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.

BioPharm International Supplements

Direct Precipitation of Contaminants from Cell Culture: Two Case Studies

Caprylic acid also has been used successfully to remove host cell contaminants directly from cell culture. In two HuMAb case studies with CHO fed-batch cultures, caprylic acid precipitation resulted in significant reductions of CHOP and DNA contaminants. In HuMAb case study 1, CHO cell culture reached a peak cell density of 12 x 106 cells/mL and a productivity of ~4 g/L. In HuMAb case study 2, cell densities were almost 10 fold (~120 x 106 cells/mL) than in the HuMAb 1 study and the titer was also much higher: 14 g/L.

Figure 4. Optimization of pH for direct precipitation of contaminants from CHO cell culture (caprylic acid concentration 1%; mixing time 1 h)
The results of contaminant removal from cell culture in the first case study are shown in Figure 4. Precipitation with 1% caprylic acid with a mixing time of 1 h followed a pattern similar to that seen in clarified bulk precipitation (Figure 1). However, the only difference was a pH shift: cell culture required a pH level of 6.0 instead of pH 6.5 as in the case of clarified bulk to achieve the same level of CHOP reduction (Figures 1 and 4). This reduction also can be achieved simply by increasing the caprylic acid concentration, but the cost of caprylic acid should be taken into consideration.

Table 1. Scale-up of caprylic acid precipitation in clarified bulk and cell culture (pH 4.5 and 1% caprylic acid with a mixing time of 2 h)
To precipitate CHOP from cell culture with a peak cell density of 120 x 106 cells/mL, 1% caprylic acid at pH 4.5 with a mixing time of 2 h was tested. The CHOP level was significantly reduced, from 3.2 x 104 to 12 ng/mg of antibody (Table 1). However, this experiment can be further optimized to define the most suitable pH level by keeping the caprylic acid level and mixing time constant. Based on the earlier results in cell culture, the right pH appears to be approximately pH 5.5.

The target CHOP value to be achieved through precipitation also can be guided by purification process capability. Optimized CEX capture for case study 1 can lower the CHOP concentration from >1,000 ng/mg to <10 ng/mg. For example, if we aim to achieve a CHOP level of ~1,000 ng/mg, a pH of 5.5 may be sufficient for HuMAb 2, whereas for HuMAb 1, pH 6.0 will be optimum (Figure 4). If precipitation is performed at relatively low pH, it is important to monitor the stability of the filtered process intermediate after depth filtration (see the section on stability below).

Post Precipitation Filtration

Figure 5. Caprylic acid precipitation of contaminants from clarified bulk
Caprylic acid precipitates form three layers after centrifugation. The top layer is a mixture of residual caprylic acid and the CHOP precipitate (Figure 5), and the bottom layer, the sediment, is enriched with precipitated DNA. Therefore, centrifugation may not be a practical option at large scale, in which case optimized depth filtration may be necessary.

Although depth filtration is commonly used to remove the biomass or contaminant precipitate, high cell density cell cultures pose several challenges for depth filtration. The significant costs involved in building filtration trains with multiple pre-filters makes a clarification step much more expensive than using a simple filtration assembly. By precipitating contaminants directly in cell culture, we can reduce costs by combining two processing steps for the recovery of product. In other words, the clarification of cells and removal of caprylic acid precipitate can be achieved by one depth filtration step. The removal of contaminant precipitates and scale-up are crucial for making caprylic acid precipitation a feasible procedure for large-scale manufacturing. In this study, caprylic acid precipitation has been successfully scaled up 2,000-fold in clarified bulk procedure and 200-fold in cell culture (Table 1).

In a high density cell culture with continuous mixing mode with 1% caprylic acid, filtration capacity can reach ~80 L/m2. When the settlement of the precipitate is performed before filtration, capacity can be increased to as high as ~150 L/m2, which is almost as high as the filtration capacity of cell culture clarification. Another alternative is to add a filter aid to the mixture after precipitation is completed, followed by depth filtration. Filter aids such as Celpure (Advanced Minerals, Santa Barbra, CA) can help increase flux and filter capacity by separating the solids in the feed8 and increasing permeability. Using a filter aid before depth filtration for high solid contents, as in the case of caprylic acid precipitate, improves the quality of the feed stream as measured by turbidity units. The filtration capacity can exceed 1,000 L/m2 for clarified bulk or ~500 L/m2 for bioreactor cell culture contents after precipitation with the addition of Celpure. Provided there is enough space to hold solids, filtration capacity is dependent on the filter pore size, filter aid amount, flow rate, and pressure.

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