liquid formulations of mab Flashcards
what are MABs?
Biopharmaceuticals and also “biosimilar”
antibody drugs are available, they are
efficient with few side effects but the size
and poor diffusion characteristics of those
therapeutics agents are challenges for
successful antibody formulations.
are all antibodies the same?
no each is different
what are the probelms with monoclonal antibodies?
Stability issues
Aggregation and precipitation
what factors should be considered for formulations of antibody drugs?
Conformational stability
Colloidal stability
Storage stability
Physicochemical properties
why is colloidal stability important?
To avoid aggregation and denaturation
why are the physiochemical properties important?
Safe and effective antibody liquid formulations
For formulations:
pHs of the solutions
Ionic strength
Additives: salts, sugars and surfactants
what are the two formulations of monoclonal antibodies?
1- lyophillised form- to be reconsituted
2- liquid form- usually one shot injections
what are MAB focused on?
Generally focused on oncology
They are designed to actively target tumour
cells
Example: Alemtuzumab, monoclonal
antibody (concentrate for I.V. infusion
30mg/ml) that leads to lysis of B lymphocytes
and used for chronic lymphocytic leukaemia
why is the formulation of antibodies a challenge?
Protein aggregation is
affected to a great extent by
protein concentration
how do you determine if mab are suitable and stable?
Turbidity determination
Secondary and tertiary structure analyses …
Determination of molecular hydrodynamic
diameters (Size of aggregates)
Chromatographic analysis (size-exclusion,
cation exchange chromatography)
Viscosity determination …
Thermodynamic analysis …
what are the thermodynamic principles of microcalimetry?
Microcalorimetry is used to study the thermal
transition (stability) of biological macromolecules (e.g. antibodies) in dilute solutions.
Note: Calorimetry (DSC) can be used to study
conformational stability of concentrated solutions.
It measures thermodynamic parameters that control protein-folding-unfolding transitions
It measures heat changes in biomolecules due to increase or decrease temperature
Native (N) Temperature Denatured (D)
what model kinetics does MAB not follow?
monoclonal antibodies’ thermal transitions
are not following two-state model kinetics
what is the best MAB formulation?
Antibodies with high Tm, Tonset Less aggregate
formation
For antibodies, the denaturation and aggregation
are irreversible.
Slight structural changes can induce aggregation.
Aggregates can be investigated by DLS
how does HSDC work?
The system is referred to high sensitivity or
microcalorimetry as it can measure very small
change in heat capacity
Biomolecules solution is heated at a constant rate
Hence, heat change associated with thermal
denaturation
what does the HSDSC consist of?
The system consists of two identical coin shaped cells enclosed in an adiabatic
jacket, one sample cell and the one reference cell.
Cells are made of inert metal
Filled with about 1.4 ml of a
solution.
what happens during scanning?
During scanning the two cells are heated with
a constant power input via their main heaters.
The temperature difference between the two cells is constantly measured and the cell feed back system, according to the existence of endothermic or exothermic process, increases or decreases
power to the sample cell to keep difference in
temperature close to zero. The power added or
subtracted is a measure of the difference between
the heat capacity of the sample and that of the
reference.
what are the thermodynamic paramaters that are important?
Thermodynamic or conformational stability is
defined as the difference in free energy (∆G)
between the folded (N) and unfolded state (D)
how are protein native structures stabilised?
by various
interactions which depend on temperature, ∆H
what is tm?
he transition midpoint temperature, Tm
(for a protein following two-state model kinetics).
50% of a protein is unfolded and it is measured as
the maximum excess heat capacity function.
The higher the Tm, the more stable the protein.
what is the denature heat capacity change?
The denaturation heat capacity change, ∆Cp.
The heat capacity of a protein in a solution
depends on the protein covalent, non-covalent,
and solvent interactions. The change in the heat capacity upon unfolding is mainly due to increase in the hydration.
what is the calorimetric enthalpy change?
The calorimetric enthalpy change, ∆Hcal. It is the
energy required to heat the system to the required
temperature. It is calculated from the area under
the transition peak.
what is half width of the transition?
The half width of the transition, ∆T1/2. It is a
measure of homogeneity in the thermal stability
of protein molecules.
what is the vants hoff enthalpy change?
The vant’s Hoff enthalpy change, ∆HvH. It is used
in conjunction with ∆Hcal for estimation of the size
of the cooperative units, m (m = ∆HvH/∆Hcal)
