47- insulin iii purification

Question 1

how does ion exchange chromatogrpahy work. what are electrostatic interactions with resin, and how do molecules adhere to resin

Answer 1

• Electrostatic interactions with resin (solid)
  
  • principle is the same, you just change the materials in the solid and the liquid phase
  • resin is a kind of plastic that is completely engineered
  • can separate based on charge
• Molecules adhere to resin according to charge
  
  • Highly charged stick tightly
  • Partly charged stick partly
  • Neutral do not stick

Question 2

how does sulfonic acid get ionized with ion exchange chromatography

Answer 2

look at notes

Question 3

what are the various types of resin

Answer 3

• anionic resin
• catonic resin
• look at notes

Question 4

why is the mobile phase usually a buffer. why would you change pH during the procedure

Answer 4

• Mobile phase is usually a buffer
  
  • Controls the ionization state of proteins
• Possible to change pH during the procedure
  
  • Alters ionization as the separation proceeds

Question 5

how does the general ion exchange column work

Answer 5

notes pootie renas of the future

Question 6

how does size exclusion gel work

Answer 6

• Stationary phase contains fixed-size pores
• Small molecules fit inside the pores
  
  • Move slowly through the gel
• Large molecules don’t fit into the pores
  
  • Move quickly through the gel

Question 7

what is size exclusion column how do they work

Answer 7

notes again

Question 8

what is HPLC and what does it use/require

Answer 8

• High pressure liquid chromatography
  
  • Uses very small particle size
  • Large surface area contact between mobile phase and stationary phase
  • Requires high pressure to force the mobile phase through stationary phase

Question 9

how did insulin production work in 1970s

Answer 9

• Clarification
  
  • dialysis or centrifuge
• Precipitation
• Crystallization and recrystallization (Zn+2)
  
  • 98% purity
• HPLC
  
  • Ion exchange
  • Size exclusion
  • 99.99% purity
• Recrystallization (Zn+2)
  
  • 99.9999% purity
• Side effects due to animal-source contamination rare by late 1970’s

Question 10

what are possible manufacturing methods for proteins

Answer 10

• Extraction from animals

- Chemical synthesis

Question 11

what are challenges to protein drug manufacture using animal sources

Answer 11

• Proteins not human
  
  • Similar but not the same
  • amino acid sequence of the animal = won’t work in exactly the same way
• Purity
  
  • Impurities are biological
  • Similar properties, hard to remove
  • Allergy due to other proteins
    
    • if you can’t get of all the proteins then that increases risk of allergy
  • Viruses (infection)
• Need large amounts
  
  • Supply may be limited (hormones)
  • need lots of animals = supply bottleneck

Question 12

why is chemical synthesis better than extraction from animals

Answer 12

• Human protein only
  
  • don’t have be stuck w what nature makes
• Could solve purity problems
  
  • Easier to separate big/small molecules

Question 13

what is the general structure of insulin

Answer 13

• 51 amino acids
• Small protein composed of 2 chains (A and B) connected by 2 disulfide bonds
• A chain
  
  • 21 amino acids
  • Intrachain disulfide bond
    
    • connects two cysteines
• B chain
  
  • 30 amino acids
    
    • connects two cysteines

Question 14

compare human, porcine, and bovine sequence

Answer 14

• in the B chain they are different
  
  • human & porcine: human has a T at position 30 and porcine has an A at position 30
  • human and bovine: human has a T at position 30 and bovine has an A at position 30. human has a G and H at 8 and 10. bovine has an A and V at 8 and 10
• not large difference but ideally we want human insulin