Process-Scale Precipitation of Impurities in Mammalian Cell Culture Broth - If certain engineering challenges can be addressed, precipitation may prove to be a valuable tool for antibody

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Process-Scale Precipitation of Impurities in Mammalian Cell Culture Broth
If certain engineering challenges can be addressed, precipitation may prove to be a valuable tool for antibody purification


BioPharm International Supplements


Cationic Detergents

Both CTAB and DB were tested extensively, initially using only the NS0-derived antibody. Although neither compound reduced the amount of HCP, both were effective in the precipitation of DNA. The best results obtained with CTAB (5 mM) were a 164,000-fold reduction in DNA levels, down to ~190 pg DNA/mg antibody, which is significantly better than that obtained with the ammonium sulfate standard.


Figure 2. Best results obtained from domiphen bromide precipitation of impurities in CHO-produced monoclonal antibody cell culture broth
DB (5 mM) performed even better than CTAB, reducing DNA to undetectable levels at all pH values tested, with very high recovery of MAb in the supernatant (>95%). Additional experiments were performed on a CHO-produced antibody and the results are summarized in Figure 2. There was no significant reduction in HCP levels, but DNA levels were below the level of detection for the assay (note that HCP levels are shown as a fold reduction, whereas DNA levels are actual values). For all of the DB concentrations and pH values tested, the starting value was >11 x 106 pg DNA/ mg antibody, showing that DB is very effective at precipitating DNA while leaving the antibody product in solution.

Caprylic Acid Precipitation

Initial experiments were performed using an NS0-produced antibody at low pH values (3.4–4.8) and a caprylic acid concentration of 30 mM, based on information from the literature. Although there was no reduction in HCP levels, DNA levels were undetectable at the higher pH values tested. Additional testing for both NS0- and CHO-produced antibodies at pH 4.0–6.0 and caprylic acid concentrations of up to 100 mM confirmed that, for the NS0-produced antibody, DNA was significantly reduced at the lower pH values while HCP was not affected. However, for the CHO-produced antibody, both HCP and DNA were significantly reduced at all pH values tested.


Figure 3. HCP fold reduction values for caprylic acid precipitation using both a CHO- and NS0-produced antibody
Additional experiments confirmed that although DNA values were reduced below the level of detection for both NS0- and CHO-produced antibodies, the impact of caprylic acid on HCP levels differed significantly according to the cell line (Figure 3). For the CHO-produced antibody, the reduction in HCP levels continued to increase in line with the caprylic acid concentration. However, for the NS0-produced antibody, there were no further significant improvements in HCP clearance at caprylic acid concentrations above 100 mM. The yield for the CHO-produced samples averaged 92%, and caprylic acid precipitation performed at pH 6.0 with 500 mM caprylic acid resulted in the best overall performance (650-fold reduction in HCP to 160 ng/mg antibody, and 6,475-fold reduction in DNA to 700 pg/mg antibody. These results were significantly better than those obtained using ammonium sulfate precipitation.


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