PR3152 IC8

Question 1

what are the major challenges to protein drugs?

Answer 1

1) stability
2) antigenicity
3) drug delivery

Question 2

what causes antigenicity challenges in proteins?

Answer 2

foreign particles from non-human host may induce immunogenic response

Question 3

what causes stability challenges in proteins?

Answer 3

1) protein extraction/recover
2) protein purification
3) post protein purification (protein storage)
- proteolysis due to enzymes from contamination
- storage in solution may also lead to degradation (due to presence of specific amino acids that contribute to destabilisation + water is weka nucleophile:hydrolysis) = need to freeze dry

Question 4

differences in potency and purity of proteins over time in storage?

Answer 4

potency will decrease faster in storage vs purity as the protein unfolds and loses its conformation

Question 5

what are the mechanisms leading to protein instability?

Answer 5

1) physical
- aggregation

2) chemical
- deamination
- oxidation
- proteolysis
- disulfide exchange

Question 6

explain the mechanism of aggregation contributing to protein instability

Answer 6

N <=> unfolded (U) => A
protein stability expressed as gibbs energy change which represents the diff in energy between the native and unfolded stages.
- unfolding can be reversible or irreversible.
- subsequent aggregation is irreversible and will lead to denaturation = immunogenicity.

Question 7

factors leading to aggregation of proteins?
describe how the proteins aggregate to each other

Answer 7

1) temp
2) vortexing
3) ionic strength
4) chemical modifications (or degradation)
5) pH

aggregation occurs due to the hydrophobic forces between the unfolded proteins.

Question 8

what are the physical factors leading to protein aggregation?

Answer 8

temp
ph
adsorption
shearing/shaking
non-aqueous solvents
repeat freeze thaw
photodegradation

Question 9

how does temperature affect protein stability?

Answer 9

temperature disrupts the non-covalent interactions causing unfolding and a loss of 3d conformation
+
aggregation = denaturation

Question 10

how does pH affect protein stability?

Answer 10

pH affects the ionisation of side chains of amino acid residues leading to disruption of the ionic forces the stabilise the 3d conformation

additionally = chemical stability e.g., with
Asp = hydrolysis
Asn & Gln = deamination

Question 11

how does adsorption affect protein stability

Answer 11

proteins attach to the surface of materials leading to change of the secondary or tertiary structure leading to loss/destabilisation.

Question 12

how does shearing and shaking affect protein stability?

Answer 12

both cause airbubbles to form = liquid=air separation = protein align to the surface and unfolds to maximise surface to hydrophobic air residues

Question 13

how does repeat freezing and thawing affect protein stability?

Answer 13

disrupts the 3d confromation by formation of ice crystals

Question 14

how does non aqueous solvent affect protein stability?

Answer 14

hydration shell disrupted exposing the hydrophobic core = unfolding

Question 15

how does photodegradation affect protein stability?

Answer 15

exposure to LIGHT causes protein aggregation and/or protein cleavage (by UV)

Question 16

(chemical instability) deamidation of proteins?

Answer 16

most common
Asn and Gln

Question 17

(chemical instability) hydrolysis of proteins?

Answer 17

acidic and basic ph
Asp-gly
Asp-pro
particularly labile

Question 17

(chemical instability) oxidation of proteins?

Answer 17

Side chains of His (H), Met (M), Cys (C), Trp (W), Tyr (Y)
catalysed by metallic ions
thio groups of M and C particularly susceptible

Question 17

(chemical instability) disulfied form/break of proteins?

Answer 17

disulfide bonds in Cys

Question 18

two strategies to modify the stabiltiy of LIQUID proteins

Answer 18

1) substitution and chemical modification (internal changing of structural characteristics without compromising activity)

2) changing the properties of the solvents, additives (external)

Question 19

substitution and chemical modification methods for improving liquid protein stability?

Answer 19

1) amino acid substitution
2) introduction of disulfied bond (increase stability)
3) PEGylation (increase circulation time)
4) acylation (increase circulation time) (attachment of fatty acid to a.a. residues = lipophilic = upon storage will avoid water = maintain protein stability)

Question 20

solvent/additive methods for improving liquid protein stability?

Answer 20

Stabilizers
* Sugars, polyols

Solubility enhancers
* Lysine, arginine, surfactants

Anti adsorption and anti aggregation agents
* Albumin, surfactants

Buffer components
* Phosphate salts (Na2HPO4 , NaH2PO4

Preservatives and anti oxidants
* Inert gas, thimerosal, phenol, benzylalcohol

Question 21

what occurs during upstream processing of biopharmaceutical manufacturing?

Answer 21

1) selection of host cells for recombinant protein making

2) selection of the 1 transfected cell that possesses the best cell growth properties and highest protein yield = development of the master cell line.

Question 22

Advantages of ecoli as the host cell for recombinant protein

Answer 22

1) well characterised - easier genetic manipulation

2) produce high expression levels of recombinant proteins

3) grows rapidly on simple and inexpensive media

Question 23

DISadvantages of ecoli as the host cell for recombinant protein

Answer 23

1) recombinant protein accumulates intracellularly
- if low yield: will dissolve in the cell and require additional processing steps to purify.
- if high yield: will form insoluble aggregates (inclusion bodies) that require additional refolding.

2) requires post translational modification e.g., glycosylation

3) presence of lipopolysaccharides (LPS) on surface of E.coli that can act as pyrogens

Question 24

what are the extra steps in e.coli protein production (to combat inclusion bodies)

Answer 24

1) isolation
- of INCLUSION BODIES

2) solubilisation
- denaturation

3) refolding
- outside the cell
- by dilution or dialysis
- involves enzymes and factors

afterwards, to separate the folded from misfolded protein

Question 25

why is CHO (Chinese hamster ovary) cells preferred for protein production?

Answer 25

1) CHO capable of growing and adapting to cell culture suspension

2) few virus can propagate = low risk

3) can grow in serum free media = ensures reproducibility + less contaminants

4) can undergo post-translational modification, eg glycosylation is human like = more human compatible and bioactive

5) can be manipulated by genetic engineering = higher yield

Question 26

Choice of host cells to make recombinant proteins depends on size and characteristics of protein

Answer 26

Large proteins 100 kDa = CHO

Small proteins 30 kDa = Ecoli

Post translational modification essential? = CHO

Production of protein where solubility and native folding
is crucial? = CHO

High yields, low cost = Ecoli

Question 27

what are the downstream processes for protein pharmaceuticals?

Answer 27

centrifugation OR filtration (cell harvesting)

precipitation AND/OR liquid-liquid extraction

chromatography

viral inactivation

polishing chromatography (remove impurities)

viral filtration

diafiltration (separate based on MW)

Question 28

what are the methods to ensure QC and product quality in protein pharmaceuticals

1) bioassay/potency testing

2) immunoassay

3) mass spectrometry

4) peptide mapping

5) amino acid analysis

6) n-terminal sequencing

7) isoelectric focusing

Answer 28

1) bioassay/potency testing
- good: potency
- bad: expensive, slow, X purity

2) immunoassay
- good: faster and cheaper than bioassay
- bad: X potency, X purity

3) mass spectrometry
- for contaminant detection

4) peptide mapping
- for protein identification
- for contaminant detection

5) amino acid analysis
- for small peptide or polypeptides < 10KDa

6) n-terminal sequencing
- for protein identfication

7) isoelectric focusing
- determine sialic acid content in glycoproteins

Question 29

what are the safety testing methods in protein pharmaceuticals?

Answer 29

1) SDS PAGE (sodium dodecyl sulphate polyacrylamide gel
electrophoresis)

2) Isoelectric focusing Dye binding methods (colorimetric assays)
- also used to monitor homogeneity of sialic acid content in glycoproteins

3) DNA hybridization (e.g. dot blots)

4) Rabbit pyrogen test (bioassay)

5) Limulus amoebocyte lysate (LAL) test

6) Viral assays
In vivo bioassays

Question 30

explain viral assays

Answer 30

To test for (i) specific viruses capable of contaminating source materials, and (ii) unknown/uncharacterized viruses not widely available or employed

• Immunoassays using antibodies specific for panel of viruses can be
  employed.
• Bioassays: (i) involving incubation of product with cell lines sensitive to range of virus (e.g. Vero cells), or (ii) injection of product into animals for stimulation of antibody production and subsequent testing of specificities of antibodies raised in these animals against a panel of viruses
• Virus specific DNA probes (Dot blots or PCR based assays)

Question 31

final characteristics of biosimilars influenced by:

Answer 31

manufacturing process (type of host cell, development of genetically modified cell for production, cell culture system, production process, purification process, formulation of recombinant protein)

Question 32

requirements for biosimilar approval by US FDA

Answer 32

Extensive in vitro studies demonstrating similarity to a reference biologic.

Non clinical and clinical studies demonstrating comparable
pharmacokinetics (PK), clinical efficacy, safety and immunogenicity.

Note
The methods/assays used to establish comparability of biosimilars to their reference products should be sufficiently specific and sensitive to
be able to detect the differences between the two products