A biological, immunological and physico-chemical comparison of the current clinical batches of the recombinant FSH preparations Gonal-F and Puregon

G. Horsman1,4, J.A. Talbot2, J.D. McLoughlin2, A. Lambert3 and W.R. Robertson3

1 Departments of Clinical Biochemistry and 2 Diabetes and Endocrinology, Hope Hospital, Salford, M6 8HD, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The immunopotency and in-vitro biopotency of clinical batches of Gonal-F® and Puregon® (recombinant human follicle stimulating hormones) were compared and their carbohydrate chains investigated for charge heterogeneity and internal carbohydrate complexity. Immunopotency (IU/pmol) for both Gonal-F and Puregon was 0.35 ± 0.01 and biopotency (ED50, pmol/l) was similar, being 7.3 ± 0.6 and 5.4 ± 0.2 respectively. Charge distributions were essentially the same with no difference either in median isoelectric point (pI) (between 4.26 and 4.50), or in the bulk of material fractionated between pI 4 and 5 (66.0 ± 1.8% Gonal-F and 72.0 ± 1.8% Puregon). However, there were minor differences in charge at extremes of pI, Gonal-F being slightly more acidic: 18.2% Gonal-F versus 9.8% Puregon at pI 3.5–4.0 (P = 0.03) and 6.7% Gonal-F versus 10.7% Puregon at pI 5.0–5.5 (P = 0.03). Carbohydrate complexity was the same: 9.3 versus 10.9 (complex), 76.6 versus 78.6 (intermediate) and 14.1 versus 10.5% (simple). In summary, Gonal-F and Puregon have similar immunopotency, in-vitro biopotency and internal carbohydrate complexity, differing slightly in charge heterogeneity, Gonal-F having more acidic glycoforms. We conclude them to be intrinsically very similar, expecting no difference in clinical efficacy on the basis of respective structure.

Key words: FSH/glycoforms/Gonal-F/Puregon/rFSH


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
FSH is a dimeric glycoprotein with an {alpha} protein subunit common to LH and thyroid stimulating hormone from the pituitary gland and human chorionic gonadotrophin (HCG) from the placenta. The {alpha} subunit is bound non-covalently to a ß subunit unique to each hormone which together confer biological specificity. There are two carbohydrate chains N-linked to asparagine residues on each FSH subunit (Baenziger and Green, 1988Go) and these comprise ~28% of the hormone mass. There is marked variability in both the oligosaccharide composition, branching pattern and overall charge of these carbohydrate structures (Green and Baenziger, 1988aGo,bGo) giving rise to a large array of `glycoforms' of FSH.

The relationship between charge and in-vivo bioactivity is a complex one. It has long been appreciated that the more acidic sialic acid-rich glycoforms have a prolonged half-life in the serum (Blum and Gupta, 1985Go), and therefore, as might be expected, have a greater in-vivo bioactivity (Mulders et al., 1997Go). However, in vitro where half-life is not a concern, there is now direct evidence using purified recombinant FSH (rFSH) glycoform species to suggest that the less acidic forms are the more bioactive in an animal model (Vitt et al., 1998Go) and for Puregon glycoforms on a molar basis, in human pre-ovulatory and granulosa-lutein cells (Harris et al., 1998Go). Overall, whereas the less acidic species may have greater receptor binding, it seems that a slow clearance rate is the principal determinant of in-vivo bioactivity which is consequently greater for the more acidic forms (de Leeuw et al., 1996Go). Furthermore, deglycosylated variants of FSH act as hormone antagonists, and forms possessing immuno- but not bioactivity do occur naturally and may be important in gonadal function by causing receptor blockade (Dahl et al., 1988Go). The evidence regarding the endocrine control and physiological importance of these glycoforms has recently been reviewed (Lambert et al., 1998Go). In brief, glycoform synthesis, secretion and the composition of the mixture in blood is related to FSH concentration (probably mediated via changes in gonadotrophin-releasing hormone pulsatility) and oestradiol concentrations (Wide and Naessén 1994Go; Chappel, 1995Go; Ulloa-Aguirre et al., 1995Go; Wide et al., 1996Go; Anobile et al., 1998Go). The particular acidity of the FSH glycoforms in blood varies considerably at different stages of the menstrual cycle most markedly at mid-cycle when there is an increase in the less acidic, less complex species (Padmanabhan et al., 1988Go; Wide and Bakos, 1993Go; Zambrano et al., 1995Go; Anobile et al., 1998Go). Further, there is a wealth of data (see Lambert et al., 1998) on changes in the mixture of secreted glycoforms of FSH and LH in a variety of pathologies and such observations have led to speculation as to whether the different forms may have different biological activities.

The increasing evidence surrounding the regulation of FSH glycoforms and the possibility of their having different functions could have important implications for the use of FSH in assisted reproduction programmes in which rFSH is playing an increasingly important part. Such products have the advantages of ease of supply in unlimited quantity, and the stringent quality control procedures employed in their production result in minimal between-batch variability as compared with the significant batch-to-batch differences seen in some urinary preparations (Rodgers et al., 1995Go).

Two rFSH preparations are in current clinical use, Gonal-F® (Ares Serono, Geneva, Switzerland) and Puregon® (NV Organon, Oss, The Netherlands). Both are produced from Chinese hamster ovary cells transfected with the human FSH subunit genes. Gonal-F is subsequently purified from the cell culture supernatant by an ultrafiltration followed by five chromatographic stages including reversed-phase highperformance liquid chromatography (HPLC) with an immunoaffinity procedure being the principal purification step (Howles, 1996Go). The chromatographic stages in the purification of Puregon include anion and cation exchange, hydrophobic interaction and size exclusion (Olijve et al., 1996Go). Both preparations have proven efficacy in clinical trials (Recombinant FSH Study Group, 1995; Out et al., 1995Go; Bergh et al., 1997Go). We have previously characterized an early batch of Puregon with regard to in-vitro biopotency, charge distribution (Lambert et al., 1995Go) and internal carbohydrate complexity (Harris et al., 1996Go) and found clear differences between these parameters in naturally occurring and urinary FSH preparations used in assisted reproduction programmes.

The aims of this study were to extend our earlier findings by determining whether or not the clinical material in the current batches of Gonal-F and Puregon is different, comparing them with regard to their respective immunopotency and bioactivity in vitro, and assessing the heterogeneity of their attached carbohydrate chains with respect to charge and internal carbohydrate complexity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Sertoli cell bioassay for FSH
Sertoli cells were isolated from 8–10 day old rats following a previously published method (Van Damme et al., 1979Go) with some modifications. Briefly the testes were removed, decapsulated, cut into small pieces and dispersed by mechanical agitation (rotating paddle, 400 r.p.m. at 37°C) in the presence of collagenase (0.03% w/v) in Dulbecco's modified Eagle's medium (DMEM) for 30 min. The cells were washed with DMEM three times by centrifugation (300 g, 5 min), resuspended in Iscove's serum-free medium (10 ml/testis) supplemented with bovine serum albumin (BSA; 0.1%; ICN Biochemicals, High Wycombe, Bucks, UK), penicillin (100 IU/ml; Life Technologies Ltd., Paisley, UK) and streptomycin (100 µg/ml, Life Technologies Ltd.) before being incubated in 96-well plates (200 µl/well) for 48 h at 37°C in an atmosphere of 95% air/5% CO2. After this period the medium was replaced with fresh culture medium (100 µl final volume) containing the androgen substrate 19{alpha}-hydroxyandrostenedione (7.5 µmol/l), the phosphodiesterase inhibitor isobutylmethylxanthine (0.125 mmol/l; Sigma Chemical Co., Poole, Dorset, UK) and FSH (0–1000 IU/l) and the plates were incubated for a further 24 h. The majority of contaminating germ cells were removed on exchange of culture medium. After 72 h in culture, samples (50 µl) were assayed for oestradiol by specific radioimmunoassay. The effect of increasing concentrations of Puregon on oestradiol secretion by rat Sertoli cells is shown in Figure 1Go. Similar dose–response relationships were obtained for Gonal-F (batch B3228) and Puregon (batch 9F073) in three separate experiments. Dilutions of Gonal-F when measured against the linear portion of the Puregon standard curve gave parallel responses. Furthermore, a comparison of the slopes of the linear portion of the curves for Gonal-F and Puregon showed no significant difference (Student's t-test).



View larger version (11K):
[in this window]
[in a new window]
 
Figure 1. Representative curve for the dose response relationship for oestradiol production (mean ± SD, n = 3 replicates) in the rat Sertoli bioassay system for Puregon (similar curve for Gonal-F not shown for clarity). The responses shown are absolute oestradiol production; unstimulated oestradiol concentrations, being below the assay detection limit (125 pmol/l), did not permit data expression as increase over basal.

 
Oestradiol radioimmunoassay
Oestradiol was measured by a standard in-house radioimmunoassay using oestradiol-6-(O-carboxymethyl) oximino-2[125I] iodohistamine (Amersham Pharmacia Biotech code IM135, Little Chalfont, Bucks UK) and anti-17ß-oestradiol (Advance Biotechnologies, Leatherhead, Surrey, UK). The cross-reactivities of this antiserum were: oestradiol, 100%; oestrone <2.0%; oestriol, 0.08%; progesterone <0.02%; androstenedione 0.056%; dehydroepiandrosterone 0.12%; testosterone <0.02%. Quality control samples (250, 750, 2500, 7500, 15 000 and 25 000 pmol/l) were assayed in duplicate in each assay. Intra- and inter-assay coefficients of variation were <10% and <17% respectively over the range 250–25 000 pmol/l.

Chromatofocusing of FSH preparations
All buffers were prepared in distilled deionized water and filtered through a 0.2 µm filter. The starting buffer for chromatofocusing was bis(2-hydroxyethyl)imino-Tris(hydroxymethyl)methane (bis-Tris, 7.14 mmol/l, pH 7.0–7.2; Sigma). The elution buffer was Polybuffer 74 (Amersham Pharmacia Biotech) used at a 1:35 dilution at pH 3.0–3.2. A saturated solution of iminodiacetic acid was used to titrate buffers to the correct pH and all buffers were degassed for a minimum of 4 h prior to use.

Ampoules of FSH preparations (Gonal-F batch B3228, Puregon batch 9F073) were reconstituted in degassed bis-Tris (10 ml, 7.14 mmol/l) containing 0.1% BSA at pH 7.0–7.2. A final dilution was made in bis-Tris such that 2.0 IU FSH in 8.5 ml was loaded onto the chromatofocusing Mono P column (4 ml) which was used in conjunction with a fast performance liquid chromatographic system (Amersham Pharmacia Biotech). The column was washed with NaCl (0.5 ml, 2 mol/l) to exchange the storage counter ion, prior to an equilibration with 30–50 ml bis-Tris (0.5 ml/min flow rate) until the column pH was equal to that of the start buffer. The sample (8.5 ml) was loaded onto the column and elution buffer was pumped at a flow rate of 0.5 ml/min to generate the pH gradient. Fractions (0.95 ml) were collected into tubes containing 50 µl 2% BSA in distilled deionized water, such that the final concentration of BSA was 0.1%, and their pH was adjusted to pH 7.4 with dilute NaOH. The run was continuous until the pH of the eluent was the same as that of the limit buffer. After completion of the pH gradient, NaCl (0.5 ml, 2 mol/l) was injected on to the column and eluted with Polybuffer (10 ml). All fractions, including the material below pI <3.25 (salt peak), were frozen and stored at –40°C until estimation of FSH immunoreactivity by Delfia® (Wallac, Milton Keynes, UK). The mean percentage recovery of FSH after chromatofocusing was 78.1 (95% confidence interval, 74.7–81.5; n = 6).

Concanavalin A affinity chromatography
Concanavalin A (con A) interacts with N-linked oligosaccharide structures according to their branching properties, such that tri-antennary, tetra-antennary and bisecting oligosaccharides (complex forms) do not bind to con A, bi-antennary and truncated hybrids (intermediate complexity forms) bind weakly, and high mannose and hybrid oligosaccharide (simple forms) bind firmly to con A (Cummings and Kornfeld, 1982Go).

Phosphate buffer (0.05 mol/l, pH 7.4) containing 0.1% BSA was used to prepare all eluents. Eluent (i) was phosphate buffer only, eluent (ii) was phosphate buffer containing 10 mmol/l {alpha}-D-methylglucopyranoside ({alpha}-MG; Sigma) and eluent (iii) was phosphate buffer containing 300 mmol/l {alpha}-D-methylmannopyranoside ({alpha}-MM, Sigma). Column regeneration solution (a) was 0.1 mol/l borate (pH 8.5), regeneration solution (b) was 0.1 mol/l sodium acetate/1 mol/l sodium chloride (pH 4.5) and the column prewash solution consisted of 1 mol/l sodium chloride, 5 mmol/l calcium chloride dihydrate, 5 mmol/l magnesium chloride and 5 mmol/l manganese (II) chloride (all chemicals from Merck, Poole, Dorset, UK). The storage and equilibration buffer was phosphate buffer only.

Before use, the lectin columns (2.5 ml con A agarose, Sigma) were equilibrated with five column volumes of prewash solution followed by 0.05 mol/l phosphate buffer. 1 IU/100 µl of each of the recombinant gonadotrophin preparations was loaded onto the column and allowed to interact for 10 min at room temperature. Unbound FSH was collected by passing 5 ml of eluent (i) down the column in 0.5 ml fractions. Weakly and firmly bound FSH was collected in a similar way by passing 25 ml of eluent (ii) and 13 ml of eluent (iii) respectively down the column. A total of 86 fractions was collected and stored frozen at –40°C for subsequent FSH immunoassay. Recoveries ranged from 73–113% (91.7 ± 2.6, mean ± SEM, n = 7). The column was regenerated using five column volumes of regeneration solutions (a) and (b) in sequence followed by prewash solution. The column was stored capped and upright in 0.05 mol/l phosphate buffer at 4°C.

FSH immunofluorimetric assay
For estimation of FSH in the chromatofocusing and con A fractions, the matrix of the FSH standard (second IRP, NIBISC code 78/549) was adjusted to be identical to that of the fractions. Serial fractions were assayed at multiple dilutions in order to ensure that the glycoforms generated parallel standard curves to the FSH 78/549. The fractions did dilute in a parallel fashion. For example, analysis of variance revealed no differences between serial dilutions of unfractionated rFSH, material at pI 4.76–5.00 and in the salt peak (pI <3.25). Furthermore the Delfia assay has been demonstrated not to show differential recognition of purified FSH glycoforms (Oliver et al., 1999Go). FSH was measured using the Delfia immunofluorimetric assay. Quality control samples were assayed in duplicate in each assay. Inter-assay coefficients of variation were 12.2% at 1.6 IU/l and 7.8% at 25 IU/l.

Calculation of molar immuno- and biopotencies and statistical analysis
A 75 IU ampoule of each of the preparations contains a known amount of protein, 5.9 µg for Gonal-F [personal communication, Serono Labs (UK) Ltd] and 7.5 µg for Puregon (from stated specific activity of 10 000 IU/mg in product data sheet), allowing the amount of protein in 10 kIU/l stock solutions of each of the preparations to be calculated. Furthermore, expression of this in molar terms is possible from knowledge of the published data on respective mean molecular weight by sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis, namely 42 000 for Gonal-F (Siebold, 1996Go) and ~42 500 for Puregon (Olijve et al., 1996Go). Published matrix-assisted laser desorption ionization (MALDI) mass spectrometry data for both these preparations are not available currently. The FSH immunoreactivity in these stock solutions as measured by Delfia thus determined the immunopotency per (pico)mole. These specific activities and molecular weights were similarly used in the expression (as ED50) of in-vitro bioactivity in molar terms.

All data are expressed as mean ± SEM and there were three or more replicates in each experiment. FSH immunoreactivity in the pooled 0.25 pH unit samples following chromatofocusing was compared using Student's unpaired t-test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Immunopotency and biopotency of Gonal-F and Puregon
Gonal-F and Puregon had similar immuno- and biopotencies of 0.35 ± 0.01 compared with 0.35 ± 0.01 IU/pmol and 7.3 ± 0.6 compared with 5.4 ± 0.2 pmol/l (ED50) respectively.

Charge distribution of Gonal-F and Puregon
The immunoreactive FSH distribution found for Gonal-F and Puregon is shown in Figure 2Go. The distributions were largely similar, with the bulk of the material focusing between pI 4.0 and 5.0, the percentages of the total FSH recovered for Gonal-F and Puregon over this range being 66.0 ± 1.8 and 72.0 ± 1.8 respectively. In each case the median pI fell within the 4.26–4.50 pH fraction. There were small differences in the extremes of the distributions with more Gonal-F focusing between pI 3.5 and 4.0 than Puregon (18.2 ± 1.7 versus 9.8 ± 1.8%; P = 0.03) and less Gonal-F than Puregon focusing between pI 5.0 and 5.5 (6.7 ± 1.2 versus 10.7 ± 0.2%; P = 0.03).



View larger version (30K):
[in this window]
[in a new window]
 
Figure 2. Distribution of immunoreactive FSH determined by Delfia following chromatofocusing (right-hand panels) for Gonal-F (upper panels) and Puregon (lower panels). Data are presented as the mean percentage ± SEM (n = 3) FSH recovered relative to the total eluted. The left-hand panels show the relative percentages recovered relative to pI 4.25.

 
Carbohydrate complexity of Gonal-F and Puregon
There were no differences between the two preparations, with the proportions of complex, intermediate and simple forms for Gonal-F and Puregon being respectively 9.3 ± 1.3 versus 10.9 ± 1.6%, 76.6 ± 2.4 versus 78.6 ± 9.0%, and 14.1 ± 4.2 versus 10.5 ± 1.0%.

The relationship of the charge distribution and internal carbohydrate complexity of Gonal-F and Puregon to those of the forms seen in the menstrual cycle is shown in Table IGo.


View this table:
[in this window]
[in a new window]
 
Table I. The percentage of FSH with isoelectric point (pI) >4.25 and the percentage of complex, intermediate and simple glycoforms (determined by Delfia) following chromatofocusing and concanavalin A affinity chromatography respectively in the recombinant FSH preparations Gonal-F and Puregon, and in the early-mid follicular (EF; day 7), late follicular (LF; day 11), mid-cycle (MC; day 14) and luteal (L; day 21) phases of the menstrual cycle
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have shown no significant differences between these two recombinant gonadotrophin preparations in terms of their immunopotency, biopotency in vitro and carbohydrate complexity. Furthermore their charge was broadly similar particularly with regard to the bulk of material recovered, although Gonal-F was slightly more acidic than Puregon. This difference may well result from differences in purification procedures of the rFSH, employing immunoextraction for Gonal-F in addition to ultrafiltration and chromatographic steps (see Howles, 1996).

In addition there is negligible between-batch variability for Gonal-F (B3228 used here) and the previous clinical batch (B3209) (Horsman et al., 1999Go). Similarly for Puregon 9F073 (Talbot et al., 1999Go) varies little in terms of immunopotency, and, when compared using similar methodologies with our previous published data on the pre-clinical batch (Org 32489), with respect to in-vitro biopotency, charge distribution (Lambert et al., 1995Go) and carbohydrate complexity (Harris et al., 1996Go). These findings lend weight to the argument that the rFSH preparations are highly consistent between batches.

Our data from the rFSH preparations reveal that they are most similar to the forms found in blood around mid-cycle with regard to charge but differ entirely with regard to carbohydrate complexity, in that there is a predominance of intermediate complexity forms in the recombinant preparations and throughout the menstrual cycle (Anobile et al., 1998Go) of complex forms. The reason for this may be that Chinese hamster ovary cells do not have the enzymatic functions to construct the complex carbohydrate structures found in the human. This difference appears, however, to be of no clinical importance since both Gonal-F and Puregon are highly bioactive and clinically effective in vivo. Moreover, most of the evidence on the possibility of the different forms of FSH having different bioactivity relates to the various charged species.

The techniques used do have their limitations, especially the rat Sertoli cell bioassay system. Not only does this utilize a single FSH response (oestradiol production) but there is also a well-recognized inherent imprecision. Our calculations on ED50 will also be affected by the relative imprecision of the published molecular weight data used to calculate biopotency. With these provisos, our studies have shown similar biopotency in vitro and very little structural difference between the material in the ampoules of Gonal-F (B3228) and Puregon (9F073) and we have produced no data which might lead us to expect any differences in efficacy in-vivo or tolerability. However, there are reasons why this may not be so, including differences in pharmaceutical formulation and patient group response. Direct clinical comparisons, preferably cross-over trials, are needed to resolve this issue, and while we were performing our study such a comparison was done in 44 patients undergoing IVF–embryo transfer and randomized to receive either Gonal-F or Puregon treatment. There was no significant difference in any efficacy measure or incidence of systemic adverse events in each patient group (Brinsden et al., 2000Go).

In summary, Gonal-F and Puregon are similar in terms of immunopotency, in-vitro biopotency and internal carbohydrate complexity, but differ slightly in charge heterogeneity with Gonal-F having slightly more acidic glycoforms. We conclude that these two recombinant hormone preparations are intrinsically very similar, and would not expect any difference in clinical efficacy on the basis of their respective structures.


    Acknowledgments
 
The authors wish to thank Ian Nickson and Richard Neal for assistance with the concanavalin A affinity chromatography assays. Funding for the study was provided by The Salford Royal Hospitals Trust and North Manchester Healthcare.


    Notes
 
3 Present address: Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Oxford OX3 9DU, UK Back

4 To whom correspondence should be addressed at: Department of Clinical Biochemistry, Hope Hospital, Salford M6 8HD, UK.E-mail: grahamhorsman{at}mcmail.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anobile, C.J., Talbot, J.A., McCann, S.J. et al. (1998) Glycoform composition of serum gonadotrophins through the normal menstrual cycle and in the post-menopausal state. Mol. Hum. Reprod., 4, 631–639.[Abstract]

Baenziger, J.U. and Green, E.D. (1988) Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lupotropin, follitropin and thyrotropin. Biochim. Biophys. Acta, 947, 287–306.[ISI][Medline]

Bergh, C., Howles, C.M., Borg, K. et al. (1997) Recombinant human follicle stimulating hormone (r-hFSH; Gonal-F) versus highly purified urinary FSH (Metrodin HP): results of a randomized comparative study in women undergoing assisted reproductive techniques. Hum. Reprod., 12, 2133–2139.[Abstract]

Blum, W.F. and Gupta, D. (1985) Heterogeneity of rat FSH by chromatofocusing: studies on serum FSH released in vitro and metabolic clearance rates of its various forms. J. Endocrinol., 105, 29–37.[Abstract]

Brinsden, P., Akagbosu, F., Gibbons, L.M. et al. (2000) A comparison of the efficacy and tolerability of two recombinant human follicle-stimulating hormone preparations in patients undergoing in vitro fertilization–embryo transfer. Fertil. Steril., 73, 114–116.[ISI][Medline]

Chappel, S.C. (1995) Heterogeneity of follicle stimulating hormone: control and physiological function. Hum. Reprod. Update, 1, 479–487.[Abstract]

Cummings, R.D. and Kornfeld, S. (1982) Fractionation of asparagine-linked oligosaccharides by serial lectin-agarose affinity chromatography. A rapid, sensitive, and specific technique. Biol. Chem., 257, 11235–11240.[Abstract/Free Full Text]

Dahl, K.D., Bicsak, T.A. and Hsueh, A.J. (1988) Naturally occurring antihormones: secretion of FSH antagonists by women treated with a GnRH analog. Science, 239, 72–74.[ISI][Medline]

De Leeuw, R., Mulders, J., Voortman, G. et al. (1996) Structure–function relationship of recombinant follicle stimulating hormone (Puregon). Mol. Hum. Reprod., 2, 361–369.[Abstract]

Green, E.D. and Baenziger, J.U. (1988a) Asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. Part I. J. Biol. Chem., 263, 25–35.[Abstract/Free Full Text]

Green, E.D. and Baenziger, J.U. (1988b) Asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. Part II. J. Biol. Chem., 263, 36–44.[Abstract/Free Full Text]

Harris, S.D., Anobile, C.J., McLoughlin, J.D. et al. (1996) Internal carbohydrate complexity of the oligosaccharide chains of recombinant human follicle stimulating hormone (Puregon, Org 32489): a comparison with Metrodin and Metrodin-HP. Mol. Hum. Reprod., 2, 807–811.[Abstract]

Harris, S.D., Lambert, A. and Robertson, W.R. (1998) In-vitro biopotency of two glycoform mixtures of differing acidity derived from recombinant FSH measured using a human granulosa-lutein cell bioassay. Hum. Reprod., 13 (Abstract Book 1), O-202.

Horsman, G., Talbot, J.A., McLoughlin, J.D. et al. (1999) A physico-chemical, immunological and biological comparison of two clinical batches of the recombinant FSH preparation. Gonal-F (1999) Proceedings of the ACB National Meeting, p. 35.

Howles, C.M. (1996) Genetic engineering of human FSH (Gonal-F). Hum. Reprod. Update, 2, 172–191.[Free Full Text]

Lambert, A., Rodgers, M., Mitchell, R. et al. (1995) In-vitro biopotency and glycoform distribution of recombinant human follicle stimulating hormone (Org 32489), Metrodin and Metrodin-HP. Hum. Reprod., 10, 1928–1935.[Abstract]

Lambert, A., Talbot, J.A., Anobile, C.J. et al. (1998) Gonadotrophin heterogeneity and biopotency: implications for assisted reproduction. Mol. Hum. Reprod., 4, 619–629.[Abstract]

Mulders, J.W.M., Derksen, M., Swolfs, A. et al. (1997) Prediction of the in vivo biological activity of human recombinant follicle stimulating hormone using quantitative isoelectric focusing. Biologicals, 25, 269–281.[ISI][Medline]

Olijve, W., De Boer, W., Mulders, J.W.M. et al. (1996) Molecular biology and biochemistry of human recombinant follicle stimulating hormone (Puregon®). Mol. Hum. Reprod., 2, 371–382.[Abstract]

Oliver, R.L., Kane, J.W., Waite, A. et al. (1999) Do immunoassays differentially detect different acidity glycoforms of FSH? Clin. Endocrinol., 51, 681–686.[ISI][Medline]

Out, H.J., Mannaerts, B.M., Driessen, S.G. et al. (1995) A prospective, randomized, assessor-blind, multicentre study comparing recombinant and urinary follicle stimulating hormone (Puregon versus Metrodin) in in-vitro fertilization. Hum. Reprod., 10, 2534–2540.[Abstract]

Padmanabhan, V., Lang, L.L., Sonstein, J. et al. (1988) Modulation of follicle-stimulating hormone bioactivity and isoform distribution by estrogenic steroids in normal women and in gonadal dysgenesis. J. Clin. Endocrinol. Metab., 67, 465–473.[Abstract]

Recombinant Human FSH Study Group (1995) Clinical assessment of recombinant human follicle-stimulating hormone in stimulating ovarian follicular development before in vitro fertilization. Fertil. Steril., 63, 77–86.[ISI][Medline]

Rodgers, M., McLoughlin, J.D., Lambert, A. et al. (1995) Variability in the immunoreactive and bioactive follicle stimulating hormone content of human urinary menopausal gonadotrophin preparations. Hum. Reprod., 10, 1982–1986.[Abstract]

Siebold, B. (1996) Physicochemical characterization of recombinant follicle stimulating hormone. Hum. Reprod., 11, 109–115.[Abstract]

Talbot, J.A., Horsman, G., McLoughlin, J.D. et al. (1999) A biological and physico-chemical comparison of two batches of the recombinant FSH preparation Puregon (1999). Proceedings of the ACB National Meeting, p. 35.

Ulloa-Aguirre, A., Midgley, A.R. Jr, Beitins, I.Z. et al. (1995) Follicle-stimulating isohormones: characterization and physiological relevance. Endocr. Rev., 16, 765–787.[ISI][Medline]

Van Damme, M.P., Robertson, D.M., Marana, R. et al. (1979) A sensitive and specific in-vitro bioassay method for the measurement of follicle-stimulating hormone-activity. Acta Endocrinol., 91, 224–237.[Medline]

Vitt, U.A., Kloosterboer, H.J., Rose, U.M. et al. (1998) Isoforms of recombinant follicle-stimulating hormone: Comparison of effects on murine follicular development in vitro. Biol. Reprod., 59, 854–861.[Abstract/Free Full Text]

Wide, L. and Bakos, O. (1993) More basic forms of both human follicle-stimulating hormone and luteinising hormone at mid-cycle compared with the follicular and luteal phase. J. Clin. Endocrinol. Metab., 76, 885–889.[Abstract]

Wide, L. and Naessén, T. (1994) 17ß-Oestradiol counteracts the formation of the more acidic isoforms of follicle-stimulating hormone and luteinizing hormones after menopause. Clin. Endocrinol., 40, 783–789.[ISI][Medline]

Wide, L., Albertsson-Wikland, K. and Phillips, D.J. (1996) More basic isoforms of serum gonadotrophins during gonadotrophin-releasing hormone agonist therapy in pubertal children. J. Clin. Endocrinol. Metab., 81, 216–221.[Abstract]

Zambrano, E., Olivares, A., Mendez, J.P. et al. (1995) Dynamics of basal and gonadotrophin-releasing hormone-releasable serum follicle-stimulating-hormone charge distribution throughout the human menstrual cycle. J. Clin. Endocrinol. Metab., 80, 1647–1656.[Abstract]

Submitted on February 1, 2000; accepted on May 26, 2000.