(12) United States Patent
`Lam et al.
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 6,171,586 B1
`Jan. 9, 2001
`
`US006171586B1
`
`(54) ANTIBODY FORMULATION
`
`(75) Inventors: Xanthe M. Lam, San Francisco; James
`Q. Oeswein, Moss Beach, both of CA
`(US); Boonsri Ongpipattanakul,
`Bangkok (TH); Zahra Shahrokh, San
`Francisco; Sharon X. Wang, San
`Mateo, both of CA (US); Robert P.
`Weissburg, Greenville, DE (US); Rita
`L- Wong, San Mateo, CA (Us)
`
`_
`_
`(73) Asslgnee' ginelljléech’ Inc" South San Franclsco’
`(
`)
`Under 35 U.S.C. 154(b), the term of this
`patent Shall be extended for 0 days_
`
`( * ) Notice:
`
`(21) APPL NO; 09/097,171
`_
`(22) Flled?
`Jun- 12, 1998
`,
`,
`J
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`(60) P _
`1532mm“ app lea Ion
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`'
`
`13
`’
`
`7
`(51) Int. Cl. ........................ .. A61K 39/395; C12P 21/08
`
`(52) US. Cl. ................................... .. 424/1301; 424/1521;
`424/1411; 424/1541; 424/173.1; 530/388.75
`
`_
`(58) Fleld of Search ............................ .. 424/ 130.1, 152.1,
`424/1361, 1411, 1541, 1731, 133-1; 530/38875
`.
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`_
`(Llst contlnued on next page.)
`
`Primary Examiner—Patrick Nolan
`Assistant Examiner—Marianne DiBrino
`(74) Attorney, Agent, or Firm—Lee K. Tan; Wendy M. Lee
`(57)
`ABSTRACT
`
`A stable aqueous pharmaceutical formulation comprising a
`therapeutically effective amount of an antibody not sub
`jected to prior lyophiliZation, a buffer maintaining the pH in
`the range from about 4.5 to about 6.0, a surfactant and a
`polyol is described, along With uses for such a formulation.
`
`29 Claims, 25 Drawing Sheets
`
`Ex. 1003 - Page 1 of 56
`
`AMGEN INC.
`Exhibit 1003
`
`

`

`US 6,171,586 B1
`Page 2
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`
`* cited by examiner
`
`Ex. 1003 - Page 2 of 56
`
`

`

`US. Patent
`
`Jan. 9, 2001
`
`Sheet 1 0f 25
`
`US 6,171,586 B1
`
`
`
`<_..07.
`
`VHDUmvf—EMn—Pv—HZmEZEU—EUHUHmmmgmmflmrfivmm0§<E>UmH
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`
`Ex. 1003 - Page 3 of 56
`
`Ex. 1003 - Page 3 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 2 0f 25
`
`US 6,171,586 B1
`
`4 _
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`—— hydrophobicity
`
`— - — - - ?exibility l
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`
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`
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`
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`
`Ex. 1003 - Page 4 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 3 0f 25
`
`US 6,171,586 B1
`
`0.05 -
`
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`FIG. 3A
`
`Storage Temperature (°C)
`FIG. 3B
`
`Ex. 1003 - Page 5 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 4 0f 25
`
`US 6,171,586 B1
`
`
`
`2595 650122
`
`w a. B
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`
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`
`Storage Temperature (°C)
`FIG. 4
`
`Ex. 1003 - Page 6 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 5 0f 25
`
`US 6,171,586 B1
`
`E
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`FIG. 5B
`
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`30°C
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`40
`
`Ex. 1003 - Page 7 of 56
`
`

`

`US. Patent
`
`Jan. 9, 2001
`
`Sheet 6 0f 25
`
`US 6,171,586 B1
`
`26500
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`0o522$8.68.32
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`Ex. 1003 - Page 8 of 56
`
`Ex. 1003 - Page 8 of 56
`
`
`
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 7 0f 25
`
`US 6,171,586 B1
`
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`FIG. 8
`
`Ex. 1003 - Page 9 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 8 0f 25
`
`US 6,171,586 B1
`
`-20°C
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`__
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`-70°C
`lllllljlllllllllllllllllllj
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`10
`15
`20
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`FIG. 9
`
`Ex. 1003 - Page 10 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 9 0f 25
`
`US 6,171,586 B1
`
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`25
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`20
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`FIG. 11
`
`Ex. 1003 - Page 11 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 10 0f 25
`
`US 6,171,586 B1
`
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`Ex. 1003 - Page 12 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 11 0f 25
`
`US 6,171,586 B1
`
`5:23
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`
`Ex. 1003 - Page 13 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 12 0f 25
`
`US 6,171,586 B1
`
`6
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`
`Ex. 1003 - Page 14 of 56
`
`

`

`U.S. Patent
`US. Patent
`
`Jan. 9, 2001
`Jan. 9, 2001
`
`Sheet 13 0f 25
`Sheet 13 0f 25
`
`US 6,171,586 B1
`US 6,171,586 B1
`
`
`
`FEG. 16
`FIG. 16
`
`Ex. 1003 - Page 15 of 56
`
`Ex. 1003 - Page 15 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 14 0f 25
`
`US 6,171,586 B1
`
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`.T..\.'l
`
`Ex. 1003 - Page 16 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 15 0f 25
`
`US 6,171,586 B1
`
`1
`
`m 00 6 4
`
`
`
`- _ - - n
`
`
`
`-
`
`- -
`
`_
`
`El 40°C
`[I —70°C
`control
`
`0 0 0
`
`38 “Ron 52: °\.
`
`5“
`FIG. 18A
`
`
`
`I n h - _
`
`0 0
`7
`
`
`
`88 “Eva 5.2: @e
`
`El 40°C
`El -70°C
`
`4.1
`
`4.1
`
`4.5
`
`4.5
`
`4.5
`
`FIG. 18B
`
`Ex. 1003 - Page 17 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 16 0f 25
`
`US 6,171,586 B1
`
`105 -
`
`8
`a
`
`‘i
`
`.E
`G
`E
`
`at 85_ +15°C
`-—*—-30°C
`——e——40°C
`
`80
`0
`
`|
`10
`
`|
`20
`
`I
`30
`
`I
`40
`
`l
`50
`
`Time (weeks)
`FIG. 19A
`
`10
`
`\ \ d1 \ a
`‘I
`
`.8
`i 80
`X
`2
`5 6°‘
`E
`a!
`
`I
`
`I
`
`40
`
`1
`
`50
`
`I
`
`30
`20
`Time (weeks)
`FIG. 19B
`
`\
`\\
`\
`-o—5°c \
`40" —-I-15°C \\
`—n--40°C
`‘
`—e—30°C
`I
`
`29
`
`0
`
`10
`
`Ex. 1003 - Page 18 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 17 0f 25
`
`US 6,171,586 B1
`
`
`
`
`
`MAC-1 Specific Activity
`
`1.1
`
`0.9 L
`
`+
`
`i‘
`
`0.8 — +
`
`0.7 _
`
`l
`
`0.6 I
`
`l
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`I
`
`l
`
`I
`
`I
`
`I
`
`I
`
`1 I
`
`I
`
`I
`
`l
`
`l
`
`I
`
`I J
`
`0
`
`10
`
`30
`20
`Time (weeks)
`FIG. 19C
`
`40
`
`50
`
`Ex. 1003 - Page 19 of 56
`
`

`

`U.S. Patent
`
`Jan. 9, 2001
`
`Sheet 18 0f 25
`
`US 6,171,586 B1
`
`beta-lactamase
`
`EcoRl
`
`phoA promoter
`STII signal
`Light Chain
`
`STH signal
`
`Heavy Chain-GCN4 leu zipper
`
`ColE1 origin
`
`lambda to terminator
`
`tet
`
`FIG. 20
`
`Ex. 1003 - Page 20 of 56
`
`

`

`US. Patent
`
`Jan. 9, 2001
`
`Sheet 19 0f 25
`
`US 6,171,586 B1
`
`1
`
`GAATTCAACT
`
`TCTCCATACT
`
`TTGGATAAGG AAATACAGAC
`
`ATGAAAAATC TCATTGCTGA
`
`61
`
`GTTGTTATTT
`
`AAGCTTTGGA
`
`GATTATCGTC ACTGCAATGC
`
`TTCGCAATAT GGCGCAAAAT
`
`121
`
`GACCAACAGC
`
`GGTTGATTGA
`
`TCAGGTAGAG GGGGCGCTGT
`
`ACGAGGTAAA GCCCGATGCC
`
`181
`
`AGCATTCCTG
`
`ACGACGATAC
`
`GGAGCTGCTG CGCGATTACG
`
`TAAAGAAGTT ATTGAAGCAT
`
`241
`
`CCTCGTCAGT
`
`AAAAAGTTAA
`
`TCTTTTCAAC AGCTGTCATA
`
`AAGTTGTCAC GGCCGAGACT
`
`301
`
`TATAGTCGCT
`
`TTGTTTTTAT
`
`TTTTTAATGT ATTTGTAACT
`
`AGAATTCGAG CTCGCCGGGG
`
`CA TTT CTT CTT
`F
`L
`L
`
`AG
`
`ATC
`I
`
`GCG
`A
`
`TAC
`Y
`
`GCT
`A
`
`GAT ATC
`D
`I
`
`K G
`
`H A
`
`TT
`
`ATCCTCTAGA
`
`GGTTGAGGTG ATTTT
`
`361
`'23
`
`413
`'14
`
`GCA
`A
`
`TCT
`S
`
`ATG
`H
`
`TTC GTT TTT TCT
`F
`V
`F
`S
`
`ATG AAA AAG AAT
`
`N A
`
`AC
`
`K A
`
`CT
`
`CA
`
`N G
`
`CC
`
`T T
`
`GC
`
`A C
`
`TG
`
`I T
`
`CC
`
`S A
`
`GT
`
`TCT
`S
`
`GTG
`V
`
`GGC
`G
`
`GAT
`D
`
`AGG
`R
`
`AAC
`N
`
`AAT
`N
`
`TAT
`Y
`
`CTG
`L
`
`AAC
`N
`
`CTA CTG ATT TAC TAT
`v
`L
`I
`.
`
`TTC TCT GGT TCT
`G
`S
`
`GGT
`
`A A
`
`TC
`
`S G
`
`AC
`
`I A
`
`AA
`
`K C
`
`GC
`
`D C
`
`CG
`
`P T
`
`CT
`
`L C
`
`AG
`
`Q G
`
`CT
`
`A C
`
`CT
`
`GC
`
`ACT
`
`GAC
`
`CAG
`
`ATG
`M
`
`ACC
`T
`
`CAG TCC CCG AGC
`Q
`S
`P
`S
`
`TC
`
`ACC ATC
`T
`I
`
`ACC TGT CGT GCC
`T
`C
`R
`A
`
`Q G
`
`V
`
`TGG
`
`TAT
`Y
`
`CAG AAA CCA GGA
`O
`K
`P
`G
`
`H A
`
`T
`
`CC TCC ACC
`S
`
`CTC CAC TCT GGA
`L
`B
`S
`G
`
`GTC
`
`461
`3
`
`509
`19
`
`557
`35
`
`605
`51
`
`S A
`
`S A
`
`P A
`
`TC
`
`I G
`
`GT
`
`N C
`
`CTG CAA CCG GAG
`Q
`P
`E
`
`AT
`
`ACT
`
`CTG CCG CCG ACG
`L
`P
`P
`T
`
`TTC
`
`GA
`
`ACT
`
`GTG GCT
`
`GCA CCA TCT
`A
`P
`S
`
`653
`67
`
`TCT GGG
`5
`G
`
`ACG
`
`GAT TAC ACT CTG
`D
`Y
`T
`L
`
`701
`83
`
`TTC GCA
`F
`A
`
`ACT
`
`TAT TAC TGT CAG
`Y
`I
`C
`0
`
`749
`99
`
`GGA CAG GGC
`Q
`
`ACC AAG GTG GAG
`T
`K
`V
`E
`
`ACC
`
`CAA
`
`ATC
`
`TCT GGA ACT
`G
`T
`
`GCC
`
`AGA GAG GCC
`A
`
`GTA
`
`GAT
`
`GAG
`
`CAG
`
`TTG
`
`E T
`
`D A
`
`AC
`
`TC
`
`TAT
`
`CCC
`
`AA
`
`TCG
`
`GGT
`
`AAC TCC
`S
`
`GAG AGT
`
`F C
`
`N C
`
`TC
`
`797
`115
`
`345
`131
`
`893
`147
`
`GTC TTC ATC
`
`TTC CCG CCA TCT
`F
`P
`P
`S
`
`TCT GTT GTG
`
`TGC CTG CTG AAT
`C
`L
`L
`N
`
`CAG TGG AAG
`
`GTG GAT AAC GCC
`V
`D
`N
`A
`
`CAG
`Q
`
`AGC
`S
`
`CTC AGC
`L
`5
`
`ACC ACC
`
`AAA
`K
`
`GTC
`V
`
`TAC
`Y
`
`GCC TGC
`
`Y C
`
`T A
`
`AA
`
`AC
`
`E G
`
`V
`
`K C
`
`CC
`
`P
`
`TC
`
`ACA
`T
`
`AAG
`K
`
`AGC
`S
`
`TTC
`P
`
`AAC
`N
`
`0 A
`
`L G
`
`AC
`
`CC
`
`ACC
`
`TAC
`
`S G
`
`AG
`
`E T
`
`CG
`S
`
`D T
`
`AC
`
`Y A
`
`GC
`
`S
`
`941
`163
`
`GTC
`V
`
`ACA
`
`GAG
`
`CAG GAC AGC AAG
`O
`D
`S
`K
`
`989
`179
`
`CTG
`L
`
`ACG
`T
`
`CTG
`
`AGC AAA GCA GAC
`S
`K
`A
`D
`
`1037
`195
`
`GAA
`E
`
`GTC
`V
`
`ACC
`T
`
`CAT CAG GGC CTG
`H
`O
`G
`L
`
`1085
`211
`
`AGG
`R
`
`GGA
`G
`
`GAG
`E
`
`TGT
`C
`
`TAA G CTGATCCTCT ACGCCGGACG CATCGTGGCG
`
`FIG. 21A
`
`Ex. 1003 - Page 21 of 56
`
`Ex. 1003 - Page 21 of 56
`
`

`

`US. Patent
`
`Jan. 9, 2001
`
`Sheet 20 0f 25
`
`US 6,171,586 B1
`
`1131 CTAGTACGCA AGTTCACGTA AAAACGGTAT CTAGAGGTTG AGGTGATTTT
`'23
`
`ATG AAA
`H
`K
`
`1187 AAG AAT ATC GCA TTT CTT CTT GCA TCT ATG TTC GTT TTT TCT ATT GCT
`'21 K
`N
`I
`A
`F
`L
`L
`A
`S
`H
`F
`V
`F
`S
`I
`A
`
`1235 ACA AAC GCG TAC GCT GAG GTT CAG CTG GTG GAG TCT GGC GGT GGC CTG
`‘5 T
`N
`A
`Y
`A
`E
`V
`O
`L
`V
`E
`S
`G
`G
`G
`L
`
`1283 GTG CAG CCA GGG GGC TCA CTC CGT TTG TCC TGT GCA ACT TCT GGC TAC
`12 V
`O
`P
`G
`G
`S
`L
`R
`L
`S
`C
`A
`T
`S
`G
`Y
`
`1331 ACC TTT ACC GAA TAC ACT ATG CAC TGG ATG CGT CAG GCC CCG GGT AAG
`28 T
`F
`T
`E
`Y
`T
`H
`B
`W
`H
`R
`Q
`A
`P
`G
`K
`
`1379 GGC CTG GAA TGG GTT GCA GGG ATT AAT CCT AAA AAC GGT GGT ACC AGC
`44 G
`L
`E
`W
`V
`A
`G
`I
`N
`P
`K
`N
`G
`G
`T
`S
`
`1627 CAC AAC CAG AGG TTC ATG GAC CGT TTC ACT ATA AGC GTA GAT AAA TCC
`60 H
`N
`O
`R
`F
`H
`D
`R
`F
`T
`I
`S
`V
`D
`K
`S
`
`1475 ACC AGT ACA GCC TAC ATG CAA ATG AAC AGC CTG CGT GCT GAG GAC ACT
`76 T
`S
`T
`A
`Y
`M
`Q
`H
`N
`S
`L
`R
`A
`E
`D
`T
`
`1523 GGC GTC TAT TAT TGT GCT AGA TGG CGA GGC CTG AAC TAC GGC TTT GAC
`92 A
`V
`Y
`Y
`C
`A
`R
`W
`R
`G
`L
`N
`Y
`G
`F
`D
`
`1571 GTC CGT TAT TTT GAC GTC TGG GGT CAA GGA ACC CTG GTC ACC GTC TCC
`108 V
`R
`Y
`F
`D
`V
`W
`G
`O
`G
`T
`L
`V
`T
`V
`S
`
`1619 TCG GCC TCC ACC AAG GGC CCA TCG GTC TTC CCC CTG GCA CCC TCC TCC
`124 S
`A
`S
`T
`K
`G
`P
`S
`V
`F
`P
`L
`A
`P
`S
`S
`
`1667 AAG AGC ACC TCT GGG GGC ACA GCG GGC CTG GGC TGC CTG GTC AAG GAC
`140 K
`S
`T
`S
`G
`G
`T
`A
`A
`L
`G
`C
`L
`V
`K
`D
`
`1715 TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC CTG ACC
`156 Y
`F
`P
`E
`P
`V
`T
`V
`S
`W
`N
`S
`G
`A
`L
`T
`
`1763 AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA CTC TAC
`172 S
`G
`V
`K
`T
`P
`P
`A
`V
`L
`Q
`S
`S
`G
`L
`Y
`
`1811 TCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGC TTG GGC ACC CAG
`188 S
`L
`S
`S
`V
`V
`T
`V
`P
`S
`S
`S
`L
`G
`T
`Q
`
`1859 ACC TAC ATC TGC AAC GTG AAT CAC AAG CCC AGC AAC ACC AAG GTC GAC
`20‘ T
`Y
`I
`C
`N
`V
`N
`H
`K
`P
`S
`N
`T
`K
`V
`D
`
`1907 AAG AAA GTT GAG CCC AAA TCT TGT GAC AAA ACT CAC ACA TGC CCG CCG
`220 K
`K
`V
`E
`P
`K
`S
`C
`D
`K
`T
`H
`T
`C
`P
`P
`
`1955 TGC CCA GGA CCA GAA GTG CTG GGC GGC CGC ATG AAA CAG CTA GAG GAC
`236 C
`P
`A
`P
`E
`L
`L
`G
`G
`R
`M
`K
`O
`L
`E
`D
`
`2003 AAG GTC GAA GAG CTA CTC TCC AAG AAC TAC CAC CTA GAG AAT GAA GTG
`252 K
`V
`E
`E
`L
`L
`S
`K
`N
`Y
`H
`L
`E
`N
`E
`V
`
`2051 GGA AGA GTC AAA AAG CTT GTC GGG GAG CGC TAA
`268 A
`R
`L
`K
`K
`L
`V
`G
`E
`R
`
`GCATGCG ACGGCCCTAG
`
`2101 AGTCCCTAAC GCTCGGTTGC CGCCGGGCGT TTTTTATTGT TAA
`
`FIG. 213
`
`EX. 1003 - Page 22 of 56
`
`Ex. 1003 - Page 22 of 56
`
`

`

`US. Patent
`
`Jan. 9, 2001
`
`Sheet 21 0f 25
`
`US 6,171,586 B1
`
`
`Strain W
`
`W3110
`
`K—12 F' lambda' lNrmD-rmE1
`
`l
`
`1A2
`
`W3110 AthA
`
`l
`
`701
`
`wa11o AfhuA AphoA A(argF-Iac)
`
`l
`
`1609
`
`W3110 AfhuA AphoA A(argF-Iac) deoC
`
`l
`
`2353
`
`M110 AfhuA AphoA A(argF-Iac) deoC AdegP
`
`l
`
`3336
`
`W3110 AfhuA AphoA A(argF-Iac) deoC AdegP iIvG
`
`l
`
`4982
`
`W31 10 AthA AphoA A(argF-lac) deoC AdegP iIvG AfucP
`
`l
`
`49A5
`
`W3110 AfhuA AphoA A(argF-Iac) deoC AdegP ilvG AfucP AmalE
`
`FIG. 22
`
`Ex. 1003 - Page 23 of 56
`
`Ex. 1003 - Page 23 of 56
`
`

`

`US. Patent
`
`Jan. 9, 2001
`
`Sheet 22 0f 25
`
`US 6,171,586 B1
`
`VIAL FROM
`
`WORKING CELL BANK
`
`OR
`
`MASTER CELL BANK
`
`PRIMARY INOCULUM.
`SHAKE FLASK
`MEDIUM
`
`14-18 hours
`temperature controlled
`
`I
`
`7-17 hours
`
`SECONDARY INOCULUM
`
`SECONDARYMEDIUM . transferredat10-2500550
`
`'
`
`temperature and pH controlled
`
`PRODUCTION VESSEL.
`PRODUCTION MEDIUM
`
`I
`
`I
`
`temperature and pH controlled
`controlled nutrient feeds
`harvested at 60-84 hours
`
`HARVEST BY CENTRIFUGATION
`
`I
`
`FREEZING OF CELLS
`
`FIG. 23
`
`Ex. 1003 - Page 24 of 56
`
`Ex. 1003 - Page 24 of 56
`
`

`

`US. Patent
`
`Jan. 9, 2001
`
`Sheet 23 0f 25
`
`US 6,171,586 B1
`
`2.01
`
`2
`
`1.99
`1.93
`1.97
`
`’g‘
`2
`g
`a
`g 1.96
`a,
`3
`
`1.95
`
`1.94
`
`1.93
`
`
`
`102.3
`
`100
`
`97-7
`95.5
`93-3
`
`°\°
`cg:
`3
`91.2 0
`.3.
`89.1 H
`
`87.1
`
`85.1
`
`.10
`
`o
`
`10
`
`20
`30
`Time (days)
`
`4o
`
`50
`
`so
`
`12
`'6
`.fi
`g
`g
`C
`:3
`2‘1
`O)
`o
`
`.1
`
`.\°
`c
`8
`E.
`E:
`N
`a
`3‘
`
`'-1o
`
`0
`
`1o
`
`20
`
`so
`
`40
`
`50
`
`so
`
`Time (days)
`
`FIG. 243
`
`EX. 1003 - Page 25 of 56
`
`Ex. 1003 - Page 25 of 56
`
`

`

`US. Patent
`
`Jan. 9, 2001
`
`Sheet 24 0f 25
`
`US 6,171,586 B1
`
`
`
`35.05:..x.
`
`
`
`
`
`E:03.03”.06omEo><
`
`\4
`
`“‘\‘\-§s6
`
`mM Trehalose
`
`FIG. 25
`
`Formulation
`
`FIG. 26
`
`Ex. 1003 - Page 26 of 56
`
`Ex. 1003 - Page 26 of 56
`
`
`
`

`

`US. Patent
`
`Jan. 9, 2001
`
`Sheet 25 0f 25
`
`US 6,171,586 B1
`
`%monomer
`
`%monomer
`
`100
`
`98
`
`96
`
`5 T— 2 wks
`El
`
`
`
`§
`
`1
`
`2
`
`3
`
`4
`
`Formulation
`
`FIG. 27
`
`105
`
`100
`
`95
`
`90
`
`85
`
`so
`
`75
`
`
`
`u m
`
`H
`
`mm...
`
`mom 2—8’ C
`—EJ
`- 30°C
`— e - 40°C
`- -E- - Reference control 40°C
`
`0
`
`100
`
`200
`
`300
`
`400
`
`500
`
`600
`
`700
`
`Time (days)
`
`FIG. 28
`
`Ex. 1003 - Page 27 of 56
`
`Ex. 1003 - Page 27 of 56
`
`

`

`US 6,171,586 B1
`
`1
`ANTIBODY FORMULATION
`
`RELATED APPLICATION
`
`This is a non-provisional application claiming priority
`under Section 119(e) to provisional application 60/053,087,
`filed on Jun. 13, 1997, the contents of which are incorpo-
`rated herein by reference.
`
`FIELD OF THE INVENTION
`
`This invention is directed to a stable aqueous pharmaceu-
`tical formulation comprising an antibody.
`BACKGROUND OF THE INVENTION
`
`In the past ten years, advances in biotechnology have
`made it possible to produce a variety of proteins for phar-
`maceutical applications using recombinant DNA techniques.
`Because proteins are larger and more complex than tradi-
`tional organic and inorganic drugs (i.e. possessing multiple
`functional groups in addition to complex three-dimensional
`structures), the formulation of such proteins poses special
`problems. For a protein to remain biologically active, a
`formulation must preserve intact the conformational integ-
`rity of at least a core sequence of the protein’s amino acids
`while at
`the same time protecting the protein’s multple
`functional groups from degradation. Degradation pathways
`for proteins can involve chemical instability (i.e. any process
`which involves modification of the protein by bond forma-
`tion or cleavage resulting in a new chemical entity) or
`physical instability (i.e. changes in the higher order structure
`of the protein). Chemical
`instability can result from
`deamidation,
`racemization, hydrolysis, oxidation, beta
`elimination or disulfide exchange. Physical instability can
`result
`from denaturation, aggregation, precipitation or
`adsorption, for example. The three most common protein
`degradation pathways are protein aggregation, deamidation
`and oxidation. Cleland et al Critical Reviews in Therapeutic
`Drug Carrier Systems 10(4): 307—377 (1993).
`Included in the proteins used for pharmaceutical applica-
`tions are antibodies. An example of an antibody useful for
`therapy is an antibody which binds to the CD18 antigen.
`CD18 is the common [3 subunit of three heterodimeric
`membrane integrins restricted to leukocytes that mediate
`trafficking and adhesion to the vascular endothelium, par-
`ticularly at sites of inflammation (for reviews see Hynes, R.
`0. Cell, 69:11—25 (1992); Stoolman, Cell, 56:907—910
`(1989); Jutila et al. Transplantation 48(5): 727—731 (1989);
`Springer, T. A., Nature 346:425—434 (1990); and Albelda
`and Buck, FASEB J. 4:2868—2880 (1990)). The heterodimer
`containing CD18 and CD11b (also called MAC-1) is found
`primarily on neutrophils, monocytes, and some lymphocytes
`whose normal interaction with ICAM-1 on vascular endot-
`
`helium mediates adhesion and “rolling” of cells along the
`vasculature. In severe hemorrhagic trauma with concurrent
`decrease in cardiac output and ischemia, early (within 30
`min) neutrophil activation (in response to released
`cytokines) and up-regulation of MAC-1 increases neutrophil
`“stickiness”. This precedes extravasation and release of
`proteases and superoxides that ultimately lead to further
`tissue damage and increased vascular permeability
`(Hernandez et al, Am. J. Physiol, 253(3 Pt 2): H699-H703
`(1987)). Reperfusion following resuscitation exacerbates the
`edema and necrosis, and leads to multi-organ failure and
`death. Early treatment with monoclonal antibodies to CD18
`in a partally-severed,
`ischemic rabbit ear trauma model
`alleviated tissue necrosis following reattachment (Vedder et
`al., J. Clin. Invest. 81:939—944 (1988)). A humanized anti-
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`body showed efficacy in reducing multi-organ damage and
`death in a rhesus monkey model of decreased cardiac output
`(created by depletion of 2/3 of blood volume for ~2 hours
`(Mileski etal., Surgery, 108(2):206—212 (1990)). These stud-
`ies point to the therapeutic potential of anti-CD18 antibodies
`for acute treatment of hemorrhagic shock.
`Another antigen of interest for targeting with antibodies is
`the CD20 antigen, also known as “Bp35”. CD20 is a human
`B cell marker which is expressed during early pre-B cell
`development and remains until plasma cell differentiation.
`The CD20 molecule may regulate a step in the activation
`process which is required for cell cycle initiation and
`differentiation and is usually expressed at very high levels on
`neoplastic B cells. Thus, the CD20 surface antigen can be
`targeted for
`treating B cell
`lymphomas. US. Pat. No.
`5,736,137 issued Apr. 7, 1998 describes the chimeric anti-
`body “C2B8” which binds the CD20 antigen and its use to
`treat B cell lymphoma.
`There is a need in the art for a stable aqueous pharma-
`ceutical formulation comprising an antibody, such as an
`anti-CD18 or anti-CD20 antibody, which is suitable for
`therapeutic use.
`SUMMARY OF THE INVENTION
`
`the invention provides a stable aqueous
`Accordingly,
`pharmaceutical formulation comprising a therapeutically
`effective amount of an antibody not subjected to prior
`lyophilizabon, a buffer maintaining the pH in the range from
`about 4.5 to about 6.0, a surfactant and a polyol. Preferably
`the formulation is stable at a temperature of about 2—8° C.
`for at least one year, and/or is stable at a temperature of
`about 30° C. for at least one month and/or is stable following
`freezing and thawing of the formulation.
`The invention also relates to an article of manufacture
`
`comprising a container holding a stable aqueous pharma-
`ceutical formulation comprising a therapeutically effective
`amount of an antibody not subjected to prior lyophilizabon,
`a buffer maintaining the pH in the range from about 4.5 to
`about 6.0, a surfactant and a polyol.
`In yet a further aspect, the invention relates to a method
`for stabilizing an antibody in an aqueous pharmaceutical
`formulation by combining a therapeutically effective
`amount of an antibody not subjected to prior lyophilization,
`a buffer maintaining the pH in the range from about 4.5 to
`about 6.0, a surfactant and a polyol.
`In a still further aspect, the invention concerns a method
`of treating a mammal comprising administering a therapeu-
`tically effective amount of the aqueous pharmaceutical for-
`mulation disclosed herein to a mammal, wherein the mam-
`mal has a disorder requiring treatment with the antibody in
`the formulation. Where the antibody binds CD18, examples
`of disorders to be treated include hemorrhagic shock, ther-
`mal
`injury (such as that resulting from burns), stroke
`(including ischemic and hemorrhagic stroke) and myocar-
`dial infarction. For an anti-1L8 antibody, disorders to be
`treated include inflammatory disorders such as adult respi-
`ratory distress syndrome (ARDS), hypovolemic shock,
`ulcerative colitis, and rheumatoid arthritis. Where the anti-
`body binds CD20, disorders to be treated include B cell
`lymphomas.
`These and further aspects of the invention will be appar-
`ent to those skilled in the art.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIGS. 1A and 1B depict the amino acid sequence of
`rhuMAb CD18 heavy chain (FIG. 1A; SEQ ID NO:1) and
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`US 6,171,586 B1
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`light chain (FIG. 1B; SEQ ID N022). The sequence in italics
`in FIG. 1A (SEQ ID N023) is that of the leucine zipper.
`FIGS. 2A and 2B are hydroflex plots of rhuMAb CD18
`heavy chain (FIG. 2A) and light chain (FIG. 2B). Kyte-
`Dooliutle hydrophobicity calculations averaged with a win-
`dow of 6 amino acids were made on the protein sequence.
`Flexibility values were estimated from the product of the
`hydrophobicity of each residue and its side chain volume
`(again averaged over a window of 6 residues). Asn-Gly and
`Asn-Ser motifs in flexible regions are more likely to dea-
`midate than those in more rigid structures. The CDRs are
`also shown. Most of the heavy chain methionines and heavy
`chain Asn 84 are near the CDR’s.
`
`FIGS. 3A and 3B show the effect of storage temperature
`on light scattering in different rhuMAb CD18 formulations
`after 5 weeks storage at
`the designated temperatures as
`measured by average absorbance in the range of 340—360
`nm (FIG. 3A), or the ratio ofA278 over A252 nm (FIG. 3B).
`RhuMAb CD18 in formulation F3 is prone to the formation
`of insoluble aggregates.
`FIG. 4 shows the effect of storage temperature on protein
`concentration of rhuMAb CD18 formulations measured by
`absorbance at 278 nm corrected for vehicle absorbance at
`320 nm.
`
`FIGS. 5A and 5B depict the effect of storage temperature
`on the stability of rhuMAb CD18 formulation F2 (FIG. 5A)
`and formulation F5 (FIG. 5B) assayed by size exclusion
`chromatography (SEC). A smaller MW species (see arrows)
`appeared which was more pronounced at pH 6 (FIG. 5B)
`compared to pH 5 (FIG. 5A).
`FIGS. 6A and 6B represent the effect of storage time and
`temperature on the stability of rhuMAb CD18 showing total
`peak area (FIG. 6A), and % main peak (FIG. 6B) for all
`formulations, assayed by SEC. No significant change in total
`peak area was noted. F1 and F5 maintained the lowest %
`main peak.
`FIG. 7 is an Arrhenius plot of the % main peak area from
`SEC of rhuMAb CD18 formulated in acetate with trehalose
`
`at pH 5 (F2). Activation Energy=19=6 kcal/mole.
`FIG. 8 shows the effect of storage for 5 weeks at 40° C.
`on different rhuMAb CD18 formulations, assayed by hydro-
`phobic interaction chromatography (HIC). The early eluting
`peaks at 17.5 min and the shoulder at the leading edge of the
`main peak increased compared to controls at —70° C. F2
`showed the least increase in both components (see FIG. 9).
`FIG. 9 shows the effect of storage for 5 weeks at different
`temperatures on formulation F2, assayed by HIC. A pre-
`main peak became apparent compared to —70° C. control.
`FIG. 10 depicts the effect of storage temperature on the
`stability of rhuMAb CD18 (5 week data) assayed by HIC
`showing total peak area recovered. A trend towards loss in
`area was noted in all formulations with increasing storage
`temperatures, except for F5 which started lower to begin
`with.
`
`FIG. 11 shows the effect of storage for 5 weeks at different
`temperatures on formulation F2, assayed by reverse phase-
`hydrophobic liquid chromatography (RP-HPLC). A