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Methods for Inactivation of Viruses and Bacteria in Cell

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Methods for Inactivation of Viruses and Bacteria in Cell (19) TZZ __T (11) EP 2 864 471 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 5/00 (2006.01) 08.03.2017 Bulletin 2017/10 (86) International application number: (21) Application number: 13733496.7 PCT/US2013/046756 (22) Date of filing: 20.06.2013 (87) International publication number: WO 2013/192395 (27.12.2013 Gazette 2013/52) (54) METHODS FOR INACTIVATION OF VIRUSES AND BACTERIA IN CELL CULTURE MEDIA METHODEN ZUT INAKTIVIERUNG VON VIREN UND BAKTERIEN IN ZELLKULTUR-MEDIEN PROCÉDÉS D’INACTIVATION DES VIRUS ET DES BACTÉRIES DANS LES MILIEUX DE CULTURE CELLULAIRE (84) Designated Contracting States: (74) Representative: Brodbeck, Michel AL AT BE BG CH CY CZ DE DK EE ES FI FR GB F. Hoffmann-La Roche AG GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Patent Department PL PT RO RS SE SI SK SM TR Grenzacherstrasse 124 4070 Basel (CH) (30) Priority: 20.06.2012 US 201261662349 P 15.03.2013 US 201313844051 (56) References cited: • "Animal Cell Culture Media" In: Vijayasankaran (43) Date of publication of application: et al.: "Encyclopedia of Industrial Biotechnology: 29.04.2015 Bulletin 2015/18 Bioprocess, Bioseparation, and Cell Technology.", 15 April 2010 (2010-04-15), John (73) Proprietor: F. Hoffmann-La Roche AG Wiley & Sons, XP002708444, DOI: 4070 Basel (CH) 10.1002/9780470054581.eib030, cited in the application the whole document (72) Inventors: • DePalma, Angelo: "Quantifying Cell Culture • SHIRATORI, Masaru, Ken Media Quality", , 15 January 2011 (2011-01-15), South San Francisco, CA 94080 (US) XP002708445, Retrieved from the Internet: • KISS, Robert, David URL:http://online.liebertpub.com/doi/pdfpl South San Francisco, CA 94080 (US) us/10.1089/gen.31.02.14 [retrieved on • PRASHAD, Hardayal 2013-08-01] South San Francisco, CA 94080 (US) • SCHLEH MARC ET AL: "Susceptibility of Mouse • IVERSON, Raquel Minute Virus to Inactivation by Heat in Two Cell South San Francisco, CA 94080 (US) Culture Media Types", BIOTECHNOLOGY • BOURRET, Justin PROGRESS, vol. 25, no. 3, May 2009 (2009-05), South San Francisco, CA 94080 (US) pages 854-860, XP002708460, ISSN: 8756-7938 • KIM, Michael • CAO XIAOLIN ET AL: "Identification and root South San Francisco, CA 94080 (US) cause analysis of cell culture media precipitates • CHARANIYA, Salim in the viral deactivation treatment with South San Francisco, CA 94080 (US) high-temperature/short-time method.", PDA JOURNALOF PHARMACEUTICAL SCIENCE AND TECHNOLOGY / PDA 2013 JAN-FEB, vol. 67, no. 1, January 2013 (2013-01), pages 63-73, XP002708446, ISSN: 1948-2124 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 864 471 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 864 471 B1 Description FIELD OF INVENTION 5 [0001] The invention provides for methods of viral inactivation using high temperature short time (HTST) treatment and adjustment of various parameters such that generation and depositions of precipitate is reduced and/or minimized. BACKGROUND OF THE INVENTION 10 [0002] Viruses are potential contaminants in drug manufacturing processes, particularly in cases where biologic drugs are derived from mammalian cell cultures. A source of viral contaminants can be the media used for cell culture or the cell lines producing the biologics of interest. Current approaches to prevent viral contamination of biologic drugs during the manufacturing process includes high temperature short time (HTST) cell media treatment for the inactivation of viruses that may be introduced into cell culture media by raw materials and is amplified during the culturing process 15 (Schleh, M. et al. 2009. Biotechnol. Prog. 25(3):854-860 and Kiss, R. 2011. PDA J Pharm Sci and Tech. 65:715-729). It has been reported that temperatures in excess of about 85°C are needed for HTST to be an effective virus inactivation method, with temperatures in excess of about 95°C needed to inactivate parvovirus, a common cell culture viral con- taminant that has been documented as occurring in cell culture processes, and which is resistant to many chemical and physical inactivating agents (Schleh et al.). 20 [0003] Although HTST treatment has proven to be highly effective in the inactivation of viruses, precipitation or formation of precipitates can occur in various cell culture media when subjected to this treatment. This precipitation leads to an accumulation of residue on the surfaces within the HTST system and can contribute to fouling of the equipment such that it can no longer heat up the media to the target temperature for proper inactivation of viral contaminants. Additionally, such precipitation can also foul the filters typically used downstream of the HTST system for the final processing to 25 remove microorgranisms, such as bacteria, from the medium. Such filter fouling can lead to inability to complete the medium processing step prior to the cell culture process. In some instances the precipitate may also impact the per- formance of the cell culture media and prevent efficient production of biologic drugs from the cultured cell lines. To prevent precipitation, the temperature can be lowered but successful viral inactivation may be negatively affected. Furthermore, precipitate formation during HTST cell media treatment can result in frequent cleaning or repair of equipment 30 used for HTST treatment during the manufacturing process which contributes significantly to the cost of processing. Therefore, there is a need for methods to prevent precipitate formation during HTST treatment without adversely affecting the efficacy of this treatment in the removal or inactivation of viral contaminants. [0004] The invention described herein addresses these needs by providing methods to effectively inactivate viral contaminants in cell culture media using HTST treatment with adjusted processing parameters that results in the reduction 35 or prevention of precipitate formation. BRIEF SUMMARY OF THE INVENTION [0005] The invention provides for methods, processes, systems and compositions for inactivating viral contamination 40 and/or other contaminants in cell culture media by using high temperature short time (HTST) treatment in combination with adjustments of various parameters, such as pH and/or calcium and/or phosphate concentration in the media. Furthermore, methods, processes, systems and compositions reducing the fouling of equipment and filters used for HTST treatment are disclosed as well. [0006] Accordingly, in one aspect, the invention provides for methods for inactivating virus or bacteria in cell culture 45 media while the media maintains suitability for cell culture, said method comprising (a) subjecting the cell culture media to high temperature short time (HTST) treatment; and (b) adjusting one or more parameters selected from the group consisting of pH, calcium level and phosphate level, whereby precipitate formation is suppressed, wherein the HTST treatment comprises raising the temperature of the media to at least about 85 degrees Celsius for a sufficient amount of time to inactivate virus or bacteria in the media, 50 a) wherein the pH is adjusted when the media comprises calcium and phosphate, the pH is adjusted in preparing the media prior to HTST treatment to a suitable low level, and the pH is adjusted following HTST treatment to a suitable level for cell culture; b) wherein the calcium level is adjusted when the media comprises phosphate, the calcium level is reduced such 55 that formation of complexes comprised of calcium and phosphate is suppressed, and the calcium level is adjusted following HTST treatment to a suitable level for cell culture; or c) wherein the phosphate level is adjusted when the media comprises calcium, the phosphate level is reduced such that formation of complexes comprised of calcium and phosphate is suppressed, and the phosphate level is adjusted 2 EP 2 864 471 B1 following HTST treatment to a suitable level for cell culture. [0007] In other aspects, the invention provides for methods for inactivating virus in cell culture media comprising subjecting the cell culture media to high temperature short time (HTST) treatment wherein the media has a pH of between 5 about pH 5.0 to about pH 6.9 during HTST treatment. In another aspect, the invention provides for methods for inactivating virus in cell culture media comprising subjecting the cell culture media to high temperature short time (HTST) treatment wherein the media has a pH of between about pH 5.0 to about pH 7.2 during HTST treatment. In some embodiments, the media has a pH of between about pH 5.3 to about pH 6.3 during HTST treatment. In other embodiments, the media has a pH of about pH 6.0 during HTST treatment. In any of the embodiments, the HTST treatment comprises raising 10 the temperature of the media to at least about 85 degrees Celsius for a sufficient amount of time to inactivate the virus or potential virus in the media. In some embodiments, the temperature of the media is raised to at least about 93 degrees Celsius for a sufficient amount of time to inactivate the virus or potential virus in the media. In some embodiments, the temperature of the media is raised to at least about 95, 97, 99, 101 or 103 degrees Celsius for a sufficient amount of time to inactivate the virus or potential virus in the media. In some embodiments, the pH of the media is lowered to 15 between about pH 5.0 to about pH 6.9 during HTST treatment prior to polypeptide production phase. In some embodi- ments, the pH of the media is then brought to between about 6.9-7.2 for the polypeptide production phase.
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