P6

What two methods can allow for protein engineering?

Recombinant DNA technology, chemical modification of the protein

Example of a fusion protein?

Etanercept, TNFalpha antagonist

What is Etanercept made up of?

Fc region of hum IgG and extracellular domain of TNF receptor

What section of insulin are crucial for binding?

B24-26, B28-29 for dimerization and binding to IR

When does insulin form hexamers?

The presence of zinc ions

What are the modifications for long acting insulin?

Promote hexamer formation, reduce solubility, promote binding to plasma proteins

What are the modifications for short acting insulin?

Prevent formation of dimers and hexamers, increase solubility

What is the other name of Humalog?

Insulin lispro, switch lysine and proline aa, are keen to dimerise causing disruption and promotion of monomer form

What is the other name for NovoRapid?

Insulin aspart, change proline to aspartic acid, change to charge causing repulsion and reducing formation of the monomer

What is the other name of Lantus?

Insulin glargine, add aspartic acid and a glycine to change isoelectric point, protein less soluble at bio pH , take longer to go into solution

What is the other name of levemir?

Determir insulin, on termnial lysine add c14 fatty acid, binds to more serum proteins and self associated

Describe the characteristics of insulin Fiasp?

Insulin Aspart, formulated with vitamin B3, promotes insulin in monomeric form

Describe the structure characteristics of insulin delgludec?

What size of proteins can be lost in pores?

5000 Daltons via pores 6-12 nm

What are the benefits of PEG of proteins?

Improved solubility, reduced protein binding, improved BA, avoidance of phagocytosis, shielding of antigenic sites, reduced toxicity, proteolytic resistance, reduced clearance, improved stability, can attach other molecules

How can PEG negatively affect protein activity?

Via random PEGylation, if there is an interaction with a site needed for activity

How can site specific PEG be carried out?

Using different pH, thiols with PEG-maleimide, enzyme catalysed reactions

What can PEGylated insulin have?

Reduced diffusion rate, reduced renal clearance, improved stability vs proteases

Describe the characteristics of spray drying

Continuous process, gentler than freeze drying, forms power, particle size can be adjusted to change dissolution rate, can be donw aseptically, may allow RT storage

What is a the benefit of using polymer and lipid based nanoparticles?

Can be modified with shielding molecules and targeting ligands

What is the problem with proteins and polymeric controlled drug delivery systems?

Problem with interfaces, soluble in organic solvents which doens’t work for biologicals, often multiple emulsions