Purification Strategies to Process 5 g/L Titers of Monoclonal Antibodies - Altering the order of operations, using new resins, and increasing dynamic binding capacity can obviate the need for major fa
Decreasing Buffer Consumption and Using In-Line Dilution
Table 3. Maximum expression level of a MAb that can be purified based on different process or facility modifications
As discussed above, it is important to take a holistic view of the process and carry out multiple model iterations that allow
bottlenecks to be identified and removed, and subsequent bottlenecks to be identified. In examining the entire purification
process, Table 3 shows the maximum titer that can be purified downstream by each unit operation. The baseline Protein A step
can accommodate a titer of 1.3 g/L, but modification of the equilibration and various wash phases to minimize the Protein
A buffer volumes increases this to 1.6 g/L. Table 4 shows an example of one equilibration phase and three separate wash phases
in each Protein A chromatography cycle, where wash 1 and wash 3 also consist of equilibration buffer. In the baseline process,
five column volumes are used for equilibration, three for wash 1, and three for wash 3. Studies have shown that this can be
reduced to three column volumes for equilibration, two for wash 1, and two for wash 3 without affecting yield, purity, or
column re-use, resulting in an overall reduction of four column volumes of equilibration buffer per cycle. For example, for
a 140-L column operating eight cycles to process each cell culture batch, the revised process would reduce the consumption
of equilibration buffer by 4,480 L per batch.
Table 4. Operational sequence of a Protein A chromatography step and example phase durations in units of column volume (CV).
Since buffer tank volumes are a significant constraint throughout the purification process as antibody mass increases, one
solution is to use concentrated buffers in volume-constrained tanks and then dilute each buffer to the desired concentration
in-line to the chromatography column. This method uses various types of equipment (pumps, metering devices, and pH and conductivity
sensors) to blend a concentrated buffer with water to achieve the desired concentration, which has been shown to attain the
target concentration ±2%.7 The primary limitations of in-line buffer dilution are the ability to concentrate buffers sufficiently to fit into available
tank volumes and the installation and validation of the in-line dilution equipment. As shown in Table 3, the maximum titer
accommodated in the Protein A step increases from 1.3 g/L in the baseline case to 1.9 g/L using optimized phase durations
and a 1.25x in-line buffer concentration factor. Using 2x concentrated buffers for the Protein A step would increase the capacity
still further, accommodating titers of up to 2.9 g/L. The cation and anion exchange chromatography steps are also able to
process more antibody through the use of in-line dilution and large column volumes. Although this approach requires capital
expenditure and plant down time for installation, in-line dilution enhances the capacity and flexibility of a manufacturing
facility. One additional benefit may be the option to move away from using fixed, stainless steel tanks for buffer storage
and move to concentrated buffers in disposable bag systems.
