CA2030782A1

CA2030782A1 - Lyophilized and reconstituted red blood cell compositions

Info

Publication number: CA2030782A1
Application number: CA002030782A
Authority: CA
Inventors: Raymond Paul Goodrich, Jr.; Christine Marie Williams; Roger W. Hackett
Original assignee: Individual
Current assignee: Terumo BCT Inc
Priority date: 1989-04-10
Filing date: 1990-04-10
Publication date: 1990-10-11
Also published as: EP0392813A3; EP0392813A2; EP0420977A1; IL94059A0; WO1990012582A1; EP0420977A4; US5178884A; JPH04502770A

Abstract

57) Abstract A process and medium are disclosed for the lyophilization of red blood cells which comprises the use of solutions includ-ng monosaccharide hexoses and pentoses, and biocompatible amphipathic polymers to permit the reconstitution of viable red ?lood cells.

Description

, j ~ DEC 07 '9~ 04:09PM FLEHR,HOH~CH,TEST 2030782 P.4 WO 9O/12S82 PCl'/US90~01827 NOVEL LYOBHILIZED AN~ ~Eco~sTITvTED
~ED BLOOD CELL COMPOS~TlONS
FIELD o~ TH~ INVENTTON

Th$~ ~nv~nt$on r-lat-c to ~h~ g-n-ral fi-ld o~
blooho~l~try ~nd ~-dlcal cl-nc-~, ~nd p-o~r$cally to novol lyophlliz-d and r-con~titut-d r-d blood c-ll oo~po~tJon-s ~c ~ppl~cstion i~ a contlnuation-in-p~rt o~
c~pon~lng commonly a~iyn-d s~r NO. 36~,386, ril-~
Jun- 2, 1~9~ whlch ~ conelnuatlon of cop~ndlng commonly a-~ign-d s-r No 335,55~, f$1-d April lo, ll~oC~ Wh~h ~.o ~ ~ontlnu~t~or~-~n p~r~s o~ O--r.
Nm~ ,7~ l~d May 19, 1999, nd 237, 603, fil-~
Augu-t 2~, 19~8 th~ olo~ur~ Or w~lc~ ar-lncorporate~ ~-r-ln by r-~-r-nc- ln th-ir ~ntlr~ty BAc~cgQuN~ ~F 5H~ ~Ny~NTT~

~od ~ ~ au-~o~ t~ Or tho ~ n bo;ly~ l~nd h~
a pr-domlnane ~ol- th- d-llvery o~ oxyg-n Sro~ t~o lun~ to p r~ph-ral tl-~u-~ Th~ rol- 1- carrl~d out ~y rythrôoyt~ , r-d blood cellr ~RBC) ~n- OXSrc~-n ~- turn~ n~c~ to tn~ p-r~ph~rAl <~ t~r-~
tho lun~- by ~n oxch~ngo-dlr~u~ion ~y~t-m brought 2C about ~y ~ r~d, lron-conta~nin~ prot-~n c~llod h-ooglo~ln Wh-n h-moglob~n co~b~n-- w~th oxyg-~, - DEC 07 '90 04:10PI~ FLEHR,HOH~3flCH,TEST 2030782 P,5 WO90/~2 ~CT/US90/0~82 oxy~-moglobin ~ ~orm~d ~nd wh~n oxyg-n ~- g~-n up to th- tl--ua~, th~ oxyh-~oglobin i~ r-duc-d to d~oxyh-aoglo~ln ~h- rod c~ bran- 1~ oompo~ of two ~a~or tructur~l unltJ, th~ ~o~br~no bll~y~r and A
cyto~k~l-ton A llpl~ bllay-r and ~ntogral ~ombr~ne prot-ln~ ~onm thc ~-~brano bllayer, whlch h-- llttl-~tructural ~trongth ~nd Sr-go-nt- ~eadlly by v-~lculatlon ~hc oth~r ra~or co~pon-nt, th~
o ~-~br~n~ k l-ton, ~t-bll$~ tho ao~br~n- b~l-y~r nd pro~id-~ t-ne- to d-~or~t~on Th-cyto~ ton ~s l$nk-d to th- btlay-r $n tb-rythrocyta ~-~bran-, poJ-lbly by llpl~-proe-ln a~
w 11 a~ prot-in-prot-$n a~ocl-tlon~ ~h~
h-~o~lobin, ~nd oth-r ~BC co~ponont-, ar- cont~n-d wlthln th- r-d o~ bran-~n ~ult~, bon- ~rrow 1~ aotlv~ ln th- ~or~-tion o~
n~w r-d blood o-lla. Onc- rythrocyt-- nt-r th~
blood, th-aa c-ll~ h-v- ~n a~ r~g~ tlm- o~ about ~20 daya ~n ~n ~v-r~g- p-r-on, about 0 ~3% oS th-rythrocyt-- ~r- d--troy-d ~ch day by ph~gocyto~
h-~oly-i~ or ~ ch~n~o~l d~g- ln th- body, and th-d-pl-t~ C-ll~ S- ron-w-d ~ro~ th- bon- ~arrow A wl~- v~rl-ty o~ ln~url-~ ~nd ~-dlc-l proc-dur--~S r qulr- tb~ tr~n ~u-lon o~ wbo~- blood or v~rl-ty o~ blood oo~pon-nt~ Fv-ry F~tl-nt do-- not r-qulr-~bol- blood ~n~, ln t~ct, th pr ~-nc- o~ ~11 o~ th-bloo~ oo~on ~ c~n c~u - n-~o~l ~robl-o~
8-p~r~t- ~lood ~r~otlon- o n b- tor-d undcr tho~-p clal oon~ltlon- ~ ~t ult-d to ~--ur- tb-lr ~lolo~c~l ~ctlvlty t th- t~a~ o~ tr~n-~u~lon Fo~
X~pl-, wh~n donor blood 1- r-a-lv-~ ~t proc-~ng o-nt-~ ~yt~rooYt-- ~r- -p~t-d ~nd ~tor-d by v~rlou~ ~tbo~ 8ucb o-ll- ~r- ~tor~l- ln cltr~t--- DEC 07 '90 04:10PI`1 FLEHR,HOHElflCH,TEST 2030782 P.6 W090~12SB2 PCT/US90/01827 pho-phat~-d-xtro~- ~t 4~C for up to f~v~ w--ks, g~n-r~lly 8~ ~ un$t Or p~ok~d ~rythrocyt-- h~vlng a volumQ Of from 200 to 300 ~1 ~nd a h~m~tocrlt ~lu~
(-xpr-e~d a~ corpu~cular volu~ p~rc-nt) o~ 70 to 90 Erythrooyt-- ~ay al-o b~ tr~tod with ~lyc-rol and then Sroz-n at ~rom -30~ to -196~C and to~ed for up to ~-~-n yoar~ ~n a glyc-Fo~ ~olutlon, ~ut ~u~t b~
X-pt froz~n ~t low t-~p-ratur~ $n ord-r to ~ur~iv~
>uffic~-ntly ~or tran-Su~lon Bot~ tho~ thodJ
r~gulr- c~r-rul ~aln~-nanc- oS torag~ t-~p-~atur- to ~vold di-ruptlon of th- d--ir-d b~ologlo-l ~ctivity of th- orythroeyt-~, ~nd p~ovld~ a tw~nty-four ~our urviv~l t~ for at l---t 70~ o~ th~ tran-fu--d C-118, which i~ con-i~er~d to be an w c-pt-bl~ el for u-- in tran~lu~lon practic- in ocoraanoe wlth th- Am rioan A~oclatlon of Blood Bank tandard~
~t ha- thu- boon d--ld-ratum to obt~tn r-con-titutabl- r-d blood c-ll~ whloh can b- tor-d at ~l~h ~tor~g- t-mp-ratur~a (4 C to room t-~p-ratur--) wlth good ~h-lf llf- 6uoh r~d blood c-ll compo-lt~on~ would ~ac$1itat- th- vallability of rythrocyt-~ Sor a-dical purpo--~

Prlor to th- pr-~-nt ln~-ntion, lt has b--n b~ v~d to b- l~po~-~bl- to fr--~--dry aryth~ocyto~ ln a ~-nn-r whlch p-r~lt- th- r oon~tltutlon of th- c~
to for~ ryt~rooyt-- ~itb n lntaot oyto-~-l-ton and b~ologlo~lly-~otlv- ~oglo~ln, ~ iabl- r-d blood o~ Vla~lo ~C'- ¢~n b- c~ar-ct-rl~-d by on- or nor~ ot th- ~ollowlngs capabll~ty o~
ynth--l~ln~ ASPs c-ll aorphology Pso valu-~s oxy, ~-t ~nd h-4 v~lu--t ~CV, MCH, ~nd MCNC ~alu-~t c~ll an~y~ otl~ltyt an~ ~ y~yQ urvl~al Wh-n ~9C'-h~v- b-~n lyophlllz-d aecordtng to pr-viouo ~ thodo, ~or xa~pl- ln ~th-r ~n aqu-ou- or phO~ph~t--~utr-r-d alin~ ~P9S) olution, th- r-con-tltut-d - DEC 07 ' 90 04: 1 lPM QEHR, HOHE~CH, TEST 2 0 3 0 7 8 2 WO 90/12S82 PCrlUS90/n1827 - 4 .
cellc ~r~ damag-d to th- xt-nt th~t th~ c~ re not c~pabl~ o~ m~t~bollzln~ or ~ynth~izing ATP, and th- o-ll b~mo~lobin c~nnot tr~n-port oxyg-n Clutar~ld~hyd--~ix-d rythrocyt~-, wh~ch h~ b~
S lyoph~liz~d ~nd rooonst$tutod, h~ ound u~
prim~rlly ~n ~qglutlnation a---y-S~MM~RX OF ~ NV~NT~N

~h- co~po-ition~ prov~d-d by th~ pre~-nt inv-nt~on allow for ~tor~ge und-r nor~l cond~tion~, while m~$nt~1n~ng tho tructur- o~ th- c~ nd th~
biologlc~l actlv~ty of th- h-moglobln ~h~
co~po~tion~ of th- pr---nt i m ontlon ~y bo ~-con~tttut~d nd u--d on th-r~p-utlc l-v-l ~rlo~ly, tho compo-ition~ r~ ~d- ~y lm~r~ing a ~S p~ur~lity oS ~ythrocyt~s, ~nd~or h-mo~o~-~, ln pby~olog~c bu~f~r-d ~qu-ou~ olutiDn conta~n~n~ ~
car~ohydr~te, ~nd a ~lolo~lcally eomp~tlble polym-r, prorer~bly havl~g amphlp~thlc prop-rti-- By th-t-rm amphip~th~c ~t i- m-~nt th~ r- hy~rophobic and bydrophll~c port~on~ on ~ingl~ ~ol-eulo ~hi~
~mm-r~lon 1~ follow-d by fr--zlnq th~ ~olution, ~nd drylng th~ ~ro~-n ~olutlon ~o yl~ld nov-l fr--z--dr~-d rythrocyt-~ oont~lnlng l~- than 10~, and pr-f-rably ~bout 3% or l--- by w-lght of moi~tu~-, whlch, ~b-n r-oon-tltut-d, p~oduc- ~ iqnltlcant p-rc-nt~g- of vl~bl~, tr~n~ru-ably us-ful rod blood o~ , OF b- o-oJ-~ N thod- of r-oon-tltutlon of th- r ~ bloo~ o-ll- or homo~o~-~ ar~ o pro~ld~d ~0 F W ~ p o-phorouc-31 NMR p-otru~ t 1~2 M~s o~
~ol~d b~rlu~ d~-thylphoJph~t-DEC 07 ' 90 04: 12PM FLEHR, HOH8~H, TEST 2 0 3 0 7 8 ~
.

WO 90/12~82 PCI/VS90~01112 F~G. 2 1~ a pho~p~orou~-31 NMR p-etrum o~ hydr~tod ~imyrl-toyl p~o~phatidylchollno ~ultll~ r ~--lcl-~ .
~C 3 i~ a pho~phorou--31 NMR pectru~ o~ ~r--z--drl-d r-d blood e-ll~ without tha eryoprot-etant of tbo lnv-ntlon r2c . 4 i- ~ pho-phorou--31 ~MR ~p-ctru~ of ~r-~
~rl-d r-d blood c-ll- tr-~tod ~lth th~ e~yoprot-et~nt for~ulation aceording to Ex-~pl- 3 _~

~h- c~rbohydr~ utlliz-d to pr-pAr~ ~rythroeyt~ an~
or h-mo-om- oompo~ition~ aeeor~lng to th~ lnv-nt~on 1~ biolog~c~lly eomp~tibl- wlth th~ e~yth~ocyt~ or h-~o-omo~, that 1~, non-dl~ruptl~e to th- e-lla or h-~o-o~ a ~br~n-, ~nd on- wh~eh p-~m--t--, or 1-eapabl- oS p~r~-at~ng, th- ~e~br~no of th-rythroeyt-J or h-~o-om-~ Sho earbohydr-t- may b~
~-l-et-d Srom the group eon-~ating of ~ono-aech~rld~ ine- dl-aeeharld-~ d~ not app-~r ~o ~0 p-rm-~t- tho m ~br~n- to ~ny iqnirloant ext-nt Mono~aecharld- p-nto--- ~nd h-xo~e~ ~r- pr-~-rr-d a~
~ a ~n~l cone-ntration o~ ~om ~bout 7 0 to 37 5 w-l~ht ~ ln pho-p~t- buSS-rod ~al~no ~P~S~, pr-~o~bly about 26~ Xylo--, gluoo--, rl~o~o, ~nno-- ~nd ~ruc*o-- ~r- ~p~oy-~ to part~cular a~vantag-S~- Inv-nt~on wlll b- h-r ~t-r d~crlb~d ln ¢onn-otlon wlth rythrocyt-- ~JC~ ut lt ~111 b~
un~-r-tood lt 1~ al-o ~ppllo~l- to ~-~o-o~--~h- polym-r ~ay b- pr---nt in t~- ~olutlon ~n concontr~t~on- of ~ro~ a 2inal conc-ntration of about DEC 07 '90 04:13PM FLEHR, HOHaf~CH, TEST P, 9 - 203~782 WO 90~ SS2 PCT/US90/0182 lX w-ig~t ~ Up tD ~tur~t~o~, and ha~ A mol~cul~r w-igbt in ~h- r~nq~ oS rrom ~bout 5X to ~bou~ 360X
Pr-~rably, th- poly~er h~ ~ ~ol-cul~r w-lght ln the ~nng- o~ ~ro~ a~out 2 5X to about ~o~, ~o-t S ~re~r~ly Sro~ a~out 5X to 50X, an~ 1~ pr---nt ln oonc-ntrat~on oS ~o~ ~out 3 6 wolght ~ up to th~
ll~lt of olu~illty oS th~ poly~-r ln th- ~olution ~olym~r~ ct-d from th~ group con~l-ting of polyv~nylpyr~olldono ~PVP) nnd polyvinylpyrrolldon-doriv~tlvo-, an~ ~xtran ~nd ~-xtran ~-rlvatlv-~
provid~ ~gnlrlo~nt ~dv~nt~ Ho-t pr-f-rr-d th- ~e or polyvlnylpyrroll~ono ~an amphlpathlc polym~r) of a~-rag- ~ol-cul~r w-lght Or ln th- rang~
of 10-40X ~n ~n ~mount ln the rang~ oS 12-20% w~ht by volum~ ln th- ~o~utlon prlor to lyophlll2~tion A~no ~old b-c-d poly~ r- (~ rot-ln~ oxtr~n~
or hydroxy-thyl ~t-scb ~y l~o b~ ~mploy~ Otb-r ~phlp~thi~ poly~ r- ~y b- u--d, uch a- polox-m~r-ln ~ny of th-lr varlou- Sor~- ~h- u-e o~ th-c-rbobyd~t--polyo~r ~olutlon ln t~- lyophlll~tion Or r-d blOod o-ll- allow- for th- r-cov-ry o~ l~t~ct c~ gnl~$c~nt p-rc~nt~g~ oS w~ic~ oontain blologic~lly-~ctlv- h-mo~lobln W~ not lnt-nding to b bou~d by any tb~ory, th~ aaphlp~thlc prop~rt~
oS th- poly~-r allow th-~ to bind t~ t~- c-ll a-~bran- whll- p~otootln~ th- a-~br~n- urf~c~ by xt-n~lon o~ th- hydrophllle ~o~tlon ~neo th- qu-ou-nvlronn-nt 5hl- ~ y ~ll-vl~t- th- d-~ag- to th-o~ r~n ~bloh o~u-o- otb-r ~robl-~, uch ~-c~ ggr- ptlon a poly~nlon ~ay b u--d ~n tb- ~a~b~n~ of th-con~t~tUc-~ ~JC~- ~t a~y ~ ~ny polyanlon wblch 1- non-d~-r~ptlv~ to tb- c-ll n-~br-n- oS th-rythrocyt--, nd polyanlon- h~lng mult~pl-~ho~ t-, ~ulS-t- or c~rboxyl~t- gro~pa ar-pr-S-rr-d ~n~ a~y b- ~r ~-nt ln ~ount- o~ ~rom 0 0 DEC 07 '90 04:14PM FLEHR,HOH~ 1CH,TEST 203~7~2 P.1~1 _7_ w-lgbt p-rc-nt up to otur~t~on ln ~ ol~tion althouqb ~nal oonc-ntrot$on~ in the rong- oS ~ro~
bout 0 1 woight t up to obout 10 w-ight ~ 1-od~ont~g~ous, Mor- pr~fcr~bly, the poly~n~on i~ a compound havlng ~ultiplo ~n~onlc ~roup- which Are pho~pb~t-, ~ul~-t- or corboxyl~t- groupc Sp-olflcolly, poly~lon- ~ o~-d fro~ tho group oon-~-tlng o~ ~yropho~phat-, tripolypho~phat-, pho-phoryl~t-d ino-ltol- (ln¢lu~l~g 10 trlpho~pho~no~ltld~ ond lno-itol hex~pho-Ph-t-), 2,3-dlpho~phoglyc~r~t , ~d-no~n- trlphotphate, and h-p~r~ y b- mploy~d to ~gn~2~c-nt ~dYant~q-~h- ~o~t proforrod ~no~ltol ~-X-phO~ph-t-conc-ntr~tion r-ng~ 0-90 ~M
15 A~ hown by tb~ d~t- ~-t ~orth below, the de~crlb~d ~o~utlon- provld~ modi~ which p~rmit r-d blood cell~ to bo ~b~-ct~d to th~ ~tr ~ o~
2rc-zlng, ublimot~on nd r-con-tltutlon ~nd to ~or~
fr~ dri-d rod blOOd o~ w~lch may ~e 20 r-con-tltutc~ to yl-ld ooll~ which or~ copobl- of Sunctlonlng o- rythrocyt-- in ~a~m~l~

All p-rc-ntog-- -t ~ortb h-~-in ar~ ~xpr-~ed o~
w~l~ht p-rc-ntag-- indlcotlng w-i~ht of tb- ~olut~
v-rcu- thO total w-lgbt o~ th- ~olutlon unlo-~
25 ot~-rw~-- lndlo~tod, ~nd w-iqht/~olu~- p-ro-ntag-c lndlo~to wolght Or olut- v-r-u- tot~l volu~- o~ th-~olutlon ' A- not-d ~bo~-, th- proc--~ of th- lnv-ntion p~ov~d-c ~-dlu~ ~or th- lyophlll~atlon ~nd r-con-tltutlon of 30 lntact and blolo~lc-lly--ot~v- ~yth~ocyt-- Whll-th- ~ o~ th- lnv-ntlon ~r- nov-l lt wlll ~-und-r-tood th~t ~pp~r~tu- ~nd r-lat-d t-chniqu~c ar-~nown by tho~- o~ ~111 ln th- art ~or t~
lyop~ atlon oS varlou- ~at-r~ nd o-ll- ln DEC 07 '90 04:14P~1 ~LEHR,HOHBP/CH,TEST P.ll 203~782 W090/12582 PCI/IJS90~82 p~r~lcul~r, ~nd only th~ ap~ci f ~ c ~mp~ratur~- ~nd ~ppar~tu~ ~ploy-d in eb~ x~mpl~ are d-~crlbed hcr-~n ~rom thi~ d~crlpt~on, one of ordin~ry kill ln th- ~rt w~ll b- capable o~ employlng the ~ed~A of S th- lnventlon ln a p~ooos~ tor the ~re-ze-~ryinq ~n~
recon~tlt~tlon o~ ~nt~ct, Yl~lc rc~ blood c~
Th~ t-rm lyophlllz~tlon ~ bro~dly d~finod ~-Sro~zlng a ~ub~t~nc- ~n~ th-n roduclng th~
conc-ntr~tion oS on- of th- olut-s, na~-ly w~t-r, b~
lo ublim~tiDn ~nd d--orptlon, to l-v-l~ wh~ch wlll no long-r upport blolo~c~l or ehomlcal r-action~
~su~lly, th- dry~ng t~p 1- ~ooo~pll-h~d ln ~ hlgh v~cuum ~ow~v-r, wlth re~poct to th- ~tor~g- of c-ll~ ~n~ p~rtlcul~rly crythrocyt~s, th- xt-nt of d~y~ng (th~ ~ount of r--ldu~l moistur~ oS
~rlt~c~l lo~orS~nc- in th bility o~ c-ll- to wlth~t~nd long-t-rm stor~g- ~t rDom t-~p~r~tur- ~n th- ~-thod o~ th- lnv-ntion, c~ ~ay b~ lyophlll2-d tD ~ r -ldu-l w~t-r cont-nt o~ lo~ th~n 10 w-l~ht ~, pr-f-~bly l--~ th~n 3~, ~n~ ctll~ ~- r-con~tl~ut-d to tr~n~fu~bl~, th-r~p-utlc~lly u~-ful c-lls C~
wlth ~bout 3 w-l~ht ~ w~t-r cont-nt ~d- in aecord~nc- wlth th- pr-~-nS ln~ntlon h-v- b--n tor-d Sor up to two w-~k- ~t room t-~p-r~tur-, ~nd ~t 4 C ~o~ long-r th~n lght ~onth~, wlthout d-oompo-ltlon ~hl- S-r xo- d - th- curr-nt A A
t~nd~rd ~o~ ~ro~-n or ~ rrl~-r~t-d o-ll- o~ lx t ~ C or 1--- ~h~n on- ~y t roo~ t-~p-r~tur-~lthout ~-coapo~ltlon 5h- lyop ll~t~on olutlon w~ll b- bu~-r-d ~n eb~
~ n~- o~ p~ oS 7 0 to 7 ~ pr S-r~ly ~y ~ po-p~
bu~ -d ~lln- olutlon ~ typla~l p o~pb~t--bu~r~r-d ~lln- ~o~utlon wlll oomprl-~ ~ono- ~nd di-b~c od~un pho-~b~t- (u-u~lly ~round ~o ~M), od~u~

I;EC 07 '90 04:15P~ FLEHR, HOHB~CH, TEST 2 0 3 ~ 7 8 2 P. 12 WO90/1~82 PCT/US90/01827 _9_ chlorl~e ~ubually about 150 mM) and lO ~M adQnDslno ~hi- olutloa ~alntaln~ th- pH at around 7 2 A pr-f-rr-d pho~phat~-bu~f-r~ alln4 ~olutlon to b-u-od - tha lyophlll~tlon bu~f-r will eompri-~
py~uvat~, lno~ln-, d-nln-, pot~ium chlorid-, ~odlum ehlor~d-, ~nd dipota~$u~ pho~ph~t-, all of wh~eh will -rv- a- A b~o ~alt buf~-r at a pN o~
about 2 2 In addition thl- lyophlllz~tlon bu f ~r w~ll eont~in ~n-l con¢!ntr~tlon Or ~bout 30%
w~ight by volum~ o~ a ~ono-~ochar~d-, ~r-f-~ably 26~
(1 44 M) glueo--, and a ~lnal eonc-ntration o~ ~out 16~ w~ight by volum o~ polyvlnylpyrrol~don- (av-r~q-~ol~cular w~ght Or 40X~
Anoth-r ~-atur- Or th- in~-ntlon i~ that th-~yop~ z-tlon burf-r -FV-- - a o~yop~ot-ctant Th~ blood e-ll- dri-d wlth thi- prot-ct~nt xh~b~t a pho~phorou~-31 NM~ ~paetrum w~th a lln-~hap~ p~tt-rn oharaet-r~tle o~ a ~-mor~n- ~truetur~ oclat~d ~n a b~layor in a g~l pha~ nd indleat-~ that ~o~
~ ~br~n~ moblliey ~- r-t~ln-d ~- hown ~y ~t-r-ooh-~lc~l altoration in th~ pho~pbolipid h-ad~roup~, On tb- oth-r h~nd, e-ll- dr~-d wltbout th- protootant how a lin-Jhapo patt-rn $ndicat~ng lo-~ o~ m-mbran- bllay-r tructur-, vld-ne- o~
2~ ho-~qroup rlq$~lty, an~ po--ibl- ~or~atlon o~
h-xagon~l ph~-- llpld truotur- wh$oh 1~
Inco~pat~lo wlth c-ll ~ lllty Addit~on~lly, th-pho-p~orou--31 NMR lln-wl~th ror o-ll- drl-d with th-protoctant $- r-duc-d b~ at l-~-t B~ compar-d to tb-lln-widtb for ~lallar o-ll~ dr$od wlthout th-prot-otant ~h$~ roductlon ln pho-phorou--31 NKR
lln-w~dth ~-y vary, d p-n~lng upon th~ partlcul~r ~a~pl~, rro~ B~ to bout ZO~

DEC 07 ' 90 04: 16Ptl FLEHR, HOH~FlCH~ TEST 2 0 3 0 7 8 2 P, 13 -WO90/12S~2 PCT/~S90/018~7 ~h- ~ryt~roeyt-- wlll pr~r~ ly ~ pr~p~r~ rrom whol- blood ccntrlrug~tion~ ov~l o~ tb- pl-~ma ~up~rn~tant an~ r-~u-p~ndlng the p~ t~ ~n P~S
~hl- w-~h cyclc ~ y be r~poatod 2-3 t$n~, th-n tho pae~od c~ ar- dllutod w~th tho ~yoph~ atlon buffer ~crlb~d abov- o that th~ ~lnal d~lut~d con¢-ntr~t~on of c~bohydr~t- ~n~ polymor ~r-aalnta~n-d ln t~- n-c~ y r~ng-~

Alt-rn~tlv-ly, eo~o-rcl~lly v~ bl- p-c~d blood ~0 o~ ay b~ u--d, ~hloh typlo~ly ~- prcp-~-d ~n CPDA tco~-rclal olutlon cont~lnlng eler~t-, phoaphat~, ~-xtro~- ~nd ~d-nln-) Upo~ lyophll~z~t~on by conv-ntlonal t-chnlqu-~ to aol-tur~ eontont of 1-~ than 10%, ~nd pr-~-r~bly lo-- than 3~, th- lyophlllz-d c-ll- aay b- ~alntain~d und-r v-cuu~ ~n vacuum-t~g~t oontalner-, or und-r nltrog-n or oth-r ln-rt ga-, ~t roo~ t-mp-r~tur-- ~or Xt-nd-d Foriod- of ti~ ~n aD--nc- D~ t~DUt ~gnl~ic~nt d~gr-datlon o~ tn-lr ~-~is~bl- proportlo~
wh-n r con~t~tut~ for u~ tr~n~u-~bl~ e~ It 1~ ~ partlcul~r ~v~nt~q- of th- pr-~-nt lnv-nt~on th~t th~ lyophlllz-d o-ll- ~y b- ~tor-d ~t room t-~p-~-tur- ~or Kt-nd d p-rlod~ o~ ti~-, thu~
o~vl~tlng tb~ n -d ~or low to~p-r~tur- r-frlg-r-t$on ~blob 1J r-~ulrod lor torln~ lyophlll~-d r d blood o~ pr p-r ~ ~y ~ tb d - o~ tb- prlor ~rt lt 1- ~ ~u~th-r n~v~nt~- o~ ~h pr---n~ ~n~ ntlon tb-t th- lyo p l~l~-d ~ d ~lo d o ll- ~y b rooon tltYt d ~t nor ~1 t -p r~tur ~ r-~t~r ~b~n ~out ~-C up to bout ~7 C, ~hloh oorr-~pond-to no~o~l ~n~n k d y tonp~r~tur , ~n~ pr-f-r bly ~t roo~ t np-r~tur- ~bout 22 C) ~h- r-con-tieutlon dlu~ 1~ pr-~-r~blY ~olutlon co~rl-ln~ ~ poly~-~
or ~lxtut- of ~olyo-r- b~vlng ~ ~ol-oul~r w-lght o~

DEC 07 '90 04:17P11 FLEHR,HOHBflCH,TEST 20307~2 P.14 WO 90/12582 PCI`/US90~01~27 from ~bout 2 . 5K to 360 X, pr-r-r~bly 5X to ~bout 3~0X, pro-ont ~n ~ conc-ntr~tlon in th~ r~n~ of about ~2 to 30~ w~lght by ~olu~ ~hi~ poly~-r m~y b- th- ~ poly~r utll~2-d to lyophll$z- th~ r-d bloo~ c-ll~ cr$bod ~bov~ Hono~ th~ polym~r~
polyvlnylpyrrolldono, hy~roxy-thyl ~t~roh, ~n~
~-xtr~n ~r- p~rtleularly pr~f-rr-d nd ~o~t pro~-rr-d ~- polyvlnylpyrrol$don- pr-~-nt ln ~ eono-ntratlon or ~bout ~9% wo$ght by ~olum- ln th~ r-con-titutlon olutlon ~h- r~con~tltutlon olution wlll b-bu~r-r-d ~galn typleAlly by pho~ph~t--buff-r-d ~alln-a~ d--er$b~d h~r-lnabo~- to ~lnt~n ~ pH within tb~
r-ngo Or ~bout 7 0 to 7 4 Tho ~o~t p~rtlcul~rly pr~f-rr-d poly~r i- polyvlnyl~yrrolidon~ o~ ~n ~v-rag~ ~ol-eul~r w-$ght or About lOX
Th- ~o-t pr f-rr-d r-oon~tltut$on buSS~r w~ll b- a Jolutlon comprl~ln~ pyruv~t-, lno-ln-, ad-nin~, pota--ium ohlorld-, odlum chlorid- ~nd dipota--ium pbo pbat-, ~11 o~ whlch rorO ~ b~-lc ~alt bu~ at a pH of ~bout 7 2, whlch al-o eontaln- ~bout 19% w~ght ~y volua- o~ polyvlnylpyrroll~on- ~av-r-g~ ~ol-eul~r w lght ~bout lOX) ~h- r-eon-tltutlon ~olutlon m~y al~o optionally eont~n a ~ono-~ceh~rld-, pr ~-r~bly pr-~-nt ln th-conc-ntr~t~on r~ng~ or ~bout 7 0 to 31.5~ w-lght by ~olu~- ~h- ~r r-rr d ~ono~cob~rl~ - xylo--, glYeo~-, rlbo~ nno - and ~rueto--Sn tb- mo~t ~r ~-rr d mbodln~nt, tb- lyop~ -d rythrocyta~ oan bo r-oon-tltut-d ~y ~x~g wlth ~n qual volu~- o~ th- r oon-tltut~on buSf-r at a t-~p ratur- o~ about 3~ C ~nd ~x-d untll tully hydrat-d By n-qual" lt 1- ~ant that th- volum- 1 th- am- a- t~- ~tartin~ volum- ~rlor to lyopblll~atlon DEC 07 '90 04:17PM FLEHR,HOH13f:CH,TEST 2030782 P.15 -WO ~/t2S42 rCT/US90/0~8~, ~h-n, lt 1- pr-f-rr-d th~t th~ r-hydr~t-d rythrooyt-~ b- w~-h-d aoeordlng tD th- followin~
proc-dur~ ~t ~ r-~llz-d, ~ow-~-r, th~t one- the rythrocytes ~rQ roeon~tltut~d ~ith ~eeonstitutlon g buSS-r th-y are ln ~ tran-Su~ly-u~-fu~ ~o~m, but th~ eo~bin~tlon of ~-hlng~ d~or$bo~ h-r~ f~-r ~r-pr ~-rr-d, ~p-elrie~lly ~or u~- o~ th- e-~l- Sor el~nle~l purpo---ArtOr epar~tlng th~ rythroeyt~ Srom tb-~0 r-con-t~tutlon buSS-r ~y o~ntr~ug~t~on tho r--u~t~ng rythroeyt-- u~u~lly ~n th- Sorm oS
p~ll-t, ~r- pr-S-ra~ly r-~-p-nd-d ~n (~pproxlm~t~
tb- ~olum~ u~-d ln th- r-con~t~tution) ~ w~Jhln~
buS~-r oomprl-ln~ th- b~-le ~lt bu~r t pH ~ 2, d-~eslb-d ~bo~-, rurtb~r oont~ln~ng ~bout 16~ w~i~ht by volum- po~yvlnylpyrrolldon- ~mol-cul~r w~lght bout ~OX). S-p r t~on by eontr~fu~t~on eoapl-t-~
th~ t po~t-r-bydr~t~o~ t-p, ~ w~-h~n~ ~t-p For th- ~oeond ~t-p, th- w-~h-d c~ ar- r--u-p-nd~
~n th- w~blng ~u~-r ~nd ~ glycolytic lnt-r~di~ry ~olutlon ~GI~ dd-~. ~b~ total volum- 1-pr-~-r-bly t~n t~- - th- volu~- o~ pac~c-d c-ll~
pr---nt. Th- ~rolum- o~ w~-h~ng bur~-r ~hould b-u2Sloi-nt to ~-ct ~ t-n-~old dilu~cion o~ th-voluo- o~ add-d a~s. ~h- e~s wlll cont~in th~
~oll w ln~s J~DP
~tAD
d-rln ~-no~ln-lno ln 11-;~0 . _pEC 07 '90 04:18PM FLEHR,HOHa~CH,TEST 2030782 P 16 WO90/~2S82 PCT/~'S90/01827 Tho c~ in thl~ ~u~p-n-ion ~r~ th~n incub-t-d, pr-f~rably ~t room t~p-r~tur~, for about 30-60 mi~ut~ It wlll b~ r~ z~d th~t the dur~tion ~nd ~-mp~r~tu~- of the incub~tlon ~y vary from th---S conditlon~ ~ long ~ lt l~ not dotrlm~nt~l to thQ
c~ Whll- not lntonding to bO bound ~y ~ th-ory, lt l- b~ vod th~t th~ ln¢ub~t~on r~to~o~ cor~ctor-lmport~nt ror A~P ynth~ to th~ o~ lo~t durln~
lyophll~z~tlon A~t-r ~ ~uf~ic~-nt p-rlod oS incu~tlon, th-u-p~n-lon i~ c-ntrlSug~d to co~pl~t~ th~ ond po-t-hydrot~on t-p ~he third post-r~hydr~tlon t~p 1~ ~n ~-o-o-motic pul-lng ~t~p in whlch th- ~ythrocyt~s ~r-r--u-p-~ded ~n an ~-o-o~motlc olution compri-~ng ~no~ltol h~xapho-ph~t- ~HP) ~at a ~ln~l conc-ntr~tlon oS ~bout 0 ~ to 7% wt/vo~) pr-r-~-bly 20-90 mM, ~nd poly~thyl-n~ ~lyeol ~av-rag- mol-cul~r w-lght Srom 1 XD to S X~, pr-S-rably about 3 3 XD~, t ~ pn 0~ ~bout 7 2 Oth-r polyanlons moy bo u~d ln pl~c~ Or or ln co~bln~t~on w~th IHP, euch ~, ~d-no~lne trlpho-pb~t~, pyropho~ph~t- or tr~polypho~ph~to ~hl- ~olutlon h~- ~n o-mot~c pr-~-ur- oS a~out 311 mO- ~rtor ~pprox~m~t-ly ~ 10-~0 ~ol~ d~lut~on, by ~dln~ w~hlng bu~-r o th~t th- opprox~-t- volum- l- th~t u--d ln th-rocon~t~tutlon, th- ~u-p-n-lon 1~ c-ntri2ug-d to co~pl-t- th- thlrd po t-hy~r~t~on t-p rOr th- ~ourt~ po~t-hydratlon t~p th~ pack-d rythrocyt-~ ~r- lncubAt-d ~or ~bout 30 ~lnut-~ at bout 3~C b Sor b lng r--u-p-nd-d ln a p~o~p~t~-buS~-r~d-~luco~--ad-no-ln- ~olut~on tP~S-eA), pr S-r~bly cont~inln~ a~out 5 ~M g~uco~ nd ~bout 10 rM ~d-no~ln~ t-p 1- conduct-d ~t ~bout DEC 07 ' 90 04: l9PM FLEHR, HOHE~CH, TEST 2 0 3 0 7 8 2 P . 17 W~ gomS82 rCT/USg0/0l82 37 C C~ntr~u~tlon co~pl~to~ th~ ~ourth t-p ~h-wa~h 1- tb~n rap-~t-d u-lng th~ PBS-CA olution ~t room e-mp~r~tur- ~ a Slrth po~t-hy~r~tlon ~tep Art-r ~-pa~atlon by c-ntrl~uga~on, tbl~ tinal ~t~p ~ co~pl-t-~

~h- r-con-tltutod CQl~- ac¢ordlnq to th~ pr---nt ln~-ntion hav~ ehar-ct-rl~tlc- w~loh rcnd-r th~m tr~n-~u~ablo ~nd u~-ful ~or thor~poutlc purpo--- ln that tb-lr prop-rtl-- ar- ~lm~lar to tbat o~ nor~l ~ ~ not pr-~ouJly lyophll$z-d) red blooa o~
Typlcally racon~titut-d r-d blood o-ll- accordlng to th- pr--~nt inv-ntion h-v- an oxyh-mo~lobln cont-nt gr-at-r th~n bout 90% o~ tb-t in nor~al r-d blo~d c~ o~oglob~n r-cov-ry prlor to any waJh~ng ~S t-p 1- typleally ln th- rang- o~ 80 to 8S~ Tb~
ov-rall h-~o~lobln r-cov-ry lnoludlnq th- po~t-hyd~at~on ~a~h~ng t-p~ ~- about 20 to 30%. ~h~
~orpholo~y of th- r-c~n-tltut-d c-ll- ~oco~dln~ to th- pr--ont lnv-ntlon ~by cannlng l-ctron ~icro~oop-) typ~c-lly how~ no hol-~ or ~p~, ~n~
~l-cocytic or to~atocytlc ~orpholo~y ~h- oxyg-n carryln~ e~paelty oS nor~al r~ blood c-ll- ~a~
~-a~ur-d by ~50, th- pr-~ur- at whlch 50% of th-oxy~cn ~oS~oulo- ar- bound) w-~ ~-a~ur-d to b- ln th~
~S rang- Or ~bout 26 to ~8 ~-v-r~ 6 7)~ wlth an a~r~ 111 oo f~lol-nt Or ~.9S. ~h- typlcal P
for rythrocyt-- lyo p lll~-d and r con-tltut-d ~ceordln~ to th pr --nt lnv-ntlon 1- bout 27 5 ~cr~9-) v~tb n ~v-~ $11 oo-~lcl~nt o~ 2 ~8 A--~y~ o~ A~P ln t~- ~ oon~tltut d c-ll- lndle~t~ ATP
l v-l- ~u~ tl~g noro-l ATP to ADP a t~oll-~
Norn~ lutln~tlon o~ r-~ ~loo~ o~ d-~ooordlng to th- pr --nt lnv ntlon $~ o typlc~lly ~oun~

DEC 07 '90 ~34:20PM FLEHR,HOH13~CH,TEST 203~782 P.18 WO90/12~82 PC~/US90/01827 B-v~ng d-scrlb-d th~ pr-f~rr~d ~mbo~lm-nt~ Or th~
pr-~en~ ln~nt~on, th~ ro~lowlng ~xamples ar~
provld-d by way of lllu~tr~tlon but are not lnt-nd-d to llm~t th- lnv-ntlon $n any way llYLeLE_~
R d blood e~ ar- o-ntrlfug~d ~o~ lo mlnut-- at 1,500 ~CF ~n a Ni~tr~l 3000 e-ntrltug- Pla-~ an~
bu~fy eo-t ar- ~-aov-d ~h- o-ll~ ar- ~ub--qu-ntly ~a-h-d 2-3 tl~ w~tb PBS-CA ~Dulb~ceo PBS, s~M
glueo~- ~o 90 g/l), and lo~M ~d~no~ino (2 67 g/l) ) ~h- e-ll- ~r~ mlx-d ln ~ fr~-zlng n a-k wlth lyophtliz~tion bu~r, tb~t w~ pr~-w~rmed to 37 C, at ~ ~-m~toer~t of 10~ and lneubat-d for 30 ~nut-~
~t 37~C ~ nd ~u~-r ~r- ~lx-d l~m-d~t-ly to pr-v-nt ~ggr-gat~on of e-ll-S~- lyophll~zatlon ~u~r-r 1~ ollow~
10 ~M pyruv~t- 1 100 q~l 10 ~M lno~ln- 2 68z g/l S mM ~d-nln- 0 676 ~ b~J;e ~lt ao 75 aM NaCl 4 380 ~/1 burrar 10 ~M N~2HP04 1 420 ~Jl lpH 7 2 1 6 M Clueo-- 28~ 3 g/l 16% w~ Pl~don- C-30 160 ~/1 (polyvlnyl Zs pyrrol~don-, 40 XD) ~h- ~r -~ln~ rl~ 1- th-n rot~t-d ln liquid n~tro~n to ~orm ~n v-n ~-11, os, ~lt-rnatl~-ly, pl-o-d on ~ r--z-r ~t -85~C ~H OH) ~nd ~otat-d to ~or~ a h-ll an~ pl-c-d ln l~uld nltro~-n S~- ~a~pl~- ar~
tb-n tr~n-~rr d to ~ Laboonoo b-n¢btop fr--~--dry-r 5~ pl-- ~r- llow d to dry ov-nnl~ht, or untll th- ~mpl- 1- thoroug~ly drl-d to about 3% by w ight ~ol~tur- oont-nt (3 33~ ~ 0 13 (grav~-trlc)~ 2.388 0 2810 (Xar~ -ch-r)~ ~o r con~tltut- th- ~rl-d ~pl--, an qual volum- o~ pr--warm-d r-con~tltue~on ,~EC 07 '90 04:21PM FLEHR,HOH~CH,TEST 2030q. 8~ P.lg WOgO/1~82 PCT/US90/01827 ~u~-r ~ ~dd-d ln ~ w~t~r b~th a~ 37~C ~h- ~mpl-1~ ~w~rl-d untll fully hydr~t-d Th- r-con-t~tutlon buf~-r i~ ollow~
b~-lc ~lt bu~-r lpH 7,Z~
19% ~/v Pl~don- C~ 90 9/1 (polyvlnyl~yrrol$don-, ~o XD) Sh- r-con~t~tut-d o~ r- o~ntrl u~-~ ln th-Ml-tr~l 3000 c-ntrlfug~ ~t 1700 scS for 10 ~ln (t-mp~r~tur~ ottln~ 20'C~

Sh~ p-ll-t 1~ ~ ~u~p-nd-d in ~hlng bu~r ~nd c-ntrl~sg-d und-r th- ~m- condltlon~
Sh- W~hlng bu~ - follow~s ~lc ~lt bufS-r IF~ 7 16~ tw/v) Pl--don- C-30 160 ~/1 ~polyvlnylpyrrolldon-, ~o XD) ~h- c~ r~ r~-u-p-nd-d ln th~ v~-h bu~f-r ~nd the ess buff-r w-~ ~dd-d Suffiol-nt w~h buff~ u~--to ~$1ut- th~ ClS bu~-r ~y ~bout t-n-~old T~- CIS
buf~-r l~ ~ rollow-s /100 ~1 A~P 1.~ 67.2 NAD ~0.0 331 7 d-n~n- ~0.0 6~.6 ~d-no~ln- 50 0 133 6 lno~ln~ 50.0 ~3~ 1 N~HP0~ ~0.0 1~2 0 c.cl2 s . o 73 . 5 ~0 ~qC12 6h20 5 0 101 7 Sh- u~p n~$on ~- lnou~t-~ ~or 30-60 ~lnut-~, th-n o~ntrl~ug-d ~t ~700 RCr ~ 10 ~lnut--- DEC 07 '90 04:ZlP~1 FLEHR,HOH13~1CH,TEST P.Z0 - 203~2 WOg0/1~82 PCT/US90/01827 ~h~ p~ t ~ th~ r--u~p~nd~d ~n ~n INp-~olutlon ~311 mO-) ~t ~ ~0-50 ~o~d ~llut~on, ~nd o-ntrl~ugod at 500 RCF ror 10 ~lnut-~
~b- IHP ~olution ~ follow~t ~HP ~o~-cA~o~lu~ ~lt (pN ~ 2) 33 3 g/l ~h~xacalelum alt ~y b- u-~d (27 ~M) or pur cld ~orm of ~P) Polyothyl~n-glyeol ~3 3kD) lO g/l ~O 300-310 ~O-~b- ~up~rnit~"e ~ dr~wn Or~ ~nd tb- p~ t i~
lneubat-~ for 30 ~in at 37'C ln ~lth~r D-x--~l$n- or P~S-OA ~pr--wa~m-d to 37 C) ~nd o-ntrlru9-d Thi~
t-p ~ r~p-~t~d wlth D-x-~ n ~or PBS-GA) ~t R. S .
~o d-t-rmln- b-mo~lobin r-co~-ry, a Ja~pl- 1-e-ntrifug~d ~or 2 ~lnut-- ~t 9000 rpm ln ~n ~pp-nd~rS contrl~u~ - p-~l-t ~nd up-rnat~nt ~r- -p~r~tod ~nd 180 ~1 of d ~ wat-r 1- ~dd-d to th- p-ll-t~, whleh ~r- ly~-d by r~gorou~ly vort-xlng ~o ~eh ~a~pl~ dd-d 1 ~1 ~r~b~in~ r-~g-nt, and aSt-r ~t~nd~ng ~t ~ ~ Sor 15 mlnut~, th-~orb~nco~ ~t ~40 ~m ar- t~k~n Rooov-ry - A540 p-ll-t~ ~540 p-ll-t I As40 ~up-~n~t~nt, rtablllty of th- c-ll~ ean b- m-~ur-~ by inoub~tln~
~ a~plo ~t 100-200-~old d~lut~on in P~S-GA, autologou~ pl~-~a or whol- blood for 30-60 ~inut-~ ~t 37 C ~-~oglob~n r-cov-ry ~ d-t-rmln-d by th-p-ctro p oto~ trlc ~ thod d--cr~b-d bov- or ~y ra~lolab-ll~ng or tb c ~l- On ~v-rag- ?o-~0~ o~
th- c-ll- urvl~- thl~ tr--tn-nt ~t~r on- hour ~h- ~ ~ur d n-t h-~oqlobln r-cov-ry ~-v-l~ ~r- on av-r q o 22-23~ ~ 2 6 Sh~ ASP l-v-l- ln th- r-con~tltutod o~ w-r~
~ y-d u~ln~ ~ ~ig~o A~P dl~gno~tlo X~t, Proc-dur-PEC 07 ~90 04:ZZPM FLEHR,HOH8~CH,TEST 2030782 P.Z1 WO 90/1~S82 K~US90~01827 No 3~6, for ~uantlt~tlv~, nzym~t~c d~ n-tlon Or A~P in blood at 3~0 nm Th~ a--ay w~s conduct-d on thr-~ dl~r-nt r-oon~titut~d human blood ~ples with on~ m-~-ur~mont e~x-n b~foro lncubation with th~
S CIS bu~f-r ~n~ cond ~a-uro~-nt ta~c-n ~tar lneu~tlon wlth th- OIS bu~r B-for a~-r C~S Sncub~tlon CI~ Ineub~tlon Sa~pl- No 1 0 5S ~ol/g 2 23 ~ mol/g S~pl-- No 2 ~ ol/q 4.4B 1~01/~
~mpl- ND. 3 0.65 I~ol/~ 2.54 l~mol/g ~h- pr-c-ding data ~how- tb~t lncu~tion in th- CIS
- burr-r lncrc~o~ th- ATP l-v-l~ by around 3- tD 4-fold ~h- a~ay ~thod w~- Sollow-d ~ p~r th~
l~ in-truce~on- ln th- Xlt, ~o~lrlad ~or u-- o~ l ml ~a~pl-~ r~th-r th-n 3 ml ~a~pl-J
~b~ Jollowlng e-ll in~ie-- w r~ o ~ ur-d on blood ~plo~ be~or~ an~ art~r th~ G~S lneub~t~ons MCY (~oan eorpu-eul-r volu~-)t MCB t~-~n eorp~-eular ~o h-~oglobln) t nd MCHC ~-an corpu~cul-s~ h-mo~lob~n cont-ne) ~or~ A~t-~

~5 ~CV0~ 0OUnt J~l- A '~9-0 U 61.3 ~1 J ~ 66.7 C - 6~
D - 73.2 ~O ~ . 5 6~
F g~ 2 ,~EC 07 '9~1 04:23Ptl FLEHR,HOH13RCH,TEST 2 0 3 0 ~ 8 2 WO90~12S82 PCT/VS90~0182 Lit-ra~lv- valu-- o~
non-lyop~ um~n (~0-9~) MC~l Hl~b(~/dl~ x 10 S ~a~opl~ A 28 pg13.8 pg J 16.9 12.3 C
D - ~6.7 E Z6.9 13.5 F Z9.1 16.9 Llt-r~tlv- v~lu-~ of non-lyophil~z-d hum~n t2~-31) MCHC . Ha~H~L~Q

~5 8~mpl- A ~4.0~ 22.6%
B 20.6 18.4 C - 21.S
D - 22.9 E 33.0 20.0 F 31.4 20.6 ~lt-r~t$~- valu~ O~
non-lyopblllz-d hu~an (32-36) ~ 2 Fro-h hu~n blood w~ lyophlll~-d and pro~ptly r-eon-tltut-d ~ollow$ng th- proc-dur- d--crl~-d ln Ex~pl- 1. B-~or- lyophll~z~t;on ~-v-ral k y ~lycolyt~c n~y~-~ crlt~c~l to norm-l r-d blood cel~
tunotlonlng w-r- ~-~ay-d ~or ct~v~ty by conv-nt~on~l proo-dur -. ~h- Foconctltutcd c~ w r- a~--y-d tor actlvl'cy b~ ~o~ - -n~y~o~. ~h- r-~ult- ~r-~o~n ~-low.

C 07 '90 04:Z3PI`1 FLEHR,HOHB~CH,TEST 20307~ P.23 WO 90/12582 PC~ S90~0~827 -ao-LYOPhIL~ZA~10~
U~;:
(unit~ /g Hb) Hoxo~ a-e 1. 20 ~ 0.12 0 . 68 Pho~phoglueos- ~oa-r~e 4S 3 ~ 6 0 32 Pho~p~ofruoto~ln~ 9 7 ~ 2 2 l9 S
Aldol~ 39 ~ 0 34 1 97 ~rlo-- Pho-ph~te I~oa r~- 2903 ~ 7~ Z~67 lo Glye-r~ld-hyd- 3-Pho~ph~t-D-bydrog-nas~ 244 ~ ~2 ~2 Dipho-p~oglyc-r~
m~t~- B . 4 3 ~ 2 . 2 3 3 . 9 4 ~5 Pho~phoglyc~r~t~ X~nA~- 349 ~ 47 ~ 2~6 Pho-phoglye-ro~ut~- 17 3 ~ 6 7 20 9 Enol~- 4 96 ~ O ~g 3 43 ~yruv~- Xin-~- lS ~ 2 14 13 6 I~ct~to D bydrog-n~- 141 ~ 56 4 146 ~h--- r-~lt~ ~ndie~t- th~ ~u~Siei-nt l-v-l- o~
~lood n~y~ r- pr~ t in th~ roeon~titut-d e-ll~
~or eoll-~unet~onlng 8-~pl-- oS drl-d rod bloo~ e-ll- w r- x-~ln~d ln th-pr---no- ~nd ~b--no- ot tb- oryoprot-et~nt ~oraul-t~o~ ~bi- ~or~ul~tlon oon~l~t-d o~ th-~ollowln~
2 00 aM pot---1u~ ohlor1d-1 47 ~M pot~--lu~ p o-ph-te 9~.~9 ~M o~u~ oh~orld-10 ~M ~od~u~ pho-ph~t-1.60 ~ glueo~-16 00 ~(w~ Polyvlnyl w rrolidon~ [40X MW ~vg ) I:EC 07 '90 04:Z4PM FLEHR,HOH13RCH,TEST 203~78~ P,24 WO90~ 2 PCT/VS90/0~827 ~2~-Th- c~ w~r- rroz~n ~t -80 ~ iu- ~t ~ 30~
H~matocrlt in tb~ approprl~to buff~r and ploc~d o~ a lyopbil~z~r for ~2 hour- Th~ ~ampl-- ~lowly quilibrat-d to room t-mp~ratur~ durln~ thl~ perlod Tb- ol~d ~atori~l wa- r~o~d ~nd pl-c-d ln ~ 10 mm dlam-t~r ~MR ~pl- tub- 6~pler w~r- xa~$n~d u-ing B ~62 MNz NMR ~p-ctrom-t-r w~th oro~
pol~rl~tlon Sa~pl-~ w-r- oxamin-d u~lng a pho-phorou--31 prob-A r~gid pho-ph~t~ wlll normally produc- ~ p~ctrum a~
d-p~ct-d ln Fig. 1 for olid b~riu~ diothyl~ho-yhat-Th- hap- ~ ~nd~cativ- of a rigid pho~yhat~ ~roup wlt~ no axial motlon ~ho lln-wldth l~ ~gual to 188 pp~ Nor~l l$qu~d-ery-tallln- pho~phollp~d- provld~
a l~n-~hap~ which ~ dr-~tlc~lly alt-r-d Thl- i~
d-plct~d ln ~l~ur- 2 tor hydr~t~d dimyr~toyl pbo~phat$dylcholin- (DMPC) ~ultllaa-ll~r v-~lc108.
~-adgroup ~otion 1~ $ndicat-d w$th a r-duetlon ln wldth to 6D-80 ppm Th- p-ctru~ Sor Sr -z--dr~-d r-d blood e~ wlthout ~y prot-ct~nt 1- d-plct-d ln rlgur- 3 Th- h~p-~- lndlo~tlv- o~ t~- lo-- Or ~o~- b~l-y-r tructur-~nd po-~$bl- Sor~atlon o~ b-xagon~l ph~- lipld Sructur- (lnd~cut-d by t~- ~ax$~u~ p-aX ~t ~out 2 2S XN~) Ih- lln-wldth t~05 ppm) lnd$eat-- t~at th~
group l~ rlgld ~h- p~tt-rn i6 qult d~tlnct ~o~ r d ~lood c-ll- drl-d wlth th- prot-et~nt-aecord~n~ ~o th- ln~-ntlon, d ~lo~-d ln Flgur- ~, ~hlo~ 1~ c~r~ot-rl~tlc or llpld ln ~ c~ t~t-Not- th~t th- 2 X~ p-~k ln Flgur~ 4 ~- r-duc-d to ~
hould~r, lndloatlng b--no- o~ or t l-~-t r-du~tlon o~ h-xn~on~l p a~- llPld t~uotur- Tb- lin-wldth ls r-duc-d to 9~ ~ ~pm lndlo~tlng o~- ~obll~ty or th~t - DEC 07 '90 04:Z5PM FLEHR,HOH13QCH,TEST 2030782 P.25 WO 9O/12S82 Pcr/usgo/ot ~t-r-och~ioal ~lt~rat~on ln t~ h-~dgrOUP h~
o¢curr-d ~h- r-~ulte ~y b- u~arl2-d a~ ~ollow~
~ ~h _ Barlum Dl-thylpho-ph~t- 1~8 ppm ~ t ~ g~oup t~lg. ~) Hy~r~t-d DMPC 10-20 ~pm Llqul~-ory~t-llln-~ ) bll-y-r ~lgh ~oblllty ~y~rat-~
Dlpalmltoyl Pho~phat~dylocholln- 60-~0 ppm G-l pha-- bllay-r R-duc-d moblllty r~ ~-d C-ll--Prot~ctant 105 ppm Lo-- o~ bll~y-r (~10 3~ ~tructur-~-~uo-t ~ t~
~ ad ~ 95. 5 ppm C-l ph--- b~l~y-r ~ 4) ~nt-r~-dl~t-~ob~llty ~h- ro~ult~ ln~le-t- that cc~l- dr~-d ~n th- pr-J-nc-o~ th- prot-ctant olut~on ~lnt-ln th~
cbar~et-rlctio- o~ n-~br-n- tructur- ~oclat-d wl~
2S ~y~rat-d c-l~- ln a ~-1 pha-- ~h- o-ll- drl-d w~thout t~- prot-ctant ehow ~ld~nc- o~ th- lo-~ o~
o~- bllay-r truotur- nd po--lbl- ~o~-tlon o~
h-xngon~l ph-~- llpld ~tructur- whlch 1~ lnoo~p~tl~l-~ltb vlnblllty. 5n-~- o-ll~ o ~ nor t- p~tt rn ob~r~ct-r~tlc o~ -v-r ~y r--trlot-d p o~pbollpld h ~group ~otlon ~roa th ~or golng d-~erlptlon, on~ ~Xlll-d ln th-rt o~n ~ ~dlly -c-~t-~n tbat -~-ntl~l oh~r~ot-rl-tlcJ o~ th- lnv-ntlon ~n~, wlthout ~5 dop~rtln~ ~rom th- plrlt ~n~ cop- th-r o~, o~n ~pt th- lnv-ntlon to ~rlouJ u--g-- ~nd oon~ltlon-Cb-ng-~ ln ~o~o ~nd ~ub tltutlon o~ qulv~l-nt~

DEC 07 '90 04:26P~ FLEHR,HOH8RCH,TEST 2 ~ 3 $ 7 ~ 2 P.Z6 9O/~2~82 P ~ /USgO/0~27 cont~mpl~t~d a- clrcum-tanc~ y ~ugg--t or r-nd-r ~xp-di-nt, and although p-cl~lc t~r~ h-v~ n ~ploy~d h-r-in, th-y ar- lnt-nd-d ln a d~-crlpt~-~n-- and not for purpo~-- Or limitatio~

Claims

WHAT IS CLAIMED IS:

1. A lyophilized erythrocyte- and/or hemosome-containing composition containing about 3% by weight or less of moisture prepared by lyophilization of an aqueous suspension of erythrocytes in a phosphate-buffered saline solution at a pH in the range of 7.0 to 7.4 comprising a final concentration of about 7 to 37.5% by weight of a monosaccharide or mixture of monosaccharides capable of permeating the membranes of erythrocytes, and a final concentration of about 0.7 by weight percent up to the saturation point of the solution of a polymer or mixture of polymers having a molecular weight in the range of about 1K
to 360K.

2. A composition according to Claim 1 wherein said polymers are amphipethic.

3. A composition according to Claim 1 wherein said polymers have a molecular weight in the range of about 2.5K to 360K.

4. A composition according to Claim 3 wherein said monosaccharide is selected from the group consisting of xylose, glucose, ribose, mannose and fructose.

5. A composition according to Claim 4 wherein said monosaccharide comprises glucose.

6. A composition according to Claim 5 wherein said final glucose concentration in said solution is about 26% by weight.

7. A composition according to Claim 3 wherein said polymer is selected form the group consisting of polyvinylpyrrolidone, hydroxyethyl starch and dextran.

8. A composition according to Claim 7 wherein said polymer comprises polyvinylpyrrolidone.

9. A composition according to Claim 8 wherein the final concentration of said polyvinylpyrrolidone in said solution is in the range of 12 to 20% by weight and has an average molecular weight of 10-40K.

10. A composition according to Claim 9 wherein said solution comprises about 14.4% by weight of polyvinylpyrrolidone.

11. A process of reconstituting lyophilized erythrocytes and/or hemosomes to a transfusably-useful state, wherein said lyophilized erythrocytes or hemosomes consist essentially of a composition according to Claim 1 comprising the step of:
mixing said compostion with a sufficient volume of a phosphate-buffered saline reconstitution solution having a pH in the range of about 7.0-7.4 at a temperature in the range of about 15-50°C, said reconstitution solution comprising a final concentration of about 0.7% by weight up to the saturation concentration of a polymer or mixture of polymers having a molecular weight in the range of about 1K to 360K, to form erythrocytes in a transfusably-useful state characterized by ?0-?5%
hemoglobin recovery compared to the pre-lyophilized state, and normal erythrocyte morphology.

12. A process according to Claim 11 wherein said polymers are amphipathic.

13. A process according to Claim 11 wherein said polymers have a molecular weight in the range of about 2.5K to 360K.

14. A process according to Claim 11, 12 or 13 further comprising the steps of:
separating said erythrocytes from said reconstitution solution by centrifugation and washing said erythrocytes by at least one wash cycle by resuspending said erythrocytes in a washing buffer solution comprising phosphate buffer, 16% weight by volume of said polymer at pH in the range of about 7.0-7.4 and separating said erythrocytes from said washing buffer solution by centrifugation.

15. A process according to Claim 14 further comprising the steps of resuspending and incubating for approximately one hour at 37°C said erythrocytes in said washing buffer containing an additional 10%
vol/vol of glycolytic intermediary solution comprising phosphate buffer, calcium chloride, magnesium chloride, ADP, NAD, adenine, adenosine and inosine at pH of about 7.0-7.4 and separating said erythrocytes from said washing buffer and glycolytic intermediary solution by centrifugation; and resuspending and incubating said erythrocytes at about 20-40°C for less than about 10 minutes in an iso-osmetic solution comprising a final concentration of about 0.1 to 10% weight by volume of a polyanion selected from the group consisting of inositol hexaphosphate, adenosine triphosphate, pyrophosphate, and tripolyphosphate, 0.1 to 5.0% weight by volume of a 3-?KD polyethylene glycol at a pH in the range of about 7.0-7.2 at a dilution factor of 10-50;
separating said erythrocytes from said iso-osmotic solution by centrifugation; then incubating said erythrocytes at about 37°C for about 30 minutes;

resuspending said erythrocytes in a phosphate buffered saline or dextrose-saline solution, and separating said erythrocytes centrifugation.

16. A method according to Claim 15 further comprising the step of resuspending said erythrocytes in a solution comprising phosphate buffered solution or dextrose-saline solution and monosaccharide at a temperature of about 20-40°C, and separating said erythrocytes by centrifugation.

17. A transfusably useful erythrocyte-containing composition made according to the process of Claim 11.

18. A transfusably useful erythrocyte-containing composition made according to the process of Claim 14.

19. A transfusably useful erythrocyte-containing composition made according to the process of Claim 15.

20. A transfusably useful erythrocyte-containing composition made according to the process of Claim 16.

21. A process of reconstituting a lyophilized composition comprising erythrocytes and/or hemosomes comprising the step of contacting said erythrocytes and/or hemosomes at a temperature greater than about 17°C with an aqueous solution of a polymer or mixture of polymers having a molecular weight of from about 1K to about 360K which is present in a final concentration in the range of 12 to 30% by weight.

22. A process according to Claim 21 wherein said polymers are amphipathic.

23. A process according to Claim 21 wherein said polymers have a molecular weight in the range of about 2.5K to 360K.

24. A process according to Claim 21, 22 or 23 wherein said polymer is selected form the group consisting of polyvinylpyrrolidone, hydroxyethyl starch and dextran.

25. A process according to Claim 24wherein said polymers comprises polyvinylpyrrolidone of average molecular weight of 40K.

26. A process according to Claim 25 wherein said polyvinylpyrrolidone is present in said solution in a final concentration of about 14.4% by weight.

27. A process according to Claim 21, 22 or 23 wherein said solution further comprises a monosaccharide in a final concentration of about 7.0 to 37.5% by weight.

28. A process according to Claim 15 wherein said polyanion comprises inositol hexaphosphate.

29. A composition according to any of Claims 1 through 10 comprising hemosomes.

30. A composition according to any of Claims 1 through 10 comprising erythrocytes.

31. A reconstituted, transfusably-useful erythrocyte-containing and/or hemosome-containing composition characterized by a P5D in the range of about 26-28, an oxyhemoglobin content greater than about 90% of that of normal red blood cells, discocytic or stomatocytic morphology, and normal ATP
metabolism.

32. A composition according to Claim 31 comprising erythrocytes.

33. A composition according to Claim 32 further comprising an additive comprising a polymer or mixture of polymers having a molecular weight of from about 1K to about 360K in a concentration of about 12 to 30% by weight.

34. A composition according to Claim 33 wherein said polymers are amphipathic.

35. A composition according to Claim 33 wherein said polymers have molecular weights in the range of about 2.5K to 360K.

36. A composition according to Claim 33, 34 or 35 wherein said polymer is selected from the group consisting of polyvinylpyrrolidone, hydroxyethyl starch and dextran.

37. A composition according to Claim 36 wherein said polymer comprises polyvinylpyrrolidone of average molecular weight of 40K.

38. A composition according to Claim 37 wherein said polyvinylpyrrolidone is present in said solution in a final concentration of about 14.4% by weight.

39. A composition according to Claim 32 wherein said solution further comprises a monosaccharide in a final concentration of about 7.0 to 37.5% by weight.

40. A composition according to Claim 32 wherein said composition further comprises a polyanion selected from the group consisting of inositol hexaphosphate, adenosine triphosphate, pyrophosphate and tripolyphosphate.

41. A composition according to Claim 40 wherein said polyanion comprises inositol hexaphosphate.

42. A composition according to Claim 32 further comprising polyethylene glycol of a molecular weight in a range of about 3 to 5 KD.

43. A lyophilized erythrocyte-containing and/or hemosome-containing compostion characterized by moisture content of 10% or less, and a phosphorous-31 NMR lineshape characteristic of a bilayer in a gel phase.

44. A composition according to Claim 43 comprising erythrocytes.

45. A composition according to Claim 44 further comprising an additive comprising a polymer or mixture of polymers having a molecular weight of from about 1K to about 360K in a concentration of about 12 to 30% by weight.

46. A composition according to Claim 45 wherein said polymers are amphipathic.

47. A composition according to Claim 45 wherein said polymers have molecular weights in the range of about 2.5K to 360K.

48. A composition according to Claim 45, 46 or 47 wherein said polymer is selected from the group consisting of polyvinylpyrrolidone, hydroxyethyl starch and dextran.

49. A composition according to Claim 48 wherein said polymer comprises polyvinylpyrrolidone of average molecular weight of 40K.

50. A composition according to Claim 44 wherein said solution further comprises a monosaccharide.

51. A composition according to Claim 45, 46 or 47 further characterized by a phosphorous-31 NMR
spectrum linewidth reduced by at least 8% as compared to the linewidth of a corresponding erythrocyte-containing composition lyophilized without said additive.