Biologics and Insulin Flashcards
Why are biologics considered to be versatile?
They can replace disease tissue as well as modifying disease tissue
Why biologics over small molecules in terms of binding?
Therapeutic protein are more specific compared to small molecules which can bind at other sites and cause toxicity
Do biologics require frequent or less frequent dosing?
Less frequent - they have longer circulation times compared to small molecule drugs
How are immunogenic effects of biologics addressed?
Humanisation
How is biologic bioequivalence risk managed?
Supportive data for structural and functional characterisation
What is biologic bioequivalence not the same as?
Having 2 small drug formulations that become bioavailable at the same rate and extent after administration at the same dose
In terms of hydrophobicity, when are most globular proteins stable?
When the loops with the hydrophobic side chains are buried in the interior of the protein. Unfolding leads to aggregation and colloidal instability
Are proteins stable at their isoelectric point?
Many are, but they still aggregate
Describe the process of protein aggregation
Hydrophobic side chains are predominately internal
Side chains become exposed, and in aq environment, bond with those on other molecules
Unfolding protein molecules aggregate
What are the potential causes of chemical degradation of exposed susceptible side chains?
Oxidation
Deamidation
Hydrolysis
What are the factors inducing conformation change/instability?
Extremes of pH Shear Forces Air/water interfaces Adsorption to solid surfaces Freezing, drying and re-hydration Elevated temperatures and pressures
How do amino acids stabilise biologic formulations?
Preferential hydration, preferential exclusion
Decrease protein-protein interaction
Increase solubility and reduce viscosity
How do polymers stabilise biologic formulations?
Competitive adsorption
Steric exclusion
Preferential exclusion, preferential hydration
How do polyols stabilise biologic formulations?
Preferential exclusion
Accumulation in hydrophobic regions
How do salts stabilise biologic formulations?
Preferential binding and interaction with protein bound water
How do surfactants stabilise biologic formulations?
Competitive adsorption at interfaces
Reduce denaturation at air/water interfaces
Interfere with ice/water interface on freezing
How does acylation with a fatty acid stabilise proteins?
Increasing binding affinity to serum albumin, resulting in longer acting insulin, glucagon and interferon
How does PEGylation stabilise stabilise proteins?
Reduces plasma clearance rate - so less frequent administration.
But some proteins can become less active when PEGylated
What is the purpose of surface engineering?
To remove sites on protein surface that are likely to cause aggregation
What effect do mutations have on proteins?
They alter surface structure and polarity
How do denaturants work?
They interact with the polypeptide backbone of the protein.
Higher interaction when unfolded, there is a positive concentration difference between local and bulk domain
How do protectants work?
They form an osmolyte strong interaction with water.
Lower interaction and higher exclusion when unfolded, there is a negative concentration difference between the local and bulk domain
Define exclusion
The thermodynamic mechanism to explain stabilisation by excipients
What is the degree of preferential exclusion and increase in chemical potential proportional to?
It is directly proportional to the protein molecule surface area exposed to solvent
How does the process of exclusion work?
Minimises the thermodynamically unfavourable effect of preferential exclusion by favouring the state with the smallest surface area
In terms of chemical potential, what happens to unfolded/denatured proteins?
There is an increase in chemical potential in those with a greater surface area
Do unfolded proteins have low energy?
No
They have high energy, they have the potential to go back to their native stative or aggregate. Excipients used can play a role in whether they aggregate or not.
What happens to biologic at low temperatures?
Low temperatures extend shelf life, cold denaturation is usually reversible, as temperature drops, solent properties change.
What happens when biologics are frozen?
They are more stable due to the lower temperature
But repeated thawing and freezing causes aggregation by pH and concentration changes, and provision of nucleation points for aggregation on ice-water interfaces
What kind of substances are used for cyroprotection?
Sugars, polyhydric alcohols, oligosaccharides, amino acids
What is the process of cyroprotection?
Works by preferential exclusion, lowering cold denaturation temperature and stabilising osmotic stresses, whereas surfactants interfere with ice/water
How should frozen vials be treated when thawing?
They should be gently mixed to avoid mis-dosing
Why should frozen vials be gently mixed?
Concentration gradients are formed during freezing and remain if thawed without mixing
What is the main advantage of freeze drying?
Formulations have greater long term stability that protein solutions
What is the disadvantage of freeze drying?
Proteins go through reversible conformational changes during transition into the lyophilised state that exposes buried regions and makes them prone to aggregation - this can happen in reconstitution
Reactions and denauturation can continue when lyophilised.
How can denaturation be avoided in lyophilised formulations?
Put them in a fridge to reduce denaturation rates
Make them sealed to avoid water vapour absorption
Describe recombinant human insulin
- Monomer
- Exists naturally in a hexameric structure
- Hexamer has globular protein structure
- 2 axial Zn ions coordinated by 6 histidine side chains
How is fast acting prandial insulin engineered?
Mutation of one or more amino acids in protein sequence to disrupt assembly through:
- Conversion to dimeric and monomeric
- Diffuses faster and improves transport
What is the benefit of fast acting prandial insulin engineering?
Makes the insulin faster acting on sub-cut administration, there is rapid absorption at mucosal barriers and a rapid response from infusion pumps
What is early basal intermediate insulin formulated with?
Protamine - to create suspensions forming crystals, when speed of dissociation and absorption varies in the same patient
What does the long acting insulin glargine need?
Dissolution of isoelectric precipitates formed after injection which causes variability
Describe long acting insulin detemir
Less variable, comes from fatty acid modification
Reversible stabilisation avoids precipitation and dissolution
Binds to albumin, when absorption rate is only slightly affected by blood levels, it circulates for longer
How are mAbs produced?
Select B cell clones to form a mAb
Fuse with myeloma cell to form a hybridoma
Optimise hybridoma growth and mAb
What is the disadvantage of mouse antibodies?
They cause immunogenic reactions, they are cleared rapidly and lack human Fc effector functions.
What is a chimeric mAb?
It has a mouse variable gene
What is a humanised mAb?
Has mouse antigen binding loops (CDRs)
How are fully human antibodies produced?
Through mice
- 4 mouse IgG gene is replaced with human transcends in transgenic mouse
- Mouse is immunised to raise immune response
- B cells are selected, a hybridoma is produced and bioreactor cell culture produces human antibodies
What happens after antibodies are administered?
They distribute mainly within the central compartment, penetration inside cells is limited by high molecular weight and hydrophilicity
What is the primary route of degradation and elimination?
Primary routes are by renal clearance and proteolytic catabolism after receptor mediated endocytosis in the cells or reticule endothelial system (RES)
