A newly developed synthetic affinity ligand for antibody adsorption is being

both ligands are immobilised to the same chromatography beads. The

synthetic matrix has shown comparable binding capacity when studied using

batch equilibrium experiments in the laboratory. The two matrices were then

packed into chromatography columns and the breakthrough curves obtained

are shown below.

i$)$ Give binding capacities $(g{l}^{-1}),$ throughput $(g{h}^{-1})$ and productivity $(g{h}^{-1}{l}^{-1})$

assuming loading to $2\mathrm{.}5\%$ breakthrough for both columns. $($Answers should be

given to $2$ significant figures$).$

ii$)$ From the data shown in the breakthrough curve why did the batch equilibrium

experiments show the capacity of the two matrices to be equal?

iii$)$ Why are equilibrium experiments unsuitable to accurately determine capacity

of chromatography matrices?

iv$)$ An error in the laboratory means it is uncertain which breakthrough curve

corresponds to which matrix. Data have also been gathered on the affinity of

antibodies for the $2$ matrices, the data are shown below. Using this data which

breakthrough curve corresponds to the synthetic matrix and which to protein

A$,$ giving reasoning.

Operating Conditions: Linear Velocity in the column $150cm{h}^{-1}($for the

whole column cycle$),$ Column Height $15cm,$ Column diameter $1\mathrm{.}6cm,$

antibody concentration in the load material $2g{l}^{-1}.$

Column cycle: Load $($X$?$ column volumes$)+$ wash $(3$ cv$)+$ elution $(2cv$

regeneration $(6$ cv$)+$ equilibration $(4cv.$

Affinity Data

Synthetic Ligand $K_D-1{10}^{-6}M$

Protein $A{K}_D-1{10}^{-9}M$

$(K_D-$ dissociation rate $/$ association rate$)$