Are you looking for a way to improve productivity in your affinity chromatography step? Have you ever applied the concept of variable loading to gain productivity? By varying residence time (RT) over the loading phase, productivity from an affinity chromatography step can be significantly improved.
Improved productivity through variable loading
MabSelect SuRe LX is a high-capacity protein A affinity medium (resin) that can bind approximately 60 g MAb/L medium. To best utilize its high capacity, the medium should be operated at 6 min RT.
To maximize productivity (g MAb/L medium/h) over a MAb capture step using MabSelect SuRe LX, the concept of variable loading was investigated. The study included 1) determination of dynamic binding capacity (DBC) at various RTs, 2) determination of optimal RT during load, 3) experimental analyses using design of experiments (DoE), and 4) theoretical modelling.
DBC at various RTs
DBC varied between 15 and 58 g MAb/L medium over the RT range studies (Fig 1). When combining two relatively short RTs, 2 min followed by 4 min (average RT of 2.6 min), a higher DBC was obtained relative to using a single RT of 3 min. A higher DBC was also obtained when combining two longer RTs, 4 min followed by 10 min (average RT of 6.1 min), relative to using a single RT of 6 min.
Fig 1. DBC at different RTs. Blue dots represent single RT and red dots dual RTs (2 min followed by 4 min, and 4 min followed by 10 min) RTs. Dual RTs are plotted as the average.
Optimal RT during sample load
By dividing the loading phase and using different flow rates for each phase, the average RT can be shortened, while capacity is kept nearly constant. In a three-step loading procedure, sample is initially loaded at a short RT (i.e., high flow rate). When 80% of the capacity at 10% breakthrough is reached, the RT is increased (i.e., intermediate flow rate). Again, when 80% of the breakthrough capacity is reached at this particular residence time, the residence time is once again increased (i.e., low flow rate).
A design was created where the shortest RT was varied between 1.2 to 2 min followed by an intermediate RT from 2.5 to 4.5 min and a final RT of 6 min. Pool volume, MAb recovery, MAb aggregates, host cell protein, and MAb charge variants were used as response parameters. To calculate the increase in productivity when performing loading at varying RTs, the productivity at a single residence time of 6 min was used as reference. The results showed no significant difference between RTs (including the reference at 6 min) for any of the analyzed parameters. Using DoE data, surface plots were constructed to visualize the optimal combination of RTs in terms of load time and productivity (Fig 2).
Fig 2. Surface contour plots of the effects of variable RTs on (A) load time, and (B) productivity. Two RTs were varied (x- and y-axis), while the third RT was always 6 min.
Theoretical modelling, using DoE methods similar to those in the experimental studies, was performed to evaluate the possibility to optimize the variable RTs using a minimum number of experiments (Fig 3). The results from the theoretical modelling were found to be similar to those obtained in the experimental study (Table 1).
Fig 3. Surface contour plot for optimization of productivity increase based on theoretical modelling.
Table 1. Comparative model parameters from the experimental and theoretical analyses of the effect of variable RT on productivity.
|Residence time|| |
Experimental model (min)
Theoretical model (min)
By using variable RTs (including RTs shorter than 6 min) during the loading phase, productivity could be increased by nearly 40% relative to a reference run at a single RT of 6 min.
More details about this study can be obtained from the application note Optimizing productivity on high-capacity protein A affinity medium.