Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 1 of 24 PageID #: 433
`Case 1:18-cv-01363-CFC Document1-10
`Filed 09/04/18
`Page 1 of 24 PagelD #: 433
`
`
`
`EXHIBIT J
`EXHIBIT J
`
`
`
`

`

`Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 2 of 24 PageID #: 434
`
`(12) U n i t ed States P a t e nt
`Basey et al.
`
`US006417335B1
`US 6,417,335 Bl
`(io) Patent No.:
`Jul. 9,2002
`(45) Date of Patent:
`
`(54) PROTEIN PURIFICATION
`
`OTHER PUBLICATIONS
`
`(75)
`
`Inventors: Carol D. Basey, Winters; Greg S.
`Blank, Menlo Park, both of CA (US)
`
`(73) Assignee: Genentech, Inc., South San Francisco,
`CA (US)
`
`( * ) Notice:
`
`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.: 09/680,148
`
`(22) Filed:
`
`Oct. 3, 2000
`
`Related U.S. Application Data
`
`(62) Division of application No. 09/304,465, filed on May 3,
`1999.
`(60) Provisional application No. 60/084,459, filed on May 6,
`1998.
`Int. CI.7
`(51)
`(52) U.S. CI
`
`C07K 1/18; C07K 16/00
`530/387.1; 530/387.3;
`530/387.7; 530/388.85; 530/389.7; 530/416
`(58) Field of Search
`530/416, 387.1,
`530/387.3, 387.7, 388.85, 389.7
`
`(56)
`
`References Cited
`
`Carter et al., "Humanization of an anti-piss™7*2 antibody
`for human cancer
`therapy" Proc. Natl. Acad.
`Sci.
`89:4285^1289 (May 1992).
`Gagnon, P., "Ion Exchange Chromatography" Purification
`Tools for Monoclonal Antibodies, Tucson: Validated Biosys-
`tems, Inc., Chapter 4, pp. 57-86 (1996).
`Graf et al., "Ion exchange resins for the purification of
`monoclonal antibodies from animal cell culture" Biosepa-
`ration 4 (1) :7-20 (Feb. 1994).
`Harris,R., "Chromatographic Techniques for the Character(cid:173)
`ization of Human MAbs" (Slides presented at the Waterside
`Monoclonal Conference held at the Omni Waterside Hotel in
`Harborside-Norfolk, Virginia on Apr. 22-25,1996) pp. 1-7.
`Mhatre et al., "Purification of antibody Fab fragments by
`cation-exchange chromatography and pH gradient elution"
`Journal of Chromatography A 707 (2) :225-231 (Jul. 2 1,
`1995).
`
`Neidhardt et al., "Rapid, two-step purification process for
`the preparation of pyrogen-free murine immunoglobulin G1
`monoclonal antibodies" Journal of Chromatography 590 (2)
`:255-261 (1992).
`Sofer et al. Handbook of Process Chromatography: A Guide
`to Optimization, Scale-up, and Validation, San Diego:Aca-
`demic Press pp. 65-80 (1997).
`Tishchenko et al., "Effect of salt concentration gradient on
`separation of different types of specific immunoglobulins by
`ion-exchange chromatography on DEAE cellulose" Journal
`of Chromatography B 706 (1) : 157-166 (Feb. 27, 1998).
`
`U.S. PATENT DOCUMENTS
`
`* cited by examiner
`
`5,110,913 A * 5/1992 Coan et al
`5,112,951 A * 5/1992 Beidler et al
`5,118,796 A * 6/1992 Prior et al
`5,821,337 A
`10/1998 Carter et al.
`
`530/416
`530/416
`530/416
`
`Primary Examiner—David Saunders
`(74) Attorney, Agent, or Firm—Wendy M. Lee
`
`(57)
`
`ABSTRACT
`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`EP
`WO
`WO
`
`333574
`556083
`460426 Bl
`WO 89/05157
`WO 96/33208
`
`9/1989
`8/1993
`9/1997
`6/1989
`10/1996
`
`A method for purifying a polypeptide by ion exchange
`chromatography is described which involves changing the
`conductivity and/or pH of buffers in order to resolve a
`polypeptide of interest from one or more contaminants.
`
`9 Claims, 7 Drawing Sheets
`
`STEP A
`BIND POLYPEPTIDE
`TO ION EXCHANGER
`LOADING BUFFER
`(e g. 50 mM NaCI or
`5.0 pH)
`
`STEPB
`ELUTE CONTAMINANTS)
`INTERMEDIATE BUFFER
`(e.g. 70 mM NaCI or
`
`STEPC
`EQUILIBRATE
`ION EXCHANGER
`WASH BUFFER
`(e.g. 50 mM NaCI or
`5.0 pH)
`
`STEPD
`ELUTE POLYPEPTIDE
`ELUTION BUFFER
`(e.g 95 mM NaCI or
`6.0 pH)
`
`STEPE
`REGENERATE
`ION EXCHANGER
`REGENERATION
`BUFFER
`(e.g. 1.0 MNaCI)
`
`

`

`Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 3 of 24 PageID #: 435
`
`U.S. Patent
`
`Jul. 9, 2002
`
`Sheet 1 of 7
`
`US 6,417,335 Bl
`
`STEP A
`BIND POLYPEPTIDE
`TO ION EXCHANGER
`LOADING BUFFER
`(e.g. 50 mM NaCI or
`5.0 pH)
`
`STEP A
`BIND POLYPEPTIDE
`TO ION EXCHANGER
`LOADING BUFFER
`(e.g. 50 mM NaCI or
`8.0 pH)
`
`STEPB
`ELUTE CONTAMINANT(S)
`INTERMEDIATE BUFFER
`(e.g. 70 mM NaCI or
`5.4 pH)
`
`STEPB
`ELUTE CONTAMINANT(S)
`INTERMEDIATE BUFFER
`(e.g. 70 mM NaCI or
`7.6 pH)
`
`STEPC
`EQUILIBRATE
`ION EXCHANGER
`WASH BUFFER
`(e.g. 50 mM NaCI or
`5.0 pH)
`
`STEPD
`ELUTE POLYPEPTIDE
`ELUTION BUFFER
`(e.g. 95 mM NaCI or
`6.0 pH)
`
`STEPE
`REGENERATE
`ION EXCHANGER
`REGENERATION
`BUFFER
`(e.g. 1.0 MNaCI)
`
`FIG.-1
`
`STEPC
`EQUILIBRATE
`ION EXCHANGER
`WASH BUFFER
`(e.g. 50 mM NaCI or
`8.0 pH)
`
`STEPD
`ELUTE POLYPEPTIDE
`ELUTION BUFFER
`(e.g. 95 mM NaCI or
`7.0 pH)
`
`STEPE
`REGENERATE
`ION EXCHANGER
`REGENERATION
`BUFFER
`(e.g. 1.0 MNaCI)
`
`FIG..2
`
`

`

`Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 4 of 24 PageID #: 436
`
`U . S. P a t e nt
`
`Jul. 9,2002
`
`Sheet 2 of 7
`
`US 6,417,335 Bl
`
`WASH
`BUFFER
`50 mM NaCI
`
`LOADING
`BUFFER
`50 mM NaCI
`
`INTERMEDIATE
`BUFFER
`70 mM NaCI
`
`ELUTION
`BUFFER
`95 mM NaCI
`
`REGENERATION
`BUFFER
`1.0 M NaCI
`
`FIG.S
`
`

`

`Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 5 of 24 PageID #: 437
`
`U.S. Patent
`
`Jul. 9, 2002
`
`Sheet 3 of 7
`
`US 6,417,335 Bl
`
`i
`
`i i i
`
`| i i i
`
`i | i
`
`i i
`
`i
`
`|
`
`'
`
`i
`
`'
`
`'
`
`| '
`
`'
`
`'
`
`'
`
`I '
`
`i
`
`i I i
`
`i i i
`
`I i i i
`
`o
`cr
`m
`z
`LU
`O
`LU
`CE
`
`•^mMi^mmm^tAmMS^ii^im^m
`K ^ v t g ^ ^ i ^ ^ c : ^ ^ ^ ^ ^ ; ^^
`
`fc^^x^zzzmm
`
`O
`o
`CL
`
`E
`
`L i-
`
`y
`
`^
`
`<
`
`^^
`
`E f?Z<? s
`
`o
`<
`DC
`U_
`>
`X
`Q.
`<
`DC
`CD
`O
`
`O
`DC
`X
`o
`
`CD
`
`O
`o
`0.
`LU
`DC
`Q_
`
`Q.
`LU
`H
`CO
`X
`CO
`<
`
`I o
`o
`GC
`I
`
`o
`
`• AVERAGE
`p 40 mg / m
`
`5 f
`
`E E _E E E
`0 ) 0 ) 0 ) 0 ) 0)
`E E E E E
`m o
`ID o
`LO
`•^
`C\J
`CM
`CO
`CO
`
`• b 0
`
`i
`
`i i
`
`i
`
`o
`oo
`
`i
`
`| i
`o
`
`i
`
`|
`
`i i
`o
`CD
`
`I
`I I I
`i i i |
`o
`
`I I I I I I
`o
`o
`
`ID
`
`M"
`
`I I I
`o
`CO
`
`I I I I I
`I I
`o
`oo
`o
`CM
`
`'
`
`I I '
`
`I '
`
`Q
`_]
`LU
`>-
`
`

`

`Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 6 of 24 PageID #: 438
`
`U.S. Patent
`
`Jul. 9, 2002
`
`Sheet 4 of 7
`
`US 6,417,335 Bl
`
`~\
`
`i
`
`r
`
`i—'—•—'—r n
`
`1
`
`r
`
`mm&^^m^^
`k g ^ ^ o ^ ^ ^ ^^
`\{C&^^^
`
`s
`z
`
`:^
`
`co
`
`5
`
`i
`
`CO CI
`
`1 22
`
`m
`
`E E E E E
`0 ) 0 ) 0 ) 0 ) 0)
`E E E E E
`m o un o m
`C\J
`OJ
`CO
`CO
`T-
`
`• E 0E E
`
`E
`O)
`E
`o
`
`•
`
`Q
`
`< 3
`
`•
`
`-[—i—i—i—i—i
`o
`oo
`
`o
`o
`
`i
`
`r T
`0
`
`CD
`
`1
`
`r
`
`i — • — • —r
`o
`CM
`
`2
`
`-^
`
`~i
`
`1
`
`r
`
`X<
`
`

`

`Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 7 of 24 PageID #: 439
`
`U . S. P a t e nt
`
`Jul. 9,2002
`
`Sheet 5 of 7
`
`US 6,417,335 Bl
`
`1 1 1 1 I 1 1 1 1 I 1 1 1 1 I 1 1 1 1 I 1 1 1 1 I 1 1 1 1
`
`o
`o
`CO
`
`o
`10
`CM
`
`O
`h- o
`CM
`
`\
`
`E E E E E£
`
`D5 CD D) O) O) O)
`EE EE EE
`m o m o m o
`1- (M CvJ co co •<*
`1 1 1 • 1 1
`
`t t t fi
`
`\
`
`\
`
`+
`
`\
`
`^ C*
`
`> C" ^ —.
`
`• • —y ^ m m mm
`
`.
`
`<^ - .. O -—
`
`. = 2.
`
`I
`
`o
`CO
`
`in
`cvi
`
`I I I I I I I I I I I I I I I I I I I I I I I I I I I I
`o
`d
`
`o
`
`ID
`
`in
`d
`
`LJJ
`
`o
`<f
`
`oco
`v-' CM
`m
`<
`
`

`

`Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 8 of 24 PageID #: 440
`
`U . S. P a t e nt
`
`Jul. 9,2002
`Jul. 9, 2002
`
`Sheet 6 of 7
`Sheet 6 of 7
`
`US 6,417,335 Bl
`1
`
`in
`
`0
`CTi
`
`in
`
`O
`CO
`
`H
`CO
`
`CO
`
`J
`
`CO
`>*
`EH
`
`CO
`
`Q
`
`CO
`0
`0
`
`m
`
`T-l
`EH
`>
`co
`U
`a
`CO
`2:
`0
`CO
`0
`J
`<
`2
`Q
`O
`>
`in
`
`0
`>
`>
`CO
`<
`E-
`O
`CO
`«
`
`0
`w
`Q
`
`CO
`
`ft.
`
`CL.
`
`M
`fr,
`>
`CO
`a,
`<
`<
`>
`^
`
`Di
`
`J^
`H
`in
`0
`
`>
`
`Ui
`H
`0
`0
`0
`u*
`E-"
`a.
`cu
`E-
`
`EH
`
`r-l
`
`>
`in
`cr>
`u
`<
`
`>H
`>
`«
`K
`«
`U
`
`>H
`Q
`<
`^
`
`CO
`1
`cc
`1 J
`
`S
`O
`>
`^
`<
`w
`P:
`cu
`
`>H
`
`Cu
`2
`2
`
`U
`r-l
`
`0
`Oi
`
`O
`f-H
`CM
`
`CO
`
`:*:
`
`EH
`>
`ft.
`
`CO
`
`CO
`
`0
`a
`K
`
`EH
`
`5
`N 1
`C3
`
`O
`O
`O
`^
`J«
`^
`<:
`u,
`Q
`
`Cd
`a.
`0
`
`CO
`
`CO
`in
`M
`
`0
`
`a,
`<
`«
`O
`CU
`^
`a
`0
`>«
`
`<
`>
`<
`
`>
`a
`0
`w
`<
`oi
`0
`
`t^
`>
`<£
`
`Q
`
`0
`
`< x
`o
`I-
`X
`(3
`
`EH
`
`J
`
`tH
`
`tl.
`a
`E-
`
`0
`
`to
`a:
`CO
`O
`CO
`
`Cn
`
`CO
`a.
`>
`U
`CO
`>*
`J
`tt,
`
`CO
`<
`co
`>-"
`M
`
`O m
`
`0
`
`<
`w
`J
`
`CO
`
`CO
`
`Oi
`
`CO
`0
`e«
`s
`a
`M
`«H Q
`
`

`

`Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 9 of 24 PageID #: 441
`
`U . S. P a t e nt
`
`Jul. 9,2002
`
`Sheet 7 of 7
`
`US 6,417,335 Bl
`
`m
`5 j
`O
`^
`
`o
`
`O.
`
`O
`
`O
`>
`
`S(J
`3:
`
`^
`
`a
`
`i
`
`i
`o
`
`C
`><o
`
`c
`
`:
`
`S
`w
`
`U
`>
`
`o
`SQ
`W
`
`m
`E W
`M
`
`in
`CSUJ
`Q
`
`o
`t- W
`>
`
`in
`JH Q
`
`o
`VOW
`
`in
`2°
`
`a
`
`w
`
`o
`SM
`
`W
`a
`
`u
`
`<
`
`J
`
`w
`
`>
`
`o
`
`QK
`'J
`
`PS
`
`w
`
`w
`
`ow
`
`«
`Q ^O
`^
`
`' a.
`
`w
`
`2
`
`cu
`
`f
`
`c
`
`<
`
`f
`
`^
`H
`
`tu
`>t
`U
`
`ft
`u
`
`,J
`
`H>
`
`i
`
`>M
`
`>
`
`E-
`
`J
`
`-
`
`t
`
`f
`
`f
`
`^
`
`z*
`>
`o < u :
`t-
`
`>«
`
`&
`
`tJ
`
`tW
`E
`
`a
`
`^^
`
`:
`
`«
`
`W>
`
`Q
`
`f
`
`t>
`
`>
`e
`
`5
`
`o
`M^
`
`Q
`
`M
`SQ
`
`in
`r -w
`
`it
`
`e
`
`o
`f NW
`^H
`
`-
`
`>
`
`W
`
`<
`
`J
`
`in
`v o<
`^
`
`wo
`
`<
`
`i
`
`2
`
`-
`
`in
`mS
`CN
`J
`0'
`
`o
`r-ico
`CN
`
`ft
`E
`
`M
`o
`o Z ll
`tn
`>
`
`w
`• cO
`
`ro
`O
`«
`
`K
`
`0,
`m
`o > ft
`
`r
`
`01
`OU
`
`M
`om
`r nj
`n_ ' ^^
`•<
`
`i
`
`j
`
`c
`
`<
`
`<
`
`-Uc
`
`o
`
`f
`
`H
`
`r
`
`<
`0
`
`>
`
`M
`
`w
`
`>
`
`f
`
`e
`
`H
`
`>
`
`c
`
`a
`
`a
`
`>
`
`W
`
`E
`
`:
`
`S
`
`2
`^
`
`n
`
`-
`
`t
`
`i
`
`a:
`ft
`
`t^
`
`H
`
`t
`
`e
`
`o
`SW
`n
`
`w
`
`>
`
`uu
`
`hv
`I
`
`s
`u
`
`<
`
`f
`
`&<
`
`«
`
`>w
`
`f
`
`t
`
`>
`
`C5
`
`X1J
`
`^
`
`:
`
`m
`
`Dj
`j
`c
`
`n
`
`ft
`O
`
`>
`
`O
`
`w
`
`>
`
`o
`
`^
`
`f
`
`t
`
`Q
`
`C
`
`O
`
`L
`S
`
`.
`>
`
`<
`o
`
`O JH
`• -•
`
`in
`i n«
`' -,
`
`<
`
`i
`V O JM
`
`o
`
`E
`
`>
`
`Z
`
`n
`
`H
`
`<
`
`O
`
`'
`O
`E
`
`<
`
`D
`
`f
`
`-
`
`J<
`c2
`>
`W>
`
`'
`O
`
`a:
`
`M
`C
`
`M>
`U
`
`<
`^
`
`t
`
`QS
`
`wo
`tO
`
`f
`
`J
`o
`
`ft
`i n.
`C D>
`^
`
`^<C5
`^
`
`J
`W
`o ^ i n,
`m r t;
`o.
`
`>r
`
`r
`
`cr\U}
`^-,
`
`JH
`o
`
`r - fa
`,, H '
`
`r
`
`t s^
`*^
`
`O
`
`U
`
`Q
`
`J
`
`J
`
`O
`
`E
`
`W
`
`f
`
`^
`
`I
`
`t
`
`f
`
`JQ
`
`J>
`
`tS
`
`S
`
`W
`
`«
`
`«W
`
`>«
`
`U>
`
`E
`
`-
`
`'
`
`S
`
`'
`
`O
`c
`
`i
`n
`
`f
`j
`
`i
`
`(
`
`)
`
`<
`(
`
`t
`^
`
`]
`
`c
`>
`
`>
`
`S
`
`H
`;
`
`-
`
`>
`
`O
`
`j
`
`H
`
`i
`
`E
`
`»<
`
`O
`
`O
`u
`
`t
`>
`j
`0
`
`>
`
`l td
`
`'
`
`<
`
`O
`O
`W
`
`E
`
`H
`
`-
`
`j
`
`<
`
`>
`
`>
`
`f
`
`<2
`tfa
`
`w
`
`o
`
`u:
`
`f
`3
`U
`t
`
`W
`
`»
`
`-
`
`W
`
`H
`
`J
`
`t>
`
`f
`
`tW
`
`ft
`HQ
`
`3:
`
`W
`
`o
`
`e
`
`e-
`
`JJ
`«
`; > - i c o > <i
`
`w
`i
`O
`3
`
`>w
`O^
`ZJ
`fc
`
`d
`
`«
`
`J E n
`^
`„
`v oS
`CI
`
`t
`
`t,
`,«
`
`i°
`oW
`
`'*
`
`W
`
`«J
`roUS
`rH
`
`«)
`" ^W
`
`i-l
`cnE^
`
`rH
`0 0O
`rH
`
`VO
`fNE-<
`<N
`
`rH
`r ~X
`CN
`
`«> ^
`^3
`m
`
`

`

`Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 10 of 24 PageID #:
`442
`
`US 6,417,335 Bl
`
`PROTEIN PURIFICATION
`
`This is a divisional of application Ser. No. 09/304,465
`filed May 3,1999 which claims priority under 35 USC §119
`to provisional application Ser. No. 60/084,459 filed May 6, 5
`1998, both disclosures of which are hereby incorporated by
`reference.
`
`BACKGROUND OF THE INVENTION
`
`1°
`
`30
`
`1. Field of the Invention
`This invention relates generally to protein purification. In
`particular, the invention relates to a method for purifying a
`polypeptide (e.g. an antibody) from a composition compris(cid:173)
`ing the polypeptide and at least one contaminant using the
`method of ion exchange chromatography.
`2. Description of Related Art
`is
`The large-scale, economic purification of proteins
`increasingly an important problem for the biotechnology
`industry. Generally, proteins are produced by cell culture, -.
`using either mammalian or bacterial cell lines engineered to
`produce the protein of interest by insertion of a recombinant
`plasmid containing the gene for that protein. Since the cell
`lines used are living organisms, they must be fed with a
`complex growth medium, containing sugars, amino acids, -,
`and growth factors, usually supplied from preparations of
`animal serum. Separation of the desired protein from the
`mixture of compounds fed
`to the cells and from
`the
`by-products of the cells themselves to a purity sufhcient for
`use as a human therapeutic poses a formidable challenge.
`Procedures for purification of proteins from cell debris
`initially depend on the site of expression of the protein.
`Some proteins can be caused to be secreted directly from the
`cell into the surrounding growth media; others are made
`intracellularly. For the latter proteins, the first step of a 35
`purification process involves lysis of the cell, which can be
`done by a variety of methods, including mechanical shear,
`osmotic shock, or enzymatic treatments. Such disruption
`releases the entire contents of the cell into the homogenate,
`and in addition produces subcellular fragments
`that are 40
`difEcult to remove due to their small size. These are gener(cid:173)
`ally removed by differential centrifugation or by
`filtration.
`The same problem arises, although on a smaller scale, with
`directly secreted proteins due to the natural death of cells
`and release of intracellular host cell proteins in the course of 45
`the protein production run.
`Once a clarified solution containing the protein of interest
`has been obtained, its separation from the other proteins
`produced by the cell is usually attempted using a combina(cid:173)
`tion of different chromatography techniques. These tech- 50
`niques separate mixtures of proteins on the basis of their
`charge, degree of hydrophobicity, or size. Several different
`chromatography resins are available for each of
`these
`techniques, allowing accurate tailoring of the purification
`scheme to the particular protein involved. The essence of 55
`each of these separation methods is that proteins can be
`caused either to move at different rates down a long column,
`achieving a physical separation that increases as they pass
`further down the column, or to adhere selectively to the
`separation medium, being then differentially eluted by dif- so
`ferent solvents. In some cases, the desired protein is sepa(cid:173)
`rated from
`impurities when
`the impurities specifically
`adhere to the column, and the protein of interest does not,
`that is, the protein of interest is present in the "flow-
`through".
`
`Ion exchange chromatography is a chromatographic tech(cid:173)
`nique that is commonly used for the purification of proteins.
`
`65
`
`In ion exchange chromatography, charged patches on the
`surface of the solute are attracted by opposite charges
`attached to a chromatography matrix, provided the ionic
`strength of the surrounding buffer is low. Elution is generally
`achieved by increasing the ionic strength (i.e. conductivity)
`of the buffer to compete with the solute for the charged sites
`of the ion exchange matrix. Changing the pH and thereby
`altering the charge of the solute is another way to achieve
`elution of the solute. The change in conductivity or pH may
`be gradual (gradient elution) or stepwise (step elution). In
`the past, these changes have been progressive; i.e., the pH or
`conductivity is increased or decreased in a single direction.
`
`SUMMARY OF THE INVENTION
`The present invention provides an ion exchange chro(cid:173)
`matographic method wherein a polypeptide of interest is
`bound to the ion exchange material at an initial conductivity
`or pH and then the ion exchange material is washed with an
`intermediate buffer at a different conductivity or pH, or both.
`At a specific point following this intermediate wash, and
`contrary to ion exchange chromatography standard practice,
`the ion exchange material is washed with a wash buffer
`where the change in conductivity or pH, or both, from the
`intermediate buffer to the wash buffer is in an opposite
`direction to the change in conductivity or pH, or both,
`achieved in the previous steps. Only after washing with the
`wash buffer, is the ion exchange material prepared for the
`polypeptide molecule of interest to be eluted by the appli(cid:173)
`cation of the elution buffer having a conductivity or pH, or
`both, which differ from the conductivity or pH, or both, of
`the buffers used in previous steps.
`
`This novel approach to ion exchange chromatography is
`particularly useful in situations where a product molecule
`must be separated from a very closely related contaminant
`molecule at full manufacturing scale, where both purity and
`high recovery of polypeptide product are desired.
`Accordingly, the invention provides a method for purify(cid:173)
`ing a polypeptide from a composition comprising
`the
`polypeptide and a contaminant, which method comprises the
`following steps performed sequentially:
`(a) binding the polypeptide to an ion exchange material
`using a loading buffer, wherein the loading buffer is at
`a first conductivity and pH;
`(b) washing the ion exchange material with an interme(cid:173)
`diate buffer at a second conductivity and/or pH so as to
`elute the contaminant from the ion exchange material;
`(c) washing the ion exchange material with a wash buffer
`which is at a third conductivity and/or pH, wherein the
`change in conductivity and/or pH from the intermediate
`buffer to the wash buffer is in an opposite direction to
`the change in conductivity and/or pH from the loading
`buffer to the intermediate buffer; and
`(d) washing the ion exchange material with an elution
`buffer at a fourth conductivity and/or pH so as to elute
`the polypeptide from the ion exchange material. The
`first conductivity and/or pH may be the same as the
`third conductivity and/or pH.
`Where the ion exchange material comprises a cation
`exchange resin, the conductivity and/or pH of the interme(cid:173)
`diate buffer is/are preferably greater than the conductivity
`and/or pH of the loading buffer; the conductivity and/or pH
`of the wash buffer is/are preferably less than the conductivity
`and/or pH of the intermediate buffer; and the conductivity
`and/or pH of the elution buffer is/are preferably greater than
`the conductivity and/or pH of the intermediate buffer.
`Preferably, the conductivity and/or pH of the wash buffer
`is/are about the same as the conductivity and/or pH of the
`loading buffer.
`
`

`

`Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 11 of 24 PageID #:
`443
`
`US 6,417,335 Bl
`
`Preferably elution of the contaminant and of the polypep(cid:173)
`tide is achieved by modifying the conductivity of the inter(cid:173)
`mediate buffer and of the elution buffer, respectively, while
`keeping the pH of these buffers approximately the same.
`The invention also provides a method for purifying a 5
`polypeptide from a composition comprising the polypeptide
`and a contaminant, which method comprises the following
`steps performed sequentially:
`(a) binding the polypeptide to a cation exchange material
`using a loading buffer, wherein the loading buffer is at 1°
`a first conductivity and pH;
`(b) washing the cation exchange material with an inter(cid:173)
`mediate buffer at a second conductivity and/or pH
`which is greater than that of the loading buffer so as to
`elute the contaminant from the ion exchange material; 15
`(c) washing the cation exchange material with a wash
`buffer which is at a third conductivity and/or pH which
`is less than that of the intermediate buffer; and
`(d) washing the cation exchange material with an elution
`buffer at a fourth conductivity and/or pH which is
`greater than that of the intermediate buffer so as to elute
`the polypeptide from the ion exchange material.
`In addition, the invention provides a method for purifying
`an antibody from a composition comprising the antibody
`and a contaminant, which method comprises loading the
`composition onto a cation exchange resin, wherein the
`amount of antibody loaded onto the cation exchange resin is
`from about 20 mg to about 35 mg of the antibody per mL of
`cation exchange resin and, optionally, further comprising
`eluting the antibody from the cation exchange resin. The
`method preferably further comprises an intermediate wash
`step for eluting one or more contaminants from the ion
`exchange resin. This intermediate wash step usually pre(cid:173)
`cedes the step of eluting the antibody.
`The invention further provides a composition comprising
`a mixture of anti-HER2 antibody and one or more acidic
`variants thereof, wherein the amount of the acidic variant(s)
`in the composition is less than about 25% and preferably less
`than about 20%, e.g. in the range from about 1% to about
`18%. Optionally, the composition further comprises a phar-
`maceutically acceptable carrier.
`
`,
`
`45
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is a flow diagram showing how one could perform
`cation exchange chromatography by altering conductivity
`(e.g. to the NaCl concentrations of Example 1 below) or by
`altering pH (e.g. to the pH values as shown in the flow
`diagram).
`FIG. 2 is a flow diagram showing how one could perform 50
`anion exchange chromatography by altering conductivity
`(e.g. to the NaCl concentrations as depicted in the figure) or
`by altering pH (e.g. to the pH values as shown).
`FIG. 3 is an absorbance trace from a cation exchange
`chromatography run of Example 1 at full manufacturing 55
`scale. Points at which the column is washed with the
`different buffers described herein are marked with arrows.
`FIG. 4 depicts recombinant humanized anti-HER2 mono(cid:173)
`clonal antibody (rhuMAb HER2) recovered in each chro(cid:173)
`matography fraction (calculated as the percentage of the sum 60
`total of all fractions of the relevant chromatography). Flow
`through, wash steps, and prepool fractions are all effluent
`samples collected from the onset of load to the initiation of
`pooling. The pool fraction is the five column volume effluent
`sample of elution starting at the leading shoulder's inflection 65
`point. The regeneration fraction contains effluent captured
`from the end of pooling to the end of regeneration.
`
`FIG. 5 shows the quality of rhuMAb HER2 in each cation
`exchange chromatography pool sample as evaluated by
`carboxy sulfon cation exchange high pressure liquid chro(cid:173)
`matography (CSx HPIEX). Peaks a, b, and 1 are deamidated
`forms of rhuMAb HER2. Peak 3 is nondeamidated rhuMAb
`HER2. Peak 4 is a combination of C-terminal Lysine con(cid:173)
`taining and iso-aspartate variants of rhuMAb HER2.
`FIG. 6 shows the absorbance (280 nm) profiles of the
`0.025 M MES/0.070 M NaCl, pH 5.6 wash for each chro(cid:173)
`matography. The mass of rhuMAb HER2 applied to the
`cation exchange resin effects the peak's absorbance level at
`the apex as well as the amount of buffer required to reach the
`apex. Due to minor peaks which occur (as best seen in the
`30 mg/mL load) in this wash, the apex is defined as
`absorbance levels of at least 0.5 absorbance units (AU).
`FIGS. 7A and 7B show the amino acid sequences of
`humMAb4D5-8
`light chain (SEQ ID NO:l) and
`humMAb4D5-8 heavy chain (SEQ ID NO:2), respectively.
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENTS
`
`Definitions
`
`The "composition" to be purified herein comprises the
`polypeptide of interest and one or more contaminants. The
`composition may be "partially purified" (i.e. having been
`subjected to one or more purification steps, such as Protein
`A Chromatography as in Example 1 below) or may be
`obtained directly from a host cell or organism producing the
`polypeptide (e.g. the composition may comprise harvested
`cell culture fluid).
`As used herein, "polypeptide" refers generally to peptides
`and proteins having more than about ten amino acids.
`Preferably, the polypeptide is a mammalian protein,
`examples of which include renin; a growth hormone, includ(cid:173)
`ing human growth hormone and bovine growth hormone;
`growth hormone releasing factor; parathyroid hormone;
`thyroid stimulating hormone; lipoproteins; alpha-1-
`antitrypsin; insulin A-chain; insulin B-chain; proinsulin;
`follicle stimulating hormone; calcitonin; luteinizing hor(cid:173)
`mone; 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(cid:173)
`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-P; platelet-derived
`growth factor (PDGF); fibroblast growth factor such as
`aFGF and bFGF; epidermal growth factor (EGF); transform(cid:173)
`ing growth factor (TGF) such as TGF-alpha and TGF-beta,
`including TGF-pi, TGF-pi, TGF-P3, TGF-P4, or TGF-P5;
`insulin-like growth factor-I and -II (IGF-I and IGF-II);
`des(l-3)-IGF-I (brain IGF-I), insulin-like growth factor
`
`

`

`Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 12 of 24 PageID #:
`444
`
`US 6,417,335 Bl
`
`binding proteins (IGFBPs); CD proteins such as CD3, CD4,
`CDS, CD19 and CD20; erythropoietin; osteoinductive fac(cid:173)
`tors; immunotoxins; a bone morphogenetic protein (BMP);
`an interferon such as interferon-alpha, -beta, and -gamma;
`colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, 5
`and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superox(cid:173)
`ide dismutase; T-cell receptors; surface membrane proteins;
`decay accelerating factor; viral antigen such as, for example,
`a portion of the AIDS envelope; transport proteins; homing
`receptors; addressins; regulatory proteins; integrins such as 10
`C D l l a, C D l l b, C D l l c, CD18, an ICAM, VLA-4 and
`VCAM; a tumor associated antigen such as HER2, HER3 or
`HER4 receptor; and fragments and/or variants of any of the
`above-listed polypeptides. Most preferred is a full length
`antibody that binds human HER2.
`
`15
`
`A "contaminant" is a material that is different from the
`desired polypeptide product. The contaminant may be a
`variant of the desired polypeptide (e.g. a deamidated variant
`or an amino-aspartate variant of the desired polypeptide) or
`another polypeptide, nucleic acid, endotoxin etc.
`A "variant" or "amino acid sequence variant" of a starting
`polypeptide is a polypeptide that comprises an amino acid
`sequence different from that of the starting polypeptide.
`Generally, a variant will possess at least 80% sequence
`identity, preferably at least 90% sequence identity, more
`preferably at least 95% sequence identity, and most prefer(cid:173)
`ably at least 98% sequence identity with the native polypep(cid:173)
`tide. Percentage sequence
`identity
`is determined,
`for
`example, by the Fitch et al., Proc. Natl. Acad. Sci. USA
`80:1382-1386 (1983), version of the algorithm described by
`Needleman et a l, /. Mol. Biol. 48:443-453 (1970), after
`aligning the sequences to provide for maximum homology.
`Amino acid sequence variants of a polypeptide may be
`prepared by introducing appropriate nucleotide changes into
`DNA encoding the polypeptide, or by peptide synthesis.
`Such variants include, for example, deletions from, and/or
`insertions into and/or substitutions of, residues within the
`amino acid sequence of the polypeptide of interest. Any
`combination of deletion, insertion, and substitution is made
`to arrive at the final construct, provided
`that the final
`construct possesses the desired characteristics. The amino
`acid changes also may alter post-translational processes of
`the polypeptide, such as changing the number or position of
`glycosylation sites. Methods for generating amino acid
`sequence variants of polypeptides are described in U.S. Pat.
`No. 5,534,615, expressly incorporated herein by reference,
`for example.
`
`20
`
`30
`
`35
`
`40
`
`45
`
`An "acidic variant" is a variant of a polypeptide of interest
`which is more acidic (e.g. as determined by cation exchange
`chromatography)
`than
`the polypeptide of
`interest. An
`example of an acidic variant is a deamidated variant.
`A "deamidated" variant of a polypeptide molecule is a
`polypeptide wherein one or more asparagine residue(s) of
`the original polypeptide have been converted to aspartate, 55
`i.e. the neutral amide side chain has been converted to a
`residue with an overall acidic character. Deamidated
`humMAb4D5 antibody from the Example below has Asn30
`regions
`thereof
`in CDR1 of either or both of the \L
`converted
`to aspartate. The
`term "deamidated human 6Q
`DNase" as used herein means human DNase that is deami(cid:173)
`dated at the asparagine residue that occurs at position 74 in
`the amino acid sequence of native mature human DNase
`(U.S. Pat. No. 5,279,823; expressly incorporated herein by
`reference).
`The term "mixture" as used herein in reference to a
`composition comprising an anti-HER2 antibody, means the
`
`65
`
`presence of both the desired anti-HER2 antibody and one or
`more acidic variants thereof. The acidic variants may com(cid:173)
`prise predominantly deamidated anti-HER2 antibody, with
`minor amounts of other acidic variant(s). It has been found,
`for example, that in preparations of anti-HER2 antibody
`obtained from recombinant expression, as much as about
`25% of the anti-HER2 antibody is deamidated.
`In preferred embodiments of the invention, the polypep(cid:173)
`tide is a recombinant polypeptide. A "recombinant polypep(cid:173)
`tide" is one which has been produced in a host cell which has
`been transformed or transfected with nucleic acid encoding
`the polypeptide, or produces the polypeptide as a result of
`homologous recombination. "Transformation" and "trans-
`fection" are used interchangeably to refer to the process of
`introducing nucleic acid into a cell. Following transforma(cid:173)
`tion or transfection, the nucleic acid may integrate into the
`host cell genome, or may exist as an extrachromosomal
`element. The "host cell" includes a cell in vitro cell culture
`as well a cell within a host animal. Methods for recombinant
`production of polypeptides are described in U.S. Pat. No.
`5,534,615, expressly incorporated herein by reference, for
`example.
`
`The term "antibody" is used in the broadest sense and
`specifically covers monoclonal antibodies (including full
`length monoclonal antibodies), polyclonal antibodies, mul-
`tispecific antibodies (e.g., bispecific antibodies), and anti(cid:173)
`body fragments so long as they exhibit the desired biological
`activity.
`The antibody herein is directed against an "antigen" of
`interest. Preferably, the antigen is a biologically important
`polypeptide and administration of the antibody to a mammal
`suffering from a disease or disorder can result in a thera(cid:173)
`peutic benefit in that mammal. However, antibodies directed
`against nonpolypeptide antigens (such as tumor-associated
`glycolipid antigens; see U.S. Pat. No. 5,091,178) are also
`contemplated. Where the antigen is a polypeptide, it may be
`a transmembrane molecule (e.g. receptor) or ligand such as
`a growth factor. Exemplary antigens include those polypep(cid:173)
`tides discussed above. Preferred molecular targets for anti(cid:173)
`bodies encompassed by the present invention include CD
`polypeptides such as CD3, CD4, CDS, CD19, CD20 and
`CD34; members of the HER receptor family such as the
`EGF receptor, HER2, HER3 or HER4 receptor; cell adhe(cid:173)
`sion molecules such as LFA-1, Macl, pl50,95, VLA-4,
`ICAM-1, VCAM and av/b3 integrin including either a or b
`subunits thereof (e.g. anti-CDlla, anti-CD18 or anti-CDllb
`antibodies); growth factors such as VEGF; IgE; blood group
`antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl
`receptor; CTLA-4; polypeptide C etc. Soluble antigens or
`fragments thereof, optionally conjugated to other molecules,
`can be used as immunogens for generating antibodies. For
`transmembrane molecules, such as receptors, fragments of
`these (e.g. the extracellular domain of a receptor) can be
`used as the immunogen. Alternatively, cells expressing the
`transmembrane molecule can be used as the immunogen.
`Such cells can be derived from a natural source (e.g. cancer
`cell lines) or may be cells which have been transformed by
`recombinant techniques to express the transmembrane mol(cid:173)
`ecule.
`
`The term "monoclonal antibody" as used herein refers to
`an antibody obtained from a population of substantially
`homogeneous antibodies, i.e., the individual antibodies
`comprising the population are identical except for possible
`naturally occurring mutations that may be present in minor
`amounts. Monoclonal antibodies are highly specific, being
`directed against a single antigenic site. Furthermore, in
`contrast to conventional (polyclonal) antibody preparations
`
`

`

`Case 1:18-cv-01363-CFC Document 1-10 Filed 09/04/18 Page 13 of 24 PageID #:
`445
`
`US 6,417,335 Bl
`
`which typically include different antibod