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
Table 5. Impact of water required to adjust the conductivity for loading on an anion exchange chromatography resin used in
platform process A
Newer anion exchange resins on the market have been designed for improved process performance at higher load conductivities.
Table 5 compares two anion exchange resins, Q Sepharose Fast Flow (QSFF, GE Healthcare, Uppsala, Sweden), which has been a
standard in antibody purification for some time, and Capto adhere (GE Healthcare), a new multimodal anion exchange resin introduced
in 2007. In the flow-through process modeled in the platform process A example, QSFF requires a conductivity of 11 mS/cm or
less to achieve optimal yield and requires 1,700 mL of water for each liter of the input pool to achieve this load conductivity.
In contrast, Capto adhere was designed to have an optimal operation window at higher conductivities than previous anion exchange
resins. At a load conductivity equal to that of QSFF (11 mS/cm), Capto adhere has a significantly poorer yield, but as the
conductivity increases, the yield improves and is equal to that of QSFF at a conductivity of 22 mS/cm. The benefit to plant
fit is the reduced volume of water (from 1,700 mL/L to 0 mL/L) needed to condition the load conductivity, removing the bottleneck
caused by the pool tank volume limitation. As an alternative, TFF can be used to diafilter the pool, achieving the desired
pH and lower conductivity, but this involves adding a new unit operation to the process. In addition, this increases the capital
costs of installation, plant down time, and running costs. There must also be space available to install a TFF system in the
existing plant.
Protein A Process Optimization for Facility Fit (Process B)
Figure 4
The Protein A process plays an important role in purification when the anion exchange chromatography step is next, as in platform
process B. Protein A process parameters such as load density, elution buffer, and pooling criteria can have a significant
effect on downstream pool volumes. The pH of the Protein A pool must meet low pH criteria for viral inactivation (pH ≤3.6),8 ideally without requiring additional acid titration. Process optimization must focus on minimizing the number of cycles,
the pool volume per cycle, the volume of titrant required for pool pH adjustment, and the adjusted pool conductivity. Although
maximizing the Protein A load density reduces the number of cycles, modifying the elution buffer components to minimize the
pH and conductivity adjustment before the next chromatography step presents another alternative to address pool tank limitations.
Figure 4 shows the impact of four different Protein A elution buffers on volume of titrant (Tris base) required to titrate
the Protein A pool to pH 7.5 for loading onto the anion exchange step, and the resulting pool conductivity. There is a considerable
difference in the amount of Tris base needed to adjust the pool pH to 7.5, ranging from 7 to 54 mL of Tris base per L of pool,
depending on the Protein A elution buffer. Even more significantly, the acetic acid elution buffer resulted in the highest
pool conductivity (~7 mS/cm), whereas the two glycine-based elution buffers resulted in pool conductivities close to 3 mS/cm.
The lower conductivity load for the anion exchange step resulted in a reduction of HCP from 800 ng/mg to <5 ng/mg in the anion
exchange pool for the two glycine buffers but only to 40 and 620 ng/mg, respectively, for the acetic acid and citrate elution
buffers. Therefore, the glycine-based buffers offer the potential for a two-chromatography step process while the citrate
buffer was eliminated because of its interference with the anion exchange process.9
Table 6. Impact of Protein A elution buffer in platform process B on conditioning operations required for downstream chromatography
operations. Facility bottleneck is the load pool to the cation exchange step (marked in red).
The need to model the entire downstream process to understand the effects on the performance of each unit operation is illustrated
in Table 6. These results show that there is a slight impact on the overall pool volume for the final adjusted Protein A pool
(~3,300 L compared to ~3,000 L) and that this increase is not constrained by available tank volumes. Because the anion exchange
step is operated in the flow-through mode, there is an overall increase in volume in the anion exchange pool tank with all
conditions resulting in ~4,000 L, which again is not constrained by tank capacity. However, due to the higher ionic components
present in the acetic acid elution pool, a significantly large volume of water is needed to adjust this pool (450 mL/L) for
loading onto the subsequent cation exchange step compared to either of the glycine-based elution buffer pools where no conductivity
adjustment is needed. This may represent a bottleneck for a manufacturing facility because the adjusted anion exchange pool
requires a tank with twice the capacity. Therefore, the glycine-based buffers deliver a robust process with lower impurity
levels while also removing downstream pool tank bottlenecks by reducing pH titrant volumes and pool conductivities.
