Wk 16: Characterisation of (concentrated) liquid formulations of monoclonal antibodies Flashcards
What are factors to be considered for liquid formulations of antibody drugs to avoid aggregation + denaturation?
- Conformational stability
- Colloidal stability
- Storage stability
- Physicochemical properties
What are the factors to be considered in the formulation of antibody drugs?
- pH of solution
- Ionic strength
- Additives: salts, sugars + surfactants
What are the types of monoclonal antibodies?
- Lyophilised form: reconstituted
- Liquid form: injection
What is an example of a clinically approved antibody?
- Alemtuzumab
- Lysis of b lymphocytes
- For: chronic lymphocytic leukaemia
How are suitability + stability of formulations investigated?
- Turbidity determination
- Secondary + tertiary structure analyses
- Determination of molecular hydrodynamic diameters
- Chromatographic analysis
- Viscosity determination
- Thermodynamic analysis
What is microcalorimetry?
- Used to study thermal transition of antibodies in dilute solutions
- Measures thermodynamic parameters that control protein-folding-unfolding transition
- Measures heat changes in biomolecules when inc/dec temp
At what temperature produces aggregation in antibodies?
Low Tm - denaturation + aggregation irreversible
How does high sensitivity differential scanning calorimetry work?
- Biomolecules solution heated at constant rate
- Heat change = thermal denaturation
What is the apparatus set up of HSDSC calorimeter?
- 2 identical cells enclosed in adiabatic jacket, 1 sample cell, 1 reference
- Cells = inert metal filled w/ 1.4ml sol
- When scanning, the cells are heated w/ constant power input
- Temp diff. btw the cells are constantly measured
- Power added/subtracted = diff. btw heat capacity of sample + reference
When is the temperature at which D=N is the midpoint transition temp?
∆G= zero and Keq= 1
What is Tm?
- Transition midpoint temp
- High = more stable protein
What is ∆Cp?
- Denature heat capacity change
- Depends on: covalent, non-covalent + solvent interactions
- Change during unfolding due to inc in hydration
What is the ∆Hcal?
- Calorimetric enthalpy change
- Energy needed to heat system to required temp
- Area under transition peak
How do you estimate the size of the cooperative units? (m)
(m = ∆HvH/∆Hcal)
