PR3152 IC8 Flashcards
what are the major challenges to protein drugs?
1) stability
2) antigenicity
3) drug delivery
what causes antigenicity challenges in proteins?
foreign particles from non-human host may induce immunogenic response
what causes stability challenges in proteins?
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
differences in potency and purity of proteins over time in storage?
potency will decrease faster in storage vs purity as the protein unfolds and loses its conformation
what are the mechanisms leading to protein instability?
1) physical
- aggregation
2) chemical
- deamination
- oxidation
- proteolysis
- disulfide exchange
explain the mechanism of aggregation contributing to protein instability
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.
factors leading to aggregation of proteins?
describe how the proteins aggregate to each other
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.
what are the physical factors leading to protein aggregation?
temp
ph
adsorption
shearing/shaking
non-aqueous solvents
repeat freeze thaw
photodegradation
how does temperature affect protein stability?
temperature disrupts the non-covalent interactions causing unfolding and a loss of 3d conformation
+
aggregation = denaturation
how does pH affect protein stability?
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
how does adsorption affect protein stability
proteins attach to the surface of materials leading to change of the secondary or tertiary structure leading to loss/destabilisation.
how does shearing and shaking affect protein stability?
both cause airbubbles to form = liquid=air separation = protein align to the surface and unfolds to maximise surface to hydrophobic air residues
how does repeat freezing and thawing affect protein stability?
disrupts the 3d confromation by formation of ice crystals
how does non aqueous solvent affect protein stability?
hydration shell disrupted exposing the hydrophobic core = unfolding
how does photodegradation affect protein stability?
exposure to LIGHT causes protein aggregation and/or protein cleavage (by UV)
(chemical instability) deamidation of proteins?
most common
Asn and Gln
(chemical instability) hydrolysis of proteins?
acidic and basic ph
Asp-gly
Asp-pro
particularly labile
(chemical instability) oxidation of proteins?
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
(chemical instability) disulfied form/break of proteins?
disulfide bonds in Cys
two strategies to modify the stabiltiy of LIQUID proteins
1) substitution and chemical modification (internal changing of structural characteristics without compromising activity)
2) changing the properties of the solvents, additives (external)
substitution and chemical modification methods for improving liquid protein stability?
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)
solvent/additive methods for improving liquid protein stability?
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
what occurs during upstream processing of biopharmaceutical manufacturing?
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.
Advantages of ecoli as the host cell for recombinant protein
1) well characterised - easier genetic manipulation
2) produce high expression levels of recombinant proteins
3) grows rapidly on simple and inexpensive media
DISadvantages of ecoli as the host cell for recombinant protein
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
what are the extra steps in e.coli protein production (to combat inclusion bodies)
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
why is CHO (Chinese hamster ovary) cells preferred for protein production?
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
Choice of host cells to make recombinant proteins depends on size and characteristics of protein
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
what are the downstream processes for protein pharmaceuticals?
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)
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
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
what are the safety testing methods in protein pharmaceuticals?
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
explain viral assays
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)
final characteristics of biosimilars influenced by:
manufacturing process (type of host cell, development of genetically modified cell for production, cell culture system, production process, purification process, formulation of recombinant protein)
requirements for biosimilar approval by US FDA
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
