change like?
Formation of incorrect structures
- aggregation
What are chemical changes like?
- break peptide backbone
- modification of important residues
- change protein shape
E.g. hydrolysis, oxidation, deamidation, glycation, disulphide bond rearrangement
How does aggregation of therapeutic proteins occur?
- folded monomer (become partly unfolded, folded clusters)
- partly unfolded monomers (become party unfolded clusters)
- become nuclei
- nuclei can combine = oligomonomers and soluble filaments
= soluble agglomerated aggregates
What operations may denature or aggregate proteins?
- Freezing/thawing – proteins are usually
refrigerated so care is needed - Agitation (interfaces)
- Sonication
- Contact with silicone oil
- Low or high pH
- Low or high salt
- Specific salts
- heat
What is an examples of aggregation?
Cooking an egg
- water-soluble
- breaks -SH
- reforms as S-S
- insoluble
What interfaces can cause aggregation?
- air-water (vials, IV bags)
- oilr-water (silicone-coated syringes)
- hydrophobic surfaces (IV set & bag)
What are possible consequences of aggregation/denaturation?
• Altered solubility
• Hypo-potency
• Hyper-potency
• Off target binding (Adverse events, faster clearance)
• Patient may generate neutralizing antibodies (ATAs) (Makes drug ineffective, may break tolerance)
• Cross react with endogenous protein
• Or no effect
What is gene therapy? Eg?
Replacing a faulty gene to cure a disease
- cystic fibrosis
- muscular dystrophy
- sickle cell anaemia
- huntington’s disease
History of gene therapy:
1989 - first approved gene therapy trial (ADA deficiency)
1999 - patient dies from severa inflammatory reaction
2003 - 4/10 get leukaemia
2003 - first gene therapy product (gendicine)
2006 - oncorine reaches market
2012 - glybera approaves - first western gene therapy product
What is done in gene therapy: delivering genes?
- therapeutic gene inserted into plasmid in bacterium + bacterial chromosome
- virus infects cells & deliver its DNA, not replicate (therapeutic gene replaces viral genome)
What is gendicine?
- rAd-p53 injection
- head and neck carcinoma treatment
- safe and effective
What is glybera?
- developed by uniQure, approaved by EU Nov 2012
- treatment of lipoprotein lipase deficiency (LPLD)
- inc fat levels in blood
- administeres as one tine series of small i.m. injections in leg
- AAV vector serotypes, particular propensity for muscle cells
What are current genetic therapy targets?
- cancer (antigen, anti-angiogenesis, antibodies, TSG, suicide genes, immunotherapy)
- monogenic disease (gene mutation, CF, sickle cell anaemia)
- infectious diseases (target viral/bacterial genes or host receptor genes)
- cardiovascular disease (anti-angiogenesis)
