Process Minimization Is Critical
The high consumable burden of utilizing disposable filtration units and disposable adsorption units can be dramatically reduced by minimizing the number of unit operations required. The ability to load the bioreactor contents directly onto the adsorption unit eliminates the need for all clarification operations and results in substantial reduction in capital expenditure and consumables savings.
Rhobust MabDirect MM disposable units are presanitized and prequalified for immediate application in manufacturing operations. The bioreactor contents can be loaded directly onto the Rhobust MabDirect MM unit, and the captured monoclonal antibody washed and eluted as a purified, clarified solution. The column can be cycled several times in order to process a whole disposable bioreactor in one day.
Equally as important as the disposable units is the simplicity of the operating system. The disposable units do not generate any backpressure, so disposable tubing can be used safely with peristaltic pumps to transfer liquids through the adsorption unit. After processing, the column and associated tubing can be disposed of, eliminating the need for all cleaning-in-place (CIP). The operating system, Rhobust Flex, can be scaled up from laboratory columns to production columns by changing pump tubing diameter and pump head (Figure 3).
Crude feedstock processing data from a mammalian cell bioreactor containing 90 million cells per mL expressing a mAb with a final titer of 4.5 g/L was recently presented at the “Recovery XIII” conference. The data was obtained from a side-by-side comparison made between the Rhobust MabDirect MM system and a traditional clarification scheme and processing on a protein A packed bed adsorbent.
The yield by MabDirect MM capture was 10% higher than clarified capture, and the quantity of host cell protein contamination of the eluted mAb was 50 µg/mg for the protein A adsorbent and 70 µg/mg for the Rhobust MabDirect MM capture. This similarity of host cell protein levels illustrates how effective the clarification is during capture on Rhobust MabDirect MM.
The mixed-mode adsorbent is stable to 1 M NaOH, which enables easy and effective between-cycle cleaning. The loading capacity on the mixed-mode adsorbent in this case was 30 g/L.
The benefits of unclarified feedstock processing with this technology is exemplified by the production of therapeutic intravenous immunoglobulin. Fraction II and III from the traditional Cohn fractionation procedure generates a precipitate of IgG that, when resolubilized, contains sticky particulates and has a concentration of approximately 10 g/L IgG. Clarification of this solution results in a yield loss of valuable IgG.
This feedstock is similar to the mAb feedstock described earlier, with high solids densities and high concentrations of IgG. It can be loaded directly onto a second-generation EBA Rhobust system and the production of a purified and clarified IgG solution enabled in one step. Minimal interaction occurs between solids and adsorbent as monitored by the quantity of proteinaceous material removed by hot caustic CIP.
Upfront Chromatography’s Rhobust processing platform offers a method of extracting biotherapeutics and biomolecules from unclarified feedstock, thereby reducing processing time and cost. The development of Rhobust MabDirect MM single-use units now means that the advantages of EBA technology can be combined with the benefits of reduced CIP, materials, and process downtime, to provide a simple, effective, streamlined, and robust downstream processing system.