Currently the purification of monoclonal antibodies for therapeutic purposes is
reliant on protein A affinity chromatography. The rapid growth of this class of
therapeutic and their high value makes the understanding of protein A
chromatography an important target. There is a range of commercially available
protein A chromatography media. The main differences between these media are
the support matrix type, the pore size, the particle size, the amount of ligand
attached to the matrix and the kind of protein A modification. The differences in
these factors give rise to differences in compressibility, chemical and physical
robustness, diffusion resistance and binding capacity of the adsorbents. The ideal
media would have high specificity, high mass transfer and binding capacity, low
non-specific adsorption and ligand leakage, incompressibility, resistance to alkaline
condition for sanitization, chemical stability and cost effectiveness. Current resins
offer a compromise, which balances what is achievable in respect of these features
giving rise to an array of different solutions. Measurement of these parameters is
often complex and agreed standards have yet to be determined. The objective of
this study is to further develop understanding of these measurements for the
assessment of the matrix performance.
This thesis employs a suite of techniques to characterise commercial and prototype
adsorbents. The adsorbents that will be looked at are MabSelect (GE Healthcare),
MabSelect Xtra (GE Healthcare), Prosep Ultra (Millipore), Protein A immobilised on
4CL Sepharose (GE Heatlhcare) in house and a prototype adsorbent with a Protein
A mimic ligand (Millipore). Both down-scaled techniques of fixed bed
chromatography, together with supporting analysis of equilibrium and dynamic
behaviours are used. The latter will adopt standard and novel ‘wet chemistry’
approaches together with the increasingly adopted techniques of laser scanning
confocal microscopy. Experiments are carried out using hIgG to study the static
capacity, adsorption equilibrium and dynamic capacity of adsorbents. Other
techniques will be used to study the kinetic uptake and desorption rates of
adsorbents in different conditions. A novel approach using confocal microscopy is
used to further understand the adsorption behaviour of individual beads of different
sizes.
The main results that were drawn from these techniques are that MabSelect Xtra
had the highest static capacity of 61.8mg/ml. It also showed the highest dynamic
capacity at 2 mins, 4 mins and 8 mins residence time (0.66cm Omnifit column, bed
height 6cm) when compared to other adsorbents. This is mainly due to the more
porous nature of the MabSelect Xtra beads, which increased the surface area
available for Protein A ligand immobilisation. From the adsorption equilibrium data
the Kd values ranged from 181nM to 36nM. Such low values are expected by
affinity adsorbents such as these. The uptake rate curves were similar for all the
adsorbents. Hence the difference in particle size, pore size, the type of ligand or the
material of the adsorbent itself did not have an effect on the uptake rate when
carried out in a batch mode. A similar behaviour was shown for the desorption
curves. The confocal analysis using a flow cell showed that all the adsorbents
showed a shrinking core effect except for the prototype where the hIgG didn’t
penetrate into the bead and was only attached to the surface of the bead. It was
found that the adsorption rate to the centre of each bead was linear. The different
particle sizes within any particular type of matrix and also across different matrix did
not result in different diffusion rates. From the adsorption curves produced it was
seen that smaller beads reached saturation much faster than larger beads at any
given time. This technique can have great benefits in understanding how individual
beads of different adsorbents behave in different circumstances