OPTIMIZATION OF FLOW-THROUGH PURIFICATION
A further screening in 96-well plate format was performed for all these media to identify the chromatographic conditions to
reduce the aggregate levels to <1%. The starting material had very low levels of HCPs, so these were not considered in this
The MAb was loaded on the chromatography media in a range of pH values and NaCl concentrations (Table 3). After 60 min of
incubation with the sample (5.3 g/L), the flow-through fractions were collected and the concentration of monomer and aggregates
in the fractions was determined by SEC.
Table 3. Screening conditions for flow-through purification
Using the monomer and aggregate concentration in the starting material (C
, Cini,a) and the concentration in the flow-through fraction (Cm,Ca), the binding capacity for monomer (Qm) and for the aggregates (Qa) can be calculated according to the following equation:
For the strong anion exchanger (Capto Q), the highest capacity for both monomer and aggregates was observed at high pH values
and the lowest NaCl concentration (data not shown). For all conditions, the monomer capacity was higher than the aggregate
capacity. The capacity for the weak anion exchanger (Capto DEAE) leveled off at higher pH levels (8.8–9.2, data not shown).
This is thought to be because of the nature of the DEAE ligand, which loses its charge around this pH, while the quaternary
ammonium ions remain charged at these conditions.
The calculations with the multimodal medium (Capto adhere) revealed the highest capacity for both monomer and aggregates at
high pH values and low NaCl concentrations, as shown in Figure 1. For phosphate, the capacity decreased with increasing NaCl
concentration, and the opposite trend was seen for citrate. Given that this was a flow-through step, the objective was to
identify conditions when the aggregate capacity was much higher than the monomer capacity. For all conditions, the monomer
capacity was higher than the aggregate capacity, which would mean a low monomer yield in a flow-through step.