Genetic Engineering & Biotechnology News

SEP15 2017

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28 | SEPTEMBER 15, 2017 | GENengnews.com Bioburden Control in Continuous Capture of Monoclonal Antibodies Continued from page 27 CO N T I N U O U S B I O P R O C E S S I N G important to determine whether such a column can withstand many cycles of purifi- cation of a continuous process. Most impor- tantly, a system that would run continuously will be able to maintain its bioburden-free status. A study showing the continuous cy- cling of a gamma-irradiated prepacked Pro- tein A Opus column was conducted. A 1.2 x 20 cm prepacked Protein A Opus column stored in 2% benzyl alcohol, pH 7.5, was gamma irradiated to 30 kGy radiation dose. The column was aseptically connected to a perfusion bioreactor–ATF system surge bag. The clarified culture media coming from the bioreactor was loaded onto the column with the help of a peristatic pump, while the chromatography steps were executed us- ing an ÄKTA™ Explorer (GE Healthcare) system fitted with sterile 0.2-µm filters on buffer lines. A sample collection manifold was attached to column outlet for daily col- lection and bioburden measurement. The column was run continuously for seven days. The results show consistency of purifi- cation between cycles, and most importantly the maintenance of a bioburden-free system (Table 3). There was a slight increase over time in leached Protein A, however, it was still within acceptable limits. Conclusions Finding solutions for bioburden control within downstream capture and purification steps allows for continuous processes to be adopted more easily. A sterilized, prepacked Opus chromatography column can be asep- tically connected to a continuous upstream perfusion bioreactor—Xcell™ ATF system— and biological products can be directly and continuously captured and purified. Gamma sterilization of prepacked Opus columns does not affect the physical integrity of the columns. An appropriate storage solution allows preservation of the properties of Protein A capture resin upon treatment with gamma radiation. Sterility is also maintained over an extended period of continuous use. The Protein A capture step is probably the most critical, yet sensitive (due to rela- tive fragility of Protein A) step in the down- stream purification of mAbs. Demonstrating that a prepacked Protein A Opus column can be sterilized with gamma irradiation and successfully integrated in a closed upstream- downstream unit opens the door to an integrated continuous biomanufacturing process. Further downstream chromatog- raphy steps could be gamma irradiated for bioburden control, and a truly closed, fully integrated unit could continuously produce biological drugs. Figure 3. Loss of binding capacity of gamma-irradiated Protein A resin exposed to 0.5 M hydroxide compared with non-irradiated Protein A resin. Table 2. % Yield Log Reduction HCP Log Reduction DNA Residual ProA (ng/mg) Pre - Gamma 100% 2.9 3.7 1.2 30kGy - Gamma 97% 2.6 3.6 3.0 Table 2. Preservation of purification properties of Protein A resin irradiated to 30 kGy of gamma radiation compared with a non-irradiated Protein A resin. ProA = Protein A; HCP = host-cell protein. Table 3. Day # % Yield ProA Leaching (ppm) Log Reduction HCP Bioburden (CFU) 1 83.2 3.8 2.6 0 2 79.9 2.7 2.7 0 3 N/A* 0.3 2.3 0 4 80.5 N/A* 2.6 0 5 83.6 10.7 2.3 0 6 81.7 10.8 2.4 0 7 83.8 9.7 2.6 0 * N/A = Not analyzed Table 3. Consistency of purification of monoclonal antibody over gamma-irradiated Protein A Opus column seven days of continuous use. ProA = Protein A; HCP = host-cell protein. Dana C. Pentia, Ph.D. (dpentia@repligen. com), is senior scientist, downstream bio- processing, and James R. Peyser (jpeyser@ repligen.com) is senior director, bioprocess development, at Repligen. Preservation of Purification Properties Consistency of Purification of Monoclonal Antibody

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