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IC8 Recombinant protein Flashcards

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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
Mechanisms causing instability of protein pharmaceuticals: chemical instability
1. Deamination 2. Oxidation 3. Disulfide bond breakage & formation 4. Hydrolysis
26
susceptible amino acids of deamination
Asn & Gln
27
susceptible amino acids of Oxidation
His, Met, Cys, Trp, Tyr
28
susceptible amino acids of Disulfide bond breakage & formation
Cys
29
susceptible amino acids of Hydrolysis
Asp-Gly & Asp-Pro
30
Oxidation: how it works
Catalysed by transition metal ions at/ near metal binding sites of proteins Reactive oxygen species generated → drives oxidation
31
Disulfide bond breakage & formation: how it works
Occurs between 2 cysteine residues → sulfhydryl groups between cysteine molecule joined together to form S-S
32
Disulfide bond breakage & formation: effects on stability
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
33
Methods for stabilisation & formulation of protein pharmaceuticals (liquid)
1. Substitution & chemical modifications (Internal) 2. Changing properties of solvent & additives (external)
34
Methods for stabilisation & formulation of protein pharmaceuticals (liquid) 1. Substitution & chemical modifications (Internal): methods
a. Amino acid substitution/ modification b. Introduction of disulfide bonds c. PEGylation (conjugation) d. Acylation (conjugation)
35
Methods for stabilisation & formulation of protein pharmaceuticals (liquid) 1. Substitution & chemical modifications (Internal): requirements
Internal changing of structural characteristics without compromising activity ⇒ improves protein stability
36
Methods for stabilisation & formulation of protein pharmaceuticals (liquid) 1. Substitution & chemical modifications (Internal): Introduction of disulfide bonds - how it works
Stabilise folded form of proteins
37
Methods for stabilisation & formulation of protein pharmaceuticals (liquid) 1. Substitution & chemical modifications (Internal): PEGylation (conjugation) - how it works
Chemical attachment of polyethylene glycol (PEG) Can keep in native form for longer periods Increase circulation time in blood
38
Methods for stabilisation & formulation of protein pharmaceuticals (liquid) 1. Substitution & chemical modifications (Internal): acylation (conjugation) - how it works
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
Methods for stabilisation & formulation of protein pharmaceuticals (liquid) 2. Changing properties of solvent & additives (external) - methods S, SE, AA, B, PA
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
Process of making recombinant protein
Upstream & downstream
41
Upstream process of making recombinant protein
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
Upstream process of making recombinant protein requirements of host cell
ensure purity & no trace of host cell components in final product ⇒ safety & quality ensured
43
Upstream process of making recombinant protein cells to use
1. E.coli 2. CHO
44
Upstream process of making recombinant protein 1. E.coli indication for use
for small proteins production & to obtain high yield @ low cost (Large protein production → might cause formation of inclusion bodies)
45
Upstream process of making recombinant protein 1. E.coli advantages
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
Upstream process of making recombinant protein 1. E.coli disadvantages
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
Upstream process of making recombinant protein 1. E.coli: solubility of proteins
Soluble proteins = in native conformation insoluble proteins = incorrect conformation
48
Upstream process of making recombinant protein 1. E.coli: why formation of inclusion body occurs
proteins are synthesised rapidly & in high levels
49
Upstream process of making recombinant protein 1. E.coli: processing of inclusion body
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
Upstream process of making recombinant protein 2. CHO indications
Large protein production Post-translational modification required Crucial for solubility & native folding
51
Upstream process of making recombinant protein 2. CHO advantages
- 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
Downstream process of making recombinant protein
Purification: protein isolation, concentration & purification steps, viral inactivation steps
53
Downstream process of making recombinant protein purpose
To obtain protein of interest that is purified To remove other CHO/ E.coli proteins → trace amount in final product results in immunogenicity
54
Testing of product: 2 methods
Quality control safety testing
55
QC: purpose
confirm conformance of final product to predetermined specifications Must be done for every batch of products
56
QC: methods
1. Bioassays/ potency testing 2. Immunoassays 3. Mass spectrometry 4. Peptide mapping 5. Amino acid analysis 6. N-terminal sequencing 7. Isoelectric focusing
57
QC: bioassay purpose
assess activity of product in a biological system; whether the product can work on the substrate
58
QC: bioassay - how it works
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
QC: bioassay Drawbacks
Time consuming High cost Do not reveal purity of final product → no information about contaminant (safety profile not considered)
60
QC: Immunoassays - how it works
Use of antibodies to quantify product → ELISA, agglutination
61
QC: Immunoassays advantage
straightforward, fast, less costly
62
QC: Immunoassays disadvantage
Quantity of product ≠ biological activity of product do not reveal purity of final product (cannot detect presence of contaminants)
63
QC: mass spectrometry - how it works
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
QC: peptide mapping purpose
product identification & detection of protein contaminants
65
QC: peptide mapping - how it works
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
QC: peptide mapping disadvantage
Does not tell activity of product
67
QC: Amino acid analysis - how it works
Protein hydrolysed into amino acids → separated by ion exchange chromatography & quantified.
68
QC: Amino acid analysis indication
characterising peptide or small polypeptide product of < 10kDa
68
QC: N-terminus sequencing purpose
identification of protein product
69
QC: N-terminal sequencing - how it works
Sequencing of the first 20-30 amino acids of the protein at the N-terminus
70
QC: Isoelectric focusing purpose
determine sialic acid content in glycoproteins
71
Safety testing: purpose
Assessment of presence of impurities; prevention of immunogenicity issues
72
Safety testing: methods
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
ST: SDS-PAGE - how it works
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
ST: SDS-PAGE detection
Detection of product variants possible using product-specific Ab in Western blot
74
ST: isoelectric focusing - how it works
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
ST: isoelectric focusing (secondary purpose)
monitor homogeneity of glycoproteins’ sialic acid content * sialic acid on glycan charged → affects pI of glycoprotein
76
ST: DNA hybridisation purpose
For detection of DNA contaminants in ng range
77
ST: rabbit pyrogen test - how it works
Detection of pyrogen by injecting product into healthy rabbits Increased temperature = presence of pyrogen
78
ST: LAL test - how it works
Endotoxin stimulated coagulation of amoebocyte fraction in blood of horseshoe crabs (Limulus)
79
ST: LAL test advantages
less variable, more sensitive, faster, cheaper
80
ST: LAL test disadvantages
only detects endotoxin-based pyrogens
81
ST: viral assays - purpose
TEST FOR: specific viruses capable of contaminating source materials AND unknown/uncharacterised viruses not widely available or employed
82
ST: viral assays - how it works
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
ST: In vivo bioassays purpose
general safety testing → ie injection into healthy mice
84
Biosimilars definition
Biologic that is almost an identical (ideally identical) alternative version of original biologic (innovator/ reference biologic) manufactured by different country
85
Biosimilars - variability (how it occurs)
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
Biosimilars: chemical drugs (pdn of generic drugs)
No issue with production of generic drugs → analytical criteria based on chemical compositions
87
Biosimilars: biologics with low MW
More straightforward development of biosimilars ⇒ approval of several biosimilars
88
Biosimilars: mABs with post-translational modification problem of biosimilarity
impossible to engineer a biosimilar 100% identical to innovator biologic ⇒ ONLY CAN produce a highly similar biosimilar
89
Biosimilars: mABs with post-translational modification reasons for variability
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
Biosimilars: requirements for approval
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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