Formulation

Question 1

ICH Q5 is guidelines for what

Answer 1

quality of biotechnological products

Question 2

Q5A

Answer 2

viral safety evaluation of products derived from cell lines

Question 3

Q5B

Answer 3

analysis of the expression construct in cells used for production of rDNA derived protein products

Question 4

Q5C

Answer 4

stability

Question 5

Q5D

Answer 5

derivation and characterization of cell substrates used for production

Question 6

ICH Q6B specifications for

Answer 6

test procedures and acceptance criteria

includes in process controls, bulk drug, final product and stability specifications

Question 7

how are biotechnological products sterilized

Answer 7

filtration
prefiltration remove bioburden and other particulates
sterilize by filtering through 0.2um membrane filters

Question 8

3Es you need to sterilze

Answer 8

equipment
excipients
environment

Question 9

ways to sterilize the facilities

Answer 9

dry heat autoclave >160C for 30min
chemical treatment
gamma radiation

Question 10

1 endotoxin unit =

Answer 10

100pg of ecoli LPS

amount in 10^5 bacteria

Question 11

____ EU/kg body weight can cause _____ symptoms

Answer 11

5

fever, low BP, increased Hr, decreased urine output

Question 12

explain pyrogen induced fever

Answer 12

viruses and bacteria stimulate phagocytic cells which release prostaglandin E2
activates the hypothalamus to increase the temperature set point

Question 13

max EU units for the following

1. intrathecal injection
2. non intrathecal injection
3. sterile water

Answer 13

1. 0.2 EU/kg body weight
2. 0.5
3. 0.25-0.5 EU/mL

Question 14

pyrogens can be detecting using rabbits becuase they have a similar endotoxin tolerance but whats the problem with this

Answer 14

cant quantify

Question 15

what is the limulis amebocyte lyase test

Answer 15

horse shoe crab blood forms clots when exposed to endotoxin

take amebocyte extract from the blood and mix with samples to test to determine pyrogen levels

Question 16

benefits of LAL pyrogen test

Answer 16

fast <30 min

highly sensitive - up to 0.005EU

Question 17

how can pyrogens be removed

Answer 17

charcoal - offers hydrophobic interactions

ion exchange chromatography

Question 18

explain how ion exchange chromatography works

Answer 18

cations line the column

negatively charged endotoxins stick to the sides and proteins are expelled

Question 19

4 ways to remove pyrogens from equipment

Answer 19

oxidation - hydrogen peroxide
heat - 250 for 30 min
acid base hydrolysis - cleave lipid A from polysacharide
sodium hydroxide - clean ion exhange

Question 20

examples of physical instability

Answer 20

adsorption
precipitate formation
denaturation
aggregation

Question 21

excipients used to prevent physical instability

Answer 21

osmotic agents
buffer components
solubility enhancers
antiaggregate and antoadsorbant

Question 22

formation of aggregation can be due to

Answer 22

electrostatic or hydrophobic interactions

formation of covalent bridge with disulfide bonds or ester linkages

Question 23

addition of lysine or arginine solubilizes

Answer 23

tPa

Question 24

addition of sodium dodecylsulfate surfactant solubilizes

Answer 24

non glycosylated IL2

Question 25

anti adsorptive agents reduce

Answer 25

adsorption of active proteins to interfaces ex. water, container, administrative utensils

Question 26

example of irreversible aggregation

Answer 26

hydrophobic parts of aqeuous phase adsorb to hydrophobic interface

Question 27

what do surfactants do

Answer 27

adsorb to hydrophobic interface with their own hydrophobic groups to make the surface hydrophilic to the aqeuous phase

Question 28

example of antiadsorbant in insulin

Answer 28

albumin

Question 29

how do you prevent fibrillar precipitates formation

Answer 29

low concentrations of phospholipids and surfactants | proper pH

Question 30

what iis the most important part of the formation process

Answer 30

adjusting pH with buffer

Question 31

buffer systems in biotech

Answer 31

citrate acetate phosphate

Question 32

example of temporary pH changes causing aggregation

Answer 32

in freeze drying if one of the buffers crystalizes and the other doesnt itll change the pH drop pH

Question 33

example of osmotic agents and what they do

Answer 33

change the tonicity of parenteral products | glucose, dextrose, saline

Question 34

how cna sugars and polyhydric alcohol stabilize the protein structure

Answer 34

enhance interaction of solvents with the protein so its hydrated and then enhances the proteisn tendency to self associate

Question 35

salts to improve physcial stability

Answer 35

decrease denaturation and aggregation by ion binding to the protein

Question 36

polyalcohol to improve physical stability

Answer 36

stabilizze by selective solvation

Question 37

surfactants to improve physical stabilityu

Answer 37

prevetn adsorption of proteins to interfaces

Question 38

some cautions of excipients

Answer 38

multivalent - some of the components may not be compatible | animal source may be contaminated - albumin

Question 39

examples of chemical instability

Answer 39

``` oxidation racemization deamidation proteolysis disulphide exchange ```

Question 40

methionine, cysteine, tryptophan, tyrosine, and histidine are readily oxidized prevent this by

Answer 40

add antioxidants ascorbic acid or acetylcystiene

Question 41

genetic modification of proteins to improve chemical stability

Answer 41

site directed mutagenesis | chemically reactive amino acids are replaced with ones that arent

Question 42

chemical modification to improve chemical instability

Answer 42

coupling with polyethyl glycol

Question 43

preservative needed in vials used for multiple injections some examples

Answer 43

bacteriostatic in nature | mercury containing - thiomerosal, phenol

Question 44

common excipients in a freeze dried prep

Answer 44

bulking agent - mannitol collapse temp modifier - dextran, albumin (increase collapse temp) lycoprotectant - sugars, albumin (protect physical structure)

Question 45

what happens during crystallization step of freeze drying

Answer 45

temp may increase temporarily pH and ionic strength may change protein denaturation

Question 46

small crystal with ___ cooling

Answer 46

fast | required for porous solids and fast sublimation rates

Question 47

large crystal with ____ cooling

Answer 47

low

Question 48

temparature in freezing stage

Answer 48

-15 for crystallization | usually gets to -40 before the pressure is reduced for sublimation

Question 49

explain what happens in primary drying

Answer 49

crystalized and water not bound to protein is removed by sublimation pressure increase temp can drop

Question 50

explain waht happens in secondary drying

Answer 50

water bound to protein is removed | temp is slowly increased to remove bound water and chamber pressure still reduced

Question 51

critical endpoint for the stability of freeze dried products

Answer 51

1% residual water left in the cake

Question 52

stability of protein solution depends on

Answer 52

ionic strength pH presence of stabilizers temperature

Question 53

proteins can be stores as

Answer 53

aqeuous solution freeze dried tablet