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`EXHIBIT 16
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`

`

`Case 1:18-cv-01363-CFC Document 87-1 Filed 03/22/19 Page 2 of 31 PageID #:
`11304
`I IIIII IIIIIIII Ill lllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111
`US009441035B2
`
`c12) United States Patent
`Carvalhal et al.
`
`(IO) Patent No.:
`(45) Date of Patent:
`
`US 9,441,035 B2
`Sep.13,2016
`
`(54) CELL CULTURE MEDIA AND METHODS OF
`ANTIBODY PRODUCTION
`
`4,927,762 A
`5,122,469 A *
`
`5/1990 Darfler
`6/1992 Mather
`
`(71) Applicant: Genentech, Inc., South San Francisco,
`CA (US)
`
`(72)
`
`Inventors: Veronica Carvalhal, South San
`Francisco, CA (US); Natarajan
`Vijayasankaran, South San Francisco,
`CA (US); Lauren Brown, South San
`Francisco, CA (US); Thomas DiRocco,
`South San Francisco, CA (US); Nathan
`McKnight, South San Francisco, CA
`(US)
`
`(73) Assignee: GENENTECH, INC., South San
`Francisco, CA (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 40 days.
`
`(21) Appl. No.: 14/211,467
`
`(22) Filed:
`
`Mar. 14, 2014
`
`(65)
`
`Prior Publication Data
`
`US 2014/0308273 Al
`
`Oct. 16, 2014
`
`Related U.S. Application Data
`
`(60) Provisional application No. 61/801,247, filed on Mar.
`15, 2013.
`
`(51)
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`Int. Cl.
`C07K 16/22
`C12N 5/00
`C12N 5/10
`A61K 39/395
`C07K 16/00
`(52) U.S. Cl.
`CPC ............... C07K 16/22 (2013.01); C07K 16/00
`(2013.01); C07K 2317/14 (2013.01); C07K
`2317/24 (2013.01); C07K 2317/76 (2013.01);
`Cl2N 2510/02 (2013.01)
`( 58) Field of Classification Search
`CPC .. C07K 16/22; C07K 16/00; C07K 2317/14;
`C07K 2317/76; C07K 2317/24; C12N
`2510/02; C12N 2800/107; C12N 2800/10;
`C12N 2500/00; C12N 2500/76; C12N
`2500/80; C12N 2500/10; C12N 2500/25;
`C12N 2511/00; C12N 5/068
`See application file for complete search history.
`
`(56)
`
`References Cited
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`2005/0026229 Al
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`2/2005 Reiter et al.
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`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`
`12/1990
`0073657 Bl
`12/1990
`0402226 Al
`(Continued)
`
`OTHER PUBLICATIONS
`
`Li et al., mAbs 2(5): 466-477, Sep./Oct. 2010.*
`(Continued)
`
`Primary Examiner - Phuong Huynh
`(74) Attorney, Agent, or Firm - Morrison & Foerster LLP
`
`(57)
`
`ABSTRACT
`
`Cell culture media are provided herein as are methods of
`using the media for cell culture and antibody production
`from cells. Compositions comprising antibodies and frag(cid:173)
`ments thereof, produced by the methods herein are also
`provided.
`
`84 Claims, No Drawings
`
`

`

`Case 1:18-cv-01363-CFC Document 87-1 Filed 03/22/19 Page 3 of 31 PageID #:
`11305
`
`US 9,441,035 B2
`Page 2
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`(56)
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`435/404
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`435/71.1
`
`Cl2N 5/0018
`435/69.3
`
`C07K 5/081
`435/69.6
`
`C07K 14/765
`435/346
`
`FOREIGN PATENT DOCUMENTS
`
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`EP
`EP
`WO
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`

`

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`
`

`

`Case 1:18-cv-01363-CFC Document 87-1 Filed 03/22/19 Page 5 of 31 PageID #:
`11307
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`US 9,441,035 B2
`Page 4
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`Case 1:18-cv-01363-CFC Document 87-1 Filed 03/22/19 Page 7 of 31 PageID #:
`11309
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`US 9,441,035 B2
`
`1
`CELL CULTURE MEDIA AND METHODS OF
`ANTIBODY PRODUCTION
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This application claims the priority benefit of U.S. Pro(cid:173)
`visional Patent Application Ser. No. 61/801,247, filed Mar.
`15, 2013, the contents of which are incorporated herein by
`reference in its entirety.
`
`FIELD OF THE INVENTION
`
`The present invention relates to cell culture media for use
`in culturing a mammalian cell comprising a nucleic acid
`encoding bevacizumab, or a fragment thereof, and to meth(cid:173)
`ods of using the media in bevacizumab production as well as
`compositions and kits comprising the bevacizumab, or a
`fragment thereof, produced by the methods provided herein.
`
`BACKGROUND OF THE INVENTION
`
`2
`media compositions that enhance the amount ( e.g., enhance
`the titer) of bevacizumab, or a fragment thereof, produced
`from a mammalian cell in cell culture, as well as composi(cid:173)
`tions comprising bevacizumab, or a fragment thereof, pro-
`5 duced by the methods described herein.
`Accordingly, in one aspect, the invention provides a
`method of producing bevacizumab, or a fragment thereof,
`comprising the step of culturing a mammalian cell compris(cid:173)
`ing a nucleic acid encoding bevacizumab or fragment
`10 thereof in a cell culture medium, wherein the cell culture
`medium comprises two or more components selected from
`the group consisting of copper, insulin, and cystine, and
`wherein the cell produces bevacizumab, or a fragment
`thereof. In a further embodiment, the cell culture medium
`15 comprises copper and insulin. In another further embodi(cid:173)
`ment, the cell culture medium comprises copper and cystine.
`In yet another further embodiment, the cell culture medium
`comprises insulin and cystine. In still another further
`embodiment, the cell culture medium comprises copper,
`20 insulin, and cystine. In any of the embodiments herein, the
`cell culture medium can further comprise a plant-derived
`hydrolysate, an animal-derived hydrolysate or both a plant(cid:173)
`derived hydrolysate and an animal-derived hydrolysate. In
`some of the embodiments herein, the cell culture medium
`25 comprises copper at a concentration selected from the con(cid:173)
`centrations listed in Table 1. In some of the embodiments
`herein, the cell culture medium comprises insulin at a
`concentration selected from the concentrations listed in
`Table 1. In some of the embodiments herein, the cell culture
`medium comprises cystine at a concentration selected from
`the concentrations listed in Table 1. It is understood that any
`combination of amounts of copper, insulin and/or cystine,
`e.g., the amounts provided in Table 1, are intended the same
`as if each and every combination of amounts were specifi-
`35 cally and individually listed. In any of the embodiments
`herein, the cell