module 6

Question 1

what are biologic drugs

Answer 1

• vaccine, protein, antibody, nucleic acid
• high molecular weight
• manufactured using living things
• generally injectable

Question 2

describe diabetes

Answer 2

• family of metabolic disorder
• characterized by high blood sugar
• cardiovascular, kidney, skin ulcer, eye damage
• top 10 disease in Canada

Question 3

how are blood glucose concentrations regulated

Answer 3

• 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

Question 4

function of insulin

Answer 4

• regulates metabolism of carbs, fats, and proteins

Question 5

what do high and low insulin levels mean

Answer 5

• high means low blood sugar (glucose to glycogen)
• low means high blood sugar (glycogen to glucose + protein catabolism)

Question 6

describe type 1 diabetes

Answer 6

• 10% of population
• starts in childhood
• autoimmune component

Question 7

describe type w diabetes

Answer 7

• 90% of population
• insulin resitance
• pancreas produces insulin but cells do not respond
• autoimmune component
• obesity

Question 8

when was insulin first marketed

Answer 8

• 1922-1923
• failed because impurities and not enough amount

Question 9

first experiment with insulin

Answer 9

• surgically altered dogs to enable insulin isolation
• pancreatic ducts tied off causing cell death
• leaving behind islets of langerhans
• isolated insulin extract from islets

Question 10

sources of commercial animal derived insulin

Answer 10

• pancreas harvested
• bovine: cows
• porcine: pig
• equine: horse
• ichthyic: fish

Question 11

what were the issues with animal sources

Answer 11

• proinsulin(allergenic): overtime patients could become allergic, require diff animal source
• glucagon
• somatostatin
• proteases: slowly destroy other proteins in drug, limit shelf life

Question 12

what were general insulin isolation methods in 1920s

Answer 12

• truck load of pancreases
• homogenization
• clarification (2)
• serial precipitation (2)
• chromatography (3)
• recrystallization

T H C S I P I S R

Question 13

describe homogenization

Answer 13

• break open organ/cells containing protein to be extracted
• blender, sanitation, osmotic shock

Question 14

describe clarification

Answer 14

• separate liquid fraction of biological isolate from solid fraction
• 2 methods: dialysis and centrifugation

Question 15

describe clarification: dialysis

Answer 15

• 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

Question 16

describe clarification: centrifugation

Answer 16

• centripetal force creates artificial gravity
• causes solids in suspension to sink quickly to bottom where they form pellets
• liquid decanted from solid

Question 17

describe serial precipitation

Answer 17

• to isolate compounds

Question 18

describe serial precipitation: isoelectric precipitation

Answer 18

• 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

Question 19

describe serial precipitation: denaturing precipitation

Answer 19

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

Question 20

describe chromatography: ion exchange

Answer 20

• 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

Question 21

describe chromatography: size exclusion

Answer 21

• 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

Question 22

describe recrystallization

Answer 22

• 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

Question 23

what is HPLC

Answer 23

High Pressure Liquid Chromatography

Question 24

what are challenges ti drug manufacturing using animal sources

Answer 24

• proteins not human
• purity
• large amounts

Question 25

describe the general structure of insulin

Answer 25

- small oriteubm 2 chains - A: 21 amino acids, intrachain disulphide bond - B: 30 amino acids - connected by 2 disufide bonds

Question 26

similarities between human and animal sequences

Answer 26

- A chains the same - Porcine is 1 amino acid different in B chain

Question 27

what are the limitations to chemical synthesis

Answer 27

- small quantities - large waye - only 20 amino acid chain length

Question 28

what are the goals of protein drug manufacture

Answer 28

- high purity - large amounts - human - low cost and waste

Question 29

what was the first synthetic gene

Answer 29

- somatostatin (expressed in coli)

Question 30

first genetically modified drug

Answer 30

recombinant insulin

Question 31

first recombinant drug

Answer 31

- humulin (thought to be safe but rather better supply)