Early process schemes
For several HuMAbs, with expression levels as high as 500 mg/L, a cation capture step with 15 to 20 mg/mL capacity was developed,
and then the process was scaled up successfully 10,000 fold with step recovery of 85 to 95%. These early process schemes consisted
of three columns and an in-process tangential flow filtration (TFF) step. As HuMAb expression levels approached 4 g/L, we
needed to integrate our upstream and downstream platform technology development to reduce the constraints posed by existing
purification schemes. The next generation process was a result of a carefully crafted cation capture step with a high binding
resin (40 to 100 mg/mL resin) and well-designed orthogonal polishing step(s). This setup was comparable to the affinity processes
both in terms of yield and purity (Figure 1).
Figure 1. Ion exchange purification schemes for human monoclonal antibody production processes
Product quality and step yield with cation capture–based purification schemes for various HuMAbs are presented in Table 1.
Process conditions were optimized for various process schemes with different HuMAbs. In the interest of brevity, we do not
list details of resin type, column size, or other aspects. Cation capture resulted in >97% purity and 82% recovery for various
Table 1. Product purity and step yield for the cation exchange capture step
Eliminating the In-Process Diafiltration Step
Although column chromatography accounts for the lion's share of costs in a purification process, intermediate diafiltration
step(s) add substantial costs and time. In certain cases, optimizing process conditions on the columns before and after the
in-process tangential flow filtration (TFF) step can be accomplished by selecting appropriate buffer species that are compatible
with both columns. Dual buffer systems can be developed for a transition between ion exchange columns, and doing so may eliminate
a TFF step. We have found that membrane chromatography is a viable alternative for processing large volumes if dilution is
necessary to decrease conductivity of the load to a polishing step.
Updating the Polishing Steps
In addition to modifying the capture step, we took a multi-pronged approach to modifying the other chromatography steps with
the goal of improving process time, economics, and validation. Innovative approaches for reducing or substituting existing
polishing steps of generic purification processes are attempted by using state-of-the-art technologies, including disposable
chromatography. Smooth transition of chromatography steps without prior conditioning of column feeds by a diafiltration step
was accomplished by modifying buffer conditions, which made simple dilution possible. Under these conditions, flow-through
membrane chromatography was handy because of its high flow rates.
These membranes not only offered fast processing time but also retained a contaminant clearance capability equivalent to column
chromatography. Table 2 shows the comparison between Q resin and Q membrane chromatography. Each antibody has optimized process
conditions. We show that the resin- and membrane-based methods are comparable in the performance of recovery and that Q membrane
chromatography is a good substitute for resin chromatography with the same ligand, and can enable faster processing and easier
Table 2. Performance comparison of anion exchange (AEX) chromatography showing that Q column and Q membrane chromatography
are approximately equal