Case 1:18-cv-01363-CFC Document 1-19 Filed 09/04/18 Page 1 of 56 PageID #: 627
`Case 1:18-cv-01363-CFC Document1-19
`Filed 09/04/18
`Page 1 of 56 PagelD #: 627
`
`
`
`EXHIBIT S
`EXHIBITS
`
`
`
`

`

`(12) United States Patent
`Gawlitzek et al.
`
`(io) Patent No.:
`(45) Date of Patent:
`
`US 8,512,983 B2
`Aug. 20, 2013
`
`U S008512983B2
`
`(54) PRODUCTION OF PROTEINS IN
`GLUTAMINE-FREE CELL CULTURE MEDIA
`
`(76)
`
`Inventors: Martin Gawlitzek, Redwood City, CA
`(US); Shun Luo, Irvine, CA (US);
`Christina Teresa Petraglia, San Ramon,
`CA (US)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 151 days.
`
`(21) Appl.No.: 12/852,377
`
`(22) Filed:
`
`Aug. 6, 2010
`
`(65)
`
`Prior Publication Data
`US 2011/0091936 Al
`Apr. 21,2011
`
`Related U.S. Application Data
`(60) Provisional application No. 61/232,889, filed on Aug.
`11, 2009.
`
`(2006.01)
`
`(51) Int.Cl.
`C12P 21/06
`(52) U.S. Cl.
`USPC ........................................................ 435/69.1
`(58) Field of Classification Search
`USPC ........................................................ 435/69.1
`See application file for complete search history.
`
`(56)
`
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`
`Primary Examiner — Karen Cochrane Carlson
`(74) Attorney, Agent, or Firm — Connie Wong; Christopher
`De Vry; Arnold & Porter LLP
`
`ABSTRACT
`(57)
`The present invention relates generally to glutamine-free cell
`culture media supplemented with asparagine. The invention
`further concerns the production of recombinant proteins,
`such as antibodies, in asparagine-supplemented glutamine-
`free mammalian cell culture.
`
`25 Claims, 25 Drawing Sheets
`
`

`

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`lished Feb. 28, 2001 (English translation).
`
`* cited by examiner
`
`

`

`U.S. Patent
`
`Aug. 20, 2013
`
`Sheet 1 of 25
`
`US 8,512,983 B2
`
`Gutarric AdcW
`
`GutamcAcic^lO
`
`;o
`
`oc
`
`5
`Cl
`
`Aspartic Acid
`
`Qutarrine
`
`10
`
`Qutanrine
`
`10
`
`FIG. 1
`
`

`

`U.S. Patent
`
`Aug. 20, 2013
`
`Sheet 2 of 25
`
`US 8,512,983 B2
`
`Qutarric Acid=1
`
`Qutarric Acid=10
`
`o C
`
`OQ.
`<!
`
`;g
`
`o C
`
`OQ.
`
`Qutarrine
`
`10
`
`Qutarrine
`
`10
`
`FIG. 2
`
`

`

`U.S. Patent
`
`Aug. 20, 2013
`
`Sheet 3 of 25
`
`US 8,512,983 B2
`
`Qutanric Add=1
`
`GutanicAcid=10
`
`T5
`
`O
`'■e
`03Q.
`3
`
`Aspartic Acid
`
`Qutarrine
`
`10
`
`Qutarrine
`
`10
`
`FIG. 3
`
`

`

`U.S. Patent
`
`Aug. 20, 2013
`
`Sheet 4 of 25
`
`US 8,512,983 B2
`
`1000 | ............
`900 -----------
`800 -----------
`27 700 -----------
`E. 600 ------------
`|
`500 -----------
`i-
`400 -----------
`300 -----------
`200
`100 — [ i f f -
`0 -i—-------
`OmM Asn
`
`Asn
`
`2.5mM
`
`15mM
`15mM
`10mM
`10mM
`7.5mM
`7.5mM
`2.5mM
`Asn
`Asn
`Asn
`Asn
`Asn
`Asn
`Asn
`(Cases formulated with OmM Glutamine, OmM or 5mM Glutamate, 10mM Aspartate)
`
`FIG. 4
`
`

`

`U.S. Patent
`
`Aug. 20, 2013
`
`Sheet 5 of 25
`
`US 8,512,983 B2
`
`Bivariate Fit of Titer Day 11 By Aspartate Glutamine=0
`
`....... Smoothing Spline Fit, lambda=1 Asparagine==2.5
`^ — Smoothing Spline Fit, lambda=1 Asparagine==7.5
`— — Smoothing Spline Fit, lambda=1 Asparagine==10
`— Smoothing Spline Fit, lambda=1 Asparagine==15
`
`Smoothing Spline Fit, lambda=1 Asparagine==2.5
`
`R-Square
`0.573894
`Sum of Squares Error
`73461.63
`Smoothing Spline Fit, lambda=1 Asparagine==7.5
`
`R-Square
`0.65596
`Sum of Squares Error
`11556.31
`Smoothing Spline Fit, lambda=1 Asparagine==10
`
`R-Square
`0.408718
`Sum of Squares Error
`25684.65
`Smoothing Spline Fit, lambda=1 Asparagine==15
`
`R-Square
`Sum of Squares Error
`
`0.220438
`26400.64
`
`FIG. 5
`
`

`

`U.S. Patent
`
`Aug. 20, 2013
`
`Sheet 6 of 25
`
`US 8,512,983 B2
`
`Apomab: Titer
`
`0 OmM Gin, 1mM Glu, 10mM Asn, 10mM Asp
`
`■ 10mM Gin, ImMGIu, 10mMAsn, 10mMAsp
`
`O)
`E
`
`1400
`
`1200
`
`1000
`
`800
`
`600
`
`400
`
`200
`
`0
`
`4
`
`7
`Time (day)
`
`14
`
`FIG. 6A
`
`

`

`U.S. Patent
`
`Aug. 20, 2013
`
`Sheet 7 of 25
`
`US 8,512,983 B2
`
`aVEGF V1.2: Titer
`
`600
`
`EOmMGIn, ImMGIu, 10mMAsn, 10mMAsp
`
`500
`
`■ lOmMGIn, ImMGIu, 10mMAsn, 10mMAsp
`
`400
`
`O)
`E 300
`
`200
`
`Time (day)
`
`FIG. 6B
`
`

`

`U.S. Patent
`
`Aug. 20, 2013
`
`Sheet 8 of 25
`
`US 8,512,983 B2
`
`BR3-Fc: Titer
`
`4
`
`7
`Time (day)
`
`14
`
`FIG. 6C
`
`

`

`U.S. Patent
`
`Aug. 20, 2013
`
`Sheet 9 of 25
`
`US 8,512,983 B2
`
`Apomab Clone: DMEM/F12 Titer
`
`ElOmMGIn, 1mM Glu, lOmMAsn, 10mMAsp
`
`■ 10mMGIn, ImMGIu, lOmMAsn, 10mMAsp
`
`250
`
`200
`
`150
`
`■■= 100
`
`Time (day)
`
`FIG. 7A
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 10 of 25
`
`U S 8,512,983 B2
`
`aVEGF V 1.2: DMBVI F12 Titer
`
`0 OmMGIn, 1mM Glu, 10mMAsn, lOmMAsp
`
`■ 10mMGIn, ImMGIu, 10mMAsn, lOmMAsp
`
`3 150
`O)
`E
`
`4
`
`7
`Time (day)
`
`12
`
`FIG. 7B
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 11 of 25
`
`U S 8,512,983 B2
`
`Apomab: Qp
`
`QOmMGIn, 1mM Glu, lOmMAsn, 10mMAsp
`
`■ 10mMGIn, ImMGIu, 10mMAsn, 10mMAsp
`
`3.0
`
`2.0
`
`1.0
`
`0.0
`
`4
`
`7
`Time (day)
`
`14
`
`FIG. 8A
`
`n■a
`®o
`oo
`
`® O)
`
`EQ.
`
`o
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 12 of 25
`
`U S 8,512,983 B2
`
`aVEGF V1.2: Qp
`
`EDOmM Gin, ImMGIu, 10mMAsn, 10mMAsp
`
`■ 10mMGIn, 1mM Glu, 10mMAsn, 10mMAsp
`
`1.0
`
`0.5
`
`0.0
`
`4
`
`7
`Time (day)
`
`14
`
`FIG. 8B
`
`®o
`00
`
`® O
`
`)
`
`EQ
`
`.
`
`O
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 13 of 25
`
`U S 8,512,983 B2
`
`BR3-Fc: Qp
`
`0 OmM Gin, 1mM Glu, 10mMAsn, 10mMAs
`>P
`■ 10mMGIn, ImMGIu, 10mMAsn, 10mM4
`^sp
`
`4.0
`
`3.5
`
`3.0
`
`n2 2.5
`® o
`*3 2.0
`O)
`£ 1.5
`Q.o
`
`1.0
`
`0.5
`
`0.0
`
`1J1 1
`
`4
`
`7
`Time (day)
`
`14
`
`FIG. 8C
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 14 of 25
`
`U S 8,512,983 B2
`
`Apomab: DM EM F12 Qp
`
`EjOmMGIn, 1mM Glu, lOmMAsn, 10mMAsp
`
`■ lOmMGIn, 1mM Glu, lOmMAsn, 10mMAsp
`
`4.0
`
`3.5
`
`3.0
`
`4
`
`7
`Time (day)
`
`12
`
`FIG. 9A
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 15 of 25
`
`U S 8,512,983 B2
`
`aVEGF V1.2: DM EM F12 Qp
`
`2.0
`
`ED OmM Gin, 1mM Glu, 10mM Asn, 10mM Asp
`
`■ 10mMGIn, 1mM Glu, 10mMAsn, 10mMAsp
`
`Time (day)
`
`FIG. 9B
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 16 of 25
`
`U S 8,512,983 B2
`
`Apomab: Viability
`
`100 T
`
`4
`
`7
`Time (day)
`
`14
`
`FIG. 10A
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 17 of 25
`
`U S 8,512,983 B2
`
`FIG. 10B
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 18 of 25
`
`U S 8,512,983 B2
`
`BR3-Fc: Viability
`
`100 i......................................................................
`
`90
`
`4
`
`7
`Time (day)
`
`14
`
`FIG. 10C
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 19 of 25
`
`U S 8,512,983 B2
`
`Apomab: DMEM F12 Viability
`
`100 T
`
`4
`
`7
`Time (day)
`
`12
`
`FIG. 11A
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 20 of 25
`
`U S 8,512,983 B2
`
`aVEGFVI .2: DM EM F12 Viability
`
`90
`
`4
`
`7
`Time (day)
`
`12
`
`FIG. 1 IB
`
`

`

`U.S. Patent
`
`Aug. 20, 2013
`
`Sheet 21 of 25
`
`US 8,512,983 B2
`
`FIG. 12A
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 22 of 25
`
`U S 8,512,983 B2
`
`aVEGF V1.2: NH4+ Concentration
`
`□ OmM Gin, ImMGIu, 10mMAsn, 10mMAsp
`
`■ 10mM Gin, ImMGIu, 10mMAsn, 10mMAsp
`
`10
`
`8
`
`6
`
`4
`
`2
`
`0
`
`®oco
`
`o+
`
`4
`
`7
`Time (day)
`
`14
`
`FIG. 12B
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 23 of 25
`
`U S 8,512,983 B2
`
`FIG. 12C
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 24 of 25
`
`U S 8,512,983 B2
`
`Apomab: DM EM F12 NH4+Concentration
`
`14 T
`
`4
`
`7
`Time (day)
`
`12
`
`FIG. 13A
`
`

`

`U.S. Patent
`
`Aug. 20,2013
`
`Sheet 25 of 25
`
`U S 8,512,983 B2
`
`FIG. 13B
`
`

`

`1
`PRODUCTION OF PROTEINS IN
`GLUTAMINE-FREE CELL CULTURE MEDIA
`
`RELATED APPLICATIONS
`
`This application claims the benefit under 35 USC § 119 to
`U.S. Provisional Application 61/232,889 filedAug. 11,2009.
`
`FIELD OF THE INVENTION
`
`The present invention relates generally to glutamine-free
`cell culture media. The invention further concerns the pro­
`duction of recombinant proteins, such as antibodies, in
`glutamine-free mammalian cell culture.
`
`BACKGROUND OF THE INVENTION
`
`5
`
`to
`
`15
`
`Mammalian cells have become the dominant system for the
`production of mammalian proteins for clinical applications,
`primarily due to their ability to produce properly folded and 20
`assembled heterologous proteins, and their capacity for post-
`translational modifications. It is conventional to have
`glutamine in cell culture media during recombinant produc­
`tion of heterologous proteins,
`including antibodies.
`L-glutamine is an essential amino acid, which is considered 25
`the primary eneigy and nitrogen sources for cells in culture.
`Most commercially available media are formulated with free
`L-glutamine which is either included in the basal formula or
`added to liquid media formulations at the time of use. Thus,
`all mammalian cell culture media contain glutamine except 30
`those for glutamine synthetase transfected cell lines, such as
`GS NS0 and GS CHO cell lines, where the cells themselves
`produce the glutamine needed for growth. Glutamine is
`widely used at various concentrations typically from 1 to 20
`mM in base media and much higher concentration in feeds for 35
`fed-batch process. For example, the concentration of
`L-glutamine is 0.5 mM in Ames’ Medium and 10 mM in
`MCDP Media 131. DMEM/Ham’s Nutrient Mixture F-12
`(50:50) is often used as a starting formulation for proprietary
`media used with Chinese Hamster Ovary (CHO) cells. 40
`L-glutamine in DMEM/Ham’s Nutrient Mixture F-12 is 2.5
`mM. L-glutamine concentration in Serum-Free/Protein Free
`Hybridoma Medium is 2.7 mM. L-glutamine in DMEM,
`GMEM, IMDM and H-Y medium is 4 mM, of which IMDM
`is often used as a starting formulation for proprietary hybri- 45
`doma cell culture media. It is generally held that hybridoma
`cells grow better in concentrations of L-glutamine that are
`above the average levels found in media. (Dermis R. Comad,
`Glutamine in Cell Culture, Sigma-Aldrich Media Expert)
`It was shown that glutamine is the main source of ammonia 50
`accumulated in cell culture (see review by Markus Schneider,
`et. al. 1996, Journal of Biotechnology 46:161-185). Thus,
`lowering glutamine in cell culture media significantly
`reduced the accumulation of NH4+ level, resulting in lower
`cytotoxicity (see Markus Schneider, et. al. 1996, supra). 55
`Reduced NH4+ cytotoxicity resulted in higher cell viability,
`thus extended culture longevity. Based on an estimated
`glutamine consumption study using CHO cells, it was sug­
`gested that cells may consume glutamine at a rate of 0.3-0.4
`mM per day (Miller, et. al. 1988, Biotechnol. Bioeng. 32: 60
`947-965). Altamirano et al. (2001, J. Biotechnol. 110:171-9)
`studied the effect of glutamine replacement by glutamate and
`the balance between glutamate and glucose metabolism on
`the redistribution of CHO cells producing recombinant
`human tissue plasminogen activator (rhut-PA). When 65
`glutamine was replaced with glutamate and balanced with
`glucose catabolism (carbon and nitrogen ratio, C/N ratio),
`
`US 8,512,983 B2
`
`2
`cell metabolism was found redistributed and forced to utilize
`carbon and energy source more favorably to production of
`rhut-PA. It was also reported that CHO cells in adherent
`cultures can grow in the absence of added glutamine due to
`endogenous glutamine synthetase activity that allowed cells
`to synthesize glutamine from glutamic acid in the medium
`(Sanfeliu and Stephanopoulos, 1999, Biotechnol. Bioeng.
`64:46-53). However, compared to control cultures in
`glutamine-containing media,
`the cell growth rate
`in
`glutamine-free media was slower with an increased fraction
`of cells distributed in the G0/G1 phase. The depletion of both
`glutamine and glutamic acid did cause cell death.
`
`SUMMARY OF THE INVENTION
`
`The present invention is based, at least in part, on the
`unexpected finding that not only can recombinant proteins be
`produced in a mammalian host cell using a glutamine-free
`production medium without any significant adverse effect, in
`fact the use of a glutamine-free medium in the production
`phase significantly increases cell viability, culture longevity,
`specific productivity and/or the final recombinant protein
`titer.
`The present invention is also based on the unexpected
`finding that the addition of asparagine to a glutamine-free
`production medium can further enhance the cell viability,
`culture longevity, specific productivity and/or the final
`recombinant protein titer in a mammalian host cell using a
`glutamine-free production medium without any significant
`adverse effect.
`In one aspect, the invention concerns a process for produc­
`in