L29 - Biologicals

Medicinal products which are produced/derived from a living system - whole organism, iolated cells - aka biopharmaceuticals

• blood, blood fractionation products • vaccines • therapeutic proteins • nucleic acids & gene therapies • cell therapies

• 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)

• 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)

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

• ligand binds to antibody • ligand binds to protein • antibody binds to receptor = prevent harmful physiological effect

Herceptin (trastuzumab) is a mAb binds to HER2 - prevents ligand from binding = no cell growth, as not activated

E.g. Rituximab - used to treat certain autoimmune disorders and blood cancers

Relies largely on recombinant DNA methods - get mRNA from gene of interest - reverse transcriptase to get DNA RNA hybrid - DNA pol for cDNA

• 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

• mouse • chimeric (half human, half mouse) • humanised (with complementarity determining regions • human

Bigger vat

• 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

• Hydrophobic interactions (80% internal) • Electrostatic (repulsions, ion pairing) • H-bonding (– Inter- and intramolecular) • VDW forces • Steric effects • Hydration • Disulphide bridges

• conformational change • chemical changes

Formation of incorrect structures - aggregation

• break peptide backbone • modification of important residues • change protein shape E.g. hydrolysis, oxidation, deamidation, glycation, disulphide bond rearrangement

• 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

• 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

Cooking an egg - water-soluble - breaks -SH - reforms as S-S - insoluble

• air-water (vials, IV bags) • oilr-water (silicone-coated syringes) • hydrophobic surfaces (IV set & bag)

• 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

Replacing a faulty gene to cure a disease - cystic fibrosis - muscular dystrophy - sickle cell anaemia - huntington’s disease