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`Case 1:18-cv-00095-CFC Document1-3 Filed 01/12/18 Page 1 of 681 PagelD #: 870
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`EXHIBIT V
`EXHIBIT V
`
`

`

`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 2 of 681 PageID #: 871
`Case 1:18-cv-00095-CFC Document 1-3 Taal
` inna
`
`
`
`US 009428548B2
`
`(12) United States Patent
`US 9,428,548 B2
`(10) Patent No.:
`Aug. 30, 2016
`(45) Date of Patent:
`Brown et al.
`
`(54)
`
`(75)
`
`ENHANCED PROTEIN PURIFICATION
`THROUGH A MODIFIED PROTEIN A
`ELUTION
`
`Inventors: Arick Brown, South San Francisco, CA
`(US): Christopher J. Dowd. South San
`Francisco, CA (US); Asha Nandini
`Radhamohan, South San Fracisco, CA
`(US)
`
`2/1998 Robinson et al.
`5,721,108. A
`31998 Hudziak et al.
`5,725,856 A
`4/1998 Anderson etal.
`5,736,137 A
`2/2006 Jiao et al.
`2006'0039901 Al
`3/2007 Godavarti et al
`2007/0072307 Al
`7/2008 Lindner et al,
`2008/0164211 Al
`7/2008 Pan
`20080167450 Al
`8/2008 Fahrner et al,
`2008/0193981 Al
`2010/0331527 Al* 12/2010 Davis et al.
`............... §30/387.3
`
`POREIGN PATENT DOCUMENTS
`
`(73)
`
`Assignee:
`
`Genentech, Inc., South San Francisco,
`CA (US)
`
`Appl. No.:
`
`13/393,525
`
`PCT Piled:
`
`Sep. 1, 2010
`
`PCT No::
`
`PCT/US2010/047448
`
`6/ L986
`0 183 070 A2
`EP
`O/L9RG6
`0.183 070 A3
`EP
`EP
`0 1835 070 Bl
`6/1986
`EP
`0 244 234 A2
`[1/1987
`Notice:—Subject to any disclaimer, the term ofthis
`FP
`0 244 234 AS
`LL/L9R7
`patent
`is extended or adjusted under 35
`EP
`0 244 234 BI
`LL/1987
`U.S.C. 154(b) by 547 days.
`EP
`0 244 234 B2
`1L/1987
`EP
`0 308 936 A?
`3/1989
`EP
`0308 936 A3
`3/1989
`EP
`0 3508 936 Bl
`3/1989
`EP
`0 402 226 Al
`12/1990
`EP
`0 420 397 BI
`4/199]
`RU
`21456)0 Cl
`2/2000
`Wo
`S7/00195 Al
`{/1987
`Wo
`90/03430 Al
`4/1990)
`wo
`91}/00360 Al
`1/199]
`Wo
`92/20373. Al
`(1/1992
`Wo
`93/04173 Al
`3/1993
`wo
`93/08829 Al
`5/1993
`WO
`95/1918) Al
`7/1995
`wo
`92/23865 Al
`9/1995
`Wo
`96/2701) Al
`1996
`Wo
`96/30046 Al
`10/1996
`Wo
`96/40210 Al
`12/1996
`wo
`97/26912 A2
`7/1997
`WO
`97/26912 A3
`T1997
`
`§ 371 (c)Q),
`(2). (4) Date:
`
`Jul, 16, 2012
`
`PCT Pub. No.: WO2011/028753
`
`PCT Pub. Date: Mar. 10, 2011
`
`Prior Publication Data
`
`US 2013/0041139 Al
`
`Feb, 14, 2013
`
`(87)
`
`(65)
`
`(60)
`
`(51)
`
`(52)
`
`(58)
`
`(56)
`
`Related U.S. Application Data
`
`Provisional application No. 61/238,867, filed on Sep.
`1, 2009, provisional application No, 61/253,438,filed
`on Oct. 20, 2009.
`
`(2006.01 )
`
`tnt. Ch
`CO7K 1/22
`U.S. Cl.
`EPS Aavpiekdaeerteiepestecrsemnn seca! OAR TZ22 (O18 OT}
`Field of Classification Search
`None
`See application file for complete search history,
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`RE30.985 E
`4,515,893 A
`4,560,655 A
`4,657,566 A
`4,676,980 A
`4,767,704 A
`4816,567 A
`4,927,762 A
`5.091.178 A
`S091313 A
`5,122,469 A
`5,434,615 A
`$622,700 A
`3,672,347 A
`5,693,762 A
`5.714.338 A
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`
`Primary Examiner — Daniel E Kolker
`Assistant Examiner — James Rogers
`(74) Atterney, Agent, or Firm — Morrison & Foerster LLP
`
`(37)
`
`ABSTRACT
`
`invention provides methods for purifying 4
`The present
`polypeptide comprising a CH2/CH3 region, comprising
`binding the polypeptide to Proiein A and eluting with a pH
`gradient starting al a low pH.
`
`73 Claims, 21 Drawing Sheets
`
`

`

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`Wo
`WO
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`WO
`WO
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`9806248 AD
`98/06248 A3
`98/23761 Al
`OR/4533L A2
`O8/45331 AS
`98/51793 AL
`99/01556 A2
`99/O1556 Ad
`OO/7S348 Al
`OO/FS348 Cl
`01/00245 A2
`01/00245 AS
`0140309 A2
`0140309 AS
`0140309 Cl
`2008/025747 Al
`WO-2008/085988 Al
`
`2/1998
`2/1998
`6/1998
`10/1998
`10/1998
`IL 1998
`1/1999
`1/1999
`12/2000
`12/2000
`1/2001
`12001
`6/2001
`6/2001
`6/200)
`3/2008
`7/2008
`
`OTHER PUBLICATIONS
`
`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 3 of 681 PageID #: 872
`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 3 of 681 PagelD #: 872
`
`US 9,428,548 B2
`Page 2
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`

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`
`

`

`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 5 of 681 PageID #: 874
`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 5 of 681 PagelD #: 874
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`U.S. Patent
`
`Aug, 30, 2016
`
`Sheet 1 of 21
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`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 6 of 681 PageID #: 875
`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 6 of 681 PagelD #: 875
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`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 7 of 681 PagelD #: 876
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`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 15 of 681 PagelD #: 884
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`U.S. Patent
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`Aug. 30,2016
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`Sheet 11 of 21
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`US 9,428,548 B2
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`Figure 10
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`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 16 of 681 PageID #: 885
`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 16 of 681 PagelD #: 885
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`U.S. Patent
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`Aug, 30, 2016
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`Sheet 12 of 21
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`US 9,428,548 B2
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`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 17 of 681 PageID #: 886
`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 17 of 681 PagelD #: 886
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`U.S. Patent
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`Aug, 30, 2016
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`Sheet 13 of 21
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`US 9,428,548 B2
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`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 18 of 681 PageID #: 887
`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 18 of 681 PagelD #: 887
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`Aug, 30, 2016
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`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 20 of 681 PageID #: 889
`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 20 of 681 PagelD #: 889
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`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 21 of 681 PagelD #: 890
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`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 22 of 681 PageID #: 891
`Case 1:18-cv-00095-CFC Document 1-3 Filed 01/12/18 Page 22 of 681 PagelD #: 891
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`Anti-VEGFantibody#1andRVLPElution:LRVperfraction
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`Fractions
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`1
`ENHANCED PROTEIN PURIFICATION
`THROUGH A MODIFIED PROTEIN A
`ELUTION
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`‘This patent application is submitted under 35 U.S.C. §371
`as a U.S. national stage application of International Patent
`Application No. PC'T/US2010/047448,filed on Sep. 1, 2010
`which claims priority to U.S. Provisional Patent Application
`No. 61/238,867,
`filed Sep. 1, 2009 and ULS. Provisional
`Patent Application No, 61/253,438 filed Oct. 20, 2009, the
`disclosure of each of which are incorporated herein by
`reference in their entirety.
`
`FIELD OF THE INVENTION
`
`‘The field ofthis invention relates generally to methods for
`purifying a polypeptide comprising a C,,2/C,,3 region, com-
`prising binding the polypeptide to Protein A andeluting with
`a pHgradient.
`
`BACKGROUND OF THE INVENTION
`
`The large-scale, economic purification of proteins is
`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 proteinofinterest by insertion ofa 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 lactors, usually supplied from preparations of
`animal serum, Separation of the desired protein from the
`nuxture of compounds fed to the cells and from the by-
`products of the cells themselves to a purity suflicient 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.
`Someproteins 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 ofa
`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 ofthe cel] into the homogenate,
`and in addition produces subcellular fragments that are
`difficult to remove due to their small size. These are gener-
`ally removed by differential centrifugation or by filtration.
`The same problemarises, although on a smaller scale, with
`directly secreted proteins due to the natural death ofcells
`and release of intracellular host cell proteins in the course of
`the protein production run.
`Once a clarified solution containing the protein ofinterest
`has been obtained.
`its separation from the other proteins
`produced by the cell is usually attempted using a combina-
`tion of different chromatography techniques. Affinity chro-
`matography, which exploits a specific interaction between
`the protein to be purified and an immobilized capture agent,
`is commonly used for some proteins (e.g., proteins for use
`as a human therapeutic). Protein A is a useful adsorbent for
`aflinity chromatography of proteins, such as antibodies.
`which contain an Pe region. Protein A is a 41 kD cell wall
`protein from Staphylococcus areas which binds with a high
`allinity (about 10-" M to human IgG) to the Fe region of
`antibodies. However, since proteins tend to aggregate or
`
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`the desired protein (i.c.. monomer) is
`become misfolded,
`often co-purified with other impurities from these aflinity
`columns, such as protein aggregates, by-products ofthe cells
`themselves (i.e,. host cell impurities), or virus filter foulant,
`Other techniques have been developed to further separate
`these impurities and mixtures of proteins on the basis oftheir
`charge, degree of hydrophobicity. or size, such as ton
`exchange chromatography, hydrophobic interaction chroma-
`tography. or size exclusion chromatography. Several difler-
`ent chromatographyresins or sorbents are available for each
`ofthese techniques, allowing accurate tailoring of the puri-
`fication scheme {o
`the particular protein involved. The
`essence of each ofthese separation methods is that proteins
`can be caused either to move at different rates down a long
`solid phase (e.g,. column), achieving a physical separation
`thal increases as they pass further down the solid phase. or
`to adhere selectively to the separation medium, being then
`differentially eluted by different solvents. However. eachof
`these methods requires additional bullers. resins or sorbents,
`and other resources for further purification, and this in turn
`results in longer processing lime and higher cost. Thus, more
`efhcient and economical methods for purifying protein
`monomers are needed.
`Methods of purifying polypeptides from aggregates, mul-
`timers, and modified proteins using a protein A column and
`eluting with a pH gradient elution system was described in
`U.S. patent application Ser. No. 12/008,160.
`All publications, patents, and patent applications cited
`herein are hereby incorporated by reference herein in their
`entirety for all pur