Genetic Engineering & Biotechnology News

SEP15 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Page 64 of 77 | SEPTEMBER 15, 2017 | 27 bioburden, an experiment was set up includ- ing a gamma-irradiated column in a closed system. The column was aseptically con- nected to a bottle containing sterilized Luria broth that was recirculated within the closed system for 8 weeks. The broth was sampled weekly for sterility. All collected samples were sterile, indicating the system main- tained sterility for the 8-week period. As a control, the recirculated broth was spiked with Escherichia coli to verify that it was favorable to bacterial growth, and growth was observed. The results demonstrated the capability of a gamma-irradiated Opus column in delivering the bioburden control needed for lengthy continuous processes. Preserving Protein A Functionality upon Gamma Irradiation Protein A is the most commonly used capture step for mAb purification. Ionizing radiation, such as gamma, can severely im- pact a protein's structure and function. 1,2 A method that sterilizes the resin while main- taining the functionality of Protein A resin is critical. To study this, Protein A was immo- bilized to crosslinked agarose and gamma irradiated in several storage solutions. Fol- lowing gamma irradiation, static binding capacity for polyclonal hIgG was measured and compared with a non-irradiated control sample. Static binding capacity was deter- mined by incubating Protein A resin with a concentrated solution of polyclonal human IgG for 30 minutes. Afterward, the resin was washed with neutral pH buffer and eluted with low pH solution. The amount of IgG eluted was measured and static binding calculated. The Protein A resin irradiated in a solution containing 2% benzyl alcohol at pH 7.5 did not show 3 a change in static binding capacity, while samples stored in other solutions had a capacity decrease of up to 24% (Figure 2). A mAb purification process operated continuously will require many column cy- cles; therefore, resin lifetime is an important consideration. While bioburden is controlled with gamma irradiation, cleaning of the col- umn of other contaminants such as host-cell proteins and DNA is still necessary at regu- lar intervals. The most used and preferred method of cleaning the capture column is sodium hydroxide solution at 0.1 M–0.5 M concentration. Even hydroxide-stable variants of Protein A will lose functionality in time after repeated or lengthy exposure to hydroxide. A gamma-irradiated Protein A resin should not be more unstable than a non-gamma-irradiated one. A study was performed to test the effect of gamma ir- radiation on hydroxide stability and overall purification performance. Protein A resin stored in 2% benzyl alcohol, pH 7.5 solution was gamma irradi- ated with a 30 kGy dose. Results showed that gamma irradiation of a Protein A resin (according to this method) had a minimal effect on the hydroxide stability as com- pared with a control (Figure 3). Purification properties of the gamma-irradiated Protein A resin were maintained as demonstrated by similarity in the purity of the elution pool compared to the pool from a non-irradiated resin. It can be seen in Table 2 that the reduction in host-cell proteins (HCP) and DNA concentration was similar between non-irradiated and irradiated resin (30 kGy), the yield was also similar between the two samples. Leached Protein A, while slightly increased in the gamma-irradiated sample, was still within acceptable limits. The data presented herein demonstrate that Protein A resin characteristics can be preserved when gamma irradiating the resin stored in 2% benzyl alcohol, pH 7.5, leading to the conclusion that a prepacked Protein A Opus column stored in this solution and gamma irradiated to 25–30 kGy of irradiation can be suitable for the capture of mAbs. It is CO N T I N U O U S B I O P R O C E S S I N G Figure 2. Storage solution effect on preservation of binding capacity of Protein A resin irradiated with 30 kGy of gamma radiation. Figure 1. Pressure drop over a column pre- and post-gamma irradiation to 30 kGy as a function of linear flow. Table 1. Packed bed performance of a 20 x 20 cm Opus column pre- and post-gamma irradiation to 30 kGy. HETP = height equivalence to the theoretical plate. ➜ Table 1. Pre-Gamma Post-Gamma HETP (N/m) 3055 2935 Peak Asymmetry 1.3 1.4 Packed Bed Performance (HETP/Peak) Bioburden Control in Continuous Capture of Monoclonal Antibodies Continued from page 24

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