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PHA223 > protein preparations > Flashcards

protein preparations Flashcards

1
Q

What are proteins composed of?

A

amino acids (base) linked by amide bonds

fibrous and globular

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2
Q

examples of fibrous proteins

A

mechanical functions

  • hair
  • skin
  • bones
  • connective tissue
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3
Q

examples of globular proteins

A

enzymes
antibodies
inhibitors

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4
Q

How many AA does insulin have?

A

55 AA (peptide)

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5
Q

What formulations are proteins given in?

A

injection

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6
Q

How are therapeutic proteins formed?

A

extracted from natural sources

OR

engineered in a lab
- recombinant deoxyribonucleic acid (rDNA) technology or gene cloning

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7
Q

examples of biopharmaceutical proteins

A 
vaccines
recombinant proteins
blood products
enzymes
mAb
nucleic acid-based therapeutics
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8
Q

Brand name for recombinant human insulin

A

Humulin

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9
Q

What challenges are there with proteins/insulin?

A

producing stable and biological active therapeutic protein for LT storage

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10
Q

When AA join what do they form?

A

proteins

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11
Q

How do AA join?

A

by formation of a peptide bond

1 molecule of water is released (condensation)

COOH—NH2

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12
Q

What are peptide bonds susceptible to?

A

hydrolysis

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13
Q

What is primary structure of a protein?

A

linear sequence of AA

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14
Q

What is secondary structure of proteins?

A

polypeptide chain folds and turns by H bonding

-> gives alpha helices and beta sheets

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15
Q

What is tertiary structure of proteins?

A

folded, native or 3D structure

  • biologically active in this shape
  • protein can have biological effect
  • polypeptide chains become functional
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16
Q

What is quaternary protein structure?

A

Ig or antibody structures (large molecules)

several protein chains packed together

held together by H bonds, van der Waals forces

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17
Q

chemical instability of folded/native proteins

A

deamidation (Glu, Asp)
oxidation (His, Met, Cys)
peptide bond hydrolysis
disulfide exchange

-> leads to irreversible denaturation

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18
Q

physical instability of folded/native proteins

A 
temperature
pressure
pH (ionisation)
surface adsorption (contsiner/package)
aggregation

-> reversible denaturation

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19
Q

challenge with proteins - aggregation

A

aggregation is a problem in high conc formulations

may lead to loss of efficacy/safety

based on surrounding environmnt (pH, ionic strength, temp, excipients, stress)

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20
Q

deatured proteins

A

unfolded

not active

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21
Q

What happens if you remove water from proteins?

A

forms dry proteins

inaccurate native structure due to protein protein interactions

-> not active if dry, need water (reconstitute back to native structure)

22
Q

Why are surfactants used in protein formulations?

A

act as a stabiliser during processing to prevent destruvtive effect of heat/shear

23
Q

Whay are sugars added as an excipient?

A

sugars prevent interactions between protein molecules

help proteins stay in native structure

24
Q

2 excipients used to protect proteins from air-liquid interface effect

A

sugars

surfactants

25
Q

preferential hydration of proteins

A

water molecules have higher affinity for protein than excipients

26
Q

What is preferential binding of proteins?

A

excipients have higher binding to the surface of the protein

excludes water

27
Q

How to make proteins stable?

A

co-solute omitted from surface of protein

interactions with water molecules more desirable than interactions with additive

formation of a ‘hydration shell’ around protein required for protein activity

28
Q

What is a ‘hydration shell’?

A

water molecules interact with protein surface and bind

more desirable than interaction with additive

shell required for protein activity

29
Q

How are proteins in liquid form given?

A

injections

solutions for nebulisation

30
Q

What maintains protein stability in liquid form?

A

additives

31
Q

examples of additives

A 
sugars
polymers
cyclodextrins
salts (pH, ionic strength)
non-ionic surfactants
32
Q

examples of sugars used as additives

A

trehalose

mannitol

sucrose

33
Q

What pH is insulin soluble at?

A

pH < 5

34
Q

What form of proteins is more stable for LT storge?

A

solid forms

35
Q

excipients in powder protein formulations

A

bulking agents (trehalose, lactose)

salt (NaCl)

36
Q

properties of injection formulations

A

sterile
isotonic
clear after reconstitution
low viscosity

37
Q

What techniques are used to produce proetins in dried powder forms?

A

freeze drying
spray freeze drying
spray drying
supercritical fluid technology

38
Q

difference between freeze drying and spray freeze/spray/supercritical fluid tech

A

freeze drying - cannot produce dried protein particles with controlled particle size

the others - can produce protein particles with controlled particle size (< 5 micro m)

39
Q

What can spray drying modify?

A

particle size and shape

particle surface texture

40
Q

What does spray drying do?

A
  • particles dissolved/suspended in a liquid

- converts contents of solution/suspension into powder in 1 step

41
Q

steps of the spray drying process

A
  1. atomisation of the liquid as fine droplets into a hot air stream by spray nozzle
  2. atomisation creates a large SA and rapid evaporation of the solvent
  3. after droplets dry, the powder is separated from the air stream by cyclone separator
42
Q

What properties of the drying chamber affect droplet stability?

A

protein denaturation
water content
hot gas in contact with protein droplets

43
Q

What properties of the air intake affect droplet stability?

A

pH, protein, additives concs
flow rate
temperature

44
Q

What properties of the dry product affect droplet stability?

A

LT stability
packaging
storage conditions
particle shape

45
Q

What spray drying variables affect the resulting particle size?

A
  1. spray pattern
  2. conc of solute
  3. air flow system
46
Q

Why is protein denaturation minimal?

A

cold water is circulating around the protein solution

AND

presence of protein solution droplets in drying chamber is for a short time

47
Q

What types of particles can spray drying produce?

A

solid particle
hollow particle
cenosphere

48
Q

What additives are good/bad to stabilise proteins?

A

good - poly vinyl pyrrolidone

bad - methacrylic acid (destabilises freeze dried proteins)

48
Q

What additives are good/bad to stabilise proteins?

A

good - poly vinyl pyrrolidone

bad - methacrylic acid (destabilises freeze dried proteins)

49
Q

What is Circular Dichroism used for?

A

characterises secondary and tertiary structure of proteins in solutions

PHA223 (43 decks)

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