Integrated continuous biomanufacturing platform with ATF perfusion and one column chromatography operation for optimum resin utilization and productivity.

A new integrated continuous biomanufacturing platform for continuous production of antibodies at fixed cell volumes and cell concentrations for extended periods with immediate capture is presented. Upstream antibody production has reached technological maturity, however, the bottleneck for continuous biomanufacturing remains the efficient and cost-effective capture of therapeutic antibodies in an initial chromatography step. In this study, the first successful attempt at using one-column continuous chromatography (OCC) for the continuous capture of therapeutic antibodies produced through alternating tangential flow perfusion is presented. By performing upstream media optimizations, the upstream perfusion rate was reduced to one vessel volume per day (vv/d), increasing antibody titer and reducing the volume of perfusate. In addition, process improvements were performed to increase productivity by 80% over previously reported values. In addition, a real-time method for evaluating column performance to make column switching decisions was developed. This improved productivity coupled with the use of a single-column improved process monitoring and control in OCC compared to multi-column systems. This approach is the first report on using a single column for the implementation of an integrated continuous biomanufacturing platform and offers a cost-effective and flexible platform process for the manufacture of therapeutic proteins.

Physicochemical surrogates for in vitro toxicity assessment of liposomal amphotericin B.

Pharmaceutical toxicity evaluations often use in vitro systems involving primary cells, cell lines or red blood cells (RBCs). Cell-based analyses ('bioassays') can be cumbersome and typically rely on hard-to-standardize biological materials. Amphotericin B (AmB) toxicity evaluations are primarily based on potassium release from RBCs and share these limitations. This study evaluates the potential substitution of two physicochemical AmB toxicity approaches for the bioassay: Ultraviolet-visible spectroscopy (UV-vis) and in vitro drug release kinetics. UV-vis spectral analyses indicated that liposomal AmB's (L-AmB) main peak position (λmax) and peak ratio (OD346/OD322) are potential toxicity surrogates. Similarly, two first-order release parameters derived from USP-4 in vitro drug release analyses also provided linear relationships with toxicity. These were the initial, overall drug release rate and the ratio of loose to tight AmB pools. Positive slopes and high correlation coefficients (R2 > 0.9) characterized all interrelations between physicochemical parameters and toxicity. These tests converted the manufacturing variables' nonlinear (i.e., curvilinear) relationships with in vitro toxicity to linear responses. Three different toxicity attenuation approaches (2 manufacturing, 1 formulation), covering formulation composition and process aspects, support this approach's universality. These data suggest that one or more spectral and kinetic physicochemical tests can be surrogates for L-AmB in vitro toxicity testing.

Impact of media and antifoam selection on monoclonal antibody production and quality using a high throughput micro-bioreactor system.

Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology-derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer-based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro-scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro-scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX-Cell Advanced, and OptiCHO media, and 204, C, EX-Cell, SE-15, and Y-30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX-Cell Advanced, and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25-35 × 106 cells-d/mL, while maintaining specific antibody production (Qp > 2 pg/cell-d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX-Cell, and SE-15 were capable of providing adequate control of foaming while antifoam 204 and Y-30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:262-270, 2018.