Amgen Inc. v. F. Hoffmann-LaRoche LTD et al

Filing 547

DECLARATION of Craig H. Casebeer in Support of Motion for Summary Judgment of No Inequitable Conduct by Amgen Inc.. (Attachments: #1 Exhibit Ex 1#2 Exhibit Ex 2#3 Exhibit Ex 3#4 Exhibit Ex 4#5 Exhibit Ex. 5#6 Exhibit Ex 6#7 Exhibit Ex 7#8 Exhibit Ex 8#9 Exhibit Ex 9#10 Exhibit Ex 10#11 Exhibit Ex 11#12 Exhibit Ex 12#13 Exhibit Ex 13#14 Errata Ex 14#15 Exhibit Ex 15#16 Exhibit Ex. 16#17 Exhibit Ex 17#18 Exhibit Ex 18#19 Errata Ex 19#20 Exhibit Ex 20#21 Exhibit Ex 21-1#22 Exhibit Ex 21-2#23 Exhibit Ex 22#24 Exhibit Ex 23#25 Exhibit Ex 24#26 Exhibit Ex 25#27 Exhibit Ex 26#28 Exhibit Ex 27#29 Exhibit Ex 28#30 Exhibit Ex 29#31 Exhibit Ex 30#32 Errata 31#33 Errata Ex 32#34 Exhibit Ex 33#35 Exhibit Ex 34#36 Exhibit Ex 35#37 Exhibit Ex 36#38 Exhibit Ex 37#39 Exhibit Ex 38-1#40 Errata Ex 38-2#41 Exhibit Ex 39#42 Exhibit Ex 40#43 Exhibit Ex 41)(Gottfried, Michael)

Amgen Inc. v. F. Hoffmann-LaRoche LTD et al

Doc. 547 Att. 36

Case 1:05-cv-12237-WGY

Document 547-37

Filed 06/22/2007

Page 1 of 6

EXHIBIT 35

Casebeer Decl to Motion for SJ re IC - Public

Dockets.Justia.com

Case 1:05-cv-12237-WGY

Document 547-37

Filed 06/22/2007

Page 2 of 6

to=ed . B :bct -,

Acta

a2 ( ·9 83) 1/2,

S 202

- : 2C6

:id ;at .on of erytnroocietin oy monoclonal antibocy' .SkSAK :, YA'IAG."A, Ji0 NI1O ClI!A :eoar_-lent of Food Science and Tea+noloQv, Faculty :r.tro0uc : :cn
'w0

;'

culture . Kyot .: University, Kyoto 606, Japan :'EPO), !fav beep -eDorLae
.,et

'actors, burs : oronnting activity and erythroooietin it-tar cells and :ar;et of EPC :S

wr,C'n s : :mulate e .'yVirci tell differentiation . Burst promoting act : vity acts on early
:b .ii tcet erv'l-Cl :

eg , ca ; ;y rn,benL " '!t Ore cursor tells . Recently, a factor wn,cn is dist,nc - 'r'bm And acs 'ate ,r. e^y_ ro,c differentiation, ;r,mar'ly on erythroblasts, has been
e :cr :et : : ·. s beiievea t.-at P0 Mays a :ROom,r nL .'Oil in reeuiat :ng bo',esis because o` a ;ooc correlation be :weer. serum ;eve's of EPO &no rates

re more rat~rec

"nor--no .
C. n =

y~,ro . of e y :r.ro .

:yte 'orma : :on . -e crob~ers in studying are onysiology and the bioeneh,str; of erytnropcies , s have .eer cumbersome, insensitive . and relatively unsoec .fic assay .*gods . and :acx o= a simple purification method :o provide suitaole our* :'eoara : :oh of :ne norrm ne . . nocionai antibodies have been spawn to be a powerful tool 'or our. .f4 :n .' s paper cat,en, cnarac :!r'_a : :on, &no ouant ;tative analysis of macromolecules .

we reocr: :he procuCt :n of hyoridomes that secrete monoclonal antibodies ace :rst !tum n urinary E=0 arc .saiat ;0n of EPO from an urine concentrate witr an immuncacsoroent column . Materials enc `* :boas

A1 were oescr ·b ea elsewhere (2-4) . Results and Oisc_ssion pvr'"'ca - : : rmucan urinary e-vthrooolet :n - Muean EPO %as purified from :ne r 7acid : Summary of purification of hucan urinary erythropoietin. Steo Urine concentrate OEAE-cellulose Phenyl-Seonarose wyaroxylaoat : :a Seonacex 5- ;0 Suifoprooyi-Seonaoex :mounoaasoroent co i u ns a ca . ns t contaminants Preoarat :ve 5 :Spolyacy iam,ce gel e l ec :ronoores, s Total protein (mg) Total activity (units) Soecific activity (units/mg) Purification

30939 161 2323 672 63 16 3
1

18060 1596840 978750 83660 2490 8160 690 380

58 99 421 1245 30 510 230 380

c 21 51 88 396 655

urine of aolastic Anemia patients by column cnroratograony with OEAE-cellulose . Phenyi-

· A cart of this oa :er pas been published (S . Yanagawa et al . . Ann c . Sic! . :hem . .

47, 131-1316

Aborev,at :on : EPO, erytnrooeietin .

(1983)) aria the rest has been subs : :ed for publication .

AF 987

Case 1:05-cv-12237-WGY
J

Document 547-37

Filed 06/22/2007

Page 3 of 6

.Segnarose C .4B . Hydroxylaoat : e, Seonadex G- .OC . Suifoorooyi-Seuharax . a11 . ant'.boules against contaminants &no suoseouently by preparative SDS-oalyacryitande gel elec :ro-

S 20?

pnoresiS (3) .

Analysis wit -, S :S-polyacrylamloe ;ti elec ;rOoforesiS revealed one protein

Una and, the distribution of

:?0

activity measure : after extraction of sliced geis was ml un ne i7abie i) Soec :`ic ac-'vity of ' ° Fe inc 'X-tnymid :ne .,etnoes . "as lower _liar.

e,uctly suoeri osea to the protein . About 55O-fold purification was ac .ievec &no -ne ag of Purified _a0 was oota,nec 'rom =e E?0 was cetrmthem 60,03 be aoout 38,0C by -n v, ·

A slm :lar value was also obtained by in vivo assay method . -his value .1 _.e value of 70 .40 rooor ·e a by Miyaxe tt a

wno isolates human urinary EPO w1 : .`n

conventional purification . met .^ocs . "he pure E?0 oculned here was prepared by extract. mg protein from SDS-oolyecrylamioe gel in the ias : sea of our-,f 4 ut on inc there denatured protein wit^, less or ho

EPO activity as possibly inciuoeo .

.t 1s

also very

iitely that the olfference in specific activity is cue, at least in tar :, to a1f`ere^. : . E-ytroooietin-soecifi_ 'lvor'dama - Fusion of spleen cells from ? e mice i-munlz-

procedures for proteinn measurement . ea with the pure EPO with XS- . ryeloea cells yieioea 258 growing nypr'.domus of Vie total seeoea 264 wells . Cultures demonstrating cell growth were tested for supernatant ant :bodies by a solid-phase anttSocy-binding assay, detecting the presence of an EPO specific antibody as an VO antibody oiotinylatea ant' .-mouse IgG avidin biotinyllatea seroxidase cooclex . Seven strongly positive (S1-S7) &no 12 weaaly were found . producing other class of infiuneglooulir positive (WI-02' cultures .t was possible last besides these pOSit,ve cultures there were hyor :comas , because :he ant,-mouse igG was usec for v is

screening . Screening of the supernatants by soiid-onase antibody-binding assay using the mixture of rabbit ana -rouse IgG, IgM . and :gA excluded this ooss,bllity . All positive cultures .are expanded in 24-well tissue culture Plates . of the supernatants with to oinCing assay before tease snowed that all of S1-S7 and four screening further enlargement of culture in a and

(W2 . W6, 48 . &no W9) of Wl-W9 .ere still Posit-

ive . The negative cultures were discarded . Cells from cultures 51-57 . .'2 . W6, w8 .

W9 . were infected into mice for production of ascites and each ascitic fluid was examined with respect to iesmunogioouitn progucrien wit·. SDS-oolyacrylamide gel ei.ec'roonoresis

(Fig . :) .

The strong production was found in cultures $2 . $3, $7, and W8 .
S7, and W8 was fixed . The Purified EPO was

Small antibody columns were trade containing Affi-Gel 10 on which i :munoglobulin pur,fiee from each asci :lc `iuld of 52, S3,

1

2 3 . ·:4 S .-(ii7

6 :9'10

:= ;

:3

Fig . : . SOS-ooiyacrylam, :e gel diet t osnor'esls of asctt,c fluids of nyoricomas . lyoridocells were 'n3ectea into mouse `Cr production of ascites and each asc,a c fluid was subjected tb SDS-ociyacrylamloe ,el eiec ;roonoreSiS . Protein Danes were -eveelec with silver Mr stncares ;94 .0, sa,ninev vanes ! onosonoryiase 3 ; 67,000, bovine serum album n ; '3 .0, ovaibumin ; 30,000 carbonic annycrase ; soybean tryps,n imloitor ; 14,400 . s20,0 lac :0aibumnnl ; 2 . 1SCitiC f1uld of hyDrldom S6 . 8 ; 57, Si . 3 ; S4 . 4 ; S3, 5 ; S4 . 5 ; S5, 9 ; W2, 10 ; 6 . 1 ; . .'S, 12 ; 9 . Arrow nears with 53K and 23K indicate neavy chain and light chain of IgG, respectively .

AF 9 8 8

Case 1:05-cv-12237-WGY

Document 547-37

Filed 06/22/2007

Page 4 of 6

S

204 :V=hs

avolka on we

and ties the columns we-t wasnec wi :.n 2 SS ;Posztnatt :u ·' fe ·e c_ ;Her, Wine ;, 'olilowec wit . · ins :H Z . . eiut*.or , zu"e" ' : . :!i act--- , e :, ; .I N&C', ) . The eluzec frac-zrs -ere ieutr&I ,_tc of _ e IMI tt& . va 1 ;t
;-4

Iris-base . -he .-On * tiCuoition wit- . - . Is _ :u : : on :plum -nice c- ntz ,.neo .:ono.
nc act .-vizy was ~zunc
~ .n

;oietl-n acvvity in ins . suffer

ac- ,. vi :y -as ' : we-e, ·o

frac-ons &no , r ei ,.jted ones as rteasurea .

when ery -_nrOOCIeZ :M was applied on _e --rt?- :1

:'·oral ar.t-. , ocy a ;a-.ns : .
: _!Ute SZ fraC-_-nS .
;n

,. nea ply transglut-aminase !,manuscr , zt in 2reeoarzvcr :I ., !C'I . --ne : :MI7*jry,, ~ i .tile :, - FD ac vity :asset directly _,-purr ins

activity was -iaC-_ve-eC -n :ne

:l C :Iumn tut . . . . 1~ _e !0 'ec activity acoearec , n
S7,

the

ne tiutec fraC--.onS . -EF 0 . :m:muno9loou'ti,.IS
Yi : y

:hat we artibocy :rocucec I y 'iyltr'comia SZ .incs -1--n . nyoricomas $ : . anc
appeared -,n tie f1bw-v!iuqn 4ractions and
no

;a roes not seem to zinc *iv, EEFO, because significant act ,.
ac : ,. vity was found in
.1y

:he eluted
SDS-

fractions . These ocseryavons were validated

&Maiys*,s of the both fract-,ons on

poiyacryliamice gel eiec-.r%- ;nores-.s (figure not shown) .
and Q .
EPC.

.then : Po was aq ;liec or. the -

: :lumps containing -.n6iuno9io0uiins from trafts ;d hyaridor.a . $3 . $7 . ut-Iml MaSe-di reczed
or : ;ain -as ounc only in the flaw-tnr- u9n fractions but not in th eluted . frac :*.cns . On we owe- nanc . tie E?O band was tetic-ei oniy in the eluted 'raC : :Ons
Wit'i

one column -,.ace -.- from antibody from SZ "yoricom4 of tie mixture of immuno: Althougn hyor , comas of $1, S4, and $6 -oor*!y zrocuced 'mmunogloouli .^.s pier :t was found Lriat EEPO activity aooearec -.rt

, , I looulins
SS .

they were injected ntc :nice, immuncadsorzent :ciumns were made with their immuno;iobulins and tested for :i riding cf .PC .
$4

the flow-tnrougn 'ractloms .rite

and SS but rot ,with Si and S6 columns . '4i= S, anc
:--oazie of :inoinq with EPO .

S6 columns the activity was recovered in the eluted fractions . Thus we nave o_ t5 :red
three hyoricoffia c ;;Itures 3rocuc ,,ng ant'.0ocies

:n parallel with screening and ProaagatIon of fused cells . hybridomas; IS',- . 7) , which were strongly positive with the binding assay -ere cloned by limiting dilution
in microt-.ter plates with muse %hyinocyte as feeder '.ager . Coning was started at

the Close of transfe- of --eilis from original 95--ell cultures into 24-well cultures . nifty coils %*-e seecec per 96 wells for cloning . ':hen results were obtained wit:: the
immuncaosoroent -Icumns, -_ :;iturt of all clones axes^ , Sl, $2 . and S6 was :eased .

Hytridoma-growing wells -ere tested for - ?0-an - *. :ccy production in the culture suoerE z ,.atants with the bincing assay . ;iybricom clones
'r - m i T'(0

positive wells of eava

ce l

were ;rczagatec in ar-;e scale cultures and r .eczac into 'Ouse f or oroauc - :on of asc -,t ,. c fluid . Analysis of asc ,, - Ic fluics -ntn =S-oo ilyacrylamide oel tiec - "-

phores ,ts revealed tut $Z produced in large i=un - -f *=unogloculin . wnile Si and SS :, a _Z -'·o re selected with a critarion .

were very poor in the or"urv.ort . qtecloninq vz n : - - nange the property of escr clone : with respect to anti :ocy ;rocuc :*.on . One of of rabid growth has been quite stable in high-ant -._ zcy production and is used throughout in tn-S ',aoorat--ry . Subclass of :q'C produced by hyDrIdoMS were ceterminto with Oucnterlony coupleirrnunociffusion metrioc by using rabbit antise? -.= rZiSed against a&" subclass of =use
:96s . * !t was found hat ; ;G from cloned hyoridomes of St . S2, and S6 were jgG

and igG . . respectively

AF

09

Case 1:05-cv-12237-WGY

Document 547-37

Filed 06/22/2007

Page 5 of 6

5 bur · 4 iUt'gn Of "uman urinary ervthroooie :'n with "nr.noadsorbent Cblusi ; vies : otherwise incica :e : . Ail

Z05

procedures for Ourification were perfor-ea at 0-4' : . ;more than 0 .5 unit/ml of urine) was collected

urine will mien EPO frtm anemic oatienu

in mottles conuining soa'um az ;de during cold seasons (Seotemoer-March) .
t ollec .e c urine was

The
to store

roti, Use .
: . e rs- ve .

Tie ' ;r , ne was : .hawec, filtered Jnoer Suc :'on, and concentrated by ultrahollow-%iSer cevice (Amicon) with a nominal :-Ir cutoff of 10,000 .

:rougnt into the laboratory every wets and frozen at -20'C

tra : .an on. I

-emovai of ;ow noiecuiar weignt materials in the final stage of concentra:'dies rtoeating ad .ition Of coil distilled water and :rated at one time and tne :,bout 70 liter of urine was

: :or. was e,- :r-ec :y severe' suosecuent :oncentra :'an .
treataront toot about =6 h

:c comalete . The urine concentrate was iyoonilizee . About

~ .! g of pry materials

as acained from l liter of urine .

protein was measured wit'+ :oomassi Brilliant Blue binding assay (Bio-Rad), using :P0 activity was measured with in vitro laoeiied thymicine ovaibumin as a stanGerc . -ooratcries . Canaca ;snee : Oiasma EPO steo-III, a units/mo protein) . ?-ate in
' Mg , .

Urine concentrate `ram ca .

-abie : : Summary :f erytnroocietin purification with immunoaasoroent column

70 liter human urine was useo as starting material .

incoroorat .on using fetal mouse liver as target cells or with in vivo radioactive 'e Standard :?0 was f-cm Connaught Aesearcn inccr :oration method, using starved rats (2) .

Step

Activity in vitro (units)
x 10

Yield

.3 10 75 63

Soec'fic activity in vitro ,unitsrmgl 23 5940 880

.. Pur-,f- a- ,.on

Soec'fic activity in vivo ;uri s ; ma ; 1 .4

;tine concentrate iwanunoaasorpent column Seonaaex 0-100

340 lO 5 .5

792 594 50

2580 3830

590 8160

Urine concentrate, wnicn came from ca .

70

liters of patients with &plastic anemia

was aopliec on a cOiumn in which EPO-specific monoclonal antibody fixed on

Affi-Gel

10 was pacxea . The column was extensively washed with PES and then developed with
0 .214 acetate buffer, pn

2 .510 .15M

NaCl . Fractions elutes with the pH Purification with the

2 .5

buffer were

immediately neutralized with 3 .414 Tris-base .

4 =unaadsoroent

column was tremendously effective ; most of protein in :he urine concentrate emer ;ee
in the flow-througn fractions without being adsorbed &me
ON

2 .5 buffer . Twentysix nunored-fold purification was achieved by

5?0

was elutco Sharoly by the
:.his

single step

&no 75 : of 7e activity was recovered (Table ill . EPO purified with : .:e iimunoadsoroent column was recagnizea as a Cain band with
Ar

35 .0

on SOS-ooiyacryiamide gel elec :rcnoresis Out Same Other oratain bands which was a main contaminant were seen . Furtherccre

including the Mr 20 .0 nrotein

this preparation was tinge : with faint brown . Further purification with a Seonadex G-100 revealed two peaks of
E .0 0 activity was recovered

orouin .

i t was

found by measuring the activity in the cooled preoarati :n of eac, peat that most of the
'n the second peak and about 5 : of the total recovered

activity appeared in void fractions .

Faint bran appeared in void fractions and the Analysis of Seonaeex 5-100 fracadns

pealed EPO preparation was cuite transparent . :a

with SOS-polyacrylamice tel electeognoresis demonstrate_ tit c :ntaminanu including

AF 9 9 0

20,0 protein, which were found in the EPO preparation

Case 1:05-cv-12237-WGY

Document 547-37

Filed 06/22/2007

Page 6 of 6

3 206 the main c ontasinaht . m r purified with vie irsaunoacsoroent column were not detec ed in any frac :ions of the second peak . However . :hey contained faint put a tectaole protein camconents in ieadinn side of the E?0 main band with Mr 35,000 . Activity measurement in the extracts fro . sliced gels indicated that ;here is V0 activity in the leading side as well as in _he As inown in 'sole main band . suggest',ng neterogeneity of E?0 protein . purification of human urinary EPO with an tarmunoadsoroent and suseduent Seoneoex G-100 column The soecific .ac : ;vity of pure E?C was 88 .0 provides us pure E?0 :n a nign yield . I units/mg of protein with 4n vitro - :i-thymidine incorporation method and 81 .50 with in vivo starved rat mehod (se--- Table ii) . These values are higher than 70 .40 of ne -isolated human urinary EPO wiLn conventions ; purification methods'by Miyake e : although the cifferences seem unimportant from limited reliaoility of the EPO assay methods . References Krystal . G : Ex: . Hematol ., 11, 18-31 (1983) Yanagawa, S . . :i . ;lanta . R . Sasaki, H . Chiba . 11 . Itaca, and H . Okada : Agric . Biol . Chem ., 47, 1:311-1315 (1903) Yanagawa . S . . S . vocoyama . K . Hi race, R . Sasaki, H . Chiba, M . Ueca . &no M . Goto sucmitted for ouclicatioh Yanagawa, S . . < . i!irace . H . Ohnota . R . Sasaki, H . Chiba, M . Ueda . and M . Goto suomitted far Duplication Miyake, T . . K .-A . Kung and E . Goldwasser . Bi ol . Chem . . 252, 5558-5564 (1977)

AF 99 1