P1 Spring Pceutics 3/09 (Lecture 4)

Front 1

Example of one type of beads used in chromatography?

Back 1

sepharose

Front 2

Purpose of chromatography?
And by what criteria?

Back 2

To separate proteins.

By size, shape, weight, degree of hydrophobicity, affinity to beads, ionic strength.

Front 3

HPLC?

Back 3

High performance liquid chromatography

Front 4

Types of chromatographic techniques?

Back 4

1.Size exclusion chromatography
2.Reverse phase HPLC
3.HIC (hydrophobic interaction chromatography)
4.Ion exchange chromatography

Front 5

Reverse phase HPLC MoA?

Back 5

•Matrix is hydrophobic,
•Hydrophobic eluant (the more hydrophobic the protein is, the more it is attracted to matrix, and the more hydrophobic the eluant must be to wash it out)
•Eluant added in increasing hydrophobicity
•Less hydrophobic proteins elute out first.

Front 6

HIC MoA?

Back 6

•Hydrophobic matrix,
•Aqueous/salty eluant,
•Eluant added in decreasing saltiness,
•Least hydrophobic proteins elute out first

Front 7

Factors that affect production and purification of proteins?

Back 7

1.N terminal and C terminal heterogeneity (during production)
2.Chemical modifications (during purification)
3.Glycosylation (lack of, during production)
4.Proteases (during purification)
5.Inclusion bodies (during production)

Front 8

N terminal and C terminal heterogeneity?

Back 8

•Methionines (MET) inserted in protein cause addition start codons
•Stop codons inserted as well?
•Aminos are lost at one of the terminal ends
•Causes non functional protein to do incorrect conformation (typically)

Front 9

What chemical mods are problematic?

Back 9

•Deamidation
•Hydrolysis of peptide bonds
•Oxidation of sulhydryls groups on aminos
•Proteases

Front 10

How do includion bodies form in bacteria?

Back 10

When they produce too much protein.

Granules of proteins form.

Front 11

What do non glycosylated proteins tend to do?

Back 11

aggregate and precipitate

Front 12

List protein solubility enhancers, and protein examples for each

Back 12

1.pH buffer (all proteins are sensitive to pH)
2.lysine and arginine (inc sol of tissue plasminogen activator, TPA)
3.surfactant (eg SDS, to inc sol of non glycosylated IL-2)

Front 13

Name an anti-aggregation/anti-adsorption agent

Back 13

Albumin

Front 14

In general, how can a change in pH affect proteins?

Back 14

•Decrease solubility
•Decrease physical stability
•Decrease chemical stability

Front 15

List buffer systems

Back 15

•Phosphate
•Acetate
•Citrate

Front 16

What makes some proteins susceptible to oxidative degradation?

Back 16

Proteins rich in:

•Methionine
•cysteine
•Tryptophan
•Histidine
•Tyrosine

Front 17

List ways to reduce oxidation of proteins in solution

Back 17

1.anti-oxidants: ascorbic acid, acetylcysteine
2.replace oxygen with inert gas

Front 18

When is adding preservatives especially important?

Back 18

For multi-use containers (due to inc contamination possibility)

Front 19

Storage forms of proteins

Back 19

1.Aqueous solution
2.Freeze dried (powder, lyoized?)
3.Tablet

Front 20

Process of freeze drying?

Back 20

1.Freezing
2.Primary drying step
3.Secondary drying step

Front 21

What temp is used during step 1 of freeze drying?

Back 21

For amorphous proteins: Tg (glass transition temp)

For non amorphous proteins: Te (eutectic temp)

Front 22

What is Tg?

Back 22

For amorphous proteins, its the temp that changes the amorphous phase to a glasslike/crystalline phase.

Front 23

What is Te?

Back 23

•For non amorphous proteins, its the temp that changes liquid phase to solid phase.
•At Te, you will have a mix of unbound water crystals and solute crystals.

Front 24

What are bulking agents, give examples.

Back 24

During freeze drying, they prevent loss of solute taken away as water vapor leaves.

eg Mannitol + glycine

Front 25

What are Lyoprotectants, give examples.

Back 25

During freeze drying, they protect the physical structure of protein.

eg Sugars, albumin

Front 26

What's accomplished during step 1, 2, and 3?

Back 26

Step 1: freezing only

Step 2: removal of unbound crystallized water

Step 3: removal of crystallized water bound to protein and excipients

Front 27

What temps/pressures are used at each stage?

Back 27

Step 1: Below Tg or Te (generally --40 deg C), normal pressure

Step 2: Same temp, but lower pressure

Step 3: Slowly increase temp to --20 deg C (normal pressure?)

Front 28

List therapeutic Abs

Back 28

•Remicade
•Rituxan
•Herceptin

Front 29

Whats remicade for, and MoA?

Back 29

Indication: rheumatoid arthritis, Crohn's disease.

MoA: anti-TNF

Front 30

Whats rituxan for, and MoA?

Back 30

Indication: lymphoma

MoA: targets B cells

Front 31

Whats herceptin for, and MoA?

Back 31

Indication: breast cancer

MoA: targets EGF-Rc (epidermal growth factor receptor)

Front 32

What enzyme cuts Ab into two Fab segments and one Fc fragment?

Back 32

Papain

Front 33

What enzyme cuts Ab into one F(ab`)2 fragment and two Fc fragments?

Back 33

Pepsin

Front 34

Whats a HYBRIDOMA comprised of?

Back 34

Tumor cell + B cell (Ab forming cell)

Front 35

Example of a MAb made of mouse Ab?

Back 35

Muromonab cd3

Front 36

Example of a MAb made of human Ab (and mouse CDRs)?

Back 36

Rituximab

Front 37

Example of a MAb made of chimeric Ab?

Back 37

Infimab (made of mouse and human Ab parts)