Evaluation of monoclonal antibody and phenolic extraction from transgenic Lemna for purification process development.
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abstract
Several pharmaceutical protein products made in transgenic plant hosts are advancing through clinical trials. Plant hosts present a different set of impurities from which the proteins must be purified compared to other expression hosts such as mammalian cells. In this work, phenolic compounds present in extracts of monoclonal antibody (mAb)-expressing Lemna minor were examined. Two different extraction pHs were evaluated to assess the effect of extraction condition on the concentration of mAb and phenolics in the extracts. The extract prepared at pH 4.5 had an enriched level of mAb relative to native protein when compared to a pH 7.5 extract although similar overall mAb was extracted at either pH. Slightly more mAb was recovered from the pH 3 elution of the pH 4.5 extract run on a MabSelect column than was recovered from the pH 7.5 extract. Phenolic levels in extracts were assessed by spectrophotometry, Folin-Ciocalteu assay and by profiling on RP-HPLC. The Folin-Ciocalteu assay results did not agree with those obtained by the other two methods. Therefore phenolic levels were quantified by RP-HPLC comparing the total area of phenolic peaks to those of reference phenolic compounds. The pH 7.5 extract had 22% less phenolics than the pH 4.5 extract. Acidic precipitation of the pH 7.5 extract resulted in further reduction of phenolics originally present in the pH 7.5 extract. The total phenolics present in the extracts were effectively removed by incubation of extracts with a commercially available anion exchange resin, Amberlite IRA-402. We anticipate that early removal of phenolic compounds will prolong the life of more expensive affinity columns used for the purification of potential pharmaceutical proteins and should therefore be considered in process development involving proteins extracted from transgenic plant hosts.
