IC8 Recombinant protein Flashcards
Protein pharmaceuticals
Advantages
High specificity & activity ⇒ high potency
Relatively low concentrations ⇒ lesser SE
Protein pharmaceuticals
challenges
- antigenicity
- stability (biological, chemical, physical)
- drug delivery
Protein pharmaceuticals
challenges: antigenicity reasons
Foreign proteins may induce immunogenic response from human host
Protein pharmaceuticals
challenges: antigenicity LT effects
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
Protein pharmaceuticals
challenges: stability - how methods of destabilising
(1) denaturation, (2) covalently modifying protein, (3) partially degrading it
loss of proper 3D confirmation ⇒ loss of biological activity
Protein pharmaceuticals
challenges: stability - when destabilisation occurs
- Protein recovery from its source (extraction procedures)
- Protein purification process
- Post-protein purification (protein storage)
Protein pharmaceuticals
challenges: stability - when destabilisation occurs
post-protein purification problems
- Proteolysis due to enzymes associated with bacterial contamination; bacteria produces proteases that hydrolyses protein
- Storage of proteins in solution → protein degradation (specific amino acids contribute to destabilisation) ⇒ hence protein products usually stored in freeze-dried form (solid)
Protein pharmaceuticals
challenges: stability - shelf-life
before reconstitution ~2-3 years
after reconstitution ~7 days to 1 month
storage temperature @ 2-8℃
Protein pharmaceuticals
challenges: stability - changes in potency
generally decreases over time due to unfolding of protein ⇒ reduced clinical effects
Mechanisms causing instability of protein pharmaceuticals
physical & chemical
Mechanisms causing instability of protein pharmaceuticals:
physical - Protein aggregation
progression
native <-> unfolded -> aggregated
Mechanisms causing instability of protein pharmaceuticals:
physical - Protein aggregation
Expression of protein physical stability
difference in free energy ∆G between N & U states
Mechanisms causing instability of protein pharmaceuticals:
physical - Protein aggregation
Unfolding reversibility conditions
reversible: If remove unfavourable conditions
irreversible: Continuous exposure to unfavourable conditions
Mechanisms causing instability of protein pharmaceuticals:
physical - Protein aggregation
Aggregated proteins: how it occurs
Subsequent aggregation of denatured molecules
Mechanisms causing instability of protein pharmaceuticals:
physical - Protein aggregation
Aggregated proteins: impact
irreversible denaturation
Aggregated proteins have altered immunity & may arouse immunogenicity
Mechanisms causing instability of protein pharmaceuticals:
physical - Protein aggregation
Aggregated proteins: Hydrophobic force
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
Physical factors affecting protein stability
T, P, A, SS, NA, FT, P
- Temperature
- pH
- Adsorption
- Shaking & shearing (agitation)
- Non-aqueous solvents
- Repeated freeze-thaw
- Photodegradation
Physical factors affecting protein stability (T)
Increased temp promotes protein unfolding by disrupting non-covalent forces that stabilise protein’s conformation ⇒ encourages denaturation
Denatured proteins aggregate → irreversible denaturation.
Physical factors affecting protein stability (P)
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
Physical factors affecting protein stability (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)
Physical factors affecting protein stability (SS)
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
Physical factors affecting protein stability (NA)
Protein hydration shell may be disrupted
Protein hydrophobic core exposed when polarity of aqueous solvent decrease → Protein unfolds ⇒ LR: protein degradation
Physical factors affecting protein stability (FT)
Formation of sharp ice crystals → can pierce through 3D conformation of protein
Polypeptide chain encouraged to unfold ⇒ protein degradation
Physical factors affecting protein stability (P)
Risk of protein aggregation upon exposure to light ⇒ important to store in amber bottle
Mechanisms causing instability of protein pharmaceuticals:
chemical instability
- Deamination
- Oxidation
- Disulfide bond breakage & formation
- Hydrolysis
susceptible amino acids of deamination
Asn & Gln
susceptible amino acids of Oxidation
His, Met, Cys, Trp, Tyr
susceptible amino acids of Disulfide bond breakage & formation
Cys
susceptible amino acids of Hydrolysis
Asp-Gly & Asp-Pro
Oxidation: how it works
Catalysed by transition metal ions at/ near metal binding sites of proteins
Reactive oxygen species generated → drives oxidation
Disulfide bond breakage & formation: how it works
Occurs between 2 cysteine residues → sulfhydryl groups between cysteine molecule joined together to form S-S
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
Methods for stabilisation & formulation of protein pharmaceuticals (liquid)
- Substitution & chemical modifications (Internal)
- Changing properties of solvent & additives (external)
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)
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
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
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
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
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]
Process of making recombinant protein
Upstream & downstream
Upstream process of making recombinant protein
- Host cells transfected with recombinant DNA (carrying desired gene)
- 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) - ONLY 1 transfected cell with best cell growth properties & highest protein yield ⇒ development of master cell line
Upstream process of making recombinant protein
requirements of host cell
ensure purity & no trace of host cell components in final product ⇒ safety & quality ensured
Upstream process of making recombinant protein
cells to use
- E.coli
- CHO
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)
Upstream process of making recombinant protein
1. E.coli advantages
- facilitates genetic manipulation (high success rate)
- High expression levels of recombinant protein (up to 30% of total cellular protein)
- Grows rapidly on simple & inexpensive media → doubles every 20 mins
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
Upstream process of making recombinant protein
1. E.coli: solubility of proteins
Soluble proteins = in native conformation
insoluble proteins = incorrect conformation
Upstream process of making recombinant protein
1. E.coli: why formation of inclusion body occurs
proteins are synthesised rapidly & in high levels
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
Upstream process of making recombinant protein
2. CHO indications
Large protein production
Post-translational modification required
Crucial for solubility & native folding
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
Downstream process of making recombinant protein
Purification: protein isolation, concentration & purification steps, viral inactivation steps
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
Testing of product: 2 methods
Quality control
safety testing
QC: purpose
confirm conformance of final product to predetermined specifications
Must be done for every batch of products
QC: methods
- Bioassays/ potency testing
- Immunoassays
- Mass spectrometry
- Peptide mapping
- Amino acid analysis
- N-terminal sequencing
- Isoelectric focusing
QC: bioassay purpose
assess activity of product in a biological system; whether the product can work on the substrate
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
QC: bioassay Drawbacks
Time consuming
High cost
Do not reveal purity of final product → no information about contaminant (safety profile not considered)
QC: Immunoassays - how it works
Use of antibodies to quantify product → ELISA, agglutination
QC: Immunoassays advantage
straightforward, fast, less costly
QC: Immunoassays disadvantage
Quantity of product ≠ biological activity of product
do not reveal purity of final product (cannot detect presence of contaminants)
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
QC: peptide mapping purpose
product identification & detection of protein contaminants
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
QC: peptide mapping disadvantage
Does not tell activity of product
QC: Amino acid analysis - how it works
Protein hydrolysed into amino acids → separated by ion exchange chromatography & quantified.
QC: Amino acid analysis indication
characterising peptide or small polypeptide product of < 10kDa
QC: N-terminus sequencing purpose
identification of protein product
QC: N-terminal sequencing - how it works
Sequencing of the first 20-30 amino acids of the protein at the N-terminus
QC: Isoelectric focusing purpose
determine sialic acid content in glycoproteins
Safety testing: purpose
Assessment of presence of impurities; prevention of immunogenicity issues
Safety testing: methods
- SDS-PAGE
- Isoelectric focusing dye binding methods (colorimetric assays)
- DNA hybridisation
- Rabbit pyrogen test
- Litmus amoebocyte lysate (LAL) test
- Viral assays
- In vivo bioassays
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
ST: SDS-PAGE detection
Detection of product variants possible using product-specific Ab in Western blot
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
ST: isoelectric focusing (secondary purpose)
monitor homogeneity of glycoproteins’ sialic acid content
* sialic acid on glycan charged → affects pI of glycoprotein
ST: DNA hybridisation purpose
For detection of DNA contaminants in ng range
ST: rabbit pyrogen test - how it works
Detection of pyrogen by injecting product into healthy rabbits
Increased temperature = presence of pyrogen
ST: LAL test - how it works
Endotoxin stimulated coagulation of amoebocyte fraction in blood of horseshoe crabs (Limulus)
ST: LAL test advantages
less variable, more sensitive, faster, cheaper
ST: LAL test disadvantages
only detects endotoxin-based pyrogens
ST: viral assays - purpose
TEST FOR:
specific viruses capable of contaminating source materials AND
unknown/uncharacterised viruses not widely available or employed
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
ST: In vivo bioassays purpose
general safety testing → ie injection into healthy mice
Biosimilars definition
Biologic that is almost an identical (ideally identical) alternative version of original biologic (innovator/ reference biologic) manufactured by different country
Biosimilars - variability (how it occurs)
occurs even within batches of same product
- variability of biological expression system & manufacturing process
- Process of manufacturing (upstream & downstream) influences nature of final product
Biosimilars: chemical drugs (pdn of generic drugs)
No issue with production of generic drugs → analytical criteria based on chemical compositions
Biosimilars: biologics with low MW
More straightforward development of biosimilars ⇒ approval of several biosimilars
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
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.
Biosimilars: requirements for approval
- Extensive in vitro studies demonstrating similarity to a reference biologic.
- Non-clinical & clinical studies demonstrating comparable pharmacokinetics (PK), clinical efficacy, safety & immunogenicity
