Precipitation of Process-Derived Impurities in Non-Protein A Purification Schemes for Antibodies - Precipitation prior to capture chromatography offers a simple, robust, and economical method to


Precipitation of Process-Derived Impurities in Non-Protein A Purification Schemes for Antibodies
Precipitation prior to capture chromatography offers a simple, robust, and economical method to remove CHO host cell proteins and DNA.

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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.

Figure 1. Optimizing pH for the precipitation of contaminants by caprylic acid in clarified bulk (caprylic acid 1%; mixing time 1 hour)
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 2. Optimizing caprylic acid concentration for the precipitation of contaminants in clarified bulk.
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 3. Impact of mixing time on the efficiency of a precipitation step (pH 4.5, caprylic acid concentration 1%)
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).

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 C).

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