Recombinant Protein Production by CHO Cells Cultured in a Chemically Defined Medium

Gorfien, Stephen F.; Dzimian, Joyce L.; Tilkins, Mary Lynn; Godwin, Glenn P.; Fike, Richard M.

doi:10.1007/978-94-011-5161-0_42

Cited by 7 publications

(4 citation statements)

References 2 publications

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“…Low molecular weight dextran sulfate (5 kDa) is a conventional anti-clumping agent that has been used by the manufacturer of 293 SFMII in past serum-free media formulations (Gorfien, 2004). To determine whether dextran sulfate inhibited ABLVp G-mediated viral infection, culture medium supplemented with 2-fold serial dilutions of 5 kDa molecular weight dextran sulfate was added to 293T cell monolayers prior to infection with rVSV reporter viruses.…”

Section: Resultsmentioning

confidence: 99%

Host cell tropism mediated by Australian bat lyssavirus envelope glycoproteins

et al. 2013

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Australian bat lyssavirus (ABLV) is a rhabdovirus of the lyssavirus genus capable of causing fatal rabies-like encephalitis in humans. There are two variants of ABLV, one circulating in pteropid fruit bats and another in insectivorous bats. Three fatal human cases of ABLV infection have been reported with the third case in 2013. Importantly, two equine cases also arose in 2013; the first occurrence of ABLV in a species other than bats or humans. We examined the host cell entry of ABLV, characterizing its tropism and exploring its cross-species transmission potential using maxGFP-encoding recombinant vesicular stomatitis viruses that express ABLV G glycoproteins. Results indicate that the ABLV receptor(s) is conserved but not ubiquitous among mammalian cell lines and that the two ABLV variants can utilize alternate receptors for entry. Proposed rabies virus receptors were not sufficient to permit ABLV entry into resistant cells, suggesting that ABLV utilizes an unknown alternative receptor(s).

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Section: Resultsmentioning

confidence: 99%

Host cell tropism mediated by Australian bat lyssavirus envelope glycoproteins

et al. 2013

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“…A Chinese Hamster Ovary subclone (D'Anna et al, 1997;D'Anna et al, 1996;Deaven and Petersen, 1973;Puck et al, 1958. ) that has been adapted to a commercially available chemically-defined, protein free-media (Gorfien et al, 1998) was evaluated to determine if it met the requirements described above. Figure 3.…”

Section: Cell Line Selectionmentioning

confidence: 99%

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et al. 2002

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UCOE vectors contain non-tissue specific chromatin-opening-elements that permit rapid expression of a protein in anintegration independent manner. Efficient expression can bederived from a single copy of an integrated gene site resulting ina higher percentage of cells expressing the marker gene in theselected pool in comparison to standard non-UCOE containingvectors. This, in combination with the utilization of a serum-free, suspension adapted parent cell line allows for rapidproduction of large quantities of protein in a short period oftime. Utilizing this system more than 300 mg of a recombinantantibody has been produced in less than 1 month from transfectionpools in shake flask. Selected subclones have been scaled intosmall bioreactors in less than 2 months, producing significantquantities of monoclonal antibody using a protocol generic for theparent cell line. The increased efficiency obtained with the UCOEvector reduces the number of transfectants which need to bescreened in order to obtain high productivity subclones.Transfection of a standard host cell line, preadapted to grow in alarge-scale setting, allows for rapid cell line developmentdecreasing the transition time from research into development andmanufacturing. Alternatively, the traditional approach of using aparent cell line which requires serum-free and suspensionadaptation after transfection further increases the need forscreening a large number of subclones, because many of thesubclones will not be able to grow under conditions that allowlarge-scale protein production. The use of a preadapted cell linecan reduce the time required to develop a cell line from months toweeks.

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“…Figures and show results of the purification strategy to capture hIgG 1 from CHO cell supernatant using a size‐exclusion desalting column followed by MMM chromatography. The desalting operation was used to remove chaotropic salts reported in the cell culture medium, since our previous study has shown that chaotropes interrupt protein binding to the MMM. From a protein sample load volume study with the desalting column, it was found that a 5 mL sample CHO cell supernatant volume gave sufficiently high peak resolution.…”

Section: Resultsmentioning

confidence: 99%

Antibody purification from CHO cell supernatant using new multimodal membranes

Wang

Zhou

Gowtham³

et al. 2017

Biotechnology Progress

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This contribution describes strategies to purify monoclonal antibodies from Chinese hamster ovary (CHO) cell culture supernatant using newly designed multimodal membranes (MMMs). The MMMs were used for the capture step purification of human IgG following a size-exclusion desalting column to remove chaotropic salts that interfere with IgG binding. The MMM column attained higher dynamic binding capacity than a Protein A resin column at an equivalent residence time of 1 min. The two-step MMM chromatography process achieved high selectivity for capturing hIgG from the CHO cell culture supernatant, though the desalting step resulted in product dilution. Product purity and host cell protein (HCP) level in the elution pool were analyzed and compared to results from a commercial Protein A column. The product purity was >98% and HCP levels were <20 ppm for both purification methods. In addition, hIgG could be eluted from the MMM chromatography column at neutral pH, which is important for limiting the formation of aggregates; although slow elution dilutes the product. Overall, this paper shows that MMMs are highly effective for capture step purification of proteins and should be considered when Protein A cannot be used, e.g., for pH sensitive mAbs or proteins lacking an Fc binding domain. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:658-665, 2017.

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Recombinant Protein Production by CHO Cells Cultured in a Chemically Defined Medium

Cited by 7 publications

References 2 publications

Host cell tropism mediated by Australian bat lyssavirus envelope glycoproteins

Host cell tropism mediated by Australian bat lyssavirus envelope glycoproteins

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Antibody purification from CHO cell supernatant using new multimodal membranes

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