module 6 Flashcards
what are biologic drugs
- vaccine, protein, antibody, nucleic acid
- high molecular weight
- manufactured using living things
- generally injectable
describe diabetes
- family of metabolic disorder
- characterized by high blood sugar
- cardiovascular, kidney, skin ulcer, eye damage
- top 10 disease in Canada
how are blood glucose concentrations regulated
- using insulin, hormone produced by pancreas
- regulates metabolism of carbs, fats, and proteins
- promotes absorption of blood glucose by fat and liver cells convert glucose into glycogen or fat
function of insulin
- regulates metabolism of carbs, fats, and proteins
what do high and low insulin levels mean
- high means low blood sugar (glucose to glycogen)
- low means high blood sugar (glycogen to glucose + protein catabolism)
describe type 1 diabetes
- 10% of population
- starts in childhood
- autoimmune component
describe type w diabetes
- 90% of population
- insulin resitance
- pancreas produces insulin but cells do not respond
- autoimmune component
- obesity
when was insulin first marketed
- 1922-1923
- failed because impurities and not enough amount
first experiment with insulin
- surgically altered dogs to enable insulin isolation
- pancreatic ducts tied off causing cell death
- leaving behind islets of langerhans
- isolated insulin extract from islets
sources of commercial animal derived insulin
- pancreas harvested
- bovine: cows
- porcine: pig
- equine: horse
- ichthyic: fish
what were the issues with animal sources
- proinsulin(allergenic): overtime patients could become allergic, require diff animal source
- glucagon
- somatostatin
- proteases: slowly destroy other proteins in drug, limit shelf life
what were general insulin isolation methods in 1920s
- truck load of pancreases
- homogenization
- clarification (2)
- serial precipitation (2)
- chromatography (3)
- recrystallization
T H C S I P I S R
describe homogenization
- break open organ/cells containing protein to be extracted
- blender, sanitation, osmotic shock
describe clarification
- separate liquid fraction of biological isolate from solid fraction
- 2 methods: dialysis and centrifugation
describe clarification: dialysis
- use continuous diffusion through semi permeable membrane
- placed between mixture and buffer
- membrane allows solutes to pass through while keeping solids behind
- solutes pass from high concentration (mixture) to low concentration
describe clarification: centrifugation
- centripetal force creates artificial gravity
- causes solids in suspension to sink quickly to bottom where they form pellets
- liquid decanted from solid
describe serial precipitation
- to isolate compounds
describe serial precipitation: isoelectric precipitation
- causes certain proteins to precipitate from solution, other proteins left behind, BY CHANGING IONIZATION
- changes pH of solution which alters the charge state of proteins depending on amino acid side chain patterns
- if net number of charges is equal then protein is at isoelectric point
- isoelectric point: neutral and insoluble in water, precipitates leaving other substances behind
describe serial precipitation: denaturing precipitation
Causes certain proteins to precipitate from solution, other proteins left behind, BY CHANGING CONFORMATION
- pH, solvent polarity (add non polar or salts), heat
- done in several ways, conditions made to cause proteins to change conformation (denature) which changes solubility and leads to precipitation
- changed pH of solution alters the charge state of proteins depending on amino acid side chains, change in ionization changes non bonding interactions that maintain conformation
- adding alcohols or other non polar solvents directs VDW in the centre, may expose electrostatic interactions or h bonds in centre to water which can weaken these interactions causing protein to change shape
- heating solution can increase moeclulue motion, if protein flexes enough to allow water to penetrate into NP portions, can disrupt electrostatic interactions or h bonds in centre, weaken these interactions causing protein to change shape
describe chromatography: ion exchange
- pass mixture in solution through ion exchange column using buffer at specific pH
- pH sets ionization state of each protein in mix
- solid phase on column is covered in ionized groups (+/-) attract opp charged proteins
- neutral molecules or molecules w same charge not attracted and pass quickly through column
- molecules with opposite charges pass slowly.
- speed determined by overall charge
- anionic resin for cationic exchange
describe chromatography: size exclusion
- molecules separated according to size
- pass mixture through size exclusion gel
- gel contrans pores of a set size
- small molecules fit and stuck
- pass through gel slowly
- large not fit and go quickly
- intermediate partly and speed depending on how much they get stuck
describe recrystallization
- dissolve mixture in minimum hot solvent
- solvent should dissolve impurties
- solvent show dissolve desire molecules well at high temp and poor at low
- after dissolving, solution cools, crystals of desired molecule precipitate from solution
- impurities remain in solution
- works well if desired is bulk not minor component
what is HPLC
High Pressure Liquid Chromatography
what are challenges ti drug manufacturing using animal sources
- proteins not human
- purity
- large amounts
describe the general structure of insulin
- small oriteubm 2 chains
- A: 21 amino acids, intrachain disulphide bond
- B: 30 amino acids
- connected by 2 disufide bonds
similarities between human and animal sequences
- A chains the same
- Porcine is 1 amino acid different in B chain
what are the limitations to chemical synthesis
- small quantities
- large waye
- only 20 amino acid chain length
what are the goals of protein drug manufacture
- high purity
- large amounts
- human
- low cost and waste
what was the first synthetic gene
- somatostatin (expressed in coli)
first genetically modified drug
recombinant insulin
first recombinant drug
- humulin (thought to be safe but rather better supply)
