19 Follicle-Stimulating Hormone

Tom Sam and Renato de Leeuw

INTRODUCTION including pharmaceutically active proteins such as

LH. Recombinant DNA technology allows the repro-
About 15% of all couples experience infertility at some
ducible manufacturing of FSH preparations of high
time during their reproductive lives. Nowadays, infer-
purity and specific activity, devoid of urinary contami-
tility can be treated by the use of assisted reproductive
nants. Recombinant FSH is produced using a Chinese
technologies (ART), such as in vitro fertilization (IVF)
Hamster Ovary (CHO) cell line, transfected with the
and intracytoplasmic sperm injection (ICSI). A com-
genes encoding for the two human FSH subunits (van
mon element of these programs is the treatment with
Wezenbeek et al. 1990; Howles 1996). The isolation pro-
Follicle Stimulating Hormone (FSH) to increase the
cedures render a product of high purity (at least 99%),
number of oocytes retrievable for the IVF or ICSI pro-
devoid of LH activity and very similar to natural
cedure (multifollicular development). Patients suffer-
FSH. Recently, a novel recombinant FSH derived from
ing from female infertility because of chronic
a cell line of human fetal retinal origin (follitropin
anovulation may also be treated with FSH, then with
delta) was introduced (Olson et al. 2014).
the aim to achieve monofollicular development.
Currently, there are several clinically approved
Natural FSH is produced and secreted by the
recombinant FSH-containing drug products on the
anterior lobe of the pituitary, a gland at the base of the
various markets. The most widely approved products
brain. Its target is the FSH receptor at the surface of the
are Gonal-F®, manufactured by Merck Serono S.A.,
granulosa cells that surround the oocyte. FSH acts syn-
and Puregon®, with the brand name of Follistim® in
ergistically with oestrogens and Luteinizing Hormone
the USA and Japan, manufactured by N.V. Organon,
(LH) to stimulate proliferation of these granulosa cells,
now part of Merck Sharp and Dohme. Regulatory
which leads to follicular growth. As the primary func-
authorities have issued two distinct International
tion of FSH in the female is the regulation of follicle
Non-proprietary Names (INN) for the three corre-
growth and development, this process explains why
sponding recombinant FSH drug substances, i.e. folli-
deficient endogenous production of FSH may cause
tropin alfa (Gonal-F®), follitropin beta (Puregon®/
infertility. In males, FSH plays a pivotal role in
Follistim®) and follitropin delta (Rekovelle®). In addi-
spermatogenesis.
tion, a few other recombinant-FSH preparations, ‘bio-
FSH preparations for infertility treatment are tra-
similar’ to follitropin alfa, were developed and are
ditionally derived from urine from (post) menopausal
available under the names: Bemfola®, Ovaleap®
women. As over 100,000 L of urine may be required for
(Rettenbacher et al. 2015).
a single batch, many thousands of donors are needed.
Hence, the source of urinary FSH is heterogeneous and
the sourcing cumbersome. Moreover, in addition to FSH IS A GLYCOPROTEIN HORMONE
FSH, these urinary preparations contain impurities
Follicle-stimulating hormone belongs to a family of
structurally related glycoproteins which also includes
luteinizing hormone (LH), chorionic gonadotropin
(CG), collectively called the gonadotropins and
T. Sam
thyroid-stimulating hormone (TSH, also named thyro-
GlobalPharmCMC, Heesch, The Netherlands tropin). These hormones belong to the ‘cystine-knot
protein family’. FSH is a hetero-dimeric protein con-
R. de Leeuw (*)
DeLeeuw Consultancy, Heesch, The Netherlands sisting of two non-covalently associated glycoprotein
e-mail: renato@deleeuwconsultancy.nl

© Springer Nature Switzerland AG 2019

D. J. A. Crommelin et al. (eds.), Pharmaceutical Biotechnology, https://doi.org/10.1007/978-3-030-00710-2_19
430 T. SAM AND R. DE LEEUW

Figure 19.1 ■ A three-dimensional model of FSH. The ribbons represent the polypeptide backbones of the α-subunit (green rib-
bon) and the β-subunit (blue ribbon). The carbohydrate side chains (violet and pink space filled globules) cover large areas of the
surface of the polypeptide subunits

subunits, denoted α and β. The α-subunit contains five essential for its biological activity since they (1) influ-
intra-subunit disulfide bonds and is identical for all ence FSH receptor binding, (2) play an important role
these glycoproteins, and it is the β-subunit (having six in the signal transduction into the FSH target cell, and
intra-subunit disulfide bonds) that provides each hor- (3) affect the plasma residence time of the hormone.
mone with its specific biological function. Recombinant FSH contains approximately one
The glycoprotein subunits of FSH consist of two third carbohydrate on a mass per mass basis. The car-
polypeptide backbones with carbohydrate side chains bohydrate side chains are composed of mannose,
attached to the two asparagine (Asn) amino acid resi- fucose, N-acetyl-glucosamine, galactose and sialic acid.
dues on each subunit. The oligosaccharides are Structure analysis by 1H-NMR-spectroscopy on oligo-
attached to Asn-52 and Asn-78 on the α-subunit (92 saccharides enzymatically cleaved from follitropin
amino acids), and to Asn-7 and Asn-24 on the beta, reveals minor differences with natural FSH. For
β-subunit (111 amino acids). The glycoprotein FSH instance, the bisecting GlcNAc residues are lacking in
has a molecular mass of approximately 35 kDa. For the recombinant molecule, simply because the FSH-
the FSH preparation to be biologically active, the two producing CHO cells do not possess the enzymes to
subunits must be correctly assembled into their three- incorporate these residues. Furthermore, the carbohy-
dimensional hetero-dimeric protein structure and drate side-chains of recombinant FSH exclusively con-
post-translationally modified (Fig. 19.1). tain α2–3 linked sialic acid, whereas in the natural
Assembly and glycosylation are intracellular pro- hormone α1–6 linked sialic acid occurs, as well.
cesses that take place in the endoplasmatic reticulum However, all carbohydrate side-chains identified in
and in the Golgi apparatus. This glycosylation process recombinant FSH are moieties normally found in other
leads to the formation of a population of hormone iso- natural human glycoproteins. The amino acid
forms differing in their carbohydrate side-chain com- sequences of the α-subunit and the β-subunit of the
position. The carbohydrate side-chains of FSH are recombinant FSH derived from human fetal retina, are
 19 FOLLICLE-STIMULATING HORMONE 431

identical to the sequence of natural human FSH and products are different. For Puregon®/Follistim® a series
CHO-derived FSH products in clinical use, while the of chromatographic steps, including anion and cation
sialic acid content is higher (Olson et al. 2014). exchange chromatography, hydrophobic chromatogra-
Whereas FSH only contains N-linked carbohy- phy and size-exclusion chromatography is used.
drates, human chorionic gonadotropin (hCG) also car- Recombinant FSH in Gonal-F® is obtained by a similar
ries 4 O-linked (at serine or threonine residues) process of five chromatographic steps, but also includes
carbohydrates, all located at the Carboxy Terminal an immunoaffinity step using a murine FSH-specific
Peptide (CTP) of its beta subunit. This glycosylated monoclonal antibody (from European Public
CTP is the major difference with the beta subunit of LH Assessment Report, EPAR Gonal-F 2011). In both pro-
and is demonstrated to be responsible for the much duction processes, each purification step is rigorously
longer plasma residence time of hCG compared to nat- controlled in order to ensure the batch-to-batch consis-
ural LH. (Matzuk et al. 1990). tency of the purified product.

PRODUCTION OF RECOMBINANT FSH DESCRIPTION OF RECOMBINANT FSH

The genes coding for the human FSH α-subunit and ■ Structural Characteristics
β-subunit were inserted in cloning vectors (plasmids) Like urinary sourced (natural) FSH, the recombinant
to enable efficient transfer into recipient cells. These versions exist in several distinct molecular forms (iso-
vectors also contained promoters that could direct hormones), with identical polypeptide backbones but
transcription of foreign genes in recipient cells. CHO with differences in oligosaccharide structure, in par-
cells were selected as recipient cells since they were ticular in the degree of terminal sialylation. These iso-
easily transfected with foreign DNA, and are capable hormones can be separated by chromatofocusing or
of synthesizing complex glycoproteins. Furthermore isoelectric focusing on the basis of their different iso-
they could be grown in cell cultures on a large scale. To electric points (pI, as has been demonstrated for folli-
construct a FSH-producing cell line, N.V. Organon, the tropin beta (De Leeuw et al. 1996), Fig. 19.2). The typical
manufacturer of Puregon®/Follistim®, used one single pattern for FSH indicates an isohormone distribution
vector containing the coding sequences for both sub- between pI values of 6 and 4. To obtain structural infor-
unit genes (Olijve et al. 1996). Merck Serono S.A., the mation at the subunit level, the two subunits were sep-
manufacturer of Gonal-F®, used two separate vectors, arated by RP-HPLC and treated to release the N-linked
one for each subunit gene (Howles 1996). Following carbohydrate side-chains. Fractions with low pI values
transfection, a genetically stable transformant produc- (acidic fractions) displayed a high content of tri- and
ing biologically active recombinant FSH was isolated. tetrasialo oligosaccharides and a low content of neutral
For the CHO cell line used for manufacturing and monosialo oligosaccharides. For fractions with a
Puregon®/Follistim® it was shown that approximately high pI (basic fractions) value the reverse was found.
150–450 gene copies were present. The β-subunit carbohydrate side chains appeared to be
To establish a master cell bank (MCB) identical more heavily sialylated and branched than the
homogeneous cell preparations of the selected clone
are stored in individual vials and cryopreserved until
needed. Subsequently a working cell bank (WCB) is 10
established by the expansion of cells derived from a recFSH
single vial of the MCB and aliquots are put in vials and 3,000
pH
8
cryopreserved, as well. Each time a production run is
recFSH, IU/ml

started cells from one or more vials of the WCB are

pH

2,000 6
cultured.
Both recombinant FSH products are isolated from
cell culture supernatants. These supernatants are col- 1,000 4
lected from a perfusion-type bioreactor containing
recombinant FSH-producing CHO cells grown on 2
microcarriers. This is because the CHO cell lines used 0 10 20 30
are anchorage-dependent cells, which implies that a Basic Fraction Acidic
proper surface must be provided for cell growth. The
reactor is perfused with growth-promoting medium Figure 19.2 ■ Isohormone profile of recombinant follicle stim-
ulating hormone (follitropin β beta) after preparative free flow
during a period that may continue for up to 3 months
focusing (De Leeuw et al. 1996). The FSH concentration was
(see also Chap. 4). The down-stream purification pro- determined by a two-site immunoassay that is capable of quanti-
cesses for the isolation of the two recombinant FSH fying the various isohormones equally well
432 T. SAM AND R. DE LEEUW

α-subunit carbohydrate side chains. The low pI value 500

Starting material
isohormones of follitropin beta have a high sialic acid/
galactose ratio and are rich in tri- and tetra-antennary pl 5.49 basic
100
N-linked carbohydrate side chains, as compared with pl 5.22
the side chains of the high pI value isohormones. pl 4.99
One of the tools for further characterization is the

FSH, IU/ml
immunoassay. Due to the specific recognition pl 4.75
10
characteristics of the antibodies used, this assay deter- pl 4.51
mines FSH-specific structural features and provides a pl 4.27 acidic
relative measure for the quantity of FSH, as it is not
sensitive to the differences in glycosylation. 1

■ Biological Properties of Recombinant FSH

Isohormones
0.1
A FSH preparation can be biologically characterized
0 40 80 120 160 200
with several essentially different assays, each having
Hours after injection
its own specific merits (Mannaerts et al. 1992). The
receptor binding assay provides information on the
Figure 19.3 ■ Kinetic behavior of FSH isoforms after a single
proper conformation for interaction with the FSH intramuscular injection (20 IU/kg) in beagle dogs
receptor. Receptor binding studies with calf testis
membranes have shown that FSH isoform activity in
follitropin beta decreases when going from high to low isoforms was higher than the absorption rates of all
pI isoforms. The in vitro bioassay measures the capa- other isoforms. The AUC and the clearance for the fol-
bility of FSH to transduce signals into target cells (the litropin beta preparation, being a mixture of all isohor-
intrinsic bioactivity). The in vitro bioactivity, assessed mone fractions, corresponded with the centre of the
in the rat Sertoli cell bioassay, also decreases when isohormone profile (Fig. 19.3). In contrast, the elimina-
going from high to low pI isoforms. The in vivo bioas- tion of the follitropin beta preparation occurred at a rate
say provides the overall bioactivity of an FSH prepara- similar to that of the most acidic fractions, indicating
tion. It is determined by the number of molecules, the that the elimination rate is largely determined by the
plasma residence time, the receptor binding activity removal of the most acidic isoforms from the plasma.
and the signal transduction. Interestingly, in contrast to Thus, for follitropin beta isohormone fractions, a
the receptor binding and in vitro bioassays, the in vivo clear correlation exists between pI value and pharma-
biological activity determined in rats shows an approx- cokinetic behavior. Increasing acidity leads to an
imate 20-fold increase between isoforms with a pI increase in the extent of absorption and elimination
value of 5.49, as compared to those with a pI of 4.27. half-life and to a decrease in clearance.
These results indicate that the basic isohormones
exhibit the highest receptor binding and signal trans-
PHARMACEUTICAL FORMULATIONS
duction activity, whereas the acidic isohormones are
the more active forms under in vivo conditions. This Recombinant FSH preparations distinguish themselves
notion also warrants pharmacokinetic studies to fur- from the earlier urinary FSH preparations by their high
ther characterize the biological properties of FSH purity (at least 99%). However, pure proteins are rela-
preparations. tively unstable and are therefore often lyophilized,
unless some specific stabilizing measures can be taken.
■ Pharmacokinetic Behavior of Recombinant FSH FSH preparations are available in different strengths
Isohormones and presentation forms, both as freeze-dried products
The pharmacokinetic behavior of follitropin beta and its (powder, cake) and as solution for injection. Follitropin
isohormones was investigated in beagle dogs that were alfa was originally formulated with sucrose (bulking
given an intramuscular bolus injection of a number of agent, lyoprotectant), sodium dihydrogen phosphate/
FSH isohormone fractions, each with a specific pI value. disodium hydrogen phosphate, phosphoric acid and
With a decrease in pI value from 5.49 (basic) to 4.27 sodium hydroxide (for pH adjustment). In 2002,
(acidic), the AUC increased and the clearance decreased, L-methionine (antioxidant) and polysorbate 20 (to pre-
each more than tenfold (Fig. 19.3). A more than twofold vent adsorption losses) were added to the single dose
difference in elimination half-life between the most formulation. Follitropin beta is formulated with
acidic and the most basic FSH isohormone fraction was sucrose, sodium citrate (stabilizer), polysorbate 20
calculated. The absorption rate of the two most acidic (lyoprotectant and agent to prevent adsorption losses),
 19 FOLLICLE-STIMULATING HORMONE 433

and hydrochloride/sodium hydroxide (for pH adjust- with 100–200 IU of recombinant FSH followed by
ment). The lyophilized preparations are to be reconsti- maintenance doses of 50–350 IU. The availability of a
tuted before use to obtain a ready-for-use solution for surplus of collected oocytes allows the vitrification of
injection. In addition to the freeze-dried presentation embryos for replacement in frozen-thawn embryo
form, a solution for injection with several strengths of transfer (FTET) cycles. Similar treatment regimens are
follitropin beta could be developed. To stabilize the recommended for Gonal-F®.
solutions 0.25 mg of L-methionine had to be added. After subcutaneous administration, follitropin
Furthermore, the solution in the cartridge contains beta has an elimination half-life of approximately 33 h
benzyl alcohol as preservative. For follitropin alfa a (Voortman et al. 1999). Steady-state levels of follitropin
multidose solution for injection in a pre-filled pen beta are therefore obtained after four to five daily doses
became available in 2004. This solution contains polox- reaching therapeutically effective plasma concentra-
amer 188 instead of polysorbate 20 and m-cresol has tions of FSH. Follitropin β is administered via the sub-
been added as preservative. cutaneous route with good local tolerance.
The Puregon®/Follistim® solution for injection is Bioavailability is approximately 77%. In a large fraction
available in vials and is very suitable for titration of patients treated with follitropin β, no formation of
because of the large range of available strengths as antibodies against recombinant FSH or CHO-cell
expressed in IU’s. Pen injectors have been developed derived proteins was observed. Injections of the follitro-
with multidose cartridges containing solution for injec- pin β preparations can be given by the patient herself or
tion, giving the patient improved convenience. her partner.
The solutions for injection should be stored in the
refrigerator for a maximum of 3 years with the con-
A NEWLY DEVELOPED FSH ANALOG
tainer kept in the outer carton to protect the solution
from light. The patient can keep the solutions at room The need for daily injections of FSH, especially in
temperature for a maximum of 3 months. The multi- combination with GnRH agonists, is a burden for the
dose solution of follitropin α has a shelf-life of 2 years women treated in an ART regimen. Therefore, several
and can be stored for 1 month at room temperature. different approaches have been undertaken to arrive
at FSH preparations that need fewer injections, such as
slow release formulations, addition of N-linked carbo-
CLINICAL ASPECTS hydrates and other chemical modifications including
Recombinant FSH products on the market have been pegylation (Fauser et al. 2009). An elegant approach
approved for two female indications. The first indica- pioneered by Irving Boime and collaborators (Fares
tion is anovulation (including polycystic ovarian dis- et al. 1992; LaPolt et al. 1992), is based on the longer
ease) in women who are unresponsive to clomiphene in vivo half-life of hCG compared to LH. Using genetic
citrate (an estrogen receptor modulator). The second engineering, the beta subunit of FSH was extended by
indication is controlled ovarian hyperstimulation to one or two CTPs of hCG. It was demonstrated that
induce the development of multiple follicles in medi- fewer injections with preparations containing such
cally assisted reproduction programs, such as in vitro molecules were needed to induce similar pharmaco-
fertilization (IVF) and intracytoplasmatic sperm injec- dynamic effects in laboratory animals. Subsequently, a
tion (ICSI). In addition, recombinant FSH may be used new cell line was generated by Organon (now part of
in men with congenital or required hypogonadotropic Merck Sharp & Dohme) that produced corifollitropin
hypogonadism to stimulate spermatogenesis. alfa (the INN of this molecule), an FSH analog in
For the treatment of anovulatory patients (aiming which the beta subunit was extended by a single CTP
at monofollicular growth) it is recommended to start (28 amino acids). Thorough biochemical analysis dem-
Puregon®/Follistim® treatment with 50 IU per day for onstrated the expected amino acid sequence of the
7–14 days and gradually increase dosing with steps of alpha subunit and the extended beta subunit, but
50 IU if no sufficient response is seen. This gradual revealed two additional O-linked glycosylation sites
dose-increasing schedule is followed in order to pre- in corifollitropin alfa (Henno van den Hooven, Ton
vent multifollicular development and the induction of Swolfs, personal communication) compared to the 4–5
ovarian hyperstimulation syndrome (a serious condi- sites reported in hCG (Fig. 19.4). Nonclinical evalua-
tion of unwanted hyperstimulation). In the most com- tion demonstrated that the receptor binding and trans-
monly applied treatment regimens in IVF, endogenous activation profile of this new molecular entity was
gonadotropin levels are suppressed by a GnRH agonist specific and comparable to that of rec-FSH without
or by the more recently approved GnRH antagonists intrinsic TSH-receptor or LH-receptor activation.
(Cetrotide® and Orgalutran®/ganirelix acetate injec- However, the in vivo half-life was increased 1.5- to
tion). It is recommended to start Puregon® treatment 2-fold in the species tested and a 2-fold to 4-fold
434 T. SAM AND R. DE LEEUW

Figure 19.4 ■ A three-

dimensional model of corifolli-
tropin alfa. The ribbons
represent the polypeptide back-
bones of the α-subunit (green
ribbon) and the β-subunit (blue
ribbon). The carbohydrate side
chains (pink and yellow space
filled globules) represent
N-linked and O-linked carbohy-
drates. The sialic acid carbohy-
drates are depicted in yellow.
The arrows indicate additional
O-linked carbohydrate sites.
Courtesy MLCE Kouwijzer and
R Bosch

increase of bioactivity was found across all in vivo (Norman et al. 2011), despite being a fusion protein.
pharmacodynamic parameters tested (Verbost et al. Hence, by virtue of its ~twofold increased in vivo half-
2011). These observations were corroborated by a very life, corifollitropin alfa has demonstrated to provide a
extensive data set obtained in a broad panel of clinical valuable alternative for FSH by acting as a sustained
trials (phase I, II and III), including the largest com- follicle stimulant. Elonva® is approved (EU) for
parator controlled trial of its kind in fertility (the com- Controlled Ovarian Stimulation (COS) in combination
parator being recFSH) (Devroey et al. 2009; Fauser with a GnRH antagonist for the development of mul-
et al. 2010). A single subcutaneous dose of corifollitro- tiple follicles in women participating in an assisted
pin alfa (Elonva®) can be used to initiate and sustain Reproductive Technology (ART) program. It is sup-
multifollicular growth for 7 days while the efficacy plied in pre-filled syringes equipped with an automatic
and safety of this novel biopharmaceutical were simi- safety system to prevent needle stick injuries after use
lar to that of daily injections with recombinant and is packed together with a sterile injection needle.
FSH. Whereas normally more than 7 days of FSH treat- Each prefilled syringe contains 0.5 mL solution for
ment has to be given after the first injection, in about injection (Fig. 19.5).
one third of the women treated with FSH-CTP no FSH provides a great example of the evolution of
additional FSH treatment was needed. biopharmaceuticals, starting from the natural form
Dedicated clinical research revealed no clinically (urine-derived), via close imitations thereof (recombi-
relevant immunogenicity against the FSH analog nant FSH) towards further improved biopharmaceuti-
 19 FOLLICLE-STIMULATING HORMONE 435

Plunger Syringe Solution Syringe cap Needle cap Needle Needle shield Figure 19.5 ■ Pre-filled
syringe with corifollitropin alfa
solution to be assembled with a
needle assembly. The syringe
is equipped with an automatic
Label perforation safety system to prevent nee-
dle sticking

cals (FSH analogs, corifollitropin alfa being the only Matzuk MM, Hsueh AJ, Lapolt P, Tsafriri A, Keene JL, Boime
CTP form that made it to the market). Such develop- I (1990) The biological role of the carboxy-terminal
ments in pharmaceutical biotechnology are clearly to extension of human chorionic gonadotropin (cor-
the benefit of the patients in need for effective, safe, rected) beta-subunit. Endocrinology 126:376–383
Norman RJ, Zegers-Hochschild F, Salle BS, Elbers J, Heijnen E,
and convenient treatment options.
Marintcheva-Petrova M, Mannaerts B (2011) Repeated
ovarian stimulation with corifollitropin alfa in patients
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