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Cambridge, MA, United States

Doolan P.,Dublin City University | Barron N.,Dublin City University | Kinsella P.,Dublin City University | Clarke C.,Dublin City University | And 6 more authors.
Biotechnology Journal | Year: 2012

Fed batch culture processes are often characterized by decreasing cell culture performance as the process continues, presumably through the depletion of vital nutrients and the accumulation of toxic byproducts. We have similarly observed that cellular productivity (Qp) often declines during the course of a fed batch process; however, it is not clear why some cell lines elicit this behavior, while others do not. We here present a transcriptomic profiling analysis of a phenotype of sustained Qp (S-Qp) in production Chinese hamster ovary (CHO) culture, in which a marked drop in Qp levels ("non-sustained" (NS) phenotype) in two cell lines irrespective of viability levels was compared to two cell lines that consistently displayed high Qp throughout the culture ("sustained" (S) phenotype). Statistical analysis of the microarray data resulted in the identification of 22 gene transcripts whose expression patterns were either significantly negatively or positively correlated with long-term maintenance of Qp over the culture lifespan. qPCR analysis of four of these genes on one of each (NS2, S2) of the cell lines examined by microarray analysis confirmed that two genes (CRYAB and MGST1) both replicated the microarray results and were differentially regulated between the NS and S phenotypes. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Chon J.H.,PERCIVIA | Zarbis-Papastoitsis G.,Eleven Biotherapeutics
New Biotechnology | Year: 2011

Sales of monoclonal antibody (mAbs) therapies exceeded $ 40 billion in 2010 and are expected to reach $ 70 billion by 2015. The majority of the approved antibodies are targeting cancer and autoimmune diseases with the top 5 grossing antibodies populating these two areas. In addition over 100 monoclonal antibodies are in Phase II and III of clinical development and numerous others are in various pre-clinical and safety studies.Commercial production of monoclonal antibodies is one of the few biotechnology manufacturing areas that has undergone significant improvements and standardization over the last ten years. Platform technologies have been established based on the structural similarities of these molecules and the regulatory requirements. These improvements include better cell lines, advent of high-performing media free of animal-derived components, and advances in bioreactor and purification processes. In this chapter we will examine the progress made in antibody production as well as discuss the future of manufacturing for these molecules, including the emergence of single use technologies. © 2011 Elsevier B.V. Source

Percivia | Date: 2011-04-09

The present invention relates to a method for clarification of, and removal of host cell proteins from, a cell broth consisting essentially of viable cells, a culture medium and a secreted desired biological substance having an overall positive charge in the cell broth by contacting the cell broth with a particulate anion exchanger, allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, separating the resulting cell pellet from the supernatant layer. The present invention further relates to a method for the recovery of a secreted desired biological substance from the cell broth by extracting the secreted desired biological substance from the supernatant layer.

Kuczewski M.,PERCIVIA | Fraud N.,Sartorius Stedim North America | Faber R.,Sartorius Stedim Biotech GmbH | Zarbis-Papastoitsis G.,PERCIVIA
Biotechnology and Bioengineering | Year: 2010

Membrane chromatography has already proven to be a powerful alternative to polishing columns in flowthrough mode for contaminant removal. As flow-through utilization has expanded, membrane chromatography applications have included the capturing of large molecules, including proteins such as IgGs. Such bind-and-elute applications imply the demand for high binding capacity and larger membrane surface areas as compared to flow-through applications. Given these considerations, a new Sartobind PhenylTM membrane adsorber was developed for large-scale purification of biomolecules based on hydrophobic interaction chromatography (HIC) principles. The new hydrophobic membrane adsorber combines the advantages of membrane chromatography-virtually no diffusion limitation and shorter processing time-with high binding capacity for proteins comparable to that of conventional HIC resins as well as excellent resolution. Results from these studies con-firmed the capability of HIC membrane adsorber to purify therapeutic proteins with high dynamic binding capacities in the range of 20mg-MAb/cm3-membrane and excellent impurity reduction. In addition the HIC phenyl membrane adsorber can operate at five- to ten-fold lower residence time when compared to column chromatography. A bind/elute purification step using the HIC membrane adsorber was developed for a recombinant monoclonal antibody produced using the PER.C61 cell line. Loading and elution conditions were optimized using statistical design of experiments. Scaleup is further discussed, and the performance of themembrane adsorber is compared to a traditional HIC resin used in column chromatography. © 2009 Wiley Periodicals, Inc. Source

Kuczewski M.,PERCIVIA | Schirmer E.,PERCIVIA | Lain B.,PERCIVIA | Zarbis-Papastoitsis G.,Eleven Biotherapeutics
Biotechnology Journal | Year: 2011

Advances in single-use technologies can enable greater speed, flexibility, and a smaller footprint for multi-product production facilities, such as at a contract manufacturer. Recent efforts in the area of cell line and media optimization have resulted in bioreactor productivities that exceed 8 g/L in fed-batch processes or 25 g/L in high-density cell culture processes. In combination with the development of single-use stirred tank bioreactors with larger working volumes, these intensified upstream processes can now be fit into a single-use manufacturing setting. Contrary to these upstream advances, downstream single-use technologies have been slower to follow, mostly limited by low capacity, high cost, and poor scalability. In this study we describe a downstream process based solely on single-use technologies that meets the challenges posed by expression of a mAb (IgG1) in a high-density suspension culture of PER.C6® cells. The cell culture harvest was clarified by enhanced cell settling (ECS™) and depth filtration. Precipitation was used for crude purification of the mAb. A high capacity chromatographic membrane was then used in bind/elute mode, followed by two membranes in flow-through (FT) mode for polishing. A proof of concept of the entire disposable process was completed for two different scales of the purification train. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

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