Precipitation Method Optimization
At low pH, the hydrophobicity of the octyl moiety of caprylic acid dominates and makes acidic proteins in the solution precipitate.
Antibodies with basic pIs, however, have sufficient charge to counteract that hydrophobicity and remain in the supernatant.
Thus, precipitation is carried out by first adjusting the pH to the appropriate level and then adding caprylic acid while
mixing the contents. In this study, effective precipitation conditions were optimized primarily with clarified CHO cell culture
supernatant and then extended directly to cell culture with limited development.
To develop a robust and scalable contaminant precipitation step, three major parameters—pH, caprylic acid concentration, and
mixing time—must be thoroughly studied. Caprylic acid concentration and pH are interdependent. Caprylic acid shows increasing
efficiency in removing CHO host cell proteins (CHOP) and DNA as pH decreases. For example, cell culture harvest by precipitation
at neutral pH does not remove sufficient levels of CHOP (Figure 1). However, controlling pH alone without adding caprylic
acid does not remove significant amounts of CHOP; in the pH range of 4.0–7.0, CHOP levels were reduced by only ~20% in the
absence of caprylic acid. The precipitation phenomenon exhibits two distinct phases of contaminant removal: first, a sharp
(~2 log) decline of CHOP from pH 7.0 to pH 6.5, followed by steady 4-fold reduction to pH 4.0 (Figure 1).
Figure 1. Optimizing pH for the precipitation of contaminants by caprylic acid in clarified bulk (caprylic acid 1%; mixing
time 1 hour)
The decision about whether to lower the pH further depends on the molecule's stability as well as the type of chromatography
used in subsequent purification steps. In general, antibodies are less stable at lower pH. On the other hand, at higher pH,
the binding capacity of the CEX column used in the following step will be significantly reduced. Therefore, conditions must
be optimized to ensure the stability of the product and at the same time to maintain the high binding capacity of the resin.
Caprylic acid concentration shows a threshold value for contaminant precipitation at any constant pH level (Figures 2a and
2b). Increasing the caprylic acid concentration from 0.1 to 0.5% at both pH 5.0 and 4.5 results in a ~2 log reduction of CHOP,
followed by a negligible decline in CHOP precipitation. However, CHOP reduction was shown to be much more efficient at the
lower pH level (4.5), even when the caprylic acid concentration was as low as 0.2% (Figure 2b). And at pH 4.5, product quality
is not affected (see the stability discussion below). Therefore, in this case study, a caprylic acid concentration of 0.2
to 0.5% was considered effective.
Figure 2. Optimizing caprylic acid concentration for the precipitation of contaminants in clarified bulk.
Another process parameter that can influence precipitation efficiency is the length of time that the cell culture contents
and the caprylic acid are mixed. Prolonging the mixing time from 30 to 120 min can significantly improve the removal of DNA
and CHOP, with the longer mixing time needed particularly for the efficient removal of DNA (Figure 3).
Figure 3. Impact of mixing time on the efficiency of a precipitation step (pH 4.5, caprylic acid concentration 1%)
Overall, these three parameters (pH, caprylic acid concentration, and mixing time) must be tested interdependently. Other
operating parameter ranges (e.g., temperature and mixing speed) also must be optimized for consistent performance of the precipitation
step during scale-up. For the current studies, all the experiments at all scales were conducted at ambient temperature (20–25