Case 1:18-cv-01363-CFC Document 1-17 Filed 09/04/18 Page 1 of 27 PageID #: 574
`Case 1:18-cv-01363-CFC Document1-17
`Filed 09/04/18
`Page 1 of 27 PagelD #: 574
`
`
`
`EXHIBIT Q
`EXHIBIT Q
`
`
`
`

`

`Case 1:18-cv-01363-CFC Document 1-17 Filed 09/04/18 Page 2 of 27 PageID #: 575
`
`US007485704B2
`
`(12) United States Patent
`Fahrner et al.
`
`(io) Patent No.:
`(45) Date of Patent:
`
`US 7,485,704 B2
`Feb.3, 2009
`
`(54) REDUCING PROTEIN A LEACHING DURING
`PROTEIN A AFFINITY CHROMATOGRAPHY
`
`(75) Inventors: Robert L. Fahrner, San Mateo, CA
`(US); Amy Laverdiere, San Francisco,
`CA (US); Paul J. McDonald, San
`Francisco, CA (US); Rhona M.
`O'Leary, San Francisco, 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 257 days.
`
`(21) Appl.No.: 10/877,532
`
`(22) Filed: Jun. 24, 2004
`
`(65) Prior Publication Data
`
`US 2005/0038231 Al Feb. 17, 2005
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/490,500, filed on Jul.
`28, 2003.
`
`(51) Int. CI.
`C07K16/00 (2006.01)
`(52) U.S. CI 530/413; 530/412; 530/387.1
`(58) Field of Classification Search None
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
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`Schuler et al., "Development and optimization of a single-step pro-
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`Chromatography pp. 61-70 (1991).
`Tu et al., "Temperature affects binding of murine monoclonal IgG
`antibodies to protein A" Journal of Immunological Methods 109:43-
`47(1988).
`van Sommeren et al., "Effects of temperature, flow rate and compo-
`sition of binding buffer on adsorption of mouse monoclonal IgG!
`antibodies to protein A Sepharose 4 Fast Flow" Preparative Bio-
`chemistry 22(2):135-149 (Jun. 1992).
`Carter et al., "Humanization of an Anti-pl85HER2 Antibody For
`Human Cancer Therapy" Proc. Natl. Acad. Sci. USA 89:4285-4289
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`Kim et al., "The Vascular Endothelial Growth Factor Proteins: Iden-
`tification of Biologically Relevant Regions by Neutralizing
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`Factor Monoclonal Antibody for the Therapy of Solid Tumors and
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`* cited by examiner
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`Dreger; Goodwin Procter LLP
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`FOREIGN PATENT DOCUMENTS
`
`(57)
`
`ABSTRACT
`
`WO
`wo
`wo
`wo
`wo
`wo
`wo
`
`95/22389
`WO 96/30046
`WO 98/23645
`WO 98/23761
`WO 98/45331
`WO 01/00245
`WO 03/041859
`
`8/1995
`10/1996
`6/1998
`6/1998
`10/1998
`1/2001
`5/2003
`
`A method for reducing leaching of protein A during protein A
`affinity chromatography is described which involves reduc-
`ing temperature or pH of, or by adding one or more protease
`inhibitors to, a composition that is subjected to protein A
`affinity chromatography.
`
`12 Claims, 6 Drawing Sheets
`
`

`

`Case 1:18-cv-01363-CFC Document 1-17 Filed 09/04/18 Page 3 of 27 PageID #: 576
`
`U.S. Patent
`
`Feb.3, 2009
`
`Sheet 1 of 6
`
`US 7,485,704 B2
`
`£
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`

`Case 1:18-cv-01363-CFC Document 1-17 Filed 09/04/18 Page 4 of 27 PageID #: 577
`
`U.S. Patent
`
`Feb.3, 2009
`
`Sheet 2 of 6
`
`US 7,485,704 B2
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`

`

`Case 1:18-cv-01363-CFC Document 1-17 Filed 09/04/18 Page 5 of 27 PageID #: 578
`
`U.S. Patent Feb. 3,2009 Sheet 3 of 6 US 7,485,704 B2
`Feb. 3,2009
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`Case 1:18-cv-01363-CFC Document 1-17 Filed 09/04/18 Page 6 of 27 PageID #: 579
`
`U.S. Patent
`
`Feb.3, 2009
`
`Sheet 4 of 6
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`US 7,485,704 B2
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`

`Case 1:18-cv-01363-CFC Document 1-17 Filed 09/04/18 Page 7 of 27 PageID #: 580
`
`U.S. Patent Feb. 3,2009 Sheet 5 of 6 US 7,485,704 B2
`
`DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYR
`YTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK
`(SEQ ID NO: 3)
`
`FIG.SA
`
`EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPN
`SGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDY
`WGQGTLVTVSS (SEQ ID NO: 4)
`
`DIQMTQSPSSLSASVGDRVTITCRASKTISKYLAWYQQKPGKAPKLLIYSGSTL
`QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPLTFGQGTKVEIKR
`(SEQ ID NO: 5)
`
`FIG.-6A
`
`EVQLVESGGGLVQPGGSLRLSCAASGYSFTGHWMNWVRQAPGKGLEWVGMIHPS
`DSETRYNQKFKDRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARGIYFYGTTYF
`DYWGQGTLVTVSS (SEQ ID NO: 6)
`
`DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSL
`HSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKR
`(SEQ ID NO: 7)
`
`FIG. .7A
`
`EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTY
`TGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHW
`YFDVWGQGTLVTVSS (SEQ |D N0: 8)
`FIG.-7B
`
`

`

`Case 1:18-cv-01363-CFC Document 1-17 Filed 09/04/18 Page 8 of 27 PageID #: 581
`
`U.S. Patent
`
`Feb.3, 2009
`
`Sheet 6 of 6
`
`US 7,485,704 B2
`
`E
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`
`FIG.. 9
`
`

`

`Case 1:18-cv-01363-CFC Document 1-17 Filed 09/04/18 Page 9 of 27 PageID #: 582
`
`US 7,485,704 B2
`
`REDUCING PROTEIN A LEACHING DURING
`PROTEIN A AFFINITY CHROMATOGRAPHY
`
`This is a non-provisional application claiming priority
`under 35 USC §119 to provisional application No. 60/490, 5
`500 filed Jul. 28, 2003, the entire disclosure of which is
`hereby incorporated by reference.
`
`BACKGROUND OF THE INVENTION
`
`10
`
`1. Field of the Invention
`The present invention concerns protein purification. In par-
`ticular, the invention concerns a method for reducing leaching
`of protein A during protein A affinity chromatography by
`reducing temperature or pH of, or by adding one or more 15
`protease inhibitors to, a composition that is subjected to pro-
`tein A affinity chromatography.
`2. Description of Related Art
`The large-scale, economic purification of proteins is
`increasingly an important problem for the biotechnology 20
`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 25
`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 sufficient for use 30
`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 intracel- 35
`lularly. For the latter proteins, the first step of a 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 addi- 40
`tion produces subcellular fragments that are difficult to
`remove due to their small size. These are generally removed
`by differential centrifugation or by filtration. The same prob-
`lem arises, although on a smaller scale, with directly secreted
`proteins due to the natural death of cells and release of intra- 45
`cellular host cell proteins in the course of the protein produc-
`tion 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 combination 50
`of different chromatography techniques. These techniques
`separate mixtures of proteins on the basis of their charge,
`degree of hydrophobicity, or size. Several different chroma-
`tography resins are available for each of these techniques,
`allowing accurate tailoring of the purification scheme to the 55
`particular protein involved. The essence of 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, 60
`being then differentially eluted by different solvents. In some
`cases, the desired protein is separated 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." 65
`Affinity chromatography, which exploits a specific inter-
`action between the protein to be purified and an immobilized
`
`capture agent, may also be an option for some proteins. Pro-
`tein A is a useful adsorbent for affinity chromatography of
`proteins, such as antibodies, which contain an Fc region.
`Protein A is a 41 kD cell wall protein from Staphylococcus
`aureas which binds with a high affinity (about 10~8M to
`human IgG) to the Fc region of antibodies.
`U.S. Pat. Nos. 6,127,526 and 6,333,398 (Blank, G.)
`describe an intermediate wash step during protein A affinity
`chromatography using hydrophobic electrolytes, e.g., tetram-
`ethylammonium chloride (TMAC) and tetraethylammonium
`chloride (TEAC), to remove the impurities, but not the immo-
`bilized protein A or the protein of interest, bound to the
`protein A column.
`
`SUMMARY OF THE INVENTION
`
`The present invention concerns a method of purifying a
`protein which comprises a CH2ICH'S region, comprising
`reducing the temperature of a composition comprising the
`protein and one or more impurities subjected to protein A
`affinity chromatography in the range from about 3° C. to
`about 20° C, wherein protein A leaching is reduced.
`Preferably the protein is an antibody, e.g. one which binds
`an antigen selected from the group consisting of HER2, vas-
`cular endothelial growth factor (VEGF), IgE, CD20, CD40,
`CD1 la, tissue factor (TF), prostate stem cell antigen (PSCA),
`interleukin-8 (IL-8), epidermal growth factor receptor
`(EGFR), HER3, HER4, a4p7 or a5p3. In another embodi-
`ment, the protein is an immunoadhesin, such as a TNF recep-
`tor immunoadhesin.
`The invention also concerns a method of purifying a pro-
`tein which comprises a CH2ICH'S region by protein A affinity
`chromatography comprising:
`(a) subjecting the protein to protein A affinity chromatogra-
`phy and measuring leached protein A in a composition
`comprising the protein which is recovered from the protein
`A affinity chromatography;
`(b) if protein A leaching is detected in step (a), reducing the
`temperature of a composition comprising the protein and
`one or more impurities subjected to protein A affinity chro-
`matography in the range from about 3° C. to about 20° C,
`such that protein A leaching is reduced.
`The invention further provides a method for reducing
`leaching of protein A during protein A affinity chromatogra-
`phy comprising reducing protease activity in a composition
`subjected to protein A affinity chromatography, wherein the
`composition comprises a protein which comprises a CH2ICH'S
`region and one or more proteases.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 depicts protein A leaching as a function of tempera-
`ture for various antibody products on PROSEP A•. Leached
`protein A is shown in ng/mg (ng protein A per mg antibody).
`Temperature on the x-axis refers to the temperature of the
`water bath. The column was equilibrated and washed with 25
`mM Tris, 25 mM NaCl, 5 mM EDTA, pH 7.1, washed with 25
`mMTris, 25 mMNaCl, 0.5 MTMAC, 5 mMEDTApH 5.0or
`7.1, eluted with either 25 mM citrate pH 2.8, orO.l Macetic
`acid pH 2.9, regenerated with 0.1 M phosphoric acid, and
`stored in 0.2 M sodium acetate, 2% benzyl alcohol pH 5.0.
`Trastuzumab was run on a bed height of 20 cm, loaded to 20
`g Trastuzumab/L resin, washed with TMAC pH 5.0, eluted
`with25mM citrate pH2.8,andpooledfrom0.1AU to 2 CV's.
`Humanized 2C4 was run on a 20 cm bed height column,
`loaded to 15 g humanized 2C4 per liter resin, washed with
`TMAC pH 7.1, eluted with 25 mM citrate pH 2.8, and pooled
`
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`10
`
`from 0.1 AU to 2 CV's pool volume. Humanized VEGF
`antibody was run on 14 cm bed height, loaded to 20 g human-
`ized VEGF antibody per liter of resin, washed with TMAC pH
`5.0, eluted with 0.1 M acetic acid pH 2.9, and pooled from 0.2
`AU to 2 CV's pool volume. Humanized GDI la antibody was 5
`run on a 14 cm bed height, loaded to 20 g humanized GDI la
`antibody per liter of resin, washed with TMAC pH 7.1, eluted
`with 0.1 M acetic acid pH 2.9, and pooled from 0.2 AU to
`2CV's.
`FIG. 2 depicts a comparison of temperature dependent
`protein A leaching from PROSEP A• and PROSEP vA•
`with Trastuzumab, humanized 2C4, and humanized GDI la
`antibody. Leached protein A is shown in ng/mg (ng protein A
`per mg antibody). Temperature on the x-axis refers to the 15
`temperature of the water bath. All columns were 0.66 cm in
`diameter and either 14 cm or 20 cm in height. One lot of
`harvested cell culture fluid (HCCF) was used for each pair of
`runs. The column was equilibrated and washed with 25 mM
`Tris, 25 mM NaCl, 5 mM EDTA, pH 7.1, washed with 25 mM 20
`Tris, 25 mMNaCl, 0.5 MTMAC, 5 mMEDTApH 5.0 orV.l,
`eluted with either 25 mM citrate pH 2.8, or 0.1 M acetic acid
`pH 2.9, regenerated with 0.1 M phosphoric acid, and stored in
`0.2 M sodium acetate, 2% benzyl alcohol pH 5.0 at 40 CV/hr.
`Humanized CD 11a antibody was run on a 14 cm bed height, 25
`loaded to 20 g humanized CD1 la antibody per liter of resin,
`washed with TMAC pH 7.1, eluted with 0.1 M acetic acid pH
`2.9, and pooled from 0.2 AU to 2CV's. Humanized 2C4 was
`run on a 20 cm bed height column, loaded to 15 g humanized
`2C4 per liter resin, washed with TMAC pH 7.1, eluted with 25
`mM citrate pH 2.8, and pooled from 0.1 AU to 2 CV's pool
`volume. Trastuzumab (from pilot plant at 400L scale at con-
`centration of 0.57 mg/ml) was run on a bed height of 20 cm,
`loaded to 20 g Trastuzumab/L resin, washed with TMAC pH 35
`5.0, eluted with 25 mM citrate pH 2.8, and pooled from 0.1
`AU to 2 CV's.
`FIG. 3 depicts protein A leaching at pilot scale versus
`temperature. Leached protein A is shown in ng/mg (ng pro-
`tein A per mg antibody). Temperature on the x-axis refers to 40
`the set temperature of the HCCF tank. The column was
`packed with 1.26 L PROSEP vA•, 9 cm in diameter by 20
`cm in height. Trastuzumab HCCF was at 0.59 mg/ml, and the
`temperature of the HCCF in the tank was maintained at 10,15,
`20,25, or 30° C. The column was loaded to 20 g Trastuzumab
`per liter of resin. Temperature was measured in the HCCF
`tank, between the pump and the column, and at the outlet to
`the column. The column was equilibrated and washed with 25
`mM Tris, 25 mM NaCl, 5 mM EDTA, pH 7.1, washed with 25
`mM Tris, 25 mM NaCl, 0.5 M TMAC, 5 mM EDTA pH 5.0,
`eluted with either 25 mM citrate pH 2.8, regenerated with 0.1
`M phosphoric acid, and stored in 0.2 M sodium acetate, 2%
`benzyl alcohol pH 5.0. A sample of each HCCF was taken and
`run at lab scale on a 0.66 cm diameter by 20 cm high column
`packed with PROSEP vA• using the same buffers as at pilot
`scale, represented on the graph by the circles.
`FIGS. 4A-B show the light chain amino acid sequence
`(SEQ ID NO:l) and heavy chain amino acid sequence (SEQ
`ID NO:2), respectively, of Trastuzumab (HERCEPTIN®). 60
`FIGS. 5A-B depict the amino acid sequences of the vari-
`able light (SEQ ID NO:3) and variable heavy (SEQ ID NO:4)
`domains, respectively, of a humanized 2C4.
`FIGS. 6A-B depict the amino acid sequences of the vari-
`able light (SEQ ID NO:5) and variable heavy (SEQ ID NO:6) 65
`domains, respectively, of a humanized CD 11a antibody RAP-
`TIVA•.
`
`50
`
`55
`
`FIGS. 7A-B depict the amino acid sequences of the vari-
`able light (SEQ ID NO:7) and variable heavy (SEQ ID NO:8)
`domains, respectively, of a humanized VEGF antibody
`AVASTIN•.
`FIG. 8 depicts the effect of EDTA and temperature on
`Protein A leaching.
`FIG. 9 depicts the effect of 4-(2-aminoethyl)-benzene-
`sulfonyl-fluoride, hydrochloride (AEBSF) (PEFABLOC®),
`a serine protease inhibitor, on Protein A leaching
`
`DETAILED DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`
`Definitions:
`When used herein, the term "protein A" encompasses pro-
`tein A recovered from a native source thereof, protein A
`produced synthetically (e.g. by peptide synthesis or by
`recombinant techniques), including variants or derivatives
`thereof which retain the ability to bind proteins which have a
`CH2ICH'i region. Protein A can be purchased commercially
`from Repligen, Pharmacia and Fermatech.
`"Protein A affinity chromatography" refers to the separa-
`tion or purification of substances and/or particles using pro-
`tein A, where the protein A is generally immobilized on a
`solid phase. A protein comprising a 0^2/0^3 region may be
`reversibly bound to, or adsorbed by, the protein A. Examples
`of protein A affinity chromatography columns for use in pro-
`tein A affinity chromatography herein include protein A
`immobilized onto a controlled pore glass backbone, includ-
`ing the PROSEP A• and PROSEP vA• columns (Millipore
`Inc.); protein A immobilized on a polystyrene solid phase,
`e.g. the POROS 50A• column (Applied BioSystems Inc.);
`or protein A immobilized on an agarose solid phase, for
`instance the rPROTEIN A SEPHAROSE FAST FLOW• or
`MABSELECT• columns (Amersham Biosciences Inc.).
`By "solid phase" is meant a non-aqueous matrix to which
`the protein A can adhere or be covalently bound. The solid
`phase may comprise a glass, silica, polystyrene, or agarose
`surface for immobilizing the protein A, for instance. The solid
`phase may be a purification column, discontinuous phase of
`discrete particles, packed bed column, expanded bed column,
`membrane, etc.
`Herein, "leaching" refers to the detachment or washing of
`protein A (including fragments thereof) from a solid phase to
`which it is bound. Leaching may result from various mecha-
`nisms such as mechanical shearing, low pH exposure, pro-
`teolytic activity etc.
`An "impurity" is a material that is different from the
`desired protein product. The impurity may be a viral impurity,
`a variant of the desired protein or another protein, nucleic
`acid, endotoxin etc. Specific examples of impurities herein
`include proteins from the host cell producing the desired
`protein (e.g. Chinese Hamster Ovary proteins, CHOP, where
`the host cell is a CHO cell), protease(s), leached protein A etc.
`"Proteases" are proteolytic enzymes including, but not lim-
`ited to, serine, cysteine, metallo- and aspartic proteases. Pro-
`teases present in a composition comprising a protein of inter-
`est may be derived from a recombinant host producing the
`protein, or from a natural source of the protein. Examples of
`proteases include thermolysin, trypsin, chymotrypsin, plas-
`min, kallikrein, thrombin, papain, plasmin, cathepsin B,
`renin, chymosin etc.
`"Protease activity" refers to the enzymatic activity of one
`or more proteases. Such activity may be measured indirectly
`by measuring leaching of protein A, for instance. The activity
`may be reduced by reducing temperature of a composition
`
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`
`comprising the protease(s), and/or by adding one or more
`protease inhibitors to the composition etc.
`A "protease inhibitor" is a compound or composition
`which reduces, to some extent, the enzymatic activity of
`protease(s). Examples of protease inhibitors include phenyl- 5
`methylsulfonyl fluoride (PMSF), 4-(2-aminoethyl)-benzene-
`sulfonyl-fluoride, hydrochloride (AEBSF) (PEFABLOC®
`SC), leupeptin, pepstatin, benzamidine, a metal ion chelator
`such as EDTA or imidazole for inhibiting metalloprotease
`activity etc. The preferred protease inhibitors inhibit metal- 10
`loprotease activity (e.g. EDTA) and/or inhibit certain serine
`protease activities.
`The protein of interest herein is one which comprises a
`CH2ICH'S region and therefore is amenable to purification by
`protein A affinity chromatography. The term "C^Z/Cf^ 15
`region" when used herein refers to those amino acid residues
`in the Fc region of an immunoglobulin molecule which inter-
`act with protein A. In preferred embodiments, the 0^2/0^3
`region comprises an intact CH2 region followed by an intact
`0^3 region, and most preferably comprises a Fc region of an 20
`immunoglobulin. Examples of CH2ICH'S region-containing
`proteins include antibodies, immunoadhesins and fusion pro-
`teins comprising a protein of interest fused to, or conjugated
`with, a CH2ICH'i region.
`The term "antibody" is used in the broadest sense and 25
`specifically covers monoclonal antibodies (including full
`length monoclonal antibodies), polyclonal antibodies, multi-
`specific antibodies (e.g., bispecific antibodies), and antibody
`fragments so long as they retain, or are modified to comprise,
`a CH2ICH'S region as herein defined. 30
`"Antibody fragments" comprise a portion of a full length
`antibody, generally the antigen binding or variable region
`thereof. Examples of antibody fragments include Fab, Fab',
`F(ab')2, and Fv fragments; single-chain antibody molecules;
`diabodies; linear antibodies; and multispecific antibodies 35
`formed from antibody fragments.
`The term "monoclonal antibody" as used herein refers to
`an antibody obtained from a population of substantially
`homogeneous antibodies, i.e., the individual antibodies com-
`prising the population are identical except for possible natu- 40
`rally occurring mutations that may be present in minor
`amounts. Monoclonal antibodies are highly specific, being
`directed against a single antigenic site. Furthermore, in con-
`trast to conventional (polyclonal) antibody preparations
`which typically include different antibodies directed against 45
`different determinants (epitopes), each monoclonal antibody
`is directed against a single determinant on the antigen. The
`modifier "monoclonal" indicates the character of the anti-
`body as being obtained from a substantially homogeneous
`population of antibodies, and is not to be construed as requir- 50
`ing production of the antibody by any particular method. For
`example, the monoclonal antibodies to be used in accordance
`with the present invention may be made by the hybridoma
`method first described by Kohler et al., Nature 256:495
`(1975), or may be made by recombinant DNA methods (see, 55
`e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies"
`may also be isolated from phage antibody libraries using the
`techniques described in Clackson et al., Nature 352:624-628
`(1991) and Marks et al, J. Mo/. Biol. 222:581-597 (1991), for
`example. 60
`The monoclonal antibodies herein specifically include
`"chimeric" antibodies (immunoglobulins) in which a portion
`of the heavy and/or light chain is identical with or homolo-
`gous to corresponding sequences in antibodies derived from a
`particular species or belonging to a particular antibody class 65
`or subclass, while the remainder of the chain(s) is identical
`with or homologous to corresponding sequences in antibod-
`
`ies derived from another species or belonging to another
`antibody class or subclass, as well as fragments of such anti-
`bodies, so long as they exhibit the desired biological activity
`(U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl.
`Acad. Sci. USA 81:6851-6855 (1984)).
`The term "hypervariable region" when used herein refers
`to the amino acid residues of an antibody which are respon-
`sible for antigen-binding. The hypervariable region com-
`prises amino acid residues from a "complementarity deter-
`mining region" or "CDR" (i.e. residues 24-34 (LI), 50-56
`(L2) and 89-97 (L3) in the light chain variable domain and
`31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain
`variable domain; Kabat et al. Sequences of Proteins of Immu-
`nological Interest, 5th Ed. Public Health Service, National
`Institutes of Health, Bethesda, Md. (1991)) and/or those resi-
`dues from a "hypervariable loop" (i.e. residues 26-32 (LI),
`50-52 (L2) and 91-96 (L3) in the light chain variable domain
`and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy
`chain variable domain; Chothia and Lesk J. Mol. Biol. 196:
`901-917 (1987)). "Framework" or "FR" residues are those
`variable domain residues other than the hypervariable region
`residues as herein defined.
`"Humanized" forms of non-human (e.g., murine) antibod-
`ies are chimeric antibodies which contain minimal sequence
`derived from non-human immunoglobulin. For the most part,
`humanized antibodies are human immunoglobulins (recipi-
`ent antibody) in which hypervariable region residues of the
`recipient are replaced by hypervariable region residues from
`a non-human species (donor antibody) such as mouse, rat,
`rabbit or nonhuman primate having the desired specificity,
`affinity, and capacity. In some instances, Fv framework
`region (FR) residues of the human immunoglobulin are
`replaced by corresponding non-human residues. Further-
`more, humanized antibodies may comprise residues which
`are not found in the recipient antibody or in the donor anti-
`body. These modifications are made to further refine antibody
`performance. In general, the humanized antibody will com-
`prise substantially all of at least one, and typically two, vari-
`able domains, in which all or substantially all of the hyper-
`variable loops correspond to those of a non-human
`immunoglobulin and all or substantially all of the FR regions
`are those of a human immunoglobulin sequence. The human-
`ized antibody optionally also will comprise at least a portion
`of an immunoglobulin constant region (Fc), typically that of
`a human immunoglobulin. For further details, see Jones et al.
`Nature 321:522-525 (1986); Riechmann et al. Nature 332:
`323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596
`(1992).
`As used herein, the term "immunoadhesin" designates
`antibody-like molecules which combine the "binding
`domain" of a heterologous "adhesin" protein (e.g. a receptor,
`ligand or enzyme) with the effector functions of an immuno-
`globulin constant domain. Structurally, the immunoadhesins
`comprise a fusion of the adhesin amino acid sequence with
`the desired binding specificity which is other than the antigen
`recognition and binding site (antigen combining site) of an
`antibody (i.e. is "heterologous") and an immunoglobulin con-
`stant domain sequence. The immunoglobulin constant
`domain sequence in the immunoadhesin is preferably derived
`from yl, Y2, or ^4 heavy chains since immunoadhesins com-
`prising these regions can be purified by protein A affinity
`chromatography (Lindmarket al, J. Immunol. Meth. 62:1-13
`(1983)).
`The term "ligand binding domain" as used herein refers to
`any native cell-surface receptor or any region or derivative
`thereof retaining at least a qualitative ligand binding of a
`corresponding native receptor. In a specific embodiment, the
`
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`receptor is from a cell-surface polypeptide having an extra-
`cellular domain which is homologous to a member of the
`immunoglobulin supergenefamily. Other receptors, which
`are not members of the immunoglobulin supergenefamily but
`are nonetheless specifically covered by this definition, are 5
`receptors for cytokines, and in particular receptors with
`tyrosine kinase activity (receptor tyrosine kinases), members
`of the hematopoietin and n