ABSTRACT
In human monoclonal antibody (HuMAb) processes, nonaffinity purification schemes have been developed as viable alternatives
to affinity schemes, especially when cell culture expression levels have been increased significantly. Primary recovery by
tangential flow filtration (TFF) for concentration and diafiltration plays a crucial role in a nonaffinity purification platform
with cation exchange as a capture step. A nonaffinity purification scheme, including an optimized primary recovery TFF step,
is comparable to Protein A-based purification processes in overall process efficiency, product recovery, and quality.
Tangential flow filtration is a common unit operation either for cell culture clarification and concentration or just for
concentration of the cell culture supernatant in biopharmaceutical manufacturing where affinity or nonaffinity capture is
used. As a platform approach, exploring nonaffinity capture as a viable alternative to Protein A has yielded efficient purification
schemes. These nonaffinity schemes with three, two, or one chromatography steps have been successfully developed for HuMAbs.1,2 Cell culture harvest needs to be preconditioned by concentration and buffer exchange to the appropriate pH and conductivity
to have a uniform and maximum load on CEX column. For this primary recovery step to fulfill both concentration and diafiltration,
a TFF step is generally optimized for different process parameters. During the diafiltration step, significant slow precipitation
from the cell culture harvest is formed as the pH and conductivity are decreased. Development and optimization of TFF is crucial
for the nonaffinity purification platform not only for the optimal loading condition on CEX, but also for determining the
cost efficiency of the purification schemes.
The Role of Primary Recovery TFF in Non-Protein A Purification Schemes
Binding to CEX resin requires below-neutral pH and low conductivity for HuMAbs. The lower the pH, the higher the binding capacity,
thereby, reducing capture chromatography cycles. However, the low pH requirement can lead to challenges because of gradual
precipitation during diafiltration. Therefore, assessing the influence of several process parameters is essential to optimize
the TFF step during primary recovery.
Primary recovery TFF plays a more significant role in the non-affinity scheme than in the affinity scheme not only because
it is a batch-volume reduction and feed conditioning step for the capture, but it is also a partial purification step.
Batch Volume Reduction
During the primary recovery TFF, the clarified harvest bulk is concentrated several fold, which will reduce batch processing
time on capture chromatography especially for high-binding CEX resins. With more high-binding CEX resins competing in the
market, development scientists have the luxury of evaluating and choosing the highest binding resins with high processing
flow rates.3 Even for the resins that can be operated at very high flow rate, the load time would be significant without volume reduction
at primary recovery step.
Feed Conditioning to Capture Chromatography
Figure 1. Reduction of diafiltration time by using a lower pH and conductivity buffer
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During TFF, the diafiltration endpoint for pH and conductivity are typically kept equivalent to those at the CEX load to avoid
further dilution or titration between the primary recovery operations and capture chromatography step. Usually, the diafiltration
end-point is when the conductivity and pH of the retentate reach those of the diafiltration buffer and it typically takes
about five diafiltration volumes (DV), which involves most of the processing time in this unit operation. Instead, the diafiltration
time can be reduced if the diafiltration buffer with lower pH and conductivity than the end-point requirements is used. For
example, if the load for CEX is set at pH 5.9 and 4.3 mS/cm, it takes only 3 DVs by using a diafiltration buffer of pH 5.7
and 3.5 mS/cm (Figure 1) instead of 5 DVs with a diafiltration buffer of pH 5.9 and 4.3 mS/cm. Because it takes a longer time
for 1 DV as flux decreases towards the end of diafiltration, buffer with lower pH and conductivity significantly shortens
the diafiltration time (~114 minutes in Figure 1). Buffer costs can be reduced by 40% when diafiltration is decreased from
5 to 3 DVs which translates into savings of process time as well as raw material.
