Case 1:18-cv-01363-CFC Document 1-20 Filed 09/04/18 Page 1 of 96 PageID #: 683
`Case 1:18-cv-01363-CFC Document1-20
`Filed 09/04/18
`Page 1 of 96 PagelD #: 683
`
`
`
`EXHIBIT T
`EXHIBIT T
`
`
`
`

`

`(12) United States Patent
`Kao et al.
`
`cio) Patent n o .:
`(45) Date of Patent:
`
`u s 8,574,869 B2
`Nov. 5,2013
`
`US008574869B2
`
`(75)
`
`( * ) Notice:
`
`(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)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`(21) Appl.No.: 13/354,223
`Jan. 19, 2012
`(22) Filed:
`Prior Publication Data
`(65)
`US 2013/0017598 Al
`Jan. 17, 2013
`Related U.S. Application Data
`(63) Continuation of application No. 12/217,745, filed on
`Jul. 8, 2008, now abandoned.
`(60) Provisional application No. 60/948,677, filed on Jul. 9,
`2007.
`(51) Int.Cl.
`C12P1/00
`C12NS/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
`U.S. PATENT DOCUMENTS
`
`2004/0029229 Al *
`2004/0138424 Al
`2006/0143549 Al
`2007/0292411 Al
`2009/0053786 Al
`
`2/2004 Reeves etal. ...
`7/2004 Takedaetal.
`6/2006 Yasumoto et al.
`12/2007 Salcedo etal.
`2/2009 Kao etal.
`
`FOREIGN PATENT DOCUMENTS
`
`WO
`
`7/1997
`97/26357
`OTHER PUBLICATIONS
`
`435/69.1
`
`Kock et al. (Biochem Soc Trans. Apr. 2004;32(Pt 2):273-5).*
`Christiansen et al. (Biochemistry 1998, 37, 12611-12623).*
`Bobovnikova et al., “Characterization of Soluble, Disulfide Bond-
`Stabilized Prokaryotically Expressed Human Thryotropin Receptor
`Ectodomain” Endocrinology 138(2):588-593 (1997).
`Chaderjian et al., “Effect of Copper Sulfate on Performance of a
`Serum-Free CHO Cell Culture Process and the Level of Free Thiol in
`the Recombinant Antibody Expressed” Biotechnology Progress
`21(2):550-553 (2005).
`Gromer et al., “The Thioredoxin System: From Science to Clinic”
`Medicubak Research Reviews 24(l):40-89 (Jan. 2004).
`Kao et al., “Mechanism of Antibody Reduction in Cell Culture Pro­
`duction Processes” Biotechnology and Bioengineering 107(4):622-
`632 (Nov. 2010).
`
`Kerblat et al., “Importance of Thioredoxin in the Proteolysis of an
`Immunoglobulin G as Antigen by Lysosomal Cys-Proteases” Immu­
`nology 97(l):62-68 (1999).
`Li et al., “Low Level Formation of Potent Catalytic IgG Fragments
`Mediated by Disulfide Bond Instability” Molecular Immunology
`33(7-8):593-600 (1996).
`Liu et al., “Study of Tioredoxin” Journal of Northeast Agricultural
`University 34(23):219-225 (Jun. 30, 2003).
`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
`Mammalian Thioredoxin System” Free Radical Biology & Medicine
`31(11):1287-1312 (2001).
`Powis et al., “Properties and Biological Activities of Thioredoxins”
`Annual Review of Pharmacology and Toxicology 41:261-295
`(2001).
`Powis et al., “Thioredoxin Redox Control of Cell Growth and Death
`and the Effects of Inhibitors” Chemico-Biologica [Interactions 111-
`112:23-34(1998).
`Salas-Solano et al., “Optimization and Validation of a Quantitative
`Capillary Electrophoresis Sodium Dodecyl Sulfate Method for Qual­
`ity Control and Stability Monitoring of Monoclonal Antibodies”
`Analytical Chemistry 78(18):6583-6594 (2006).
`Smith et al., “Specific Cleavage of Immunoglobulin G by Copper
`Ions” International Journal of Peptide and Protein Research
`48(l):49-55 (1996).
`Starks et al., “Atomic-Resolution Crystal Structure of Thioredoxin
`From the Acidophilic Bacterium Acetobacter Aceti” Protein Science
`16(l):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(l):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 of Thioredoxin 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
`ies” Biotechnol. Prog. 18:509-513 (2002).
`Lillig, C.H. etal. (Jan. 2007). “Thioredoxin and Related Molecules—
`From Biology to Health and Disease,’"Antioxidants & Redox Signal­
`ing 9(t):25-47.
`Lydersen, B.K. et al. (Nov. 1994). “Acid Precipitation of Mammalian
`Cell Fermentation Broth,” Ann. N.Y. Acad. Sci. 745:222-231.
`Roman, B. et al. (Sep. 2005). “Development of a Robust Clarification
`Process for MAb Purification,” Case Study presented at the
`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
`gation and Membrane Technology,” Biotechnol. Prog. 24(3):488-
`495.
`
`* cited by examiner
`Primary Examiner — Suzanne M Noakes
`Assistant Examiner — Jae W Lee
`(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
`fide-containing polypeptides. In particular, the invention con­
`cerns the prevention of disulfide bond reduction during har­
`vesting of disulfide-containing polypeptides,
`including
`antibodies, from recombinant host cell cultures.
`10 Claims, 40 Drawing Sheets
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 1 of 40
`
`US 8,574,869 B2
`
`Ladder OHours
`
`1 Hours
`
`3Hours
`
`21 Hours 25Hours 29Hours
`
`[kDa]
`
`240-
`
`150-
`
`95­
`
`63­
`
`4 6 -
`
`Dialysis Experiment
`FIG. 1
`
`

`

`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
`
`[kDaj
`
`95­
`
`63­
`
`4 6 -
`
`Dialysis Experiment
`FIC i ?
`X X VJ • jLat
`
`

`

`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 3 of 40
`
`US 8,574,869 B2
`
`Free Thiol Levels from Dialysis Experiment
`FIG. 3
`
`(jiM)
`
`Free Thiol Concentration
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 4 of 40
`
`US 8,574,869 B2
`
`Thioredoxin System
`
`Trx
`
`Trx'
`
`NADPH+H1
`
`NADP
`
`First Reaction in Pentose Phosphate Pathway
`
`NADPH
`
`/
`
`\ 6-Phosphogluconolactone
`
`G lucose-6-phosphate dehydrogenase
`
`NADP~
`
`Glucose-6-phosphate
`
`(Mg-ADP)+ +H+
`
`Hexokinase
`
`Glucose
`
`(Mg-ADP)2+
`
`First Reaction in Glycolysis
`
`Thioredoxin System and Other Reactions Involved in Antibody Reduction
`FIG. 4
`
`

`

`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
`
`1 5 0 -
`
`-
`
`4 , m '*<
`
`r!«,'
`
`95-»
`
`6 3 - ..............
`
`46 - ' ■ ■•■■■......
`
`28­
`
`1
`
`5
`
`'
`
`L
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`In Vitro Activity of Thioredoxin System
`FIG. 5
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 6 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`Ladder
`
`OHours
`
`0,5Hours 1 Hours
`
`2Hours
`
`3Hours
`
`21 Hours 23Hours
`
`*7_—
`/
`
`.. ....
`
`-
`1., f:-\4'‘4r
`
`L
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`In vitro Activity of Thioredoxin System Inhibited by Aurothioglucose
`FIG. 6
`
`

`

`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 7 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`Ladder OHours 0.5Hours 1 Hours
`
`2Hours
`
`3Hours 21 Hours 23Hours
`
`ST’*' "* •* ► f
`
`~7
`I
`4.5~
`
`ssa&tesSa ssaassaa
`
`mssssxss wmmmmm ssssssgss aasss
`
`,
`
`,
`
`L
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`In vitro Activity of Thioredoxin System Inhibited by Aurothiomalate
`FIG. 7
`
`

`

`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 8 of 40
`
`US 8,574,869 B2
`
`Ladder OHours 0,5Hours 1 Hours
`
`2Hours
`
`3Hours 21 Hours 23Hours
`
`[kDa]
`
`240-
`
`L
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`In vitro Activity of Thioredoxin System
`FIG. 8
`
`

`

`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
`
`150- '
`
`..
`
`
`
`
`
`t.irnrmiWHRMl
`
`9 5 - ...-
`
`..
`
`33-
`
`
`
`-
`
`4 6 - .............
`
`28-
`
`15-
`
`.
`
`* * * * *
`
`« * « ? «
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`In wfro Activity of Thioredoxin System Inhibited by CUSO4
`FIG. 9
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 10 of 40
`
`US 8,574,869 B2
`
`Ladder OHours O.SHours 1 Hours
`
`2Hours
`
`19Hours 21 Hours 23Hours
`
`[kDa]
`
`240-
`
`150”
`
`9 5 - —
`
`6 3 - ............. -
`
`4 6 —
`
`....
`
`2 8 - —
`
`Ocrelizumab Reduction
`FIG. 10
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 11 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`2 4 0 -
`
`150 —
`
`95-
`
`63—
`
`46—
`
`28-
`
`15-
`7-
`4.5 —
`
`Ladder
`
`OHours
`
`0.5Hours 1 Hours
`
`2Hours
`
`19Hours 21 Hours 23Hours
`
`p & H twn^rfi m sm sawgg *
`
`M R
`
`L
`
`1
`
`3
`
`inhibition of Ocreiizumab Reduction In HCCF by Aurothioglucose
`FIG. 11
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 12 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`Ladder OHours 0,5Hours 1 Hours
`
`2Hours
`
`19Hours 21 Hours 23Hours
`
`2 4 0 - ........
`
`
`
`
`
`'
`
`...............................
`
`- j g g _ __.........
`
`
`
`;
`
`•
`
`’
`
`'
`
`'
`
`9 5 - ...... ■ ~~
`
`6 3 -
`
`4 6 - ■■■■....-.....
`
`28- ...... -....."
`
`
`
`
`
`
`
`■ ■■
`
`Inhibition of Ocrelizumab Reduction In HCCF by Aurothiomalate
`FIG. 12
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 13 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`240-
`
`150-
`
`95-
`
`63­
`
`46­
`
`28­
`
`15­
`
`4 .5 -
`
`Ladder OHours
`
`1 Hours
`
`2Hours
`
`4Hours
`
`19Hours 21 Hours
`
`.......-
`
`nM. K> » >
`
`t a M M N W M n l l '
`
`• 4K&
`
`i**> *
`
`Losing Reduction Activity in HCCF
`
`F I G . 1 3
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 14 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`240­
`
`150­
`
`9 5 ­
`
`6 3 ­
`
`4 6 ­
`
`2 8 ­
`
`15­
`
`7 _
`/
`4 .5 -
`
`Ladder
`
`OHours
`
`1 Hours
`
`2Hours
`
`3Hours
`
`4Hours
`
`19Hours 21 Hours
`
`23Hours
`
`. ...
`
`✓
`
`Vf*--' e' 9
`
`- -
`
`The Lost Reduction Activity in HCCF Restored by Addition of NADPH
`FIG. 14
`
`

`

`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
`
`The Lost Reduction Activity in HCCF Restored by Addition of Glucose-6-Phosphate
`FIG. 15
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 16 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`240-
`
`150-
`
`95­
`
`63­
`
`46­
`
`28­
`
`15­
`
`7­
`4.5-
`
`Ladder OHours
`
`1 Hours
`
`2Hours
`
`3Hours
`
`19Hours 21 Hours
`
`...............r ' r i s n & m - " " " "
`
`Ocrelizumab Reduction
`FIG. 16
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 17 of 40
`
`US 8,574,869 B2
`
`Ladder OHours
`
`1 Hours
`
`2Hours
`
`3Hours
`
`19Hours 21 Hours
`
`[kDaj
`
`240-
`
`150 —
`
`95 —
`
`63 —
`
`46­
`
`2 8 -
`
`15-
`
`EDTA Inhibits Ocrelizumab Reduction
`FIG. 17
`
`

`

`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]
`
`240-
`
`150-
`
`95­
`
`63­
`
`46­
`
`28-
`
`15-
`
`The Lost Reduction Activity in Run 8 HCCF Restored by Addition
`of Glucose-6-Phosphate but No Inhibition of Reduction by EDTA
`FIG. 18
`
`

`

`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 19 of 40
`
`US 8,574,869 B2
`
`150kDa Peak
`
`%
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 20 of 40
`
`US 8,574,869 B2
`
`FIG. 20
`
`150kDa Peak
`
`%
`
`

`

`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 21 of 40
`
`US 8,574,869 B2
`
`Light Chain
`
`45
`30
`15
`1
`D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q D V N T A V A W Y Q Q K P G K A P K
`46
`60
`75
`90
`L L I Y S A S F L Y S G V P S R F S G S R S G T D F T L T I S S L Q P E D F A T Y Y C Q Q
`91
`105
`H Y T T P P T F G Q G T K V E I K
`
`FIG. 21
`
`Heavy Chain
`
`45
`30
`15
`1
`EVQLVE S G G G L V Q P G G S L R L S C A A S G F N I K D T Y I H W V R Q A P G K G L
`46
`60
`75
`90
`E W V A R I Y P T N G Y T R Y A D S V K G R F T I S A D T S K N T A Y L Q M N S L R A E D
`91
`105
`120
`TAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
`
`FIG. 22
`
`

`

`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 22 of 40
`
`US 8,574,869 B2
`
`FIG. 23
`
`>•, CZ
`rtz o03
`CZ 03
`03
`X3 13
`"O
`03CZ
`CD
`X3 Z3
`03 —
`CD Z3
`00 o
`
`3>s X3
`T3
`CO CO
`o TO
`E CD
`J3
`If)
`O SZ
`3
`co
`
`o0
`
`03
`03 cz
`33 E
`"co
`CD ©a
`CL in
`E
`,CD _CZ
`1— CO
`JZ 03
`C l aS
`CXI Eo CO
`X3
`00
`CL
`
`CD
`
`CO
`m
`2 . = CO
`E- <D >-
`E - a =5
`.CD
`"O
`? C D
` "O Z3
`I
`CL CD —
`_ CD 3
`O
`
`O~
`
`D
`
`czo
`CO
`
`“O
`CD
`
`>%
`CD
`cn
`Z3 cz
`CD
`CO “O
`03 CD
`Q. cz
`E T3
`,<D CD
`h -
`03
`00 o
`
`<=a
`
`v
`
`J
`
`CD 13
`_> X3
`cz
`CD
`O O 2CD
`O
`cz
`Z3 CD o
`T3 CO
`O sz
`o
`CL a
`zz. TJ
`L
`
`OC
`
`EZ
`
`3
`
`CD
`_>
`a
`jD
`CD
`
`£Z
`CZ CO
`CD
`CO CD
`H - CO
`CD
`CO
`E
`CO
`Z3 CL
`Z3
`O CD
`o Q_
`cz
`—
`
`tn s—
`CD
`cd - a
`CO CD
`CD ^
`CD COo_ CD
`>
`_03
`Q_ O_CD
`CD
`CO
`
`co
`
`T3
`<D
`aj
`m
`
`Typical Batch or Fed-Batch Culture Process
`
`

`

`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 23 of 40
`
`US 8,574,869 B2
`
`FIG. 24
`
`t=24
`
`t=5
`
`t=3
`
`t=2
`
`t=1:30
`
`t=1
`
`t=0;45
`
`t=0:30
`
`t=0:15
`
`t=0
`
`Ladder
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 24 of 40
`
`US 8,574,869 B2
`
`FIG. 25
`
`t=24
`
`t=5
`
`t=3
`
`t=2
`
`{=1:30
`
`t=1
`
`t=0:45
`
`t=0:30
`
`t=0:15
`
`M)
`
`Ladder
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 25 of 40
`
`US 8,574,869 B2
`
`vj
`
`v_l*
`
`FIG JL
`
`JL
`
`t=24
`
`t=5
`
`t=3
`
`t=2
`
`t=1:30
`
`t=1
`
`t=0:45
`
`t=0:30
`
`t=0;15
`
`t=0
`
`Ladder
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 26 of 40
`
`US 8,574,869 B2
`
`FIG. 27
`
`t=24
`
`t=5
`
`t=3
`
`t=2
`
`{=1:30
`
`t=1
`
`t=0:45
`
`t=0;30
`
`t=0:15
`
`t=0
`
`Ladder
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 27 of 40
`
`US 8,574,869 B2
`
`FIG. 28
`
`t=24
`
`t=5
`
`t=3
`
`t=2
`
`{=1:30
`
`t=1
`
`t=0:45
`
`t=0:30
`
`{=0:15
`
`t=0
`
`Ladder
`
`

`

`Case 1:18-cv-01363-CFC Document 1-20
`712
`
`Filed 09/04/18
`
`Page 30 of 96 PagelD #:
`
`U.S. Patent
`U.S. Patent
`
`Nov. 5, 2013
`Nov.5, 2013
`
`Sheet 28 of 40
`Sheet 28 of 40
`
`US 8,574,869 B2
`US 8,574,869 B2
`
` 8t~
`
`FIG. 29
`
`S6-
`
`eg
`
`97—-
`
`
`
`[eqy]lequ)
`
`
`
`ver}Gtc=oI0€-L=}=Sy-0=)0E-0=}GL'0=IO=Jeppe)
`
`
`
`PeomehesnitiesSeas7wweons84ORS.RSISESCCDNOSPSAODUIROIRE
`
`
`
`
`
`BOURSESSuesbmermatconbin‘tennsorssassOP088AESREESEORL.
`
`
`
`LANIER,“SSMUMtenoLaiis.Subeustapessomenaesefesthe
`
`Myusieascaensmesnantn
`
`
`
`—0S)
`
`
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 29 of 40
`
`US 8,574,869 B2
`
`FIG. 30
`
`t=24
`
`t=5
`
`t=3
`
`t=2
`
`t=1:30
`
`i=1
`
`t=0:45
`
`t=0:30
`
`t-0:15
`
`t=0
`
`Ladder
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 30 of 40
`
`US 8,574,869 B2
`
`CO
`Q
`
`o
`
`oo
`CO
`
`CD
`
`LOCO
`I
`
`FIG. 31
`
`Ii
`
`h -
`
`I
`LO
`
`t=24
`
`t=5
`
`t=3
`
`t=2
`
`t=1:30
`
`t=1
`
`{=0:45
`
`1=0:30
`
`t=0:15
`
`t=0
`
`Ladder
`
`co
`Q
`
`!
`LO
`CD
`
`c o
`CD
`
`co
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 31 of 40
`
`US 8,574,869 B2
`
`FIG. 32
`
`1=24
`
`1=5
`
`t=3
`
`1=2
`
`t=1:30
`
`t=1
`
`1=0:45
`
`t=0:30
`
`i=0:15
`
`t=G
`
`adder
`
`

`

`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 32 of 40
`
`US 8,574,869 B2
`
`FIG. 33
`
`t=24
`
`t=5
`
`t=3
`
`t=2
`
`t=1:30
`
`t=1
`
`t=0:45
`
`t=0;30
`
`t=0:15
`
`t=0
`
`Ladder
`
`

`

`U.S. Patent
`
`Nov. 5, 2013
`
`Sheet 33 of 40
`
`US 8,574,869 B2
`
`CO
`cn>
`
`coco
`I
`
`CO
`
`CO
`CM
`
`CO
`i
`
`i
`
`OL
`
`O
`
`CDO
`
`CO
`CM
`
`jl >"~
`ii
`
`M "
`CO
`d
`
`CMi!
`
`CO
`IE
`
`COif
`
`CM
`IE
`
`OC
`
`O
`
`LO
`
`CO
`IE
`
`O
`
`oC
`
`oI
`
`E
`
`COI!
`
`0>-OT3 03
`
`CD
`Q
`
`Si
`
`co
`LO
`
`CO
`CO
`
`COco
`
`CO
`
`CO
`CM
`
`I
`LO
`
`in
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 34 of 40
`
`US 8,574,869 B2
`
`FIG. 35
`
`{=24
`
`t=5
`
`t=3
`
`t=2
`
`t=1:30
`
`t=1
`
`t=0:45
`
`t=0:30
`
`t=0:15
`
`t=0
`
`Ladder
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 35 of 40
`
`US 8,574,869 B2
`
`FIG. 36
`
`{=24
`
`t=5
`
`t=3
`
`t=2
`
`t=1:30
`
`t=1
`
`t=0:45
`
`t=0:30
`
`t=0:15
`
`t=0
`
`Ladder
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 36 of 40
`
`US 8,574,869 B2
`
`t=24
`
`t=5
`
`t=3
`
`t=2
`
`t=1:30
`
`t=1
`
`t=0:45
`
`t=0:30
`
`t=0:15
`
`t=0
`
`Ladder
`
`

`

`Case 1:18-cv-01363-CFC Document 1-20
`721
`
`Filed 09/04/18
`
`Page 39 of 96 PagelD #:
`
`U.S. Patent
`U.S. Patent
`
`Nov. 5,2013
`Nov.5, 2013
`
`Sheet 37 of 40
`Sheet 37 of 40
`
`US 8,574,869 B2
`US 8,574,869 B2
`
`[eqy)[eqy]
`
`
`
`
`Qe—vavusnanwsneonaticeamOF
`
`pZ=]G=}Es}z=]O€:,=]l=]Gp'O=}OE0=}—S}0=3O=]}—Jeppey
`
`FIG. 38
`
`S6-
`
`
`
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 38 of 40
`
`US 8,574,869 B2
`
`FIG. 39
`
`t=22
`
`t=5
`
`t=3
`
`t=2
`
`t=1:30
`
`t=1
`
`t=0:45
`
`t=0:30
`
`t=0:15
`
`t=0
`
`Ladder
`
`

`

`Case 1:18-cv-01363-CFC Document1-20
`723
`
`Filed 09/04/18
`
`Page 41 of 96 PagelD #:
`
`U.S. Patent
`U.S. Patent
`
`Nov. 5, 2013
`Nov. 5,2013
`
`Sheet 39 of 40
`Sheet 39 of 40
`
`US 8,574,869 B2
`US8,574,869 B2
`
`msnnone
`
`15
`
`95
`
`
`
`
`
`“yr
`
`+0.1mMDHEA
`
`FIG.40
`
`>6— |
`
`ayy
` 2
`
`
`
`UIEmemes
`940womenGRUaaitvavon
`
`:8s5:5:= :
`
`[kDa]
`
`150-
`
`95—
`
`8
`
`46—
`
`

`

`U.S. Patent
`
`Nov. 5,2013
`
`Sheet 40 of 40
`
`US 8,574,869 B2
`
`[kDa]
`
`Ladder
`
`t=0
`
`t=1
`
`t=3
`
`t=4
`
`150
`
`95­
`
`63­
`
`46­
`
`28­
`
`1 5 -
`
`L
`
`1
`
`2
`
`3
`
`4
`
`FIG. 41
`
`

`

`US 8,574
`
`1
`PREVENTION OF DISULFIDE BOND
`REDUCTION DURING RECOMBINANT
`PRODUCTION OF POLYPEPTIDES
`
`,869 B2
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`5
`
`This application is a continuation of U.S. application Ser.
`No. 12/217,745, filed Jul. 8, 2008, which is a non-provisional
`application filed under 37 CFR 1.53(b)(1), claiming priority 10
`under 35 USC 119(e) to provisional Application No. 60/948,
`677 filed Jul. 9, 2007, the contents of which are incorporated
`herein by reference.
`
`FIELD OF THE INVENTION
`
`15
`
`The invention concerns methods and means for preventing
`the reduction of disulfide bonds during the recombinant pro­
`duction of disulfide-containing polypeptides. In particular,
`the invention concerns the prevention of disulfide bond reduc- 20
`tion during harvesting of disulfide-containing polypeptides,
`including antibodies, from recombinant host cell cultures.
`
`BACKGROUND OF THE INVENTION
`
`25
`
`In the biotechnology industry, pharmaceutical applications
`require a variety of proteins produced using recombinant
`DNA techniques. Generally, recombinant proteins are pro­
`duced by cell culture, using either eukaryotic cells, such as
`mammalian cells, or prokaryotic cells, such as bacterial cells, 30
`engineered to produce the protein of interest by insertion of a
`recombinant plasmid containing the nucleic acid encoding
`the desired protein. For a protein to remain biologically
`active, the conformation of the protein, including its tertiary
`structure, must be maintained during its purification and iso- 35
`lation, and the protein’s multiple functional groups must be
`protected from degradation.
`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 40
`assembled heterologous proteins, and their capacity for post-
`translational modifications. Chinese hamster ovary (CHO)
`cells, and cell lines obtained from various other mammalian
`sources, such as, for example, mouse myeloma (NS0), baby
`hamster kidney (BHK), human embryonic kidney (HEK- 45
`293) and human retinal cells, such as the PER.C6® cell line
`isolated from a human retinal cell, which provides human
`glycosylation characteristics, and is able to naturally produce
`antibodies that match human physiology, have been approved
`by regulatory agencies for the production of biopharmaceu- 50
`tical products.
`Usually, to begin the production cycle, a small number of
`transformed recombinant host cells are allowed to grow in
`culture for several days (see, e.g., FIG. 23). Once the cells
`have undergone several rounds of replication, they are trans- 55
`ferred to a larger container where they are prepared to
`undergo fermentation. The media in which the cells are grown
`and the levels of oxygen, nitrogen and carbon dioxide that
`exist during the production cycle may have a significant
`impact on the production process. Growth parameters are 60
`determined specifically for each cell line and these param­
`eters are measured frequently to assure optimal growth and
`production conditions.
`When the cells grow to sufficient numbers, they are trans­
`ferred to large-scale production tanks and grown for a longer 65
`period of time. At this point in the process, the recombinant
`protein can be harvested. Typically, the cells are engineered to
`
`2
`secrete the polypeptide into the cell culture media, so the first
`step in the purification process is to separate the cells from the
`media. Typically, harvesting includes centrifugation and fil­
`tration to produce a Harvested Cell Culture Fluid (HCCF).
`The media is then subjected to several additional purification
`steps that remove any cellular debris, unwanted proteins,
`salts, minerals or other undesirable elements. At the end of the
`purification process, the recombinant protein is highly pure
`and is suitable for human therapeutic use.
`Although this process has been the subject of much study
`and improvements over the past several decades, the produc­
`tion of recombinant proteins is still not without difficulties.
`Thus, for example, during the recombinant production of
`polypeptides comprising disulfide bonds, especially multi­
`chain polypeptides comprising inter-chain disulfide bonds
`such as antibodies, it is essential to protect and retain the
`disulfide bonds throughout the manufacturing, recovery and
`purification process, in order to produce properly folded
`polypeptides with the requisite biological activity.
`
`SUMMARY OF THE INVENTION
`
`The instant invention generally relates to a method for
`preventing reduction of a disulfide bond in a polypeptide
`expressed in a recombinant host cell, comprising supplement­
`ing the pre-harvest or harvested culture fluid of the recombi­
`nant host cell with an inhibitor of thioredoxin or a thiore-
`doxin-like protein.
`In one embodiment, the thioredoxin inhibitor is added to
`the pre-harvest culture fluid.
`In another embodiment, the thioredoxin inhibitor is added
`to the harvested culture fluid.
`In a further embodiment, the thioredoxin inhibitor is a
`direct inhibitor of thioredoxin.
`In all embodiments, the thioredoxin inhibitor may, for
`example, be an alkyl-2-imidazolyl disulfide or a naphtho­
`quinone spiroketal derivative.
`In a further embodiment, the thioredoxin inhibitor is a
`specific inhibitor of thioredoxin reductase.
`In a still further embodiment, the thioredoxin inhibitor is a
`gold complex, where the gold complex may, for example, be
`aurothioglucose (ATG) or aurothiomalate (ATM). While the
`effective inhibitory concentration may vary, it typically is
`between about 0.1 mM and 1 mM. Similarly, the minimum
`effective inhibitory concentration varies depending on the
`nature of the polypeptide and overall circumstances, and is
`typically reached when the ATG or ATG concentration is at
`least about four-times of thioreduxin concentration in the
`pre-harvest or harvested culture fluid.
`In another embodiment of this aspect of the invention, the
`thioredoxin inhibitor is a metal ion, where the metal ion,
`without limitation, may be selected from the group consisting
`of Hg2+, Cu2+, Zn2+, Co2+, and Mn2+. When the metal ion is
`added in the form of cupric sulfate, the effective inhibitory
`concentration generally is between about 5 pM and about 100
`pM, or between about 10 pM and about 80 pM, or between
`about 15 pM and about 50 pM. The minimum inhibitory
`concentration of cupric sulfate also varies, but typically is
`reached when cupric sulfate is added at a concentration at
`least about two-times of thioredoxin concentration in the
`pre-harves or harvested culture fluid.
`In different embodiment, the thioredoxin inhibitor is an
`oxidizing agent, e.g., an inhibitor of G6PD, such as, for
`example, pyridoxal 5'-phosphate, 1 fluoro-2,4 dinitroben­
`zene, dehydroepiandrosterone (DHEA) or epiandrosterone
`(EA); cystine or cysteine. Typical effective inhibitor concen­
`
`

`

`US 8,574,
`
`3
`trations of DHEA are between about 0.05 mM and about 5
`mM, or between about 0.1 mM and about 2.5 mM.
`In a further embodiment, the thioredoxin inhibitor is an
`inhibitor of hexokinase activity, including, without limita­
`tion, chelators of metal ions, such as, for example, ethylene- 5
`diamine tetraacetic acid (EDTA). EDTA is typically added
`and effective at a concentration between about 5 mM and
`about 60 mM, or about 10 mM and about 50 mM, or about 20
`mM and about 40 mM.
`In other preferred embodiments, the inhibitor of hexoki- 10
`nase activity is selected from the group consisting of sorbose-
`1- phosphate, polyphosphates, 6-deoxy-6-fluoroglucose,
`2- C-hydroxy-methylglucose, xylose, and lyxose.
`Other inhibitors include cystine, cysteine, and oxidized 15
`glutathione which are typically added at a concentration at
`least about 40-times of the concentration of the polypeptide in
`question in the pre-harvest or harvested culture fluid.
`In a still further embodiment, the thioredoxin inhibitor is an
`siRNA, an antisense nucleotide, or an antibody specifically 20
`binding to a thioredoxin reductase.
`In another embodiment, the thioredoxin inhibitor is a mea­
`sure indirectly resulting in the inhibition of thioredoxin activ­
`ity. This embodiment includes, for example, air sparging the
`harvested culture fluid of the recombinant host cell, and/or 25
`lowering the pH of the harvested culture fluid of the recom­
`binant host cell.
`In various embodiments, indirect means for inhibiting
`thioredoxin activity, such as air spaiging and/or lowering of
`the pH, can be combined with the use of direct thioredoxin 30
`inhibitors, such as those listed above.
`In all embodiments, the polypeptide may, for example, be
`an antibody, or a biologically functional fragment of an anti­
`body. Representative antibody fragments include Fab, Fab',
`F(ab')2, scFv, (scFv)2, dAb, complementarity determining 35
`region (CDR) fragments, linear antibodies, single-chain anti­
`body molecules, minibodies, diabodies, and multispecific
`antibodies formed from antibody fragments.
`Therapeutic antibodies include, without limitation, anti-
`HER2 antibodies anti-CD20 antibodies; anti-IL-8 antibodies; 40
`anti-VEGF antibodies; anti-CD40 antibodies, anti-CDlla
`antibodies; anti-CD18 antibodies; anti-IgE antibodies; anti-
`Apo-2 receptor antibodies; anti-Tissue Factor (TF) antibod­
`ies; anti -human a 4|37 integrin antibodies; anti-EGFR antibod­
`ies; anti-CD3 antibodies; anti-CD25 antibodies; anti-CD4 45
`antibodies; anti-CD52 antibodies; anti-Fc receptor antibod­
`ies; anti-carcinoembryonic antigen (CEA) antibodies; anti­
`bodies directed against breast epithelial cells; antibodies that
`bind to colon carcinoma cells; anti-CD38 antibodies; anti-
`CD33 antibodies; anti-CD22 antibodies; anti-EpCAM anti- 50
`bodies; anti-GpIIb/IIIa antibodies; anti-RSV antibodies; anti-
`CMV antibodies; anti-HIV antibodies; anti-hepatitis
`antibodies; anti-CA 125 antibodies; anti-av|33 antibodies;
`anti-human renal cell carcinoma antibodies; anti-human
`17-1A antibodies; anti-human colorectal tumor antibodies; 55
`anti-human melanoma antibody R24 directed against GD3
`ganglioside; anti-human squamous-cell carcinoma; and anti­
`human leukocyte antigen (HLA) antibodies, and anti-HLA
`DR antibodies.
`In other embodiments, the therapeutic antibody is an anti- 60
`body binding to a HER receptor, VEGF, IgE, CD20, CD1 la,
`CD40, or DR5.
`In a further embodiment, the HER receptor is HER1 and/or
`HER2, preferably HER2. The HER2 antibody may, for
`example, comprise a heavy and/or light chain variable 65
`domain sequence selected from the group consisting of SEQ
`ID NO: 16, 17, 18, and 19.
`
`869 B2
`
`4
`In another embodiment, the therapeutic antibody is an
`antibody that binds to CD20. The anti-CD20 antibody may,
`for example, comprise a heavy and/or light chain variable
`domain sequence selected from the group consisting of SEQ
`ID NOS: 1 through 15.
`In yet another embodiment, the therapeutic antibody is an
`antibody that binds to VEGF. The anti-VEGF antibody may,
`for example, comprise a heavy and/or light chain variable
`domain sequence selected from the group consisting of SEQ
`ID NOS: 20 through 25.
`In an additional embodiment, the therapeutic antibody is an
`antibody that binds CD 11a. The anti-CDlla antibody may,
`for example, comprise a heavy and/or light chain variable
`domain sequence selected from the group consisting of SEQ
`ID NOS: 26 through 29.
`In a further embodiment, the therapeutic antibody binds to
`a DR5 receptor. The anti-DR5 antibody may, for example, be
`selected from the group consisting of Apomabs 1.1, 2.1, 3.1,
`4.1, 5.1, 5.2, 5.3, 6.1, 6.2, 6.3, 7.1, 7.2, 7.3, 8.1, 8.3, 9.1, 1.2,
`2.2, 3.2, 4.2, 5.2, 6.2, 7.2, 8.2, 9.2, 1.3, 2.2, 3.3, 4.3, 5.3, 6.3,
`7.3, 8.3, 9.3, and 25.3, and preferably is Apomab 8.3 or
`Apomab 7.3, and most preferably Apomab 7.3.
`In other embodiments of the method of the present inven­
`tion, the polypeptide expressed in the recombinant host cell is
`a therapeutic polypeptide. For example, the therapeutic
`polypeptide can be selected from the group consisting of a
`growth hormone, including human growth hormone and
`bovine growth hormone; growth hormone releasing factor;
`parathyroid hormone; thyroid stimulating hormone; lipopro­
`teins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain;
`proinsulin; follicle stimulating hormone; calcitonin; luteiniz­
`ing hormone; glucagon; clotting factors such as factor VIIIC,
`factor IX, tissue factor, and von Willebrands factor; anti­
`clotting factors such as Protein C; atrial natriuretic factor;
`lung surfactant; a plasminogen activator, such as urokinase or
`human urine or tissue-type plasminogen activator (t-PA);
`bombesin; thrombin; hemopoietic growth factor; tumor
`necrosis factor-alpha and -beta; enkephalinase; RANTES
`(regulated on activation normally T-cell expressed and
`secreted); human macrophage inflammatory protein (MIP-1 -
`alpha); a serum albumin such as human serum albumin;
`Muellerian-inhibiting substance; relaxin A-chain; relaxin
`B-chain; prorelaxin; mouse gonadotropin-associated pep­
`tide; a microbial protein, such as beta-lactamase; DNase; IgE;
`a cytotoxic T-lymphocyte associated antigen (CTLA), such as
`CTLA-4; inhibin; activin; vascular endothelial growth factor
`(VEGF); receptors for hormones or growth factors; Protein A
`or D; rheumatoid factors; a neurotrophic factor such as bone-
`derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5,
`or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor
`such as NGF- (3; platelet-derived growth factor (PDGF); fibro­
`blast growth factor such as aFGF and bFGF; epidermal
`growth factor (EGF); transforming growth factor (TGF) such
`as TGF-alpha and TGF-beta, including TGF-|31, TGF-|32,
`TGF-|33, TGF-|34, or TGF-|35; insulin-like growth factor-I
`and -II (IGF-I and IGF-II); des(l-3)-IGF-I (brain IGF-I),
`insulin-like growth factor binding proteins; CD proteins such
`as CD3, CD4, CD8, CD19, CD20, CD34, and CD40; eryth­
`ropoietin; osteoinductive factors; immunotoxins; a bone mor­
`phogenetic protein (BMP); an interferon such as interferon-
`alpha, -beta, and -gamma; colony stimulating factors (CSFs),
`e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g.,
`IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface
`membrane proteins; decay accelerating factor; viral antigen
`such as, for example, a portion of the AIDS envelope; trans­
`port proteins; homing receptors; addressins; regulatory pro­
`teins; integrins such as CDlla, CDllb, CDllc, CD18, an
`
`

`

`US 8,574
`
`5
`ICAM, VLA-4 and VCAM; a tumor associated antigen such
`as HER2, HER3 or EIER4 receptor; and fragments of said
`polypeptides.
`In all embodiments, the recombinant host cell can be an
`eukaryotic host cell, such as a mammalian host cell, includ- 5
`ing, for example, Chinese Elamster Ovary (CEIO) cells.
`In all embodiments, the recombinant host cell can also be a
`prokaryotic host cell, such as a bacterial cell, including, with­
`out limitation, E. coli cells.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`10
`
`FIG. 1. Dialysis Experiment: Digital gel-like imaging
`obtained from Bioanalyzer analysis (each lane representing a
`time point) demonstrating that ocrelizumab (rhuMAb 2H7— 15
`Variant A) inside the dialysis bag remained intact during the
`incubation period.
`FIG. 2. Dialysis Experiment: Digital gel-like imaging
`obtained from Bioanalyzer analysis (each lane representing a
`time point) showing that ocrelizumab outside the dialysis bag 20
`was reduced during the incubation period. This is evidenced
`by the loss of intact antibody (-150 kDa) and the formation of
`antibody fragments depicted in the Figure. At the 48-hour
`time point (Lane 7), the reduced antibody appeared to be
`reoxidized, presumably as a result of loosing reduction activ- 25
`ity in the Harvested Cell Culture Fluid (HCCF). The band
`appearing just above the 28 kDa marker arose from the light
`chain of antibody. There was a significant amount of free light
`already present in the HCCF before the incubation began. The
`presence of excess free light chain and dimers of light chain in 30
`the HCCF is typical for the cell line producing ocrelizumab.
`FIG. 3. Free Thiol Levels from Dialysis Experiment: Puri­
`fied ocrelizumab in phosphate buffered saline (PBS) was
`inside the dialysis bag and HCCF containing ocrelizumab
`