In recent years biopharmaceutical manufacturing has demonstrated major improvements in MAb production, exhibiting product titers as high as 25 g/L often associated with very high cell densities (1). High-density cell cultures with >150 million cells/mL pose a great challenge in clarification and further downstream processing because of a need to remove a large amount of biomass and increased levels of contaminants from cell debris generated during cell culture and harvesting. Production of biological substances (MAbs, in particular) usually involves processing a complex cell culture broth from which desired biological substances must be isolated and purified while maintaining high overall product recovery and quality.

The eXtreme-Density (XD) cell culture process is a continuous process in which both cells and product are retained in a stirred-tank bioreactor using suspension culture of Crucell's (www.crucell.com) PER.C6 human cells (1,2). This is accomplished by the use of a modified alternating tangential-flow perfusion system from Refine Technology (www.refinetech.com) in which fresh medium is continuously supplied and waste by-products are continuously removed and discarded. Cell densities of >150 million viable cells per milliliter of culture and product titers of >25 grams of MAb per liter of culture are possible. Because it retains product inside a bioreactor, the XD process produces a much lower harvest volume (only that contained in the bioreactor), which allows for less downstream processing than with traditional perfusion processes (1).

PRODUCT FOCUS: Monoclonal antibodies and other biologics
PROCESS FOCUS: Production and early downstream processing
WHO SHOULD READ: Manufacturing and process development
KEYWORDS: HIGH-DENSITY CELL CULTURE, CLARIFICATION, ANION EXCHANGE, PURIFICATION, PER.C6 CELLS
LEVEL: INTERMEDIATE

Traditionally, centrifugation and a combination of filtration techniques (tangential-flow filtration and depth filtration) have been widely accepted as workhorses for clarifying these complex cell culture broths (3,4,5). However, improvement of mammalian cell culture processes is providing for total cell densities far beyond traditional levels of 20 × 10⁶ cells/mL for CHO cells (6) to >150 × 10⁶ cells/mL for PER.C6 cells (1,2). Thus, limitations of both centrifugation and filtration techniques are apparent by the high (≤40%) solids content of such harvests.

Centrifugation can be applied to process feed streams with high levels of solids, for instance. However, product recovery can be low because of increased pellet volumes and a need to desludge frequently (especially in large-scale continuous centrifugation). Additionally, cell disruption from shear forces generated during centrifugation can further decrease the efficiency of harvest clarification and potentially cause product damage and/or entrapment (4,7).

Depth filters are advantageous because they remove contaminants (8), and many come in single-use format, reducing the need for cleaning and validation (9). However, depth filters are currently unable to handle high-solids feedstreams and are often used in series with centrifugation. TFF can handle high solids loading, but this technique can exhibit poor yield because of polarization of solids at the membrane surface when processing highly dense feed streams. Excessive product dilution and cell lysis caused by shear forces can also limit the utility of TFF.

Flocculation of cell culture harvests has also been widely used to enhance clarification throughput and downstream filtration operations (10,11,12). Current techniques include the use of soluble polyionic polymers (such as DEAE dextran, acryl-based polymers, and polyethylene amine) and inorganic materials such as diatomaceous earth and perlites, which remove cells and cell debris (13). However, polymers must subsequently be removed from process streams, which requires monitoring and quantification by in-process and product-release assays. If IEX chromatography is included as a purification step in the downstream process, binding capacities will be greatly affected by the charged nature of flocculants. The high viscosity of polycation stock solutions presents an additional process challenge.

The goal of our work was to develop an alternative method to centrifugation and known flocculation techniques and apply commonly used micro- or depth-filtration steps for clarification of high-density cell harvests. The method presented here is advantageous over classical flocculation because the polyionic polymer is attached to an insoluble matrix and consequently removed along with cells and cell debris. In this approach, IEX matrices are used to induce and enhance the settling of cells in situ. With a much lower cell density than the starting material, the partially clarified supernatant is recovered for further processing (e.g., by depth filtration). Because the matrices have ionogenic groups, this method also potentially reduces contaminants such as HCP and DNA. Reduction of impurities at this early stage of downstream processing can greatly increase the efficiency of subsequent unit operations (e.g., affinity or IEX chromatography) and thus reduce the overall number of downstream steps.

We developed an enhanced cell settling (ECS) technique for clarification of high-density cell cultures of PER.C6 cells in four distinct stages. First, we screened a range of IEX matrices in terms of their chemical functionality, particle size, and particle density to identify those matrices with the best performance. Second, we investigated the optimal initial cell density (X_t) and the amount of matrix needed to observe ECS. Third, because the technique is based on IEX, we had to determine the effect of cell culture broth conductivity on the extent of settling and contaminant removal. Last, we scaled up the technique to harvest 2-L bioreactors and depth-filtered the subsequent process stream.