(289a) Mammalian Cell Culture Broth Clarification and Affinity Capture of Secreted Antibodies in a Novel Single Integrated Process Using a Compact Settler

Authors: 
Kompala, D. - Presenter, Sudhin Biopharma Co
Enhanced sedimentation on inclined surfaces has been exploited successfully to separate the larger live and productive mammalian cells (>12 microns in diameter) from the smaller (<8 microns diameter) dead cells and cell debris. The selective recycle of live and productive Chinese hamster ovary (CHO) cells to the continuously fed or perfused bioreactor results in a high cell concentration/density inside the bioreactor, while the dead cells and cell debris are continuously removed from the top of the "inclined settler". This selective cell retention device has been scaled up initially in a multiplate rectilinear design and used by several biomanufacturing companies, such as Bayer, Biomarin, Eli Lilly, Roche, etc. This original lamellar design requires a large footprint for the large production scale (>3000L) perfusion bioreactor and is quite inefficiently scaled up.

Sudhin Biopharma Company has now scaled up the inclined surfaces in a more compact cylindrical and conical design, which houses over 6 x more inclined surfaces on a given footprint, compared to the original lamellar inclined settler design. With its more efficiently scalable patent-pending design, this compact settler has been initially demonstrated to settle live yeast Pichia pastoris cells (3 - 5 microns in size) from the fermentation broth and return them to the continuous bioreactor, achieving very high cell densities (O.D. >800) over two months long culture duration. In mammalian cell cultures applications, this compact settler again demonstrates selective retention of live and productive CHO cells while the dead cells and cell debris are continuously removed along with the secreted proteins in the harvest stream.

A novel application for the compact settler is the clarification of mammalian cell culture broth at the end of a fed-batch bioreactor culture, which is more commonly used in the biomanufacturing industry. This device is currently being evaluated at a collaborating large biopharmaceutical manufacturing company to potentially replace the primary clarification device of a continuous centrifuge and reduce the requirement of depth filtration membrane used as a secondary clarification device. Current results from our own cell culture broth clarification experiments carried out in a single use disposable plastic settler will be presented in this talk.

Another novel extension of the compact settler's usage is to contact the mammalian cell culture broth containing the secreted antibody product directly with protein A coated affinity capture beads inside the settler, without any need for prior clarification or filtration of the cell culture broth. After sufficient contacting time for antibody molecules to be captured by the protein A beads suspended inside the settler, all the cells, cell debris, and unbound host cell proteins can be washed out of the settler by increasing the top effluent rate from the settler. After adequate washing, bound antibodies can be released by the elution buffer and the pure antibodies can be readily harvested in the top effluent stream. Subsequently regeneration of protein A beads are carried out inside the settler by pumping the equilibration buffer into the settler. Latest experimental results from the sequential batch operations will be presented.

It is relatively simple to convert the above sequence of batch affinity capture, wash, elution and regeneration steps inside a single compact settler into a continuous flow process of protein A beads undergoing capture, wash, elution and regeneration in a series of settlers, with the beads flowing from one settler to the next in a truly continuous cross-flow contacting pattern with different buffers. This continuous affinity capture on protein A beads suspended inside a series of compact settlers will be a more efficient usage of the more expensive protein A beads than the current packed protein A bead affinity column chromatography, commonly used in the industry today. The relative advantages and disadvantages of the novel integrated process of affinity capture of antibodies inside compact settlers will be discussed.