liquid formulations of mab

Question 1

what are MABs?

Answer 1

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.

Question 2

are all antibodies the same?

Answer 2

no each is different

Question 3

what are the probelms with monoclonal antibodies?

Answer 3

Stability issues
 Aggregation and precipitation

Question 4

what factors should be considered for formulations of antibody drugs?

Answer 4

Conformational stability
Colloidal stability
Storage stability
Physicochemical properties

Question 5

why is colloidal stability important?

Answer 5

To avoid aggregation and denaturation

Question 6

why are the physiochemical properties important?

Answer 6

Safe and effective antibody liquid formulations
For formulations:
pHs of the solutions
Ionic strength
Additives: salts, sugars and surfactants

Question 7

what are the two formulations of monoclonal antibodies?

Answer 7

1- lyophillised form- to be reconsituted
2- liquid form- usually one shot injections

Question 8

what are MAB focused on?

Answer 8

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

Question 9

why is the formulation of antibodies a challenge?

Answer 9

Protein aggregation is
affected to a great extent by
protein concentration

Question 10

how do you determine if mab are suitable and stable?

Answer 10

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 …

Question 11

what are the thermodynamic principles of microcalimetry?

Answer 11

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)

Question 12

what model kinetics does MAB not follow?

Answer 12

monoclonal antibodies’ thermal transitions
are not following two-state model kinetics

Question 13

what is the best MAB formulation?

Answer 13

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

Question 14

how does HSDC work?

Answer 14

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

Question 15

what does the HSDSC consist of?

Answer 15

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.

Question 16

what happens during scanning?

Answer 16

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.

Question 17

what are the thermodynamic paramaters that are important?

Answer 17

Thermodynamic or conformational stability is
defined as the difference in free energy (∆G)
between the folded (N) and unfolded state (D)

Question 18

how are protein native structures stabilised?

Answer 18

by various
interactions which depend on temperature, ∆H

Question 19

what is tm?

Answer 19

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.

Question 20

what is the denature heat capacity change?

Answer 20

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.

Question 21

what is the calorimetric enthalpy change?

Answer 21

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.

Question 22

what is half width of the transition?

Answer 22

The half width of the transition, ∆T1/2. It is a
measure of homogeneity in the thermal stability
of protein molecules.

Question 23

what is the vants hoff enthalpy change?

Answer 23

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)