Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 1 of 275 PageID #: 28404
`
`C.A. No. 17-1407-CFC
`(CONSOLIDATED)
`
`
`IN THE UNITED STATES DISTRICT COURT
`FOR THE DISTRICT OF DELAWARE
`
`GENENTECH, INC. and CITY OF HOPE, )
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`Plaintiffs,
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`AMGEN INC.,
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`Defendant.
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`____________________________________)
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`GENENTECH, INC.,
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`Plaintiff and
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`Counterclaim Defendant,
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`v.
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`AMGEN INC.,
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`Defendant and
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`Counterclaim Plaintiff.
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`____________________________________)
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`C.A. No. 18-924-CFC
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`APPENDIX TO GENENTECH’S LETTER-BRIEF CONCERNING
`CONSTRUCTION OF “FOLLOWING FERMENTATION” AND
`SUPPORTING DECLARATION OF DR. HANSJÖRG HAUSER
`
`
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 2 of 275 PageID #: 28405
`
`U.S. Patent No. 8,574,869
`Bruce Alberts et al., MOLECULAR BIOLOGY OF
`THE CELL, Chapter 3 (4th Ed. 2002) (“Molecular Biology of
`the Cell”)
`Mullan et al., Disulphide bond reduction of a
`therapeutic monoclonal antibody during cell culture
`manufacturing operations, BMC Proc., 22(5) Suppl
`8:P110 (2011) (“Mullan 2011”)
`Trexler-Schmidt et al., Identification and Prevention of
`Antibody Disulfide Bond Reduction During Cell Culture
`Manufacturing, Biotechnol Bioeng., 106(3):452-61 (2010)
`(“Trexler-Schmidt 2010”)
`Kao et al., Mechanism of Antibody Reduction in Cell
`Culture Production Processes, Biotechnol Bioeng.,
`107(4):622-32 (2010) (“Kao 2010”)
`Mun et al., Air Sparging for Prevention of Antibody
`Disulfide Bond Reduction in Harvested CHO Cell
`Culture Fluid, Biotechnol Bioeng., 112(4):734-42
`(2015) (“Mun 2015”)
`Hutterer et al., Monoclonal Antibody Disulfide
`Reduction During Manufacturing, MAbs., 5(4):608-13
`(2013) (“Hutterer 2013”)
`Chung et al., Effects of Antibody Disulfide Bond
`Reduction on Purification Process Performance and
`Final Drug Substance Stability, Biotechnol Bioeng.,
`114(6):1264-1274 (2017) (“Chung 2017”)
`Fahrner et al., Industrial Purification of Pharmaceutical
`Antibodies: Development, Operation, and Validation of
`Chromatography Processes, Biotechnology and Genetic
`Engineering Reviews, 18:1, 301-327 (2001) (“Fahrner 2001”)
`Birch and Racher, Antibody production, Advanced Drug
`Delivery Reviews 58(5-6):671-85 (2006)
`Webster’s Dictionary, “Fermentation”
`FDA Biotechnology Inspection Guide (excerpts)
`Persson et al., Mammalian Cell Fermentation, Production of
`Biologicals from Animal Cells in Culture (1991) (“Persson
`1991”)
`
`Appx1
`Appx96
`
`Appx127
`
`Appx130
`
`Appx140
`
`Appx151
`
`Appx160
`
`Appx166
`
`Appx177
`
`Appx205
`
`Appx220
`Appx223
`Appx228
`
`2
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 3 of 275 PageID #: 28406
`
`Bödeker et al., Production of recombinant factor VIII from
`perfusion cultures: I. Large-scale fermentation, Animal
`Cell Technology (1994) (“Bödeker 1994”)
`Ozturk et al., Real-time Monitoring of Protein Secretion in
`Mammalian Cell Fermentation: Measurement of Monoclonal
`Antibodies Using a Computer-Controlled HPLC System
`(BioCad/RPM), Biotechnol Bioeng., 48:201-206 (1995)
`(“Ozturk 1995”)
`Kemp G., O’Neil P., Large-Scale Production of Therapeutic
`Antibodies: Considerations for Optimizing Product Capture
`and Purification, in: Subramanian G. (eds) Antibodies (2004)
`(“Kemp 2004”)
`Dwivedi, Validation of Cell Culture-Based Processes and
`Qualification of Associated Equipment and Facility, in:
`Ozturk, Cell Culture Technology for Pharmaceutical and Cell-
`Based Therapies (“Dwivedi 2006”)
`US 2007/0141687 (Porro)
`Kaufmann et al., Influence of low temperature on productivity,
`proteome and protein phosphorylation of CHO cells,
`Biotechnol Bioeng., 63(5): 573-582 (1999)
`Matijasevic et al., Hypothermia causes a reversible, p53-
`mediated cell cyle arrest in cultured fibroblasts, Oncol Res.,
`10(11-12): 605-610 (1998)
`Roobol et al., ATR (ataxia telangiectasia mutated- and Rad3-
`related kinase) is activated by mild hypothermia in
`mammalian cells and subsequently activates p53, Biochem J.,
`435(2):499-508 (2011)
`Hunt et al., Low-Temperature Pausing of Cultivated
`Mammalian Cells, Biotechnol Bioeng., 89(2):157-63, (2004)
`Yoon et al., Effect of Low Culture Temperature on Specific
`Productivity and Transcription Level of Anti-4-1BB Antibody
`in Recombinant Chinese Hamster Ovary Cells, Biotechnol
`Prog., 19(4):1383-6 (2003)
`Roobol et al., Biochemical insights into the mechanisms
`central to the response of mammalian cells to cold stress and
`subsequent rewarming, FEBS J., 276(1):286-302 (2009)
`
`Appx234
`
`Appx240
`
`Appx246
`
`Appx272
`
`Appx307
`Appx344
`
`Appx354
`
`Appx360
`
`Appx374
`
`Appx381
`
`Appx385
`
`3
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 4 of 275 PageID #: 28407
`
`Supplement to Roobol et al., Biochemical insights into the
`mechanisms central to the response of mammalian cells to
`cold stress and subsequent rewarming, FEBS J., 276(1):286-
`302 (2009)
`McGraw-Hill Dictionary of Scientific and Technical Terms
`(6th ed.), “Fermentation”
`Deposition of Jeffrey John Chalmers (excerpts)
`Amgen 2011 Annual Report and Financial Summary
`(excerpts)
`Deposition of Stuart Watt (excerpts)
`
`Appx402
`
`Appx408
`
`Appx411
`Appx448
`
`Appx450
`
`
`
`
`
`4
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`

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`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 5 of 275 PageID #: 28408
`
`I 1111111111111111 11111 lllll lllll lllll 111111111111111 111111111111111 IIII IIII
`US008574869B2
`
`c12) United States Patent
`Kao et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8,574,869 B2
`Nov. 5, 2013
`
`(75)
`
`(54) PREVENTION OF DISULFIDE BOND
`REDUCTION DURING RECOMBINANT
`PRODUCTION OF POLYPEPTIDES
`Inventors: Yung-Hsiang Kao, San Mateo, CA
`(US); Michael W. Laird, San Ramon,
`CA (US); Melody Trexler Schmidt, San
`Carlos, CA (US); Rita L. Wong,
`Redwood City, CA (US); Daniel P.
`Hewitt, Sunnyvale, 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 O days.
`(21) Appl. No.: 13/354,223
`Jan.19,2012
`(22) Filed:
`Prior Publication Data
`(65)
`
`US 2013/0017598Al
`
`Jan. 17, 2013
`
`Related U.S. Application Data
`
`(63) Continuation of application No. 12/217,745, filed on
`Jul. 8, 2008, now abandoned.
`
`(51)
`
`(60) Provisional application No. 60/948,677, filed on Jul. 9,
`2007.
`Int. Cl.
`C12P 1100
`C12N5/02
`(52) U.S. Cl.
`USPC ............................................. 435/41; 435/325
`( 58) Field of Classification Search
`None
`See application file for complete search history.
`
`(2006.01)
`(2006.01)
`
`(56)
`
`References Cited
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`U.S. PATENT DOCUMENTS
`
`2004/0029229 Al *
`2004/0138424 Al
`2006/0143549 Al
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`2/2004 Reeves et al. ................ 435/69.1
`7/2004 Takeda et al.
`6/2006 Yasumoto et al.
`12/2007 Salcedo et al.
`2/2009 Kao et al.
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`Serum-Free CHO Cell Culture Process and the Level of Free Thiol in
`the Recombinant Antibody Expressed" Biotechnology Progress
`21(2):550-553 (2005).
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`Mun et al., "BIOT 245-Air Sparging of Harvested Cell Culture Fluid
`(HCCF) to Prevent Antibody Disulfide Bond Reduction" Abstracts of
`Papers American Chemical Society 238:245 (Aug. 2009).
`Nordberg et al., "Reactive Oxygen Species, Antioxidants, and the
`Manunalian Thioredoxin System" Free Radical Biology & Medicine
`31(11):1287-1312 (2001).
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`Annual Review of Pharmacology and Toxicology 41:261-295
`(2001).
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`and the Effects of Inhibitors" Chemico-Biologica llnteractions 111-
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`Salas-Solano et al., "Optimization and Validation of a Quantitative
`Capillary Electrophoresis Sodium Dodecyl Sulfate Method for Qual(cid:173)
`ity Control and Stability Monitoring of Monoclonal Antibodies"
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`Smith et al., "Specific Cleavage of Immunoglobulin G by Copper
`Ions" International Journal of Peptide and Protein Research
`48( 1 ):49-55 ( 1996).
`Starks et al., "Atomic-Resolution Crystal Structure of Thioredoxin
`From the Acidophilic Bacterium Acetobacter Aceti" Protein Science
`16(1):92-98 (Jan. 2007).
`Teilum et al., "Disulfide Bond Formation and Folding of Plant
`Peroxidases Expressed as Inclusion Body Protein in Escherichia coli
`Thioredoxin Reductase Negative Strains" Protein Expression and
`Purification Academic Press 15(1):77-82 (1999).
`Trexler-Schmidt et al., "Identification and Prevention of Antibody
`Disulfide Bond Reduction During Cell Culture Manufacturing"
`Biotechnology and Bioengineering 160(3):452-461 (2006).
`Urig et al., "On the Potential ofThioredoxin Reductase Inhibitors for
`Cancer Therapy" Seminars in Cancer Biology 16(6):452-465 (
`2006).
`Wipf et al., "New Inhibitors of the Thioredoxin-Thioredoxin
`Reducatase System Based on a Naphthoquinone Spiroketal Natural
`Product Lead" Bioorganic & Medicinal Chemistry Letters( 11 ):2637-
`2641 (2001).
`Zhang et al., "Free sulfhydryl in recombinant monoclonal antibod(cid:173)
`ies" Biotechnol. Prog. 18:509-513 (2002).
`Lillig, C.H. eta!. (Jan. 2007). "Thioredoxin andRelatedMolecules(cid:173)
`From Biology to Health and Disease," Antioxidants & Redox Signal(cid:173)
`ing 9(1):25-47.
`Lydersen, B.K. et al. (Nov. 1994). "Acid Precipitation ofManunalian
`Cell Fermentation Broth," Ann. NY Acad. Sci. 745:222-231.
`Roman, B. et al. (Sep. 2005). "Development ofa Robust Clarification
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`BioProcess International 2005 Conference&Exhibition, Sep. 19-22,
`2005, Boston, MA, four pages particularly p. 4.
`Roush, D.J. et al. (2008). "Advances in Primary Recovery: Centrifu(cid:173)
`gation and Membrane Technology," Biotechnol. Prag. 24(3):488-
`495.
`
`* cited by examiner
`Primary Examiner - Suzanne M Noakes
`Jae W Lee
`Assistant Examiner -
`(74) Attorney, Agent, or Firm - Morrison & Foerster LLP
`ABSTRACT
`(57)
`Provided herein are methods for preventing the reduction of
`disulfide bonds during the recombinant production of disul(cid:173)
`fide-containing polypeptides. In particular, the invention con(cid:173)
`cerns the prevention of disulfide bond reduction during har(cid:173)
`including
`vesting of disulfide-containing polypeptides,
`antibodies, from recombinant host cell cultures.
`10 Claims, 40 Drawing Sheets
`
`Appx1
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 6 of 275 PageID #: 28409
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 1 of 40
`
`US 8,574,869 B2
`
`Ladder
`
`OHours
`
`1 Hours
`
`3Hours
`
`21 Hours 25Hours 29Hours
`
`..... •11un ., •• ,, • • • ll(cid:127) UIDltl
`
`[kDa]
`
`150 -
`
`95-
`
`63-
`
`46-
`
`28-
`
`15-
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`Dialysis Experiment
`FIG. 1
`
`Appx2
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 7 of 275 PageID #: 28410
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 2 of 40
`
`US 8,574,869 B2
`
`Ladder
`
`OHours
`
`1 Hours
`
`3Hours
`
`21 Hours 25Hours 29Hours 48Hours
`
`[kDa]
`
`150-
`
`95-
`
`63-
`
`46-
`
`28- ,,,,,,,,,,,,,,
`
`15- -
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`Dialysis Experiment
`FIG. 2
`
`Appx3
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 8 of 275 PageID #: 28411
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 3 of 40
`
`US 8,574,869 B2
`
`900.0
`
`800.0
`
`700.0
`
`600.0
`
`500.0
`
`400.0
`
`300.0
`
`200.0
`
`100.0
`
`0.0
`
`0
`
`C
`0
`_,_.
`cu ,_
`C
`Q)
`0
`C
`0
`0
`
`0
`
`(1)
`(l)
`'-
`LL
`
`+ Outside Dialysis Bag
`-0- Inside Dialysis Bag
`
`10
`
`20
`
`30
`
`40
`
`50
`
`Time (hours)
`
`Free Thiol Levels from Dialysis Experiment
`FIG. 3
`
`Appx4
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 9 of 275 PageID #: 28412
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 4 of 40
`
`US 8,574,869 B2
`
`NADPH+H+
`
`/s
`P" I s
`
`Thioredoxin System
`/S
`I
`TrxR
`"-S
`
`/SH
`
`Trx
`"-SH
`
`/s
`I
`Trx
`"s
`
`/SH
`TrxR
`"-SH
`
`First Reaction in Pentose Phosphate Pathway
`
`NADPH
`
`6-Phosphogluconolactone
`
`GI ucose-6-phosphate
`
`(Mg-ADPt +H+
`
`Glucose
`
`(Mg-ADP) 2+
`
`First Reaction in Glycolysis
`
`Thioredoxin System and Other Reactions Involved in Antibody Reduction
`FIG. 4
`
`Appx5
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 10 of 275 PageID #: 28413
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 5 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`Ladder OHours 0.5Hours 1 Hours
`
`2Hours
`
`3Hours
`
`21 Hours 23Hours
`
`240- ---~-----·•·«• ~-·---, .. •----------------------· - - -
`
`7 - -LIii lift!UlillUUNJllll!JUF IJU l!llli lillll!JUll:Uti••111111u;11 !ff 1 L IJJthu ••·-•
`4.5--~~___......,. ................... ,rr~ii',tl,ill'{l·••r•111i1
`L
`2
`4
`3
`5
`6
`7
`
`In Vitro Activity of Thioredoxin System
`FIG. 5
`
`Appx6
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 11 of 275 PageID #: 28414
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 6 of 40
`
`US 8,574,869 B2
`
`Ladder OHours O 5Hours 1 Hours
`
`2Hours
`
`3Hours
`
`21 Hours 23Hours
`
`[kDa]
`
`95- ,,
`
`63-
`
`46
`
`28-
`
`15-
`
`7 - I.JliJUll _,.. I !lll!LC M.111t.llJ,JL111,•,Y••~4••·~'.~;:,,,,, ~-• lit um • 1 t 1 1uJJ• Mime•
`4.s-•••••••••~•••aau(cid:127) 1111111Cil11J1tninnll¢:ti11·11n•
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`In vitro Activity of Thioredoxin System Inhibited by Aurothioglucose
`FIG. 6
`
`Appx7
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 12 of 275 PageID #: 28415
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 7 of 40
`
`US 8,574,869 B2
`
`Ladder
`
`OHours 0.5Hours 1 Hours
`
`2Hours
`
`3Hours
`
`21 Hours 23Hours
`
`[kDa]
`
`150-
`
`95-
`
`63-
`
`46-
`
`28-
`
`15-
`
`11111 111.u1u1111 UUJIIJIIM lJJllil~Xn• ••i•• •1n·11
`
`7 - (cid:127)
`4 '5 - MiillilNJ) l·illllll llti'l'ir• •n-•roiW 1r1111111u11t'illll
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`In vitro Activity of Thioredoxin System Inhibited by Aurothiomalate
`FIG. 7
`
`Appx8
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 13 of 275 PageID #: 28416
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 8 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`150-
`
`95-
`
`46-
`
`28-
`
`15-
`
`7-
`4.5-
`
`Ladder OHours 0.5Hours 1 Hours
`
`2Hours
`
`3Hours
`
`21 Hours 23Hours
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`In vitro Activity of Thioredoxin System
`FIG. 8
`
`Appx9
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 14 of 275 PageID #: 28417
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 9 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`Ladder OHours 0.5Hours 1 Hours
`
`2Hours
`
`3Hours
`
`21 Hours 23Hours
`
`240- - - - - - - - - - - - - - - - · - · ___
`
`llo_l\_l,i. ....... tit«
`
`j~ ... _ ___ , ,_
`
`95-
`
`63-
`
`46- · ·
`
`28- C
`
`15-
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`In vitro Activity of Thioredoxin System Inhibited by CuSO 4
`FIG. 9
`
`Appx10
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 15 of 275 PageID #: 28418
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 10 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`Ladder OHours 0.5Hours 1 Hours
`
`2Hours
`
`19Hours 21 Hours 23Hours
`
`95 - -~
`
`28 - -
`
`1 i : ; - -
`
`,v 7--
`
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`
`-
`
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`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`Ocrelizumab Reduction
`FIG. 10
`
`Appx11
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 16 of 275 PageID #: 28419
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 11 of 40
`
`US 8,574,869 B2
`
`Ladder OHours 0.5Hours 1 Hours
`
`2Hours
`
`19Hours 21 Hours 23Hours
`
`[kDa]
`
`150
`
`95----
`
`46
`
`28
`
`15--,..--
`
`7-
`4.5-
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`Inhibition of Ocrelizumab Reduction In HCCF by Aurothioglucose
`FIG. 11
`
`Appx12
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 17 of 275 PageID #: 28420
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 12 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`95-
`
`63-
`
`46-
`
`28-
`
`15-
`
`7-
`4.5-
`
`Ladder
`
`OHours 0.5Hours 1 Hours
`
`2Hours
`
`19Hours 21 Hours 23Hours
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`Inhibition of Ocrelizumab Reduction In HCCF by Aurothiomalate
`FIG. 12
`
`Appx13
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 18 of 275 PageID #: 28421
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 13 of 40
`
`US 8,574,869 B2
`
`Ladder
`
`OHours
`
`1Hours
`
`2Hours
`
`4Hours
`
`19Hours 21 Hours
`
`[kDa]
`
`150-
`
`95-
`
`63-
`
`46-
`
`28-
`
`15-
`
`7--lll ... I
`4.5- -.....-,-·--
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`Losing Reduction Activity in HCCF
`FIG. 13
`
`Appx14
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 19 of 275 PageID #: 28422
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 14 of 40
`
`US 8,574,869 B2
`
`Ladder
`
`OHours
`
`1 Hours
`
`2Hours
`
`3Hours
`
`4Hours
`
`19Hours 21 Hours 23Hours
`
`[kDa]
`
`150-
`
`95-
`
`63-
`
`46-
`
`28-
`
`15-7--
`
`·r ~@ :;v·,
`
`4.5-
`
`P.1 li.l~HUI lULll,e lltUlU
`•n r1 ii •Fi 1$ 11 lliiUI l81
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`The Lost Reduction Activity in HCCF Restored by Addition of NADPH
`
`FIG. 14
`
`Appx15
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 20 of 275 PageID #: 28423
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 15 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`Ladder OHours
`
`1 Hours
`
`2Hours
`
`3Hours
`
`4Hours
`
`19Hours 21 Hours 23Hours
`
`63- ,,.
`
`46-
`
`..... .
`
`15- · ... ,, .. " · 7--
`
`L
`
`:·.:.,1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`The Lost Reduction Activity in HCCF Restored by Addition of Glucose-6-Phosphate
`FIG. 15
`
`Appx16
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 21 of 275 PageID #: 28424
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 16 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`19Hours 21 Hours
`3Hours
`2Hours
`1 Hours
`0Hours
`Ladder
`240- ________ ........_ _____________ _
`
`150-
`
`63-
`
`46-
`
`28-
`
`7-
`4.5-
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`Ocrelizumab Reduction
`FIG. 16
`
`Appx17
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 22 of 275 PageID #: 28425
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 17 of 40
`
`US 8,574,869 B2
`
`Ladder
`
`OHours
`
`1 Hours
`
`2Hours
`
`3Hours
`
`19Hours 21 Hours
`
`[kDa]
`
`150-
`
`95-
`
`63-
`
`46-
`
`15-
`
`·••~•--
`
`·•11',n'tllillllt'• Ii ·r1•-·
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`EDTA Inhibits Ocrelizumab Reduction
`FIG. 17
`
`Appx18
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 23 of 275 PageID #: 28426
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 18 of 40
`
`US 8,574,869 B2
`
`Ladder OHours
`
`1 Hours
`
`2Hours
`
`3Hours
`
`19Hours 21 Hours
`
`[kDa]
`
`1
`
`-
`
`95-
`
`63-
`
`46-
`
`28-
`
`15-
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`The Lost Reduction Activity in Run 8 HCCF Restored by Addition
`of Glucose-6-Phosphate but No Inhibition of Reduction by EDTA
`
`FIG. 18
`
`Appx19
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 24 of 275 PageID #: 28427
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 19 of 40
`
`US 8,574,869 B2
`
`..::.::::
`ro
`(l)
`CL
`(1j
`0
`..::.::::
`0
`LO
`
`~
`
`mo
`90
`80
`70
`60
`50
`40
`30
`20
`10
`0
`0
`
`-0-20 mM EDTA
`-A-30 mM CuS04
`-o-pH 5,5 (acetic acid)
`-+- No additions
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`HCCF Hold Time (hr)
`
`FIG. 19
`
`Appx20
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 25 of 275 PageID #: 28428
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 20 of 40
`
`US 8,574,869 B2
`
`100
`90
`
`-o-Air Sparged
`-+-Nitrogen sparged
`
`80
`~ 70
`cu
`<D
`60
`0...
`m
`50
`0
`~
`0
`LO
`..,-
`
`40
`30
`
`~ 0
`
`20
`10
`0
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`HCCF Hold Time (hr)
`
`FIG. 20
`
`Appx21
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 26 of 275 PageID #: 28429
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 21 of 40
`
`US 8,574,869 B2
`
`Light Chain
`
`45
`30
`15
`1
`DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPK
`
`90
`75
`60
`46
`LLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQ
`
`105
`91
`HYTTPPTFGQGTKVEIK
`
`FIG. 21
`
`Heavy Chain
`
`45
`30
`15
`1
`EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGL
`
`90
`75
`60
`46
`EWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAED
`
`120
`105
`91
`TAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
`
`FIG. 22
`
`Appx22
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 27 of 275 PageID #: 28430
`
`Typical Batch or Fed-Batch Culture Process
`
`Seed Train
`Multiple Passages in
`Selective Medium
`
`lnoculum Train
`Multiple Passages in
`Non-Selective Medium
`
`Production
`Non-Selective
`Production Medium
`
`dal
`
`D l
`
`T-.T-.iJ-.f!J-. i ~•
`
`G
`
`Temperature
`Seeding density
`Culture duration
`
`d02, pH, Temperature
`Seeding densi
`Culture duration
`
`__...,_
`
`__...,_
`
`Harvest
`__.,..
`
`d02, pH, Temperature
`Parameter shifts & timing
`Osmolality
`Batch feed addition
`Seeding density
`Culture duration
`
`FIG. 23
`
`~
`00
`•
`~
`~
`~
`
`~ = ~
`
`z 0
`
`~
`
`~
`Ul
`N
`
`0 ....
`
`~
`
`rJJ =(cid:173)
`('D a
`0 ....
`
`N
`N
`
`.i;...
`0
`
`d r.,;_
`00
`tit
`-....l
`~
`00
`0--,
`
`\0 = N
`
`Appx23
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 28 of 275 PageID #: 28431
`
`[kDa]
`
`[kDa]
`
`Ladder
`
`t:::0
`
`t:::0:15
`
`t:::0:30
`
`t:::045
`
`t:::1
`
`t:::1 :30
`
`t:::2
`
`t:::3
`
`t:::5
`
`t:::24
`
`240- - - - - - - - - - - - - - - - - - - - - - - - - ----- ••>M•~•~•••-- --,~•-"'•-•-------- 240
`
`~
`00
`•
`~
`~
`~
`
`~ = ~
`
`150 - ·--1 (cid:127) I-•IUIIIH (lllllil 1111111(cid:127) 11111111n11~: illlllJHll 11 fJ!iMllM itlil~~-~
`
`z 0
`
`~
`~Ul
`N
`
`0 ....
`
`~
`
`-150
`
`-95
`
`95-
`
`63-
`
`46-
`
`28 - ---"-~'""'
`
`15-
`
`7-
`4.5-
`
`L
`
`2
`
`(cid:127)-3
`
`····•· , ••••• fl"•"'• ll11t•n~llf.h 11c1n1w111 r · 1111• - 63
`-46
`
`i>M<•¾f41a!4>#~,'f,\¾~~~J¥;;--rt:tM1t-lti~
`
`-28
`
`-15
`
`-7
`
`¾~}i8'\:lil~/i1N4•~¼V,,il
`~~· - 4. 5
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`FIG. 24
`
`('D
`
`N
`~
`
`rJJ =(cid:173)
`('D ....
`0 ....
`
`.i;...
`0
`
`d r.,;,
`00
`tit
`-....l
`~
`00
`0--,
`
`\0 = N
`
`Appx24
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 29 of 275 PageID #: 28432
`
`[kDa]
`
`[kDa]
`
`Ladder
`
`l=0
`
`15
`
`t=0:30
`
`t=045
`
`!=1:30
`
`t=2
`
`t=3
`
`t=5
`
`t=24
`
`2 40 - WwNff-w.,w--•M •••••••m•••·••••••••••••·•••
`
`,.,. •••••·•·••-'•' ••••=••.cw='-•M• =•••M••••••••w•••• ww,ww,ww,w,w-•••• Mw,wMo<wwww•,•-•ww •••••••~•-"-·•W-• -•,w=<.wwwwwww0· •••••=•-••••=•• Wo$o=w••••--•- - 2 40
`
`150 _ o
`
`Hr.· ii Tlll'l(cid:127)
`
`l•:rurm ,: . • 1: TIJl[III II II r 11111111111111•trnllllllll!IIUIHl . • lllll]l _150
`
`~
`00
`•
`~
`~
`~
`
`~ = ~
`
`z 0
`
`~
`~Ul
`N
`
`0 ....
`
`(,H
`
`95- ,,,w,,,.,.,...., ......... .
`
`63-
`
`46 - *'''"¾••····--"'
`
`28 - ·-·"--
`
`15- •-••w--
`
`7-
`4.5-
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`FIG. 25
`
`-95
`
`-63
`
`-46
`
`-28
`
`-15
`
`-----4.5
`
`-7
`
`9
`
`10
`
`('D
`
`N
`.i;...
`
`rJJ =(cid:173)
`('D ....
`0 ....
`
`.i;...
`0
`
`d
`rJl.
`00
`tit
`-....l
`~
`00
`0--,
`
`\0 = N
`
`Appx25
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 30 of 275 PageID #: 28433
`
`Ladder
`
`t=0
`
`5
`
`t=0:30
`
`t=0:45
`
`t= 1
`
`t=1 :30
`
`!=2
`
`!=5
`
`1=24
`
`2 4 0 - -••-M-•=•= -sc~m,,mm,,,m,,,,,, _,,,-,,,,mw~,.w,,, _,,,-,,_,_M,,,~
`
`_" __ ,_,,,-,_ '"''"'''"''''"""·'' - 2 40
`
`~
`00
`•
`~
`~
`~
`
`~ = ~
`
`150-
`
`95-
`
`63-
`
`46-
`
`28
`
`15-
`
`7-
`
`•·-11r11 •-·r·1_ -]nr ·dll1rt11(cid:127) 11m••• m111mu11_1:1ur1urrn 1111•11111111;1111rm111llll!I(cid:127) · -150
`
`-95
`
`-63
`
`-46
`
`-28
`
`15
`
`-7
`-4.5
`
`L
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`FIG. 26
`
`z 0
`
`~
`~Ul
`N
`
`0 ....
`
`~
`
`('D
`('D
`
`rJ'1 =(cid:173)
`.....
`N
`Ul
`
`0 ....
`
`.i;...
`0
`
`d r.,;_
`00
`tit
`-....l
`~
`00
`0--,
`
`\0 = N
`
`Appx26
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 31 of 275 PageID #: 28434
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 26 of 40
`
`US 8,574,869 B2
`
`cu
`~.
`0
`
`0
`~.;
`N
`I
`
`I
`
`LD a,
`I
`
`C'0
`(0
`1
`
`(0
`-tj-
`I
`
`co
`N
`I
`
`LD
`
`~
`
`I
`
`"'
`
`! I • I
`I
`I
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`
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`
`½'
`~.1.•.· ·.·.
`;
`\,
`/
`
`..
`
`· .. ·1 · . : . ·.·
`
`.1;
`
`,t .. I .
`
`~
`N
`lL
`
`lD
`lL
`
`(Y)
`
`lL
`
`N
`lL
`
`C)
`(Y)
`
`~
`
`lL
`
`lL
`
`LO
`'ST
`C)
`lL
`
`0
`(Y)
`ci
`lL
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`~
`
`LD
`ci
`lL
`
`C)
`
`lL
`
`CT)
`u
`-a
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`0
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`I
`0
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`
`I
`0
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`
`~
`
`I
`LD
`CT)
`
`I
`co
`N
`
`I
`LO
`
`I
`I"-
`
`I
`LD
`-tj-
`
`Appx27
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 32 of 275 PageID #: 28435
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 27 of 40
`
`US 8,574,869 B2
`
`L.f)
`~
`
`co
`N
`I
`
`I I
`
`0
`-tj-
`N
`I
`
`0
`L.f)
`~
`
`I
`
`(0
`(D
`I
`
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`-tj-
`I
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`
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`
`D
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`
`I
`D
`'st
`N
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`I
`0
`l{")
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`
`I
`LO
`0)
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`I
`(0
`(0
`
`I
`(D
`-tj-
`
`I
`co
`N
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`I
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`
`~
`
`(cid:127) _j
`
`'
`" "
`
`I
`LO
`-tj-
`
`I
`I"'-
`
`Appx28
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 33 of 275 PageID #: 28436
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 28 of 40
`
`US 8,574,869 B2
`
`LO m
`I
`
`co
`N
`I
`
`LO
`
`~
`
`I
`
`c::,
`LO
`
`~
`
`.
`
`I I
`I .
`. I 1
`I~-.
`I . .
`I
`I
`
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`lL
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`N
`lL
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`LO
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`c::,
`lL
`
`c::,
`11_.
`
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`
`lL
`
`I
`c::,
`'St"
`N
`
`I
`0
`LO
`
`I
`LO
`O'l
`
`I
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`
`I
`11.D
`"11"
`
`I
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`N
`
`I
`lD
`~
`
`Appx29
`
`

`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 34 of 275 PageID #: 28437
`
`[kDa]
`
`2 40 -
`
`Ladder
`
`t=0
`
`t=0:15
`
`t=0:45
`
`1=1
`
`t=1:30
`
`t=2
`
`!=3
`
`t=S
`
`!=24
`
`"'-"''''"''°'"-'"' ,,,,,~,,,~,,,,,,,, __ ,,,,,"'''''''''"'
`
`,,,,,,,.,,,.,,,,,,, ,,,,.,,,-,,,,,,,,, .,,,,,,,_,,,,,,,,,,,
`
`-
`
`,,,,, __ ,,,,,,, ,,,,,,m, '''''''''''' ,,,,,,,,,,,,,,,,,,m
`
`[kDa]
`
`_ M,w.M,,h,,,_ 1111ar:mrn11111r1mmn:1w 11::··,••r• rm LILI •,,. JL r •.,•'IIUlL!liJI!i,..ll.llllUI!lllll'''',.••',.IE _150
`
`150
`
`95-
`
`63-
`
`46-
`
`28 - ""''*'''"'"'''"'""'
`
`15-
`
`7-
`
`4.5-
`
`illll II.
`
`li!!al
`
`L
`
`4
`
`FIG. 30
`
`-95
`
`-63
`
`-46
`
`-28
`
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`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 35 of 275 PageID #: 28438
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`U.S. Patent
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`Nov. 5, 2013
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`Sheet 30 of 40
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`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 36 of 275 PageID #: 28439
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`U.S. Patent
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`Nov. 5, 2013
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`Sheet 31 of 40
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`US 8,574,869 B2
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`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 37 of 275 PageID #: 28440
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`U.S. Patent
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`Nov. 5, 2013
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`Sheet 32 of 40
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`US 8,574,869 B2
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`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 38 of 275 PageID #: 28441
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`U.S. Patent
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`Nov. 5, 2013
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`Sheet 33 of 40
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`US 8,574,869 B2
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`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 39 of 275 PageID #: 28442
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`U.S. Patent
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`Nov. 5, 2013
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`Sheet 34 of 40
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`US 8,574,869 B2
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`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 40 of 275 PageID #: 28443
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`U.S. Patent
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`Nov. 5, 2013
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`Sheet 35 of 40
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`US 8,574,869 B2
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`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 41 of 275 PageID #: 28444
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`U.S. Patent
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`Nov. 5, 2013
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`Sheet 36 of 40
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`US 8,574,869 B2
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`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 42 of 275 PageID #: 28445
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 37 of 40
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`US 8,574,869 B2
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`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 43 of 275 PageID #: 28446
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`U.S. Patent
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`Nov. 5, 2013
`
`Sheet 38 of 40
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`US 8,574,869 B2
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`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 44 of 275 PageID #: 28447
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 39 of 40
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`US 8,574,869 B2
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`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 45 of 275 PageID #: 28448
`
`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 40 of 40
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`US 8,574,869 B2
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`

`Case 1:18-cv-00924-CFC Document 376 Filed 09/27/19 Page 46 of 275 PageID #: 28449
`
`US 8,574,869 B2
`
`1
`PREVENTION OF DISULFIDE BOND
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