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endo y3s1 > IC8 Recombinant protein > Flashcards

IC8 Recombinant protein Flashcards

1
Q

Protein pharmaceuticals
Advantages

A

High specificity & activity ⇒ high potency
Relatively low concentrations ⇒ lesser SE

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2
Q

Protein pharmaceuticals
challenges

A
  1. antigenicity
  2. stability (biological, chemical, physical)
  3. drug delivery
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3
Q

Protein pharmaceuticals
challenges: antigenicity reasons

A

Foreign proteins may induce immunogenic response from human host

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4
Q

Protein pharmaceuticals
challenges: antigenicity LT effects

A

Loss of efficacy due to development of Ab in the patient’s body against an exogenous protein

Can counter by increasing dose, but might cause SE

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5
Q

Protein pharmaceuticals
challenges: stability - how methods of destabilising

A

(1) denaturation, (2) covalently modifying protein, (3) partially degrading it

loss of proper 3D confirmation ⇒ loss of biological activity

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6
Q

Protein pharmaceuticals
challenges: stability - when destabilisation occurs

A
  1. Protein recovery from its source (extraction procedures)
  2. Protein purification process
  3. Post-protein purification (protein storage)
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7
Q

Protein pharmaceuticals
challenges: stability - when destabilisation occurs
post-protein purification problems

A
  1. Proteolysis due to enzymes associated with bacterial contamination; bacteria produces proteases that hydrolyses protein
  2. Storage of proteins in solution → protein degradation (specific amino acids contribute to destabilisation) ⇒ hence protein products usually stored in freeze-dried form (solid)
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8
Q

Protein pharmaceuticals
challenges: stability - shelf-life

A

before reconstitution ~2-3 years
after reconstitution ~7 days to 1 month

storage temperature @ 2-8℃

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9
Q

Protein pharmaceuticals
challenges: stability - changes in potency

A

generally decreases over time due to unfolding of protein ⇒ reduced clinical effects

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10
Q

Mechanisms causing instability of protein pharmaceuticals

A

physical & chemical

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11
Q

Mechanisms causing instability of protein pharmaceuticals:
physical - Protein aggregation

progression

A

native <-> unfolded -> aggregated

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12
Q

Mechanisms causing instability of protein pharmaceuticals:
physical - Protein aggregation

Expression of protein physical stability

A

difference in free energy ∆G between N & U states

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13
Q

Mechanisms causing instability of protein pharmaceuticals:
physical - Protein aggregation

Unfolding reversibility conditions

A

reversible: If remove unfavourable conditions
irreversible: Continuous exposure to unfavourable conditions

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14
Q

Mechanisms causing instability of protein pharmaceuticals:
physical - Protein aggregation

Aggregated proteins: how it occurs

A

Subsequent aggregation of denatured molecules

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15
Q

Mechanisms causing instability of protein pharmaceuticals:
physical - Protein aggregation

Aggregated proteins: impact

A

irreversible denaturation
Aggregated proteins have altered immunity & may arouse immunogenicity

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16
Q

Mechanisms causing instability of protein pharmaceuticals:
physical - Protein aggregation

Aggregated proteins: Hydrophobic force

A

major force for protein unfolding & aggregation

*Due to exposure of hydrophobic surfaces → result of chemical degradation/ modifications
Might also be due to unfavourable physical & chemical factors occurring simultaneously

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17
Q

Physical factors affecting protein stability

T, P, A, SS, NA, FT, P

A
  1. Temperature
  2. pH
  3. Adsorption
  4. Shaking & shearing (agitation)
  5. Non-aqueous solvents
  6. Repeated freeze-thaw
  7. Photodegradation
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18
Q

Physical factors affecting protein stability (T)

A

Increased temp promotes protein unfolding by disrupting non-covalent forces that stabilise protein’s conformation ⇒ encourages denaturation
Denatured proteins aggregate → irreversible denaturation.

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19
Q

Physical factors affecting protein stability (P)

A

Proteins unfold at extreme pHs due to changes in ionisation status of side chains of amino acid residues
Causes confirmation changes → protein starts unfolding ⇒ aggregation

Disruption of distribution of ionic attractive & repulsive forces that stabilise protein’s conformation

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20
Q

Physical factors affecting protein stability (A)

A

Proteins can be adsorbed to many surfaces & interfaces → especially plastic
Significant change in secondary structure & tertiary structure → change in 3D conformation
Loss of proteins or destabilisation of proteins (due to aggregation)

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21
Q

Physical factors affecting protein stability (SS)

A

Incorporation of air into protein solution, creates air/liquid interface
Alignment of proteins along such interfaces → unfolding of protein to maximise exposure of hydrophobic residues to air ⇒ partial or complete protein denaturation

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22
Q

Physical factors affecting protein stability (NA)

A

Protein hydration shell may be disrupted
Protein hydrophobic core exposed when polarity of aqueous solvent decrease → Protein unfolds ⇒ LR: protein degradation

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23
Q

Physical factors affecting protein stability (FT)

A

Formation of sharp ice crystals → can pierce through 3D conformation of protein
Polypeptide chain encouraged to unfold ⇒ protein degradation

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24
Q

Physical factors affecting protein stability (P)

A

Risk of protein aggregation upon exposure to light ⇒ important to store in amber bottle

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25
Q

Mechanisms causing instability of protein pharmaceuticals:
chemical instability

A
  1. Deamination
  2. Oxidation
  3. Disulfide bond breakage & formation
  4. Hydrolysis
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26
Q

susceptible amino acids of deamination

A

Asn & Gln

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27
Q

susceptible amino acids of Oxidation

A

His, Met, Cys, Trp, Tyr

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28
Q

susceptible amino acids of Disulfide bond breakage & formation

A

Cys

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29
Q

susceptible amino acids of Hydrolysis

A

Asp-Gly & Asp-Pro

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30
Q

Oxidation: how it works

A

Catalysed by transition metal ions at/ near metal binding sites of proteins
Reactive oxygen species generated → drives oxidation

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31
Q

Disulfide bond breakage & formation: how it works

A

Occurs between 2 cysteine residues → sulfhydryl groups between cysteine molecule joined together to form S-S

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32
Q

Disulfide bond breakage & formation: effects on stability

A

Sometimes needed to favour stability in some proteins

sometimes detrimental to protein stability:
Destroys activity of proteins; more so if cysteine residue in reduced form is required for activity

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33
Q

Methods for stabilisation & formulation of protein pharmaceuticals (liquid)

A
  1. Substitution & chemical modifications (Internal)
  2. Changing properties of solvent & additives (external)
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34
Q

Methods for stabilisation & formulation of protein pharmaceuticals (liquid)
1. Substitution & chemical modifications (Internal): methods

A

a. Amino acid substitution/ modification
b. Introduction of disulfide bonds
c. PEGylation (conjugation)
d. Acylation (conjugation)

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35
Q

Methods for stabilisation & formulation of protein pharmaceuticals (liquid)
1. Substitution & chemical modifications (Internal): requirements

A

Internal changing of structural characteristics without compromising activity ⇒ improves protein stability

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36
Q

Methods for stabilisation & formulation of protein pharmaceuticals (liquid)
1. Substitution & chemical modifications (Internal): Introduction of disulfide bonds - how it works

A

Stabilise folded form of proteins

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37
Q

Methods for stabilisation & formulation of protein pharmaceuticals (liquid)
1. Substitution & chemical modifications (Internal): PEGylation (conjugation) - how it works

A

Chemical attachment of polyethylene glycol (PEG)
Can keep in native form for longer periods
Increase circulation time in blood

38
Q

Methods for stabilisation & formulation of protein pharmaceuticals (liquid)
1. Substitution & chemical modifications (Internal): acylation (conjugation) - how it works

A

Chemical attachment of fatty acids to residues on protein surface
FA:
Lipophilic; makes overall complex more lipophilic ⇒ expels water
Maintains protein stability

Increase circulation time in blood

39
Q

Methods for stabilisation & formulation of protein pharmaceuticals (liquid)
2. Changing properties of solvent & additives (external) - methods

S, SE, AA, B, PA

A

Stabilisers: sugars, polyols
Solubility enhancers: Lysine, arginine, surfactants
Anti-adsorption & anti-aggregation agents: Albumin, surfactants
Buffer components: Phosphate salts (Na2HPO4, NaH2PO4)
[Prevents extreme acidic/ alkaline conditions]
Preservatives & antioxidants: Inert gas, thimerosal, phenol, benzyl alcohol
[Prevents oxidation]

40
Q

Process of making recombinant protein

A

Upstream & downstream

41
Q

Upstream process of making recombinant protein

A
  1. Host cells transfected with recombinant DNA (carrying desired gene)
  2. Each transfected cell is different from each other in terms of number of copies of plasmids transfected
    (Higher number of copies of plasmids = higher amount of protein expressed)
  3. ONLY 1 transfected cell with best cell growth properties & highest protein yield ⇒ development of master cell line
42
Q

Upstream process of making recombinant protein
requirements of host cell

A

ensure purity & no trace of host cell components in final product ⇒ safety & quality ensured

43
Q

Upstream process of making recombinant protein
cells to use

A
  1. E.coli
  2. CHO
44
Q

Upstream process of making recombinant protein
1. E.coli indication for use

A

for small proteins production & to obtain high yield @ low cost
(Large protein production → might cause formation of inclusion bodies)

45
Q

Upstream process of making recombinant protein
1. E.coli advantages

A
  1. facilitates genetic manipulation (high success rate)
  2. High expression levels of recombinant protein (up to 30% of total cellular protein)
  3. Grows rapidly on simple & inexpensive media → doubles every 20 mins
46
Q

Upstream process of making recombinant protein
1. E.coli disadvantages

A

Recombinant protein accumulates intracellularly

Lack the ability to perform post-translational modifications (ie. glycosylation) –> Cannot use E.coli if glycosylation required by human protein for therapeutic fx

Presence of lipopolysaccharides (LPS) on its surface → act as pyrogens
- Have both lipophilic & polar ends → difficult to remove in downstream processes
- Fever inducing; when introduced into bloodstream → may lead to inflammatory response, shock or multi-organ failure ⇒ death

47
Q

Upstream process of making recombinant protein
1. E.coli: solubility of proteins

A

Soluble proteins = in native conformation
insoluble proteins = incorrect conformation

48
Q

Upstream process of making recombinant protein
1. E.coli: why formation of inclusion body occurs

A

proteins are synthesised rapidly & in high levels

49
Q

Upstream process of making recombinant protein
1. E.coli: processing of inclusion body

A

Isolation of inclusion bodies
Insoluble products; if protein in native formation → will be soluble

Solubilisation of protein with denaturant
Breaks up non-covalent interactions in unfolded polypeptide chain → protein unfolds to primary polypeptide chain ⇒ soluble

Refolding of protein outside cell
Native conformation → soluble
Incorrectly folded protein → insoluble, will form aggregate

50
Q

Upstream process of making recombinant protein
2. CHO indications

A

Large protein production
Post-translational modification required
Crucial for solubility & native folding

51
Q

Upstream process of making recombinant protein
2. CHO advantages

A
  • Capable of adapting & growing in suspension culture → ideal for large scale culture
  • Pose less risk → few human viruses can propagate in them ⇒ less likely to have cross infections
  • Can grow in serum-free media → ensures reproducibility between different batches of cell culture [independent of human component]
  • Allow post-translational modifications to recombinant proteins
  • Can be manipulated by genetic engineering techniques to produce a higher yield of recombinant proteins
52
Q

Downstream process of making recombinant protein

A

Purification: protein isolation, concentration & purification steps, viral inactivation steps

53
Q

Downstream process of making recombinant protein
purpose

A

To obtain protein of interest that is purified
To remove other CHO/ E.coli proteins → trace amount in final product results in immunogenicity

54
Q

Testing of product: 2 methods

A

Quality control
safety testing

55
Q

QC: purpose

A

confirm conformance of final product to predetermined specifications
Must be done for every batch of products

56
Q

QC: methods

A
  1. Bioassays/ potency testing
  2. Immunoassays
  3. Mass spectrometry
  4. Peptide mapping
  5. Amino acid analysis
  6. N-terminal sequencing
  7. Isoelectric focusing
57
Q

QC: bioassay purpose

A

assess activity of product in a biological system; whether the product can work on the substrate

58
Q

QC: bioassay - how it works

A

Activity of product → “units of activity” per vial/dose of the product
* Chosen at random
* Quantitative measure against a “standard” preparation of known activity

59
Q

QC: bioassay Drawbacks

A

Time consuming
High cost
Do not reveal purity of final product → no information about contaminant (safety profile not considered)

60
Q

QC: Immunoassays - how it works

A

Use of antibodies to quantify product → ELISA, agglutination

61
Q

QC: Immunoassays advantage

A

straightforward, fast, less costly

62
Q

QC: Immunoassays disadvantage

A

Quantity of product ≠ biological activity of product
do not reveal purity of final product (cannot detect presence of contaminants)

63
Q

QC: mass spectrometry - how it works

A

Each protein made have unique mass spectrum

Comparing mass spectrum of each batch of final product (against a highly pure “standard”) will allow identification of possible contaminants in the samples

Presence of extra peaks in results → likely additional ingredients that do not belong to human recombinant

64
Q

QC: peptide mapping purpose

A

product identification & detection of protein contaminants

65
Q

QC: peptide mapping - how it works

A

Protein product hydrolysed using reagents specific in cleaving specific peptide bonds (e.g. cyanogen bromide, trypsin) → gives unique peptide fingerprint (by mass spec, 2D gel electrophoresis, RP-HPLC)
* Predictable location of cleavage & what kind of peptides will be produced

66
Q

QC: peptide mapping disadvantage

A

Does not tell activity of product

67
Q

QC: Amino acid analysis - how it works

A

Protein hydrolysed into amino acids → separated by ion exchange chromatography & quantified.

68
Q

QC: Amino acid analysis indication

A

characterising peptide or small polypeptide product of < 10kDa

68
Q

QC: N-terminus sequencing purpose

A

identification of protein product

69
Q

QC: N-terminal sequencing - how it works

A

Sequencing of the first 20-30 amino acids of the protein at the N-terminus

70
Q

QC: Isoelectric focusing purpose

A

determine sialic acid content in glycoproteins

71
Q

Safety testing: purpose

A

Assessment of presence of impurities; prevention of immunogenicity issues

72
Q

Safety testing: methods

A
  1. SDS-PAGE
  2. Isoelectric focusing dye binding methods (colorimetric assays)
  3. DNA hybridisation
  4. Rabbit pyrogen test
  5. Litmus amoebocyte lysate (LAL) test
  6. Viral assays
  7. In vivo bioassays
73
Q

ST: SDS-PAGE - how it works

A

High resolution electrophoretic separation of proteins based on molar mass (for SDS-PAGE) or protein folding (for native PAGE)
Visualisation of separated proteins by protein stains

73
Q

ST: SDS-PAGE detection

A

Detection of product variants possible using product-specific Ab in Western blot

74
Q

ST: isoelectric focusing - how it works

A

Separation of proteins by isoelectric point (pI)
Can be used with SDS-PAGE in 2D electrophoresis → provide added dimension of separation to detect contaminants

75
Q

ST: isoelectric focusing (secondary purpose)

A

monitor homogeneity of glycoproteins’ sialic acid content
* sialic acid on glycan charged → affects pI of glycoprotein

76
Q

ST: DNA hybridisation purpose

A

For detection of DNA contaminants in ng range

77
Q

ST: rabbit pyrogen test - how it works

A

Detection of pyrogen by injecting product into healthy rabbits
Increased temperature = presence of pyrogen

78
Q

ST: LAL test - how it works

A

Endotoxin stimulated coagulation of amoebocyte fraction in blood of horseshoe crabs (Limulus)

79
Q

ST: LAL test advantages

A

less variable, more sensitive, faster, cheaper

80
Q

ST: LAL test disadvantages

A

only detects endotoxin-based pyrogens

81
Q

ST: viral assays - purpose

A

TEST FOR:
specific viruses capable of contaminating source materials AND
unknown/uncharacterised viruses not widely available or employed

82
Q

ST: viral assays - how it works

A

Use of Immunoassays using Ab specific for panel of viruses (to do as a panel)

incubation of product with cell lines sensitive to range of virus (ie: Vero cells) OR
injection of product into animals for stimulation of antibody production & subsequent testing of specificities of Ab raised in the animals against a panel of viruses

83
Q

ST: In vivo bioassays purpose

A

general safety testing → ie injection into healthy mice

84
Q

Biosimilars definition

A

Biologic that is almost an identical (ideally identical) alternative version of original biologic (innovator/ reference biologic) manufactured by different country

85
Q

Biosimilars - variability (how it occurs)

A

occurs even within batches of same product

  1. variability of biological expression system & manufacturing process
  2. Process of manufacturing (upstream & downstream) influences nature of final product
86
Q

Biosimilars: chemical drugs (pdn of generic drugs)

A

No issue with production of generic drugs → analytical criteria based on chemical compositions

87
Q

Biosimilars: biologics with low MW

A

More straightforward development of biosimilars ⇒ approval of several biosimilars

88
Q

Biosimilars: mABs with post-translational modification
problem of biosimilarity

A

impossible to engineer a biosimilar 100% identical to innovator biologic ⇒ ONLY CAN produce a highly similar biosimilar

89
Q

Biosimilars: mABs with post-translational modification
reasons for variability

A

Pattern of glycosylation & amount of glycosylation dependent on cell production system (i.e. type of host cells used for culture)

For the same monoclonal Ab for innovator & biosimilar biologic produced in same host cells, glycosylation may be different → other factors like cell culture conditions can influence glycosylation.

90
Q

Biosimilars: requirements for approval

A
  1. Extensive in vitro studies demonstrating similarity to a reference biologic.
  2. Non-clinical & clinical studies demonstrating comparable pharmacokinetics (PK), clinical efficacy, safety & immunogenicity

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