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Tutorials : Sep 15, 2008 (Vol. 28, No. 16)

mAb Capture from Unclarified Feedstock

Reinvention of Expanded Bed Adsorption Can Simplify Downstream Processing
  • Allan Lihme
  • ,
  • Rob Noel

The new age of disposable biopharmaceutical manufacturing technologies offers greater operational flexibility and time and cost savings for multiproduct facilities—benefits that have seen a greater acceptance over the last few years. The advantages of less cleaning and validation and greater flexibility, however, are marred by higher consumable costs and greater output of solid waste.

New technology developed by Upfront Chromatography overcomes these disadvantages and enables the capture of monoclonal antibodies (mAbs) and other therapeutics directly from the bioreactor, eliminating the need for all clarification unit operations. Cost and time savings result from the removal of all disposable clarification steps such as centrifugation or microfiltration, depth filtration, fine filtration, and sterile filtration.

Reinvented EBA

Upfront’s universal processing platform, Rhobust™, is a second-generation expanded bed technology that facilitates crude feedstock processing (Figure 1). The principle behind expanded bed adsorption (EBA) is to allow chromatography beads to fluidize in the feed stream, which is pumped at low pressure into the bottom of the column and out through the top. The expanded bed allows particulate impurities in the feed stream to pass freely—and at high flow rates—through the system without any clogging or pressure build up.

A key feature of the Rhobust platform is the use of tungsten carbide densified agarose beads, which provide greater operational flow rates for a given expansion of the adsorption unit compared to first-generation EBA materials. More importantly, tungsten carbide-agarose beads provide a greater operational buffer to process changes such as solution viscosity variation and bed disruption by particulates. Denser beads (3 g/mL) make for a more user-friendly operation.

There are two other critical innovations in second-generation EBA—dedicated hardware designs and mixed-mode (MM) ligand chemistry. It is the combination of all these developments that are the basis for the robust performance of EBA.

Processing unclarified mammalian cells, microbial lysates, or high cell density yeast feedstocks requires free movement of the particulates through the adsorbent and hardware. Upfront’s Rhobust platform provides a fluid distribution system that removes the requirement for column meshes, frits, or porous plates that would otherwise hinder the passage of particulates and subsequently cause cleaning issues and preferential flow issues.

The development of mixed-mode ligand chemistry has also enhanced the effectiveness of second-generation EBA—adsorbents with added selectivity limit any interactions with cell debris or sticky particulates. Mixed-mode ligands are small and stable compounds that interact with proteins with a combination of binding moieties. During the ligand development process, all ligands chosen for library screening have low intrinsic toxicity and are stable in NaOH solutions at high temperature. The selectivity of the adsorbent is optimized by adjusting the loading pH and conductivity of the separation.

This technology has now been adapted into small-scale disposable devices (Figure 2) for production of monoclonal antibodies for use in toxicity studies and Phase I to Phase III trials. Two ligand chemistries are available for Rhobust MabDirect adsorbents—protein A and a mixed-mode capture adsorbent. Thereafter, the same basic technology can be applied to full-scale manufacturing with fully cleanable and reusable process equipment.

Disposable Chromatography

The advantages of single-use unit operations such as bioreactors and buffer-preparation and product-hold bags are well known. Many of the economic advantages apply to disposable filtration and adsorption operations as well. For instance, disposable adsorption units reduce cleaning procedures and materials (i.e., NaOH, water, steam, and acid) and reduce facility downtime by removing steps that do not add value such as pre- and post-sanitization and packing.

The higher consumable cost of disposable unit operations is more than offset by the increased flexibility in the production facility, which is more difficult to quantify in terms of dollar value but imparts a valuable increase in the number of projects or contracts that a facility can undertake.

Many contract manufacturing organizations and pilot plant operations already effectively dispose of their protein A adsorbent after it has been used. This eliminates cross-contamination issues, but the need to maximize the operational value of costly adsorbents still remains. Rhobust MabDirect MM aims to lower the overall consumable costs as well as capital and operational costs of single-campaign mAb processing while increasing the much valued operational flexibility.

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).

Performance Data

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.