Biologicals 1 PDB

Question 1

What is deamidation?

Answer 1

Amide group on asparagine or glutamine residue converted in to different FG.

Question 2

What factors affect the likelihood of deamidation occurring?

Answer 2

PH

Location of asparagine residue. If in beta sheet or alpha helix then protected. Also protected if next to AA with large side chain

Question 3

Which amino acids are most likely to undergo oxidation

Answer 3

Methionine (ox, Arsenal, London, met line)

Cysteine, (ox, Arsenal, ozil, sissy, cysteine)

Question 4

Methionine undergos oxidation to?

Answer 4

Reversible oxidation to sulphoxide

Further oxidation to sulphone

Question 5

Cysteine undergos oxidation to?

Answer 5

Sulphenic acid

Cystine in the presence of another cysteine (disulphide bridge forms)

Question 6

Oxidation is mostly likely with which AAs

Answer 6

Methionine

Cysteine

Question 7

What does methionine oxidise in to?

Answer 7

Sulphoxide (reversible). Think of the met line smelling of sulphur. And then remember what reaction it is (oxidation) = sulphoxide

Sulphoxide to sulphone. Met like smells grim and then you lose your cell phone = sulphone.

Question 8

What does cysteine oxidise in to?

Answer 8

Sulphenic acid (Again sulph but remember this is cystEINE). Therefore sulpENIC acid

In presence of another cysteine it oxidises to cystine

Question 9

What determines the level of oxidation?

Answer 9

Location of the residue

Question 10

Hydrolysis is catalysed by?

Answer 10

Acid or base

Question 11

Name a bond that is susceptible to hydrolysis?

Answer 11

X-Aspartic acid-Y

E.g aspartic acid - proline

Question 12

How are deamidation and hydrolysis involved in insulin degradation?

Answer 12

Certain asparagine residues undergo deamidation in to aspartic acid which is then hydrolysed

Question 13

Asparagine residues at which position in insulin are susceptible to deamidation?

Answer 13

A-21 and B-3

Question 14

CystINE can be converted in to?

Answer 14

Dehydroalanine

AND

Thiocyststeine

Question 15

Dehydroalanine can react with what residues on what AA?

Answer 15

Primary amine on lysine

Question 16

Give an example of how breaking / switching a disulphide bonds can have consequences for a protein

Answer 16

RNAse A. Terminal disulphide bonds broken then melting temp drops by 40 degrees. Can be significant

Question 17

What are the consequences of a raised temperature on proteins?

Answer 17

Denaturation

Melting can lead to exposure of hydrophobic residues. These can then form aggregates with other proteins.

Chemical reactions are quicker at higher temperatures

Question 18

Are thermophillic or mesophillic proteins more susceptible to heating?

Answer 18

Thermophillic!

Question 19

How can pH effect proteins?

Answer 19

Can lead to deamidation but also changes ionisation which can lead to change in a structure of proteins as changes in electrostatic forces (attraction / repulsion)

Question 20

Name 6 physical factors to think about when storing proteins:

Answer 20

Temperature

Ph

Adsorption

Presence of salts and metal ions

Concentration

Interfaces

Question 21

How can the presence of metal ions or salts effect proteins?

Answer 21

Iron ions from stainless steel led to oxidation of herceptin

Question 22

Increased concentration of protein leads to a increased or decreased chance of aggregation?

Answer 22

Increased!

Question 23

Deamidation of asparagine can lead to?

Answer 23

Asparagine can be converted in to aspartic acid (PH 1-2). Aspartic acid has a different charge and therefore different folding

Can also be converted in to aspartimide under ph 5-12. Then hydrolysis in to aspartic acid or iso-aspartic acid

Question 24

How is aspartimide formed?

Answer 24

Deamidation of asparagine under basic conditions.

Question 25

Give some examples of consequences of aggregation:

Answer 25

Hypo or hyper potency Altered solubility Off target binding and therefore adverse effects Anti therapeutic antibody production - Can lead to production of antibodies against endogenous proteins e.g insulin - Can lead to reduced compliance - Can make drug ineffective Aggregates can block lungs or cause strokes May have no consequences

Question 26

Proteins may need to be formulated with:

Answer 26

Solubility enhancers (amino acids, surfactants, sugars, polymers) Buffers to maintain physiological PH (citrate, phosphate or acetate) Anti-absorption /anti-aggregation agents (surfactants or albumin) Preservatives and anti oxidants (ascorbic acid) Cake formers Osmotic agents

Question 27

Proteins have a layer of water around their outside (water of hydration) and bulk water. When solutes are added what can happen?

Answer 27

Can have preferential binding of solute to surface of protein disrupting folding (bad) Can have preferential exclusion of the solute. I.e the water is around the outside of the protein still (best)

Question 28

What agents can be used to prevent preferential binding of solutes to the surface of proteins?

Answer 28

Amino acids, sugars, glycerol, PEG (sterically prevents solutes from interacting with surface)

Question 29

Why would amino acids be added to a protein formulation to aid stability?

Answer 29

Methionine is preferentially oxidised Arginine and glutamine can neutralise charges and stabilise proteins

Question 30

Why would e.g human serum albumin (HSA) be added to a protein formulation?

Answer 30

Degraded preferentially in comparison to the protein

Question 31

Why are cyclodextrins added to proteins?

Answer 31

Hydrophobic pocket can trap and bind hydrophobic residues so it can't aggregate with other hydrophobic residues

Question 32

What are polysorbates?

Answer 32

Ampiphilic emulsifiers Ampiphilic means they have a hydrophobic portion and hydrophilic portion

Question 33

Give an example of a polysorbate

Answer 33

Sorbitan derivatives 3 OH on srobitan replaced with PEG 1 OH replaced with PEG and a fatty acid

Question 34

What do polysorbates do?

Answer 34

Mediate preferential exclusion of solutes Hydrophobic end binds hydrophobic portions of protein and hydrophilic portion bonds hydrophilic portions of protein (keeps it in solution) Form film that prevents protein interaction with air Also preferentially interacts with sides of iv bags

Question 35

Polysorbates can undergo what degradation processes?

Answer 35

Auto oxidation and hydrolytic degradation

Question 36

Polysorbates can undergo degradation to what compounds?

Answer 36

Reactive peroxides Aldehydes

Question 37

Reactive peroxides are a degradation product of polysorbates. What effects can they mediate and how can there action be prevented?

Answer 37

Can react with tryptophan and methionine residues therefore put things in there that preferentially will oxidise (methionine and cysteine)

Question 38

Aldehydes are a degradation product of polysorbates. What effects can they mediate?

Answer 38

Can interact with lysine Increased immunogenicity Formaldehyde and acetaldehyde can cause allergy and are carcinogenic

Question 39

What is the relation between freeze drying and PH?

Answer 39

When buffers are freeze dried, the conjugate acid and the conjugate base will crystallise at different rates. If more of the conjugate acid crystallises (H2Po4-) then get higher acidity. More basic if more base crystallises (HPO42-)