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`#: 1035
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`EXHIBIT AA
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`O
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`(12) Unlted States Patent
`Gawlitzek et a].
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 8,512,983 B2
`Aug. 20, 2013
`
`US008512983B2
`
`(54) PRODUCTION OF PROTEINS IN
`GLUTAMINE-FREE CELL CULTURE MEDIA
`
`.
`.
`.
`(76) Inventors. Martln GaWhtzek, Redwood City, CA
`(US); Shun L110, Irvme, 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) App1_ NO; 12/852,377
`
`_
`.
`(22) Flled-
`
`(65)
`
`US
`
`Aug- 6’ 2010
`_
`_
`_
`Prlor Publlcatlon Data
`
`21,
`
`Related U_s_ Application Data
`
`51
`(
`)
`
`(60) Provisional application No. 61/232,889, ?led onAug.
`11 2006
`’
`I t Cl
`n '
`'
`C121’ 21/06
`(52) US, Cl,
`USPC ....................................................... .. 435/691
`_
`_
`_
`(58) Fleld 0f Classl?catlon Search
`USPC ....................................................... .. 435/69.1
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`
`(Continued)
`
`Primary Examiner * Karen Cochrane Carlson
`,
`.
`_
`.
`Attorney, Agent, or Flrm i COnme Wong, ChnStOpher
`De Vry; Arnold & Porter LLP
`
`(57)
`
`ABSTRACT
`
`_
`_
`_
`The present invention relates generally to glutamme-free cell
`culture media supplemented With asparagine. The invention
`further concerns the production of recombinant proteins,
`such as antibodies in as ara ine-su lemented lutamine
`_
`’
`P g
`PP
`g
`free mammalian cell culture.
`
`25 Claims, 25 Drawing Sheets
`
`

`

`US 8,512,983 B2
`Page 2
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`
`* cited by examiner
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 5 of 460 PageID
`#: 1039
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 1 0f 25
`
`US 8,512,983 B2
`
`GutarTiC Acid=1
`
`GLIaITiC Acid=10
`
`743.75 10
`
`Asparagine
`
`80. 75
`
`Gutem're
`
`1O
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 6 of 460 PageID
`
`#: 1040
`
`FIG. 1
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 2 0f 25
`
`US 8,512,983 B2
`
`Gutam'c Acid=1
`
`GUla'T'iC ACid=1O
`
`
`
`Aspartic Acid
`
`743%‘
`
`@987 10
`Asparagine
`
`697.988 0
`
`0
`
`Gutanine
`
`10
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 7 of 460 PageID
`#: 1041
`
`FIG. 2
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 3 0f 25
`
`US 8,512,983 B2
`
`Gutan'ic Acid=1
`
`Gulam'c Acid=10
`
`O
`
`Guta‘rine
`
`1O
`
`0
`
`GlutarTine
`
`10
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 8 of 460 PageID
`#: 1042
`
`F163
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 4 0f 25
`
`US 8,512,983 B2
`
`Apomab: Flnal Tlter
`
`OmM Glutamate
`5mM Gmtamale
`
`
`
`Titer (mg/L)
`
`0') O O
`
`OmM Asn OmM Asn 2.5mM
`Asn
`
`2.5mM
`Asn
`
`7.5mM
`Asn
`
`7.5mM
`Asn
`
`10mM
`Asn
`
`10mM
`Asn
`
`15mM
`Asn
`
`15mM
`Asn
`
`(Cases formulated with OmM Glutamine, OmM or 5mM Glutamate, 10mM Aspartate)
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 9 of 460 PageID
`#: 1043
`
`FIG. 4
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 5 0f 25
`
`US 8,512,983 B2
`
`Bivariate Fit of Titer Day 11 By Aspartate Glutamine=0
`
`1000
`
`900 —
`
`800 -
`
`3 700 -
`
`o 600 —
`
`E i; 500 —
`
`400 -
`
`300 -
`
`200 -
`
`100
`
`-2
`
`|
`0
`
`|
`2
`
`|
`|
`6
`4
`Aspartate
`
`|
`8
`
`|
`10
`
`12
`
`---- -- 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
`Sum of Squares Error
`
`0.573894
`73461.63
`
`Smoothing Spline Fit, lambda=1 Asparagine==7.5
`
`R-Square
`Sum of Squares Error
`
`0.65596
`11556.31
`
`Smoothing Spline Fit, lambda=1 Asparagine==10
`
`R-Square
`Sum of Squares Error
`
`0.408718
`25684.65
`
`Smoothing Spline Fit, lambda=1 Asparagine==15
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 10 of 460 PageID
`#: 1044
`
`R-Square
`Sum of Squares Error
`
`0.220438
`26400.64
`
`FIG. 5
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 6 0f 25
`
`US 8,512,983 B2
`
`Apom ab: Titer
`
`E 0mM Gln, 1mM Glu, 10mM Asn, 10mM Asp
`
`I 10mM Gln, 1mM Glu, 10mMAsn, 10mMAsp
`
`1400
`
`1200 i
`
`1000
`
`2 800
`m
`E
`,2
`|_
`
`600
`
`L
`
`400
`
`200
`
`0 _
`
`4
`
`Time (day)
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 11 of 460 PageID
`#: 1045
`
`FIG. 6A
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 7 0f 25
`
`US 8,512,983 B2
`
`aVEGF V1.2: Titer
`
`600
`
`E1 OmM Gln, 1mM Glu, 10mM Asn, 10mM Asp
`
`500 i
`
`I 10mM Gln, 1mM Glu, 10mM Asn, 10mM Asp
`
`
`
`Titer (mg/L)
`
`400
`
`300
`
`200
`
`100
`
`Time (day)
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 12 of 460 PageID
`#: 1046
`
`FIG. 6B
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 8 0f25
`
`US 8,512,983 B2
`
`BR3-Fc: Titer
`
`El OmM G|n,1mM Glu, 10mM Asn, 10mM Asp
`
`I 10mM Gln, 1mM Glu, 10mM Asn, 10mMAsp
`
`900
`
`800 i
`
`700
`
`600
`
`500
`
`400
`
`300
`
`200
`
`100
`
`
`
`Titer (mg/L)
`
`Time (day)
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 13 of 460 PageID
`#: 1047
`
`FIG. 6C
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 9 0f25
`
`US 8,512,983 B2
`
`Apom ab Clone: DM EMIF12 Titer
`
`250
`
`El OmM Gln, 1mM Glu, 10mM Asn, 10mM Asp
`
`I 10mM Gln, 1mM Glu, 10mMAsn, 10mMAsp
`
`200 i?
`
`
`
`Titer (mg/L)
`
`150
`
`100
`
`50
`
`Time (day)
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 14 of 460 PageID
`#: 1048
`
`FIG. 7A
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 10 0f 25
`
`US 8,512,983 B2
`
`aVEGF V1.2: DM HVI F12 Titer
`
`OmM Gln, 1mM Glu, 10mM Asn, 10mM Asp
`
`I 10mM Gln, 1mM Glu, 10mMAsn, 10mMAsp
`
`250
`
`200 i
`
`
`
`Titer (mg/L)
`
`150
`
`100
`
`50
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 15 of 460 PageID
`#: 1049
`
`FIG. 7B
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 11 0125
`
`US 8,512,983 B2
`
`Apom ab: Qp
`
`E OmM Gln, 1mM Glu, 10mM Asn, 10mM Asp
`
`I 10mM Gln, 1mM Glu, 10mMAsn, 10mMAsp
`
`3.0
`
`2 1
`O. O.
`
`
`
`929:8 M6295 Q0
`
`0.0
`
`Time (day)
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 16 of 460 PageID
`#: 1050
`
`FIG. 8A
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 17 of 460 PageID
`#: 1051
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 13 0f 25
`
`US 8,512,983 B2
`
`4.0
`
`3.5 i»
`
`E1 OmM Gln, 1mM Glu, 10mM Asn, 10mM Asp
`
`I 10mM Gln, 1mM Glu, 10mMAsn, 10mM Asp
`
`5. 0. 5. 2 2 1
`
`
`
`929:8 M6295 Q0
`
`Time (day)
`
`14
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 18 of 460 PageID
`#: 1052
`
`FIG. 8C
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 14 0f 25
`
`US 8,512,983 B2
`
`Apomab: DMEM F12 Qp
`
`4.0
`
`E OmM Gln, 1mM Glu, 10mM Asn, 10mM Asp
`
`3-5 ** I10mMG|n,1mM Glu, 10mMAsn, 10mM Asp
`
`
`
`Qp (mg/198 cell-day)
`
`3.0
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`Time (day)
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 19 of 460 PageID
`#: 1053
`
`FIG. 9A
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 20 of 460 PageID
`#: 1054
`
`

`

`US. Patent
`
`Aug. 20, 2013
`
`Sheet 16 0f 25
`
`US 8,512,983 B2
`
`Apomab: Viability
`
`% Viability
`
`B OmM Gln, 1mM Glu, 10mM Asn, 10mM Asp
`
`I 10mM Gln, 1mM Glu, 10mMAsn, 10mMAsp
`
`4
`
`7
`
`Time (day)
`
`14
`
`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 21 of 460 PageID
`#: 1055
`
`FIG. 10A
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 22 of 460 PageID
`#: 1056
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 23 of 460 PageID
`#: 1057
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 24 of 460 PageID
`#: 1058
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 25 of 460 PageID
`#: 1059
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 26 of 460 PageID
`#: 1060
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 27 of 460 PageID
`#: 1061
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 28 of 460 PageID
`#: 1062
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 29 of 460 PageID
`#: 1063
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 30 of 460 PageID
`#: 1064
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 31 of 460 PageID
`#: 1065
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 32 of 460 PageID
`#: 1066
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 33 of 460 PageID
`#: 1067
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 34 of 460 PageID
`#: 1068
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 35 of 460 PageID
`#: 1069
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 36 of 460 PageID
`#: 1070
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 37 of 460 PageID
`#: 1071
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 38 of 460 PageID
`#: 1072
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 39 of 460 PageID
`#: 1073
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 40 of 460 PageID
`#: 1074
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 41 of 460 PageID
`#: 1075
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 42 of 460 PageID
`#: 1076
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 43 of 460 PageID
`#: 1077
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 44 of 460 PageID
`#: 1078
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 45 of 460 PageID
`#: 1079
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 46 of 460 PageID
`#: 1080
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 47 of 460 PageID
`#: 1081
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 48 of 460 PageID
`#: 1082
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 49 of 460 PageID
`#: 1083
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 50 of 460 PageID
`#: 1084
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 51 of 460 PageID
`#: 1085
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 52 of 460 PageID
`#: 1086
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 53 of 460 PageID
`#: 1087
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 54 of 460 PageID
`#: 1088
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 55 of 460 PageID
`#: 1089
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 56 of 460 PageID
`#: 1090
`
`

`

`Case 1:18-cv-00924-CFC-SRF Document 3-2 Filed 06/21/18 Page 57 of 460 PageID
`#: 1091
`
`
`
`
`
`
`
`EXHIBIT BB
`
`

`

`(12) United States Patent
`Kao et a1.
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 8,574,869 B2
`Nov. 5, 2013
`
`US008574869B2
`
`(75)
`
`(73) Assig