CA2181711A1 - New composition of glycoprotein isoforms having follicle stimulating activity - Google Patents

Abstract

The invention is concerned with a mixture of isoforms of a glycoprotein with follicle stimulating activity, a pharmaceutical composition comprising such isoforms as well as a method to induce follicular growth and maturation. Better results were obtained if a pharmaceutical composition with a relatively high proportion of basic isoforms were administered in controlled hyperstimulation. More oocytes could be retrieved and a lower dosage as well as a shorter treatment period sufficed.

Description

2 1 ~ 1 7 1 1 ~ WO95/19991 P~ JI'~
I
NEW COMPOSITION OF GLYCOPROTIEIN ISOFORMS
HAVING FOLL] CLE STIMULATING ACTIVITY

The present inventi~n is concerned with a L
comprising a mi~ture of isoforms of a ~ .v~"~ with follicle stimulating activit~ as well as an , u._ in a method to induce follicular growth arld Follicle stimulating llormone (FSH) produced by the anterior pituitary plays a major role in female and male .~,..' by stimulating gonadal ~ dtivll and maturation via its regulatory action on the Sertoli cell in the testes amd granulosa ceU in the ovary.
FSH is produced amd secleted by the pituitary in different molecular forms ( ' or i.soforms), which vary nn overall charge, receptor binding affinity. biological activity, amd plasma residence time. This micro ~_t~"u~ ,ity is due to differences in the amount amd/or . , of the ;. ' '~.' residues, in particular sialic acid. Multiple forms Of L ' I, have been isolated amd . I~.., t~,i~ from anterior pituitary glands, se~um amd urine of several " and ' species, mcluding man. Relatively acidic FSH isoforms, which are more heavily sialylated, exhibit lower receptor affinity amd in vi~ro biological activities thim more basic isoforrnes. However, due to their longer plaslna residence time these more acidic forms have greater in vivo biological activities (Ulloa-Aguirre et al., 1988, Hum.Reprod., 3, 491-501).
FSH is used for o~ulation induction and controlled ovarian stimulation in in vit~o fertization (IVF). The aim of controlled !, V.~ ' '- is to increase the number of retrievable mature oocytes for IVF and subsequent embryo transfer (ET). Generally, up to three embryos are replaced per transfer. As usually more tham one treatment is necessary, in most infertility clinics spare embryos or fertilized oocytes are frozen and ~,.msferred in subsequent cycles. Assuming normal rt~ dtiu." tbe more oocytes retrieved the higher the number CONFIRlvlA'rlON COPY

WO95119991 .~l/~r,~ -2 of possible transfers and thus the higher the chance of a woman to become pregnant after one treatment cycle. In case of rnale infertility, the chances of, ' ,, r~li' and thus pregnancy, also increases with the number of oocytes recovered.
S
Previously, it was shown that the majority of circulating FSH
isoforms during the follicular and luteal phase of the normal menstrual cycle, have pl values < 4.8, whereas during midcycle, or after treatment with oestrogens ~ more alkaline FSH isoforms 10 were present (F-' et al., 1988, J.~l F.. ~
67, 465-473). This indicates that the more acidic FSH isoforms play a role in the growth and .c~,.. of ovarian follicles during the follicular phase.
Hitherto for clinical purposes FSH ~ Lu,l.. have been used which have been isolated from natural sources. I ' distribution profiles of . , "~ available l,.c~ Lu,have been reported (e.g. Harlin et al, 1986, Fert. Ster., 46, 1055-1061). It appears that these FSH . consist of relatively acidic fractions.
" it now has been found that a mixture of ~ ,u~lutc .
isoforms with isoelectlic points in between and extending from the range 4.8 to 4.2., having follicle stimulating activity which consists for more than 15% of isoforms with isoelectric poirlts above 4.8 and for less than 30% of isoforms with isoelectric points below 4.2, when used in the same clinical settings, exert a better effect than the known . . .
~1~ .,u~u. ~ , Clinical studies have been performed with a: . of ~ .U~I~ ' according to the invention and obtained by l~ ' DNA methods. A separation of FSH
according to their isoelectric point (pl) revealed that the preparation according to the invention has relatively basic isoforms. The isoelectric points as described herein were determined by mearls of chromato-focusing (see example 1).
The commonly known ~ ,u~,., ' . , (of which MetrodinR used in this clinical study is an example) have a more acidic profile.
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The fact that a more basic ' profile gives better results in . u~.' is surprising because it has been thought in the literature that the in vivo blùa~a;ldb !il~ is the most import~mt factor for the follicle sbmulabng effect of the ~ .,v~ (European patent applicabon EP 388 æ3). The more basic ' have a shorter half-life than the more acidic isoforms which would make the basic isoforms less suite(f for use m IVF schemes and thus teaches away from the present invention.
It is preferred that the ~ ,u," ' - mixture according to the imvenbon contains a relabvely high proporbon of relabvely basic isoforms. This can be e~tablished by having ~ ,v~,. isoform having more than 15% of the isoforms with isoelectric points above 4.8 and less than 30% below 4.2, also preferably, isoforms mixtures can be osed wherein more than 15% of the isoforms have isoelectric points above 4.8 and less th~m 25% below 4.2, also preferably more th m 25 % of the isoforms have isoelectric poimts above 4.8 and less thdn 25% below 4.2 ,also preferably more tham 25% of the isoforms have isoelectric points above 4.8 amd less th;m 20% below 4.2 The percentage of the isoforms as used herein is defined as the relabve amoumt of ~,a~tl~, isoforms recovered after A~ also protein contents can be ~ ' by ~~' assays.
In a clinical study, as described herein, it has been found that in controlled , ~,~. ' the number of oocytes retrieved is different in the two FSH ~ al~Lv.~ used i.e. the ~ al according to the invenbon amd MetrodinR with respect to the number of oocytes retrieved More oocytes conld be retrieved from the group treated by a 1'1`l according to the invenbon. In addibon, a ~ '~
Iower FSH dosage and a shorter treatment period sufficed.
The mixture of gl~,v~ according to this invenbon may be derived from urinary origini. For natural FSH ~JIc~lal.lLivl,s, the number amd relabve amount of each isoform species depends on the source (pituitary, serum or urine), the a~e and the endocrine status of the CON~-IPA,1ATION COPY

WO 95/19991 P~. Ir.J ~
2l8171 1 donor, and the isolation procedure applied for its I ~ (Wide, 1981, ~.('1 F ' ' ~' ' ., 55, 682-688; Wide and Hobson, 1983, J.(~l F~ ., 56, 371-375).
Alternatively, the ol~ul,l might also be a 1~ ' Ol~U~.~
For .~ ' FSH (recFSH) the charge ~.L.u~ ,;ty is determined by the host cell-line chosen for its production as well as cell culture conditions. For Ol~,v~ such as FSH, a Chinese Hamster Ovary (CHO) cell line is an obvious choice since these cells are known to produce ol~,u~ X with ~ ' hl.,~ identical or closely related to those found in man (Sasaki et al.,J. Biol.Chem.,1987, 262, 12095-12076). However, also other host cell lines can be used.
A ,c ' FSH ~ iu.. according to this mvention can be produced by a CHO cell line stably transfected with a plasmid containing the two subunit genes encoding humam FSH (hFSH).
Such a CHO cell line produces intact, ~ u~- ' hFSH that is secreted into the culture medium, which is the source for further ;r..~,;.... Batches like this can be prepared as described (van Wezenbeek et al, 1990, in: From Clone to Clinic, Kluwer Academic PuWishers, 245-251). Previous preclinical studies ' ' that the receptor binding affinity amd in vilro and in vivo efficacy of recFSH are compatible to those of FSH isolated from natural sources (Mannaerts et al, 1991, F '- ' O~, 129, 2623-2630).
FSH ~1~ . according to the invention can be selected based upon their ~ profile. In addition, the profile cam be influenced by a particular host cell line choice or by adaptation of culture conditions.
As an alternative, also ol~.,u~ isoforrn nnixtures according to the invention c~m be isolated from cell lines the ol~,u~,.. gene e~pression o~ which has been turned on by site specific targetmg of a regulatory sequence to the ~ .U,VI~ gene. (PCT application WO
92/19255).
In addition to wild-type cell lines, basic ' can be obtained by expression of IC '' ' FSH in cell lines which are impaired in ol~u~:~tiu.. Such a cell line might be e.g. a cell line deficient in the enzyme N-acet~,, tlansferase or in sialic 1~1~4T101~1 COPY

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acid transport into the Gol~i (Galway et al., 1990, F ' ' ~,y, 127, 93-100) .
Other ~.... "-' of this invention are basic ~;ly~u~
isoforms mixtures obtain~d by enzymatic or chemical '~
With such a treatment parts of the ~ubvh~.' chains can be removed without affecting the amil-o acid sequence. ~ u~ ~ batches carl e.g. be treated with HF (C`hen et al, 1982, J.Biol.Chem., 257, 14446-14452). Partial d~ idlyld~iull can be performed by enzymatic hydrolysis with ' (Vaitllkaitis and Ross, 1971, J.
Metab., 33, 308-311).
Also subject of this invention are basic ~Iy~u~,.ut~,l.. hormone mi~tures baving biological i.c. follicle stimulating activity which are prepared by removing one or more N-linked r5,. ' ' chains from eitber one of the subunits. These chains can be removed by site-directed ~ of th~ gene encoding the ~Iy~u~ In this way a single amino acid cham~e can be obtained m such a way that the .' attachment site is no longer present. t'~ , ~y ~ one or several ~I,uh).' chairls carmot be attached on the IY or M subunit (Matzuk and Boime, 1988 J.Cell Bio~., 106, 1049-1059).
The mi~ture of relatilvely basic ' forms can be used according to the invention for the ' ~ of a ' to be used e.g. in the treatment of ovulation induction or controlled ovarian .. . ..
Thus, the invention relates to a 1' comprising these ~Iy~u~ isoforms admrxed vith I ' ~y acceptible auxiliairies. Methods for making ~, and admrlctures are disclosed ill l;' 's r Sciences, pp.
1463-1497 (16th ed. 1980, Mack Publ. Co of Edston, Pa, USA). For e~ample, ampoules colltaining the ~hal according to the invention may contain I to 1000 ~g of the u~ mrb~ture (e.g. 75 IU is considered a tberapeutic amount).
The invention also ]~elates to an improved method to induce folliculdr growth amd maturation wherein the 1' containing the mixture of basic E,ly~,u~l~ isoforms as described above is used. ~ccordimg to the present invention there is no need to imclude in the treatment schedule the ~ ' of more acidic isoform mixtures.
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wo ss/lsssl r~ e.~ --Another , u.. of the methûd consists of a repeated : ' of 1' ' . . containing a mixture of isoforms of a ~;lywln~' with follicle stimulating activity, each having identical, i.e. having the same isoform profile.
Thus, during a treatment the _' of only relatively basic isoforms is sufficient.
Such mixtures can also be prepared by isolation of only basic isoforms e.g. by preparative ', ' ~. Preferably such isoforms have an isoelectric point above 4.2 . It will be clear that ' of isoform mixtures can be perforr~ed on ~ ,u~l batches obtained from different origins such as ,u-.,l.~u~l;o.~., isolated from urine or .~ ' DNA cell lines which may or may not be chemically or e..~ "~, modified.
r , . - according to this invention can be used in clinical treatments in ' with e.g. GnRH antagonists or agonists and/or LH activity e.g. HCG or LH to induce ,. U v uldliU~ .

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r ~ I
C~ . of the FSI~ ,L.~. ' profile of 1~ ' human I~SH (Org 32489) and urinary FSH
S ('''~ ) In the present study the ' , profile of severai batches of Org 32489 ( ' human FSH, N.V. Organon), derived from Chinese Hamster Ovary ~:ells transfecte~i with the genes encoding for hFSH, was examined ~nd compared with tilat of ~,;dll~
availabie batches of urinaly hFSH, i.e. MetrodinR.
Purified (299%) Org 32489 bulk and finai product ~
were obtained from Diosynth and Organon (The ~. ' ' '`, .~ . Urinary hFSH, i.e. MetrodinR (Serono, Rome, Italy; 75 IUlampoule declare~i in vl vo bioactivity relative to IS 70/45), was used as reference. In total twelf Org 32489 bulk batches, five Org 32489 clinical ~ , (CP's), and twelve MetrodinR batches were used for ' ~ . . i~ll. MetrodinR batches indicate(i with an in-house CP-code ilave been used in a trial with Org 32489 CP batches to compare the clinical effic~lcy (see Exarnple 2).
By employing ' . ' ~ the different ' of hFSH were separated l~ased on their isoelectric point (pl). The :' ' of hFSEI was determined after ~ ~ by an enzyme J employing amtibodies that showedl . '' activibes towards all isoforms.
WdS performed in the range of pH 6-3 on a fast protein liquid .,t.., ~, .'.~ (FPLC) column HR 5120 (Pharmacia, Woerden, The ~ ) packed with polybuffer e~changer 94 (PBE-94; Pharmacia) ard; . '' ' with 0.034 molll L-nisbdine (Aldrich Chemie, Steinileim, FRG) adjusted to pH 6.2 with HCI.
Before use each colur~ln was eluted with 53 ml polybuffer 74 (Pharmacia) diluted 1:11 (vlv) with disblled water and adjusted to pH

3.0 with HCI (elubon buffer) unbl a pH curve ~vith a constant deciine from pH 6 to 3 was obtained. Before each C~ ,I ' run ttle column was eluted with elubor~ buffer and 2 ml 0.034 molll L-hisbdine Co~lFlR~ A T7~r,1N COr~V

WO 95/19991 2 1 ~3 1 7 ~
containing 100 ~glml human serum albumin (HSA; Behring, Marburg, FRG).
Of each hormone preparation, ..~ , 225 ru FSH in terms of in vivo bioætivity was dissolved in 3 ml ~ . ' ' buffer (stock solution) and 2 ml of this solution (150 IU) was applied to the column.
" the columm was eluted witn the elution buffer. Fractions of I ml were collected at a flow rate of I ml/min. After 53 fractions 2 ml of a 2 molll NaCI solution was applied to the column and 7 additional fractions were collected. For the ~. ~ of FSH, fraction 1, 2 and 3 and fraction 4, 5 and 6 had to be pooled. All otner fractions were adjusted up to 2.5 ml with a 1:1 n~Lxture of HAM F12 and DMEM (Gibco, Grands Islands, NY, USA) ~ with I
gll bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) (medium+). In addition, 0.3 ml of the FSH stock solution was adjusted to 2.5 ml with 2.2 ml medium+. All FSH fractions were desalted by applying each fraction to a PD-10 column (r' ), . -' ' with 12 ml meoium+. After elution with 3.5 ml medium+, the collected samples were stored at -20 C until ~ t,.l of FSH ~,LiviLy.
FSH -~,Liv~ly was measured in a two-site sandwich enzyme ~, using a B-directed capturing antibody ' antibody 4B) and an ct-directed HRP-labeled detection antibody ( ~ ' ' antibody 116B) as described previously (Mannaerts et al., 1991, Fn" '~v.~, 129, 2623-2630). This assay recogluses only intact dimers and was found to detect all FSH isoforrns equally well. The assay sensitivity in terms of IS 70145 was 0.4 IU/I
and the intra- and interassay ~ of variations were 7% and 8%, .c~p~li~ . The cross-reætivity with hLH and hCG was <0.01% and <0.01%, Ic~ Lv~,lJ
In order to quantify the ' ~, . of Org 32489 and MetrodinR, the dic~rlh~ n of FSH ~.,liv;ly after ' . ' ~ was divided in tne following six pH ranges:
> 5.3, 5.30 - 4.81, 4.80 - 4.20, CONFI~MAT~ON COPY

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4. 19 - 3.60, 3.59 - 3.00, < 3.0 (salt fraction).
These pH ranges were selected to obtain a ~y ' dividing of 5 the bcll ', :' ~i.. i;' of FSH ~,Liv;ly after of Org 32489. The amount of FSH ta~Livity which eluted within each pH range was e~pressed as percentage of total eluted FSH ~,Li~ity. In addition, recovery, i.e. total eluted FSH ~Liv;Ly expressed as pe~centage of FSH
~.,Lv;~y applied to the column, pH range, and pH top were d ' Recoveries below 75 % and above 125 % were considered not-acceptable resulting in rejection of the, , . r ' ~ run.
The recovery, pH range, pH top and the ~' of FSH
~Liv;ly after !- ~ , of the Org 32489 bullc and MetrodinR batches are shown in Table 1. R,~
, profiles of Org 32489 are shown in Figure 1. The recovery of FSH ~.,Livll~ after , r ' ~ of Org 32489 and MetrodinR was , ', being 93.7 (SD 8.9) % and 89.1 (SD 6.1) %, I~).,..Liv.,l~. Org 32489 e~hibited a bell-shaped ;' ' of FSH ~.,L~ in a pH ra~ge of 5.68 (SD 0.11) - 3.18 (SD 0.09) with a top at pH 4.53 (SD 0.08) (n=12). The FSH
~.,Liv~, profile o~ MetrodinR ranged from 5.58 (SD 0.18) to 3.07 (SD 0.05) with a top at pH 4.28 (SD 0.13) (n= 12).
The :' ' of F3H .;ly o~er the si~ pH ranges 25 showed that in ~ ,.. with Me~ûdinR, Org 32489 contained an nl '~ 2-fold highler percentage of relative basic isoformes with pl > 4.8 (20.9 % vs 12.7 %) and an ~LJ~ 2-fold lower percentage of relative aci~lic isoforms with pl < 4.2 (21.1 % vs 41.7 %) (Table I and 2). In addition, all MetrodinR batches contained more FSH ~Li~;L~ in the salt fraction (3.3 % vs 0.8 % for Org 32489), .~ FSH isoforms with a pl of less than 3Ø
In addition to the a~ove described Org 32489 bulk batches, the , profile of five clinical ~ Liu.~ was . ~i, ' The mean FSH di .~ of these CP-s was very . , ' ' to that found for the buLk batches (Table 1). Like the tvelve batches Org 32489 and Metrodin used for ' , , , the Org 32489 and Metrodin clinical ~ uaLio..s llsed to compare the clinical efficacy CONF~F~MAT~ON coPV

WO 95/19991 2 F~
I ~ 1 7 1 1 , rl (see Example 2) showed the same difference in the relative . ' of basic and acidic isoforms, i.e. the Org 32489 CP's contained more basic isoforms than the Metrodin CP's (17.120.2 % vs 12.7 % if pl >
4.8).
This, , ~~, slndy comprising " ' ,, profiles of Org 32489 and urinary hFSH, i.e. MetrodinR, revealed that Org 32489 containes more basic isoforms with pl > 4.8 and less acidic isoforms with pl < 4.2 than Metrodin.

CONFI~MA~1ON COPY

]]
r '-2 , efficacy stDldy of an Org32489 ~,.. , in patients ~ IVF
s Study .1Pc~,, The study was desiglled as a multicentrel ' 1, assessor-blind, group-; . ~, study, in which safety and efficacy of Org 32489 and MetrodinR were compared in infertile pituit~ ,, G~
subjects I ' 1;, ,, in vitro f~,.i' (IVF) and embryo transfer (ET). A~ , one thous7md subjects were included in this study with a ratio between subjects treated with Org 32489 and with MetrodinR of 3:2. The st~ldy period covered no more than 3 treatment cycles. Analysis of efficacy included f~rst treatrnent cycles only.
Inclusion of subjects l/vas based on the following entrance criteria:
Inclusion criteria - At least 18 amd at lmost 39 years of age at the time of screening;
- Cause of infertilit~ of the subject potentially solvable by IVF;
- Maximum of tbree previous I~F, gamete ' ', transfer (GIFT) or zygote '-l~. transfer (ZIFT) attempts, in which oocytes wele collected at least once;
- Normal ovulatory cycles with a mean length of between 24 and 35 days amd am ' v ' variation of plus or minu~ 3 days (but never o~tside the 24-35 days range);
- Good physical amd mental health;
- Body weight bet~een 80 and 130% of the ideal body weight;
and - Willing to give written informed consent.
Exclusion criteria - Infertility cause~ by endocrine :' ' such as h~ ,.,' polycystic ovary syndrome, and absence of ovarian function;
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2 1 ~3 ~ 7 1 1 - Male infertility deftned according to the followmg criteria. <
10X106 sperms per mL and/or < 40% normal . ' " ~J
and/or 40% normal motility;
- Contra ~ ' ~ for the use of GnRH analogues, FSH, hMG, 5 and/or hCG;
- Any ovarian and/or abdominal ' ' '~J that would interfere with adequate ultrasound ~ LiO.., - H~ (sitting diastolic blood pressure > 90 mm Hg and/or systolic blood pressure > 150 mm Hg);
- Chronic wl' .. ' , hepatic, renal, or pulmonary disease;
- History (within 12 months) or current abuse of alcohol or drugs; and - A~' of .~t~ ;OIIal drugs within three months prior to screening.
Treatment schedule Buserelin treatment was started on the first day of the menstrual cycle. The first day of the menstrual cycle was dehned as the ftrst day the subject waked up wi~h menstrual bleeding. Treatment with Org 32489 (75 lU/ampoule) or MetrodmR (75 lU/ampoule) was started 14 to 18 days after the onset of the buserelin intake when this treatment had resulted in an hJ~L~ Iu~ul,.c state (i.e., serum E2 < 50 pg/mL). In case this state had not been achieved, Org 32489 ûr Metrodin treatment was postponed and the dose of buserelin was increased to 4 x 300 ~g/day. This increased ~userelir~ dose was sustained during the whole further treatment period up to the moment FSH treatment was stopped. F-t: with buserelin was not allowed to exceed a period of five weeks. If after complete down-regulation on the frst FSH treatment day a follicular cyst ~ 20 mm had developed, this cyst was punctured before the first FSH imjection.
In each treatment cycle dosing was performed according to the schedule below:

`O wogs/lg99l 2 1 8 1 7 1 1 r~llrl~5l -Buserelin Dose 4x150 ~Lg/day Route intranasal Org 3248g/MetrodinR Dose Treatment day 1-4:
150-æ5 lUlday.
from day 5 on: dose was adjusted per subject Route i.m.
HCG (10.000 IU) was - ~ when at least three follicles 2 17 mm were present. No Inore than three embryos were replaced and frozen embryos were replaced in natural cycles or stimulated cycles. A
cycle was considered cancelled if no embrsto ~insfer had taken place.
The luteal phase was monitored by ~O l Luteal ` phase support (e.g. three iijections of 1500 IU hCG or at least 50 mg i.m. per day or 400 mg ,vl~o ~ v ~ , per day) was Oiven for at least two weeks after the injection of 10.000 IU
hCG.

The total number of oocytes recovered in the first treatment cycle was a primar~t efflcacy variable. Other analysed variables in the first treatDnent cycle were the Inumber of FSH ampoules: ' ~ for ovariam stimulation and the duration of FSH treatment. The outcome of tbese parameters were amalysed by means of Cochran's method of combining individual centre results. In addition, the Wilcoxon rank sum test and an analysis of variance (ANOVA) were applied to consolidate the outcome of Cochran's method.
Sh~ly I of first cyt iPC
Altogether 981 subjec~s started FSH treatment in 18 study-centres.
In total 907 subjects had a punctur~, 546 in the Org 32489 group and CONFI~MA7itON COPY

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218~71 1 361 m the Metrodin group. With this number of punctured subjects it was possible with probability 0.8 (the so-called power of the study) to detect at least a difference of 1.2 oocytes between the two treatment groups.
The number of oocytes recovered in each treatment group per centre is presented in Table 3. In all centres the Org 32489 group had a larger mem number of oocytes recovered than the Metrodim group.
The overall mean number of oocytes (weighted over centres) was 10.84 oocytes in the Org 3248g group and 8.95 oocytes in the Metrodin group, resulting im a treatment difference of 1.89 oocytes im favour of Org 32489. The difference of 1.89 oocytes is more tban 5 times its standard error and highly sigmficant (P < 0.0001). The resulting 95%
confidence interval indicated that on the average subjects treated with Org 32489 end up with at least 1.2 and at most 2.6 oocytes more than those treated with MetrodinR. Exclusion of immature oocytes from tbe total number of recovered oocytes did not influence the treatment effect (see Table 4).
The total number of ampoules ~ ' ci to punclured subjects is presented per centre in Table 5. The total dosage of Org 32489 r ' Cd was in 14 out of the 18 centres lower than the total dosage of MetrodinR. The overall mean FSH dosage (weighted over centres) was 28.5 ampoules in the Org 32489 group and 31.8 ampoules m the Metrodin group, resulting in a treatment difference of 3.3 ampoules which is highly statistically significamt (P<0.0001). The resulting 95%
confidence interval indicated that on the average snbjects treated with Org 32489 receive minimally 2.1 amd maximally 4.5 ampoules less th~m those treated with MetrodinR.
The duration of FSH treatment of punctured subjects is presented per centre in Table 6. The treatment duration was m 13 out of 18 centres shorter in the Org 32489 group than in the Metrodin group.
The overall mean treatment duration (weighted over centres) was 10.7 days m the Org 32489 group amd 11.3 days in the Metrodm group, resulting in a treatment difference of 0.6 days which is highly statistically sigluficant (P<0.0001). The resulting 95% confidence CON~ lA~70N COPY

`O wogS~Ig99l 2 1 8 1 7 l 1 3~
interval indicated that o~ the average fo} subjects treated with Org 32489 the treatment period is minimally 0.3 day and maximally 0.9 day shorter than for those treated with MetrodinR.
The outcome of the Wilcoxon lank sum test and the analysis of variance confirmed all of Ihe above findings.
Thus, it can be concluded that in this study controlled . u.,' by means of Org 32489 treatment s~ ' superiority over MetrodilnR with respect to tbe number of oocytes ~etrieved with a . ~ lower FSH dosage and shorter treatment period.

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woss/lsssl r~"~.,s,~
21~1711 16 ~e~d~s Figure I
Distribution of FSH ~.,li~ily after .', ' ,, of a specific Org 32489 batch. The profile of one batch is shown as example. Upper and lower panel represent sepaJate runs. (sf = salt don).Tol.311ecovery919:d9139,.~,u~.,1~.

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Table I
Mean recovery~ pH range, pH top and distribution of FSH
S C.l~liYily over pH ranges after ~ ', f~ ~ of tvelve Org 32489 bulk batches, five Org 32489 final products, i.e. clinical iu... (CP's) and tv/elve MetrodinR batches.
Org 32489 MetrodinR
Bulk: material CP's n 12 5 12 Recovery 93.7 i 8.9 % 84.2 1 3.6 % 89.1 t 6.1%
pH range 5.68 - 3.18 5.60 - 3.13 5.58 - 3.07 pH top 4.53 4.52 4.28 FSH :" il,_ iu,.:
pH range> 5.3 2.0 % 2.2 % 1.3 %
5.30- 4.81 18.9 % 18.0 % 11.4 %
4.80- 4.20 58.2 % 55.5 % 45.6 %
4.19- 3.60 18.6 % 21.7 % 33.8 %
3.59-3.00 1.7 % 2.0 % 4.6 %
<3.0 ~.8% 0.8% 3.3%

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21817~ 1 18 Table 2 Percentage basic isoforms, defined as FSH ~livily at pl > 4.8, and percentage æidic isoforms, defined as FSH
S ' ~,iJvi~y at pl < 4.2 of the Org 32489 and MetrodinR CP's used to compare the clinical efficacy (see E~ample 2).
Preparation I ' distribution % basic % acidic (pl >4.8) (pl <4.2) Org 32489:
bulkbatchesn=12 20.9 21.1 CP's . ~, efficacy study 17.1 23.9 15(n=2) MetrodinR:
Batches n= 12 12.7 41.7 CP's , ~, efficacy study 11.9 44.4 20(n=3) CONFlR~.,1A rlON COP'~

~ wo ss/lgggl 21 8 1 7 ~ I r~

Table 3 Statistical analysis (Ct)chran's approach) of the total number of oocytes recoYered Including all punctured subjtxlts.
Tot-l numo~r of ooc~ee~ recovered Group Org 32489 minus Metrodin Cycle 1 Org 3 differ~nc~ ~st~mate (p-v~lue) Ce tr~ nM,~n 5~6 361 1.89 0.~7 1.2 to 2i6 ~d1u~ted for c~nere Tese for lnter~ct~on t0.88) C~tr~
M~an 12.6~7 11.80 0.73 1.33 M~dn 12.67 a.7i 3.95 3.6 3 ni S 1. 7 i Me~n 31 7 2.~5 1.89 Me~n 15.~6 10.36 5.09 3.20 6 n12.60 9.s3 3.07 2.0 7 n7 . 2 3 s ~ 22 l 2 . 0 2 1 . o M~-n7.6~3 6.50 1.08 1.92 Me~n 10.76 9.79 0.99 l.S9 10 Me~n 11.~2 25 2.04 0.96 11 MeilD 9 . SO li 1 . 56 12 Me~n 9.36 a.62 0.76 1.9S
13 Mean 11.70 lo, j2S 1.2i 1.~7 li Mnean 12.960 12.il7 0.62 1.29 lS Mean 12.~88 9.81 2.67 1.13 16 Me~n 13.70 12.76 0.9~ 2.iO
17 M13.297 11.123 2.06 1.93 1~ Mn aan 16 . i i 1 1 1 . 6 7 i . 7 6 1 3 7 7 CON~,71,'A~IA7.70N COPY

WO 95/19991 P~ 5.'~ - --218171~ 20 Table 4 Statistical analysis (Cochran's approach) of the number of mature oocytes recovered Including all punctured subjects.
Numoer of m~ture ooeytes recovered Group Org 32iag minu~ Metrodln Cy~le 1 tre tment error of inter rdl differen~e estlmate (p-~nlue~
Cener MeAn 8 55 6376 1.79 0.33 1.1 to 2 ~d~u~ted for ~ntre Te~t for inter~tion ~0.89) C~ntr~
n12,6j78 11.29 1.18 1.31 Me~n 10.92 7.i3 3.i9 3.10 Me~n 5.75 1.60 i.l5 1.2i Me~n 10 7 2.07 2.02 Me~n 13.23 h,285 ~.ga 3.20 Me~n 10.53 8.35 2.18 1.81 Me~n 7.23 s 22i 2.02 1.01 8nj,37 i.27 0.10 1.30 Me~n 10 . 579 9 . 74 0 . 86 1. 59 10Mel~n 6.93 5.40 1.53 0.99 11Mne~n b,l27 li 1.27 12Mne~n 6.81 7.15 -0.3i 1.98 13Me21n 10.93i 9.822 1.12 1.~8 linMe~n 12.55 11.356 1.25 1.33 15Me~n 7.39 ~.85 2.s~ 0.79 16Mei~n 12.56 11.2~7 1.32 2.33 17Me~n 13,l279 11.23 2.~6 1.93 lbMe~n 8.11 2.20 2.78 ~.~rlN~lRMATlON COPY

21817~ 1 W09S119991 r(.

Table 5 Statistical analysis ((,ochran's approach) of the total FSH dose :' c;d Including all punctured subjects.
L:
Tot~l nume~r of FSg vi~ =poule~ Group org 32i89 =inu~ MeCrodin Cy=le 1 Org 12~89 M~rodin 1: timate of 3t nd~rfd 9s~icorfidelnce di~f~rence ~tim~te (p-n~lu~) ecmbie~ d ad3u~t6d for ~encre 28S-S 31 82 _3 33 0 62 (<0 0003 Te~t for internction (0 06) C ner~
Menn29.29 38.13 -8.8i 3 ~
M~n28.18 27.~3 0.65 2.80 3 M~n39.50 ~2.60 -3.10 8.10 n 36 3l6 37.86 -1.~9 S.C9 M~an30.S43 ~2.00 -11.~6 4.86 M~n26.8`7 28 l76 -1.90 2.95 Me~n27.~o47 33.83 -6.77 2.21 Me~n~9.26 i8.77 O.SO 5.66 9 n3S.04 32.79 2.~5 3.09 lO Me~n 35 47 37 2858 -2.18 3.59 11 Me~n 30 274s 38.o47 -7.32 2.75 12 Me~n 32 1336 32.~70 0.35 6.01 13 Me2n 27.6333 29 o227 -1.39 2.1S
li M~n3O 56S 31 ~262 -0.62 1.29 lS M~n23.637 28 Slg -4.S~ 4 16 M-~n23.267 23.47 -S.21 2.6S
17 M~n25.72 3 3.64 ~8 Me~n 26 4 3S S6 -6.63 3.33 CONF7~,'M~7,'0N GOPY

WO 95/19991 1~

Table 6 Statistical analysis (Cochran's approach) of the duration of the FSH treatment 5Including all punctured subjects.
our~ticn o~ F5H ~re~ A~ (d~y) 3roup org 32-89 minu~ Metrodln Cycle 1 org 32~89 Me~rodin ~ tim ~e o~ t nd~rd 95~ ~ontld;n~e di~renc~ e-tim~te (p-r~lue) C~er ~d~u~ted ~or c~ntre 105666 113267 -0.60 0.1~ -0 .7 to -0;3 7~t ~or int~r~tion 10.02) ~Q~
Me~n 11,6997 13.73 -1.75 0,65 ~n9.09 9,00 O.o~ 0.66 Me~n ~3.75 1~.00 -0.25 1.~1 M~n 11,6i 11.57 o.D6 1.15 Me n ~.92 11.39 -1.-5 0.69 Me~n 10.83 11.2- -0.-0 0 50 Me6n 12.~3 13.~26 -L.05 0.55 Me~n 13.13 13.53 -O.i3 0,96 Me~n 1l,l25 l0,26F o.~e 0.59 10nM~n 12.~2~ 12.72 -0.~9 0.-3 IlnMe~n 11,27~3 12,9i-3 -1.60 0 57 12nM, n 12,265 11.323 1.02 1.0~
1~Me~= .55 6.-5 0.09 0.35 1~Ile~n lo,60 1o,~362 -0.2~ 0.35 15nMe~n 10. l 12.02 -1.21 0.~7 ltMe~n lo.F6 11.927 Ø8C 0.~6 17nMe~n 10.06 11.377 -1.71 0.67 I&nMe~n 9,17 11.33 -I.SS o.
~ = . , - CONFI~MA7,70N COP~

Claims

Claims 1.
A pharmaceutical composition comprising a mixture of isoforms of a glycoprotein hormone with isoelectric points in between and extending from the range 4.8 to 4.2 having follicle stimulating activity, in admixture with pharmaceutically acceptable auxiliaries, characterized in that more than 15% of the isoforms have isoelectric points above 4.8 and less than 30% below 4.2.
2.
The composition according to claim 1 characterized in that more than 15% of the isoforms have isoelectric points above 4.8 and less than 25% below 4.2.
3.
The composition according to claim 2 characterized in that more than 25% of the isoforms have isoelectric points above 4.8 and less than 25% below 4.2.

The composition according to claim 3 characterized in that more than 25% of the isoforms have isoelectric points above 4.8 and less than 20% below 4.2.
5.
The composition according to claims 1-4 characterized in that the glycoprotein is recombinant follicle stimulating hormone.
6.
The composition according to claims 1-5 characterized in that the glycoprotein is modified by enzymatic or chemical treatment.
7.
The composition according to claim 5 characterized in that the recombinant follicle stimulating hormone is modified by eliminating at least one of the N-linked oligosaccharide chains.

8.
Method to induce follicular growth and maturation in females by administering according to claims 1-7.
9.
Method to induce follicular growth and maturation by administering during the ovulatory cycle identical pharmaceutical compositions of a mixture of isoforms of a glycoprotein with follicle stimulating activity characterized in that said isoforms have an isoelectric point above 4.2.