Of particular interest are the numerous descriptions of the use of short-chain fatty acids, such as caprylic acid, for the
precipitation of plasma proteins and DNA, as first described by Chanutin and Curnish in 1960.15 Several groups have developed conditions for the use of caprylic acid in antibody purification from various starting materials,
such as ascites fluid and mammalian serum.16–19 Parkinnen and Lundblad also showed that caprylic acid could inactivate enveloped viruses.20,21
Evaluating Potential Precipitants for Impurity Removal
Protocol
Table 1. Characteristics of the chemical compounds used in the precipitation evaluation
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Using this large body of work as a guide, the Mammalian BioProcess Research and Development group at Pfizer, Inc. (New York,
NY), recently completed an evaluation of several chemical compounds that could potentially be used to precipitate impurities
from mammalian cell culture broth containing a MAb. Various cell culture broths from either Chinese hamster ovary (CHO) or
NS0 cell lines were grown in an instrumented bioreactor from 1–10 liters in size. The chemical compounds discussed in this
article are listed in Table 1.
The basic experimental protocol used for the evaluation was as follows: Cells and cellular debris were removed by centrifugation
and depth filtration before the study commenced. The broth was adjusted to a final concentration of approximately 1 g antibody/L
solution, the pH was adjusted, and the potential precipitant was added. The broth was incubated for 30–60 min, then pelleted
in a Sorvall Evolution RC centrifuge for 10 min at 4,000g. The supernatant was separated from any precipitate, and if possible
the precipitate was resolubilized. The resulting fractions of clarified supernatant and resolubilized precipitate were then
tested for impurity levels using a standard Cygnus kit (Cygnus Technologies, Southport, NC) for HCP and the threshold assay
for DNA. All of the precipitating agents were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO).
Ammonium Sulfate Precipitation
Ammonium sulfate precipitation for the purification of MAbs has been extensively documented in the literature.22–24 This method was performed and used as a standard comparison for antibody purity. In this case, the antibody was precipitated
while the impurities remained in solution. After evaluating a range of ammonium sulfate concentrations, 1.8–2.0 M was chosen
and tested for impurity removal and product yield at various pH values ranging from 4.0–7.0. The resulting precipitate was
recovered by centrifugation and washed three times with ammonium sulfate solution to remove additional impurities. The precipitate
was then resolubilized and analyzed.
Figure 1. Best results obtained from ammonium sulfate precipitation using a variety of NS0- and CHO-produced antibodies.
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The best results obtained using a variety of NS0- and CHO-produced antibodies are shown graphically in Figure 1. The results
indicate a 21-fold reduction in HCP (to 8,960 ng HCP/mg antibody) from the starting broth value, and a 79-fold reduction in
DNA (to 38,800 pg DNA/mg antibody) with a yield of 91%. The fold reduction in impurities was calculated by dividing the impurity
value in the broth by the impurity value in the antibody phase (supernatant or resolubilized precipitate, as appropriate).
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