protein stability Flashcards
1
Q
what are the major adv of Protein Pharmaceuticals ?
A
- High specificity and strong potency
- Relatively low concentrations so less side effects
2
Q
what are the major challenges of Protein Pharmaceuticals ?
A
antigenicity
stability
Injection so less convenient delivery
3
Q
what can affect protein antigenicity?
A
- Pharmaceutical proteins are foreign as produced from non-human host cells like E. coli and Chinese hamster ovary CHO cells
- If downstream processes not conducted properly —> Impurities —> Immunogenic response (mild allergic reaction vs anaphylactic shock)
- Long term injection of same protein —> Patient may develop antibodies against exogenous protein —> Protein attacked —> Protein loss efficacy over time —> So can increase dosing but also means more side effects
4
Q
how can a protein loss it’s biological activity?
A
through loss of 3d conformation via:
- Denaturation
- Covalently modifying protein
- Partially degrading protein
5
Q
when can the loss of protein’s biological activity occur?
A
- Protein recovery phase
- Protein purification process
- Post-protein purification (storage)
- Bacterial contamination —> Produce proteases that conduct slow proteolysis on proteins
- Storage in solution: Water is weakly nucleophilic so can cause slow hydrolysis of protein —> protein degradation —> So store in freeze-dried form
- Solution (shelf life few days-1 month) VS Freeze-dried form (shelf life 2-3y)
6
Q
how is the potency of the protein affected?
A
- Difficult to change purity of protein because protein stays in unit dose
- On storage over 12 months, proteins start to unfold slightly —> Potency affected
- Conduct biological assays to see if stability will affect activity and potency of protein, physical and chemical assays are inadequate
7
Q
What are the mechanisms that lead to instability of proteins?
A
- Physical: Protein Aggregation
- Chemical
8
Q
what are the stages where protein aggregation can occur?
A
- N stage: Protein folded in optimal 3D conformation (native conformation)
- U stage (reversible by restoring back favourable conditions): When unfavourable conditions are introduced (eg. heat) —> Protein start to unfold to reach U stage, protein still active but start to ppt out
- A stage (irreversible): If continue to subject U stage to unfavourable conditions —> protein continue to unfold till aggregated stage, have ppt out, protein lost all or most of activity —> Immunogenicity
9
Q
why do proteins aggregate?
A
Due to intermolecular association of partially denatured protein chains —> Fully unfolded polypeptide chains tend to aggregate tgt due to hydrophobic interactions
10
Q
what are the Factors inducing aggregation?
A
- temperature
- Increase temp —> disrupt non-covalent forces —> promotes protein unfolding —> denatured protein aggregation —> irreverisble denaturation
- pH
- extreme pHs —> change in ionisation status of side chains of amino acid residues —> disrupt conformation —> protein unfold and aggregate
- Can contribute to chemical instability
- Hydrolysis of Asp residues
- Deamidation of Asn and Gln
- Ionic strength
- Adsorption (stuck)
- Proteins can be adsorbed to many surfaces (esp plastic) and interfaces —> Change in secondary or tertiary structure —> Destabilisation of protein —> Protein aggregation
- Use glass bottles
- Shaking and shearing
- Agitation —> Air introduced —> Surface of air bubbles creates an air-liquid interface that trap proteins —> Change in conformation of proteins —> Unfolding of proteins to expose hydrophobic core —> Protein aggregation
- Addition of Non-aqueous solvents
- Organic solvents: Ethanol, Propanol —> Decrease polarity of aqueous solvent containing proteins —> Decrease ability of formation of hydrogen bonds between protein and water —> Disrupt hydration shell that surrounds and hence stabilises proteins —> expose hydrophobic core —> Protein aggregation
- Repeated freeze-thaw
- Freeze —> Sharp ice crystals form and pierce through 3D conformation of protein —> decrease stability of protein —> Unfold —> and thaw and freeze and thaw —> further unfolding —> protein aggregation
- Photodegradation (exposure to light) of amino acids
- Tryptophan very susceptible, where its side chain can absorb UV rays and undergo changes
- Store proteins in amber bottle for increase stability of proteins
- Vortexing
- Chemical modification of proteins —> Expose hydrophobic sites to aggregate by hydrophobic interactions
11
Q
what other factors can cause protein aggregation?
A
Simultaneous chemical and physical factors can cause protein aggregation
12
Q
why can chemicals cause protein aggregation?
A
Proteins consist of amino acids —> reactivity of side chains of amino acids —> susceptible to chemical reactions
note: more than 1 reaction can happen simultaneously
13
Q
what determines extent of chemical reactivity of protein aggregation?
A
- Localisation of susceptible amino acid can determine extent of chemical reactivity
- For same AA that is located on protein surface, it is usually more exposed to chemical reactions as compared if located in core of protein
- Chemical reaction does not always mean loss of conformation or activity —> need to test to determine extent
14
Q
what are the Types of Chemical reactions that cause protein aggregation?
A
- Deamidation
- More susceptible amino acids: Asn and Gln
- Rmb that location matters!
- Oxidation
- Can occur to side chains of His (H), Met (M), Cys (C), Trp (W), Tyr (Y)
- Presence of transitional metal ions —> Closer proximity to metal binding sites generates reactive oxygen species —> Increase oxidation rates
- Most susceptible amino acids: Tyr and Cys
- Proteolysis
- Disulfide bond breakage and formation
- Type of oxidation that only happens to Cys due to S groups between two Cys molecules can join together to form disulfide bond
- Presence of disulfide bond —> good or bad is determined through testing
- Good: Favourable for protein conformation or activity
- Bad: Unfavourable for protein conformation (if native form required) or activity (if reduced form required)
- Hydrolysis of peptide bonds
- Especially under acidic or basic pH
- More susceptible amino acids: Asp-Gly and Asp-Pro
15
Q
what are the Ways to stabilise final liquid formulations?
A
- Substitution or chemical modification of protein
- Add different agents into final formulation
16
Q
how to do Substitution or chemical modification of protein to increase its stability?
A
note: wont affect overall activity
- Modify or Substitute susceptible AA with another AA
- Via site-directed mutagenesis
- Eg. Cys replaced by Ser
- Introduce disulfide bond to stabilise folded form
- May not work as may be unfavourable to protein (determined by testing)
- PEGlyation (very common)
- Conjugating polyethylene glycol (PEG) to a protein —> Keep in native form longer, Increase circulation time in blood
- Eg. Recombinant proteins conjugated with PEG —> PEGlyated proteins
- Acylation
- Conjugating lipophilic fatty acids to protein —> makes overall complex more lipophilic so expel water —> Upon storage, protein scared of water because water is weak nucleophile —> maintain stability
17
Q
what kind of agents to add into final formulation to increase protein stability?
A
- Stabilisers (sugar, polyols)
- Solubility enhances (Lysine, arginine, surfactants)
- Anti-adsorption and anti-aggregation agents (albumin, surfactants)
- Buffer components (phosphate salts): Prevent very acidic or alkaline conditions
- Preservatives and anti-oxidants (inert gas, thimerosal, phenol, benzyl alcohol)
18
Q
What is recombinant proteins?
A
- Artificial/ Synthetic protein synthesised by host cell, not by own body
- Immunogenicity issues —> Shift from animal to recombinant proteins
- FDA: Manufacturing steps should avoid animal products, Animal component-free media to be used in host cell culture
19
Q
what is the Impact of using recombinant DNA (rDNA) technology on biopharmaceutical products?
A
- Overcome limitations regarding source availability
- Outbreak of virus in animals doesn’t matter anymore
- Cheaper compared to natural sources
- Allows production of safer biopharmaceuticals
- No need to worry if donor is HIV or HepB infected, whether any transmission will occur
- Provides a more appropriate way to obtain protein-based products
- No need to slaughter animals, can just collect urine or placenta (useless for females after giving birth but rich in proteins) —> Not very hygienic
- Can design (customise) abundant supply of desirable mutations to produce engineered protein-based products that possess advantages
- Mutate to cause greater clinical efficacy, greater protein stability for longer shelf-life (~2y), short/longer circulation half-life
- Storage conditions may not be very ideal so should optimise protein stability
20
Q
what is the upstream method of Recombinant protein making?
A
Upstream: Yield selection and genetic modification of host cell to optimally express protein of interest (eg. recombinant human insulin in E. coli cell)
Achieving master cell line, producing large scale production of cells growing in culture medium
Application of rDNA technology occurs in upstream processing in manufacturing of biopharmaceuticals
21
Q
how is the upstream method of Recombinant protein making carried out?
A
- Choose host cells (bacteria or mammalian cell)
- Transfect (introduce) host cells with rRNA that carries desired DNA
- Each transfected host cell DIFFER from each other in terms of number of copies of plasmids (DNA molecule that carries genes, not essential for cell survival) being transfected
- Higher the number of copies, higher the amount of protein expressed by host cell —> Choose cell (master cell line) that gets transfected with highest number of copies for high protein yield
22
Q
what are the type of host cells used for the upstream method of Recombinant protein making?
A
- Prokaryotes (Bacteria): E. coli
- Eukaryotes (Yeast)
- Mammalian cells: Human, Chinese hamster ovary cells
- Insect cells: Multiply faster and less fussy nutrients than mammalian cells
- Transgenic (genetically modified) plants and animals
note: Make sure to remove all components from host cells if not quality and safety of product is compromised (eg. never remove pyrogens from E. coli)
23
Q
what are the adv of using e. coli in the upstream method of Recombinant protein making?
A
- Well characterised
- Highly susceptible to genetic manipulation —> high expression levels of recombinant proteins
- Grow rapidly on simple and inexpensive media
24
Q
what are the disadv of using e. coli in the upstream method of Recombinant protein making?
A
- Accumulation of recombinant cells intracellularly
- Protein is forcefully being made (useless to bacteria) and dividing at high speed —> High conc of recombinant proteins produced in small little bacteria cell —> High levels ppt out as protein aggregates (appear as solids) —> Trapped in E. coli (thus intracellular aka inclusion bodies)
- Inclusion bodies may not be in native conformation due to unfolding —> Inactive proteins —> need to add denaturant to solubilise (break up bonds and dissolve) proteins —> soluble primary polypeptide chain —> refold OUTSIDE of cell, in a test tube —> but refolding is by chance so contributes to wastage —> Drop in yield
- Lack ability to perform post-translational modifications that mammalian cells perform
- No glycosylation, phosphorylation
- Gram neg bacteria express lipolysaccharides (LPS) that act as pyrogens on cell surface that is difficult to remove
- Lipophilic and polar so challenging and hence expensive to remove
25
what are the adv of using cho in the upstream method of Recombinant protein making?
- Capable of adapting and growing in suspension culture —> Ideal for large scale culture
- Pose less risk as few human viruses can propagate in them, quite resistant to human virus infections
- Adapted to be less demanding in nutrients in culture media —> Dont require serum (liq fraction in blood) which is rich in growth factors to grow
- Can undergo post-translational modifications —> Allow for glycosylation of Fc domain for activity
- Can genetically manipulate as much as desired to further increase yield of recombinant proteins
26
what is the downstream method of Recombinant protein making?
Protein isolation, concentration, purification, viral inactivation, removes all impurities and contaminants
27
which of the host cells allows for protein inside it to exit via secretion?
cho
After protein is made by cells —> Still trapped intracellularly or secreted out of cell
- E. coli: No secretory pathways to secrete out recombinant protein
- CHO cell: Can secrete out (preferred) recombinant protein but may not be completely
28
how to ensure and why need to ensure full secretion of protein from host cell?
- Ensure full secretion of cell by rupturing cells and empty out all intracellular content —> stringent purification due to heavy protein load
- If have trace amounts of eg. CHO host cell proteins remaining in product to be lyophilised —> Immunogenicity (local rash vs anaphylactic shock)
29
who does quality control of recombinant protein making?
- auditors
- To ensure every batch will conform to pre-determined specifications
30
what are the Methods to determine Product quality?
note: Investigate activity and safety (purity), Can pick and mix methods
1. Potency testing/ bioassays
2. Immunoassays
3. Mass spectrometry
4. Peptide mapping
5. Amino acid analysis
6. N-terminal sequencing
7. Isoelectric focusing
31
what is the method: Potency testing/ bioassays used for determination of product quality?
- Access activity of product in a biological system
- Expressed as “units of activity” per vial/ dose of product —> Random picking to test
- Types of assays
- In vitro cell culture assays
- In vivo (whole animals) - rare
32
what is the method: Immunoassays used for determination of product quality?
Use of antibodies in ELISA or agglutination test
33
what is the method: Mass spectrometry used for determination of product quality?
- Can do together with peptide mapping for large productions worldwide
- Compare mass spectrum of protein with gold standard
- Unknown mass —> Extra ingredient —> Impurities
34
what is the method: Peptide mapping used for determination of product quality?
- Partially hydrolysed product —> Treat with protease like trypsin that will recognise specific AA to undergo peptide bond cleavage at specific locations
- Trypsin can recognise specific AA and cleave and give rise to peptide fragments that is not recorded in standard 100% pure spectrum —> Extra ingredients —> Impurities
35
what is the disadv of using (i) mass spectrometry and (ii) peptide mapping for determination of product quality?
Activity not revealed
36
what are the adv and disadv of using immunoassays for determination of product quality?
- Advantages: Straightforward, fast, less costly
- Disadvantages: Purity not revealed
37
what are the disadv of using Potency testing/ bioassays for determination of product quality?
- Time consuming
- High cost
- Purity of final product is not revealed, only potency —> Contamination and thus safety profile unknown
38
summary of drawbacks of methods used to determine product quality
Purity not revealed: Potency testing/ bioassays, Immunoassays
Activity not revealed: Mass spectrometry, Peptide mapping
39
what are the methods used to conduct Safety testing?
note: Patient dont suffer anaphylactic shock so no immunogenicity issues so no impurities, no endotoxins
- SDS-PAGE
- Isoelectric focusing
- DNA hybridization
- Rabbit pyrogen test (bioassay)
- Limulus amoebocyte lysate (LAL) test
- Viral assays
40
what is Rabbit pyrogen test (bioassay) used to conduct Safety testing?
- Outsource rabbits to prevent introducing rabbit germs into facility
- Send product sample to third party —> 3 healthy rabbits chosen to be injected and temp observed over a few days to test fo pyrogens
- Slow and expensive and animal cruelty
41
what is Limulus amoebocyte lysate (LAL) test used to conduct Safety testing?
- Alternative to sacrificing animals
- Collect amoebocyte fraction in blood of horseshoe crabs —> if have endotoxins in product, coagulation occurs
42
what is Viral assaysused to conduct Safety testing?
- Test for specific and unknown viruses
- CHO cells more resistant to HIV, HepB viruses than human cells
- CHO cells can still be susceptible to own panel of viruses —> So carry out viral inactivation —> Test for any remaining live viruses using immunoassays
- Immunoassays have antibodies that recognises against whole panel of viruses
- In vitro —> Safe testing
- PCR testing
43
what are Biosimilars?
- Generic version of biologics
- When patents expire, alternative and cheaper versions can be produced by other biotech companies as AA seq is known so can get hold of gene that exactly encode innovator biologic and same host cells —> Does not necessarily totally replace original biologic/ innovator biologic/ reference biologic
44
why need to treat biosimilars with caution?
- Each biological product **displays variability** even between different batches of the same product due to variability of biological expression system and **manufacturing process** (upstream and downstream)
- Environment that host cells (eg. CHO cells) grow in and transfection of CHO cells etc. is different —> Difference is more apparent in larger proteins
- Insulin small protein so approved quite easily
- mAbs are large proteins less easily approved
45
how to get approval for biosimilars?
- Ensure trial and in vitro results are sufficient to show similarity between reference biologics and biosimilars
- Need to invest in trials
46
what to take note of for biosimilars?
Not necessarily an automatic substitution for approved biosimilars, may only be ok for one indication out of the many indications that original biologics can treat
