Harvest and Recovery of Monoclonal Antibodies from Large-Scale Mammalian Cell Culture - Comparing primary harvest techniques adopted in commercial-scale operations for monoclonal antibody products. -

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Harvest and Recovery of Monoclonal Antibodies from Large-Scale Mammalian Cell Culture
Comparing primary harvest techniques adopted in commercial-scale operations for monoclonal antibody products.


BioPharm International
Volume 21, Issue 5

ABSTRACT

The objective of harvest and clarification unit operations is the removal of cells and cell debris to enable product capture on a chromatographic column. The first step in the harvest of monoclonal antibodies from mammalian cell culture is cell removal, followed by filtration unit operations for additional clarification. Centrifugation and microfiltration have been the primary harvest techniques adopted industrially. Although depth filtration can be used as a primary harvest method, it is more common to see this unit operation follow the primary harvest step to provide additional clarification. Flocculants are sometimes added before harvest to improve harvest and clarification operations. Filtration through absolute pore size membranes is typically the final clarification step before capture chromatography. Expanded bed chromatography has been developed as an integrated unit operation that combines harvest with product capture, but to date, practical limitations have kept this technique from being adopted in commercial-scale operations.



Several types of unit operations have been used for harvest and clarification of extracellularly expressed products from mammalian cell culture. Key techniques include centrifugation, microfiltration, depth filtration, filtration through absolute pore size membranes, the addition of flocculants to improve other harvest steps, and expanded bed chromatography. This article provides a basic review of the fundamentals of these unit operations and practical considerations during process development. Further details on these unit operations have been reviewed elsewhere.1 The final section describes how these unit operations fit together to form the harvest and clarification process for monoclonal antibody (MAb) products.

CENTRIFUGATION

Centrifugation uses the density difference between solids and the surrounding fluid. The centrifugal force accelerates the settling that would normally occur during sedimentation. Most industrial applications use disk stack centrifuges to remove cells and cell debris.2 Disk stack centrifuges offer continuous operation, making their throughput consistent with the desire to limit the time for harvest operations. Figure 1 shows the schematic for a disk stack centrifuge.

Editors note: Figure 1 for this article is not available online. To obtain a copy of the figure by fax, please contact

The basic principles of centrifugation involve a balance between the buoyant force acting on solid particles and Stokes's law, which expresses the drag force. The ratio of flow rate to the effective settling area (termed Σ) is held constant during scale-up of centrifugal operations. For a disk stack centrifuge, Σ is expressed as:



in which n is the number of disks, r 0 and r i are the outer and inner radii of the disk, and θ is the angle of the disks from the vertical.

Operating conditions are often first screened at laboratory scale in a tubular bowl centrifuge and then translated to a disk stack centrifuge. For the two centrifuges:




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