CA1047919A - Pharmaceutical insulin preparations having antidiabetic activity and process for their manufacture - Google Patents

Abstract

NEW PHARMACEUTICAL INSULIN PREPARATIONS HAVING ANTIDIABETIC
ACTIVITY AND PROCESS FOR THEIR MANUFACTURE
Abstract of the disclosure:

An insulin preparation in an aqueous suspension which com-prises a crystallized insulin, dissolved des-phenyl-alanineB1-insulin of the same species and 25 to 80 mcg of zinc ions per 100 I.U. of insuline and a process for its manufacture.

Description

~047~
The pre~e~t in~o~tion relat0~ to pha~maceutiGal in~uli~
preparation~ ha~ing antidlabetlc actl~i~y and to a proces~ ior their manu~aoture.
Up to now, t~o method~ ha~e been e~ployed ~or preparing in~ulin compoeition~ ha~ing a depot ei~ect: in one there i~
addsd to the in~ulin, zinc ion~ and retarding aeents 9 ~u~h a~
bi~-(4-amino-quinaldine-6)~ ur~a hydrochlorldo 7 gleblne or protamlne, in the other there are u~ed ~u~pension~ o~
cryetalli~ed or amorphous insuli~.
10~he onset o~ actiYity o~ the in~ulin obtained irom crg~tal ~u~pen~ion~ low and it~ ei~ec~ cont~nue2, in m~n~ ca~e~, ~or too long a time, whilst the e~fect cau~ed by a ~uapen~ion ~.
o~ amorphou~ insull~ bsgin~ immediately but wear~ o~ too rapidly.
15There~ore, cry~talli~ed and amo~phou~ in3ulin~ h~ve been combined to achie~e a more ~aYourable time-e~eot ratio.
~ o~e~er, ~u¢h a combination not alwa~ an ei~ective depot principle, becau~e, depending o~ the eatlng hablts ln the ~:
dif~erent countrie~, the onset o~ a¢ti~it~ o~ the i~ulln i8 too 810w and it~ e~ect conti~ue~ ~or too lo~g a timeO ~o r~
~olve thi~ problem, th~ combinatio~ of cr~talli~ed in~ulin with dis~olv0d in~ulln has been u~ed with su¢ca~.
With ~uch a ¢ombinatio~, howe~er, on~ ha~ to make ~ure that di~30l~sd in~uli~ ot converted i~to ¢r~stalli~ed in~uli~ durlng ~torag~. ~uch a co~ver~ion ha~ hltherto bee~
preve~t~d by using in~uli~0 o~ different ~peole~, for e~pl~
cry~tallie~d bovl~e in~uli~ a~d amorphou~ porcine i~uli~
Such a co~bl~ation ha~9 ho~o~er~ t~e disaa~antage o~
29 cau~ing the iorm~tion of antibodie~ a~ain~t the t~o 8p~Cle8 .
. 2 ~

~a\4L79~9 Some yeare ago, mono-~peoias i~aullrls we~e u~d ~or the ~ir~t tlme, thue a~auring that when resi~tance to insuliIl ooGura ~or immunological reasone9 u~e can be made oi a di~Ier~t i~ulin which had ~ot ~et been admi~lstered, ~or example porcine ingulln irlg~ead o~ bo~ine ln9ulin~, ~hi~ po~bilit~
i9, however ~ excluded when combination~ oi in~ull~ oi difierent ~pecie~ are u~ad.
We have now iou~d that cry~tal~ o~ complete i~ulin and di~solved de~-phenyl-al~nineB~ ulin of the ~sme ~peeie~
csn be m~xed to ~ield a 8table combination ~ithout sub~anw tial ri~k o~ ¢on~r0roio~ o~ tha die~olved dee~ph~ alani~e in8u}in (des-Ph~B~ ulin) into a cr~stalli~e~ ona.
Sin¢e, e~ccept :eor the mi~ing phenyl-alanlne, des-phenyl-alanineB~ ineulin has the ~ame immunoge~lc groupi~g~
a~ the corre~ponding complete lnsulin, the two i~oulins r~-pre~ent a t~o-pha~e (cr~alli~ed/amorphous) ~table ~y~tem which exhibit~ the immunological propertie~ o~ a mono specie~
in~ulin.
The total ~i~c conte~t in the solution and th~ cry~tal-liaed ~u~pen~ion~ which ha~ to be ~ithin ~he range o~ 25 and 80 mag/I.U. ineulin~ i9 0~ decis~ve i~por~anoe ior the atability o~ the preparation~
Henae, the pre~ent in~ention provide~ an in~ulin prs-paratio~ compri~ln~, in an aqueous solutlon~ cr~talli~ed in3ulin and di~olv~d de~-phenyl-alanineB1 in~ulin o~ the same ~pa¢i~ and, in t~tal, 25 - 80 m¢g zi~c lon~ per 100 I.~. insulinO
~he ratio o~ cr~talli~ed i~uli~ to di~ol~ed de~phe-

2~ in~uli~ determines the time-e~fect ra~io and ma~ range ~rom _ 3 _ ~.

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- ~ .. . . . . .

` HOE ~

60:40 and 90:10, pre~erably ~ro~ 80:20 and 90:10. The total in~ulin content of the compo~ltion i~ advantaeeou~ rom 40 to 100 I.U./ml. The p~ o~ the ~uepen~ion i~ ad~u~ted to a value in the range of from 6.8-7.2, pre~erably ~rom 6 J ~ to 7~0. The ~uepension contai~s ~rom 25 to 80 mcg o~ zinc ion~
per 100 I~.
Thi~ in~ention al~o proYides a proces~ ~or the prspara~
tion of an in~ulin compo~itlon, ~hich oompri~es mising a ouspenelon o~ inoulln cry~tals with a ~olution o~ de~-phe-1o i~Bulin o~ the ~ame ~peclaa. ~hi~ proce~ iD carried out, .
~or example~ a~ followss .~ Proparation o~ an in2ulin cr~tal ~uapen~io~
(Cry~talline component of the ~in~shed preparation).
Fir~tly, i~ulln is precipitated ~rom a buf~er containing zinc ion~ aB cry~t~le 9 whereupon in~ulin cry~tal~ are obta~ned having the uniform ~ize oi 15 - 35 ~, their zinc oont~nt being 0.3 - 1.0 %.
A~ter ~epar~tlon irom th~ Btook liquor~ the cry~talB are euspensed in a bu~fer ~olution ~hich has a volu~e ealculated suoh that the suepen0ion i~ ad~u~ted to a de~ined insuli~ `
oontent i~ the range o~ ~rom 40 to 100 I.U~/ml. ~he bu~ier eolution contal~e zi~c io~s, ior exa~ple in the ~orm o~ ~
zlnc ohlorlde, in au¢h a number that the total zina oontont ~:
o~ t~e orgetal ~u~pen~ion ad~u~ted to 40 - 100 I.~./~l 18 within tho range o~ from 30 to ao mog/100 I.U~ Moreo~er, the buffer aolution co~tai~s 3alt~, euch a~ ~odiw~ acetate, advantag~ously i~ a cono~entration o~ ~rom 0~1 ~ 0.2 ~ sodium chlorido, ad~a~tag~ou~ly in a oono~ntratio~ o~ 0.5 to 1 %, 29 a~ well a9 oo~ervatio~ agcnt~ ~uch a~ 4-h~drox~-b~n~oiG
, .:

~479~1L9 acid methyl ~9ter9 adva~tageou~ly l~ a concentration of about 0.1~q6 and i~ ad~usted wlth s0dlum h~dro~cide ~olution to a p~ OI
a value wlthin the range o~ ~ro~ 608 to 7~2~ pr~erabl~ 6~B
~o 7Ø
2.) ~
3pecies (dissolved oomponent of the ~inished preparation).
Cry~talli~ed9 dr~ de~ phe in~ulln ha~i~g a ~in¢ co~tent o~ 0.3 - 0.7 ~ are ~ ol~ed in a liquid in such a~ a~ount that the 801utio~ ha~ a de~insd de~-phe in~ulin con~ent ~ithi~
the range o~ ~rom 40 - 100 I.~./ml. ~he ~olutlon liquid oon-tain~ zinc ion~, ~or example in the ~or~ oP æ1nc ohloride, in ~UCh an amount that the total zi~¢ content o~ the solutio~ ;
i~ wi thin the range o~ from 25 - 40 ~¢~/100 I~Uo The ~olut~o~
liquid also contai~o sodium chloride and 4-hydro~benzoi¢ ~"
aoid methyl e~ter and~ to di~sol~e the de~ phe-in~ulin, ~u~ficient amount~ of acetic acid a~d hgdroc~lorio aoid. AYter cterile ~iltration, the de~-phe-in~uli~ 801utlon waa ~tand-ardi~ed with ~odium hydroxide ~olutlon to a pH o~ a value ranging ~rom 6 . 8 - 7 . 2 9 pr~Ierably from 6 . 8 - 7 . O ~, It ha~
the same content of ~odium chloride, ~odlum a¢~tate and 4-hydroxyben~olc acid methyl e~ter a~ the bu~fer ~olutlo~
d~acribed under 1. .
.~ Preparation of a depot i~BUli~ com~sitio~ read~ Por,~ E
~illed into ampo~l~a ~ .
~he crystalll~ed component prepared according to 1. i~ :
mixed with .~he dissol~ed oo~ponent dea¢r$bed u~der 2.~ in~u- ;
lin cryetal~ and di~solved de~-phe-i~sulin o~ tbe same ~pecie~ . .
being u~ed~ ~he ~rolumetric ratio OI ery~talli~ed component to 29 dls~ol~ed compone~t 1~ 60~40 to 90s10~ pre~erabl~ 80s20 to ~. , .... ; .. :, - .. ,. .. ~, :. ; ,,, ,. : --~L~)4791~
90:10. ~he total in~ulin eontent i8 40 - lO0 IO~g/ml~ ~he zinc content o~ the ~ini~hed preparation depe~d~ on the ml~ ratio o~ the two component~ and ie ~rom 25 80 mcg/100 I~o ~ the pH
valua is within the range o~ from 6~8 to 7.2., pre~erabl~ ~ro~
6.8 to 7~0O
~he preparatio~ o~ th~ pre~ent in~entlon generally haa a long-la~ti~ and equilibrised l~ulin depot ef~ect. Even a~ter ~torage for one year at 4-8C a~d at 25C, its ph~
cal condition ~nd its biological actlYitg are ~ubstantially unchanged.
The preparation may be used a~ depot in~ulin ~or the tr~atment ~ ~13~ ~ It iB e~peciall~ ~uitable ~or admini~tration to pa~ie~t0 ~or the iirst time to preYent the :
~ormation o~ antlbcdies again~t ~everal in~uli~ ~peele~
~he ~ollo~i~g E~ample~ i1lu~rate the preparation of the . ~:
compo~itio~ o~ the i~vention: :
E X A ~ P L E 1:
A. ~ .
16 Gra~ o~ pure cry~tallised bovin ln~ulln having a minimum aotivity oi 25 I.~ g, calculated on dry ~ub~tanoe were di8solved i~ 750 ml oi 0.02 ~ hydrochloric acid co~- ;
taining 0.013 % o~ zi~o io~s in the ~o~m o~ zinc chlorlde 250 ml o~ a ~olut~o~ containi~g 0~1.95 ~ o~ glacial aoetio aoid and 2.8 ~ o~ ~odium ~hloride wa~ add~d to thi~ in~ulin ~olution. The olsar mixtur~ ~a~ then ~terilia~d by sterile ~iltration o~er a mambra~e layer. Subsegu0~tl~, ~teril~ 4 . 80dium hydrox~da wa~ add~d to the ~terile-~iltered ~olutio~ ~;
in ~uch an amount that its pH alu~ rang~d ~ro~ 504 to 5~8 29 ~he:~morphou9 ln~ulin suBpen~lon obtained ~as stirred ~or - 6 - :

. ~L~
~479i9 a short ~ime a~ room temp0ra~ure u~d~r ~terile oo~di~io~ and then placed in a re~riger~tor at ~4 to ~7C ~o lnltiate cry~talli~ationO
A~ter a maxim~m ~torage time o~ ~6 to 40 houre, ~hombo-hedral crystale o~ u~i~or~ ~hape and ~iæe oi fro~ 15 to 35 microns were obtai~ed. T~e cry~tal~ ~ere centriiuged off at low speed i~ sterlle beaker0 9 ~hereupon ~mall ~morphou~
portion~ an~ 9 were ~eparated. The centri*uged or~stal~
~ere then again su~pendea in 1 l o~ ~terlle bu~fered neutral 10 in~ulin dilutio~ ~olution (compo~ed as in ~able 1) (stock su0pension).
Table 1:
Composition o~ the i~ulin dilution 901ution:
10 liter3 o~ solutlon co~tained 10 g o~ 4-h~dro~ybenzoie acid methyl e~ter = 0~1 %0 14 g of oodium aoetate . ~2 = 0.14 %
75 g o~ sodium chlorida 5 0.75 1.66 g oi æinc chloride (ZnC12) = 5.0 ~cg Zn+~/ml and eodium hydroside up to pH 6.9, the whole being ~iltered under sterile condition~.

~U~pension ~or determin$ng the in~ulin cr~0tal cont~nt i~ the stock ~u~pen~ion, 4 tlmeo 1 ~l (~ull pipette ) ~as taken ~rom the ory~tal ~uspensio~ a~ter intimate mi2ing b~ m~a~o o~ a ~agner ti¢ stirrer, and the ~o~tent o~ in~ulin wa~ dete~mi~ed accor-ding to the ~ollowi~g metho~a 1. Gravimetrical d~ter~i~ation upon dryl~g, u~ing ao~tone a~d 29 ether arld weighi~g.

H~E`~ K

2. Spectrophotom3trical dete~mination o~ th3 ultra~lole~ band at 280 ~m upon di~olut-Lon o~ th~ or~al~0

3~ Determi~ation of the nitro~e~ oonten-t 3 9epending o~ the ineuli~ content dets~mined in t~e 3toGk ~uepension~ the ~-terile d~lu~e ~olu~on ~a8 t~e~ standardioed by ~urther dilu~ion to reach ~he de0~rod in~ulln co~tent o~
40 I.U./~l-. ~a~imum yield- 10 liter~O

bo~in~in~
6.304 g o~ tho ~ry~tallieed de~-phe~bo~in i~ulln were di~solved in 3.5 1 o~ a ~olution liqui~ oi the composition ` ~ :
indi¢ated in ~able 20 ~able 2 ~.
Compo3itio~ o~ the d~-phe~olu~on li~uid 5 ~iters oi ~-olutlon contained 5 g o~ 4-h~droxybenzoic a~id methyl e~ter -- 0~
2.85 ml of acetic a~id`contairli~g 0.14 % o~ ~odium ac~tate : `
3~2) 37~5 g o~ ~od-lum chlor~:de ~ 0~75 %
21 mg o~ zin¢ ohloride (anhydrou~) ~ 2.0 mog Zn+~ml 5 ml o~ 4~ hydrochlori~ acidO
~he clear oolution o~ the de~-phe-in3ulin wa~ the~
~terili~ed by mean6 o~ a me~b~ane filter and diluted to a volume o~ 3.9 1 by adding 400 ml of a ~olution liqu~d whioh~
a~ter ~lltration o~ the insulin9 had ~ee~ pa~sed through the ~ame oterlle fi~lter~ By ~.dditio~ ~ 4~ 3~erile sodium hydro~l-de solution the pE wa~ ad~ueted to 6.9 (~o~ ptlo~ about 29 1~ ml). ~y ~urther dilution ~th 34 ml o~ a ~t~rile ~utral .
_a~

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~7~

insulin dilution 30lutionJ (oompoa.~L~lon as d~cribed i~ Table 1 ) a Iinal volume o~ 30 947 1 wa8 reached and thu~ ~tandardi~ed to an in~ulin conteIlt of 40 IOU./ml, ba~ed on a~ aotivity o~
th~ dee-ph0-ln~ulin o~ 25 I.U~/mg,.

am~oulee ~he depot coml?oxlent prepa.red acco~dlng to A, oontaining .
cr~tallised bo~in in~ul~n, svas mi~c~d wlth the componeDLt ~ropared a~cordlng to B9 oorltainl~g di~olved d0~ phe-bo~in 10 ln~ul~n9 at a vol~metric rat~o oi 8O:2O9 I;o ~ield a oompo-~ltion contai~ing 32 I.~./~l oi ¢ry~alli~0d in~t~lin ~na 13 I.U~/~l o~ diesolve~ des-phe~ eulin; both ma~ g up 40 /~o ~he Buspen~ion wa~ 1Ra under ~ierile oond~tio~o lnto bottlea ea¢h eontaini~g; 10 mlD . ~ .
~ P I E 2:
A~
A ateril~ suBpen~ion oi 16 g o~ or~alll~ea ~oroi~e aving a ~ize o~ 15 ~o 35 ~ioro~ wh~oh ~ra~ ~ryotall~
` i~ed ~ p~ 5.4 - 5.8 Irom a ~u~table buPfer (ds~orlb~a igl ~0 ~Dple 1 ) was c3~t~i~ugcd~ A~ber deo~n~in~ ~he ~ulp~r~at~t, ~ ~8tal9 we~e 3u~ponsed in 7 1 o~ a ~ teril~" bu~f~red, n~ut~al in~ulin dilute eol~io~ (compo~iLbion as deaoribed in ~sLble 3) (~tock 3usp~n~ion)0 ~able~
25 Oompo~itio~ o~ the insulin dilute ~olutlo~
.
liter~ of solution contai~e~
10 g o~ 4-hydro~cyben~oic acid m~thyl e~ter = 00 1 ~
14 g o~ sodium acetate o3~120 5 0~14 %
~`' 75 g of ~odium ch~orid~ 5 0,75 96 - q -79~a H3E 74~ 114 K

210 mg OI zinc ohloride (ZnC12) ~ 10 mcg Zn~+/ml :-and ~odium hydro:cide ~p to pH 6.9, the ~hola being ~iltered :
under sterile condlttons~

~
~ or determining the in~uli~ ¢ry~tal Co~nt i~ the ~tock suspen~ion, 4 tlme~ 1 ml (~ull pipette) ~a~ take~ ~rom the cry~tal ~uspe~ion aiter i~timate mi~ing by mean~ o~ a magneti¢
stirr~r~ and the ~ontent o~ insuli~ wa~ det~rmin~d a¢cording to the ~ollo~ing m~thods:
1. Gravimetrical determination upon dr~ing, uai~g aCoto~ and ether and weighing.
2. Spectrophotometrieal d~termination o~ the ultra~iolet band at 280 nm.
3. Determination o~ the nitroge~ cont~nt~
Preparation o~ the daPot com~one~t~contai~ing 40 I.U
Dspending on th~ in~ulin con~nt d~t~rmined in the atoek su~pension, the sterile dilute 00lutlon wa~ then ~t~ndardi~ed by ~urthar dllution to r~ach tha de~ired ineulin cont~nt o~
40 I.U./ml, Maximu~ ~iold: 'lO ~it~r~.

i,n~ulin 6.304 g oi the cr~stal1ised de~-phe-porcine ln~ulin w0ro dissolved in 3. 5 1 oi a aolution liquid oi the ¢ompooition i~dioated iIl ~able 2.
~able 2 Compositio~ o~ the d~-pha i solutio~ liquid:
5 ~itors o~ th2 solutlon contained ~: 29 5 g OI 4-hydrox~benæoi¢ a~id m~thyl a~tar a 0~1 %

~L0~79~

2.85 ml o* acetic aeid eontaining 0~14 ~ o~ ~odium aoetate . 3~2-37.5 g oi sodium chloride = 0.75 ~
42 mg o~ zinc chloride (anhydroue) = 4.0 mcg Zn~+/ml 5 ml oi 4N h~drochloric. acld.
The clear ~olution o~ the de~-phe-in~uli~ wa~ then - -~terilised b~ mean~ Or a membrane iilter and diluted to a volume o~ 3.9 l by ~ddlng 400 m} oi a ~olutlon liquid ~hich, a~ter iiltration o~ th~ in~ulin, had been passe~ through the ~ame ~terile ~ilter. B~ addition o~ 4N ~terile sodium hydroxide ~olution the pH wa~ ad~u~ted to 6.9 (co~aumption about 13 ml). By ~urther dilution with 34 ~1 o~ a st~rile ~.
neutral in~ulin dilution ~olution (compo31tion a~ d~orlbed in ~able 1) a ~inal volume o~ 3O947 l~a~ raaohed and thus standardised to a~ insulin co~tent o~ 40 I.U./ml, ba~ed o~
an activity o~ the d~-phe-insulln of 25 I.U~g.
C. Preparation o~ o~itlon r~aa~r am oula~
~he depot co~pon~nt pr~par~d aocording to A, containlng orystalll~ed porci~e lnsulin, wa~ mixed with th~ ¢omponen~ :
prep~red according to B, containing di~solved de~-phe-porcl~e ln~ulin1 ab a volumetric ratio of 80~20,: to ~ield a ¢ompo-8itlon oontai~ing 32 I.U./~l o~ cr~stalli~ed in~ulin and 8 I.U./ml of di~aol~ed d2s-pho-in~ulin, both making up 40 I.U./ml. ~e ~spen~io~ ~a8 filled und~r ~tar1le condition~
into bottIo~ ~a~h ~ontainl~g 10 ~1. :
E X A ~ P ~ E
75 Part~ of tho d~o-t ~o~po~on~:~prqpared ~coordi~g to 29 containl~g in~ulln ory0tale, ~r~ ~ised ~ith 25 parts o~ ths ~0 47 9 ~ -component prepared according to B, containing dieeolY0d des-phe-in~ulin of the ~ame specie~ whereupon a composition ~as obtained co~tal~ing 30 I.~/ml o~ crys~alli~ed insulin and 10 I.U./ml oi de~phe-insulin9 both ma~ing up 40 I.U./~l in~ulin. ~he ~uspen~ion wae ~illed under ~terile condition~
into bottle~ o~ 10 ml ea¢h.
E X A M P ~ 4:
70 Part~ of the depot component prepared aocording to A, containing cry~tallised in~ulin9 w~ro mixed with 30 parte oi the component pr~pared according to B, oontaining dis~olved de~-phe-in~ulin o~ the ~a~e ~pecies, ~hereupon B compo~itio~
~a~ obtai~ed containing 28 I.U./~l oi cr~stalli~ed in3ulin and 12 I.~ l o~ di3solved de~-phe-insulin, both making up 40 I.U./ml oi insulin~ The ~u~pension ~a~ ~illsd und~r aterile ~5 condltions i~to bottles o~ 10 ~1 eachO
E X A ~ P ~ E 5-90 Parts o~ the depot componff~t pr~pared acoordi~g to A~oontaining cryetalli3ed in~ulin ~ere misod with 10 part~ oi the component prepared aocording to B, contalnlng dissol~ed deQ-phe-insuli~ o~ the ~ame 0pe¢1e~, whereupon a compo~ition wae obtained oontaining 36 I.~./ml oi crystalli~ed in~ulin and

4 I.U./ml o~ diHsol~e~ de~-phe-lnsulin, both m~king up 40 I.U./ml o~ in~ulin. ~he ~uspen~ion wa~ $illed under ~terile condition~ into bottle~ o~ 10 ml ea~h.

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Claims (9)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An insulin preparation comprising an aqueous suspension of a crystallised insulin, dissolved des-phenyl-alanineB1-insulin of the same species and 25 to 80 mcg of zinc ions per 100 I.U. of insulin, said preparation having a pH
in the range of from 6.8 to 7.2.

2. An insulin preparation as claimed in claim 1 in which the insulin crystals have a uniform size within the range of from 15 to 35 microns.

3. An insulin preparation as claimed in claim 1, in which the ratio of crystallised insulin to dissolved des-phenyl-alanineB1-insulin is in the range of from 60:40 to 90:10.

4. An insulin preparation as claimed in claim 1, claim 2 or claim 3 in which the insulin content is from 40 to 100 I.U./ml.

5. An insulin preparation as claimed in claim 1, claim 2 or claim 3 in which the two insulin components are porcine.

6. An insulin preparation as claimed in claim 1, claim 2 or claim 3 in which the two insulin components are bovin.

7. A process for the preparation of an insulin pre-paration in which a solution of des-phenyl-alanineB1-insulin is added to a suspension of zinc-containing insulin crystals, the two insulins being of the same species, the amount of zinc ions being such that the total zinc content of the preparation is within the range of from 25 to 80 mcg/per 100 I.U. of insulin, the pH of the preparation being adjusted in the range of from 6.8 to 7.2.

8. A process as claimed in claim 7 in which the insulin crystals have a uniform particle size within the range of from 15 to 35 microns.

9. A process as claimed in claim 8 or claim 9 in which the ratio of crystallised insulin to dissolved des-phenyl-alanineB1-insulin is in the range of from 60:40 to 90 :10.